KR920008744B1 - Monoclonal antibodies to hiv and related peptides - Google Patents

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Description

Monoclonal Antibodies and Peptides for the Treatment and Diagnosis of HIV Infection

The present invention generally relates to the treatment, diagnosis and prevention of viral infections. In particular, the present invention relates to compositions and methods for the preparation of monoclonal antibodies and peptides for use in diagnosing, neutralizing and immunizing human immunodeficiency virus (HIV) infections.

The present invention, which will be described in detail below, is previously identified as part of the application of US Patent Application Nos. 898, 273, August 20, 1986 and US Patent No. 045, 026, May 1, 1987.

The pathogens that cause acquired immunodeficiency syndrome (AIDS) and its prostates, AIDS related complexes (ARC), and pharyngeal nephropathy (LAS) are novel lymphotropic retroviruses. The virus is called variously, such as LAV, HTLV-III, ARV, and most recently, HIV.

The primary concern is the spread of HIV throughout the world, preventing the spread of infection to uninfected people facing the treatment and exposure risks of infected individuals. Different treatments have focused on different stages of the virus's life cycle, which are outlined in Mitsya and Broder's 1987, Nature 325: 773. One approach is to use antibodies that bind to the virus to prevent viral invasion into the host cell or to inhibit the replication of the virus by other mechanisms. If susceptible viral components are identified by antibody intervening, manual administration or prophylactic concentration of monoclonal antibodies or immunoglobulins with the required specificity may result in sufficient antibody titers to neutralize viral infection. It is considered to be.

Envelope glycoproteins of most retroviruses are thought to react with subtractor molecules on the surface of sensitive cells and therefore measure viral infectivity against a host. Antibodies bound to glycoproteins can interfere with virus-cell interceptor interactions to neutralize the infectivity of the virus (generally, The Molecular Biology of Tumor Viruses, 534 (J. Tooze, ed., 1973) and RNA Tumor Viruses, 226, 236 (R. Weiss et al., Eds., 1982), and Gonzalez-Scarano et al., 1982, Virology 120: 42 (La Crosse Virus); Matsuno and Inouye, 1983, Infect. 39: 155 (Neonatal Calf Diarrhea Virus); and Mathews et al., 1982, J. Immunol., 129: 2763 (Encephalomyelitis Virus).

The general structure of HIV consists of a lipid-containing envelope obtained during virus budding from the membrane of a virally infected host cell and the core of ribonucleic acid protein surrounded by it. The envelope glycoproteins of HIV are first synthesized as 150,000-160,000 daltons of precursor molecules (gp 150 or gp 160) in infected cells, and then 110,000-120,000 daltons of N-terminal fragment (gp) to produce extraglycoproteins in cells. 110 or gp 120) and C-terminal fragments (gp 141) of 41,000-46,000 daltons of transition membrane envelope glycoprotein.

For the reasons mentioned above, the gp 110 glycoprotein of HIV has been the subject of numerous research as a powerful target for disrupting the life cycle of the virus. Serum from HIV infected individuals has been shown to neutralize HIV in vitro and antibodies that bind to purified gp 110 are present in serum (Robert-Guroff, et al., 1985, Nature 316: 72; Weiss et. al., 1985, Nature 316: 69; and Mathews et al., 1986, Proc. Natl. Acad. Sci, USA, 839709). Purified and recombinant gp 110 promoted the production of neutralized active antibodies when used to immunize animals (Robey et al., 1986, Proc. Natl. Acad. Sci. USA, 83: 7023; Lasky et al., 1986, Science 233: 209; and a human, Zagury et al, 1986, Nature 326: 249). It was also found that the gp 110 molecule binds to the CD4 (T4) receptor, and monoclonal antibodies that recognize a special epitope of the CD4 receptor have been shown to interfere with HIV binding, follicle formation and infectivity (McDougal et al. , 1986, Science 231: 382). Putney et al. (1986, Science 234: 1392), immunized with recombinant fusion proteins containing half of the carboxyl termini (1/2) of gp 110 molecules, induced immobilized serum antibodies and It has been shown that glycosylation is not required for neutralized antibody reactions.

Thus, subunit waxes for AIDS using HIV gp 110 molecules or portions thereof may be required. Subunit waxes are wax substitutes prepared from inactivated or attenuated viruses. Inert waxes are difficult because they may fail to kill all virus particles, and weakened viruses may mutate and regain their ability to develop. Only viral parts containing antigens or epitopes capable of inducing an immune response, ie, antibodies, ADCC and cytotoxin T-cell responses, are used to immunize the host. The main advantage of subunit waxes is that they exclude sugar virus material. Virus subunits for waxing can be produced in a variety of ways. For example, most of these molecules lack post-translational mutations (glycosylation) or other processing, but envelope glycoproteins can be expressed and purified from bacterial hosts. Such mutations can be obtained using mature nucleus expression devices such as yeast or cultured mammalian cells. Viral genes are introduced into mammalian cells using waxy viruses as vectors (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 wax virus can be constructed according to the method of Hu et al. Co-investigator [Nature 320: 537 (1986)] or Checkrabati et al. Collaborator [Nature 320: 535 (1986)]. Viral glycoproteins produced by cells infected with recombinant waxes in these devices can be appropriately glycosylated and transferred to cell tables for extraction and final isolation.

An important step in the production of the subunit waxes is to appropriately purify the desired glycoprotein from the mixture of complexes of the expression apparatus. Various methods can be used to carry out such purification. Examples include, but are not limited to, preliminary polyacrylamide gel electrophoresis, gel permeation chromatography, and various chromatographic methods (eg, ion exchange, reverse phase, immunoaffinity, hydrophobic interactions). Most of these methods are used in various combinations to obtain substantially pure formulations (Kleid, DG, et al., 1986, Science 214: 125; Cabradilla, CD, et al., 1986, Biotechnology 4: 218, Dowbenko, DJ, 1985, Proc. Nat. Acad. Sci. USA 82: 7748).

A method of reducing the number of processes required to obtain the highest purification effect of a particular viral antigen from a complex expression mixture is needed to make a subunit wax. Methods for effectively separating antigens from extra components can be performed using immunoaffinity chromatography. Also known as immunoabsorption, the technique consists of the principle of selectively absorbing an antigen into a solid aid to which a specific antibody is covalently attached. The selectively absorbed antigen is eluted from the affinity absorbent of the antibody, for example by changing the strength of the pH and / or ions of the buffer.

Polyclonal antibodies obtained from animals immunized with the desired antigen or from naturally infected individuals (see, eg, Raski et al., Co-workers) have sometimes been used as immunoabsorbers. Generally, however, these reagents require additional purification because not all of the antibodies bound to the insoluble support are specific for the antibody of interest, the yield of the desired antigen is low, and the affinity of the antibody often varies from formulation to formulation. Therefore, there are practical disadvantages such as the need to change the elution process. In addition to polyclonal antibodies, such challenges can be avoided by using monoclonal antibodies that have special effects on the desired viral antigens used in subunit wax formulations.

Murine monoclonal antibodies that bind to HIV antigens have been described. Several research groups have reported monoclonal antibodies specific for the central protein p25 (di Marzo Veronese, et al., 1985, Proc. Nat. Acad, Sci. USA 82: 5199 and Chassagne, J., et al., 1986, J. Immunol. 136: 1442). Specific monoclonal antibodies against the membrane glycoprotein gp 41 have also been reported (see, for example, di Marzo Veronese, et al., 1985, Science 229: 1402).

There is a need for a description of specific monoclonal antibodies for epitopes in well defined bands of the major envelope glycoprotein gp 110. Monoclonal antibodies that bind to these zones and reduce or eliminate the replication and deliverability of HIV have substantial therapeutic and prophylactic utility. Moreover, monoclonal antibodies can also be used to purify the desired zone of gp 110 from ruptured viruses or recombinant expression devices for use in waxing, for example. In addition, chemically synthesizing zones containing epitopes recognized by monoclonal antibodies can avoid the inherent difficulties in purifying and administering large fragments of gp 110 molecules. The present invention satisfies this and other related requirements.

Peptides that can immunologically mimic the neutralized epitopes of HIV proteins, nucleic acid probes encoding such peptides, and monoclonal antibodies reactive with these peptides, as well as other peptides that prevent HIV infection, are also provided. Such new substances are used, for example, in diagnostic assays to detect HIV infections and in the therapeutic field for treating or vaccinating such infections.

The present invention provides methods and new compositions that neutralize HIV infection, ie, prevent or inhibit the production of HIV or the conversion of cells into the host. In particular, monoclonal antibodies that respond to these bands and peptides that mimic the bands that neutralize HIV are used to diagnose, treat and vaccinate HIV infection. In this sense, the term “bandoning band” refers to an HIV that contains an amino acid fragment that defines an amino acid fragment of one or more epitopes that are combined with other antibodies of the invention or that are individually reactive with antibodies that can neutralize HIV infection. Part, especially HIV protein. Suitable neutralization assays are well known and include reduction of HIV infection in T-cell lines, reduction of plaque forming units of VSV (HIV) pseudotypes containing the envelope glycoprotein of HIV, fusion cell inhibition and virion. Includes submerger binding tests. As desired, the neutralizing activity can be compared with antibody reactivity in immunochemical tests such as immunofluorescence, immunospot and radioimmunoprecipitation assays.

On the other hand, new peptides, usually of less than about 50 amino acids, contain five or more contiguous amino acids that form epitopes that are substantially similar to epitopes located in the neutralization band of HIV gp 110 or p 25 (recorded in env or gag bands, respectively) of the HIV genome. It contains. Of particular importance are those in the extended band of amino acid residues about 301-about 336, about 278-about 319 and p25 of about 315-about 363 of gp 110, all of which originated from HIV strains designated LAV _BRUs . Amino acid residue designations are those specified in the Los Alamos Data Bank (AIDS Virus Sequence database, Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 87545).

One of ordinary skill in the present invention may identify additional analogous bands (homologs) originating from other HIV isolates depending on their position in related proteins from various isolates. It can be seen that the same LAV _BRU array data can be identified:

아미노산 The amino acid sequence of HIV isolates and LAV _BRUs can be aligned to obtain maximum homogeneity between the two sequences.

펩티드 Peptides that make up the amino acid sequence of the HIV isolate corresponding to the position of the LAV _BRU peptide that immunologically mimics the LAV _BRU protein can be identified. The peptides constituting the HIV isolate amino acid sequence thus identified will immunologically mimic the corresponding HIV isolate protein.

This method can be applied to already developed HIV strains. For example, a new strain of HIV check being shell and center the amino acid sequence of these could be aligned to the LAV _BRU to obtain the maximum analogs. Arrangement methods are well known to those skilled in the art. In order to align, homogeneity between cysteine residues needs to be kept to the maximum possible. The amino acid sequence of the new HIV strain or species corresponding to the position of the peptide described above can be synthesized and used according to the present invention.

Another method of determining the arrangement of a homologue band in other HIV strains is described by Scharf et al. In Science (1986) 233: 1076. This method uses two oligonucleotide primers that bind to an out-of-band conserved arrangement of related sequences and contain different restriction sites within each primer. Oligonucleotides produced after DNA from HIV strains have been propagated in vitro and can be asexually reproduced in vectors for analysis of the alignment, such as cassettes that display special epitopes from HIV strains. It can be mixed in wax.

In the present invention, epitopes contained in this arrangement need not cross react with antibodies against all strains or species of HIV. Peptides that surround an immunological epitope identifying one species or another serogroup are used to identify a particular species or serogroup and may help identify individuals infected with one or more species or serogroups of HIV. They can also be used in combination with other peptides from the homologue zone or other neutralization zones in therapeutic compositions.

The most important peptide is most preferably derived from the gp 110 band of the virus. In this band, the most important are the peptides recorded in the env open reading frame, extending from about 6667 to about 6774 base pairs (bp) of the LAV _BRU isolate. Thus, various homologous bands of other HIV isolates include homologous sequences obtained from the Los Alamos Data Bank (except for LAV2), as described in Table 1 below. Other peptides suitable for generating or selecting monoclonal antibodies include those recorded in the env openreading frame from about bp 7246 to about 7317 of the LAV _BRN .

TABLE 1a

TABLE 1b

Such antibodies and reactive peptides are particularly useful for immunoassays.

The p25 amino acid sequence of about 278-319 and 315-363 in the gag band of the LAV _BRU isolate is an additional neutralization band of HIV. Those skilled in the art can identify additional disabling zones of HIV based on those described herein, in particular mixing with monoclonal antibodies that react with several different HIV epitopes exhibits disabling action.

Peptide (I) designated as peptide 29 is also recorded in the env open reading frame of amino acid residues of about 308 to about 328 and has the following amino acid sequence, wherein the oligopeptides contained in the next amino acid sequence are linear epitopes within this sequence. It will include.

In the above arrangements, Y and Y ', when present, each represent up to about 20 amino acid sequences. If Y and / or Y 'are present, they may, for example, be composed of one or more amino acids from an arrangement flanking the amino acid residues 308 to 328 of the HIV envelope arrangement or from any portion of these flanking arrangements. In one embodiment, Y may consist of all or a portion of a LAV _BRU envelope amino acid sequence having residues of about 301-307 and Y 'may comprise all or a portion of a LAV _BRU envelope amino acid sequence of residues of about 329-336, wherein Can be configured.

Alternatively, truncated arrays of the peptides of the invention can be obtained. In this regard the following arrangements obtained from peptides 29 are particularly useful.

Y and / or Y ', when present, represent an array with up to about 20 amino acid residues each, if present.

Where Y and Y ', if present, represent an array with up to about 20 amino acid residues each.

As another example, the homologous bands of the particularly important ARV-2 isolates are recorded in an env open reading frame with amino acid residue numbers from about 306 to about 323, wherein the oligopeptides contained in the following amino acid sequences are linear epitopes within that array. Will include.

In the above, Y and Y ', if present, each represent up to about 20 or more amino acid residues. When Y and / or Y 'are present they may be composed of one or more amino acids from an arrangement flanking the amino acid residues 306-323 of the ARV-2 envelope arrangement or from any portion of these flanks. In particular, Y may consist of all or a portion of an HIV envelope amino acid sequence having about 299-306 residues, and Y 'may consist of all or a portion of an HIV envelope amino acid sequence having about 324-333 residues.

Alternatively, a truncated array of peptides (V) can be obtained. The following arrangement is particularly useful in this regard.

When Y and / or Y 'are present in the above formula, each represents an array with up to 20 or more amino acid residues.

Another embodiment consists of the same band as the LAV-2 isolate encoded during env open readings, wherein the number of amino acid residues is about 311-330, with the following arrangement.

Above, when Y and / or Y 'are present, each represents an array with up to 20 or more amino acid residues (see Nature 326: 662 (1987)).

A further feature of the present invention provides a novel cell line capable of producing monoclonal antibodies and a composition comprising such antibodies, wherein the antibody comprises envelope glycoprotein gp 110 or p 25, their protein precursors, and biologically expressed recombinants. The highest potency (from 10 ^2, from 10 ⁴ to about 10 ⁷ ) or higher in the neutralized zone contained in the predetermined role of the fusion protein and the synthetic peptide containing one or more epitopes within the predetermined constellation band of gp 110 or p 25 It can be recognized selectively. Hybrid cells of the present invention have a chromosome that can be identified and bacterial strain DNA is arranged to record antibodies having binding sites for epitopes on gp 110 or p 25 that are common to several or all HIV clinical isolates.

These monoclonal antibodies can be used in a wide variety of fields, including diagnosis and treatment, as well as identifying other cross-reactive antibodies such as antibody blocking. Peptides or polypeptides containing epitopes to which they react have been found to have separate use as waxing immunogens or therapeutic agents.

[Blocking peptide]

Another embodiment of the present invention, mainly for use in admixture with the above peptides or to neutralize monoclonal antibodies, is to use additional peptides or antibodies that interfere with HIV binding to the subpopulators to further mitigate HIV infectivity. Preferably monoclonal antibodies specific for epitopes contained in such blocking peptides as well as so-called "blocking peptides" capable of inhibiting virus proliferation can be used to enhance the effectiveness of the treatment against HIV infection. HIV blocking peptides are directed to the amino acid sequence of HIV essential for binding the virus to host cells, such as about 190-about 197 of LAV _BRU and about 185-about 192 env-coding amino acid residues of ARV2 and HTLV-III (BH-10). Corresponding. These include peptide T otopeptides (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) and various derivatives thereof (e.g., envelope glycoproteins (gp 110 or 120) described by the following IX and Pat et al co-investigators). Homologues (e.g., XI) (1986, proc, Natl. Acad. Sci USA 83: 8254-9258).

For example, blocking peptides having the following configuration are particularly important, with NH ₂ -terminals being acetylated and COOH terminal groups being amidated.

In each of the above peptides Y and Y ', if present, each have an amino acid sequence with up to about 20 amino acids. Epitopes or epitopes in these peptides usually have at least about 5 or more contiguous amino acids, and are used, for example, to simulate naturally occurring HIV antigen positions for the production of HIV reactive antibodies and waxes.

Production of Monoclonal Antibodies

Monoclonal antibodies can be prepared by immortalizing the expression of a nucleic acid sequence encoding an antibody specific for HIV and introducing such an arrangement, typically a cDNA encoding the antibody, into a cultivable host. Immortalized cell lines can be mammalian cell lines transformed through oncogenesis by transfection, mutation, and the like. Such cells include myeloma, lymphoma or other cell lines capable of supporting the expression and secretion of antibodies in vitro. The antibody may be a mammalian naturally occurring immunoglobulin produced by producing a hybrid cell line by transforming lymphoma, in particular splenocytes, with a virus or by fusing lymphocytes with neoplastic cells, such as myeloma. Typically, splenocytes can be obtained from animals immunized against the HIV virus or fragment thereof containing an epitope site.

Immunization protocols are well known and can make significant changes while maintaining their effectiveness (see Goding, Monoclonal Antibodie: Principles and Practice, Academic Press, 2nd edition (1986)). Bacterial fusion proteins, synthetic peptides and ruptured viruses containing antigen fragments of gp 110 or p 25 molecules can be used as immunogens. Preferably the immunogen of the ruptured virus, peptide or recombinant protein is enriched for a protein or fragment thereof that contains the epitope in which the antibody producing B cell or splenocyte is desired. In particular, solutions containing ruptured viral lysates or extracts or supernatants of biologically expressed recombinant proteins or ruptured expression vectors are enriched for the desired glycoproteins using purification methods such as, for example, polyacrylamide gel electrophoresis. You can. Lactin affinity purification is an excellent convenient method for purifying gp 110 and other glycoproteins (eg, affinity purification using lentilactin). The range of purifying glycoproteins from a solution used as an immunogen can vary widely, for example 50% or less, or usually at least 75-95%, preferably 95-99%, most preferably pure homogenate. .

The protein can be suspended or diluted in a suitable medical carrier for immunization while purifying the protein to the desired purity and can also be combined with an adjuvant. For example, a suitable technique is to absorb proteins and fragments thereof in injectable lentil lactin agarose or other macromolecular carriers. An immunogenic amount of an antigen-enhanced antigen preparation to an HIV protein comprising a gp 110 glycoprotein and a p 25 core protein or antigenic portion thereof can generally be injected at a concentration ranging from 1 ug-20 mg / kg of the host. Administration methods include injection, for example, intramuscular injection, intraperitoneal injection, subcutaneous injection, intravenous injection and the like. It can be administered once or several times, and can usually be administered at intervals of 1-4 weeks. Immunized animals were examined to produce antibodies against the desired antigen, followed by removal of the spleen, followed by transformation by isolation of spleen B-lymphocytes and fusion with myeloma cell lines. Transformation or fusion is carried out by known methods, and fusion techniques are described in various patents, for example in US Pat. Nos. 4,172,124, 4,350,683, 4,363799, 4,381,292 and 4,423,147, and the like. Please refer to the monoclonal antibody (1980) by Kenneth et al.

Immortalized cell lines can be cloned and screened according to conventional techniques and the antibody in the cell supernatant can bind to the desired gp 110 or p 25 HIV virus protein, either a recombinant fusion protein or a synthetic protein containing the desired epitope band. Was detected. Suitable immortalized cell lines can then be grown in vitro or injected intraperitoneally in a host suitable for producing ascites fluid. For example, the LAV _BRU genome of about bp 6688 to about bp 6750 (recorded peptide 29fh) or about bp7246 to about 7317 (recorded peptide 36) (bp values given according to Wayne-Hobson et al., Cell 44: 9 1985). Several antibodies of the invention known to be specific for epitopes contained within the band encoded by the band can be used to competitively screen the supernatant with the present monoclonal antibodies in a competitive assay. Thus additional immortalized hybrid cell lines with the necessary binding properties can be readily obtained from various raw materials based on the availability of the antibodies of the invention specific for specific antigens. Alternatively, these cell lines can be fused with other tumor B-cells and these B-cells can act as submergers for genomic DNA encoded against the antibody.

Rodents, in particular murine tumor B-cells are suitable, but other mammals such as lagomorpha, cattle, sheep, horses, pigs, birds and the like can also be used. Immunization of these animals can be easily performed and their lymphocytes, especially splenocytes, can be obtained for fusion.

Monoclonal antibodies distributed by transformed or hybrid cell lines are any of the group or subgroups of immunoglobulins such as IgM, IgD, IgA, IgG _1-4 or IgE. This is appropriate because IgG is the most common isotype used in diagnostic assays. Monoclonal antibodies are used in their native form or as fragments such as Fv, Fab, F (ab ') ₂ , but can usually be used in their own native form.

In order to avoid possible antigenicity in the human host of monoclonal antibodies derived from animals other than the human body, a mutant antibody can be constructed in which antigen-binding fragments of immunoglobulin molecules (several bands) are produced by human immunoglobulin due to peptide defects. It is linked to at least another protein that is not recognized as a foreign body by the human body, such as the molecule's input. This can be done by fusing exons of the human kappa or gamma always part with different bands of the animal. Various techniques for this are known in the art and are described, for example, in PCT 86/01533, EP 1711496 and EP 173494.

In addition, monoclonal antibodies of the present invention that exhibit a neutralizing effect, such as reacting with epitope sites on gp 110 or p 25 or with blocking peptides, can be mixed with components of a pharmaceutical composition to attenuate HIV infection. The composition should contain a pharmacologically effective carrier and at least one of the monoclonal antibodies of the invention in an amount suitable for treatment or prevention. The pharmacological carrier should be a compatible non-toxic substance suitable for delivering monoclonal antibodies to the patient. Sterile water, alcohols, fatty waxes and inert solids can be used as the carrier. Pharmaceutically acceptable auxiliaries (buffers, dispersants) may also be added to the pharmaceutical composition. Such compositions may contain, for example, a single monoclonal antibody that is effective against strains of HIV and for example envelope glycoproteins containing epitope sites in the band encoded by bp 6688-bp 6750. Alternatively, the pharmaceutical composition may form a 'cocktail' by containing one or more monoclonal antibodies. For example, cocktails containing monoclonal antibodies against various strains of HIV will be a universal product with therapeutic or preventive action against the majority boom of clinical isolates of HIV. This cocktail may contain monoclonal antibodies that bind to proteins or glycoproteins of HIV other than gp 110 or p 25, such as gp 41 glycoprotein or p 34 nuclease / integrase. The molar ratio of the various monoclonal antibody components is usually not more than 10 times and preferably not more than 5 times, and the molar ratio of the components of each other antibody is preferably about 1: 1-2.

The monoclonal antibodies of the invention can be used as separate dosage compositions in admixture with other antilitroviral agents, including blocking peptides. For recent developments in anti-retroviral and specific anti-HIV agents, see Mitsiya et al. (Nature 325: 773-778, 1987).

Monoclonal antibodies, peptides and pharmaceutical compositions thereof of the present invention are particularly useful for oral or parenteral administration. The pharmaceutical composition is preferably administered parenterally, ie, subcutaneously, intramuscularly or intravenously. Thus, the present invention provides a composition for parenteral administration consisting of a solution of a monoclonal antibody, peptide or a cocktail thereof dissolved in an acceptable carrier (with an aqueous carrier). Various aqueous carriers can be used, such as water, buffers, 0.4% saline, 0.3% glycine and the like. These solutions are sterilized and generally do not contain particulate matter. These compositions can be sterilized by conventional methods by known sterilization techniques. These compositions may contain pharmaceutically acceptable auxiliaries such as sodium acetate, sodium chloride, potassium chloride, sodium lactate, etc. required for appropriate medical conditions such as pH control and buffers, toxicity inhibitors and the like. The concentration of antibodies in such formulations varies widely, for example, from about 0.5% by weight or less (typically or at least about 1% by weight) to 15 or 20% by weight, mainly determined by the volume, viscosity, etc. of the liquid. It should be decided according to the type of special administration selected. Thus, typical pharmaceutical compositions for intramuscular injection can be prepared by containing 1 ml of sterile neutral water and 50 mg of monoclonal antibody. Representative compositions for intravenous injection can be prepared by containing 250 ml of sterile Ringer solution and 150 mg of monoclonal antibody. Substantial methods for preparing compositions for parenteral administration are well known to those skilled in the art and are described in detail, for example, in Remington's Pharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pennsylvania (1980).

Monoclonal antibodies and peptides of the invention are lyophilized for storage and then mixed in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulins and known lyophilization and reconstitution techniques can be used. Lyophilization and reconstitution techniques are the major causes of altering the degree of activity loss of antibodies (e.g. conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies). It is well recognized by those skilled in the art.

Compositions containing monoclonal antibodies, peptides or cocktails thereof of the present invention may be administered for the prophylaxis and / or treatment of HIV infection. The therapeutic composition is administered to a patient already infected with HIV in an amount sufficient to treat or at least partially inhibit the infection and its complications. A reasonable amount to do this is defined as the “therapeutically effective amount”. The effective amount for such uses depends on the degree of infection and the general condition of the patient's own immune tissue. Generally, however, about 1-200 mg of antibody per kilogram of body weight and a more preferred dosage is 5-25 mg per kilogram of body weight. The materials of the present invention are generally used in critically ill patients with life critical conditions. In such cases, such antibodies may be administered in substantially excess amounts, but should be prescribed by the therapist.

The prophylactic composition containing the peptide, the antibody or a cocktail thereof of the present invention enhances the patient's resistance to viral infection not only in patients already infected with HIV but also in patients who have recently been exposed to, thought to be at risk or exposed to the virus. It is administered either for inoculation or for vaccination against the virus. The amount is called the "preventive effective amount". Suitable amounts for this use will vary depending on the patient's state of health and general immunity, but are generally from 0.1 mg to 25 mg per kilogram of body weight, particularly from 0.5 mg to 2.5 mg.

Single or multiple administration of this composition can be carried out at a dosage and dosage form selected according to the therapist's prescription. In any case, the medicament provides an amount of the antibody of the invention sufficient to effectively treat the patient.

On the other hand, the use as a target-specific transport molecule can be found. Antibodies may bind to toxins to form immunotoxins or radioactive materials, or may bind to drugs that form radiopharmaceuticals or over-the-counter drugs. Methods of making immunotoxins and radiopharmaceuticals are well known (eg Cancer Treatment Reports 68: 317 (1984)).

In addition, human T-cell activators such as heterologous aggregates of monoclonal antibodies of the invention and monoclonal antibodies against CD3 antigens or Fcgamma subpopulators on T-cells may be used to inhibit cell disruption (ADCC) by the antibody. It gives the human T-cell or Fc-gamma-bearing cells (such as K cells or Neutrotyl) the ability to kill HIV-infected cells through. Such heterologous aggregates can covalently bind anti-HIV antibodies to anti-CD3 antibodies using, for example, the heterodifunctional N-succinimidyl-3- (2-pyridyldithiol propionate) ( Karpowsky et al., J. Exp. Med. 160: 1686 (1084).

The subject peptide composition itself may also find therapeutic uses that can be reduced or eliminated in infected hosts by administration. Compositions such as peptide 29, blocking peptide and peptide 126, the latter of which are described in commonly pending US patent applications 930, 785, may be combined with suitable medical carriers intravenously, subcutaneously, intramuscularly, intraperitoneally. It can be administered by the same route. Phosphate buffered saline, saline, water, potassium chloride, sodium lactate and the like can be used as the carrier.

The concentration of peptides varies widely depending on the end use, activity and dosage form. The peptides preferably have COOH-terminal amidation, NH ₂ -terminal formylation or other pharmacologically acceptable derivatives. The addition of a blocking peptide to the peptide mimicking the neutralized HIV band and / or the post-reactive antibody of the present invention can significantly improve the therapeutic effect. Other anti-HIV agents also include 3'-azido-3'-deoxythymidine, 2 ', 3'-dideoxycytidine, 2', 3'-dideoxy-2 ', 3'-dehydro Such as cytidine and the like (in addition to monoclonal antibodies that bind peptides).

[Use of Monoclonal Antibody in Immunoaffinity Tablets]

Monoclonal antibodies that are effective against gp 110 or other epitopes, in particular those that contain biologically expressed recombinant fusion proteins or their epitopes obtained from the degradation products or extracts of cultured HIV, can be used in particular for purification procedures. There is an advantage. Generally, the antibody has an affinity binding constant of about 10 ⁸ -10 ¹² M. Such antibodies can be used to purify the recombinant fusion protein from the medium of the recombination expression apparatus if the expressed protein is secreted, or from the constructs of the cleaved biological expression apparatus if it is not secreted.

Generally monoclonal antibodies capable of reacting with gp 110 or other epitopes are bound to or immobilized on a substrate or support.

The solution containing the HIV epitope is then contacted with the immobilized antibody under conditions suitable for the formation of an immunocomplex of the antibody and polypeptide comprising the epitope. Unbound material is separated from a conjugated immunocomplex in which the complex or gp 110 antigen fragment is separated from the support.

Typically monoclonal antibodies are roughly purified from isolates or cell culture supernatants prior to binding to the support.

The process is well known to those skilled in the art and may include oils with high concentrations of neutral salts. Other methods such as DEAE chromatography, gel filtration chromatography, preliminary cell electrophoresis, or Protein A affinity chromatography can also be used to purify monoclonal antibodies prior to use as immunosorbents.

The support on which the monoclonal antibody is to be immobilized should have the following general properties. That is, ⒜ the interaction with the protein should be weak to minimize nonspecific defects, 양호한 have good flow characteristics that allow fluid to pass through high molecular weight materials, and 활성화 be activated or modified to enable chemical binding of monoclonal antibodies It possesses a chemical group which can be used, and is physicochemically stable under the conditions used to bind the monoclonal antibody, and must be stable to the conditions and components of the buffer required for the uptake and elution of the antigen. Some commonly used supports include agarose, derivatized polystyrene, polysaccharides, polyacrylamide globulars, active cellulose, vitreous, and the like. There are several ways to bind antibodies to substrate supports (see Cuatrecasas, P., Advances in Enzymology 36: 29 (1972) for general). Antibodies of the invention can be bound directly to a support or alternatively through a linking group or a spacer arm.

General conditions required for immobilization of monoclonal antibodies on chromatographic supports are well known in the art (see, eg, Tijssen, P., 1985, Practice and Theory of Enzyme Immunoassay). Actual coupling methods vary slightly depending on the nature and shape of the antibody to be coupled. Monoclonal antibodies maintain optimal conditions because all of the batches have certain characteristics. Representative binding states are covalent bonds.

Suspensions of extracts or digests of HIV virus, supernatants from cultured biological expression devices or suspensions of divided cells were added to the separation matrix. This mixture is incubated for conditions and for a time sufficient to form an immune complex (typically at least 30 minutes or longer, preferably 2-24 hours). Immunocomplexes containing polypeptides with the antigenic portion of gp 110 were isolated from the mixture. For example, the mixture was removed by elution and the bound immunocomplex was washed throughout with adsorption buffer. The immunocomplex can then be eluted from the separation matrix using an eluent suitable for the particular support used, which is well known to those skilled in the art.

In addition, polypeptides containing gp 110 or other antigen moieties can be selectively removed. For example, peptides containing epitopes recognized by antibodies can be used to complete antibody binding sites that provide other elution techniques that can be performed under mild elution conditions. Selectively absorbed polypeptides containing the gp 110 antigen can be isolated from the affinity absorbent of the antibody by changing the PH and / or ionic strength of the roundworm solution. Chaotropic agents can be used to remove bound antigens. The choice of chaotropic agent, its concentration and other elution conditions depend on the nature of the antibody-antigen interaction.

However, what has been determined once cannot change what is generally needed in a polyclonal affinity separation system.

The eluted material needs to be adjusted to medical PH if low or high PH or ionic stiffness buffer is used to separate the bound gp 110 antigen from the separation matrix. Dialysis or gelfiltration chromatography is also necessary to remove excess salt used in the eluate so that the original structure can be reconstituted with gp 110 or polypeptides containing antigen fragments of gp 110.

By the method of the present invention, for example, a polypeptide containing substantially purified gp 110 or an antigen fragment thereof produced naturally by recombination expression systems in infected cell culture or cells of bacteria, yeast or cultured insects or mammals is obtained. do. gp 110 and fragments or other ordered proteins maintain at least 50% purity, typically at least 75% purity and sometimes at least 95-99% purity. There are a wide variety of ancillary uses for these molecules.

HIV gp 110 protein, polypeptides containing other antigen fragments thereof, or other proteins purified according to the present invention have found a wide variety of uses, including AIDS subunit wax preparations. Wherein the immunogen consists of, for example, an effective dose of an antigenic determinant of gp 110 or its neutralizing zone.

Other components of this formulation will include antigenic portions of HIV proteins or glycoproteins that promote the production of antibodies (suitable for neutralizing antibodies) to immunized hosts where the antibodies can prevent secondary infection by HIV.

Diagnostic Use of Monoclonal Antibodies

In addition, the monoclonal antibodies of the present invention are used for diagnostic purposes. It may or may not be labeled for use for this purpose. Typically, diagnostic assays involve the formation of complexes through the binding of monoclonal antibodies to HIV antigens. If no label is attached, the antibody can be used, for example, for aggregation analysis. In addition, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that react with monoclonal antibodies, such as antibodies specific for immunoglobulins. Alternatively, monoclonal antibodies can be directly labeled. Various types of labels can be used, such as radionuclides, fluidizers, enzymes, enzyme substrates, enzyme donors, enzyme inhibitors, ligands (especially heptenes), and the like.

Examples of obtaining various types of immunoanalytes are described in US Pat.

In general, the monoclonal antibodies and peptides of the present invention are used, for example, for enzyme immunoassay in which a subject antibody or a second antibody obtained from a different kind is bound to an enzyme. When a biological sample containing HIV antigens, such as human serum, saliva, semen, vaginal secretions or virally infected cell culture suspensions, is mixed with the antibody of the host, a binding reaction occurs between the antibody and the moiety that represents the epitope required. These protein or virus particles are separated from unbound reagents and the second antibody (labeled with enzyme) is added. Thereafter, the presence of antibody-enzyme conjugates specifically bound to the antigen is checked. It can be used by other well-known techniques.

It is also possible to provide a tool for use as a subject antibody for detecting HIV infection or for testing for the presence of HIV antigens. Thus, a monoclonal halide composition of the subject matter of the present invention can be provided, which is provided in combination with additional antibodies specific for other epitopes of HIV or in lyophilized form, alone. Antibodies that may or may not bind to the label are included in a tool containing a buffer such as tris, phosphate, carbonate, stabilizer, fungicide, inert protein, such as so-called serum albumin. Generally these materials are present at about 5% by weight or less based on the amount of active antibody, and typically at least about 0.001% by weight or more based on the antibody concentration. It is necessary to include excipients or excipients to dilute the active ingredient which may be present in about 1-99% by weight of the total composition. A second antibody that can bind to a monoclonal antibody is used, which is usually placed in a separation bottle. The second antibody is conventionally bound to the label and is formed in the same manner as the antibody formation.

Detection of the entire virus in the gp 110 or p 25 antigen or in various biological samples can be used to diagnose recent infection by the HIV virus. Biological samples include serum, saliva, semen, tissue autopsy samples (brain, skin, ramp nodules, spleens, etc.), cell culture supernatants, split mature nuclei and bacterial detection systems. Monoclonal antibodies were incubated with biological samples under conditions suitable for the formation of immune complexes, followed by testing for the presence of viruses to confirm the formation of complexes. In one embodiment, the formation of the complex is typically detected using a second antibody capable of binding to a label and binding to a monoclonal antibody formed in the same manner as the Hancheong formation method. In another embodiment, the monoclonal antibody is bound to a solid support that contacts the biological sample. After incubation, the labeled antigen is added to detect the bound antigen.

Preparation and Use of Synthetic Peptides

In particular immunologically mimicking protein epitopes are provided by the HIV retrovirus, in particular the novel peptides provided in the present invention wherein the epitopes are encoded in the env or gag of the viral genome encoding gp 110 or p 25, respectively.

To ensure variation between strains versus strains between different isolates, a selection was made between the quantitative and non-conservative substituents for conserved substituents. These peptides can be used as immunogens to inhibit or eliminate HIV antigen production in vitro or in vivo for the detection of viruses or antibodies to viruses in medical samples. The peptide may be bound to a carrier or other compound with or without a label attached to a solid surface, depending on the contents of the process record table.

In one embodiment the beneficial peptide is derived from the gp 110 zone of the virus. One of the most important ones is the band in env open reading frames of the base pair (bp) about 6688- about bp 6750 and about bp 7246- about 7317.

At least five important peptides, including blocking peptides, are at least five and sometimes at least six, eight, twelve, or at least twenty, typically less than about 50 or less than 35, and less than about 25 contained in an array encoded for HIV retroviruses. It is preferable to include the amino acid of. Preferred peptides are those in which the higher molecular weight is small enough to retain both the immunoreactive or antiviral activity of the peptide. In some instances there is a need to combine two or more non-redundant oligopeptides that provide the same sensitivity as the parent or separately to form a single peptide structure or to be used simultaneously as each peptide.

Peptides can be mutated to up to 20 percent, particularly up to 10 percent, of amino acids that are typically exchanged by introducing protected or unprotected substituents in the peptide. When the band is found to be polymorphic, it is necessary to change one or two special amino acids in order to more effectively mimic the different epitopes of other retroviral strains. Methionine can be replaced with norleucine (Nor) among various examples that provide chemical stability.

It is to be understood that the peptides used in the present invention do not need any particular HIV polypeptide sequence as long as the subject compounds can confer immunological competition with at least one protein of the HIV retrovirus strain. Thus, the peptides of the subject may bring about various changes, such as insertion, deletion and substitution of conserved or non-conservative substituents, and such changes may provide certain advantages for their use. Such a change may provide some advantage for their use. Gly, ala by conservative substituents; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; phe, tyr; And nor, met in groups such as met.

The arrangement is typically an arrangement of at least one strain of HIV retrovirus except that the peptides of the invention can be readily immobilized so that additional amino acids can be added to the short term for the purpose of providing an arm. No more than 20% difference from These arms are usually one or more amino acids, may be 50 or more amino acids, and sometimes 1-10 or more amino acids in the longitudinal direction.

Peptides in which the amino acid sequence is mutated by substitution, addition or removal of amino acid residues must maintain the antiviral activity or all immunoreactivity of the non-mutant peptide, which can be measured by various known analytical techniques. The d-isomeric form of one or more amino acids can be used to develop biological properties such as activity, isolation rate and the like.

In this regard, one, two or more amino acids may be used in combination with oligopeptides to facilitate binding of the peptides to each other in order to initiate physicochemical properties such as peptides or oligopeptides or to couple a support or peptide of higher molecular weight for the reasons described below. It can be added to the end group of the peptide.

Amino acids such as tyrosine, cysteine, lysine, glutamic acid or aspartic acid may be introduced at the C- or N-terminal end of the peptide or oligopeptide to provide useful functionalities for binding. Cysteine is particularly suitable for promoting covalent bonds to other peptides or for forming polymers by oxidation.

In addition, the peptide or oligopeptide sequence may be terminated-NH ₂ acylation, eg, acetylated or thioglycolic acid amidation, or full carboxy amidation (eg, as ammonia or methylamine) to bind or polymerize to a support or other molecule. The mutated arrangement may differ from the native arrangement in order to increase stability and hydrophobicity.

Thus, for example, in the case where Y or Y 'is present, suitable examples in the peptides I-VIII and VIII-XV described above are those wherein one or more cysteine residues in which Y or Y' have a spacer amino acid or When it consists of one or more cysteine residues. Glycine is a particularly suitable spacer.

Suitable peptides used in oxidative polymerization are when Y or Y 'is at least two cysteine residues. A suitable example when two cysteine residues are present at the same end of the peptide is when the cysteine residues are separated into one to three spacer amino acid residues (glycine is suitable). The presence of cysteine residues can enhance the hydrophobicity of the peptide, which allows the formation of dimers of the peptide or facilitates immobilization of the peptide in solid or immobilized assays.

Of particular importance is cysteine or thioglycolic acid used to acylate terminal amino groups and the like to bond two of the peptides or oligopeptides or mixtures thereof by disulfide bonds or longer bonds to form polymers containing various epitopes. It is to use a mercaptan group.

Such polymers have the advantage of increasing immunological response. When different peptides are used to prepare polymers, they have the additional ability to induce antibodies to immunoreact with different epitopes of different HIV isolates.

In order to form an antigen polymer (synthetic multimer), compounds such as bis-haloacetyl group, nitro aryl halide, etc., which are effective reagents for the thio group, are used. Thus, the bond between two mercapto groups in different peptides or oligopeptides is at least two, typically four or more, no more than about 16, and typically no more than about 14 Do not.

Subject may be used in which the peptide is bound to a soluble macromolecule (eg, 5 kDal or more) of carrier. The carrier may be a poly (amino acid) which is a naturally present or synthetic substance in which the antibody is encountered in human serum. Examples of such carriers include poly-L-lysine, such as bovine serum albumin, parenchymal denatured toxin, tyroglobulin, albumin, etc., but not chylimpet hemoci. The choice of carrier depends primarily on the final use and convenience and availability of the antigen.

In such aggregates, at least one subject per macromolecule consists of one or more molecules of peptides, and the macromolecule does not exceed about 1 per 0.5 kDal and typically does not exceed about 1 per 2 kDal. One or more different peptides can be bound to the same macromolecule. The binding method uses a known method using a reagent such as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic anhydride, succinic anhydride, glutaraldehyde and the like. Bonding may occur at the N-terminus, the C-terminus or at the intermediate to the both ends of the molecule. The peptides of the subject may be derivatized by a binding reaction and bound while bound to the support.

Various assay records well known to those skilled in the art can be used to detect the presence of antibodies to the retroviral protein or the retrovirus itself.

Importantly, the peptide is used as a labeled reagent (where the label generates a searchable signal) or the peptide is bound to the surface directly or indirectly, wherein the antibody to the peptide in the sample is bound to the surface. Will bind to the peptide. The presence of a human antibody bound to the peptide is then used either as a heterologous antibody specific for human immunoglobulin normal agents IgM and IgG, or with a labeled protein specific to the immunocomplex (eg, Rf factor of S. Orius protein A). Can be detected. An example of an analytical technique is to use a sample container (eg, a well of a microwell plate), wherein the subject polypeptide or conjugate thereof is sucked into the bottom and / or wall of the container either covalently or non-covalently. The blood or serum of a normal person diluted in a sample, appropriately buffered medium is placed in the container to form a complex between the polypeptide in the sample and the antibody of the same kind. The supernatant is removed and the container is washed to remove unspecifically bound protein. Special binding proteins with labels that specifically bind to complexes such as antisera for use with human immunoglobulins are used for detection.

Peptides can be prepared in several ways. Since peptides are relatively short in length, they can be synthesized in solution or in a solid support by known techniques. Several types of automatic synthesizers are now commercially available and can be used according to known records (e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., 1984: and Tam at al., J Am. Chem. Soc. (1983) 105: 6442).

Alternatively, hybrid DNA technology can be used, wherein the synthetic gene can be prepared using a single element encoded for a polypeptide or a substantial accessory element thereof and the single elements can be introduced into the annealing medium together so that they are redundant and hybridized. Hybridized elements can be bound to form a complete gene and inserted into an expression vector that can be readily obtained by selecting the appropriate terminal group (Maniatis et al., Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory). , 1982). Alternatively, viral genome bands encoded for peptides can be developed by asexual reproduction by known recombination DNA techniques (see Maniatis, supra).

DNA coding sequences obtained from LAV _BRU and ARV2 isolates of HIV that can be used to express peptides are as follows.

LAV _BRU : TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC CGT ATC CAG AGG GGA CCA GGG AGA GCA TTT GTT ACA ATA GGA AAA ATA GGA AAT ATG AGA CAA GCA CAT TGT

ARV-2 TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC TAT ATA GGA CCA GGG AGA GLA TTT CAT ACA ACA GGA AGA ATA ATA GGA GAT ATA AGA AAA GCA CAT TGT

Fragments in one configuration can be used to express peptide fragments, either conservative basic changes (wherein variant codons are encoded for the same amino acid), or nonconservative changes in the coding sequence. The generated amino acid is then subjected to conservative or non-conservative changes in the amino acid sequence as described above. The coding sequence can be extended to the 5'- or 3'- or both terminal groups to extend the peptide, while retaining it intact at the epitope position. For binding cancers, two or all peptides can be extended in the same chain to the same label as the enzyme or to provide antigenic activity or convenient restriction sites for asexual reproduction.

The DNA sequence itself, fragments thereof, or larger arrays (typically at least 15, preferably 18 or more) are used to identify the same band for asexual reproduction or alignment, or as nucleotide probes for detection of retroviral RNA or proviral DNA. Can be used. There are various techniques such as the Grunstein-Hogness technique, Southern technique, Northern technique, dot-blot, and their improvement techniques as well as other methods such as those described in US Pat. No. 4,358,553.

Subject peptides, including blocking peptides and homologues thereof, can be used on their own or mixed in a preventive wax. In a similar manner it can be used for waxing that contains an antigenic antibody, ie an epitope that is reactive with the genotype of the antibody of the invention and thus mimics the neutralization zone of HIV. Peptides or anti-genetic antibodies can generally be prepared by conventional methods at a concentration of lug-20 mg / kg of host. Medically acceptable media may be used as the carrier, such as sterilized water, saline, phosphate buffered saline, and the like. Surfactants such as aluminum hydroxide gels, risolecithin, pluronics, polyols, tolianions, peptides, proteins (e.g. diphtheria or cholera toxin) and oil emylzones can be used as additives. Peptides may also be combined with glucose, polypeptides or polymers for mixing into liposomes or for use in wax preparations. There is a method of injection intravenously. The administration of an immunologically effective amount can be administered once or several times, usually at 1-4 week intervals. An "immunologically effective amount" is the amount of wax that shows an increased host infection by inducing an immune response in the host.

Other features and advantages of the present invention will become more apparent in the following examples, which are not intended to limit the scope of the invention.

Example 1

[Production and Characterization of Monoclonal Antibodies]

Example 1 relates to the preparation of hybrid cell lines producing monoclonal antibodies specific for the envelope glycoprotein of HIV. This method involves the use of a lectin tablet extract of LAV _BRU bound to lectin agarose of lentils as an immunogen. Monoclonal antibodies produced by hybrid cell lines are characterized by their ability to immunospot and radioimmunoprecipitate gp 110 from purified LAV and as a biologically expressed recombinant fusion protein. In addition, monoclonal antibodies that bind to epitopes on gp 110 react with ruptured whole viruses, fusion proteins, and synthetic peptides in ELISAs and whole viruses during indirect fluorescence analysis.

The production protocol of the hybrid cell line producing the monoclonal antibody and the characteristics of the antibody are as follows.

LAV virus purified from infected CEM cells (ATCC No. CRL 8904) was purified by 50 mM Tris, PH 7.4, 0.15 M Nacl, 1.0% aprotinin, 2.0% nonidet p-40 (R) (NP-40) (octylphenoxy Cypolyethoxyethanol). The extract was purified twice by centrifugation and adjusted to 0.5% NP-40 by the addition of three times (dose) of a cleavage buffer without NP-40. Lentil lectin sepharose (Pharmacia, Piscataway, NJ) was pre-washed in cleavage buffer without NP-40 and then in adsorption buffer (50 mM Tris, PH 7.4, 0.15 M NaCl, 1.0% aprotinin, 0.5% NP-40) Parallel. The clear virus extract was adsorbed with lectin sepharose of lentils at 40 ° C. for 42 hours. Non-adsorbed material was removed by washing with excess adsorbent buffer. Elution of the adsorbed material was carried out with 0.2 M alpha methyl mannose in adsorption buffer. The eluent was dialyzed with PBS to remove sugar and the material was resorbed to the lectin cephales of lentils.

The glycoprotein-lens lectin sepharose complex was used to immunize BALB / C mice by three injections intraperitoneally at intervals of 2-3 weeks without the use of supplements. The spleens were removed from immunized birds with antibodies that purified to glycoproteins of HIV by immunospots, RIP and / or ELISA.

The protocol used for the production of cell lines generally uses that of Kohler and Milstein (Nature 256495 (1985)), which is Goldstein, L.C. It was improved by the collaborators.

(Infect. Immun. 38: 273 (1982)), B-lymphocytes of the spleen from immunized piglets were fused with NS-1 myeloma cells using 40% (w / v) polyethylene glycol. After fusion, the cell mixture was subjected to HAT medium (15% calf fetal serum, 1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin and 1.6 × 10 ⁻⁵ M Timmy to screen for growth of hybrid cells. Dean was resuspended in RPMI-1640 medium) and then placed in a 96-well microculture tray at a concentration of 1-3 × 10 ⁶ cells / ml and incubated in a humid atmosphere at 37 ° C. containing 6% CO ₂ . . The medium was replaced with 1/2 of the supernatant with fresh HAT medium. Wells were observed using an inverted microscope for cell proliferation signals. Supernatants were tested for anti-LAV antibodies when the cells reached sufficient density.

Wells containing hybrid cells producing antibodies to LAV were confirmed by ELISA measuring binding to purified whole ruptured virus or biologically expressed fusion proteins. ELISA assays for ruptured viruses were performed on LAV EIA plates (Genetic Systems, Seattle, Washington). The platelets were incubated with the cell culture at 37 ° C. for 45 minutes and washed three times with 0.05% Twin 20 (PBS-Twin) in phosphate buffered saline.

The oxidase-goat anti-rat mouse IgG (1: 2,000 dilution in PBS-Twin, Zymed Laboratories, Inc., South Sanfrancisco, California) was added (100ul per well) and the platelets were incubated for 45 minutes at 37 ° C. Washed together. Base plate (0.025 M citric acid, 0.05 M dibasic tar phosphate, PH 5.0 containing 14 mg of 0-phenylenediamine and 10 ul 30% hydrogen peroxide per 50 ml) was added and the platelets were incubated for 30 minutes in the dark at room temperature.

The reaction was stopped with 3N sulfuric acid and the colorimetric reaction was weighed with an automatic microplate recorder. The wells that yielded the desired results were then constantly diluted, expanded to 2nd asexual, and expanded.

The monoclonal antibodies secreted by the hybrid cell lines thus obtained were identified for their effectiveness or reactivity by immunospots, immunoprecipitation and ELISA using cleaved LAV virus, recombined LAV conjugate protein and synthetic LAV peptide. All measured whether the antibody is IgG ₁ isotype. The cell lines HIV-gp110-1, HIV-go110-2 and HIV-gp110-3 were deposited in the American type culture collation (ATCC) prior to the filing of this patent application, respectively. HB 9175, HB 9176 and HB 9177 were given, and these cell lines were also deposited with KAIST on February 20, 1988 and were given KCTC 8349P, 8350P and 8351P, respectively.

The recombination fusion proteins that were first tested for reactivity were labeled ENV2, ENV3, ENV4 and ENV5. Protein ENV2 was expressed from PENV2 (ATCC No. 53071), which is in the LAV band of base pairs (bp) 6598 to pb 7178 (numbered according to Wayne-Hobson et al., Cell 44: 9 (1985)) and ENV3 Is expressed from PENV3 (ATCC No. 53072), which is in the LAV band of bp 7178 to bp 7698. ENV4 is expressed from PENV4 (ATCC No. 53073), bp 7698 to bp 852 and ENV5 is expressed from PENV5 (ATCC No. 53074) and consists of a LAV band of bp 5889 to bp 7698. The production of recombinant fusion proteins is described in detail in general pending US patent applications 721, 237.

[Array of Synthetic Peptides]

Peptides I (29) and VIII (110-2-2) were arranged on benzhydrylamine (polystyrene / divinylbenzene) resins (Applied Biosystems, Inc., Foster City, California). Peptide V (177) was arranged on t-butyloxycarbonyl (Boc) -ethylbenzylcysteine-phenylacetamidomethyl (PAM) polystyrene / divinykenene resin. Symmetric anhydride coupling reactions were performed on an Applied Biosystems 430A synthesizer. Cysteine was added as the first residue in both peptides.

In the case of asparagine and glutamine, a dicyclohexylcarbodiimide coupling reaction was used in the presence of hydroxylbenzotriazole. Benzyl-based side chain protection and Boc alpha-amine protection reactions were used. Other commonly used side chain protection means include Boc (propyl) tryptophan, Boc methionine slapoxide, Boc (tosyl) arginine, Boc (methylbenzyl) cysteine, Boc (tosyl) histidine, Boc (chlorobenzyloxycarbonyl) lysine And Boc (bromobenzyloxycarbonyl) tyrosine.

When a radiolabel was applied to the peptide, an acetylated amino end group was used as 3H-acetic acid and excess dicyclohexylcarbodiimide. Deprotection and cleavage of the peptides from the resin were done by Tam's low-high 'HF protocol (Tam co-investigator). Extraction from the resin was performed with 5% acetic acid and the extract was subjected to gel filtration chromatography in 5% acetic acid.

Synthetic HIV peptides were tested for reactivity with monoclonal antibodies comprising peptides 29, 36 and 39. Peptide 29 was encoded with a LAV _BRU genome band of about bp 6688 to bp 6750. Peptide 36 was encoded in a band of about bp 7246 to bp 7317, and peptide 39 was recorded in a band of about bp 7516 to bp 7593. Peptides 36 and 39 are described in detail in US Pat. No. 4,629,783.

The blocking peptide VII-XV was necessarily arranged as above on methyl-benzhydrylamine (polystyrene / divinylbenzene) resin (Applied Biosystems, Inc., Foster City, California). The symmetric anhydrous coupling reaction was performed on an Applied Baosystims 430A synthesizer. Dicyclohexyl carbodiimide coupling in the presence of hydroxylbenzotriazole was used for asparagine. Benzyl-based side chains and Boc alpha-amine protection reactions were used for protection, while Boc (bromobenzyloxycarbonyl) was used on the other hand, especially for tyrosine side chains. Acetation was carried out only if present, with acetic anhydride or glacial acetic acid and dicyclohexylcarbodiimide. Deprotection and cleavage of peptides from resins were performed by standard “high” HF protocols (method of Stewart et al., Co-workers above). Extraction from the resin was performed with 50% acetic acid, and the extract was then subjected to gel filtration chromatography in 20% acetic acid. High performance liquid chromatography was performed on Vidac C18 column (Rainin Instrument Co., Emeryville, Calif.) Using 0.1% trifluoroacetic acid (acetonitrile credit) as required.

[Immune Spot]

Characterization by immunospots was carried out in ascites fluid or clone supernatants using purified LAV virus and recombination fusion protein as antigen. The separated antigen was first separated by polyacrylamide gradient gel electrophoresis (7.0-15.0%) and transferred to nitrocellulose separator (NCM) by electrophoresis at 25V in 25 mM sodium phosphate (PH 7.0) for 4 hours. The NCM was then blocked by incubating in PBS-to-win or blotto (5% nonfat milk in PBS) for 1 hour at room temperature. In the second step, the NCM was diluted in PBS-Twin for 1 hour at room temperature and incubated with goat anti-lemur IgG horseradish peroxidase. This culture was washed with PBS-Twin and soaked in horseradish peroxidase embolic solution (Bio-Rad Laboritories, Richmond, California) for 20 minutes. The reaction was stopped by immersion in deionized water. The reactivity of monoclonal antibodies was compared with positive inhibitory sera that reacted with purified fission virus or expressing fusion protein. The results showed that all antibodies bound to gp 110 and its precursor molecule gp 150 using a cleavage virus preparation. In addition, antibodies 110-1 and 110-2 recognized the fusion protein ENV3 and antibodies 110-3, 110-4, 110-5 and 110-6 made ENV2 an immunocomplex.

[Immunoprecipitation]

Virus extracts for radioimmunoprecipitation were prepared from infected CEM cells with LAV _BRU isolates of HIV used for continuous growth by passing through. When the initial cell pathology was confirmed, the cells were transferred to a label medium containing ³⁵ [S] -methionine (0.05 mCi / ml) or ³ [H] -glucosamine (0.025 mCi / ml), and most of the cells were decomposed and cultured. Incubated for 24 hours until the virus was digested. The virus was isolated from the cell free supernatant into small globules (1 hour at 100,000 × g) and the wash extract contained P-RIPA buffer (1.0% Triton X-100, 1.0% dioxycholate, 0.1% SDS and 1% aprotinin). Phosphate complete saline). Similar extracts were prepared from supernatants of uninfected CEM cells.

Immunoprecipitation assays were performed with 100ul of virus extract incubated with 100ul of culture supernatant obtained from hybrid cell line for 1 hour on ice. Rabbit anti-cub Ig 4 microlithium (Zymed Laborato-ries, So. San Francisco, Calif.) Was added to each sample and incubated for 30 minutes. An immunoprecipitate (100ul, Bethesda Reseavch Laboratory, Bethesda, Maryland) suspended in P-RIPA complete solution containing 1.0% ovalbumin was added to each sample and incubated for another 30 minutes. The bound complex was washed and separated by SDS-polyacrylamide gel electrophoresis (15.0% acrylamide: DATD gel). The gel was then fixed, adsorbed in Enhance (New England Nuclear, Boston, Mass.), Dried and exposed to Kodak XR-5 film. Comparative positive sera with immunoprecipitated all HIV virus proteins were reacted with virus-infected and mock-infected CEM cell supernatants as positive and negative regulators. The results showed that all six monoclonal antibodies specifically immunoprecipitated gp 110 and gp 150.

[Enzyme-linked Immunosorbent Analysis]

In order to align the gp 110 epitope recognized by the monoclonal antibody of the present invention, the reactivity between the fusion protein and the synthetic peptide biologically expressing the culture supernatant obtained from the hybrid cell line or ascites fluid was characterized in ELISA. A fusion protein or synthetic protein was carried out in the same manner as above except that the virus was replaced with purified virus as an antigen adsorbed on the surface of the micro titer well.

The protocol for using peptides as antigens is as follows. Lyophilized peptide was dissolved in 6M guanidine HCl. Immediately prior to 96 well platelets, the guanidine solution was diluted with 0.05 M carbonate / bicarbonate buffer (PH 9.6) to adjust to a final peptide concentration of less than 100 ug / ml. A 50ul dose of diluted peptide was placed in each microtiter well and platelets were incubated at 4 ° C. overnight. The excess peptide solution was shaken to allow the platelets to block bloto and proceed as above for the remaining ELISA. Similarly, the recombination protein was diluted to a final concentration of about 2 ug / ml in 0.05 M carbonate / bicarbonate buffer (PH 9.6) prior to performing the above method.

The results are shown in Table 2 below. Monoclonal antibodies produced by the cell lines HIV gp110-1 and HIV gp110-2 reacted with the virus cleaved with ENV3, ENV5, peptide 36. Antigens obtained from the cell lines HIV-gp110-3, HIV-gp-110-4 reacted with the divided virus as well as ENV2 and peptide 29.

TABLE 2

The results in Table 2 above showed that monoclonal antibodies 110-1 and 110-2 recognize antigenic determinants encoded by the pENV3 band, particularly the band of the HIV genome, which is defined by the arrangement of amino acids in peptide 36. Monoclonal antibodies gp110-1 and 110-2 bind to the peptide band of gp 110 encoded in bp 7246 to 7317 as evident by the formation of peptides 36 and ENV3 and an immunocomplex. This band of the HIV genome was found to be conserved (ie hardly changed) in the band's DNA sequence encoded by peptide 36 between different viral isolates isolated from opposite positions (Starcich et al., Cell 46637 (1986). Reference). In contrast the monoclonal antibodies gp110-3, -4, -5 and -6 bind to peptides of HIV defined by a band encoded with peptide 29 of gp 6688 to about bp 6750. The band within gp 110 defined as peptide 29 was found to contain various nucleotide substituents between different virus isolates. Monoclonal antibodies that selectively bind gp 110 polypeptides containing conserved epitopes, such as antibodies 110-1 and 110-2, have increased use in various methods, such as affinity chromatography. Alternatively, in the ELISA assay, peptide 110-2-2 reacted with sera from individuals from which LAV-2 was isolated.

Indirect Immunofluorescence Analysis

Indirect immunofluorescence assays using monoclonal antibodies against the gp 110 antigen of HIV were performed in acetone-fixed live cells. Acetone immobilized slides were prepared from LAV-infected CEM cells and incubated with diluted culture supernatant or ascites for 1 hour at 37 ° C. and incubated with culture supernatant or ascites for 1 hour at 4 ° C. prior to placing them on live cell slides and acetone fixtures. . Fluoricin isothiocyanate-labeled murine IgG was used to detect cells with reactive gp110-antigens in both methods. Monoclonal antibody HIV-gp 110-1 obtained positive results using live or acetone immobilized LAV-infected cells.

Example 2

Neutralization of HIV infectivity by anti-gp 110 monoclonal antibodies

This example characterizes gp110 and a method of neutralizing HIV infectivity using monoclonal antibodies that bind peptides within gp110. The results showed that the monoclonal antibodies gp110-3, -4, -5 and -6 exhibited neutralizing activity, and gp110-3 and -4 showed particularly high neutralizing activity.

[Disabled Analysis]

Sensitive neutralization assays have been developed to measure the effects of monoclonal antibodies on HIV infection. CEM, a CD4 + cell line extremely susceptible to HIV, was selected as a target cell for infectivity comparison. IgG fragments purified using the ascites solution or ammonium boamate precipitate prepared as in Example 1 were inactivated by heating at 56 ° C. for 30 minutes and then diluted appropriately in RPMI medium containing 10% fetal calf serum. . Suspensions of HIV strain LAV _BRU were harvested from logistic 4-day old CEM cultures, filtered on a 0.2 or 0.45 micron filter, aliquoted and frozen to -70 ° C. One aliquot was thawed and titrated to determine TCID ₅₀ . Subsequent assays were aliquoted during the new year and diluted 1: 500 in culture medium (10TCID ₅₀ ) until the concentration reached approximately 10 times the amount required to infect 50% of CEM cells in the culture. Virus suspensions were mixed with the same amount (250 ul) as monoclonal antibody preparations diluted five-fold from 1: 5 to 1: 9,765,625. The virus / antibody mixture was incubated for 45 minutes at 37 ° C. and doubled from 1: 5 to 1: 9,765,625. The virus / antibody mixture was incubated at 37 ° C. for 45 minutes to dilute twice the 200 ul sample used to inoculate a well containing 1.0 ml of about 2 × 10 ⁵ CEM cells per well. The cells were harvested, granulated and eluted with 1% Triton X-100 in PBS for about 10 minutes. The amount of virus (or viral antigen) present in the eluted cells was measured using the sensitive HIV antigen capture “sandwich” enzyme immunoassay described below. The neutralization activity was measured as a dilution table of monoclonal antibodies which suppressed antigen production up to 50% or more of the cultured antiviral control with the same type of isotype monoclonal antibody which was shown to be weak or without any neutralizing activity.

The HIV antigen capture assay mentioned above used two monoclonal antibodies directed to the p25 antigen as capture reagents. Using the gag recombination fusion proteins purified from these murine cell lines as immunogens, and using the previously designated GAG-1, GAG-2 and GAG-3 recombination fusion proteins and synthetic peptide 141, the characteristics of monoclonal antibodies such as properties and reactivity were determined. The method of identification was changed slightly to produce the same method as described. Protein GAG-1 is expressed from pGAG-1 (ATCC No. 53379), GAG-2 eye is expressed from pGAG-2 (ATCC No. 53111), GAG-3 is expressed from pGAG-3 (ATCC No. 53112) do. Methods for producing recombinant fusion proteins are described in detail in US general pending patent applications 764, 460 and 828, 828. Synthetic peptide 141 was encoded into the LAV genome band corresponding to amino acid residues 192-242. It was found that the monoclonal antibodies produced by the hybrid cell lines p25-2 and p25-3 react with the recombinant fusion proteins GAG-1, GAG-2 and GAG-3, and also the monoclonal of p25-3 was synthesized. Reacted with peptide 141.

In order to perform an antigen capture assay, the capture reagent was first adsorbed onto a solid support. Ascites fluids derived from the hybrid cell lines p25-2 and p25-3 were diluted 1: 5000 in 25 mM Tris buffer (PH 8.5) and 200ul was placed in a well of a microwell plate. The wells were sealed and incubated at 4 ° C. for about 16 hours. The solution was aspirated and removed from the wells before 0.3% bloto blocking solution was added. Blocking was performed for 15 minutes at room temperature. The blocking solution was aspirated to add a sample. 200 ul eluted cell suspension and 5.0 ul of the detection binding prepared as follows were added to each well. The well strips or platelets were aspirated from suspension and incubated at 37 ° C. for 2 hours and the wells washed four times with buffer (0.05% twin in PBS). Detection conjugates were prepared as follows. Monoclonal antibodies p25-6 and p25-7 were subjected to cyclic oxidation (Nakane, et al., J. Histochem. Cytochem. 22: 1084 (1974)) for 3 hours in a 3: 1 molar ratio (Ab: HRP) for 3 hours. ) Was horseradish peroxidase (HRP). The conjugate was diluted 1: 1500 in 0.005% antifoam A in 2.5% (W / V) nonfat milk, 0.01% thimerosal and 20 mM sodium citrate. The residue in the ELISA process was treated as in Example 1.

Assays for neutralization reactivity with antibodies gp110-3, -4, -5 and -6 are as follows. The highest titers that retained neutralizing activity were determined to be gp110-3 = 15,625, gp110-4 = 9,765,625, gp110-5 = 125 and gp110-6 = 625.

Due to the predetermined genetic variability of the band defined by peptide 29, the ability of the monoclonal antibody gp110-4 to recognize other isolates of HIV was tested. Fluorescent antibody analysis was performed as above. Antibody gp110-4 detected antigen in at least three media in the 16 HIV isolates tested.

Antibody gp110-4 was able to neutralize virus isolated for more than 15 weeks from chimpanzees inoculated with LAV _BRU as a control in the AIDS test. Monoclonal antibodies have serum antibodies that immortalize HIV in vitro and develop a measurable cell-modulated immune response to HIV infection but can immortalize the isolate. This fact indicates the lack of antigen targets in vivo for epitopes recognized by the gp110-4 antibody.

Example 3

Neutralization of HIV infectivity by cocktails of anti-gp110 and anti-p25 monoclonal antibodies

This example shows HIV infection using a combination of monoclonal antibodies that bind to peptides in p25 and p25, and monoclonal antibodies that bind to peptides in gp 110 and gp 110, which alone or exhibit no neutralization activity. Explain the neutralization of This result indicated that the monoclonal antibody gp110-2 mixed with p25-6 or p25-7 had particularly high immortalization activity.

Hybrid cell lines p25-6 and p25-7 are GAG-1, GAG-2 and GAG-3 recombinant fusion proteins and synthetic peptides specified above using purified gag recombinant fusion proteins expressed in E. Coli as an inactive isolated virus or immunogen. 15, 88, 150, 147 and 148 were used to improve the characterization of recombinant monoclonal antibodies such as their properties and activity, and were produced according to the methods described above. Synthetic peptides were encoded with the LAV _BRU genome band corresponding to the following amino acid residues. That is, peptide 15, amino acid residues 329-350; Peptide 88, amino acid residues 315-350; Peptide 150, amino acid residues 318-363; Peptide 147, amino acid residues 278-319; Monoclonal antibodies produced by peptide 148, amino acid residues 290-319, hybrid trichophytes p25-6 and p25-7 react with the recombination fusion protein GAG-3 such that p25-6 reacts with synthetic peptides 147 and 148, and p25 -7 reacts with synthetic peptides 15, 88 and 150.

The neutralization assay was performed as described above except for the use of cocktails and each monoclonal antibody was first diluted to 1: 5 in the medium and then mixed in the same proportion to a final dilution factor of 1:10. Analytical residues were treated as above.

Monoclonal antibodies gp110-2, p25-6 and p25-7 showed up to 50% immortalization activity when used alone. When used as a cocktail containing monoclonal antibody gP110-2, such as gp110-2 or p25-6 with p25-7 diluted 1:10, it was completely neutralized. Cocktails comprising monoclonal antibodies p25-6 and p25-7 had 60-90% neutralization activity.

Example 4

Immunity of Peptide 29 and Its Homologs

The ability of peptide 29 and homologue peptides, including peptide 177, to test the immune response to HIV was first tested in two strains of birds. Methods for the preparation of peptide immunogens, translational records and characterization of immune responses are described in detail below.

Peptide 29 was prepared for immunization by binding to purified tyroglobulin. Tyroglobulin is bound to N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the method described in US Pat. No. 4,629,783 (Col. 10, lines 28-21). Can be derived from Tyroglobulin as the second immune source was induced by glutaraldehyde as follows. 27 mg of swine tyroglobulin was dissolved in 1 ml of 0.1 M sodium bicarbonate, and 8.3 ul of 25% glutaraldehyde solution was added dropwise. The mixture was stirred overnight at room temperature. 1 ml of sodium carbonate / bicarbonate buffer (PH 9.3) was added to the solution followed by dialysis for 8 hours against 2 liters of the same buffer at 4 ° C. and complete change of dialysate at 4 hours. Peptide 29 was added to about 100 molar excess of induced tyroglobulin. The mixture was stirred overnight at room temperature. Unreacted glutaraldehyde was blocked with 200ul of 0.2M lysine solution and the mixture was stirred for several hours (or one night) at room temperature. Peptide-tyroglobulin conjugates were extensively dialyzed against PBS at 4 ° C.

Two strains of young rats (C57 black and BALB / C) were inoculated with peptides prepared according to the respective binding methods. All animals were about 2-4 weeks old at the time of inoculation. The inoculation route is trunk, tail bleeding, subcutaneous tissue, nose, or intraperitoneal. The inoculum consists of 25 ug of binding peptide suspended in 0.5 ml of complete Freund's binder and agonist repeatedly inoculated at 2, 3 and 5 weeks with the same immunogen suspended in incomplete Freund's binder. Serum samples from each rat were collected on the fourth day after the immunization was promoted at 3 weeks before the immunization, and at the 4th day after the final efficacy was promoted on the 4th and 5th weeks. Serum showing antibody activity to LAV was further selected for neutralizing activity, and serum in immunospots was analyzed for the radioimmunoprecipitation analyte as gp 110 with cleaved LAV antigen and radiation label.

혈청 Serum samples from immunized rats that display antibodies to the entire virus and are able to neutralize HIV as in the assay described in Example 2, and react with peptide 29 in ELISA, may be used for peptide formation in waxing. The effect of using 29 is shown.

Example 5

Immunoaffinity Isolation of GP110 Using Monoclonal Antibodies

Monoclonal antibodies against the gp 110 antigen of HIV can be used in immunoaffinity separation methods to substantially purify bacterial expression recombinant fusion proteins. If the expressed protein is secreted by bacteria, the protein can be isolated from the culture supernatant. If the protein is not secreted, it will require rupture of the bacterial cells.

The construction of plasmid pENV-5 (ATCC No. 53074) is described in pending US patent application Ser. No. 721,237. Plasmid pENV-5 encodes the major portion of the carboxyl end group of gp110 and the amino end group of gp41 of LAV inserted into the trp expression vector. E. Coli C600 was transformed by this vector expression but did not secrete gp110 fusion protein.

E. Coli C600 containing plasmid pENV-5 was grown in a medium containing tryptophan (200 ug / ml) and ampicillin (100 ug / ml) overnight at 37 ° C. while blowing air at 37 ° C. Overnight cultures were inoculated at 1: 100 in fresh minimal medium containing ampicillin (100ug / ml) but no tryptophan. These cultures were grown with aeration at 37 ° C. for 2-3 hours (up to the initial aqueous phase). Inducer 3-B-indole-acrylic acid (Sigma Chemical Co., St. Louis, Mo.) was added to a final concentration of 20 ug / ml from 20 mg / ml fresh stock in 95% ethanol. The induced cultures were grown with air blowing at 37 ° C. for 4-5 hours, then pelletized and frozen by any method. The yield of protein from pENV-5 was typically less than 1 mg / l.

Pelleted bacterial cells were treated with P-RIPA buffer (PBS containing 1% preton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate and 1% aprotinin) to lyse E. coli cells. To dissolve. The suspension was digested by sonication to distribute DNA and RNA and centrifuged to remove particles. Dilution or concentration processes are necessary to normalize the concentration of the protein.

First, the monoclonal antibody HIV-gp110-1 was buffered to pH 7.3 from ascites or cell culture supernatant at room temperature or in a cold state (NH ₄ ) ₂ SO ₄ or Na ₂ SO ₄ solution at a final concentration of 33 or 18%. Precipitated. Precipitated protein was removed by centrifugation and redissolved in PBS to precipitate twice with 33% (NH ₄ ) ₂ SO ₄ or 12-15% Na ₂ SO ₄ . This step can be repeated as necessary. The granules were again dissolved in PBS and excess salt was removed by gel filtration through a desalting matrix or by exhaust dialysis to PBS. The purified 110-1 monoclonal antibody can be coupled to the cyanogen bromide-activated sepharose. The required amount of gel was swelled in a ^10-3 MHCl solution on a glass filter (1 g of lyophilized material yielded about 3.5 ml of the final gel), washed with the same solution for 15 minutes and immediately added antibody. In general, the coupling reaction proceeds most effectively within the pH range of 8-10, but lower pH may be used if necessary to maintain the stability of the antibody. Antibodies should be dissolved in PBS or dissolved in borate buffer with high ionic strength carbonate / bicarbonate or 150 mM NaCl. The active Sepharose and antibody suspensions were gently stirred for 2-4 hours at room temperature or 1 night at 4 ° C. and washed in a glass filter with coupling buffer. The residual activator was blocked by treatment with 1.0 M ethanolamine of PH 8 for 2 hours.

The final antibody-sepharose product was washed 4 or 5 times with a high and low PH buffer solution (borate buffer, 0.1M, PH 8.5, 1M NaCl and acetate buffer, 0.1M, PH 4.0, 1M NaCl, respectively). This cleaning method removes even the smallest amount of non-covalent adsorbent. The completed immunoaffinity separation matrix was stored at 8 ° C. or lower in the presence of a suitable bacterial growth inhibitor such as 0.01% azide.

The addition of the protein suspension expressed in the immunoaffinity separation matrix allows for the selective removal of the gp110 antigen. The mixture was reacted slowly by stirring or shaking for 2-24 hours (12-18 hours are suitable). The columnar form can also be used to slowly add a suspension of expressed protein to the column, with the immunoaffinity matrix added in parallel to the column. After addition of the protein suspension, flow must be stopped to maximize production of the immunocomplex.

Unlabeled material was washed or separated with adsorption buffer by extensive washing. A glass filter in a vacuum state or a column flow path can be used. Bound material was eluted using high or low pH buffer (acetate buffer, PH 4.0 or borate buffer, PH 8.56) or chaotropic agents.

Example 6

Immunoaffinity Purification of Recombinant GP110 from Mammalian Expression Systems

It has been found that the monoclonal antibodies of the invention can be used for immunoaffinity purification of recombinant fusion gp110 expressed by mammalian cells. Mammalian cells were infected with recombinant heads (see Markett et al., J. Virol. 49: 857 (1984)) comprising an array encoding at least a portion of gp110 that induces antigenic and neutralizing antibodies.

Recombinant heads were constructed by the method described in US Pat. No. 842,984. In short, an array encoding the envelope glycoprotein of HIV was inserted into the downstream plasmid vector (pGS20) from the frontal transcription inhibitory elements. This mutant gene is identified by the sequence encoding the viral thymidine kinase (TK) gene.

The mutant plasmid vector containing wax virus promoter bound to the LAV envelope gene was used to convert E. Coli strain MC1000. Insertion of the LAV-env sequence was accomplished by in vivo recombination and was made possible by the fact that the mutant gene in the plasmid pv-env5 was encountered by the head virus sequence encoding the TK gene. This plasmid was introduced into the cells before being infected by wild-type head virus, so that recombination occurred between the TK sequence on the plasmid and the homologue sequence in the head virus genome, thereby inserting the mutant gene. American Green Monkey Kidney cells (strain BSC-40, cell line derived from BSC-1 cells, ATCC No CCL26) were used as hosts for the expression system.

Confluent BSC-40 cells were variously infected with 10 types of infection by recombinant head virus. Infection was allowed to proceed for 12 hours, collected, washed once with PBS and collected by centrifuge. Cell pellets were resuspended in elution buffer (1.0% NP 40, 2.5% sodium deoxycholate, 0.1M NaCl, 0.01M Tris-HCl, PH 7.4, 1 mM EDTA).

The eluate was purified by centrifugation. Immunoaffinity separation of the expressed recombinant fusion protein was carried out in the same manner as above for the bacterial expression system using the monoclonal antibody gp110-1. Because of the glycoproteins and processes provided by mammalian cells, the proteins produced by this expression system are very similar to the naturally produced HIV gp110.

Example 7

Inhibition of HIV Infection Using Blocking Peptides

The effect of blocking peptides on inhibiting infection of tissue culture cells by LAV _BRU strains of HIV was studied using an improved protocol for peptide T evaluation published by the above, Pert et al. Collaborators. Preferred HIV inhibition assays are performed by mixing the same dose of blocking peptide and CEM cells (2.5 × 10) ⁵ in medium (RPMI, 10% FCS and 2 mg / ml polybrene) and inoculating for 45 minutes at 37 ° C. The virus was then added at various doses (10,50,500 TCID50) and the mixture was incubated at 37 ° C. for 14 days to analyze the supernatant for the production of viral antigens (eg, p25 center).

Inhibiting CEM cells in advance with the peptide prior to adding the virus to the medium promotes the inhibitory effect in the analyte. It was found that there was a difference in the inhibitory effect depending on the dose of the virus. A small dose of the medium showed a strong activity, and a lower effect was obtained at the highest dose of the virus.

Peptide T was able to inhibit about 60-90% viral antigen production in low virus dose testing. Further experiments with peptide X-XV, typically amidated COOH-terminus and acylated NH ₂ -terminus, yielded similar results, while peptide XI was found to be particularly effective at a wide range of doses. . Inactivated antibodies can be added prior to culture or when the virus is added to obtain additional inhibitory or synergistic effects of viral antigen production.

As noted above, it will be appreciated that monoclonal antibodies and peptides, including the blocking peptides of the present invention, provide an improved method of neutralizing and / or inhibiting HIV infection. This makes it easy to develop effective prophylactic and therapeutic compositions against infection by most HIV strains (if not all). In addition, the new material could be used for diagnostic analysis and other well-known methods.

While the invention has been described in detail by way of examples in order to provide a thorough understanding of the invention, it will be appreciated that certain changes and improvements may be made within the scope of the appended claims.

[Microbial Deposit Data]

The following microorganisms forming part of the present invention were deposited in the American Type Culture Collection (20852, Maryland, United States, Rockville, Parknrow Drive 12301), and on February 20, 1988, the Korea Advanced Institute of Science and Technology (Seongbuk-gu, Seoul, Korea). Hawolgok-dong 39-1) was given the following accession number.

[Microorganisms]

Hybrid cells HB 9175, HB 9176 and HB 9177 were tested to find viability on August 26, 1986. Testing on the remaining hybrid cells revealed the viability on May 4, 1987.

Claims (26)

A composition for treating HIV infection comprising an effective amount of at least one monoclonal antibody that specifically reacts with a gp110 neutralized epitope between the amino acid sequence of about 301 to 336 of the LAV and its homologue, and a pharmacologically effective carrier. The composition of claim 1, further comprising a cocktail of monoclonal antibodies that react with the different homologs of the arrangement. The composition of claim 1, wherein the gp110 epitope is located in LAV _BRU amino acid sequence 308-328 or an analog thereof. A peptide characterized by consisting of at least six consecutive amino acids from the following HIV gp110 amino acid sequence or its analogs and containing the neutralized epitope according to claim 1.

The peptide of claim 4, wherein said contiguous amino acid defines at least one antigenic determinant capable of inducing an antibody in the host by immunization, wherein said antibody is able to neutralize HIV infection. The peptide of claim 5, wherein the peptide consists of one of the following HIV gp110 amino acid sequences or one of its analogs.

Of these, Y and Y ', if present, each have a sequence of up to about 20 amino acids. 7. The peptide according to claim 6, wherein Y or Y 'is actually composed of a binding residue selected from glycine, tyrosine, cysteine, lysine, glutamic acid or asparagine. An isolated nucleic acid sequence, which encodes a peptide according to any one of claims 4 to 7. The nucleic acid segment of claim 8, which encodes at least about 6 consecutive amino acids from HIV gp110 amino acid sequences 301 to 336 and their analogs. A waxy against HIV infection in an immunologically effective amount of one or more peptides and homologues thereof according to claim 4, wherein the peptide is mixed with a physiologically acceptable carrier or adjuvant. 11. The wax of claim 10, further comprising a cocktail of peptides of different homologs of said arrangement. An immortalized cell line that produces the monoclonal antibody of claim 1 capable of reacting with an HIV antigenic determinant contained within the gp110 amino acid sequence 301 to 336 and neutralizing HIV infection. 13. The method of claim 12, wherein ATCC No. A cell line that is HB 9175, HB 9176, HB 9177, HB 9404, HB 9405, HB 9406, HB 9409 or HB 9410. Monoclonal antibody produced by the cell line of claim 13. Monoclonal antibody that can specifically react with the antigenic determinant of HIV, characterized in that the binding of the antibody produced by the cell line of claim 13. Administering to the host an immunologically effective amount of an antigen enriched for HIV gp110 protein; Examine the immunized host for the production of antibodies reactive with the gp110 antigenic determinant, obtain antibody producing cells from the host to immortalize the cells, and examine the HIV antigenic determinant contained within the band of gp110 amino acid sequences 301 to 336. A method for producing a cell line for producing a monoclonal antibody according to claim 1, wherein the cell is produced by selecting an immortal cell line capable of producing an antibody and neutralizing HIV infection. The method of claim 16, wherein the HIV protein is a recombinant fusion protein expressed by a progressive or bacterial host, or the HIV protein is obtained from an extract or lysate of HIV. Incubating the biological sample with the peptide according to claims 4 to 7 from the neutralization zone of HIV gp110 of six or more consecutive amino acids of amino acid sequences 301 to 336; A method for diagnosing the presence of HIV in a biological sample in vitro, characterized by detecting the presence of HIV in the sample and the presence of an immune complex formed between the peptide and the antibody in the sample reactive with the peptide. . A monoclonal antibody capable of specifically reacting with a gp110 neutralized epitope in the HIV band from amino acid sequences about 301-336 of the LAV and its analogs and neutralizing HIV infection. The monoclonal antibody of claim 19, wherein said zone consists of about 308 to 328 HIV gp110 amino acid sequence and homologues of said sequence. The monoclonal antibody according to claim 19, which binds to a peptide having the following configuration and its homologue configuration:

The monoclonal antibody according to claim 20, which binds to a peptide having the following configuration and its homologue configuration:

The method of claim 22, wherein the monoclonal antibody is HIV-gp-110-3, HIV-gp-110-4, HIV-gp-110-5, or HIV-gp-110-6 or to bind to a peptide. A monoclonal antibody characterized by competing with at least one of the above monoclonal antibodies. A therapeutically effective amount of two or more monoclonal antibodies and a pharmacologically effective carrier, wherein at least one of said two or more monoclonal antibodies comprises a p25 neutralizing epitope in the HIV band from amino acid sequences 278 to 319 of LAV. A composition for use in the treatment of HIV infection, characterized in that it reacts specifically and the second antibody reacts with an epitope in p25 315 to 363, and its homologues. A therapeutically effective amount of two or more monoclonal antibodies and a pharmacologically effective carrier, wherein at least one of said two or more monoclonal antibodies is in the HIV band from amino acid sequences 278 to 319 or 315 to 363 of LAV. A composition for use in the treatment of HIV infection, characterized in that it specifically reacts with a p25 neutralizing epitope of, and the second antibody reacts with an epitope in peptide 36 of the HIV gp110 band. The composition of claim 24, wherein the first monoclonal antibody is HIV-p25-6 or HIV-p25-7 and the second monoclonal antibody is reactive with HIV-gp110-2.