JP2813630B2 - Human immunodeficiency virus envelope polypeptide and its antibody - Google Patents

Description

The present invention relates to human immunodeficiency virus or HIV (HTLV-I
It relates to the use of envelope (env) polypeptides of II) and more particularly to their use in undesirable 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 immunotoxic substances and other immunoconjugates (immunoconjugates) and their use for killing HIV or other viruses encoding cell surface antigens.

BACKGROUND OF THE INVENTION Inflammatory responses associated with various immune disorders can generally be divided into four classes. However, many autoimmune diseases included in the treatment of the present invention are difficult to classify due to their uncertain origin (eg, autoimmune diseases caused by myasthenia gravis, Graves 'disease, 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 responses are characterized by chronic production of an immune complex containing IgG and / or IgM. Class IV responses are associated with delayed-type hypersensitivity reactions, mediated by cytokines and T-lymphocytes, and are commonly found in tuberculosis, sarcoidosis, polymyopathy, granulomas and vasculitis.

Rheumatoid arthritis (RA) is a serious class III disorder
One. Synovitis is a characteristic tissue reaction of RA, in which normal thin connective tissue is replaced by overgrown synovial fibroblasts and the synovium infiltrates lymphocytes, macrophages and other immune cells. Has been invaded by In rheumatic synovial fluid, the reversal of complement components is increased, and cleavage products of complement, particularly C5a, are present in rheumatic synovial fluid. C5a
This is important because and other complement derivatives contribute to the activation of immune cells and the production of inflammatory cytokines. Synovial fluid also contains, along with kinin and LTB4, hydrolases derived from inflammatory cells, including collagenase.

Various mechanisms have been proposed to elucidate the degree of tissue destruction characteristic of rheumatoid arthritis. These mechanisms
It involves a complex interaction of activation and repression that regulates 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.

One generally accepted assumption about RA induction is that
An unknown antigen is chronically deposited on the synovium where it is subject to 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, immature transformation, and then generation of antigen-specific helpers and cytotoxic T cells. The combination of an antibody and an antigen, or the combination of an antibody-antigen complex with a rheumatoid factor, or the self-association of a rheumatoid factor, activates the complement and kinin-forming system through activation of the Hageman factor. Activate. This results in the production of pro-inflammatory products such as C5a, arachidonic acid metabolites, quinine and fibrinopeptides,
They diffuse into the synovial fluid and further into the synovial vessels. These substances increase the permeability of blood vessels and attract polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes ingest a number of immune complexes in the fluid, release lysosomal and other destructive enzymes, and generate 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 (eg, cytokins) and production that are characteristic of RA, sustain inflammatory conditions, and destroy tissue .

The enzymes that are released into the synovial fluid by cells that are present in the synovial fluid or that make up proliferative synovial foci and that are synthesized locally contribute to pathological events in the joint structure, thus binding The unique structure of 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 are prostaglandins, hydrolases, collagenases,
Produces plasminogen activator and IL-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 T and B cell activation leads to a chronic inflammatory response of the synovium. Sustained cell proliferation and influx causes the synovium to proliferate and invade its surrounding structures. Diffusion of collagenases, PGEs, 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-type hypersensitivity-type immune response.

Today's treatment for RA involves bed rest, warming, and medication. Salicylate is currently the preferred drug 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. They include indomethacin, phenylbutazone and phenylacetic acid derivatives such as ibuprofen and fenoprofen, naphthaleneacetic acid (naproxen), pyrrolealkanoic acid (tomethine), indoleacetic acid (salindac), halogenated acetranilic acid (sodium meclofenamate) , Piroxicam, zomepirac, and diflunisal. These are generally considered less effective than aspirin, but may be better tolerated in certain patients.

Other drugs that can be used for RA include antimalarials such as chloroquine, gold salts and penicillamine. These alternatives often have serious side effects, including retinal lesions and kidney and bone marrow toxicity. Because immunosuppressants such as methotrexate are toxic, they are used only for the treatment of severe, non-remitting RA. Corticosteroids also have undesirable side effects that 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,
There is a need for effective therapies and compositions for treating immune response suppression associated with Graves 'disease, Chagas' disease, and juvenile onset diabetes.

The present invention also provides a method for transplanting cells, organs or tissues,
Alternatively, the present invention relates to suppression of immune rejection. Rejection in transplants has been controlled using immunosuppressants such as cyclosporine antibiotics or steroids. Such drugs have widespread and undesirable side effects.

In other attempts, monoclonal antibodies have been used that target and neutralize subsets of lymphocytes 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 the treated patient to rats,
Since the murine constant region can activate human complement, anti-T
Subset antibodies can lyse and deplete T cells. There is a need for compositions and methods for treating rejection in tissue transplants that do not use murine immunoglobulins.

Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus that has been identified as human immunodeficiency virus (HIV) (1-6). Abnormal B cell function, abnormal antibody response, impaired monocyte function, impaired cytokine production (7-10), inhibited the function of natural killer and cytotoxic cells (11), and recognized soluble antigens, There have been reports of a number of AIDS-related immune abnormalities, such as reduced responsive lymphocyte function (12). Other AIDS-related immune abnormalities have been described (13, 14). A more serious immune injury in AIDS patients is the depletion 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 the damage in the immune response is that HIV selectively infects helper T cells, resulting in impaired function of the helper T cells and eventual depletion of the 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 also causes immunodeficiency by interfering with normal cytokine production in infected cells and leading to secondary immunodeficiencies such as IL-1 and IL-2 deficiency (18). Other HIV-induced immunodeficiencies
Lies 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.

There is an extensive description of the HIVenv protein, including amino acids and Rs encoding HIVenv from many HIV strains.
The NA sequence is also known (34). HIV pillion is covered by a membrane or envelope derived from the outer membrane of the host cell. This membrane contains (anchor) envelope glycoproteins (gp160) immobilized via its carboxy-terminal region to the lipid bilayer of the membrane. Each glycoprotein contains two segments. The N-terminal segment, termed gp120, based on its relative molecular weight of about 120 kD, 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, a fusion such as gp120 fused to the N-terminal fragment of gp160 or gp41, HIVenv with or without various glycosylations, as well as the T cell binding fragment of HIVenv,
It is intended to include all forms of gp120. HIVenv and its variants are prepared conventionally by recombinant culture (see EP-A-187041). Previously, gp120 obtained by recombinant cell culture was called rgp120. Although recombinant synthesis is preferred for safety and economic reasons, it is known that HIV env purified from viral cultures and such env preparations are also included in the definition of HIV env.

Current AIDS treatments aimed at protecting uninfected cells are:
Approximately every 4 hours, oral administration of nucleoside analogs (such as AZT and DDC) capable of blocking viral RNA replication. These drugs inhibit viral replication at concentrations of 50-500μΜ but higher concentrations ^- in (1 mM), healthy cells, also inhibits DNA polymerase required for cell division (Mitsuya and Broder, 1985). Current therapies require very large doses (up to 1 g / day) of drug.
Since 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 DNA
To be incorporated into (and exhibit its chain breaking action)
It must first be phosphorylated, which requires a kinase that is not always present in equal amounts in all cells susceptible to virus. Thus, oral administration of a nucleoside analog that is ineffective against cells that have already been infected can protect only those cells that are at risk of infection (ie, 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). The conserved region of the HIV virus coat protein (gp160) binds to the CD4 receptor, which internalizes and transports the RNA virus into cells. Once inside the cell, the virus makes a DNA copy of the RNA inside the cell with its reverse transcriptase. Nucleoside analogs act as strand breakers in viral RNA transcription to protect cells. Since they do not have the functional groups necessary for incorporation into the growing polymer to incorporate the next nucleoside into the polymer, the chain will be interrupted and have no function.

Nucleoside analogs need to be phosphorylated (converted to nucleotides) by kinases to be incorporated into nucleic acid strands (Pherman et al., 198).
6). T cells that can rapidly divide contain high levels of kinases that phosphorylate antiviral agents such as ATZ and DDC. Monocytes that divide only 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, current treatment with nucleoside analogs is ineffective at protecting monocytes from complete HIV.

Lyerly et al. (PNAS84, 4601 (1987)
Uninfected CD4 ⁺ lymphocytes bound to gp120 have been shown to be targets for antibody-dependent cellular cytotoxicity by goat serum raised against native gp120. However, gp
Human serum binding to 120 adsorbed cells did not show its destructive effect in the presence of complement. In contrast, these sera potently mediate antibody-dependent cellular cytotoxic effects.

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

Immunotoxic substances (ITs), consisting of cell-reactive antibodies linked to the A chain of littin toxin (IT-As), have been used to specifically kill various target tumor cells. Kronke et al. (Cancer Research, 46 (7), 3295 (1986)
Is a conjugate of a monoclonal antibody of the interleukin II receptor and a ricin A chain (a conjugate).
Discloses that I-infected leukocyte T cells have been killed.
See also Kronke, Brad 65 (6), 1416 (1985).

(US Pat. No. 4,714,613) show that hepatitis B surface antigen is expressed on the surface (where the surface antigen is HBV).
A method for inhibiting the growth of hepatocytes or hepatoma cells infected with the hepatitis B virus (encoded by DNA). 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 present 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.

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

SUMMARY OF THE INVENTION HIV env is useful for treating immuno-inflammatory responses and diseases, and it is an object of the present invention to administer to a patient exhibiting an immuno-inflammatory response an amount of an HIV env formulation sufficient to suppress the immuno-inflammatory response. It consists of doing.

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

HIVenv from which the functional TCB domain has been deleted 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 the T4 cell surface marker. This indicates that the antibody at least partially reduces the binding of recombinant gp120 to the T4 cell surface marker, possibly by sterically inhibiting access to the TCB domain binding site on the T4 cell surface antigen. Is indicated by being able to shut off. The antibody also provides any free HIV that is immunosuppressive in HIV-infected patients.
Since it has the advantage of being able to bind to env, it is also useful for passive immunization of 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 litin A chain bound to the monoclonal antibody, targeting a protein encoded by a virus. Thus, proteins associated with the toxic conjugate are only taken up by infected cells, such as helper T cells, and kill the cells. The present invention also provides a method for infecting a patient with a therapeutically effective amount of an immunotoxic substance containing a monoclonal antibody bound to a toxin that specifically binds to a protein such as gp160 encoded by a virus on the cell surface. It is intended to provide a method for killing cells infected with a virus, which comprises killing cells.

HIVenv or an immunosuppressive fragment thereof (including the TCB domain) was conjugated to a hapten or polypeptide capable of binding to a cell population of cells for which a cytotoxic T cell cytolytic response is desired. These conjugates are particularly useful as anti-tumor agents or for treating allergic responses.

DETAILED DESCRIPTION OF THE INVENTION As described above, HIVenv is derived from the human immunodeficiency virus envelope protein and its in vitro immunosuppressive amino acid sequence variants and derivatives obtained by covalent modification of HIVenv or variants thereof. Defined to be. Variants include those in which one or more residues of the env amino acid sequence have been substituted, those in which one or more residues of the env sequence have been deleted, and one or more residues in the env amino acid sequence. Is inserted.

Major deletion mutants of the present invention are mutants in which immune epitopes other than the TCB domain have been deleted or, conversely, TCB domains have been deleted and the remaining epitopes of HIVenv have been deleted. Deletion mutants are described in detail below, as are substitution mutants. Variants of another class are those in which a heterologous (heterologous) polypeptide for HIVenv is fused to the amino or carboxy terminus of the HIVenv 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 the TCB domain, which is an HIV immune epitope that normally does not border the TCB domain in native HIVenv, especially bordering the C-terminus of the TCB domain. Expression: FRPGGGDMRDNWRSELYKYKV is a mutant obtained by fusing with an eptope other than the sequence shown.

Fusions of the TCB domain of HIVenv with an immunogenic hapten or polypeptide are useful as components 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 bind to cytotoxic T cells targeted to the target cell if the hapten or heterologous polypeptide can bind to a target cell surface receptor or other binding partner. 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 that can bind to TGF-α are known. By linking such an antibody with HIVenv or a fragment containing the TCB domain thereof, for example by covalent cross-linking (33), or by recombinant cell culture,
Ligation is achieved by expression as an N-terminal or C-terminal fusion of the B domain containing fragment. The latter method is achieved by obtaining a DNA encoding the TGF-α antibody according to the general procedure described in EP125,023A. This DNA is ligated to the amino or carboxy terminus of gp120 or a fragment containing the TCB domain thereof using a suitable adapter and inserted into a mammalian cell expression vector as described in EP 187,041A. CHO cells and other EP187,0
Transform a suitable host cell as described in 41A. If desired, DNA encoding the variable region of the TGF-α antibody may be encoded with a TCB domain in place of the human constant region described in EP184,187, EP194,276A, EP171,496A or Bouliamne et al. (32). Ligated to DNA. The resulting fusion is, for example, immobilized TGF-α
Alternatively, when the constant region of the TGF-α antibody is retained, the antibody is recovered by adsorption to an immobilized antibody against the constant region, and formulated with a therapeutically acceptable vehicle (eg, physiological saline). , Administered to patients with TGF-α expressing tumors. This fusion produces cytotoxic T cells,
Facing tumors with TGF-α cell surface antigens, resulting in lysis of tumor cells. Similarly, when a patient fuses a TCB domain with an antigen that is producing an unwanted immune response, such as the development of allergic or post-transplant rejection, the response of the autoimmune cytotoxic T cells is reduced. The patient is administered at a dose sufficient to direct the clonal cells to contribute to the undesired response.

The TCB domain of HIVenv is the HIV strain described in EP187,041A,
The region from nucleotide about 7006 to about 7203, or other
It is thought to contain an equivalent region in HIV strains (other strains whose viral genome and HIVenv length are altered by deletions or insertions upstream yield the same nucleotide and amino acid residue numbers) Although not possible, the region encoding this portion of the TCB domain in each strain can be readily identified based on amino acid sequence homology. The TCB domain has the formula: Having the sequence shown in particular, residues 411-454, 416-443,
And sequences contained between 440-442. Table 1 below shows the amino acid sequences of TCB domains from multiple HIV strains below the right column "Origin" and the predefined mutant TCB domains in the left column. The mutant indicated by “△ 3” (mutant 1) enables this part of the TCB domain of HIV to be identified. Deletion of the gp3 residue of rgp120 by site-directed M13 mutagenesis and expression of this mutant in recombinant CHO cell culture as otherwise described in EP 187,041 resulted in selected AIDS patients. Although indistinguishable from rgp120 based on assays such as serum binding, rgp120 lost its T4 binding ability. Surprisingly, as shown in Table 1,
The deletion of the Δ3 region is less highly conserved than BH10 in many HIV strains and as a whole TCB, as can be seen by comparison to homologous sequences from other HIV strains.
The domain is found to contain the HIVenv region, which is characterized by being extremely variable (34). It was thought that only highly conserved regions had adequate certainty to ensure that the virus always recognized the T4 receptor, and thus affected host cells, but this hypothesis suggests that The study did not work. For example, residues 5-31
Is highly conserved in various HIV strains, the deletion of the first 29 residues from the amino terminus of HIVenv
It had no effect on the binding of p120 to T4.

Improve the variants designated in Table 1 as numbered 2-15: eliminate the T4 binding of rgp120. For example, 431
Substituting alanine at the position with aspartic acid,
Mutant binding decreased to about 10-15% of the binding exhibited by rgp120 having the native sequence. However, in our assay, substitution of lys430 with glutaminyl, tyr433 with phenylalanyl, and pro435pro436 with valylvalyl did not cause any change in detectable rgp120 binding. Therefore, by measuring the activity of each mutant,
It should be screened by conventional assays. In view of the role of the Ala431 residue, substitution, deletion or insertion at this site is particularly preferred, and includes histidine, tryptophan, proline, phenylalanine, glycine, leucine, isoleucine, methionine,
Valine, serine, threonine, tyrosine, glutamine,
Substitution with asparagine, lysine, glutamic acid, cysteine and arginine, or such a residue or Ala
An insertion of aspartic acid adjacent to 431 is included. The above mutants and △ 3 mutants are useful in preparing HIVenv antigens that can be used in diagnostic assays or vaccines. HIVenv 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 thus has a higher titer of anti-HIV. 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. Also, introducing a deletion or insertion into the TCB domain,
Agonist activity can be evaluated. As used herein, the term "agonist" refers to an increase in the HIVenv activity of a variant as compared to a native molecule (eg, when the variant is
Show higher affinity for T4 than native HIVenv)
Means However, it will be appreciated that this activity is an antagonist against HIV infection. HIVenv agonist analogs are useful in treating desirable immune inflammation as in the embodiments described above for intact or native HIVenv. 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, and is used in AIDS or ARC patients.
Administered to treat IV infection.

HIVenv agonist activity is described in the Examples below for binding to the T4 receptor by measuring its ability to compete with labeled rgp120 or native gp120, or a fluorescein isocyanate conjugated anti-T4A antibody. Fluorescence activated cell sorter system
Measure with This ability of a mutant candidate to compete with the labeled conjugate for T cell binding in PBMC is one measure of its activity:
As the competition for the T4 binding site with the labeled conjugate increases, the agonist activity of the mutant increases.

The HIVenvTCB domain or a variant thereof is prepared in a manner known per se. One method is Merrifield (Me
rrifield) TCB by in vitro manipulation methods such as synthesis
Synthesizing a domain. 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, 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, for example, ST
II, alkaline phosphatase, lpp and penicillinase signal. 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. The resulting fusion gene is ligated into a conventional vector such as pBR322, transfected into host E. coli, and the cells are cultured.
One could measure the amount of TCB domain secreted into the periplasm of the host cell. Alternatively, the TCB domain is synthesized as a secreted or non-secreted fusion by a recombinant cell culture method and recovered.

Antibodies capable of inhibiting or binding the TCB domain, immunize mice such as Balb / c or preferably C57 Black / 6 against gp120 and screen for clonal antibodies that, when pre-incubated with gp120, prevent binding to the T4 T cell marker. It is obtained by doing. The 5C2E5 antibody is an example of such an antibody, but it is not unique as other antibodies exhibiting the same qualitative activity can be obtained by the methods described herein. The term "qualitative" activity means that the antibody binds to the T4 binding site or to both regions of HIVenv, the latter case having rgp120 or HIVe at the T4 site.
It is a bond that sterically hinders the binding of nv. By tryptic digestion of the 44 residue TCB domain, the 5C2E5 antibody spans residues 420-430 (s
panning) was confirmed to bind to the peptide. Therefore, this domain is included in the TCB domain,
Or it is adjacent to it. The 5C2E5 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 is residue 430-4
Binds to peptides spanning 59.

The TCB domain binding of antibody 5C2E5 is specified as its ability to inhibit rgp120 binding to the T4 receptor of helper lymphocytes. Such an effect can be exhibited. Monoclonal antibodies other than 5C2E5 belong to this specific range, even if they differ in affinity, immunoglobulin class, species of origin or exact TCB sequence,
For example, a 5C2E5 antibody mutant whose antibody or amino acid sequence expressed in recombinant cell culture is predetermined, such as a chimera of the variable region of the 5C2E5 antibody and a human constant region.

The antibodies described herein may be prepared by hybridoma purification using conventional immunoglobulin purification methods, such as ethanol or polyethylene glycol precipitation, as previously used to purify IgG or IgM from stored plasma. Recovered from cell culture. The antibody thus purified is sterile-filtered and, if necessary, conjugated with a cytotoxic substance such as ritin that can be used for AIDS treatment,
Alternatively, it is conjugated to a detectable marker such as yeast or a spin label that can be used for an HIV diagnostic assay in a test sample.

A new application 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. In particular, gp160 binding immunotoxics kill HIV infected cells.

The following describes treatments intended to kill cells already infected and actively producing new viruses. Killing is achieved by immunotoxic agents that bind to the viral coat protein expressed on infected cells. Thus, the immunotoxic substance is internalized in the cells, causing the cells to die. Infected cells in which the virus genome is integrated into the DNA but do not synthesize viral proteins (ie, cells in which the virus is latent) are killed by the immunotoxic agent until the virus synthesis begins. Maybe not.

HIV-infected cells, which are actively producing the virus, were found to 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. Monoclonal antibodies specific to conserved regions on gp160 (strain-dependent) can be used as attacking molecules to deliver toxic substances to infected cells. Additionally or alternatively, a "cocktail" (cocktai) of immunotoxics to non-conserved areas.
l) "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. Since these antibodies specifically bind to antigens that are not present on uninfected cells, the method of the invention allows the antibodies to attack the cells to be destroyed very accurately. ing. 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 force a 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.

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.

When all the monoclonal antibodies specific to gp160 are not necessarily coupled to the ritin A chain (IT-A)
In addition, it does not create high levels of cytotoxic and immunotoxic substances. IT
Assays employed to predict the ability of the antibody to function as a subfield of -A were performed. This test was performed and various anti-HI
Antibodies V were screened for the ability to create IT-A effective against HIV-infected cells. This result is
It has been shown that only HIV-specific antibodies create effective IT-As. These antibodies are purified and coupled to the ricin A chain, which is used to specifically kill HIV-infected cells. Advantageously, these antibodies cross-react with some (or all) HIV strains.

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

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

Further, the toxic substance-conjugate is Fab or F (A
b ') It is created using _two fragments. Because these are relatively small in size, they penetrate tissue better,
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 on the cell surface having antigenic activity (eg, gp120, gp4
1, an epitope selected from TCB etc.) was isolated from an infinite passage hybrid cell line generated by fusion of lymphocytes and myeloma cells primed with the antigen. Advantageously, according to the invention, the gp
Monoclonal antibodies that bind to 160 bind to domains 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 which is the object 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 agent. This assay involves applying the cells to a diluent of the test antibody and then to the Fab fragment of the secondary antibody coupled to the ricin 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. Science of Vitetta et al. (Scienc)
e) 238 , 1098-1104 (1987) and Weltsman (Welt
man) et al. Cancer Res. 47 , 5552
(1987), which also fall within the scope of the present invention.

Monoclonal antibodies are highly 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 hybridoma cells in culture or from ascites induced by inoculating hybridoma cells into the peritoneal cavity of mice.

Kohler and Milstein [E
6 , 511 (1976)], has been widely used to prepare hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens. I have.

The steps and schedule of 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 immunolymphoid cells are fused with myeloma cells to generate a hybrid cell line that can be cultured and subcultured indefinitely to prepare large amounts of monoclonal antibodies. 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 type of cell to 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 used in cell culture media.
It can be maintained in vitro culture. The cell line according to the present invention comprises a known hypoxanthine-aminopterin-
It can be selected and / or maintained in a composition consisting of infinite passage cell lines in 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 on the tissue culture by a conventional method such as a precipitation method, ion exchange chromatography, and affinity chromatography.

Although the present invention has been described using a mouse monoclonal antibody, the present invention is not limited thereto. In fact, a human antibody can also be used, and it can be proved that the present invention is suitable. Such antibodies can be obtained using human hybridomas [Cote et al., Monoclonal Antibodies and Cancer Therap.
y), Alan R. Liss, p. 77 (19
85)]. Indeed, in accordance with the present invention, genes derived from mouse antibody molecules of appropriate antigen specificity as well as human antibody molecules having appropriate biological activity (eg, capable of activating human complement and mediating ADCC) To prepare a “chimeric antibody” by splicing the gene [Proc. Natl. Acad. Soc. Of Morrison et al.
ci.) 81 , 6851 (1984): Nature of Neuberger et al. (Nature) 312 , 604 (1984): Takeda (Ta)
Nature, 314 , 452 (1985)]; such antibodies are also within the scope of the present invention.

As an alternative to the cell fusion method, a monoclonal antibody according to the present invention is prepared using EBV-immortalized B cells. Furthermore, 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.

The cytotoxic portion of the immunotoxic agent can be a cytotoxin or enzymatically active toxin of bacterial, fungal, plant or animal origin, or an enzymatically active fragment of these toxins (A chain). The enzymatically active toxicities used and their fragments include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa)
Origin), ritin A chain, abrin A chain (abrin A chai
n), modeccin A chain, α-sarcin (alp
ha-sarcin), Aleurites fordii proteins (Aleurites fordii proteins), dianthin proteins (Dianthin proteins), Phytolacca americana proteins (PAPI, P
AP II and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor (sapaonaria officinalis)
inhibitor), gelonin, mitgeline (mit
ogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin)
And trichothecenes. 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. An example of such a reagent is SP
DS, IT, bifunctional derivatives of imide esters such as dimethyl adipimidate HCl, 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
-Diisocyanates, and bis-activated fluorine compounds, such as 1,5-difluoro-2,4-dinitrobenzene. The cytolytic portion of the toxin can be linked to a Fab fragment of the antibody.

It is preferred that a monoclonal antibody that specifically binds to the domain of the protein exposed on the infected cells be conjugated to the ricin A chain. Most preferably, ritin A
The chains are deglycosylated and prepared by recombinant methods. A suitable method for making ritin immunotoxicants is described in Vitetta et al., Science 238 , 1098.
(1987), which also fall within the scope of the present invention.

When using conjugates to kill infected human cells for in vitro diagnostics,
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 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.

Cytotoxic radiopharmaceutical formulations for treating infected cells include:
It 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, a fusogenic liposome is loaded with a cytotoxin and the resulting liposome is coated with an antibody that specifically binds to gp160.

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

The present invention is also directed to the use of these antibodies, which are natural forms for treating AIDS. For example, IgG2a and IgG3 mouse antibodies that bind to HIV-related cell surface antigens can be applied in vivo for AIDS treatment. In fact, gp16
Since 0 is present in infected monocytes and T-lymphocytes, the antibodies of interest and their therapeutic uses 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,
Textbook of Immunology, 2nd edition, Williams & Wilkins 2
18 (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 class
Serum complement can be activated when bound to a target cell that expresses a cognate antigen.

Generally, antibodies of the IgG2a and IgG3 subclasses, and, but not always, IgG1, are capable of mediating ADCC,
3, and antibodies of the IgG2a and IgM subclasses bind and activate serum complement. Complement activation generally requires that at least two IgG molecules bind in close proximity to the target cell. However, serum complement is activated even if only one IgM molecule binds.

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 targets 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.

Antibodies of any viral origin can be used as this embodiment in the present invention if they can bind to the gp160 epitope and activate complement or mediate ADCC. Monoclonal antibodies have the advantage of a continuous rich source. Indeed, infection is achieved by immunizing mice with gp160, establishing hybridomas producing antibodies to gp160, and then selecting hybridomas producing antibodies capable of lysing the infected cells in the presence of human complement. A panel of antibodies that can react with and lyse cells can be quickly established.

Therapeutic Use of the Antibodies of the Invention For therapeutic use in vivo, the antibodies of the invention are administered to a patient in a therapeutically effective amount [ie, an amount that restores T cell counts]. I do. Usually this is administered parenterally. The dosage and manner of administration will depend on the degree of infection, the characteristics of the particular immunotoxicant (if used), eg, 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 antibody is formulated in a unit dosage form for injection (solution, suspension, emulsion) 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. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. Such vehicles include:
Small amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives, may be included. Normal,
Antibodies are formulated containing such vehicles at a concentration of about 1 mg / ml to 10 mg / ml.

IgM antibodies are advantageous because their antigens are highly 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.

Complement activation in vivo has been shown to lead to a variety of biological effects, including the induction of inflammatory responses and macrophage activation [Uananue and Benacerraf, Textbook, of·
Textbook of Immunology, 2nd edition, Williams & Wilkins, p. 218 (19)
84)]. The increased vasodilation associated with this inflammation is
AIDS agents may increase the effect of concentrating on infected cells. Thus, antigen-antibody combinations of the type identified according to the invention can be used for many therapeutic applications. Further, purified antigens related to such antigens [Hakomori, Ann, Rev. Immunol. 2 , 103 (198
4)] or anti-idiotype antibodies [Nepom et al., Proc. Natl. Acad. Sci. 81 , 2864 (1985): Koprowski et al., Proc. Natl. Acad. Sci. 81 , 216 (198).
4)] could be used to induce 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 is useful for diagnosing HIV in a test sample. It is used as one axis in a sandwich assay to assay for HIVenv, along with polyclonal or monoclonal antibodies to other sterically free epitopes of HIVenv. For use in some specific sandwich assays, 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. Other examples of labeled antibodies include, for example, 5C
Using a labeled sheep anti-mouse IgG capable of binding to the 2E5 antibody, by per se known procedures
Detect HIVenv binding.

Finally, in another embodiment, the antibodies are immobilized on a water-insoluble support and used for immunoaffinity purification of the HIVenv, rgp120 or TCB domains, as described in more detail below. 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 characterized by a humoral and / or cytotoxic cellular response to foreign or autologous target tissue that is undesirable in view of its therapeutic relevance. Usually, immune-enhancing inflammation
(1) Antibody production and / or response of cytotoxic T cells to antigens in undesired autoimmune diseases or in graft-versus-host disease, or (2) in transplanted tissues, such as organ grafts. It is characterized by. This is because polymorphonuclear neutrophils and mononuclear leukocytes invade target tissues,
It is clinically characterized by pain, concentrated edema, possible vascular endothelial damage, and extensive production of cytokines by stimulator cells.

An HIV env composition intended for therapeutic use will provide a good treatment, taking into account the disorder to be treated, the condition of the individual patient, the site of dispersion of the env polypeptide, the manner of administration, and other factors known to the practitioner. Should be formulated and the dosage established in a manner consistent with the procedure.

HIVenv is prepared for administration by mixing the desired moderately purified HIVenv with a physiologically acceptable carrier, ie, a carrier that is nontoxic to recipients at the dosages and concentrations employed. Usually, HIVenv 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. HIVenv for use in therapeutic administration must be sterile. This is easily accomplished by sterile filtration through a (0.2 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, HIVenv should be formulated and administered for site-specific distribution. This is RA
And in the case of inflammatory bowel disease. In the former case,
HIVenv can be injected into synovial fluid or synovial lining.
It is formulated into a sterile sustained-release composition suitable for ining) or implantation into a synovial capsule. For inflammatory bowel disease,
HIVenv is formulated into suppositories using pharmaceutically acceptable oily substances well known in the art.

The sustained release formulation is selected from microcapsule particles and implantable articles. Encapsulation in liposomes involves the transfer of lipids to the joints, so synovial space (synovi
al cavity) is not preferred. However, encapsulation in liposomes is suitable for implanting sustained release HIVenv in the synovial sac. To treat RA using a sustained release HIVenv, the HIVenv is preferably placed in a biodegradable matrix or microcapsule.
A suitable material for this purpose is polylactide
ide), but other polymers of poly (α-hydroxycarboxylic acid), such as poly-D-(−)-3-hydroxybutyric acid, can also be used [EP 133,988A]. Other biodegradable polymers include poly (lactones), poly (acetals), poly (orthoesters), or poly (orthocarbonates) and the like. Here, the first consideration is that the carrier itself or its degradation products are non-toxic to the target tissue,
And that they do not exacerbate the disease. This can be assayed by performing an extended release screen in an animal model of the target disorder, or if such a model is not available, in normal animals. For examples of sustained release compositions, see, e.g., U.S. Patent No. 3,773,919, EP58,
481A, U.S. Patent No. 3,887,699, EP 158,277A, Canadian Patent 1176565, "Biopolymers" by U. Sidman et al., 22: 547 (1983), and "Chem" by R. Langer et al. .Tech, 12:98 (1982).

The dose of HIVenv to be used depends on the T4 binding affinity of the HIVenv 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 presence of anti-HIVenv antibody,
Whether HIVenv can be used to prevent disease, or the occurrence of acute autoimmune disease or transplant rejection (episodes)
Depends on many factors, including whether it can be used to treat As a general idea, the HIVenv dose to reach the target site should be about 0.5 × 10 ⁻⁸ M to 5 × 10 ⁻⁹ M. Usually, the 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 up to 50 ml at the knee joint, for example,
The exact amount of Venv is determined by the dilution in synovial fluid, as well as the empirically measured loss of HIVenv for endogenous proteases, leakage into the systemic circulation, and any anti-HIVenv present.
Is determined by the sequestration by 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 HIVenvT4 binding assay and to evaluate the efficacy of the initial dose protocol. It is.

HIVenv therapy includes other anti-inflammatory substances such as salicylates, non-steroidal anti-inflammatory drugs, penicillamine, gold salts, TGF
-Β, TNF-α or TNF-β antagonists (U.S. Pat.
SSN898,272), 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 may be formulated with HIVenv.

HIV env concentrations concentrated at sites of inflammation may exceed the highest therapeutic dose systemically. HIVe in inflammatory invasion
Assaying the nv concentration can provide guidance as to the amount of HIVenv for intravenous infusion, especially where intensive dosing is impractical. 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 required
The dosage of HIVenv preparations with low T4 binding capacity and / or low affinity for T4 increases, but the dosage of high capacity and high affinity preparations also increases. Also, when treating patients with acute rejection or episodes of inflammation, i.e., acute organ transplant rejection or arthritis flare, relatively high doses are required initially.

For emergency treatment following the onset of acute rejection, HIVenv is injected intravenously or directly at the site of inflammatory injury when symptoms develop or rejection becomes serologically apparent. Alternatively, appropriate prevention can be 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.

Example 1 The onset 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, 15, 23-
26). More specifically, McDoug et al.
al et al.) have shown that HIV can selectively bind to the T4 epitope of the T4 antigen complex (25). In addition, many of the impaired immunological functions of patients infected with the AIDS virus were associated with the selective ability of HIV to infect helper cell populations and eventually destroy them (1,
2, 9, 10). It has also been suggested that PBMCs from AIDS patients are less responsive to mitogen and antigen (particularly tetanus toxoid) stimulation, probably due to reduced helper cell function associated with HIV (12). This example is mainly concerned with measuring the activity of rgp120 in the tetanus toxoid response in normal PBMC.

Materials and Methods Recombinant HIV envelope glycoprotein (rgp120): HIV
Chinese hamster ovary cells transfected with the envelope gene expression plasmid (20)
It was used for the preparation of rgp120 used in these examples. Rgp120 was purified from the conditioned cell culture medium by immunoaffinity chromatography. This rgp120 is SDS-
It was> 98% pure as determined by 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 and add Ficoll-
Layered on a Ficoll-Hypaque gradient (sp.gr.1.08) and centrifuged at 400 × g for 40 minutes at room temperature. Remove PBMCs at the plasma-Ficoll interface and use Hank's Balanced Salt Solution (HBSS) [Gibco; Grand Island Biological C
o., Grand Island, NY)], resuspended in complete medium and counted. Cell viability as measured by trypan blue exclusion was greater than 95%.

Culture medium: RPMI 1640 medium was purchased from Gibco and used. This includes 10% heat-inactivated fetal bovine serum [Hyclone, Logan, Utah], 2 mM L-glutamine, 10 mM HEPES, penicillin / streptomycin (Gibco), and 5 × 10 ⁻⁵ M 2-Mercaptoethanol [Sigma (Sigma, St. Louis, MO)] was added. 2
-The same medium without 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 was supplemented with 10% autologous plasma, 2 mM L-glutamine and antibiotics.
A 1: 300 final dilution of TT [Dr. A. Muchmore, National C
ancer Institute, Bethesda, the presence of available from MD)] or absence, were cultured PBMC fourfold such that medium 10 ⁵ cells 200μ per well microtiter plates. Replicate cultures containing various concentrations of rgp120 were checked in parallel. After 6 days of incubation,
When the response of the peak appeared, the culture ^[3 H] - were pulsed with thymidine, processed and counted as described above.

Staining of cells with monoclonal antibodies: OKT3, OKT4, OKT4A and OKT8 conjugated to fluorescent isothiocyanate (FITC) were conjugated to Ortho Diagnostic Systems, Inc.
Systems, Inc. Raritan, NJ). OKT3, a part of cells (10 ⁶ / 50μ) conjugated with FITC,
OKT4, OKT4A, OKT8 or murine IgG2ab control (5μ)
And mixed. After incubation on ice for 40 minutes, the cells were washed twice with medium, fixed with 2% paraformaldehyde and the samples were run on a fluorescence-activated cell sorter [FACS IV, Becton, equipped with a 488 nm wavelength, 4 watt argon laser.・ Dickinson (Becotn Dickinson, Mt.View, C
A]. 10 ⁴ cells were obtained in the indicated format and measurements were made on a single cell basis and represented by a frequency histogram (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 design and analysis of variance for multiple group comparisons.

RESULTS: The effect of rg on PBMC responses to soluble specific antigens.
The effect of p120 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 normal PB
Rgp120 proliferation of PBMC in response to specific antigens in MC
Can be significantly (p = 0.05) inhibited. The degree of inhibition was in the range of 19-51% of the control value and was different in the five experiments. No inhibition was observed in experiment No. 6 (not shown).

* Quadruplicate cultures of PBMCs were incubated with TT only (control) or
10 ⁵ cells / 200 in the presence of 5 μg rgp120 / ml (+ rgp120)
Measurements were taken on a μ / well flat bottom microtiter plate. After incubation for 6 days, the cultures ^[3 H] - were pulsed with thymidine, collected, processed, and counted. Each data is average
Represents cpm and% inhibition was calculated as follows; 1- (rgp
Cpm in presence of 120 / cpm of control) × 100; p <0.05.

Interaction of rgp120 with various T-cell populations: T by HIV
-It has been suggested that cell infection begins with a selective interaction of the viral envelope glycoprotein with the T4 component of the T-helper membrane receptor complex (1-6, 1
5, 23-26). Therefore, it was of interest to determine whether rgp120 has the same selective ability to bind to human lymphoid cells. PBMC at 4 ° C. and 37 ° C. for 24 hours in the presence or absence of rgp120 (5 μg / ml)
Was incubated. The cells were washed and stained directly with FITC-conjugated OKT4 and OKT4A. FACS
Analysis showed that treatment of cells with rgp120 at 4 ° C. was OKT4
Does not inhibit the binding of. However, at 37 ° C rgp120
Treatment resulted in a change in OKT4 binding indicating a decrease in T4 + cell fluorescence. Conversely, there was no binding of OKT4A after incubating cells with rgp120 at 37 ° C. These results indicate that rgp120 is similar to HIV (2
5, 26), indicating that the T4 helper membrane receptor can occupy the T4A epitope or adjacent epitopes.

Selective binding of rgp120 to the T4A epitope
This was further investigated in a series of experiments involving treating cells with high concentrations of rgp120 (100 μg / ml) for 4 hours. The cells were then washed and FITC-conjugated OKT3, OKT4, OK
Stained with T4A and OKT8. The result of FACS analysis is PBMC
Is pretreated with rgp120, T-helper membrane surface antigen T4A
It was shown to block the epitope completely. The binding of OKT3 and OKT8 to rgp120-treated cells was similar to that of untreated cell preparations, even at high concentrations.
It was shown not to bind to T3 or T8 membrane surface antigen. rg
It is interesting that p120 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 change in the membrane structure of the T-helper receptor.

Example 2 Anti-TCB domain monoclonal antibody The present invention describes a method for obtaining monoclonal antibody 5C2E5. Female Balb / c mice were immunized with about 30 μg of 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 monoclonal antibody 5C2E5 were splenocytes from the rgp120-immunized animals described above and mouse myeloma cell line NP ₃ x63-Ag8.653.
And obtained by the conventional method of Oi et al. (37). This cell line is widely and generally available. Plate fused cell cultures in 60-well plates
Transferred to 10. About 480 wells (80%) contained live cells. Rgp120 by radioimmunoprecipitation or ELISA
Each well was screened for antibodies to
This ELISA blocked nonspecific binding sites by (a) incubating rgp120-coated microtiter wells at 1% (weight / volume) for 30 minutes at room temperature.
Conventional double antibody sandwich assay using rgp120 coated wells and (b) goat anti-mouse IgG labeled with horseradish peroxidase. Ten of the 480 wells were positive for the rgp120 antibody. These 10 wells were then screened for their ability to block rgp120 binding to human T4 helper cells. The blocking assay was performed as follows: The in vitro culture supernatant of ascites fluid, or the purified ascites preparation or supernatant, is screened by conventional assays. For example, this purification method consists of adsorbing to a column of insolubilized staphylococcal protein A and then eluting the antibody. Serial dilutions (undiluted 未 1: 1000) of the monoclonal antibody preparation 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 80 in phosphate buffered saline e) 0.06 M NaCl f) 0.25 mg / ml BSA (bovine serum albumin) g) 12.5 mM Hepes buffer pH 7.4 in polystyrene tubes and radiation iodinated rgp120 ^(- 100,000
cpm) assay buffer solution (50-100μ) and 3-5 ×
10 ⁵ CHO (Chinese Hamster Ovary) Cell Line SV
E OKT4 (clone 17) was mixed with assay buffer (100μ) and incubated at 4 ° C for 1 hour. The cells were pelleted by centrifugation, the supernatant was aspirated, the pellet was resuspended in assay buffer (1.0 ml), repelleted, aspirated, and the pellet was counted on a gamma counter.

This CHO cell line harbors the gene encoding the human T4 receptor under the control of the SV-40 early promoter.
Transformation placed in a vector encoding DHFR, transfected into CHO cells, and amplified in 500 nM methotrexate to amplify its T4 receptor gene and increase expression on transformed cell surfaces CHO cell line. 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, which contain high amounts of T4, give the assay high sensitivity.

It is important to note and use the radioiodinated rgp120. According to some other conventional methods, ¹²⁵ I-rgp120 having a binding property that renders the tracer unusable in this assay.
It turned out to be. The successful method used was as follows: 1 mg / 400 μ rgp120 (400 μ) in 1 mCi
¹²⁵ I, 0.35 mg / ml lactoperoxidase (20μ)
And 1 mM H ₂ O ₂ (20 μ), incubate at room temperature for 10 minutes, and add 1 mM β-mercaptoethanol (20 μM).
μ) was added, the reaction mixture was incubated at room temperature for 2 minutes, 0.5% w / v BSA (50μ) was added, and the reaction mixture was added to a PBS buffer containing 0.5% w / v BSA (PBS).
(BS-BSA) to 2.5 ml, applied to a G-25 column and eluted with PBS-BSA (3.5 ml). ¹²⁵ I-rgp120 was recovered in the eluate.

The only wells producing blocking antibodies cells (5C ₂₎ in the sub-clones. The same inhibitory activity article was obtained from the wells E _5. This active substance resulting from IgG was purified to a desired form by ammonium sulfate precipitation or the like. This 5C2E5
Monoclonal antibodies show a binding number of approximately 10-15
% Showed the greatest inhibition of ¹²⁵ I-rgp120T4 receptor binding. This 5C2E5 hybridoma is generally available (ATCC HB9435).

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

Transfected with pAIDSenvTrDHFR, EP 187,
Rgp120 used in this example was purified from the culture solution of CHO cells cultured as described in 041. The rgp120 was purified by immunoaffinity chromatography to near homogeneity. This purified rgp120 was purified by adding 1 g of the purified rgp120 in 0.25 M glacial acetic acid under reduced pressure oxygen atmosphere according to the method of Ingram (35).
Hydrolysis at 10 ° C. for 18 hours. Under these conditions, rgp1
The backbone of the 20 protein is truncated on both the N and C termini of aspartyl residues. The digested rgp120 was neutralized by adding Tris buffer, and the pH was adjusted to 7.

The digested rgp120 was then applied to a column of the murine monoclonal antibody 5C2E5 covalently linked to aldehyde-silica by conventional methods (36). The column was washed with a saline solution buffered to pH3. Monoclonal antibody 5C2E
5 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: The conditions under which the analysis was performed hindered accurate measurement of C and W. The eluate from the 5C2E5 column was also analyzed by N-terminal sequencing to yield only the following sequence: _I_LP_RIK
QF.

The results of this amino acid analysis and N-terminal sequencing indicate that only the peptide in the acetic acid digest of rgp120 bound to the 5C2E5 column is a 44 amino acid fragment from threonyl residue 411 to arginine residue 454 of the HIV env sequence. confirmed. This region has the following sequence: TIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSN ITGLLLTR. This peptide binds rgp1 to the T4 surface antigen of T4 + helper T cells.
A murine monoclonal antibody 5C2E that blocks binding of 20
It contains 5 epitopes and the sequence deleted in the Dalta 3 mutant described above, thus concluding that this peptide is part of the TCB domain.

Example 4 Immunotoxicants Independently or as a cocktail, coupled to deglycosylated ricin A chain (dgA) and used to kill cells infected with HIV from a wide variety of viral subtypes. A monoclonal antibody directed against an HIV-encoded protein can be obtained as follows: Heat treatment from HIV-infected human Hp (CD4 ⁺ ) cells (30 sec /
60 ゜) Serum is obtained from mice immunized with NP-40 lysate (multiple intraperitoneal injection). In addition, serum can be obtained from mice immunized with gp120 or gp150. Protein gp15
0 is the extracellular domain of gp160, with the region cleaved off to obtain gp120 and gp41. All sera are tested in the following assays: 1. Indirect immunotoxicants (IT) using HIV-infected, uninfected, or gp120-coated uninfected H9 cells
Assay 2. Radioimmunoassay (RIA)-Positive serum
Tests against purified gp120 and gp150 determine whether mice have produced 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 mice are boosted with HIV-H9, gp120 ± gp150 lysate. Sacrifice a few mice,
The spleen was fused with Sp2 / 0 or NP3X63-Ag8.653 myeloma cells. Supernatants (SNs) from wells containing growing hybridomas were (a) H9 vs. (B) HIV-H9 was tested by an indirect IT test. Positive in (b),
Hybridomas 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 was directly coupled to dgA. These IT-dgAs were used to transfer cells from AIDS patients and HIV
Tested on infected cells. Then, clinical IT was prepared.

Litin A chain antibody conjugate (1) Preparation of antibody F (ab ') ₂ and FAB' fragments Antibody preparations were prepared under the following conditions using pepsin (4500 U / ml)
(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: 2/100 (weight ratio). Terminate the digestion by raising the pH to 8.0 with 1N NaOH. Isolation of the F (ab ') fragment is performed by gel filtration on Sephacryl S-200HR (Pharmacia, Piscataway, NJ) equilibrated with 0.3 M NaCl phosphate buffer or 0.1 M citrate buffer (pH 3. The unneutralized digest is adsorbed onto the SP-Sephadex column (10 × 2 cm) equilibrated in step 7), and phosphate buffered saline (PBS, p
H7.2) by eluting the F (ab ') ₂ fragment. The yield of F (ab ') ₂ fragments is generally 35-50%.

The Fab 'fragment was obtained by dividing the F (ab') ₂ fragment obtained above
Ethylenediaminetetraacetic acid (disodium salt) (EDT
A) It can be obtained by treatment with dithiothreitol (DTT) having a final concentration of 5 mM in a 0.1 M phosphate buffer (pH 7.5) containing (PBE) 0.003 M for 1 hour at room temperature for reduction. Excess DTT is Sephadex G-25
The gel can be removed by gel filtration above and the Fab 'fragment (5 mg / m
l) The thiol group is described in Fulton et al. [Fulton et al.
J. Immunol., 136 , 3103-3109 (1986)] and an Ellman 'reagent having a final concentration of 2 mM.
(5,5'-dithio-bis (2-nitrobenzoic acid)) (DTN
B) can be induced. Unreacted DTNB can be removed by gel filtration on a Sephadex G-25 column (30 × 2 cm) equilibrated with PBE. The purity of the F (ab ') ₂ fragment and the Fab' fragment is measured by SDS-PAGE and binary diffusion and immunoelectrophoresis using anti-mouse IgG serum that does not react with both fragments of Fab 'and Fc. This preparation does not contain Fc fragments and intact IgG.

(2) Litin A chain The deglycosylated A chain (dgA) was obtained by the method of Thorpe et al., Eur. J. Biochem., 147 , 197-206 (198).
5)] Prepare according to the method described. The LD ₅₀ values of native and deglycosylated A chain per 25 g of mouse are about
0.7 mg and 0.3 mg. The IC ₅₀ values in the cell-free rabbit reticulocyte assay were determined for native and deglycosylated A
The chain has about ^10-11 to ^10-12 mol.

For binding to the antibody, the A chain is reduced with 5 mM DDT as described by Fulton et al. (Supra).

(3) Preparation of IT-As by SPDP Using IgG or F (ab ') ₂ fragment of an antibody, IT-As is prepared according to the method described in Fulton et al. PB
Solution of IgG or F (ab ') ₂ fragment in E (pH 7.5) (10m
g / ml) with SPDP dissolved in dimethylformamide to a final concentration of 1 mM. After 30 minutes at room temperature, the above solution was equilibrated with PBE and Sephadex G-25.
Filter through a column (30 × 2 cm). Induced IgG or F
The substitution ratio of the (ab ') ₂ fragment is about 3 to 4 PDP / protein molecules. The induced protein was then mixed with 1.3 mg / ml of reduced A chain dissolved in PBE and
Leave at ℃ overnight. This mixture is then purified.

(4) Preparation of Fab'As by DTNB Preparation of mouse Fac'-A by Fab 'induced by DTNB is performed on rabbit Fab'-A according to the method described in Fulton et al. Fas derived with Ellman's reagent containing one or two TNB-substituted thiol groups
The b 'fragment (see Preparation of Fab' fragment above) was dissolved in PBE (5
mg / ml) and this is mixed with the reduced A chain (1.3 mg A chain / mg Fab ') at room temperature to a final concentration of 2 mg protein / ml.
The reaction between TNB-Fab 'and the A chain is followed by an increase in absorbance at 412 nm and is completed in about 2 hours at 25 ° C. This mixture is prepared immediately.

(5) Purification of IT-As IT-As prepared with a complete antibody or a fragment thereof was purified from Knowles and Thorpe [Knowles and Thorpe, Anal.
iochem. 160 , 440-443 (1987)] with a slight modification of affinity chromatography on blue sepharose. Chromatography is performed in 0.05 M phosphate buffer (pH 7.0), and the A-chain and IT-As are eluted with 1 M NaCl prepared in the same buffer. The eluate was concentrated to 5 mg / ml by ultracentrifugation, and phosphate buffer 0.3 M
Flow through Sephacryl S-200HR equilibrated with NaCl (pH 7.2). 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) 2% papain solution the Fab fragment of Fab'-GAMIg-A Preparation and indirect immunotoxicity assay affinity purified goat anti-mouse immunoglobulin (GAMIg), in 0.1 M NaPO ₄ containing 0.1 M EDTA and cysteine It is prepared by papain digestion using By affinity chromatography on equilibrated with 0.01M NaPO ₄ (pH7.6) SEAE- Sephacel to separate the Fab fragments from the Fc fragment. Fab
The fragment is desalted on Sephadex G-25. Then reduction
The Fab 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. 261 , 5314 (198
6)] A 5 mM litin A chain prepared and tested according to the method described above.
Reduce with DTT. The A chain is desalted on Sephadex G-25 and conjugated to Fab-GAMIg-E. Then Fab-GAMIg
-A is purified by affinity chromatography on S200 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 a serial dilution of the first unbound antibody at a concentration in the range of 2 × 10 ^{−8 to} 2 × 10 ⁻¹³ M in a suitable medium and add 100 μl
Aliquots are plated in triplicate in 96-well plates. 10 ⁵ H9 with a volume of 100μ in the same medium or
HIV-H9 cells were added to each well and the plate was incubated at 4 ° C for 1 hour.
Incubate for hours. Then, Fab-GAMIg-A is added to a suitable plate at a final concentration of 1 μg / ml. Control groups included (a) untreated cells, (b) cells treated with Fab-GAMIg-A alone, (c) cells treated with antibody only, and (d) Fab-GAMIg (A chain) after treatment with antibody. Not included)
Includes cells treated with Then place the plate in 5% CO ₂
Incubate at 37 ° C for 36 hours. Cells are incubated at 37 ° C for 4
Treat with ³ H-thymidine for ８8 hours, then collect on glass fiber filters using a Titertek automatic collector. ³ H- thymidine incorporation to measure a liquid scintillation counting LKB beta counter. Results show% of ³ H-thymidine taken up by untreated cells.
Expressed by In all the controls cultured cells 80% to 100% of ³ H- thymidine by untreated cells is captured.

(8) the expected ability of monoclonal antibodies in the data analysis the indirect IT assay is based on the concentration of antibodies capable of killing 50% of the target cells (used at optimal concentrations Fab-GAMIg-A) (IC 50). For IT-As to be useful in vivo, the IC ₅₀ is 10
Must be between ^{-12 and} 10 ^-10 .

IC ₅₀ is ranked as follows:

10 ^-11 M = 4 + 10 ^-10 M = 3 + 10 ^-9 M = 2 + 10 ^-8 M = 1 + 4+ and 3+ Antibodies appear to be suitable as IT-As and are further tested in RIA against gp150 and gp120 .

(9) The wells of the RIA 96-well microtiter plate were
gp120 or gp150 in μPBS 5 μg / ml for 16 hours at 4 ° C.
Coating. The wells are decanted, washed 5 times with dH ₂ O, and blocked with 200 μl PBS-10% FCS for 24 hours at 4 ° C. Decant the wells are washed five times with dH ₂ O. Dilute serum or supernatant (SN) at 25 ° C. over 4 hours.
Except for the material, to the wells are washed five times with dH ₂ O. 10 ⁵ cpm GA
Add the MIg for 4-6 hours / 25 ° C. The sample is removed, the plate is washed 5 times with dH ₂ O, 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) polyclonal mouse serum was produced by immunizing BALB / c mice with lysates from HIV-H9 cells (11) Result monoclonal antibody _{(a) Rαgp41: IC 50 1} × 10 -8 M ( Affinity (Not purified) (b) Monoclonal antibody 7F11: +1 1F9: -5B9: -1D10: -5G9: -5C2: -6D8E9: -9F6:-Only one monoclonal antibody is slightly effective in the indirect assay Was only.

First, 50 BALB / c mice were immunized with a lysate prepared from 1 × 10 ⁸ H9 cells infected with HIV-I. 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 to gp120 (see FIG. 3).

As described above, the present invention has been described mainly with respect to the preferred embodiments. However, the present invention is not limited to the disclosed embodiments, and various modifications included in the scope of the appended claims may be made. And equivalents. The scope of the claims should be given the broadest interpretation as encompassing all such modifications and equivalents.

Reference 1. Barre-Sinoussi, F. et al., Science 220 : 868 (1983) 2. Popovic, M. et al., Science 224 : 497 (1984). 3. Gallo, RC et al., Science 224 : 500 (1984). 4. Schupbach, J. et al., Science 224 : 503 (1984). 5. Sarngadharan, MG et al., Science 224 : 506 (1984). 6.Fauci, AS et al., Annals.Intern.Medicine, 102 : 800 (198
Five). 7. Lane, HC et al., N. Engl. J. Med. 309 : 453 (1983). 8. Merray, HW et al., N. Engl. J. Med. 310 : 883 (1984). 9. Bowen, DL et al., Annals Intern. Med. 103 : 704 (1985). 10. Fauci, ASClin. Res. 32 : 491 (1984). 11. Rook, A, H. et al., J. Clin. Invest. 72 : 398 (1983). 12. Lane. HC et al., N. Eegl. J. Med. 313 : 79 (1985). 13.Ammann, AJ et al., Clin.Immunol.Immunopathol. 27 : 315.
(1983). 14. Stahl, RE et al., Am. J. Med. 73 : 171 (1982). 15. Vilmer. 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 (1985). 19. Cunningham-Rundles, S. et al., J. Clin. Immunol. 3 : 156
(1983). 20. Lasky, LA et al., Science 233 : 209 (1986). 21. Steinkamp, JA et al., Science 215 : 64 (1982). 23. Klatzmann, D. et al., Nature 312 : 767 (1984). 24. Dalgeish, AG et al., Nature 312 : 763 (1984). 25. McDougal. JS et al., Science 231 : 382 (1986). 26. McDougal, JS et al., J. Immunol. 135 : 3151 (1985). 32. Boulaimne, G. et al., Nature 312 : 643 (1984). 33. Perez, P. et al., Nature 316 : 354 (1985). 34. Modrow, S. et al., J. Virology 61 (2): 570 (1987). 35.Ingram, VMMethods in Enzymology VI Volume 831-848
(1963). 36.Roy, SK et al., Journal of Chomatography, 303 : 225-22.
8 (1984). 37. Oi, V. et al., In: Selected Methods in Cellular Immunol
ogy , B. Mischel et al., WJ Freeman Co., San Francisco, California, p.251 (1980). 38. Maddon, P. et al., Cell 47 : 333-348 (1986). 39. Dalgleish, A. et al., Abstract, International Conference o
n AIDS, Paris (1986). 40. Tersmette, A. et al., Abstract, International Conference o
n AIDS, Paris (1986).

────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (72) Inventor Timothy J. Gregory Pine Hill Road 414, Hillsborough, CA 94010, USA (72) Inventor Lawrence A. Ruskey, CA 94965, USA, Sausalito, Star Route Box No. 40 (72) Inventor Gerald Earl Nakamura, CA 94116, USA, San Francisco, Twenty-Nineth Avenue 1923 (72) Inventor Eric Jay Patzer, CA 94563, U.S.A., No. 470, Tahoes Road, Olinda (72) John S. Patton Inventor, California Lunia 94070, San Carlos, Emerald Avenue 330 (72) Inventor Ellen S. Vittetta Texas, United States 75230, Dallas, Pemberton Drive 6914

Claims (2)

(57) [Claims] 1. A human immunodeficiency virus antigen comprising a variant of a human immunodeficiency virus envelope selected from gp120 and gp160 that is incapable of binding to a T4 receptor of T4 helper lymphocytes, wherein the variant is a T4 helper lymphocyte. An antigen in which amino acid residues in the TCB domain have been substituted or deleted so that they cannot bind to the T4 receptor of lymphocytes. 2. The antigen according to claim 1, wherein the human immunodeficiency virus envelope is obtained from strain 3B and has amino acid residues 424-435 deleted.