JP3364181B2 - Human immunodeficiency virus envelope polypeptide and its antibody - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the envelope of human immunodeficiency virus or HIV (HTLV-III).
It relates to the use of polypeptides, and more particularly to their use in undesired suppressive immune responses and the resulting inflammation. The present invention also relates to the use of HIV env variants and antibodies to HIV env for vaccination, treatment in HIV infected patients, diagnostic assays for test samples, and other therapeutic indications. In particular, the invention relates to the treatment of autoimmune diseases and immunosuppression associated with transplantation. The invention also relates to immunotoxic agents and other immunoconjugates and their use for killing HIV or other viruses encoding cell surface antigens.

[0002]

BACKGROUND OF THE INVENTION Inflammatory responses associated with various immune disorders can generally be divided into four classes. However, a number of autoimmune diseases included in the treatment of the present invention include:
Classification is difficult due to its uncertain origin (eg,
Myasthenia gravis, Graves 'disease, autoimmune disease caused by Chagas' disease, and juvenile diabetes). A class I response is a rare or allergic reaction characterized by atopy or anaphylaxis. Class II responses are dependent on cytotoxic antibodies and are associated with, for example, autoimmune hemolytic anemia, and thrombocytopenia associated with systemic lupus erythematosus (SLE). Class III response is IgG
And / or is characterized by the chronic generation of immune complexes containing IgM. Class IV responses are associated with delayed-type hypersensitivity reactions, and cytokin
It is mediated by family members and T-lymphocytes and is commonly found in tuberculosis, sarcoidosis, polymyopathy, granulomas and vasculitis.

Rheumatoid arthritis (RA) is a severe class II
I is one of the abnormalities. Synovitis is a tissue reaction characteristic of RA, in which the normal thin connective tissue is replaced by hyperproliferative synovial fibroblasts and the synovium is invaded by lymphocytes, macrophages and other immune cells. Being invaded. In rheumatoid synovial fluid, complement component reversal is increased and complement cleavage products, especially C5a, are present in rheumatoid synovial fluid. This is important because C5a and other complement derivatives contribute to immune cell activation and inflammatory cytokin production. Synovial fluid also contains kinin (kini
n) and LTB4, it also contains hydrolases (including collagenase) derived from inflammatory cells.

Various mechanisms have been proposed to elucidate the extent of tissue destruction characteristic of rheumatoid arthritis. These mechanisms include complex activation and inhibition interactions that regulate the type and magnitude of the inflammatory response in RA. These mechanisms include arachidonic acid metabolites, particularly prostaglandin E2 produced by leukocytes and rheumatoid synovial fluid, bioactive amines such as histamine and serotonin, complement degradation products, eosinophil chemotactic factors, and quinine. , Interleukins, and proteolytic enzymes, especially collagenase or procollagenase activating enzymes.

Generally accepted for RA induction 1
One hypothesis is that an unknown antigen is chronically deposited in the synovium,
Therefore, it means that they are undergoing phagocytic action by type A synovial cells. B cells are induced by the donation of antigen to differentiate into plasma cells, which in turn leads to the production of antibodies, rheumatoid factors and cytokins. Activation of T lymphocytes by antigen causes synthesis of lymphokines, blastogenesis, followed by generation of antigen-specific helper and cytotoxic T cells.
The combination of an antibody and an antigen, the combination of an antibody-antigen complex and a rheumatoid factor, or the self-association of a rheumatoid factor is associated with Hagema.
n) Activate complement and quinine formation system through activation of factors. This results in the production of pro-inflammatory products such as C5a, arachidonic acid metabolites, quinines and fibrinopeptides, which diffuse into synovial fluid and further into synovial vessels.
These substances enhance the permeability of blood vessels and attract polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes are
It ingests a large number of immune complexes in the fluid, releases lysosomal and other destructive enzymes, and produces peroxide anions. This causes destruction of the hyaluronic acid polymer in the binding fluid and cartilage damage. Cytokin production in the synovium results in the further accumulation of macrophages, other immune cells, and rheumatoid synovial fibroblasts. All of these cell types can contribute to the immune cell recruitment cycle, immune medium (eg, cytokin) activation, and production that are characteristic of RA and can sustain inflammatory conditions and destroy tissues. .

Enzymes present in the synovial fluid or released in the synovial fluid by the cells that make up the proliferative synovial lesion and synthesized locally contribute to the pathological events in the joint structure. Therefore, the peculiar structure of the binding space is important. The chondrolytic lysosomal enzymes, collagenase and elastase, are primarily derived from inflammatory cells (immune cells and rheumatoid synovial fibroblasts). The proteolytic enzyme released from the stained cells plays a role in releasing the cross-linkage of collagen fibrils, and as a result, assists in the destruction of the cartilage surface and enhances the susceptibility to destruction by the enzyme. Macrophages in the synovium are prostaglandins, hydrolases, collagenases, plasminogen activators, and IL-
Produces 1. Collagenase synthesis by macrophages or rheumatoid synovial fibroblasts is greatly enhanced by the presence of IL-1 or other inflammatory proteins.
In addition to collagenase, lysosomal proteolytic enzymes also degrade proteoglycan aggregates, and once these soluble components are released from cartilage they become susceptible to subsequent enzymatic attack.

Persistence of antigen activation or dysregulation in T and B cell activation leads to a chronic inflammatory response of the synovium. Sustained cell proliferation and influx proliferates the synovium, leading to its entry into surrounding structures. Collagenase, P
Diffusion of GEs, hydrolases, and cytokins into cartilage and bone leads to the erosion of these tissues. Thus, rheumatoid arthritis is manifested as a devastating local tissue destruction resulting from a chronic inflammatory reaction brought about by a delayed hypersensitivity type immune response.

[0008] Today's treatment of RA involves bed rest, warming and medication. Salicylates are presently preferred drugs because other alternatives currently available, particularly immunosuppressants and adrenocorticosteroids, exhibit higher morbidity than the underlying disease itself. Non-steroidal anti-inflammatory drugs can also be used, many of which have effective analgesic, antipyretic and anti-inflammatory effects in patients with rheumatoid arthritis. They include indomethacin, phenylbutazone and phenylacetic acid derivatives such as ibuprofen and fenoprofen, naphthaleneacetic acid (naproxen), pyrrolealkanoic acid (tomethine), indoleacetic acid (sarindac), halogenated anthranilic acid (meclofenamate sodium). , Piroxicam, zomepirac, and diflunisal. Although they are generally considered less effective than aspirin, they may be more well tolerated in some patients.

Other drugs that can be used for RA are chloroquine,
Antimalarial agents such as gold salts and penicillamine are included.
These alternatives often result in serious side effects including retinal lesions and renal and myelotoxicity. Immunosuppressive agents such as methotrexate are toxic and are therefore used only in the treatment of severe, non-remissioning RA. Corticosteroids also have undesirable side effects, which many patients cannot tolerate well.

Chronic inflammatory diseases or abnormalities associated with class I-IV responses, especially ulcerative colitis (inflammatory bowel disease), RA or SLE (systemic lupus erythematosus), myasthenia gravis, Graves' disease, Chagas There is a need for effective treatments and compositions for the treatment of disease and suppression of immune response associated with early-onset diabetes.

The present invention also relates to suppression of immune rejection of transplanted cells, organs or tissues, or thereby. Rejection in transplants has been controlled with immunosuppressive agents such as cyclosporine antibiotics or steroids. Such drugs have a wide range of undesirable side effects.

Another attempt has been to use monoclonal antibodies that target and neutralize lymphocyte subsets that appear to be associated with transplant rejection. These antibodies are of murine origin. In the case of murine monoclonal antibodies, they generally have a low affinity effect on the target lymphocyte subsets. In addition, anti-T subset antibodies lyse and deplete T cells, because they carry the risk of increasing murine susceptibility in treated patients and the murine constant region may activate human complement. There is a risk. There is a need for compositions and methods for the treatment of rejection in tissue transplants that do not use murine immunoglobulin.

Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus identified as the human immunodeficiency virus (HIV) (1-6). Aberrant B cell function, abnormal antibody response, impaired monocyte function, impaired cytokine production (7-10), suppression of natural killer and cytotoxic cell function (11), and recognition of soluble antigens, There are reports of many immune abnormalities associated with AIDS, including reduced lymphocyte function to respond (12). Other AIDS-related immune disorders have also been described (13, 14).
A more serious immune injury in AIDS patients is the exhaustion of T4 helper / inducer lymphocytes (1, 2,
9, 10).

Despite the close observation of immunodeficiency in AIDS, the mechanism (s) of immunodeficiency is not clearly understood. There are several hypotheses. One accepted theory is that the cause of damage in the immune response is that HIV selectively infects helper T cells, resulting in helper T
The function of cells is impaired, resulting in the exhaustion of cells necessary for a normal immune response (1-
6, 15). Recently, in vivo and in vitro studies have shown that HIV also infects monocyte cells, which are known to play an essential role as accessory cells in the immune response (16,17). HIV interferes with the production of normal cytokines in infected cells and IL-1
It also causes immunodeficiency by causing secondary immunodeficiency, such as IL-2 deficiency (18). Induction of other immunodeficiencies by HIV lies in the production of factors that can suppress the immune response (19). None of these models address whether the HIV component itself, rather than the replicative virus, is involved in AIDS-related immune disorders.

The HIV env protein has been extensively described, and the amino acid and RNA sequences encoding HIV env from many HIV strains are also known (3
4). HIV virions are covered by a membrane or envelope derived from the outer membrane of the host cell. This membrane has an anchor glycoprotein (gp) anchored in its lipid bilayer via its carboxy-terminal region.
160) type is contained. Each glycoprotein contains two segments. Its relative molecular weight is about 120k
The N-terminal segment, designated gp120 on the basis of being D, projects into the aqueous surrounding environment surrounding the virion. The C-terminal segment, called gp41, spans the membrane. gp120 and gp41 are covalently linked by a peptide bond that is particularly proteolytically cleavable.
For the purposes of the present invention, HIVenv is eg gp160
Or fusions such as gp120 fused to the N-terminal fragment of gp41, HIV env with or without glycosylation, as well as any form of gp120, such as the T cell binding fragment of HIV env. HIV env and its variants are routinely prepared by recombinant culture (European Patent Publication No. 187041).
No.). Previously, g obtained by recombinant cell culture
p120 was called rgp120. Recombinant synthesis is preferred for reasons of safety and economy, but HIV env purified products from viral cultures and such env formulations are also HI.
It is known to be included in the definition range of Venv.

AI for protection of current uninfected cells
DS treatment is a nucleoside analog (AZT and DDC, etc.) that can block the replication of viral RNA about every 4 hours.
Orally. These drugs are 50-5
At a concentration of 00 μM, virus replication was blocked, but at a higher concentration (-1 mM), D required for cell division of healthy cells was
It also inhibits NA polymerase (Mitsuya and Broader, 1985). Very large amount (1g / day)
(Even) drug administration. Since the drug is ingested and absorbed by whole cells, the whole body is exposed to the drug. Its use is extremely limited by toxicity. One of the most serious side effects is anemia. Incorporation of nucleoside derivatives into DNA
In order to (and to exert its strand-breaking effect), it must be phosphorylated first, which requires a kinase that is not necessarily contained in equal amounts in all cells susceptible to virus infection. Thus, oral treatment of nucleoside analogs that are ineffective against already infected cells can protect only those infectious cells (i.e., dividing cells) that can convert high levels of nucleosides to nucleotides. it can. For this reason, this treatment has limitations and only slows the progression of the disease.

At least two types of immune system cells (monocyte cells and T lymphocytes) are infected with HIV (human immunodeficiency virus) (Streicher, 1986). Only monocytes and T cells with the CD4 receptor were thought to be infected with HIV (McDogal et al., 1986). H
Conserved region of IV virus coat protein (gp160)
Binds to the CD4 receptor that internalizes and translocates the RNA virus into the cell. Once inside the cell, the virus uses its reverse transcriptase to
Makes a DNA copy of Nucleoside analogs act as chain breakers in viral RNA transcription to protect cells. Chains are interrupted because they are incorporated into the growing polymer but do not have the functional groups necessary to incorporate the next nucleoside into the polymer.
It has no function.

Nucleoside analogs need to be phosphorylated (converted to nucleotides) by a kinase in order to be incorporated into the strand of nucleic acid (Ferman et al., 1986). T cells that can be rapidly divided are AZT and D
It contains high levels of kinases that phosphorylate antiviral agents such as DC. Monocytes that divide only in specific circumstances have kinases at relatively low levels and are not protected from infection by nucleoside derivatives. Administration of the phosphorylated form of the nucleotide analog is done in a single solution. However, they do not cross the cell membrane. Therefore, current treatment with nucleoside analogs does not
It is ineffective in protecting against IV infection.

Lyary (PNAS 84, 4
601 (1987)) is an uninfected CD bound to gp120.
4+ lymphocytes have been shown to be a target for antibody-dependent cellular cytotoxicity by goat serum raised against native gp120. However, human serum that binds to gp120 adsorbed cells did not show its disrupting effect in the presence of complement. In contrast, these sera strongly mediate antibody-dependent cellular cytotoxic effects.

Monoclonal and polyclonal antibodies specific for the HIV encoded protein have been developed (eg PCT / WO86 / 00217).

Immunotoxic substance (ITs) consisting of a cell-reactive antibody bound to the A chain (IT-As) of rittin toxin
Have been used to specifically kill a variety of target tumor cells. Kronke et al. (Cancer Research, 46 (7),
3295 (1986)) discloses that a conjugate of a monoclonal antibody to interleukin II receptor and ritin A chain kills HTLV-I infected leukocyte T cells. Kronke, "Blood" 65
See also (6), 1416 (1985).

Shobal et al. (US Pat. No. 4,714,61)
No. 3) expresses hepatitis B surface antigen on its surface (here,
(A surface antigen is encoded by the DNA of HBV). A method of suppressing the growth of hepatocytes or hepatoma cells infected with hepatitis B virus was disclosed. The method of Shoval et al. Consists of administering to infected cells complement which has immobilized a monoclonal antibody against the hepatitis B surface antigen.

That is, the present invention aims to provide an improved therapeutic method for immune inflammatory diseases. The present invention also relates to an improved method for treating AIDS with reduced side effects. The present invention also provides a method for killing non-tumorigenic cells that express viral antigens encoded on their cell surface.

Other objects, features and characteristics of the invention will be apparent upon consideration of the following description and appended claims.

[0025]

SUMMARY OF THE INVENTION HIV env is useful in the treatment of immune inflammatory responses and diseases, and an object of the present invention is to provide patients exhibiting an immune inflammatory response with an HIV env formulation in an amount sufficient to suppress the immune inflammatory response. Administering.

The HIV env polypeptide used in one aspect of the invention is the polypeptide portion of HIV env that contributes to the binding of T helper cells to the T4 cell surface marker. This part of the HIV env sequence was called the TCB domain and was located in an unexpected region containing residues 400 to 465. HIV env TC, including various analogues
The B domain is also useful in treating immune inflammation. The TCB domain is also useful in diagnostic assays for TCB domain neutralizing antibodies in samples obtained from patients.

HI with the functional TCB domain removed
Venv is useful as a vaccine to raise immunity to HIV infection.

The present invention provides a monoclonal antibody specific for one region of the TCB domain. This antibody appears to be directed against an epitope located adjacent to the sequence of the TCB domain that binds the T4 cell surface marker. This indicates that this antibody probably sterically inhibits the approach of the TCB domain binding site on the T4 cell surface antigen to the recombinant gp1.
It is demonstrated by the ability to at least partially block the binding of 20 to the T4 cell surface marker. This antibody is also useful for passive immunization of HIV-infected patients, since it has the advantage that it can bind to any free HIV env that exhibits immunosuppressive action in HIV-infected patients.

The present invention provides an immunotoxic agent containing a monoclonal antibody that specifically binds to gp160 or other virus-encoded protein, and a toxin such as ritin A chain that binds to the monoclonal antibody. , Provides a protein encoded by a virus as a target. Thus, the protein associated with the toxic conjugate is only taken up by infected cells, such as helper T cells, causing them to die. The present invention also provides a gp encoded by the virus on the cell surface.
An immunotoxic agent containing a monoclonal antibody bound to a toxin that specifically binds a protein such as 160,
The present invention provides a method for killing virus-infected cells, which comprises administering to a patient a therapeutically effective amount to kill infected cells.

HIV env or an immunosuppressive fragment thereof (containing the TCB domain) was treated against cytotoxic T
Conjugates have been formed with haptens or polypeptides that are capable of binding the cell population of cells for which a cell cytolytic response is desired. These conjugates are particularly useful as antitumor agents or in the treatment of allergic responses.

[0031]

DETAILED DESCRIPTION OF THE INVENTION As described above, HIV env was obtained with the human immunodeficiency virus envelope protein and its immunosuppressive amino acid sequence variants in vitro and covalent modifications of HIV env or its variants. It is defined as a derivative. For mutants, en
v Amino acid sequence with one or more residues replaced, env sequence with one or more residues deleted, and one or more residues inserted therein Is included.

The major deletion mutants of the present invention are TCB
These deletion mutants, which are mutants in which immune epitopes other than the domain are deleted, or conversely, in which the TCB domain is deleted and the remaining epitopes of HIVenv remain, Will be explained in detail. Variants belonging to another class are those in which the HIV env sequence or an immunosuppressive fragment thereof (eg the TCB domain) is fused at the amino or carboxy terminus with a polypeptide heterologous to HIV env (heterologous). Alternatively, the HIVenv or TCB domain can be
Covalently attached to a heterologous polypeptide or hapten. Another class of TCB domain variants is TC
The B domain is an HIV immunoepitope that is not normally bound to the TCB domain in the native HIV env, and in particular is bound to the C-terminus of the TCB domain by the formula: FRPGGGDMRDNWRSELYKY
It is a mutant obtained by fusing with an epitope other than the sequence shown by KV.

The fusion of TCB domain of HIV env with an immunogenic hapten or polypeptide is useful as a component of a vaccine to immunize patients against HIV infection. A fusion of a hapten or heterologous polypeptide with HIV env or an active fragment thereof will target cytotoxic T cells that target the surface receptor or other binding partner of the target cell if the hapten or heterologous polypeptide is capable of binding the target cell. It is useful for the purpose.
For example, membrane-bound transforming growth factor-α (TGF-α)
Is present on the surface of many solid (non-hematopoietic) malignancies. Antibodies capable of binding TGF-α are known. N-terminal or C-terminal fusion of HIV env or a TCB domain containing fragment thereof by linking such antibody to HIV env or a TCB domain containing fragment thereof, eg by covalent cross-linking (33) or by recombinant cell culture. It is bound by being expressed as a product. The latter method is described in EP125,02.
This is achieved by obtaining the DNA encoding the TGF-α antibody according to the general procedure described in 3A. This DNA was ligated with the appropriate adapter to the amino or carboxy terminus of gp120 or a fragment containing the TCB domain thereof, EP187,
It is inserted into a mammalian cell expression vector as described in 041A and the resulting vector is used to transform CHO cells or other suitable host cells as described in EP187,041A. If desired, DNA encoding the variable region of the TGF-α antibody may be prepared using EP184,
187, EP194,276A, EP171,496A or the human constant region described in Bouliamne et al. (32), is ligated with DNA encoding the TCB domain. The resulting fusion, for example, when the fixed region of the immobilized TGF-α or TGF-α antibody is retained, is recovered by adsorbing to the immobilized antibody to the constant region, It is formulated in a therapeutically acceptable vehicle (eg saline) and administered to patients with TGF-α expressing tumors. This fusion directs cytotoxic T cells to tumors bearing TGF-α cell surface antigens, resulting in lysis of tumor cells. Similarly, a patient may have fused the TCB domain with an antigen that is producing an unwanted immune response, such as the development of allergic or post-transplant rejection reactions, resulting in autoimmune cytotoxic T
The patient is administered at a dose sufficient to direct the cellular response to clonal cells that contribute to the unwanted response.

The TCB domain of HIV env is EP187,
About 7006 nucleotides of the HIV strain described in 041A.
To about 7203, or equivalent regions in other HIV strains (in other strains in which the viral genome and HIV env length are altered by upstream deletions or insertions). , It is not possible to obtain the same nucleotide and amino acid residue number, but the region coding for this part of the TCB domain in each strain can be readily identified on the basis of amino acid sequence homology). The TCB domain has the formula:

[Chemical 5] In particular, residues 411-454, 41
6-443, and the sequences contained between 440-442. Table 1 below shows the amino acid sequences of the TCB domains from multiple HIV strains below "Origin" in the right column and the pre-determined variant TCB domains in the left column. The variant indicated by "Δ3" (variant 1) makes it possible to identify this part of the TCB domain of HIV. Rg by site-directed M13 mutagenesis
Deletion of the Δ3 residue of p120 and expression of this variant in recombinant CHO cell culture as otherwise described in EP187,041 was based on assays such as binding by serum of selected AIDS patients. , But indistinguishable from rgp120, the T4 binding ability of rgp120 was lost. Surprisingly, as shown in Table 1, the deletion of the Δ3 region was more pronounced in many HIV strains than in BH10, as can be seen by comparison with homologous sequences from other HIV strains. It is found that the TCB domain as a whole is not highly conservative and contains the HIV env region, which is characterized by being highly variable (34). It was believed that only regions with a high degree of conservedness had the appropriate certainty to confirm that the virus always recognized the T4 receptor and consequently affected recipient host cells. It didn't work in the study. For example, although residues 5-31 are highly conserved in various HIV strains, HIVen
Deletion of the first 29 residues from the amino terminus of v had no effect on the binding of rpg120 to T4.

[0035]

[Table 1]

The variants numbered 2-15 in Table 1 are improved: the T4 binding properties of rgp120 are eliminated. For example, substituting aspartic acid for alanine at position 431 reduced the binding of the mutant to approximately 10-15% of the binding exhibited by rgp120 with the native sequence. However, in our assay, even if lys430 was replaced with glutaminyl, tyr433 was replaced with phenylalanyl, and pro435pro436 was replaced with valylvalyl,
No change in detectable rgp120 binding occurred. Therefore, the activity of the individual variant should be measured and screened by conventional assays.
From the role of the Ala431 residue, substitution, deletion or insertion at this site is particularly preferred, which includes histidine, tryptophan, proline, phenylalanine, glycine, leucine, isoleucine, methionine, valine, serine, threonine, Substitution with tyrosine, glutamine, asparagine, lysine, glutamic acid, cysteine and arginine, or such residues or Ala43
The insertion of aspartic acid adjacent to 1 is included.
The variants and Δ3 variants described above are useful in preparing HIV env antigens for use in diagnostic assays or vaccines. HIV env containing no TCB domain is particularly useful as a vaccine because it not only shows no immunosuppressive effect, but also retains the desired number of residual gp120 epitopes, thus it has a higher titer of anti-HIV. And lead to an extension of this titer. Other variants will be apparent to those of skill in the art. For example, to identify variants that function as HIV env agonists, substitutions are made within residues 416-442. In addition, the deletion or insertion is TC
It can be introduced into the B domain to evaluate agonist activity.
The term "agonist" as used herein refers to
It is meant that the HIV env activity of the variant is increased compared to the native molecule (eg the variant shows a higher affinity for T4 than the native HIV env). However, it will be appreciated that this activity is an antagonist to infection by HIV.
HIV env agonist analogs can be isolated from intact or native HIV e
It is a substance useful in treating undesired immune inflammation as in the embodiments described above for nv. Agonist analogs are optionally used in conjunction with other therapeutic agents useful in the treatment of HIV infection, such as tumor necrosis factor alpha, tumor necrosis factor beta, interferon and / or AZT, AIDS or ARC.
Administered to treat a patient's HIV infection.

HIV env agonist activity was determined in the following examples by measuring its agonistic ability to compete with labeled rgp120 or native gp120, or fluorescein isocyanate-conjugated anti-T4A antibody for binding to the T4 receptor. The fluorescence activated cell separation and collection device (fluorescence activate described in
d cell sorter system). This ability of mutant candidates to compete with the labeled conjugate for T cell binding in PBMC is one measure of its activity: labeling of the T4 binding site in the mutant. As the competitiveness with the conjugate increases, the agonist activity of the variant increases.

The HIV env TCB domain or variants thereof are prepared in a manner known per se. One method is to synthesize the TCB domain by in vitro engineering methods such as Merrifield synthesis. Another method is to prepare the domain with recombinant cell culture methods. Facilitates the preparation of DNA encoding its domain by conventional in vitro methods and the construction of suitable 5'and 3'adapter or linker to ligate into a conventional vector for bacterial expression. Is practical. In this one preferred embodiment, the 5'end of the DNA encoding this domain is ligated to the DNA encoding the signal sequence recognized by the host. Such signals are well known and examples include the STII, alkaline phosphatase, lpp and penicillinase signals.
Certain heterologous signals are also recognized by bacteria, such as mammalian or yeast signals. Since the DNA encoding such a signal is only about 45 to 75 nucleotides in length, it was prepared by ligating the signal DNA to the 5'end of the DNA encoding the TCB domain, The resulting fusion gene is ligated into a conventional vector such as pBR322 and used as a host E. coli.
The amount of TCB domain secreted into the periplasm of the host cell could be measured by transfecting (E. coli) and culturing the cells. Alternatively, the TCB domain is synthesized by recombinant cell culture methods as a secretory or non-secretory fusion and is recovered.

Antibodies capable of inhibiting or binding the TCB domain are clonal which prevent binding to the T4 T cell marker when mice such as Balb / c or preferably C57 Black / 6 are immunized against gp120 and preincubated with gp120. Obtained by screening the antibody. The 5C2E5 antibody is an example of such an antibody, but it is not unique as other antibodies exhibiting the same qualitative activity are obtained by the methods described herein. The term “qualitative” activity means that the antibody is HIVen.
It means that it binds to the T4 binding site or both side regions of v. In the latter case, rgp120 or HIVenv
Is a bond that sterically hinders the binding of.
Tryptic digestion of the 44 residue TCB domain confirmed that the 5C2E5 antibody binds to a peptide spanning residues 420-430. Therefore, this domain is contained within or adjacent to the TCB domain. This 5
The C2E5 antibody was found in 1 to 500 myeloma fusions with murine B cells recovered from mice immunized with gp120. Another inhibitory antibody is 7F11, which binds to a peptide spanning residues 430-459.

The TCB domain binding property of the antibody 5C2E5 is determined by the rgp for the T4 receptor of helper lymphocytes.
Specified as the ability to inhibit 120 binding. Monoclonal antibodies other than 5C2E5 capable of exhibiting such an action include affinity, immunoglobulin class,
Even if the species of origin or the exact TCB sequence is different, it falls within this particular range, for example, of the 5C2E5 antibody whose antibody or amino acid sequence expressed in recombinant cell culture is predetermined. Variant eg 5C
Examples include chimeras of the variable region of the 2E5 antibody and human constant regions.

The antibodies described herein are conventional
These immunoglobulins are recovered from hybridoma cell cultures by conventional purification methods for these immunoglobulins, such as those used to purify IgG or IgG from stored plasma, eg ethanol or polyethylene glycol precipitation.
The thus purified antibody is sterilized and filtered, and if necessary, AID
It is conjugated to a cytotoxic agent such as ritin that can be used for S treatment or conjugated to a detectable marker such as an enzyme or spin label that can be used in an HIV diagnostic assay in a test sample.

A new application example of an immunotoxic substance (immunotoxin) was discovered. It was surprisingly found that an immunotoxic agent that specifically binds to the virus-induced surface antigen of cells infected with non-neoplastic virus kills the infected cells. Specifically, gp160-binding immunotoxic agents kill HIV-infected cells.

The following is a description of treatments intended to kill cells that have already been infected and are actively producing new virus. Killing is done by immunotoxic agents that bind to viral coat proteins expressed on infected cells. Therefore, this immunotoxic substance is internalized in the cells and kills them. Infected cells whose viral genome has been integrated into DNA but have not synthesized viral proteins (ie, cells in which the virus is latent) are killed by immunotoxic agents until they begin to synthesize virus. May not be.

We have discovered that HIV-infected cells, which are actively producing virus, express the viral coat protein (gp160) on their plasma membrane. This molecule represents an infected cell-specific antigen that can be used to target and kill infected cells. gp1
A monoclonal antibody (strain-dependent) specific for the conserved region on 60 can be used as an attacking molecule to deliver toxic substances to infected cells. Additionally, or alternatively, a "cocktail" of immunotoxic agents against non-conserved regions is used. In addition, the toxicant-antibody conjugate can bind to the circulating virus or viral coat protein, thus killing the cells internalizing the virus or coat protein.

The object of the present invention is to provide a highly selected method for destroying HIV infected cells.
Since these antibodies specifically bind to antigens that are not present on the uninfected cells, the method of the invention comprises:
It allows the antibody to attack the cells to be destroyed very accurately. Unlike tumor treatment with immunoconjugates that result in the loss of antigen and develop resistance, antigen expression is always associated with active viral infection.

Without intending to force a particular theory regarding the practice of the present invention, it is believed that the expression of the target antigen on the surface of infected cells is transient. The antibody must be able to reach the site on the cell surface where the antigen is present and where it interacts. After the antibody is complexed with the antigen, a phagocytosis (endocytosis) occurs, and the toxic substance is carried into the cell.

Immunotoxic agents are particularly useful in killing monocytes / macrophages infected by the HIV virus. In contrast to transient virus production from T cells, macrophages produce high levels of virus for long periods of time. Conventional therapies are inefficient at inhibiting the production of new virus in these cells.

Not all monoclonal antibodies specific for gp160 produce high levels of cytotoxic immunotoxic agents when coupled to the ricin A chain (IT-A). The assay employed to predict the ability of an antibody to function as a subbody of IT-A was performed. This assay was performed and various anti-HIV antibodies were screened for their ability to effectively create IT-A against HIV-infected cells. The results show that only certain HIV-specific antibodies create effective IT-As. These antibodies were purified and coupled to ritin A chain,
It is used to specifically kill HIV-infected cells. Conveniently, these antibodies cross-react with some (or all) HIV strains.

Immunotoxic agents can be prepared by a variety of methods (see below for details). Several cross-linking reagents can be used to prepare conjugates to obtain stable conjugates.

A preferred embodiment deglycosylates the ritin A chain or prepares it without oligosaccharides and reduces its clearance by an irrelevant mechanism of clearance (ie liver). Another aspect is B
If the ability to bind galactose in the chain can be inhibited ("inhibitory ritins"), then all ritins (A chain + B chain) are conjugated to the antibody.

Further, the toxic substance-conjugate is Fab
Or F (Ab ') 2 fragment. Due to their relatively small size, they penetrate tissue more favorably and reach infected cells.

Antibodies According to the invention, a monoclonal antibody that specifically binds to an epitope of gp160, or a cell surface exposed fragment thereof having antigenic activity (eg gp120, gp
41, an epitope selected from TCB, etc.) was isolated from an infinite hybrid cell line generated by fusion of lymphocytes primed with antigen and myeloma cells. Conveniently, a gp160-binding monoclonal antibody according to the invention binds to a domain of this protein exposed on the cell surface. Another aspect of the invention is the use of polyclonal antibodies that specifically bind to gp160.

The antibody which is the object of the present invention can be obtained by a screening method. An assay is performed in which the monoclonal antibody is screened for its cytotoxicity as potent as the A chain containing the immunotoxic agent. This assay involves applying cells to a dilution of the test antibody, followed by ritin A.
For application to Fab fragments of a secondary antibody coupled to a chain (indirect assay). The cytotoxicity in this indirect assay is compared to that in the direct assay in which a monoclonal antibody is attached to the ritin A chain.
This indirect assay method can accurately predict the effectiveness of the monoclonal antibody as an immunotoxic substance, and thus can be used for screening a monoclonal antibody useful as an immunotoxic substance. Vitetta et al. Science 238 , 1098-1104 (198)
7), and Weltman et al., Cancer Res. 47 , 5552 (1987), which are also within the scope of the invention.

Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to conventional antibody (polyclonal) preparations which usually contain different antibodies directed against different determinants (epitopes), monoclonal antibodies are individually aimed at a single determinant on the antigen. .. Monoclonal antibodies are useful for improving the selection sensitivity and specificity of diagnostic and analytical methods that utilize antigen-antibody binding properties. The second advantage of monoclonal antibodies is that they are synthesized by the hybridoma culture and are not contaminated by other immunoglobulins. The monoclonal antibody can be prepared from the supernatant of the hybridoma cells in culture or from the ascites induced by injecting the hybridoma cells into the abdominal cavity of the mouse.

Kohler and Mils
tein) [Eur. J. Immunol. 6 , 511 (1976)], the hybridoma engineering method originally disclosed extensively for preparing hybrid cell lines secreting high levels of monoclonal antibodies against many specific antigens. It's being used.

The steps and regimens of immunizing a host animal or culturing antibody-producing cells derived therefrom are generally carried out as routine procedures established for antibody stimulation and production. When the method of the present invention is used as a basis for producing a hybrid cell line of mammals including humans, it is considered that any mammalian subject including human subjects or antibody-producing cells derived therefrom can be skillfully utilized. We used mice as a test model.

After immunization, immune lymphoid cells are fused with myeloma cells to generate hybrid cell lines that can be cultured and subcultured indefinitely to prepare a large amount of monoclonal antibodies. For purposes of this invention, immune lymphoid cells selected for fusion are lymphocytes taken from either the lymph node tissue or spleen tissue of immunized animals and their normally differentiated progeny. We prefer to use immune spleen cells for the mouse system, rather, as the immune spleen cells provide a higher concentration and a convenient source of antibody-producing cells. Myeloma cells provide the basis for continued growth of fused hybrids. Myeloma cells are tumor cells derived from plasma cells.

It is possible to fuse cells of one species to cells of another species. However, the sources of antibody-producing cells and myeloma to immunize are preferably from the same species.

This hybrid cell line can be maintained by in vitro culture in a cell culture medium. The cell lines according to the invention can be selected and / or maintained in a composition consisting of an infinite passage cell line in the known hypoxanthine-aminopterin-thymidine (HAT) medium. In fact, once the hybridoma cell line has been established, it can be maintained in various nutritionally appropriate media. Further, the hybrid cell line can be stored and protected by any of a number of conventional methods, such as cryopreservation and storage under liquid nitrogen. Frozen cell lines can be revived, cultured indefinitely, and monoclonal antibody synthesis and secretion reinitiated. The secreted antibody is recovered from the tissue culture supernatant by a conventional method such as precipitation, ion exchange chromatography, or affinity chromatography.

Although the present invention has been described using a mouse monoclonal antibody, it is not limited thereto and, in fact, a human antibody can also be used, and it can be proved that it is preferable. it can. Such antibodies can be obtained using human hybridomas [Cot (Cot
e) Monoclonal Antibodies and Cancer Therapy
Therapy), Alan R. Liss, 77
P. (1985)]. Indeed, according to the present invention, a gene derived from a mouse antibody molecule of suitable antigen specificity, as well as having a suitable biological activity (eg, activating human complement, A
A method for preparing a "chimeric antibody" by splicing a gene derived from a human antibody molecule (which can mediate DCC) [Procedures of Morrison et al.
National Academy of Science (Proc.Nat
l.Acad.Sci.) 81 , 6851 (1984): Naberger
(Neuberger) et al. Nature 312 , 604 (1)
984): Natures 314 and 45 of Takeda et al.
2 (1985)] are available; such antibodies are also within the scope of the present invention.

As another method in the cell fusion method, EBV
Immortalized B cells are used to prepare monoclonal antibodies according to the invention. In addition, other methods such as recombinant DNA for preparing monoclonal antibodies can be performed.

The most important immunochemical derivative of the antibody of the present invention is an immunotoxic substance (conjugate of antibody and cytotoxic moiety). This antibody can also be used to induce cell lysis by the natural complement reaction and interact with the normally existing antibody-dependent cytotoxic cells.

[0063] cytotoxic moiety of the immunotoxin immunotoxin are bacteria, fungi, plant or animal origin cytotoxic or enzymatically active toxins or enzymatically active fragments of these toxins (A-chain) possible. The enzymatically active toxins and their fragments used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ritin A chain, abrin A chain. Chain (abrin A chain), modeccin A chain, alpha-sarcin (alpha-sarci)
n), Aleurites Foldy Protein (Aleurites
fordii proteins), dianthin protein (dianthin p
roteins), Pitracuka americana protein (Phytola
cca americana proteins) (PAPI, PAPII and PA
PS), Momordica Carranchia Inhibitor (m
omordica charantia inhibitor), curcin,
Crotin (crotin), sapaonaria officinalis inhibitor (sapaonaria officinalis inhibitor), gelonin (gelonin), mitogellin (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin),
Enomycin and trichothecenes
enes). In other embodiments, the antibody is conjugated to a small molecule anti-cancer agent. Conjugates of monoclonal antibodies with the above cytotoxic moieties are prepared using a variety of bifunctional protein coupling agents. Examples of such reagents include SPDS, IT, bifunctional derivatives of imide esters such as dimethyl adipimidate HC.
1, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azide compounds such as bis (p-azidobenzyl) hexanediamine, bisdiazonium derivatives such as bis (p-diazoniumbenzoyl) ethylenediamine, diisocyanates such as Tolylene 2,6-diisocyanate, and bis-activated fluorine compounds such as 1,5-difluoro-2,
4-dinitrobenzene is mentioned. The cytolytic portion of the toxin can be attached to the Fab fragment of the antibody.

Suitably, a monoclonal antibody that specifically binds to a domain of the protein exposed on infected cells is conjugated to the ritin A chain. Most preferably, the ricin A chain is deglycosylated and prepared by recombinant methods. Suitable methods for creating the ricin immunotoxin is Bitteta (Vitetta) et al. Science (Science) 2
38 , 1098 (1987), which are also within the scope of the present invention.

If a conjugate is used when killing infected human cells for the purpose of in vitro diagnosis, this is usually added to the cell culture medium at a concentration of at least 10 nM. The dosage form and method of administration for in vitro applications is not critical. Aqueous dosage forms compatible with the culture or perfusion medium will usually be used. Cytotoxicity can be read by a conventional method.

Cytotoxic radiopharmaceutical formulations for treating infected cells can be made by conjugating a radioactive isotope (eg I, Y, Pr) to an antibody. It is preferred to use isotopes that emit alpha particles. The term "cytotoxic moiety" as used herein is meant to include such isotopes.

In another embodiment, fusogenic
The liposome is filled with cytotoxin, and the obtained liposome is gp
Coated with an antibody that specifically binds to 160.

Antibody-Dependent Cytotoxicity The present invention further provides (a) gp160, a subclass or isotype capable of mediating lysis of cells infected with the HIV virus (b) to which the antibody molecule is bound. And methods of using the antibodies. More specifically, these antibodies complex with cell surface proteins, activate serum complement, and / or effector cells (e.g., natural killer cells or macrophages).
FFector cell) to activate antibody-dependent cellular cytotoxicity (ADCC).

The present invention is also directed to the use of these antibodies in their natural form for the treatment of AIDS. For example, H
IgG2a and IgG3 that bind to IV-related cell surface antigens
Mouse antibodies can be applied in vivo for AIDS treatment. Indeed, since gp160 is present on infected monocytes and T-lymphocytes, the antibodies of interest and their therapeutic use have wide applicability.

It is known that the biological activity of antibodies can be measured in large part by the Fc region of antibody molecules [Uananue and Benacerraf, Textbook of Immunolog.
y), Second Edition, Williams & Wilkins
ins) page 218 (1984)]. This includes the ability to activate complement and mediate the antibody-dependent cellular cytotoxicity (ADCC) performed by leukocytes. Antibodies of different classes and subclasses differ in this respect, and according to the invention, one selects for a class of antibody having the desired biological activity. For example, IgG3 and IgG2a class mouse immunoglobulins can activate serum complement when bound to target cells expressing cognate antigens.

In general, antibodies of IgG2a and IgG3 subclasses and, but not always, IgG1, are found in ADCC.
And antibodies of the IgG3 and IgG2a and IgM subclasses bind to and activate serum complement. Activation of complement generally requires that at least two IgG molecules bind to the target cell in close proximity. However, the binding of only one IgM molecule activates serum complement.

The ability of any particular antibody to mediate lysis of target cells by activation of complement and / or ADCC can be assayed. The cells of interest are grown and labeled in vivo: the antibody is placed in cell culture with either serum complement or immune cells that can be activated by antigen-antibody conjugation. Cytolysis of target cells is detected by the label released from the lysed cells. Indeed, antibodies can be screened using the patient's own serum as a source of complement and / or immune cells. Thus, antibodies capable of activating complement in an in vitro assay or mediating ADCC can be used to treat that patient.

Antibodies of any viral origin
Can it bind to the gp160 epitope and activate complement,
Or, if it can mediate ADCC, it can be used as this aspect of the present invention. Monoclonal antibodies have the advantage of being abundant in origin. In fact
By immunizing mice with gp160, establishing hybridomas that produce antibodies to gp160, and then selecting those hybridomas that produce antibodies capable of lysing infected cells in the presence of human complement. A panel of antibodies capable of reacting and dissolving it can be quickly established.

Therapeutic Uses of the Antibodies of the Invention When used therapeutically in vivo, the antibodies of the invention are administered in a therapeutically effective amount [ie, an amount that restores T cell counts]. Administer to patient. It is usually administered parenterally. The dose and method of administration depend on the degree of infection,
It depends on the characteristics of the individual immunotoxic substance (if used), eg its therapeutic index, the patient's condition, and the patient's medical history. The immunotoxic agent is preferably administered intravenously to treat cells of the vasculature and subcutaneously and intraperitoneally to treat local lymph nodes for 1-2 consecutive weeks. If desired, tumor necrosis factor and interferon may be administered during an adjunct treatment phase, such as a combined cycle.

For parenteral administration, the antibody is formulated in a unit dosage form for injection (solution, suspension, emulsion) together with a pharmaceutically acceptable parenteral vehicle. The vehicle is essentially non-toxic and has no therapeutic effect. Such vehicles include, for example, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Fixed oils and non-aqueous vehicles such as ethyl oleate can also be used. Liposomes can be used as carriers. Such vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives. Usually, the antibody will contain about 1 mg / m 2 of such vehicle.
Formulations are included at concentrations from 1 to 10 mg / ml.

IgM antibodies are advantageous because their antigens are highly target cell-specific and rarely appear on normal cells. Since the IgG molecule is smaller than the IgG molecule, it may be more likely to concentrate in infected cells.

In vivo complement activation has been shown to lead to various biological effects, such as the induction of inflammatory responses and macrophage activation [Uananue and Benacerraf, textbook·
Textbook of Immunology, Second Edition,
Williams & Wilkins 218
P. (1984)]. The increased vasodilation associated with this inflammation is
Various anti-AIDS agents may increase the effect of focusing on infected cells. Thus, the types of antigen-antibody combinations identified according to the present invention can be used in many therapeutic applications. Furthermore, purified antigens related to such antigens [Hakomori, Ann, Rev. Immunol. 2 , 1
03 (1984)] or anti-idiotype antibody [Nepom.
(Nepom) et al., Proc. Natl. Acad. Sci. 81 , 2864 (19
85): Proc.Natl.Acad.S of Koprowski et al.
ci. 81 , 216 (1984)] could be used to induce an active immune response in human patients. Such responses include the formation of antibodies that can activate human complement and mediate ADCC, and this mechanism leads to the destruction of infected cells.

The antibody according to the present invention is used for HIV in a test sample.
It is useful in diagnosing. It is used as one axis of a sandwich assay to assay HIV env, along with polyclonal or monoclonal antibodies to other sterically free epitopes of HIV env.
For use in some specific sandwich tests,
The 5C2E5 antibody or its equivalent is attached to an insoluble support or labeled with a detectable moiety according to standard methods using other monoclonal antibodies. As another example of the labeled antibody, for example, labeled sheep anti-mouse IgG capable of binding to 5C2E5 antibody is used, and HIV env binding property is detected by a conventionally known operation method.

Finally, by way of another example, the antibody is immobilized on a water-insoluble support and, as described in more detail below, HIVenv, rgp120 or TCB.
Used for immunoaffinity purification of domains. Any known method for immobilizing monoclonal antibodies can be utilized for this purpose.

In general, inflammation to be treated with HIV env or immune-enhanced inflammation is humoral and / or humoral to foreign or autologous target tissue which is undesirable in terms of therapeutic implications.
Alternatively, it is characterized by a cytotoxic cellular response. In general, immunopotentiating inflammation is the production of antibodies against (1) host antigens in unwanted autoimmune diseases or in graft-versus-host disease, or (2) antigens in transplanted tissues, such as organ grafts. And / or is characterized by a cytotoxic T cell response. This is clinically characterized by infiltration of polymorphonuclear neutrophils and mononuclear leukocytes into target tissues, pain, focal edema, possible vascular endothelial damage, and widespread production of cytokines by stimulator cells. Attached.

HIV env compositions intended for therapeutic use take into account the disorder to be treated, the condition of the individual patient, the site of dispersion of the env polypeptide, the mode of administration, and other factors known to the practitioner, It should be formulated and doses established in a manner consistent with good treatment.

HIV env is prepared for administration by mixing HIV env, purified to the desired degree to a physiologically acceptable carrier, ie, a carrier that is nontoxic to recipients at the dosages and concentrations used. HIV env is usually combined with buffers, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates such as glucose or dextran, chelating agents such as EDTA, and other excipients. HI for use in therapeutic administration
Venv must be sterile. This means that (0.
It can be easily done by sterile filtration with a (2 micron) membrane. HIV env is usually stored as a lyophilizate for reconstitution, but is stable when stored as an aqueous formulation.

In general, where the disorder permits, HIV env should be formulated and administered for site-specific distribution. This is convenient in case of RA and inflammatory bowel disease. In the former case, HIVenv is formulated in a sterile, sustained release composition suitable for injection into synovial fluid or implantation in the synovial lining or capsule. In the case of inflammatory bowel disease, HIV env is formulated into suppositories using pharmaceutically acceptable oily substances well known in the art.

The sustained release formulation is selected from among microcapsule particles and implantable articles. Encapsulation in liposomes is not preferred for direct injection into the synovial cavity, as it entails the transfer of lipids into the joint. However, liposome encapsulation is suitable for implanting sustained release HIV env in the synovial sac. To treat RA using a sustained release HIV env, the HIV env is preferably encapsulated in a biodegradable matrix or microcapsules. A suitable substance for this purpose is
Polylactide, but poly-D-(-)
Other polymers of poly (α-hydroxycarboxylic acid) such as -3-hydroxybutyric acid can also be used [E
P 133,988A]. Other biodegradable polymers include poly (lactones), poly (acetals), poly (orthoesters), poly (orthocarbonates), and the like. Here, the first consideration must be that the carrier itself or its degradation products are non-toxic to the target tissue, and that they do not aggravate the disease. This can be assayed by performing routine screening in animal models of the target disorder, or in normal animals if no such model is available. For examples of sustained release compositions, see, for example, U.S. Pat. No. 3,773,919, EP58,481A, U.S. Pat. "Biopolymers", 22: 547 (198).
3), and R. Langer et al., "Chem.Tech" 12: 9.
8 (1982).

The dose of HIV env to be used is HI
T4 binding affinity of Venv preparation, half-life of env preparation, administration route, clinical condition of patient (presence or absence of infection, etc.), anti-
Depends on many factors, such as the presence of HIV env antibodies and whether HIV env can be used to prevent disease, or to treat acute autoimmune disease or episodes of transplant rejection . As a general rule, the HIVenv dose to reach the target site is about 0.5.
It should be from x10-8M to 5x10-9M. The HIV env concentrate in therapeutic dosage forms is usually administered by continuous infusion, sustained release infusion or empirically determined infusions. RA synovial fluid can be up to 50 ml in knee joints, for example, so HIV
The exact amount of env depends on the dilution in synovial fluid, as well as the empirically measured loss of HIV env for endogenous proteases, leakage into the systemic circulation, and existing anti-H.
Determined by sequestration by IVenv. Therefore, in order to determine an appropriate dosing regimen, it is best to take a sample of synovial fluid for the HIV env T4 binding assay during the early stages of treatment to assess the efficacy of the initial dose protocol. Is.

HIV env therapy includes other anti-inflammatory substances such as salicylates, non-steroidal anti-inflammatory agents, penicillamine, gold salts, TGF-β, TNF-α or TNF-β antagonists (pending U.S.S. .N898,272), a γ-interferon antagonist and / or an IL-1 antagonist.
Although it is not essential that such substances be included in the HIV env composition itself, these anti-inflammatory substances are
It may be formulated with Venv.

HIV env concentrations concentrated at the site of inflammation may exceed the highest therapeutic doses systemically. Assaying the HIV env concentration in the inflammatory infiltrate provides guidance on the amount of HIV env for intravenous infusion, especially when concentrated administration is not practical. The result obtained by this is an important factor for selecting an appropriate dose. If the patient's inflammatory response does not resolve, at least in part, within about 48 hours after administration, the dose is gradually increased until the desired effect is achieved. Correspondingly, the required T4 binding capacity and / or T4
The dose of HIV env preparations with low affinity for E. coli is increased, but the dose of preparations with high potency and affinity is also increased. Also, when treating patients with acute rejection or development of inflammation, ie acute organ transplant rejection or development of arthritis flare, relatively higher doses are required initially.

For urgent treatment associated with the onset of acute rejection, HIV env is injected intravenously or introduced directly at the site of inflammatory injury when symptoms develop or when rejection is serologically revealed. . Alternatively, intramuscular or subcutaneous administration can provide appropriate prophylaxis. Hereinafter, the present invention will be described in more detail with reference to Examples, but these are not intended to limit the present invention. All references cited are included as references.

[0089]

EXAMPLES Example 1 It was found that the initiation of T-helper infection by HIV involves a selective interaction between the HIV envelope glycoprotein and the T4 surface structure (1-6, 1).
5, 23-26). More specifically, McDougal et al. Have shown that HIV can selectively bind to the T4A epitope of the T4 antigen complex (25). Furthermore, many of the impaired immunological functions of patients infected with the AIDS virus have been linked to HIV's selective ability to infect and ultimately destroy helper cell populations (1, 2, 9). 10). Also, AI
It has also been suggested that PBMCs from DS patients are hyporesponsive to mitogen and antigen (especially tetanus toxoid) stimulation, presumably due to reduced helper cell function associated with HIV. This example is mainly
Rgp in tetanus toxoid response in normal PBMC
Relates to measuring the activity of 120.

Materials and Methods Recombinant HIV envelope glycoprotein (rgp120) :
Chinese hamster ovary cells transfected with the HIV envelope gene expression plasmid (20) were used for the preparation of rgp120 used in these examples. Rgp120 was purified from conditioned cell culture medium by immunoaffinity chromatography. The rgp120 was> 98% pure as determined by SDS-polyacrylamide gel electrophoresis.

Peripheral blood mononuclear cell (PBMC) isolation : Blood was collected from healthy donors using heparinized syringes. Dilute each blood sample with an equal volume of saline,
Ficoll-Hypaque Gradient (sp.gr.1.
Layered in 08) and centrifuged at 400 xg for 40 minutes at room temperature.
Take the PBMC at the plasma-Ficoll interface and use Hank (Han
k) Balanced salt solution (HBSS) [Gibco; Grand Island Biol
ogical Co., Grand Island, NY)] three times, resuspended in complete medium, and counted. Cell viability was always greater than 95% as measured by trypan blue exclusion.

Culture medium : RPMI 1640 medium was purchased from Gibco and used. To this, 10% heat-inactivated fetal bovine serum [Hyclone, Logan, Utah], 2m
M L-glutamine, 10 mM HEPES, penicillin / streptomycin (Gibco), and 5 × 10 −5 M
2-mercaptoethanol [Sigma, St. Louis, M
O)] was added. The same medium without 2-mercaptoethanol was used for polyclonal activation. RPMI 1640 containing 2% heat-inactivated fetal bovine serum was used for staining cells with monoclonal antibodies.

Tetanus toxoid (TT) induced proliferative activity :
RPMI 1640 medium used for this assay has 10%
Autologous plasma, 2 mM L-glutamine and antibiotics. TT [Mookmore] at a final dilution of 1: 300 confirmed in preliminary experiments for optimal response
(Dr. A. Muchmore, National Cancer Institute, Bethesda,
Available from MD)], or in the absence of PBMCs in quadruplicate at 105 cells in 200 μl of medium per well of a microtiter plate. Replicate cultures containing various concentrations of rgp120 were verified in parallel. After 6 days of incubation, when peak response appeared, cultures were pulsed with [3H] -thymidine, processed and counted as above.

Staining of cells with monoclonal antibody : Monoclonal antibodies (Mab) OKT3, OKT4, OK conjugated with fluorescent isothiocyanate (FITC).
T4A and OKT8 are ortho-diagnostic.
Systems (Ortho Diagnostic Systems, Inc., Rarita
n, NJ). Part of cells (106/50 μl)
Is a FITC-conjugated OKT3, OKT
4, OKT4A, OKT8 or murine IgG2ab control
(5 μl). After incubating on ice for 40 minutes, cells were washed twice with medium and fixed with 2% paraformaldehyde, and this sample was subjected to fluorescence activated cell sorter equipped with a 4 watt argon laser with a wavelength of 488 nm.
[FACS IV, Becton Dickin
son, Mt.View, CA)]. 104 cells were obtained in the indicated format and measured on a single cell basis and represented by a frequency distribution histogram (21). Dead cells and debris were excluded from forward light scatter based analysis. The results are expressed as a percentage of specifically stained cells (%; number of fluorescently labeled cells / number of analyzed cells × 100) after subtracting non-specific staining (IgG2ab-FITC control). The mean channel of intensity of stained cells was also measured, which was not shown.

Statistical analysis : Results were analyzed by Student's paired design and analysis of variance for multiple group comparisons.

Results On the response of PBMC to soluble specific antigens
The effect of rgp120 was investigated. Six experiments were performed with TT as stimulant with or without rgp120 added to the culture. The results are shown in Table 2, which shows that P in normal PBMC responds to a specific antigen.
It shows that rgp120 can significantly (p = 0.05) inhibit BMC proliferation. The degree of inhibition is 19 to 51 of the control value.
%, Which was different in the 5 experiments. Experiment N
No inhibition was observed at o.6 (not shown).

[0097]

[Table 2] * PBMC quadruplicate cultures were treated with TT alone (control) or T
105 in the presence of T + 5 μg rgp120 / ml (+ rgp120)
Cells / 200 μl / well were measured on flat bottom microtiter plates. After incubation for 6 days, the culture was [3H]-
Pulsed with thymidine, collected, processed and counted. Each data represents the mean cpm and% inhibition was calculated as follows: 1- (cpm in the presence of rgp120 / cpm of control) x 10
0; p <0.05.

Interaction of rgp120 with various T-cell populations
For : Infection of T-cells with HIV involves viral envelope glycoprotein and T-helper membrane receptor complex T
It has been suggested to begin with a selective interaction with four components (1-6, 15, 23-26). Therefore, rgp12
It was interesting to determine if 0 had the same selective ability to bind to human lymphoid cells. PBMCs were incubated in the presence or absence of rgp120 (5 μg / ml) for 24 hours at 4 ° C and 37 ° C. The cells were washed and directly stained with FITC-conjugated OKT4 and OKT4A. F
Results of ACS analysis indicate that treatment of cells with rgp120 at 4 ° C does not inhibit OKT4 binding. However, treatment with rgp120 at 37 ° C resulted in altered OKT4 binding, which was indicated by a decrease in T4 + cell fluorescence. Conversely, there was no OKT4A binding after incubation of cells with rgp120 at 37 ° C.
These results show that rgp120 is similar to HIV (25,2).
6), showing that it can occupy the T4A epitope or adjacent epitopes of the T4 helper membrane receptor.

Selective binding of rgp120 to the T4A epitope was observed at 37 ° C. for 24 hours in high concentrations of rgp120 (10).
It was further investigated in a series of experiments involving treatment of cells with 0 μg / ml). The cells are then washed and FITC
-Conjugated OKT3, OKT4, OKT4
A, and stained with OKT8. FACS analysis results showed that pretreatment of PBMCs with rgp120 completely blocked the T4A epitope of the T-helper membrane surface antigen. OKT3 and O to cells treated with rgp120
The binding of KT8 was similar to that of the untreated cell preparation,
It was shown that rgp120 does not bind to T3 or T8 membrane surface antigens, even at high concentrations. It is interesting to note that rgp120 treatment does not completely block the T4 receptor but only reduces the mean channel of fluorescence of T4 stained cells. This may indicate a partial binding and / or alteration in the T-helper receptor membrane structure.

Example 2 Anti-TCB Domain Monoclonal Antibody This example illustrates how to obtain monoclonal antibody 5C2E5. Female Balb / c mice were immunized with about 30 μg of rgp120 per dose for about 7 months. Initial 3-site subcutaneous vaccination with rgp120 in complete Freund's adjuvant was followed by 43-site subcutaneous injection in incomplete Freund's adjuvant followed by 23-site subcutaneous injection in phosphate buffered saline. . The final intraperitoneal booster injection was also performed in phosphate buffered saline.

Hybridomas secreting the monoclonal antibody 5C2E5 were used as described in Oi et al. Obtained by conventional methods. This cell line is widely and generally available. 60 fused cell cultures each
Transferred to 10 well plates. About 480 wells (8
0%) contained live cells. Each well was screened for antibodies to rgp120 by radioimmunoprecipitation or ELISA. This ELISA is
(a) rgp120 coated microtiter wells that were incubated at room temperature for 30 minutes at 1% (weight / volume) to block non-specific binding sites, and (b) labeled with horseradish peroxidase. Is a conventional double antibody sandwich assay using the prepared goat anti-mouse IgG. 10 out of 480 wells are rgp
It was positive for 120 antibodies. Then these 1
0 wells were screened for their ability to block the binding of rgp120 to human T4 helper cells. This blocking assay was performed as follows:

In vitro culture supernatants of ascites fluid or purified ascites preparations or supernatants are screened by standard assays. For example, this purification method consists of adsorbing to a column of insolubilized Staphylococcal protein A and then eluting the antibody. Serial dilutions of the monoclonal antibody preparation (undiluted to 1: 1000) were prepared in assay buffer. The assay buffer contains the following components:

A) Gibco F-12 / DMEM 50:50 mixture (medium) b) 10% strictly dialyzed fetal bovine serum c) 0.2 mM phenylmethylsulfonyl fluoride d) 0.05% Tween 80 in phosphate buffered saline e) 0.06M NaCl f) 0.25mg / ml BSA (bovine serum albumin) g) 12.5 mM Hepes buffer pH 7.4

Add 12 μl of each serial dilution (50 μl).
Radioiodinated rg in a x75 polystyrene test tube
p120 (-100,000 cpm) assay buffer solution (50-
100 μl) and 3-5 × 10 5 CHO (Chinese Hamster Ovary) cell line SVE OKT4 (clone 17) assay buffer (100 μl) and incubated at 4 ° C. for 1 hour. The cells were pelleted by centrifugation, the supernatant was aspirated, and the pellet was placed in assay buffer (1.0 ml).
Resuspended, repelletized, aspirated and the pellet counted in a gamma counter.

In this CHO cell line, the gene encoding the human T4 receptor was placed in a vector encoding DHFR under the control of the SV-40 early promoter and transfected into CHO cells,
And a transformed CHO cell line amplified in 500 nM methotrexate to amplify its T4 receptor gene and increase expression on the surface of transformed cells. Other T4 transformants suitable for use in this assay are known in the art and are readily prepared from commonly available starting materials (38, 39, 40). Such T4 transformants are preferably co-transformed with DHFR and amplified by standard methods to maximize T4 cell surface expression. Such cells, which contain large amounts of T4, give the assay high sensitivity.

It is important to note the use of radioiodinated rgp120. Several other conventional methods have been found to result in 125I-rgp120 with binding properties that render the tracer useless in this assay. The successful method used was as follows: 1 mg / 400μ
l rgp120 (400 μl) with 1 mCi of 125I, 0.3
Mix with 5 mg / ml lactoperoxidase (20 μl) and 1 mM H 2 O 2 (20 μl), incubate for 10 minutes at room temperature, add 1 mM β-mercaptoethanol (20 μl), incubate the reaction mixture for 2 minutes at room temperature, 0.5 wt / vol% BSA (50μ
l) was added and the reaction mixture was added to 0.5 wt / vol% BSA.
Diluted to 2.5 ml with PBS buffer (PBS-BSA), loaded onto a G-25 column, and PBS-BSA.
Elute with (3.5 ml). 125I-rgp120 was recovered in the eluate.

Wells producing only one blocking antibody (5
The cells in C2) were subcloned. The same inhibitory activity was obtained from well E5. This activator due to IgG was purified to the desired form, such as by ammonium sulfate precipitation. This 5C2E5 monoclonal antibody is a pellet
The number of binding, as measured by reduction in cpm, was reduced to about 10-15% of the murine monoclonal antibody control.
It showed maximal inhibition of 25I-rgp120 T4 receptor binding. This 5C2E5 hybridoma is publicly available (ATCC HB9435).

Example 3 Recovery of the TCB domain from rgp120 This example describes the method used to prepare the TCB domain portion spanning residues 411-454.

Rgp12 used in this Example from the culture medium of CHO cells transfected with pAIDSenvTrDHFR and cultured as described in EP 187,041.
0 was purified. The rgp120 was purified by immunoaffinity chromatography to near homogeneity. This purified rgp120 was subjected to 110 g in 0.25 M glacial acetic acid under reduced pressure oxygen atmosphere according to the method of Ingram (35).
It was hydrolyzed at 18 ° C for 18 hours. Under these conditions, rgp
The 120 protein backbone is truncated on both sides of the N and C termini of aspartyl residues. The digested rgp120 was neutralized by adding Tris buffer, and the pH was adjusted to 7.

Then, this digested rgp120 was treated with the conventional method (3
6) was loaded onto a column of murine monoclonal antibody 5C2E5 covalently linked to aldehyde-silica. The column was washed with a saline solution buffered at pH3. Monoclonal antibody 5C2E5 was found to bind rgp120 so as to prevent binding of rgp120 to T4 surface antigen of T4 + helper T cells (see Example 2). The eluate from the 5C2E5 column was analyzed by conventional amino acid quantitative analysis methods to give the following amino acid composition:

Amino Acid D / N T S E / Q P G A C mol / rgp 120 mol 3.0 3.9 2.8 4.2 3.4 3.1 1.9 0.8 amino Acid V M I L Y F K R mol / rgp 120 mol 1.4 1.5 5.2 3.8 1.0 1.6 2.1 2.6 This condition that was analyzed prevents accurate measurement of C and W.
It In addition, the eluate from the 5C2E5 column was N-terminal
It was also analyzed by sequencing and only the following sequences were obtained: I  
L P  R I K Q F.

The results of this amino acid analysis and N-terminal sequencing revealed that rgp120 bound to the 5C2E5 column.
The only peptide in the acetic acid digest of 4 was 4 from threonyl residue 411 to arginine residue 454 of the HIV env sequence.
It was confirmed to be a 4-amino acid fragment. This region has the following sequence: TITLPCRIKQFINMWQEVGKAM
YAPPISGQIRCSSNITGLLLTR. This peptide contains the epitope of the murine monoclonal antibody 5C2E5 that blocks the binding of rgp120 to the T4 surface antigen of T4 + helper T cells, and the sequence deleted in the dalta3 mutant described above, thus It is concluded that it is part of the domain.

Example 4 Immunotoxic Agents Independently or as a cocktail, coupled to deglycosylated ritin A chain (dgA) and killing HIV infected cells from a wide variety of viral subtypes. A monoclonal antibody directed against the HIV-encoded protein, used to :, is obtained as follows:

Immunization with heat-treated (30 sec / 60 °) NP-40 lysate derived from HIV-infected human Hp (CD4 +) cells.
Serum is obtained from (multiple ip injection) mice. Also, gp
Sera can also be obtained from mice immunized with 120 or gp150. The protein gp150 is gp120 and
gp1 having a region that is cleaved off to obtain gp41
It is the extracellular domain of 60. All sera are tested in the following assay:

1. HIV infected H9 cells, uninfected H
Indirect immunotoxic substance (IT) assay using 9 cells or uninfected H9 cells coated with gp120

2. Radioimmunoassay (RIA) -Positive sera are tested by RIA against purified gp120 and gp150 to determine if mice have antibodies to these proteins.

Sera from mice positive for either or both RIA and indirect IT assays were collected and affinity purified on Sepharose-gp120 or Sepharose-gp150. This purified antibody was tested once again in three assays. If positive, the mice were HIV-H9, ±
Perform booster treatment with gp120 ± gp150 lysate. Several mice were sacrificed and their spleens were Sp2 / 0 or N
Fused with P3X63-Ag8.653 myeloma cells. Supernatant (SN) from wells containing growing hybridomas,
(a) H9 vs. (b) HIV-H9 was tested by the indirect IT test. Hybridomas positive in (b) and negative in (a) were further subcloned and tested by RIA, indirect IT, immunoprecipitation and the like. Antibodies from positive clones were isotyped to increase antibody production. This antibody
Directly coupled to dgA. These IT-dgA, A
Tested on cells from IDS patients and HIV infected cells. Then, a clinical IT was prepared.

Litin A Chain Antibody Conjugate (1) Preparation of F (ab ') 2 and FAB' Fragments of Antibody The antibody preparation was treated with pepsin (4500 U / ml) under the following conditions.
(Sigma, St. Louis, MO) at 37 ° C. for 6 hours. pH = 3.7 (0.1 M citrate buffer), protein concentration: 2-3 mg / ml, enzyme / protein ratio: 2/1
00 (weight ratio). The digestion is terminated by raising the pH to 8.0 with 1N NaOH. Isolation of the F (ab ') fragment
Performed by gel filtration on Sephacryl S-200HR (Pharmacia, Piscataway, NJ) equilibrated with 0.3M NaCl buffer, or 0.1M.
The unneutralized digest was adsorbed on an SP-Sephadex column (10 x 2 cm) equilibrated with citrate buffer (pH 3.7), and F (a
b ') by elution of the two fragments. The yield of F (ab ') 2 fragments is generally 35-50%.

The Fab ′ fragment is the F (ab ′) 2 obtained above.
Fragment of ethylenediaminetetraacetic acid (2 sodium salt)
(EDTA) (PBE) 0.003 M in 0.1 molar phosphate buffer (pH 7.5) with a final concentration of 5 mM dithiothreitol (DTT) at room temperature for 1 hour treatment and reduction. be able to. Excess DTT can be removed by gel filtration on Sephadex G-25 and the thiol group of the Fab 'fragment (5 mg / ml) can be found in Fulton et al. [Fulton et al. J. Immunol. , 136 , 3
103-3109 (1986)] and a final concentration of 2 mM Ellman'reagent (5,5 ').
It can be induced with -dithio-bis (2-nitrobenzoic acid)) (DTNB). Unreacted DTNB can be removed by gel filtration on a Sephadex G-25 column (30 x 2 cm) equilibrated with PBE. The purity of the F (ab ') 2 fragment and the Fab' fragment was determined by SDS-PAGE and Fa
It is measured by binary diffusion and immunoelectrophoresis using anti-mouse IgG serum that does not react with both b'and Fc fragments. This preparation does not contain the Fc fragment and complete IgG.

(2) Litin A chain The deglycosylated A chain (dgA) was prepared according to Thorpe et al. [Th.
orpe et al. , Eur. J. Biochem. , 147 , 197
~ 206 (1985)]. L of native and deglycosylated A chain per 25 g mouse
The D50 values are about 0.7 mg and 0.3 mg, respectively.
The IC50 values in the cell-free rabbit reticulocyte assay are about 10-11 for both native and deglycosylated A chains.
It is 10-12 mol. To bind to the antibody, the A chain was added to 5 mM D as described in Fulton et al. (Supra).
Reduce with DT.

(3) Preparation of IT-As by SPDP IT-As is prepared by the method described in Fulton et al. (Supra) using the IgG or F (ab ') 2 fragment of the antibody. P
Solution of IgG or F (ab ') 2 fragment in BE (pH 7.5)
SPD dissolved in dimethylformamide (10 mg / ml)
Add P to give a final concentration of 1 mM. After standing at room temperature for 30 minutes, the solution is filtered on a Sephadex G-25 column (30 x 2 cm) equilibrated with PBE. The substitution rate of the induced IgG or F (ab ') 2 fragment was about 3
~ 4 molecules PDP / protein molecule. The induced protein was then reduced to 1.3 mg / m 3 of reduced A chain dissolved in PBE.
Mix with l and leave at 25 ° C for 2 hours and 4 ° C overnight. This mixture is then purified.

(4) Preparation of Fab'As with DTNB Preparation of mouse Fac'-A with DTNB-induced Fab 'is carried out according to the method described in Fulton et al. (Supra) for rabbit Fab'-A. 1-2 TNB
An Ellman's reagent-derived Fab 'fragment containing a substituted thiol group (see preparation of Fab' fragment above) was dissolved in PBE (5 mg / ml) and this was reduced A chain (1.3 mg A chain / mg Fab ').
And at room temperature to a final concentration of 2 mg protein / ml. The reaction between TNB-Fab 'and the A chain is 412 nm.
Followed by an increase in absorption at 25 ° C and is complete in about 2 hours at 25 ° C. This mixture is then immediately purified.

(5) Purification of IT-As IT-As prepared with a complete antibody or a fragment thereof was prepared as described in Knowles and Thorpe, An.
al. Biochem. 160 , 440-443 (1987)] with a slight modification and purification by affinity chromatography on blue sepharose. Chromatography is performed in 0.05M phosphate buffer (pH 7.0) and A chain and IT-As are eluted with 1M NaCl prepared in the same buffer. The eluate was ultracentrifuged at 5 mg /
Concentrate to ml and use phosphate buffer 0.3M NaCl (pH 7.
Pour onto Sephacryl S-200HR equilibrated in 2).
The peaks containing purified IT-As are collected, concentrated by ultracentrifugation to at least 0.5 mg / ml and stored in aliquots at 70 ° C. (6) Preparation of Fab'-GAMIg-A and indirect immunotoxin assay

Fab fragments of affinity-purified goat anti-mouse immunoglobulin (GAMIg) were treated with 0.1 M EDT.
Prepared by papain digestion with 2% papain solution in 0.1M NaPO4 containing A and cysteine. SE equilibrated with 0.01M NaPO4 (pH 7.6)
Fab fragments are separated from Fc fragments by affinity chromatography on AE-Sephacel. Fab fragments are desalted on Sephadex G-25. Then reduction Fab
The fragment is reacted with a 100-fold molar excess of 5,5 dithio-bis (2-nitrobenzoic acid) (DTNB, Ellman's reagent).
Fab-GAMIg substituted with Ellman's reagent is separated from free Fab-GAMIg and free Ellman's reagent by chromatography on S200.

Fulton et al. [J. Biol. Chem. Two
61 , 5314 (1986)], and the ritin A chain prepared and tested is reduced with 5 mM DTT. The A chain was desalted on Sephadex G-25 and Fab-GAMIg
Combine with -E. Then Fab-GAMIg-A was added to S2
00 and affinity chromatography on blue sepharose.

(7) Indirect immunotoxin assay Use cells with a viable cell count of greater than 95% in the logarithmic growth phase. Serial dilutions of initial unbound antibody in concentrations ranging from 2 × 10 −8 to 2 × 10 −13 M are prepared in the appropriate medium and 100 μl aliquots are plated in triplicate in 96 well plates. 100 volumes in the same medium
μl of 105H9 or HIV-H9 cells is added to each well and the plates are incubated at 4 ° C for 1 hour. Fab-GAMIg-A is then added to the appropriate plates at a final concentration of 1 μg / ml. Control group (a) untreated cells, (b) Fab-GAMIg-A only treated cells, (c)
Includes cells treated with antibody only, and (d) antibody treated followed by Fab-GAMIg (without A chain). The plate is then placed in 5% CO2 at 37 ° C for 36
Incubate for hours. Cells at 37 ° C for 4-8 hours 3
Treated with H-thymidine, then Titer Tech
ek) Collect on a glass fiber filter using an automatic collector. 3H-thymidine incorporation is measured by liquid scintillation counting on an LKB beta counter. Results are expressed as% of 3H-thymidine taken up by untreated cells. In all control cultures, 80-100% of 3H-thymidine is taken up by untreated cells.

(8) Data analysis The expected potency of monoclonal antibodies in the indirect IT assay is that the concentration of antibody capable of killing 50% of target cells (Fab-GAMIg-A used at optimal concentration) (IC5
0). The IC50 must be 10-12 to 10-10 for IT-As to be useful in vivo. The IC50 is ranked as follows. The 10-11M = 4+ 10-10M = 3+ 10-9 M = 2+ 10-8 M = 1+ 4+ and 3+ antibodies appear to be suitable as IT-As and therefore R for gp150 and gp120.
Test at IA.

(9) RIA 96-well microtiter plate
Gp120 or gp150 5 μg / m in 0 μl PBS
coating for 16 hours / 4 ° C. The wells were decanted and washed 5 times with dH2O, PBS-10% FCS2
Block with 00 μl for 24 hours / 4 ° C. The wells are decanted and washed 5 times with dH2O. Serum or supernatant (S
The N) dilution is added at 25 ° C. over 4 hours. The material is removed and the wells are washed 5 times with dH2O. GA of 105 cpm
Add MIg over 4-6 hours / 25 ° C. Samples are removed, plates are washed 5 times with dH2O, dried, cut and counted.

(10) Antibodies and sera tested (a) Rabbit anti-gp41 (b) MoAbs Nos. 1,2,3,4,5,6,7,8,10,12 (code) (c) MoAbs 7F11,1F9,5B9,1D10,5G9,5C2,6D8E9,9F6 (code) (d) HIV-H9 Polyclonal mouse serum produced by immunizing BALB / c mice with lysates from cells

(11) Results Monoclonal antibody (a) Rαgp41: IC501 × 10-8M (not affinity-purified) (b) Monoclonal antibody 7F11: +1 1F9:-5B9:-1D10:-5G9:-5C2:- 6D8E9:-9F6:-Only one monoclonal antibody was slightly effective in the indirect assay.

First, 50 BALB / c lysates prepared from 1 × 10 8 H9 cells infected with HIV-I.
Mice were immunized. Serum taken from these mice was screened by an indirect assay. 15 out of 38 surviving mice were 3 + -4 + in the indirect IT assay. After RIA and two immunizations, 3 mice significantly produced antibodies against gp120 (see Table 3).

[0132]

Table 3 Table 3 indirect IT assay and RIA results indirect IT RIA Mouse IC 50 μG / ML anti-gp120 anti -Gp15 (2 after booster) (after 5 booster) 302 4+ 2+ 304 1+ 4+ 306 1+ 4+ 310 - 3+ 312 1+ 3+ 314 3+ 4+ 316 1+ 2+ 318 1+ 3+ 320 1+ 3+ 332-2+ 334 2+ 2+ 336 4+ 4+ 338 1+ 2+ 340 1+-344 3+ 2+ 348 2+ 4+ 350 4+ 3+ 352 2+-354 2+-2+ 356 1+ 2+- 358 3+ 3+-362 4+ 4+ 4+ 364 4+ 3+ 3+ 366 2+ 3+ 4+ 368 2+ 4+ 4+ 370 4+ 4+ 4+ 372-1+-374-3 + -376 4+ 3+ 4+ 1+ or greater than 1 μg / ml .

The present invention has been described above mainly with reference to the preferred embodiments thereof, but the present invention is not limited to the embodiments of these disclosures, and conversely falls within the scope of the appended claims. It covers various modifications and equivalents. The scope of the claims should be given the broadest interpretation as including all such modifications and equivalents.

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─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12N 15/02 C12P 21/08 15/09 ZNA G01N 33/569 H C12P 21/08 33/577 B G01N 33/569 C12R 1: 91 33/577 C12N 15/00 ZNAA (C12P 21/08 C C12R 1:91) (72) Inventor Philip W Berman USA California 94402, San Mateo, Lexington Avenue 1739 (72) Inventor Timothy Jay Gregory USA California 94010 Hillsborough, Pine Hill Road 414 (72) Inventor Lawrence A. Rasky USA California 94965, Sousalito, Star Root Box 40 (72) Inventor Gerald Earl Nakamura USA California 94116, San Francisco, Twenty Avenue 1923 (72) Inventor Eric J. Patzer USA California 94563, Olinda, Tajos Road 470 (72) Inventor John S. Patton USA California 94070 , San Carlos, Emerald Avenue No. 330 (72) Inventor Ellen Es Vittetta Texas 75230, USA Pemberton Drive No. 6914 (56) Reference JP-A-61-233700 (JP, A) ( 58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 16/00-16/46 BIOSIS (DIALOG) WPI (DIALOG)

Claims (9)

(57) [Claims] 1. A monoclonal antibody 5C2E5 (ATCCHB94) defined as the ability to inhibit rgp120 binding to T4 receptor of helper lymphocytes.
An antibody having the TCB domain binding property of 35). 2. The antibody according to claim 1, which is a monoclonal antibody. 3. The antibody according to claim 2, which is conjugated with a cytotoxic substance. 4. The antibody according to claim 3, wherein the cytotoxic substance is ritin. 5. The antibody of claim 2 covalently attached to a detectable marker or a water-insoluble matrix. 6. The antibody of claim 2 in a sterile pharmaceutically acceptable carrier. 7. A portion of a human antibody or a human antibody,
The antibody according to claim 1. 8. The antibody according to claim 7, which is a monoclonal antibody. 9. The antibody according to claim 1, which comprises a chimeric antibody.