JP2001292788A - Composition comprising envelope polypeptide of human immunodeficiency virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an improved therapy for immunological inflammatory diseases, an improved therapy for AIDS reduced in adverse effects and further a method for killing a nontumorous cell expressing an encoded viral antigen on the cell surface. SOLUTION: This composition comprising a T-cell binding(TCB) domain of a human immunodeficiency virus envelope deficient in a human immunodeficiency virus immunological epitope usually adjacent to the C-terminal or the N-terminal of the TCB domain of the human immunodeficiency virus envelope is effective as a vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human immunodeficiency virus or HIV (HTLV-III) envelope (env).
It relates to the use of polypeptides, and more particularly to their use in unwanted suppressive immune responses and consequent inflammation. The invention also relates to the use of HIVenv variants and HIVenv antibodies 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 immunotoxins and other immunoconjugates (immunoconjugates) and their use for killing HIV or other viruses encoding cell surface antigens.

[0002]

BACKGROUND OF THE INVENTION The inflammatory response 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 uncertain origin (e.g.,
Myasthenia gravis, Graves 'disease, autoimmune diseases caused by Chagas' disease, and juvenile diabetes). A class I response is a rearginous 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 chronic formation of IgM-containing immune complexes. Class IV responses are associated with delayed-type hypersensitivity reactions,
And is mediated by T-lymphocytes and is commonly found in tuberculosis, sarcoidosis, polymyopathy, granulomas and vasculitis.

[0003] Rheumatoid arthritis (RA) is a severe class II disease
This is one of the abnormalities. Synovitis is a characteristic tissue reaction of RA in which normal thin connective tissue is replaced by overgrown synovial fibroblasts and the synovium is invaded by lymphocytes, macrophages and other immune cells. Is invaded. In rheumatic synovial fluid, the reversal of complement components is increased and complement cleavage products, especially C5a, are present in rheumatic synovial fluid. This is important because C5a and other complement derivatives contribute to immune cell activation and inflammatory cytokine production. Synovial fluid also contains kinin (kini
Together with n) and LTB4, they also contain hydrolases (including collagenase) derived from inflammatory cells.

[0004] Various mechanisms have been proposed to elucidate the degree of tissue destruction characteristic of rheumatoid arthritis. These mechanisms involve complex activation and suppression interactions that regulate the type and magnitude of the inflammatory response in RA. These mechanisms include arachidonic acid metabolites, especially prostaglandin E2 produced by leukocytes and rheumatic synovial fluid, biologically active amines such as histamine and serotonin, complement breakdown products, eosinophil chemotactic factors, kinin , Interleukins, and proteolytic enzymes, particularly collagenase or procollagenase activating enzymes, are involved.

[0005] The generally accepted 1 for RA induction
One assumption is that an unknown antigen is chronically deposited on the synovium,
Therefore, it is phagocytic action by type A synovial cells. B cells are induced to donate antigen and differentiate into plasma cells, which in turn leads to the production of antibodies, rheumatoid factors and cytokins. Activation of T lymphocytes by antigens causes lymphokine synthesis, blastogenesis, and then generation of antigen-specific helpers and cytotoxic T cells.
A combination of an antibody and an antigen, or a combination of an antibody-antigen complex and a rheumatoid factor, or a self-association (association) of a rheumatoid factor is caused by Hageman (Hagema
n) Activates complement and kinin-forming systems via factor activation. This results in the production of pro-inflammatory products such as C5a, arachidonic acid metabolites, kinins and fibrinopeptides, which diffuse into synovial fluid and further into synovial blood vessels.
These substances increase the permeability of blood vessels and attract polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes are
Ingests a large number of immune complexes in the fluid, releases lysosomal and other destructive enzymes, and produces peroxide anions. This results in destruction of the hyaluronic acid polymer in the binding solution and damage to the cartilage. Cytokin production in the synovium leads to further accumulation of macrophages, other immune cells, and rheumatoid synovial fibroblasts. All of these cell types can contribute to the recruitment cycle of immune cells, activation of immune media (e.g., cytokins), and production that are characteristic of RA, sustain inflammatory conditions, and destroy tissue .

[0006] Locally synthesized enzymes released into synovial fluid by cells that are present in the synovial fluid or that constitute proliferative synovial foci contribute to pathological events in joint structures. Therefore, the specific structure of the binding space is important. Cartilage-destroying lysosomal enzymes, collagenase and elastase, are mainly derived from inflammatory cells (immune cells and rheumatoid synovial fibroblasts). Proteolytic enzymes released from the stained cells are responsible for breaking down the crosslinks of the collagen fibrils, thereby helping to destroy the cartilage surface and increasing the likelihood of enzymatic breakdown. Macrophages in the synovium contain prostaglandins, hydrolases, collagenase, plasminogen activator, and IL-
Produce 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 break down proteoglycan aggregates, and these soluble components, once released from cartilage, are susceptible to subsequent enzymatic attack.

Dysregulation of the persistence of antigen activation or of T and B cell activation leads to a chronic inflammatory response of the synovium. Sustained cell growth and influx causes the synovium to proliferate and invade its surrounding structures. Collagenase, P
The diffusion of GEs, hydrolases, and cytokins into cartilage and bone causes erosion of these tissues. Thus, rheumatoid arthritis is manifested as debilitating local tissue destruction due to a chronic inflammatory response caused by a delayed hypersensitivity immune response.

[0008] The treatment of RA today includes bed rest, warming and medication. Salicylates are currently preferred drugs because other currently available alternatives, particularly immunosuppressants and adrenocorticosteroids, exhibit a 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. These include indomethacin, phenylbutazone and phenylacetic acid derivatives such as ibuprofen and fenoprofen, naphthaleneacetic acid (naproxen), pyrrolealkanoic acid (tomethine), indoleacetic acid (salindac), halogenated anthranilic acid (meclofenamate sodium) , Piroxicam, zomepirac, and diflunisal. These are generally considered less effective than aspirin, but may be better tolerated in certain patients.

Other drugs that may be used for RA include chloroquine,
Antimalarial agents such as gold salts and penicillamine are included.
These alternatives often have serious side effects, including retinal lesions and kidney and bone marrow toxicity. Because immunosuppressive drugs such as methotrexate are toxic, they are used only for the treatment of severe, non-remission RA. Corticosteroids also have undesirable side effects that many patients cannot tolerate well.

Chronic inflammatory diseases or disorders 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 therapies and compositions for the treatment of the disease and the suppression of the immune response associated with juvenile onset diabetes.

[0011] The invention also relates to the suppression of transplanted cells, organs or tissues or of immune rejection thereby. Rejection in transplants has been controlled using immunosuppressants such as cyclosporine antibiotics or steroids. Such drugs have a wide range of undesirable side effects.

[0012] In other attempts, monoclonal antibodies have been used that target and neutralize lymphocyte subsets that may be involved in transplant rejection. These antibodies are of murine origin. In the case of murine monoclonal antibodies, the affinity effect on the lymphocyte subset that they target is usually low. Also, they carry the risk of increasing the susceptibility of treated patients to rodents, and since the constant regions of the rodent can activate human complement, anti-T subset antibodies lyse and deplete T cells. There is a risk. There is a need for compositions and methods for treating rejection in tissue transplants that do not use murine immunoglobulins.

[0013] Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus that has been identified as the human immunodeficiency virus (HIV) (1-6). Abnormal B cell function, abnormal antibody response, impaired monocyte function, impaired cytokine production (7-10), suppressed natural killer cell and cytotoxic cell function (11), and recognized soluble antigens, There have been reports of many immune abnormalities associated with AIDS, such as reduced responsive lymphocyte function (12). Other AIDS-related immune abnormalities have also been described (13, 14).
A more significant immune injury in AIDS patients is the depletion of T4 helper / inducer lymphocytes (1,2,
9, 10).

[0014] 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, and
Cell function is impaired and ultimately leads to the depletion of cells required for a normal immune response (1-
6, 15). Recently, in vivo and in vitro studies have shown that HIV also infects monocyte cells known to play an essential role as accessory cells in the immune response (16,17). HIV interferes with normal cytokine production in infected cells, and IL-1
It also causes immunodeficiency by causing a secondary immunodeficiency, such as IL-2 deficiency (18). Another immunodeficiency induction by HIV is in the production of factors that can suppress the immune response (19). None of these models address whether HIV components themselves, but not replicating viruses, are involved in AIDS-related immune abnormalities.

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) envelope glycoprotein (gp) anchored to its lipid bilayer via its carboxy-terminal region.
160). Each glycoprotein contains two segments. Its relative molecular weight is about 120k
The N-terminal segment, referred to as gp120 based on its D, protrudes into the aqueous surrounding environment surrounding the virion. The C-terminal segment, called gp41, spans the membrane. gp120 and gp41 are particularly covalently linked by proteolytically labile peptide bonds.
For the purposes of the present invention, HIVenv is, for example, gp160
Alternatively, it is intended to include all forms of gp120, such as fusions such as gp120 fused to the N-terminal fragment of gp41, HIVglyc with or without glycosylation, and the T cell binding fragment of HIVenv. HIVenv and its variants are routinely prepared by recombinant culture (EP-A-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 from viral cultures and such env preparations are also HI
It is known that it is included in the definition range of Venv.

AI for the purpose of protecting non-infected cells at present
DS therapy is a nucleoside analog (such as AZT and DDC) that can block viral RNA replication approximately every 4 hours
Orally. These drugs are 50-5
At a concentration of 00 μM, it blocks virus replication, but at higher concentrations (−1 mM), the D
It also inhibits NA polymerase (Mitsuya and Broader, 1985). Very large doses (1 g / day)
). Because the drug is taken orally and absorbed by all cells, the whole body is exposed to the drug. Its use is very limited by toxicity. One of the most serious side effects is anemia. Nucleoside derivative is incorporated into DNA
It must be phosphorylated first (and exhibit its chain-breaking action), which requires a kinase that is not always present in equal amounts in all cells susceptible to virus. Thus, oral treatment with a nucleoside analog that is ineffective on cells that have already been infected may only protect potentially infected cells (i.e., split cells) that can convert high concentrations of nucleosides to nucleotides. it can. For this reason, this treatment is limited 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) (Stricher, 1986). Only monocytes and T cells with the CD4 receptor were thought to be infected with HIV (MacDogal et al., 1986). H
Conserved region of coat protein of IV virus (gp160)
Binds to the CD4 receptor, which internalizes and translocates the RNA virus into cells. Once inside the cell, the virus is turned into RNA by its reverse transcriptase.
DNA copies are made in cells. Nucleoside analogs act as strand breakers in viral RNA transcription to protect cells. The chains are interrupted because they are incorporated into the growing polymer but do not have the necessary functional groups to incorporate the next nucleoside into the polymer.
It has no function.

In order for a nucleoside analog to be incorporated into a nucleic acid strand, it must be phosphorylated (converted to nucleotides) by a kinase (Pherman et al., 1986). T cells that can be rapidly divided include AZT and D
It contains high levels of kinases that phosphorylate antiviral agents such as DC. Monocytes that only divide in special circumstances have relatively low levels of kinase and are not protected from infection by nucleoside derivatives. Administration of the phosphorylated form of the nucleotide analog is performed in a single solution. However, they do not cross cell membranes. Thus, treatment with current nucleoside analogs converts monocytes to H
It is ineffective in protecting against IV infection.

Lyerly et al. (PNAS 84, 4
601 (1987)) is an uninfected CD bound to gp120.
4+ lymphocytes have been shown to be targets for antibody-dependent cellular cytotoxicity by goat serum raised against native gp120. However, human serum binding to gp120-adsorbed cells did not show its destructive effect in the presence of complement. In contrast, these sera potently mediate antibody-dependent cellular cytotoxic effects.

[0020] Monoclonal and polyclonal antibodies specific for the protein encoded by HIV have been developed (eg, PCT / WO86 / 00217).

An immunotoxic substance (ITs) comprising a cell-reactive antibody bound to the A chain of littin toxin (IT-As)
Was used to specifically kill various target tumor cells. Kronke et al. (Cancer Research, 46 (7),
3295 (1986)) discloses that a conjugate (conjugate) of a monoclonal antibody of an interleukin II receptor and a ricin A chain killed HTLV-I infected leukocyte T cells. Kronke, "Blood" 65
See also (6), 1416 (1985).

(US Pat. No. 4,714,61)
No. 3) expresses hepatitis B surface antigen on the surface (where
(The surface antigen is encoded by HBV DNA.) A method for inhibiting the growth of hepatocytes or hepatoma cells infected with hepatitis B virus has been disclosed. The method of Shobar et al. Comprises administering to infected cells complement, which fixes a monoclonal antibody against the hepatitis B surface antigen.

That is, an object of the present invention is to provide an improved method for treating an immune inflammatory disease. The invention also relates to an improved treatment for AIDS with reduced side effects. The present invention also provides a method for killing non-tumorigenic cells expressing a viral antigen encoded on the cell surface.

[0024] Other objects, features and characteristics of the present invention will become apparent from a consideration of the following description and the appended claims.

[0025]

SUMMARY OF THE INVENTION HIV env is useful in the treatment of immuno-inflammatory responses and diseases, and it is an object of the present invention to provide a patient with an immuno-inflammatory response with an HIV env formulation in an amount sufficient to suppress the immuno-inflammatory response. To be administered.

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

HI with functional TCB domain removed
Venv is useful as a vaccine for obtaining immunity against HIV infection.

The present invention provides a monoclonal antibody specific to one region of the TCB domain. This antibody appears to be against an epitope located adjacent to the sequence of the TCB domain that binds to a T4 cell surface marker. This suggests that the antibody sterically hinders access to the TCB domain binding site on the T4 cell surface antigen, possibly resulting in recombinant gp1
Indicated by being able to at least partially block binding of 20 to T4 cell surface markers. This antibody is also useful for passive immunization of HIV-infected patients, since it has the advantage of being able to bind to any free HIV env that exhibits immunosuppressive effects in HIV-infected patients.

The present invention provides a monoclonal antibody that specifically binds to a protein encoded by gp160 or another virus, and an immunotoxic substance containing a toxin such as a ritin A chain bound to the monoclonal antibody. , And provide proteins encoded by the virus as targets. Thus, the protein associated with the toxic conjugate is only taken up by infected cells, such as helper T cells, and kills the cells. In addition, the present invention provides a method for treating gp encoded by a virus on the cell surface.
An immunotoxic substance containing a monoclonal antibody conjugated to a toxin that specifically binds to a protein such as 160,
It is intended to provide a method for killing cells infected with a virus, which comprises administering to a patient in a therapeutically effective amount to kill infected cells.

HIV env or an immunosuppressive fragment thereof (including the TCB domain) is rendered cytotoxic to it.
Conjugates were formed with haptens or polypeptides that could bind to a cell population of cells where a cytolytic response of the cells was desired. These conjugates are particularly useful as anti-tumor agents or for treating allergic responses.

[0031]

DETAILED DESCRIPTION OF THE INVENTION As described above, HIV env was obtained by the covalent modification of the human immunodeficiency virus envelope protein and its in vitro immunosuppressive amino acid sequence variants and HIV env or variants thereof. It is defined as a derivative. Mutants include en
v Amino acid sequences in which one or more residues have been substituted, env sequences in which one or more residues have been deleted, and in which one or more residues have been inserted Is included.

The main deletion mutant for the purpose of the present invention is TCB
These deletion mutants, in which immune epitopes other than the domain have been deleted or, conversely, the TCB domain has been deleted and the remaining epitope of HIVenv has been retained, these deletion mutants are, like the substitution mutants, Will be described in detail. Variants belonging to another class are those in which a heterologous (heterologous) polypeptide for HIV env is fused to the amino or carboxy terminus of the HIV env sequence or an immunosuppressive fragment thereof (eg the TCB domain). Alternatively, the HIVenv or TCB domain is
Covalently linked to a heterologous polypeptide or hapten. Another class of TCB domain variants is TC
The B domain is a naturally occurring HIV env which is an HIV immune epitope that normally does not border the TCB domain, specifically borders the C-terminus of the TCB domain: FRPGGGDMDNWRSELYKY
A mutant obtained by fusing with an epitope other than the sequence represented by KV.

A fusion of the TCB domain of HIVenv with an immunogenic hapten or polypeptide is useful as a component of a vaccine to immunize a patient against HIV infection. A fusion of a hapten or heterologous polypeptide with HIV env or an active fragment thereof will bind to a cytotoxic T cell targeted to the target cell if the hapten or heterologous polypeptide can bind to a target cell surface receptor or other binding partner. 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 that can bind to TGF-α are known. N-terminal or C-terminal fusion of HIVenv or a TCB domain-containing fragment thereof by joining such an antibody to HIVenv or a TCB domain-containing fragment thereof, for example, by covalent cross-linking (33), or by recombinant cell culture. It binds by expressing as a substance. The latter method is described in EP 125,02
This is achieved by obtaining the DNA encoding the TGF-α antibody according to the general procedure described in 3A. This DNA is ligated to the amino or carboxy terminus of gp120 or a fragment containing its TCB domain using a suitable adapter, and EP187,
The vector is inserted into a mammalian cell expression vector as described in EP 041A, and the resulting vector is used to transform CHO cells and other suitable host cells as described in EP 187,041A. If desired, the DNA encoding the variable region of the TGF-α antibody was replaced with EP184,
187, EP194,276A, EP171,496A or the DNA encoding the TCB domain instead of the human constant region described in Bouliamne et al. (32). The obtained fusion is recovered by, for example, adsorbing the immobilized antibody against the constant region, when the constant region of the immobilized TGF-α or TGF-α antibody is retained, It is formulated with a therapeutically acceptable vehicle (eg, saline) and administered to patients with TGF-α expressing tumors. This fusion directs cytotoxic T cells to tumors that have a TGF-α cell surface antigen, thus lysing the tumor cells. Similarly, when a patient fuses the TCB domain with an antigen that is producing an unwanted immune response, such as, for example, the development of allergic or post-transplant rejection, the autoimmune cytotoxic T
The patient is administered a dose sufficient to direct the cellular response to clonal cells that contribute to the undesired response.

The TCB domain of HIVenv is EP187,
Nucleotide No. 7006 of the HIV strain of No. 041A;
To about 7203, or equivalent in other HIV strains (in other strains where the length of the viral genome and HIVenv has been altered by deletions or insertions upstream). Although the same nucleotide and amino acid residue numbers cannot be obtained, in each strain the region encoding this portion of the TCB domain can be readily identified based on amino acid sequence homology. The TCB domain has the formula:

Embedded image And especially residues 411-454, 41
6-443, and 440-442. Table 1 below shows the amino acid sequences of TCB domains from multiple HIV strains, below "Origin" in the right column, and the predefined mutant TCB domains in the left column. The variant indicated by "$ 3" (variant 1) allows this part of the TCB domain of HIV to be identified. Rg by site-directed M13 mutagenesis
Deletion of the △ 3 residue of p120 and expression of this variant in recombinant CHO cell cultures as otherwise described in EP 187,041 can be performed based on assays such as binding with the serum of selected AIDS patients. , Although indistinguishable from rgp120, the T4 binding ability of rgp120 was lost. Surprisingly, as shown in Table 1, the deletion of the △ 3 region is greater than BH10 in many HIV strains, as seen by comparison with homologous sequences from other HIV strains. It can be seen that the TCB domain is not highly conserved and, as a whole, contains an HIVenv region, which is characterized by a high variability (34). Only this highly conserved region was thought to have adequate certainty to ensure that the virus always recognizes the T4 receptor and thus affects recipient host cells, but this hypothesis suggests that The study did not work. For example, despite the fact that 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 rpg120 binding to T4.

[0035]

[Table 1]

Improve the variants identified in Table 1 as numbers 2-15: ie, eliminate the T4 binding of rgp120. For example, replacing the alanine at position 431 with aspartic acid reduced the binding of the mutant to approximately 10-15% of the binding exhibited by rgp120 having the native sequence. However, in our assay, replacing lys430 with glutaminyl, tyr433 with phenylalanyl, and pro435pro436 with valylvalyl,
No change in detectable rgp120 binding occurred. Therefore, the activity of an individual mutant should be measured and screened by conventional assays.
In view of the role of the Ala431 residue, substitutions, deletions or insertions at this site are particularly preferred, including histidine, tryptophan, proline, phenylalanine, glycine, leucine, isoleucine, methionine, valine, serine, threonine, Substitution with tyrosine, glutamine, asparagine, lysine, glutamic acid, cysteine and arginine, or such a residue or Ala43
Includes an insertion of aspartic acid adjacent to 1.
The above mutants and △ 3 mutants are useful in preparing HIV env antigens for use in diagnostic assays or vaccines. HIV env without the TCB domain is particularly useful as a vaccine because it not only has no immunosuppressive effect, but also retains the desired number of residual gp120 epitopes, and is therefore a more potent anti-HIV titer. And lead to an extension of this titer. Other variants will be apparent to those skilled in the art. For example, to identify a variant that functions as an HIVenv agonist, a substitution is made within residues 416-442. In addition, TC
It can be introduced into the B domain to evaluate agonist activity.
As used herein, the term "agonist" refers to
It means that the HIV env activity of the variant is increased compared to the native molecule (eg, the variant exhibits a higher affinity for T4 than the native HIV env). However, it will be appreciated that this activity is antagonistic to infection by HIV.
HIVenv agonist analogs can be intact or native HIVe
It is a substance useful in treating undesirable immuno-inflammation as in the embodiments described above for nv. The agonist analog is optionally used with other therapeutic agents useful for the treatment of HIV infection, such as tumor necrosis factor α, tumor necrosis factor β, interferon and / or AZT, AIDS or ARC
Administered to treat an HIV infection in a patient.

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

The HIV env TCB domain or a variant thereof is prepared in a manner known per se. One method is to synthesize the TCB domain by an in vitro manipulation method such as Merrifield synthesis. Another method is to prepare the domain in a recombinant cell culture method. DNA encoding the domain is prepared by conventional in vitro methods and suitable 5 'and 3' adapters or linkers are created to facilitate ligation into conventional vectors for bacterial expression. It is practical. In one preferred embodiment, a DNA encoding a signal sequence recognized by the host is ligated to the 5 'end of the DNA encoding this domain. Such signals are well known and include STII, alkaline phosphatase, lpp and penicillinase signals.
Certain heterologous signals, such as mammalian or yeast signals, are also recognized by bacteria. Since the DNA encoding such a signal is only about 45 to 75 nucleotides in length, it is prepared and the signal DNA is ligated to the 5 'end of the DNA encoding the TCB domain and obtained. The resulting fusion gene is ligated into a conventional vector, such as pBR322, and
By transfecting (E. coli) and culturing the cells, the amount of TCB domain secreted into the periplasm of the host cell could be determined. Alternatively, the TCB domain is synthesized as a secretory or non-secretory fusion by recombinant cell culture and recovered.

Antibodies capable of inhibiting or binding the TCB domain include clonal antibodies that immunize mice, such as Balb / c or preferably C57 Black / 6, against gp120 and prevent binding to the T4 T cell marker when pre-incubated with gp120. Obtained by screening antibodies. The 5C2E5 antibody is an example of such an antibody, but it is not unique as other antibodies with the same qualitative activity can be obtained by the methods described herein. The term "qualitative" activity means that the antibody is HIVen
v means binding to the T4 binding site or both regions, and the latter case means that rgp120 or HIVenv
Are sterically hindered from binding.
Tryptic digestion of the 44 residue TCB domain confirmed that the 5C2E5 antibody bound to the peptide spanning residues 420-430. Thus, this domain is one that is encompassed by 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 430-449.

The TCB domain binding of antibody 5C2E5 was determined by rgp on the T4 receptor of helper lymphocytes.
120 as an inhibitory capacity in binding. Monoclonal antibodies other than 5C2E5 capable of exhibiting such effects include affinity, immunoglobulin class,
Even if the species of origin or the exact TCB sequence is different, it is still within this particular range, for example, the antibody expressed in recombinant cell culture or the 5C2E5 antibody whose amino acid sequence is predetermined. Mutant such as 5C
Chimeras between the variable region of the 2E5 antibody and the human constant region.

The antibody described in the present specification has been
These immunoglobulins are recovered from hybridoma cell cultures by conventional purification methods, such as those used to purify IgG or IgM from stored plasma, such as ethanol or polyethylene glycol precipitation.
The antibody thus purified is sterile-filtered, and if necessary, AID
It is conjugated to a cytotoxic substance such as litin that can be used for S therapy, or to a detectable marker such as an enzyme or a spin label that can be used in an HIV diagnostic assay in a test sample.

A new application example of an immunotoxic substance (immunotoxin) has been discovered. Surprisingly, it has been found that immunotoxic substances that specifically bind to the virus-inducible surface antigen of cells infected with a non-tumorigenic virus kill the infected cells. Specifically, gp160 binding immunotoxics kill HIV-infected cells.

In the following, treatments intended to kill cells already infected and actively producing new viruses will be described. Killing is achieved by immunotoxic agents that bind to the viral coat protein expressed on infected cells. Therefore, this immunotoxic substance is internalized in the cell, and kills the cell. Infected cells in which the viral genome is integrated into the DNA, but which do not synthesize viral proteins (ie, cells in which the virus is latent) will be killed by the immunotoxic agent until the virus synthesis begins. Maybe not.

It has been found that HIV infected cells, which are actively producing the virus, express the viral coat protein (gp160) on its plasma membrane. This molecule represents an infected cell-specific antigen that can be used to target and kill infected cells. gp1
Monoclonal antibodies specific to conserved regions on 60 (strain-dependent) can be used as attacking molecules to deliver toxic substances to infected cells. Additionally or alternatively, a "cocktail" of immunotoxic agents to non-conserved regions is used. In addition, the toxicant-antibody conjugate can bind to the circulating virus or viral coat protein and thus kill cells that internalize the virus or coat protein.

It is an object of the present invention to provide a highly screened method for destroying HIV infected cells.
Because these antibodies specifically bind to antigens that are not present on uninfected cells, the method of the present invention
The antibodies allow the cells to be destroyed to be attacked very precisely. Unlike tumor treatment with immunoconjugates that result in antigen loss and develop resistance, antigen expression always accompanies an active viral infection.

While not intending to be bound by any particular theory regarding the practice of the present invention, it is believed that the expression of the target antigen on the infected cell surface is transient. The antibody must be able to reach the site on the cell surface where the antigen is present and interacts with it. After the antibody is complexed with the antigen, phagocytosis (endocytosis) occurs and toxic substances are carried into the cells.

[0047] Immunotoxicants are particularly useful for killing monocytes / macrophages infected by the HIV virus. In contrast to transient virus production from T cells, macrophages produce high levels of virus for extended periods of time. Conventional treatments are inefficient in inhibiting the production of new viruses in these cells.

All of the monoclonal antibodies specific for gp160 do not necessarily create high levels of cytotoxic immunotoxics when coupled to litin A chains (IT-A). Assays employed to predict the ability of the antibody to function as a subform of IT-A were performed. This assay was performed to screen various anti-HIV antibodies for the ability to create IT-A effective against HIV-infected cells. This result indicates that only certain HIV-specific antibodies create effective IT-As. These antibodies are purified and coupled to litin A chain,
This is used to specifically kill HIV-infected cells. Advantageously, these antibodies cross-react with some (or all) HIV strains.

The immunotoxic substance can be prepared by various methods (see below for details). Using some cross-linking reagents, conjugates can be prepared to obtain stable conjugates.

A preferred embodiment is to deglycosylate the ricin A chain or prepare it without oligosaccharides and reduce its clearance by an unrelated clearance mechanism (ie, liver). Another embodiment is B
If the ability to bind galactose in the chains can be inhibited ("inhibited litin"), the entire ricin (A + B) is conjugated to the antibody.

Further, the toxic substance-conjugate is Fab
Alternatively, it is prepared using the F (Ab ') 2 fragment. Because of their relatively small size, they better penetrate tissue and reach infected cells.

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

The antibody of the present invention can be obtained by a screening method. An assay is performed to screen the monoclonal antibody for its cytotoxicity as strongly as the A chain containing the immunotoxic substance. This assay involves applying cells to a diluent of the test antibody, followed by ritin A
For applying to a Fab fragment of a secondary antibody coupled to a chain (indirect assay). The cytotoxicity in this indirect assay is compared to that in a direct assay in which the monoclonal antibody is conjugated to the ricin A chain.
In this indirect assay, the effectiveness of the monoclonal antibody as an immunotoxic substance can be accurately predicted, 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 also fall within the scope of the invention.

[0054] Monoclonal antibodies are very specific, being directed against a single antigenic site. Moreover, in contrast to conventional antibody (polyclonal) preparations, which usually contain various antibodies to different determinants (epitopes), monoclonal antibodies individually target a single determinant on the antigen. . Monoclonal antibodies are useful for improving the selectivity and specificity of diagnostic and analytical methods that utilize antigen-antibody binding. A second advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. Monoclonal antibodies can be prepared from supernatants of cultured hybridoma cells or from ascites induced by inoculating hybridoma cells into the peritoneal cavity of mice.

[0055] Kohler and Mils
tein) [Eur. J. Immunol. 6 , 511 (1976)]. It's being used.

The steps and schedule for immunizing a host animal or culturing antibody-producing cells therefrom are generally performed as a well-established routine procedure for stimulating and producing antibodies. By utilizing the method of the present invention as a basis for producing hybrid cell lines of mammals including humans, it is thought that any mammalian subject including human subjects or antibody-producing cells derived therefrom can be skillfully used. We used mice as a test model.

After immunization, the immune lymphoid cells are fused with myeloma cells to generate a hybrid cell line that can be cultured and subcultured without restriction, and a large amount of monoclonal antibody is prepared. For the purposes of the present invention, immunolymphoid cells sorted for fusion are lymphocytes taken from either lymph node tissue or spleen tissue of the immunized animal and their normally differentiated progeny. We use immunized spleen cells rather than the mouse spleen cells because they provide a more convenient source of antibody-producing cells at higher concentrations. Myeloma cells provide the basis for continued growth of the fused hybrid. Myeloma cells are tumor cells derived from plasma cells.

It is possible to fuse one cell type with another. However, it is preferred that the source of the immunizing antibody-producing cells and myeloma is from the same species.

This hybrid cell line can be maintained by in vitro culture in a cell culture medium. The cell line according to the invention can be selected and / or maintained in a composition consisting of an infinite passage cell line in a known hypoxanthine-aminopterin-thymidine (HAT) medium. In fact, once a hybridoma cell line is established, it can be maintained in various nutritionally appropriate media. In addition, hybrid cell lines 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 the synthesis and secretion of monoclonal antibodies restarted. The secreted antibody is recovered from the supernatant of the tissue culture by a conventional method such as precipitation, ion exchange chromatography, affinity chromatography and the like.

Although the present invention has been described using a mouse monoclonal antibody, the present invention is not limited to this. it can. Such antibodies can be obtained using human hybridomas [Cot (Cot
e) et al. Monoclonal Antibodies and Cancer Therapy
Therapy), Alan R. Liss, 77
P. (1985)]. Indeed, according to the present invention, genes derived from mouse antibody molecules of appropriate antigen specificity, as well as having appropriate biological activity (eg, activating human complement,
A method of preparing a "chimeric antibody" by splicing a gene derived from a human antibody molecule (which can mediate DCC) [Proceedings 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): Takeda et al. Nature 314 , 45
2 (1985)]; such antibodies are also within the scope of the present invention.

As an alternative to the cell fusion method, EBV
A monoclonal antibody according to the present invention is prepared using immortalized B cells. 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 (a conjugate of an antibody and a cytotoxic moiety). This antibody can also be used to induce cell lysis by the natural complement reaction and interact with 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, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), litin A chain, abrin A Chain (abrin A chain), modeccin A chain, α-sarcin (alpha-sarci
n), Aleurites Fordii Protein
fordii proteins), dianthin protein (dianthin p
roteins), Pitracuka americana protein (Phytola
cca americana proteins) (PAPI, PAPII and PA
PS), momordica carantia inhibitor (m
omordica charantia inhibitor), curcin,
Crotin, crotin, sapaonaria officinalis inhibitor (sapaonaria officinalis inhibitor), gelonin (gelonin), mitogellin (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin),
Enomycin and tricothecnes
enes). In other embodiments, the antibody is conjugated to a small molecule anti-cancer agent. Conjugates of the monoclonal antibody and the cytotoxic moiety described above are prepared using various 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-azido 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. The cytolytic portion of the toxin can be linked to the Fab fragment of the antibody.

It is preferred that a monoclonal antibody that specifically binds to the domain of the protein exposed on infected cells be conjugated to the ricin A chain. Most preferably, the ricin A chain is deglycosylated and prepared by recombinant methods. A preferred method for making littin immunotoxics is described in Vitetta et al. Science 2
38 , 1098 (1987), which are also within the scope of the present invention.

If the conjugate is used when killing infected human cells for in vitro diagnosis, it 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 are not critical. Aqueous dosage forms compatible with the culture or perfusion media will usually be used. The cytotoxicity can be read by a conventional method.

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

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

Antibody-Dependent Cytotoxic Effect The present invention further relates to (b) subclasses or isotypes capable of mediating the lysis of HIV virus-infected cells to which (b) gp160 binds the antibody molecule. And a method of using an antibody. More specifically, these antibodies complex with cell surface proteins, activate serum complement, and / or effector cells (e.g., natural killer cells or macrophages).
It belongs to a subclass or isotype that mediates antibody-dependent cellular cytotoxicity (ADCC) by activating ffector cells.

The present invention is also directed to the use of these antibodies, which are naturally occurring, for treating AIDS. For example, H
IgG2a and IgG3 binding to IV related cell surface antigens
Mouse antibodies can be applied in vivo for AIDS treatment. In fact, since gp160 is present on infected monocytes and T-lymphocytes, the antibodies of interest and their therapeutic applications are widely applicable.

It is known that the biological activity of an antibody can be largely determined by the Fc region of the antibody molecule [Uananue and Benacerraf, Textbook of Immunolog.
y), 2nd edition, Williams & Wilkins
ins) 218 (1984)]. This includes the ability to activate complement and mediate antibody-dependent cellular cytotoxicity (ADCC) performed by leukocytes. The different classes and subclasses of antibodies differ in this regard, and according to the present invention, select antibodies of the class having the desired biological activity. For example, mouse immunoglobulins of the IgG3 and IgG2a classes can activate serum complement when bound to target cells that express cognate antigens.

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

[0072] The ability of any particular antibody to mediate lysis of target cells by activating complement and / or ADCC can be assayed. The cells of interest are grown and labeled in vivo: the antibodies are placed in cell culture with either serum complement or immune cells that can be activated by antigen-antibody conjugation. Cell lysis of the target cells is detected by the label released from the lysed cells. In fact, antibodies can be screened using the patient's own serum as a source of complement and / or immune cells. Thus, antibodies that can activate complement or mediate ADCC in an in vitro assay can be used to treat that patient.

[0073] Antibodies of all viral origins
can bind to the gp160 epitope and activate complement,
Or, if it can mediate ADCC, it can be used as this embodiment in the present invention. Monoclonal antibodies have the advantage of a continuous rich source. In fact,
Mice are immunized with gp160 to establish hybridomas that produce antibodies to gp160, and then screened for hybridomas that produce antibodies capable of lysing the infected cells in the presence of human complement. A panel of antibodies that can react and dissolve it can be quickly established.

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

For parenteral administration, the antibodies are formulated in a unit dosage form for injection (solution, suspension, emulsion) with a pharmaceutically acceptable parenteral vehicle. Vehicles are essentially non-toxic and have no therapeutic effect. Such vehicles include, for example, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and 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. Typically, antibodies will provide about 1 mg / m 2 of such a vehicle.
It is formulated to be contained at a concentration of 1 to 10 mg / ml.

[0076] IgM antibodies are advantageous because their antigens are very specific for target cells and rarely appear on normal cells. Because IgG molecules are smaller than IgM molecules, they may be more likely to concentrate on infected cells.

It has been shown that complement activation in vivo leads to various biological effects, such as the induction of an inflammatory response and the activation of macrophages [Uananue and Benacerraf, textbook·
Textbook of Immunology, 2nd edition,
Williams & Wilkins 218
P. (1984)]. The increased vasodilation associated with this inflammation
Various anti-AIDS agents may increase the effect of concentrating on infected cells. Thus, antigen-antibody combinations of the type specified according to the present invention can be used for many therapeutic applications. Additionally, purified antigens [Hakomori associated with 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): Koprowski et al., Proc. Natl. Acad. S.
ci. 81 , 216 (1984)] could be used to elicit an active immune response in human patients. Such responses include the formation of antibodies capable of activating human complement and mediating ADCC, and leading to the destruction of infected cells by this mechanism.

The antibody according to the present invention can be used for HIV in a test sample.
It is useful in diagnosing. It is used as one axis in a sandwich assay to assay for HIVenv, with polyclonal or monoclonal antibodies to other sterically free epitopes of HIVenv.
For use in some specific sandwich tests,
The 5C2E5 antibody or equivalent is bound to an insoluble support or labeled with a detectable moiety according to conventional methods using other monoclonal antibodies. As another example of a labeled antibody, HIV env binding is detected by a procedure known per se, for example, using a labeled sheep anti-mouse IgG capable of binding to the 5C2E5 antibody.

Finally, in another embodiment, 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 the domain. For this purpose, any known method for immobilizing monoclonal antibodies can be used.

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

An HIV env composition intended for therapeutic use may 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. Formulations and dosages should be established in a manner consistent with successful treatment.

HIV env is prepared for administration by mixing the desired moderately purified HIV env with a physiologically acceptable carrier, ie, a carrier that is non-toxic to recipients at the dosages and concentrations employed. Typically, HIV env is 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 (0.
It can be easily done by sterile filtration through a (2 micron) membrane. HIVenv is usually stored as a lyophilized product for reconstitution, but is stable when stored as an aqueous formulation.

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

The sustained-release preparation is selected from microcapsule particles and implantable articles. Encapsulation in liposomes is not preferred for direct injection into the synovial cavity as it involves the transfer of lipids into the joint. However, encapsulation in liposomes is suitable for implanting sustained release HIVenv in the synovial sac. To treat RA using sustained release HIVenv, the HIVenv is preferably placed in a biodegradable matrix or microcapsule. Suitable substances for this purpose are:
Polylactide, 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), or poly (orthocarbonates) and the like. Here, the primary consideration must be that the carrier itself, or its degradation products, is non-toxic to the target tissue and that they do not exacerbate the disease. This can be assayed by performing routine screening on animal models of the target disorder, or if such models are not available, on normal animals. For examples of sustained release compositions, see, for example, U.S. Pat. No. 3,773,919, EP 58,481A, U.S. Pat. Biopolymers, 22: 547 (198).
3) and R. Langer et al., Chem. Tech 12: 9.
8 (1982).

The dose of HIVenv to be used is HI
The T4 binding affinity of the Venv preparation, the half-life of the env preparation, the route of administration, the clinical condition of the patient (such as the presence or absence of infection), the anti-
It depends on a number of factors, including the presence of HIVenv antibodies and whether HIVenv can be used to prevent disease, or can be used to treat acute autoimmune diseases or episodes of transplant rejection . As a general rule, the HIV env dose to reach the target site is about 0.5
Should be from 10-8M to 5-10-9M. Typically, HIV env concentrate in a therapeutic dosage form is administered by continuous infusion, sustained release infusion, or empirically determined number of infusions. RA synovial fluid can be as high as 50 ml at the knee joint, for example,
The exact amount of env is determined by the dilution in the synovial fluid, as well as the empirically measured loss of HIV env for endogenous proteases, leakage into the systemic circulation, and any anti-H present.
Determined by IVenv sequestration. Therefore, to determine the appropriate dosage regimen, it is best during the early stages of treatment to withdraw a sample of synovial fluid for an HIV envT4 binding assay and to evaluate the efficacy of the initial dose protocol. It is.

HIV env therapy may include other anti-inflammatory substances such as salicylates, non-steroidal anti-inflammatory drugs, penicillamine, gold salts, TGF-β, TNF-α or TNF-β antagonists (pending USS N898,272), γ-interferon antagonists and / or IL-1 antagonists.
It is not essential that such substances be included in the HIVenv composition itself, but these anti-inflammatory substances are
It may be formulated together with Venv.

The HIV env concentration concentrated at the site of inflammation may exceed the highest therapeutic dose systemically. Assaying HIV env levels in inflammatory invasions provides guidance as to the amount of HIV env for intravenous infusion, especially when focused administration is not practical. The result obtained is an important factor in selecting an appropriate dose. If the patient's inflammatory response does not resolve, at least in part, within about 48 hours after administration, the dosage is gradually increased until the desired effect is achieved. Correspondingly, the required T4 binding capacity and / or T4
The dosage of HIVenv preparations with low affinity for, increases the dosage of preparations with high capacity and high affinity. Also, when treating patients with acute rejection or episodes of inflammation, i.e., acute organ transplant rejection or arthritis flare, relatively high doses are initially required.

For emergency treatment following the onset of acute rejection, HIV env is injected intravenously or directly at the site of inflammatory injury when symptoms develop or rejection becomes serologically apparent. . Alternatively, prevention can be appropriately achieved by intramuscular or subcutaneous administration. 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 cited as references.

[0089]

EXAMPLE 1 The initiation of T-helper infection by HIV was found to involve 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). In addition, many of the impaired immunological functions of patients infected with the AIDS virus have been implicated in the selective ability of HIV to infect and eventually destroy helper cell populations (1,2,9). , 10). Also, AI
It has also been suggested that PBMCs from DS patients are less responsive to mitogen and antigen (particularly tetanus toxoid) stimulation, probably due to reduced helper cell function associated with HIV (12). This embodiment is mainly
Rgp in tetanus toxoid response in normal PBMC
120 to determine the activity.

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 the conditioned cell culture medium by immunoaffinity chromatography. This rgp120 was> 98% pure as determined by SDS-polyacrylamide electrophoresis.

Isolation of peripheral blood mononuclear cells (PBMC) : 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.
08) and centrifuged at 400 xg for 40 minutes at room temperature.
Remove the PBMC at the plasma-Ficoll interface and hank
k) Balanced salt solution (HBSS) [Gibco; Grand Island Biol
ogical Co., Grand Island, NY)], resuspended in complete medium and counted. Cell viability as measured by trypan blue exclusion was always greater than 95%.

Culture medium : RPMI 1640 medium was purchased from Gibco and used. 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 calf serum was used for staining the cells with the monoclonal antibody.

Tetanus toxoid (TT) -induced proliferative activity :
RPMI 1640 medium used in this assay contains 10%
Of autologous plasma, 2 mM L-glutamine and antibiotics. 1: 300 final dilution of TT [Mookmoor] confirmed in preliminary experiments to obtain optimal response
(Dr. A. Muchmore, National Cancer Institute, Bethesda,
PBMCs were cultured in quadruplicate in the presence or absence of [A. Replicate cultures containing various concentrations of rgp120 were checked in parallel. After a 6-day incubation, when a peak response appeared, the cultures were pulsed with [3H] -thymidine, processed and counted as above.

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

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

Results Affects PBMC Response to Soluble Specific Antigen
The effect of rgp120 was investigated. Six experiments were performed, using TT as a stimulant with or without rgp120 added to the culture. The results are shown in Table 2, which shows that Ps responding to specific antigens in normal PBMCs.
This shows that rgp120 can significantly (p = 0.05) inhibit BMC proliferation. The degree of inhibition was 19-51 of the control value.
% And was different in the five experiments. Experiment N
At o.6 no inhibition was observed (not shown).

[0097]

[Table 2] * Quadruplicate cultures of PBMC were prepared with TT alone (control) or T
105 in the presence of T + 5 μg rgp120 / ml (+ rgp120)
Measurements were taken on a flat bottom microtiter plate of cells / 200 μl / well. After a 6-day incubation, the culture is [3H]-
Pulsed with thymidine, collected, processed and counted. Each data represents the mean cpm and% inhibition was calculated as follows: 1- (cpm in presence of rgp120 / cpm of control) × 10
0; p <0.05.

Interaction between rgp120 and various T-cell groups
For : Infection of T-cells by HIV, virus envelope glycoprotein and T-helper membrane receptor complex
It was suggested to start with a selective interaction with the 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. PBMC were incubated at 4 ° C. and 37 ° C. for 24 hours in the presence or absence of rgp120 (5 μg / ml). The cells were washed and stained directly with FITC-conjugated OKT4 and OKT4A. F
The results of the ACS analysis indicate that treatment of the cells with rgp120 at 4 ° C. does not inhibit OKT4 binding. However, treatment with rgp120 at 37 ° C. resulted in a change in OKT4 binding as indicated by a decrease in T4 + cell fluorescence. Conversely, there was no OKT4A binding after incubating the cells with rgp120 at 37 ° C.
These results indicate that rgp120 is similar to HIV (25, 2,
6), indicating that they can occupy the T4A epitope or adjacent epitopes on the T4 helper membrane receptor.

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

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

Hybridomas secreting the monoclonal antibody 5C2E5 were ligated using the spleen cells derived from the animals immunized with rgp120 and the mouse myeloma cell line NP3x63-Ag8.653 as described in Oi et al. (37). Obtained by a conventional method. This cell line is widely and generally available. The fused cell cultures were 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 were incubated at room temperature for 30 minutes at 1% (weight / volume), rgp120-coated wells that blocked nonspecific binding sites, and (b) labeled with horseradish peroxidase This is a conventional double antibody sandwich assay using goat anti-mouse IgG. 10 out of 480 wells are rgp
Positive for 120 antibodies. Then these 1
Zero wells were screened for their ability to block rgp120 binding to human T4 helper cells. This blocking assay was performed as follows:

The in vitro culture supernatant of the ascites body fluid, or the purified ascites preparation or supernatant is screened by a conventional assay. 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 １ ： 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% severely dialyzed fetal bovine serum c) 0.2 mM phenylmethylsulfonyl fluoride d) 0.05% Tween in phosphate buffered saline 80 e) 0.06 M NaCl f) 0.25 mg / ml BSA (bovine serum albumin) g) 12.5 mM Hepes buffer pH 7.4

Each serial dilution (50 μl) was added to 12
Radiation iodinated rg in × 75 polystyrene test tube
Assay buffer solution of p120 (-100,000 cpm) (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 are pelleted by centrifugation, the supernatant is aspirated, and the pellet is washed with assay buffer (1.0 ml).
, Repelleted, aspirated and the pellet was counted on a gamma counter.

This CHO cell line was constructed by placing the gene encoding the human T4 receptor in a vector encoding DHFR under the control of the SV-40 early promoter and transfecting it into CHO cells;
And a transformed CHO cell line amplified in 500 nM methotrexate to amplify the T4 receptor gene and increase expression on the transformed cell surface. 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). Preferably, such T4 transformants are co-transformed with DHFR and amplified by conventional methods to maximize T4 cell surface expression. Such cells containing high amounts of T4 provide high sensitivity to the assay.

It is important to note that rgp120, which is radioiodinated, is used. Several other conventional methods have been found to result in 125I-rgp120 with binding properties that render the tracer unusable in this assay. The successful method used was as follows: 1 mg / 400 μ
1 rgp120 (400 μl) was added to 1 mCi of 125 I, 0.3
Mix with 5 mg / ml lactoperoxidase (20 μl) and 1 mM H 2 O 2 (20 μl), incubate at room temperature for 10 minutes, add 1 mM β-mercaptoethanol (20 μl), incubate the reaction mixture for 2 minutes at room temperature, 0.5% w / v BSA (50μ
l) was added and the reaction mixture was treated with 0.5% w / v BSA.
Diluted with PBS buffer containing PBS (PBS-BSA) to 2.5 ml, applied to a G-25 column, and
(3.5 ml). 125 I-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 active form due to IgG was purified to the desired form, such as by ammonium sulfate precipitation. This 5C2E5 monoclonal antibody is
Since the number of conjugates is reduced to about 10-15% of the murine monoclonal antibody control as measured by a decrease in cpm,
It showed the greatest inhibition of 25I-rgp120 T4 receptor binding. This 5C2E5 hybridoma is generally available (ATCC HB 9435).

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

Rgp12 used in this example was transfected from a CHO cell culture solution 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 treated with 0.25M glacial acetic acid under reduced pressure oxygen atmosphere for 110 days according to Ingram method (35).
Hydrolyzed at C for 18 hours. Under these conditions, rgp
The backbone of the 120 protein is truncated on both sides of the N and C termini of aspartyl residues. Tris buffer was added to the digested rgp120 to neutralize it, and the pH was adjusted to 7.

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

Amino acid D / NTSE / QPGAC mol / rgp 120 mol 3.0 3.9 2.8 4.2 3.4 3.1 1.9 0.8 Amino acid VMILYFKR mol / rgp 120 mol 1.4 1.5 5.2 3.8 1.0 1.6 2.1 2.6 Hinder measurement
You. The eluate from the 5C2E5 column was used at the N-terminal.
Analysis by sequencing also yielded only the following sequences: I  
L P  R I K Q F.

The results of this amino acid analysis and N-terminal sequencing indicate that rgp120 bound to the 5C2E5 column
Only the peptide in the acetic acid digest of the HIV env sequence from threonyl residue 411 to arginine residue 454
It was confirmed to be a four amino acid fragment. This region has the following sequence: TITLPCRIKQFINMWQEVGKAM
YAPPISGQIRCSSNITGLLLTR. Since this peptide contains the epitope of the murine monoclonal antibody 5C2E5, which blocks rgp120 binding to the T4 surface antigen of T4 + helper T cells, and the sequence deleted by the dalta 3 mutant described above, It is concluded that it is part of the domain.

Example 4 Immunotoxicants Independently or as a cocktail, coupled to deglycosylated ricin A chain (dgA) to kill cells infected with HIV from a wide variety of viral subtypes A monoclonal antibody directed against an HIV-encoded protein used for the following is obtained as follows:

Immunization with heat-treated (30 sec / 60 °) NP-40 lysate from HIV-infected human Hp (CD4 +) cells
Serum is obtained from mice (multiple intraperitoneal injection). Also, gp
Serum can also be obtained from mice immunized with 120 or gp150. The protein gp150 is gp120 and
gp1 with the region cut out to obtain gp41
60 extracellular domains. All sera are tested with the following assays:

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

[0116] 2. Radioimmunoassay (RIA) -Positive sera are tested by RIA against purified gp120 and gp150 to determine if mice have developed antibodies against these proteins.

Sera from mice positive for one or both of the RIA and indirect IT assays were collected and affinity purified on Sepharose-gp120 or Sepharose-gp150. This purified antibody was tested once more in three assays. If positive, the mouse was HIV-H9, ±
Booster treatment with gp120 ± gp150 lysate. Several mice are sacrificed and their spleens are Sp2 / 0 or N
It was fused with P3X63-Ag8.653 myeloma cells. Supernatant (SN) from wells containing growing hybridomas was
(a) H9 vs. (b) HIV-H9 was tested in an indirect IT test. Hybridomas that were 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
Coupled directly to dgA. These IT-dgA
Tested on cells from IDS patients and cells infected with HIV. Then, clinical IT was prepared.

Preparation of F (ab ') 2 and FAB' Fragments of Ritin A Chain Antibody Conjugate (1) Antibody
(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 0.3 M NaCl phosphate buffer, or 0.1 M
The unneutralized digest was adsorbed on an SP-Sephadex column (10 × 2 cm) equilibrated with a citrate buffer (pH 3.7), and F (a) was added with phosphate buffered saline (PBS, pH 7.2).
b ') By eluting the two fragments. The yield of F (ab ') 2 fragments is generally 35-50%.

The Fab 'fragment was obtained from the F (ab') 2 obtained above.
Fragment is prepared using ethylenediaminetetraacetic acid
(EDTA) (PBE) Obtained by treatment with dithiothreitol (DTT) at a final concentration of 5 mM in 0.1 M phosphate buffer (pH 7.5) containing 0.003 M for 1 hour at room temperature for 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) is determined by the Fulton et al. J. Immunol. , 136 , 3
103-3109 (1986)], and a final concentration of 2 mM Ellman's reagent (5,5 ').
-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 determined by binary diffusion and immunoelectrophoresis using anti-mouse IgG serum that does not react with both fragments b 'and Fc. This preparation does not contain the Fc fragment and intact IgG.

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

(3) Preparation of IT-As by SPDP Using IgG or F (ab ') 2 fragment of an antibody, IT-As is prepared by the method described in Fulton et al. P
Solution of IgG or F (ab ') 2 fragment in BE (pH 7.5)
SPD dissolved in dimethylformamide (10 mg / ml)
Add P to bring the final concentration to 1 mM. After 30 minutes at room temperature, the solution is filtered over a Sephadex G-25 column (30 × 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 1.3 mg / m of reduced A chain dissolved in PBE.
and left at 25 ° C for 2 hours and 4 ° C overnight. This mixture is then purified.

(4) Preparation of Fab'As by DTNB Preparation of mouse Fac'-A by DTNB-induced Fab 'is performed according to the method described in Fulton et al. (Supra) for rabbit Fab'-A. 1-2 TNB
The Fab 'fragment derived with an Ellman's reagent containing a substituted thiol group (see above for the preparation of the Fab' fragment) 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.
At 25 ° C. in about 2 hours. This mixture is then immediately purified.

(5) Purification of IT-As IT-As prepared with an intact antibody or a fragment thereof was purified from Knowles and Thorpe [Knowles and Thorpe, An.
al. Biochem. 160 , 440-443 (1987)] with a slight modification of affinity chromatography on blue sepharose. Chromatography is performed in 0.05M phosphate buffer (pH 7.0) and the A-chain and IT-As are eluted with 1M NaCl prepared in the same buffer. The eluate was centrifuged at 5 mg /
and concentrated to a phosphate buffer 0.3M NaCl (pH 7.
Flow through Sephacryl S-200HR equilibrated in 2).
The peak containing purified IT-As is 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

The affinity-purified goat anti-mouse immunoglobulin (GAMIg) Fab fragment was ligated with 0.1M EDT.
Prepared by papain digestion using a 2% solution of papain in 0.1 M 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. The Fab fragment is 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.

The literature by Fulton et al. [J. Biol. Chem. 2
61 , 5314 (1986)], and the tested litin A chain is reduced with 5 mM DTT. The A chain was desalted on Sephadex G-25 and Fab-GAMIg
Combined with -E. Then, Fab-GAMIg-A was replaced with S2.
Purify by affinity chromatography on 00 and Blue Sepharose.

(7) Indirect immunotoxin assay Cells with a viable cell count of greater than 95% in the logarithmic growth phase are used. Prepare serial dilutions of the initial unbound antibody at concentrations ranging from 2 × 10 −8 to 2 × 10 −13 M in a suitable medium and plate 100 μl aliquots in triplicate into 96-well plates. 100 volumes in the same medium
Add μl of 105H9 or HIV-H9 cells to each well and incubate the plate at 4 ° C for 1 hour. Then, Fab-GAMIg-A is added to a suitable plate at a final concentration of 1 μg / ml. Control groups were (a) untreated cells, (b) cells treated with Fab-GAMIg-A alone, (c)
Cells treated with the antibody alone and (d) cells treated with the antibody and then treated with Fab-GAMIg (without the A chain) are included. The plate was then placed in 5% CO2 at 37 ° C for 36 hours.
Incubate for hours. Cells are incubated at 37 ° C for 4-8 hours 3
Treatment with H-thymidine followed by Titertec
ek) Collect on glass fiber filter using 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. All control cultures take up 80-100% 3H-thymidine by untreated cells.

(8) Data Analysis The expected ability of the monoclonal antibody in the indirect IT assay is to determine the concentration of antibody capable of killing 50% of target cells (using Fab-GAMIg-A at optimal concentration) (IC5
0). The IC50 must be between 10-12 and 10-10 for IT-As to be useful in vivo. ICs 50 are ranked as follows. 10-11M = 4 + 10-10M = 3 + 10-9M = 2 + 10-8M = 1 + 4 + and 3+ Antibodies appear to be suitable as IT-As, so further R against gp150 and gp120.
Test with IA.

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

(10) Antibodies and serum 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 lysate from cells

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

First, 50 BALB / c lysates were prepared from lysates prepared from 1 × 10 8 H9 cells infected with HIV-I.
The mice were immunized. Serum from these mice was screened in an indirect assay. 15 out of 38 surviving mice were 3+ to 4+ in the indirect IT assay. RIA was performed and after two immunizations, three 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 .

Although the present invention has been described mainly with respect to the preferred embodiments thereof, the present invention is not limited to the disclosed embodiments, but rather falls within the scope of the appended claims. It is intended to cover various modifications and equivalents. The scope of the claims should be given the broadest interpretation as encompassing all such modifications and equivalents.

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Ilmer, E. et al., Lancet 1 : 753 (1984). 16. Koenig, S. et al., Science 233 : 1089 (1986). 17. Levy, JA et al., Virology 147 : 441 (1985) 18. Siegel, JP et al. , J. Clin. Invest. 75 : 1957 (198
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25-228 (1984) .37.Oi, V. et al., In: Selected Methods in Cellular Im.
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──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/06 C07K 16/28 C07K 14/705 19/00 16/28 A61K 39/395 S 19/00 C12N 15/00 ZNAA C12N 15/02 A61K 37/02 // A61K 39/395 C12N 15/00 C (72) Inventor Timothy Jay Gregory Pine Hill Road 414, Hillsboro, California 94010 United States (72) Inventor Lawrence A. Ruskey United States 94949, Sousalit, Star Root Box No. 40 (72) Inventor Gerald Earl Nakamura United States California 94116, San Francisco, Twenty Nineth Avenue 1923 (72) Author: Eric J. Patzer CA 94563, U.S.A., 470, Tahoes Road, Olinda Inventor John E. Patton CA 94070, U.S.A., San Carlos, 330 Emerald Ave. 330 (72) Inventor Ellen Es Vittetta Texas, United States 75230, Dallas, Pemberton Drive 6914

Claims (19)

[Claims] 1. The T of human immunodeficiency virus envelope
A composition comprising a TCB domain of a human immunodeficiency virus envelope that lacks a human immunodeficiency virus immune epitope that is normally in contact with the C-terminus or N-terminus of the CB domain. 2. A composition comprising a human immunodeficiency virus envelope TCB domain that lacks the human immunodeficiency virus envelope sequence in contact with the C-terminus or N-terminus of the TCB domain. 3. The composition of claim 2, wherein the TCB domain is residues 411-454 of the human immunodeficiency virus isolate 3B envelope or a fragment thereof, which is capable of binding to the T4 receptor of T4 helper cells. 4. The method of claim 1, wherein the TCB domain is of the formula: ## STR1 ##
FINMWQEVGKAMYAPPISGQIRCSS
The composition according to claim 3, which has the amino acid sequence represented by NITGLLLTRDGGNNNNNSEEI. 5. The method of claim 1, wherein the TCB domain has the formula:
The composition according to claim 3, which has an amino acid sequence represented by ISGQIRC. 6. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier and sterile. 7. A substance capable of binding to a cell surface marker
directing cytotoxic lymphocytes to a selected cell surface marker comprising preparing a conjugate of (a), and a fragment thereof (b) capable of binding to the T4 receptor of human immunodeficiency virus envelope or helper lymphocytes Method. 8. The method of claim 7, wherein the conjugate comprises a human immunodeficiency virus envelope fragment. 9. The fragment according to claim 1, wherein the fragment is of the formula: TESGNNTGSDTITLPCRIKQ
FINMWQEVGKAMYAPPISGQIRCSS
9. The method according to claim 8, which has a sequence represented by NITGLLLTRDGGNNNNNSEEI. 10. The method according to claim 7, wherein the marker binding substance is a hapten. 11. The method according to claim 7, wherein the marker binding substance comprises a variable region of an antibody. 12. The method of claim 11, wherein the antibody is capable of binding tumor necrosis factor-α. 13. A substance capable of binding to a cell surface marker
A conjugate of (a), and a fragment thereof (b) capable of binding to the human immunodeficiency virus envelope or T4 receptor of helper lymphocytes. 14. The conjugate according to claim 13, wherein the cell surface marker is a protein. 15. The conjugate according to claim 14, wherein the substance capable of binding to the protein comprises a variable region of an antibody. 16. The conjugate has the formula: TEGSNNTGSDTITLPCRIKQ
FINMWQEVGKAMYAPPISGQIRCSS
15. The conjugate according to claim 14, comprising a human immunodeficiency envelope fragment having a sequence represented by NITGLLLTRDGGNNNNNESEI. 17. The method according to claim 1, wherein the substance capable of binding to the cell surface marker and the human immunodeficiency virus envelope or a fragment thereof are covalently crosslinked.
4. The conjugate according to 4. 18. The conjugate according to claim 17, wherein the covalent bond is a peptide bond. 19. The conjugate is a fusion having the variable region of an antibody capable of binding to a cell surface marker on the N-terminal side and the human immunodeficiency virus envelope TCB domain on the C-terminal side. The conjugate according to 1.