WO1992015326A1 - Targeting complex mediated immunogenicity - Google Patents

Abstract

A method for targeting the body's immune system to pathological organisms, tissues and other undesirable materials is provided by use of an inventive targeting complex. It is particularly applicable to the treatment of HIV infections. The complex is made of a targeting element which binds to an undesirable material. The targeting element is attached to a recognition element which allows the body to identify the targeted material as harmful. The body then neutralizes the material. When necessary to avoid steric hindrance, a spacer element is used between the targeting element and the recognition element. This feature allows ambient immunoglobulins to attach to the recognition element.

Description

TARGETING COMPLEX MEDIATED IMMUNOGENICITY

This invention was made with U.S. Government support under Department of Energy Contract DE-AC03-76F00098. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention is related to the targeting of the body's immune system to pathological organisms and tissues, and other undesirable materials.

Throughout medical history, great efforts have been made to combat human and animal diseases. In the last two hundred years, two different approaches have been taken towards this end. Pioneered by the work of Louise Pasteur, scientists strove to develop or increase the body's natural immunity to such diseases. Additionally, researchers attempted to identify and develop medications which would attack pathogenic organisms while being of a comparatively low toxicity to the patient's tissues.

VIRAL PATHOGENS

Considerable difficulties have been encountered in the treatment of viral infections caused by pathogens for which no vaccine has been developed. While antibiotics have been developed which are toxic to bacterial pathogens, very few "magic bullets" have been developed to combat viruses. Interferon, which at one time held great pro ise as an anti-viral agent, has very limited clinical applications due to its high cost, variable action and toxicity.

For lack of a pharmaceutical arsenal with which to defend the body against viruses, doctors are often limited to treating viral disease symptomatically or treating secondary infections. Even this limited treatment regimen can in some cases save many patients. Examples of diseases which can be effectively treated in this manner are viral pneumonia and pertussis. Much of the pre- antibiotic death rate among patients infected with these and similar viral diseases was due to secondary bacterial infection.

The devastation caused by other viral diseases can not be circumvented through treatment of secondary effects.

Despite the availability of antibiotics, polio exacted a horrific toll prior to the development of the Salk vaccine. Other viral diseases such as viral meningitis and hepatitis continue to cause death and debilitation for lack of an effective treatment.

During influenza epidemics, there is an expected loss of life among infants, young children, the elderly, and patients with complicating medical problems. Such epidemics often repeat themselves in relatively short periods of time. This is due to pathogens having, at various generational stages, a constantly changing character with respect to their external glycoproteins. Because the body's immune system must identify such proteins before an effective defense can be mounted against the pathogens, such diversionary adaptions stymie the body's defense systems and allow sequential infections by essentially the same pathogen. Vaccination of vulnerable individuals against each variant of the flu viruses has proven only partially effective. CANCER AND AIDS

Of particular concern to the medical community are pathogens or cancer cells to which the body does not develop immunity even upon prolonged exposure to the offending agent. In the case of the AIDS virus and certain bone marrow cancers and leukemias, the disease may actually directly compromise the immune system. This further limits the body's ability to cope with the disease.

Malignant tissues regularly escape detection by the immune system by maintaining membrane surfaces which appear immunologically identical to the patient's. Other mammalian species can produce antibodies to the changed factors on human cancer cells. Ironically, the use of these targeting molecules is limited to the identification and monitoring of the cancerous tissue as it spreads throughout the body. Binding toxins to these antibodies to provide treatment has proven clinically disappointing.

Antibodies to certain factors which specifically target human cancer cells can be produced in other animals and by using hybridoma technology. The carcinoembryonic antigen (CEA) is produced by human bowel cancer cells even after these cells metastasize to other parts of the body. Exogenous antibodies are often used for monitoring the progress of the disease.

The human bowel produces a very similar factor to CEA during its fetal development. As a result, the human immune system views these identifying materials as "self", and will not attack the cancer cells despite the presence of CEA. This is typical of human cancers. The cells revert to the unspecialized predecessor forms and multiply without the inhibition typical of mature cells. IMMUNOLOGICAL THERAPIES

As a result of the limitations of specific vaccines and other medical techniques, clinicians have resorted to general immunotherapy and more specific immunotherapy techniques in combating human disease. These efforts have met with only limited success in a few isolated treatment circumstances.

?aτnma-Globulin. From the 1940's to the present day, the Gamma-globulin fraction of donor blood has been administered to patients infected or exposed to pathogens to which they lack immunity. The fraction is generally not specifically characterized, but is assumed to have some reasonable component of the desired protective antibodies.

Contraction of rubella by women during the first trimester of pregnancy puts their unborn children at great risk of developing severe birth defects. Prior to the development of the rubella vaccine, doctors would treat susceptible, pregnant women exposed to rubella with a Gamma-globulin enriched portion of donor blood in order to limit such problems. The hope was that there would be anti-viral antibodies in this blood fraction which would destroy the rubella pathogen prior to infection, or serve to lessen the severity of the disease should it be contracted.

Specific Immune Functions. In Rh disease, the mother builds up antibodies to her unborn child's blood group during pregnancy. These antibodies attack the unborn child, often causing severe disease and even death. In this case, donor anti-Rh antibodies are administered to the mother to circumvent the instigation of her immune reactions.

General Stimulation of Immune System. One cancer therapy technique employed by clinicians has been to "build" the immune system on an omnibus bases. This approach was pursued in the belief that such efforts would invigorate t\e defense mechanisms of the body. Tuberculoses antigens were often employed as the stimulating agent. This treatment effort was meant to help the body recognize the cancer cells as pathogenic to the body. Factors isolated from cancerous tissues removed from the patient to be treated during surgery were also used in this manner.

An object of the present invention is to provide a therapeutic targeting element-recognition element complex which allows the immune system to neutralize, destroy, detoxify or kill harmful pathogens.

Another object is to provide multi-pronged effective therapies for patients infected with the AIDS viruses.

Yet another object of the invention is to circumvent infection by the AIDS virus to exposed individuals by destruction of the viruses before they incorporate their genetic information into the host chromosome.

An additional object of the invention is to provide specific immunity to patients with highly depressed immune systems by redirecting general antibodies to specific target organisms and cells.

A further object of the invention is to destroy fetal cells or cell factors in the maternal bloodstream prior to her immune systems becoming sensitized to these materials. ^•

Another object of the present invention is to provide a therapy for viral, bacterial, and parasitic infections.

SUMMARY OF THE INVENTION

The present inventive method provides for the targeting of pathogens which the immune system is otherwise unable to identify using an inventive targeting complex. The inventive complex also allows existing humoral factors to clear away or otherwise neutralize a substance from the body while minimizing im unogenic sensitization to that substance.

A targeting element of inventive complex is bound to a recognition element easily identifiable as an appropriate target by the body's immune system. When the inventive targeting element binds to the target, this undesirable material is labeled with the recognition element, generally an antigenic agent.

The inventive targeting complex allows the body to locate and destroy many pathogens and other harmful materials to which the body's immune system heretofore was unable to respond, or able to mount only a limited response. The use of the inventive targeting complex exploits the general effectiveness of the body's immune system while avoiding the necessity of or complications due to direct sensitization.

TREATMENT OF AIDS

A particularly important application for the present invention is in the treatment of patients who have either been exposed to or infected with the virus responsible for AIDS (acquired immune deficiency syndrome) . This virus is known as the human immunodeficiency virus, or HIV. A well known effect of this disease is the substantial diminution of the body's capacity to mount an effective immune response to many diseases. However, a certain level of general antibody production remains intact even in full- blown cases of the disease.

The present inventive targeting complex allows for two separate treatment modes for this fatal disease. First, whatever antibody production survives the onslaught of the infection in an AIDS patient can be actively marshalled to attack HIV and/or HIV infected cells. Secondly, other life-threatening pathogenic agents that may or may not target immune system cells can be specifically targeted even with unrelated antibodies produced by the body naturally or with induction by various stimulation techniques.

Targeting HIV. Because HIV is continually produced during the course of the illness, its ongoing destruction by the body's immune system is of prime importance in therapy. Ironically, the effect of the disease is to radically limit the protective immunogenic response. The insidious cycle of HIV infection can be diminished or broken using the method of the present invention.

HIV employs a recognition site on its surface, gpl20.

This site is used by the virus to identify and bind to its victim cell's surface. HIV then enters the cell and incorporates its genetic information into the host cell chromosomes. This recognition site on gpl20 may be the only identifying protein which is produced consistently on the surface of the virus. Unfortunately, it is so situated that this site does not elicit an immune response from the body. It also is so protected that it can not be directly bound by antibodies. [See Landau, Nature, Vol. 334, pp. 159-162, 1988.]

The victim cell surface protein to which gpl20 binds has been isolated and is termed CD4. In its free form, CD4 (soluble CD4, sCD4) is bound very effectively by HIV on its gpl20 recognition site. One potential clinical application of this phenomena is to use free CD4 to bind up the attachment sites on HIV and so thwart its ability to attach to and infect target cells. [See Capor, et al.. Nature. Vol. 337, 525-531, 1989; Traunecker, et al., Nature, Vol. 339, p. 68-70, 1989.] Some successes at limiting the infectivity of other viruses had previously been obtained using similar techniques. [See Rossman, Proc. Natl. Acad. Sci. USA. Vol. 85, pp. 4625-27, 1988.]

The ability of CD4 to bind to HIV has been postulated as being the bases for a new AIDS therapy wherein CD4 is conjugated to toxins in order to target these toxins to HIV. Pseudomonas exotoxin A bound to CD4 has been produced. This complex was found to suppress protein production in HIV infected cells in vitro. [Chaudhary, et al.. Nature. Vol. 335, pp. 369-372, 1988.] There are concerns that the hybrid toxin will interfere with certain immunogenic functioning (ibid, page 370) .

Researchers have targeted HIV infected cells with CD4 bound to the toxin ricin which inhibits protein synthesis. The infected cells were killed _in vitro by this complex while two other cell lines were much less effected.

However, the mass infusion of cytotoxiσ materials into the human system is problematic. The clinical usefulness of this approach must await the results of clinical trials.

Capon et al. have developed CD4 immunoadhesins which consist of CD4 bound to a portion of antibodies, that is the Fc portion [Nature, Vol. 332, pp. 525-31, 1989]. The Fc portion of an antibody instigates the effector functions of the immune system including the complement pathway, when the antibody binds to a target. The CD4 portion of the immunoadhesin serves in place of the FAB region of natural antibodies, which is responsible for binding an antibody to its target. The large globular immunoadhesin of Capon thus represents an artificial antibody. Because the Fc region is a large, complex foreign glycoprotein, sensitization to this material can be a problem during the treatment period. Additionally, such materials can be difficult to administer, and require careful handling to avoid inactivation. The testing of this material is currently confined to diminishing HIV titres in the blood of pregnant HIV positive women in the hopes of limiting the chances of the infection spreading to their unborn child.

Traunecker et al. take an approach similar to Capon [Nature, Vol. 339, pp. 68-70, 1989]. CD4 is produced bound to an immunoglobulin constant region of mouse IgM. Multivalency was provided by this particular production technique. Again, this approach is an effort to build artificial immunoglobulin molecules to supplement those naturally occurring in the body.

The present inventive targeting complex takes a fundamentally different approach to the challenges of HIV than does Capon or Traunecker. In the present scheme, a therapeutic agent is crafted which exploits, rather than substitutes for, the existing immunogenic strengths of the patient. The material can be easily produced, as it does not require a complex Fc portion, and is highly stable. It is also much smaller than artificial antibodies, and thus provides ease of storage and administration. The present invention exploits the highly specific binding of CD4 to the AIDS virus in an entirely original and unexpected manner. Any material which can bind gpl20 is useful in the present invention, such as rCD4, VIP and peptide T, among others. A material which is easily recognized by the body as foreign is bound to CD4 or comparable material through a spacer molecule. This material is termed the recognition element. The CD4 protein of the targeting complex is termed the targeting element. When the CD4 element of the targeting complex attaches to HIV or HIV infected cells (these cells have gpl20 on their surface) the body then recognizes the virus through the recognition element as a foreign invader. The immune system of the body is then brought into play, and destroys HIV and HIV infected cells by any of several possible mechanisms. The present inventive complex differs from prior art materials in a number of ways. As explained above, researchers have bound CD4 to toxins in order to identify and destroy offending infected cells which display gpl20 on their surfaces. This requires that the toxin be placed in as close a proximity as possible to the offending body. Researchers in this area also believe that the toxic materials may need to be assimilated into the infected cell to produce the desired weakening or death of that cell.

The present targeting element has a very different action from the directed toxin research. As a result, the inventive targeting complex will differ considerably from prior art constructs. A spacer element required in the HIV aspect of this invention is lacking in the prior art. Also, the toxins selected will have a different and often contradictory purpose from those employed in the prior art and differ from them accordingly.

The recognition element of the present invention can be drawn from a vast area of immunogenic materials. While these may include some bacterial toxins, recognition elements selected for this invention will differ considerably from those used in approaches requiring the cytotoxicity of the toxins to kill the cells directly. The toxicity of these materials is actually a detriment in the context of the subject invention.

Appropriate toxins for recognition elements in the present invention would be those of very limited cytotoxicity so that the ambient immune system cells will not be compromised. Antibody recognition is all that is required of these materials, as the body's own immune system provides the bases for the destruction of the pathogen. Chemotaxis is an additional property of the recognition element which is desirable. Because of steric hinderance, immunologic binding or recognition of the toxins bound to CD4 in prior art constructs would be compromised or destroyed once bound to the target. This is due to the steric hinderance innate in the HIV interaction of the subject invention.

In one aspect of the inventive method, dinitrobenzene (DNP) is attached to sCD4. DNP generally has an extremely high antigenic reactivity in humans in proportion to its size. Additionally, because anti-DNP antibodies are ubiquitously present in human populations, a universally useful targeting complex is produced even when a patient's immune system has been damaged.

The immunogenic reaction of the body to the DNP portion of the inventive targeting complex prior to its binding HIV is avoided using the present inventive approach. It is not until a number of the inventive targeting complexes have been bound to the various target sites on HIV that the recognition elements becomes multimeric. It is only in this state that the inventive complex elicits the immunogenic response of the body. This particular example of the use of the present invention in combating AIDS has been experimentally investigated and shown to initiate the complement cascade reaction of the body. (See Example 1 below.)

An arsenal of inventive immunogenic complexes can be made available to optimally utilize the strengths of any particular patient's immune system. For instance, if a patient has maintained a high immunity to rubella despite repression of immunity, a rubella antigenic substance can be used in place of DNP. The spacer molecule may need to be selected or modified with a view to avoiding steric hinderance when different moieties are substituted or added. The specific production of certain antibodies can be elicited more effectively than others in an ailing immune system. For instance, in AIDS patients there is typically a paradoxical polyclonal B cell activation, which means that high levels of generic IgG, IgA and IgM are produced. (See Sacerdote, et al., J. of Neurscience Research. Vol. 18, pp. 102-107, 1987.) This aberrational increase in production due to HIV pathology can be exploited by choosing a recognition element to which such immunologic fractions will bind. Similarly, because NK cell activity is diminished by AIDS (ibid, page 106) , one would not employ a recognition element which would rely exclusively on these cells for efficiency.

The body can also be stimulated to produce relevant antibodies with appropriate vaccines, remembering the remaining capacities of the compromised immune system. These antibodies can then be directed to the AIDS virus using an appropriately selected recognition element for the inventive targeting complex.

Targeting HIV Infected Cells. The methodology for targeting B cells and other cells infected with HIV is much the same as described in the section above for targeting HIV itself. However, it does differ in certain ways.

The major effective means the body has for eliminating cancerous or infected cells is through the implementation of cytotoxic or "killer" T cells. Unfortunately, the activity of these cells is mediated by helper T cells which are the first victims of the HIV infection. Therefore, the subject invention must exploit the pre¬ programmed killer T cells to bring about the destruction of the pathogenic B cells. Instigation of certain aspects of the classical complement cascade is also very important in bringing about the lysis of lipid membranes of infected cells. Bacterial or parasitic identifying factors may thus prove the best choice for the recognition element when configuring the targeting complex to mediate the destruction of affected cells.

It may be more difficult to achieve a multimeric state with a limited amount of targeting complex because of the high surface area of the infected cell as compared to HIV. When several of the inventive complexes bind to the cell surface, they tend to draw together. This is called the •capping' phenomena. Thus, a multimeric target can be provided to the immune system recognition factor.

Antibody levels of a patient may provide clues as to the recognition element most likely to be successful in a particular individual. Additionally, multimeric recognition elements can prove a good choice when that element has been pre-selected to induce killer T cell activation.

Targeting Opportunistic Diseases. Another challenge in the treatment of AIDS or any other immunodeficiency disease or induced immunodeficiency state is the inability of the body to respond to an infection or cancer within the necessary time and with the required vigor. Failure of the immune system to respond quickly or effectively can debilitate or even kill a patient. Conditions under which the immune system fails to respond effectively include AIDS, chemotherapy or radiation therapy induced deficiencies, inherited immunodeficiency, among others. The present invention could be used to eliminate the normal lag time necessary to 'educate' the immune system to target a particular infectious agent.

An inventive targeting complex is assembled with a ligand, complementary nuclei acid strand, or other targeting element which will specifically bind the offending agent or cells. The recognition element of the targeting complex can be pre-selected after evaluating the patient to determine which humoral antibodies are presently at the highest levels. A cocktail of inventive targeting complexes, with various recognition elements, can be used when a high antigenic response is necessary. The inventive targeting complexes, however tailored or configured, allow the body to bring into force many antibodies against a serious infection prior to, or in the absence of, its immune system recognition of the agent.

In the case of AIDS, a number of opportunistic diseases are characteristic of the diminished immunologic capacity brought on by the HIV infection. Among bacterial and fungus infections in these patients are Pneumocystic carinii Pneumonia, Toxoplasmosis, Candidiases, Cryptococcus, and Salmonella. Some characteristic neoplasms among AIDS patients are Kaposis sarcoma and Burketts lymphomas. Among the panoply of cancers characteristic of AIDS patients, they are unusual in their poor response to chemotherapeutic efforts.

TREATMENT OF OTHER DISEASES

A wide range of diseases can be treated by use of the inventive targeting complex. Many of the evasive strategies used by pathogens can be circumvented by treating a patient with the subject invention. Furthermore, patients with compromised immune systems can have their remaining immune capacity brought directly to bare on a pathogen by correct selection of appropriate elements for the subject invention. A specific cocktail of various targeting complexes can be administered which takes advantage.of the particular strengths of a patent*s immune system.

Non-HIV viruses. A large number of viruses avoid or even completely escape the defenses of the immune system using ever-changing surface proteins. Conserved proteins are kept away from the reach of the immune recognition system by structural impediments. These defensive tactics are reviewed in more detail in the following section. They follow a pattern very similar to that of HIV described above.

As with HIV, many of these viruses have receptor recognition sites tucked into valleys on their surface to avoid detection by relatively large antibodies. The inventive complex exploits the these highly conserved receptor sites, and uses these as binding sites for the targeting element. A spacer molecule is provided to avoid steric hinderance problems with the binding of antibodies to the recognition element. In this way, such viral infections can be treated by the inventive complex.

Other viruses stuck by mounting an infection more quickly that the body's immune system can respond. Small pox, polio, rubella, rabies, and chicken pox are examples of such viruses. Although the body may ultimately build an immunity to such disease, death or permanent debilitation often result before this can occur.

The inventive targeting complex for such diseases may not require spacer molecules. The purpose of the complex is to avoid infection when exposure is known to occur, or to keep the infection at bay until the body can mount an appropriate defense. Avoiding the disease entirely is particularly important in women carrying fetuses which can be badly damaged by the disease, such as by rubella.

Other diseases, such as leprosy and polio, advance during the progress of the disease to residing in the nerve cells, where they are resistant to attack by the immune system. Again, avoidance of the infection upon exposure to the pathogene is the prime goal in such cases.

Bacterial Pathogens. Another application of the subject invention is the treatment or avoidance of bacterial diseases. Tuberculoses, salmonella and streptococcus are examples of such diseases. As with some of the viruses described above, these diseases often cause disability or death before the body is able to provide an effective immune response to these organisms. For instance, strep, can cause heart defects in children if it progresses to rheumatic fever.

Parasitic and Other Diseases. Of particular concern to the world community is a number of parasitic diseases to which vaccines have not been developed. Examples are sleeping sickness, river blindness, toxoplasmosis, and amebic dysentery. The main difficulty with these organisms is that they are in a vulnerable stage of their development for too short a time for the body to mount an effective defense against them.

For instance, the malaria trypanosome is in the free blood for too short a time for resistance to be mounted against it. From that time onward, it lives within the blood cells, protected against recognition by the cell membrane.

Using the present invention, such apparently cursory vulnerable stages in pathogenesis can be exploited to cause their destruction. Any surface structure of the trypanosome can be targeted by a targeting element, which may be a small organic molecule, or even the pathogene receptor recognition cite. The patient is treated when exposure is suspected.

CLEANSING OF ANTIGENS FROM THE BODY

There are many clinical situations where it is deleterious for the body's immune response to be stimulated. These can include allergic reactions, autoimmune diseases, transplant rejection, and reaction of the mother's immune system against cell factors in her unborn child.

Maternal Sensitization to Fetal Cells. One case when antibody production is undesirable is when mothers show an antigenic response to factors in the blood of their unborn babies. A classic example is the Rh factor, but these problems can also occur with simpler interactions such as ABO incompatibility.

The present treatment for maternal reaction to fetal antigens is to treat the mother with exogenous antibodies. This binds up and eliminates the offending antigens before the mother's immune system is able to identify the antigens and mount a response. However, there are problems inherent in such a process. The antiserum must be handled with care in order to retain its potency. It must also be deliver in large quantities in order to assure that no antigen escapes to be detected by the maternal B cells. The antiserum is inherently bulky, and must be produced by a foreign organism. Additionally, there are always certain risks inherent in introducing foreign animal products into the blood stream.

The present invention allows for a much simplified method of cleansing the maternal blood stream of fetal antibodies. As with the prior art treatment methods, care must be taken to select materials which will not traverse the placental barrier. A ligand, in some cases a small molecule, is selected which will bind to the fetal antigens. Then, the maternal antibodies to an antigen unrelated to the target bind the antigen component of the inventive immunologic complex. In this way, ambient maternal antibodies continuously eliminate the fetal antigens. The inventive complex thus avoids the sensitization of the maternal immune system to fetal factors, while using that same system to eliminate these factors.

Allergies. The development of allergies to harmless materials such as house dust and pollen represents another situation in which mounting an immune response to a material is counterproductive. A more serious situation is when such reactions progress to anaphylaxes. This can happen to people who are highly sensitive to bee stings or other materials, and can result in death.

Present methods for treating such problems include emergency injections of atropine, desensitization of the immune system through sequential administrations of offending agents, and general treatment of symptoms. Unfortunately, extreme reactions such as acute asthma can lead to death even when the whole available panoply of drugs is brought to bear against the illness.

Treatment of allergic reactions by administration of the inventive immune complex avoids many of the complications and limitations of presently available treatments. The use of a targeting agent directed against the allergin serves to eliminate the offending material from the body prior to its provoking the immune response. In cases where the lungs are the main site of the irritation, the inventive complex may be preferentially administered as an inhalant. Transdermal continuous administration may be useful when the patient is chronically exposed to the irritating antigen.

Autoimmune Diseases. Autoimmune diseases are another case where the immune system acts to the disadvantage of the body's welfare. Examples of such diseases are arthritis and lupus. Treatment of such debilitating diseases is often limited to symptomatic medications, which, in many cases, is unsatisfactory in providing relief to the patient.

The present inventive complexes can provide a limited advantage in such a situation. Humoral antigenic factors can be bound up and eliminated using the scheme provided above. Depending on the nature of the antigen, this may well limit the reaction of the body to its own tissue and other factors. Transplant Rejection. Rejection of transplanted organs and tissues is a chronic problem in achieving success in transplant procedures. Such transplants can range from kidney and liver transplants to bone marrow transplants. Great pains must now be taken to match donor tissue factors as closely as possible with the recipient's factor profile. Many needy patients die in the face of available organs for lack of such a match. Even with such matching, suppression of the immune system of the patent receiving the organ is often required. This obviously compromises an already vulnerable individual's ability to resist infection.

As with the autoimmune disease, the inventive complex can play a role in limiting transplant rejection. Foreign humoral factors can be cleansed from the blood using a properly assembled inventive complex. The interaction with the transplanted organ itself is of concern. However, if the humoral antibodies are sufficiently cleared to avoid specific sensitization, such effects may be unusual.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a flow diagram of the general targeting strategy of the subject invention.

Fig. 2 is a flow diagram which shows the targeting strategy of the inventive complex directed to gpl20, produced by Example 1.

Fig. 3 is a flow diagram which shows the targeting strategy of the inventive complex directed to streptavidin produced by Example 2.

Fig. 4 is a flow diagram which shows an alternative targeting strategy of the inventive complex directed to streptavidin, produced by Example 4. Fig. 5 are three bar graphs showing the results of tests conducted on the materials produces in Examples 1, 2, and 3 as explained therein.

DETAILED DESCRIPTION OF THE INVENTION

The targeting complex of the present invention comprises a targeting element attached to a recognition element. Optionally, a spacer molecule is provided between these two components. The elements of the complex are selected and configured in order to cause the immune system of the patient to select a specific target for neutralization by destruction, agglutination, elimination, or detoxification upon administration of the therapeutic agent.

The general process by which the inventive complex targets unwanted materials is shown in Fig. 1. Inventive complex 1 is comprised of recognition element 3 bound to target element 5 either directly or optionally through a spacer element 11. Target organism 9 has a targeting site 7. When the inventive targeting complex 1 is in the vicinity of target organism 9, the targeting complex 1 attaches to or becomes associated with the target organism 9 through targeting site 7. At this point, humoral factors in the patient's blood, such as recognition element specific antibody 13, bind to the target organism 9 through the targeting complex 1.

TARGETING ELEMENT

The choice of the targeting element determines the target specificity of the inventive complex. The targeting element can come from many different sources, and can be any number of a wide variety of substances.

Full antibodies to the target or relevant small molecules can be utilized. Often, it is advisable to employ only a portion of an antibody because of steric hinderance and dosage scheme limitations. Also, when the immunoglobulin is derived from an animal source dissimilar to the patient, there is a chance of sensitization of the patient to this material. Small molecules are a good choice because of their comparative low cost, and long halflife. The entire targeting complex can be produced as a single step using some of these techniques. Yeast, bacteria, or mamunalian monoclonals are possible sources of such production.

Many materials physically or functionally related to naturally available targeting elements can often be employed in their place. (See Table I.) Any associating or attaching material can be employed, such as various homologous nucleic acids, adherent peptides, carbohydrates including polysaccarides, or lipids, and various combinations of such materials. For instance, sialic acid, which binds influenza viruses, is a carbohydrate, having the specific form of a trisaccharide. A number of serologically differing strains of viruses may be vulnerable to the same element because of the canyon effect. Therefore, these factors' usefulness in related strains should be considered. (See Rossman, J. of Biological Chemistry. Vol. 264, 145 pp. 87-90, 1989.)

TABLE I

Receptor Related Molecule Molecules

CD4 14 Vasoactive Intestinal Peptide⁷, Peptide T⁸, Sialic

Epidermal Growth

Factor¹

Rabies Acetylcholine receptor²

Epstein Barr Complement Receptor^3,4

Rheo Beta-adrenergic receptor⁵

Rhinovirus ICAM-1⁶'¹⁰'¹¹ N-CAM, myelin- associated glycoprotein MAb¹³

Polio viruses Polio virus receptor⁹ Influenza Sialic Acid¹⁵

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White, et al.. Cell, Vol. 56, pp. 725-728, 1989. Viral Recognition Sites. A particularly elegant iteration of the present invention allows the destruction of viruses to which the unaided immunogenic system cannot mount a defense. The inventive complex targets pathogens for destruction by using existing aspects of the immune system. It also exploits the specificity of a pathogen's attack system to target that pathogen.

Often the only stable surface site on such viral pathogens is a recognition region which the virus uses to attach to a factor on a victim cell. These factors often lie in a valley on the corrugated surface of the viruses in such a way that the globular antibodies of the immune system can not reach them. Additionally, it is difficult for the B cells to acquire these factors to display them to a helper T cell or to use as a basis to transform into a memory cell in order to achieve persistent immunity.

Many of the host cell surface factors to which viruses bind have been isolated. They are generally developed for academic or assay purposes. However, the sCD4 factor to which the AIDS virus binds has been used in experimental treatment systems to target toxins to these viruses, as is described above.

In the present invention, the host cell surface factors are used as the targeting segment in order to specifically attach the recognition moiety to the pathogen. This process targets the pathogen for destruction by the body's existing immune defense. Because of the surface factors in the case of AIDS, a spacer as described below is needed in order to assure that steric hinderance will not preclude the adhesion of either the entire complex, or the immune defense reaction once the complex is associated with HIV.

In Example 1 below, the CD4 receptor is used to attach the inventive complex to the gpl20 site which is a surface f ctor on AIDS viruses. Vasoactive intestinal peptide 1- 12 shares sufficient homology with CD4 to bind to the gpl20 site as well. (See Sacerdote, et al., J. of Neuroscience Research. Vol. 18, p. 102, 1987.) This as well as other related moieties can be employed in the subject invention. Other appropriate materials include Recombinant CD4 (rCD4) , soluble CD4 (sCD4) , Vasoactive Intestinal Polypeptide (VIP) and Peptide T.

A large number of other pathogenic viruses also bind a conserved surface factor to attachment sites on host cells. Some examples of these viruses are listed in Table 1. In combating any of these viruses, the recognition site on the host cell can be used as the targeting segment in the present invention. Of course, as with the CD4 antigen, any other molecule which is sufficiently analogous to the original targeting segment to bind the pathogen may also be employed in the system.

Targeting Elements for Newly Discovered Viruses. Using recently developed technology, targeting elements can be characterized quickly for newly isolated viruses. This is a very useful approach to control new diseases when a vaccine has not yet been developed. It is also a particularly advantageous therapy when the external proteins or the virus mutate regularly, but the recognition sites are conserved. Such a pattern of mutation necessitates sequential vaccinations, and newly mutated pathogens often evade even that level of vaccination effort.

Once the virus has been isolated, it is screened using a 'library' of 10⁶ - 10⁸ different protein fragments. The members of the library that bind the target are isolated and characterized. [Scott, et al. Science, Vol. 249, p. 386, 1990.] The factor can be produced synthetically or through cloning and employed directly in the subject invention. Other Viral Factors. Other materials which bind pathogens have also been developed separate from recognition considerations. These often are developed for diagnostic purposes. Examples are Influenza binding synthetic peptides (US Patent #4,981,782 issued to Judd, et al., issued 1/1/91) and HIV-1 detecting anti-HIV F(ab)j fragment (US Patent #4,983,529 to Stewart, et al., issued 1/8/91) . Targeting elements could also be identified through efforts in synthetic organic chemistry or from screening of fermentation broths.

SPACER ELEMENT

An aspect of the present invention is a spacer element. In a number of systems, its use is optional in the present invention. This structural element serves to space the recognition element and targeting element apart from one another.

One of the most important aspects of the spacing element in the present invention is in its application to the targeting of a large number of viruses. In the infection scheme of certain viruses, a structural factor on their surface is used to identify target cells and bind the virus to these cells. Table 1 provides a number of examples of such viruses, their host cells, and the cell receptor involved.

To confound the immune system of the host organism, these virus cells constantly modify their external proteins. Because the receptor site is highly conserved, it is hidden in narrow channels on the surface. These sites can then not be reached by antibodies even when the body manages to produce them. This is called the Canyon Hypothesis (Rossmann, J. of Biological Chemistry, Vol. 264, pp. 14587-90, 1989). The present invention provides a method of attaching recognition elements to such pathogens despite the above described evasion methods. First, the targeting element is in fact the body's surface material to which the viral receptor binds. This element will fit into the viral surface indentations at the bottom of which lies the receptor site. Secondly, a spacer element is attached to the targeting element. This allows the recognition element to clear the surface of the virus and be available for immunogenic recognition by the body.

A number of qualities should be considered when selecting a spacer element for a particular use. The inventive spacer element should ideally be water soluble and reasonably stable under physiological conditions. Ease of synthesis and compatibility with the other elements of the targeting complex are other factors to consider.

Spacer molecules can serve to tailor the inventive complex to specific needs. For instance, when the complex should be inactivated in certain regions of the body or under certain conditions, the spacer molecule can be constructed to be nonfunctional or fall apart under those conditions. Also, by restricting the size of the molecule, the reaction of the body to the targeting complex can be limited. As an example, steric hinderance would not prove a problem for immunogenic cells attracted to a target by chemotaxis. Therefore, if one wished the defense reaction to be limited to such an immunogenicity mechanism, the spacer element would be of small size, or eliminated altogether.

When steric hinderance is not a problem, there is generally little need for the spacer molecule. This would be true in cases where the target has its identifying molecule protruding from its surface. Malaria organisms are an example of such a situation. In that case, it is speed of infection, rather than its immunogenic unrecognizability, that defeats the immune system. In other cases, such as rubella or chicken pox, it is the naivety of the immune system that leads to the full-blown disease and its sometimes tragic sequela. In these and similar situations, spacer elements are generally unnecessary.

RECOGNITION ELEMENT

The choice of the recognition element of the present invention is determined by the needs of the specific system in which it is to elicit the immunogenic response.

Inborn or Non-specific Reaction. Selecting a recognition element which elects a non-specific or inborn defense reaction is advisable in many cases.

The mode and degree of Immune response can be graduated to some extent by the choice of the recognition element. Thus, the careful choice of components, their configuration, and administration mode can optimize the therapeutic effect while minimizing undesired results. For instance, certain materials are likely to activate a tissue macrophage or cascade response rather that a B-cell type response. In an immunologically compromised patient, it may be prudent to employ one of the former approaches in order to avoid exhaustion of the system. In the case of cancer patients, the choice of a recognition element unlikely to occupy the killer T-cell resources of the body would be advisable.

Non-specific, inborn immune response can also be elicited against pathogenic cells in the present invention by choosing certain materials, such as bacterial toxins, for the recognition element. Such defending cells as tissue macrophages, mast cells, neutrophils, and granular monocytes can be brought into play, den acquired immune responses are compromised by disease, chemotherapy, or for other reasons, the recognition element should be chosen to illicit the non-specific, inborn immune response.

Example of factors which elicit these inborn responses are provided in Table 2. The mechanism for these reactions are not fully understood. However, as long as the patient retains the capacity for such a defense, the present invention complex serves as an appropriate treatment.

These materials and their identifying aspects are thus useful as recognition elements in the present invention. TABLE 2

Targets for Non-Specific Defense and/or Natural Antibodies

Bacteria Skarnes et al., Bact. Rev., Vol. 21, p. 273, 1957 Bacteriophages Jerne, J. Immunol., Vol. 76, p. 209, 1956

Toussaint et al., J. Immunol. Vol. 89, p. 27, 1962

Animal viruses Svehag, et al., J. Exp. Med. Vol. 119, p. 1, 1964 Starch particles Sinclair, Immunol., Vol. 1, p. 291,

1958

Protozoa Brody et al.. Blood Vol. 6, p. 453, 1960

Fungi Sewell, Immunol., Vol. 6, p. 453, 1963 Metazoal Parasites Landsteiner, The Specificity of

Serological Regions, Harvard Press, Cambridge, Mass., 1945

Erythrocytes Garbar, et al., Ann. Inst. Pasteur, Vol. 88, p. 11, 1955 Serum proteins Landy, et al., Cancer Res., Vol. 20, p. 1297, 1960 Tissue cells Lunsde et al., J. Path. Bact., Vol. 32, p. 185, 1929

Potter, et al. , Science, Vol. 162, p. 453, 1968

Spermatozoa Boydens, Nature, London, Vol. 201, p. 200, 1964 Acquired Immune Response. Optimization of the therapeutic effect of the present invention can be achieved by selecting recognition elements to which the patient has previously been sensitized but is unlikely to have need of on a short-term bases. For instance, a patient can be screened for a small-pox or polio titer. If the titer is at a high level, the patient can be optimally treated with a form of the inventive complex employing these recognition elements.

Chemical and Physical Qualities. Other factors to weigh when selecting a recognition element are the size, stability and compatibility of the material. In general, small molecules have many advantages in the invention system. For instance, one can often load multiple recognition elements on to the targeting segment when the recognition elements are small. Also, the choice of such recognition elements provides a smaller load when administering the drug. This consideration is of particularly importance in intravenous and intermuscular administration. The selection of a small sized recognition element can be of critical importance if transdermal administration is contemplated.

The stability of the recognition element can also be selected to provide the final inventive complex with desired characteristics. A long-lived recognition element may result in a longer lasting effect of each administration. This can be a useful characteristic when administration must be accomplished in an institutional setting or by intravenous methods. Often, such stabile recognition elements also enjoy a prolonged shelf-life.

The selection of a recognition element with a short survival time after administration may be appropriate where the dosages may need to be monitored closely, or where adverse side effects can develop quickly. A material may be selected which breaks down in an inappropriate environment, such as in an organ to which one wishes to avoid exposure to the intact complex. Ideally, any desired instability would make itself apparent only in the body environment, and still allow for a long shelf life, such as in a lyophilized state, or in an alcohol carrier.

The recognition element must be compatible with the carrier material for the complex, as well as with the other components of the complex. Additionally, it needs to be compatible with its route of administration and with the area of the body in which it is to be active.

The recognition element can be any of a wide panoply of materials. As can be seen from Table 2, a large variety of organic materials are suitable for eliciting non- specific defense reactions. Any immunogenic proteins of materials can be similarly employed. Recognition elements can be employed in multiples or mixed groups. For instance, to assemble an omnibus complex, a recognition element which stimulates B-cell activity could be paired with one which activates T cell reactions. In that manner, one could be assured of at least some defense response without resorting to a full testing of the immunogenic profiled of the patient. In a similar way, antigens which will be targeted by different common antibodies can be employed. In this case, deficiency of any one titer need not defeat the effects of the treatment.

The possible modes of administration of the inventive immunologic complex are varied. A carrier material will often be required, such as in intravenous or intermuscular administration. Transder al or other gradual administration will be possible, in come cases. As described above, efficacy in ameliorating lung problems may be best accomplished by administration in inhalant carrier. The efficiency of the inventive concept has been demonstrated in vitro using the nonpeptidyl and peptidyl ligands, biotin and CD4, respectively, and the antigen, dinitrobenzene (DNP) , which is recognized by approximately 1-2% of naturally occurring antibodies. This work is set out in full in Examples 1-3 below.

The inventive approach has been demonstrated in vitro using the nonpeptidyl and peptidyl ligands, biotin and CD4, respectively, and the antigen, dinitrobenzene (DNP). The CD4-DNP conjugate directs a monoclonal anti-DNP antibody to gpl20, the envelope protein of the human immunodeficiency virus (HIV) , via its functionally conserved CD4 binding domain. This arrangement is shown in the diagram in Fig. 2. The binding of soluble CD4 (sCD4) to gpl20 has been extensively characterized (K_D = 10^_9M) and has been exploited in other anti-AIDS therapeutic strategies. In the second example biotin directs the same anti-DNP antibody to the tetrameric protein streptavidin (K_D = 10^"15M) . This arrangement is shown in the diagram in Fig. 3. The antigen, DNP, elicits antibodies with 10³ - 10⁴ fold higher K_A's(2 x lo'tø'¹) than do antigens of similar size. (Karush, Adv. in Immunol.. Vol. 2 pp. 1-40, 1962.) In addition natural anti-DNP antibodies account for 1% of all antibodies of the IgM subclass, and 0.8% of the IgG subclass with K_A's 10⁴ - lO'fr¹. (Karjalainen, et al., Eur. J. Immunol.. Vol. 6, pp. 88-93, 1976; and Ortega, et al., C. Mol. Immun.. Vol. 6, pp. 88-93, 1984.) In both examples the anti-DNP antibody is recognized by the first component of the complement cascade, Clq, (Muller-Eberhard, Ann. Rev.

Biochem. , Vol. 57, pp. 321-347, 1988) demonstrating the viability of ligand mediated immunogenicity in generating active immunity against the target of interest, such as HIV or HIV infected cells. In both cases the antigenic determinant only becomes multimeric and hence able to activate complement when presented in the context of the naturally occurring target protein. EXAMPLE 1

Dinitrobenzene was derivatized with a water soluble tetraethylene glycol spacer (16A) to ensure the DNP group would be accessible for antibody binding. Monofunctionalization of tetraethylene glycol diamine with 2,4 dinitro luorobenzene, followed by formation of the isothiocyanate with thiophosgene afforded the acylating reagent which was used to introduce the DNP group onto the e-amino groups on the surface of sCD4. Limited derivitization of the e-amino groups of surface lysines on sCD4 resulted in a DNP-conjugate that retained gpl20 binding activity. The SCD4-DNP conjugate comigrated with unmodified sCD4 when analyzed by polyacrylamide gel electrophoresis with silver staining. The ultraviolet- visible spectrum of SCD4-DNP was consistent with one mole of DNP per mole of sCD4.

To test the ability of SCD4-DNP to target anti-DNP antibody to the HIV envelope protein gpl20, an enzyme- linked immunosorbant assay (ELISA) experiment was performed (Scheme 1) . Recombinant gpl20 was blotted onto nitrocellulose and the remaining protein binding sites were blocked with bovine serum albumin (BSA) . CD4-DNP was then added to the dot blot chamber, incubated for 30 minutes and the excess CD4-DNP was removed by washing with PBS. Anti-DNP antibody AN09 (Leahy, et al., Proc. Natl. Acad. Sci. USA. Vol. 85, pp. 3661-3665, 1988) was then added, followed by the same washing procedure. A second antibody, goat anti-mouse-antibody-horseradish peroxidase (GAM-Ig-HRP) , was used to detect the amount of anti-DNP antibody which bound gpl20. Formation of the antibody complex was assayed spectrophotometrically by the addition of 3,3'-diaminobenzidine tetrahydrochloride (DAB) and H₂0₂ (HRP substrates at 4°C and quantitated by densitometry of a positive exposure of the filter (Scheme 1) . (To a 3 mm diameter circle of nitrocellulose 50 μL of gpl20 (100 μg/mL) was added (30 min.) and excess protein binding sites were blocked with a 3% BSA solution (2 hr.), followed by addition of CD4-DNP. (+) CD4-DNP = 100 μL (25 μg/mL) of CD4-DNP (30 min.); (-) CD4-DNP = no CD4-DNP added. One hundred μL (8 μg/mL) of anti-DNP antibody AN09 (30 min.) was added followed by either 100 μL (1 μg/mL) of GAM-Ig-HRP (30 min.) or 100 μL (10 μg/mL) of Clq-HRP (30 min.). DAB substrate (4°C) was added to the washed nitrocellulose. Product intensity was determined by a laser densitometer scan of a film positive of the nitrocellulose.) A control lacking CD4-DNP was performed to test for non-specific binding of the anti-DNP antibody and the GAM-Ig-HRP conjugate. Controls lacking gpl20 but containing CD4-DNP showed no signal (data not shown) . In contrast, a large signal was obtained for the ELISA containing gpl20, CD4-DNP, anti-DNP antibody and GAM-Ig- HRP indicating the successful targeting of antibodies to gpl20 via non-covalently introduced epitopes.

The ability of the CD4-DNP gpl20 complex to activate complement was assayed by substitution of Clq-HRP for GAM- Ig-HRP in the experiment described above. The first component of complement, Clq, is responsible for triggering the complement cascade to destroy cells on which immune complexes form. (Muller-Eberhard, Ann. Rev. Biochem. , Vol. 57, pp. 321-347, 1988.) Clq-BRP bound specifically relative to a control lacking CD4-DNP

(Scheme 1) , demonstrating the successful formation of an immune complex on gpl20, mediated by a non-covalently introduced epitope. (The decrease in the ratio of specific to non-specific binding of Clq-HRP compared to GAM-Ig-HRP is due to the low affinity of Clq for monomeric mouse IgG2a. Clq is hexameric and thus binds to aggregated IgG with great avidity (K_A = loV) , [Emanuel, et al., Biochem. J.. Vol. 205, pp. 361-372, 1982] but binds monomeric IgG poorly (K_A = 10⁴M^_1) . [Sledge, J. Mol. Biol.. Vol. 148, pp. 191-197, 1981.] Because nitrocellulose is a solid support the anti-DNP antibodies remain fixed rather than being able to aggregate or 'cap' as they would on the surface of a cell. Goat anti-mouse- IgG antibodies typically have relatively high affinities (K_A = 10⁷-10⁹M"¹) for monomeric Ig-G and thus gives a much better signal to noise ratio in this type of assay.) These test results are seen in Fig. 5.

EXAMPLE 2

In order to demonstrate the ability of small molecules to direct anti-DNP antibodies to proteins, a biotin-DNP linker was synthesized. Biotin-DNP was synthesized by condensation of DNP-tetraethylene glycol diamine and biotin using dicyclohexylcarbodiimide. Streptavidin was bound to nitrocellulose and the sandwich assay was performed as described above (Scheme 2) . (Streptavidin (50 μL of 100 μg/mL) was substituted for gpl20. Biotin- DNP (100 μL of 10 μg/mL) was substituted for CD4-DNP.

GAM-Ig-HRP concentration was 2 μg/mL.) Again the biotin- DNP conjugate successfully targeted anti-DNP antibody to streptavidin. A functional assay substituting Clq-HRP for GAM-Ig-HRP shows that the protein sandwich can also trigger a complement response. Streptavidin contains four biotin binding sites and thus may provide a better mimic of 'capping' of antibodies on the surface of cells. Anti- DNP antibody also binds specifically to immobilized streptavidin in the presence of an excess of free biotin- DNP relative to the control lacking biotin-DNP. These test results are seen in Fig. 5.

EXAMPLE 3

In order for LMI to be viable drug therapy to prevent infection in vivo, the binding of anti-DNP antibody to viral protein must take place in the presence of bound and unbound bifunctional drug. Two populations of DNP ligands would be present, those attached to the receptor protein via a ligand binding event and those free in the serum. The unbound DNP-protein ligand will only present DNP in monomeric form (and thereby not activate complement) and is also likely to be cleared relatively quickly from the body. In order to assay sandwich formation in the presence of an excess of free biotin-DNP, the following sandwich experiment was performed (Scheme 3) . This arrangement is shown in the diagram in Fig. 4. Streptavidin was incubated on the nitrocellulose for 30 minutes, followed by a BSA blocking step. A mixture of biotin-DNP, anti-DNP and GAM-Ig-HRP was incubated at room temperature for 30 minutes and then added to the blocked nitrocellulose filter. The filter was washed three times with PBS and DAB was added to assay sandwich formation. Relative to a control lacking biotin-DNP, anti-DNP antibody binds specifically to immobilized streptavidin in the presence of an excess of free biotin-DNP (Scheme 3) . These results are seen in Fig. 5. On the surface of a target cell, the anti-DNP antibodies are likely to aggregate (cap) and further increase their avidity for DNP bound to the target protein relative to free biotin-DNP.

EXPERIMENTAL MATERIALS USED

Reagents: Soluble CD4 was obtained from Dan Littman (UCSF) and American Bio-Technologies, Inc. (Cambridge, MA) . Anti-DNP antibody (AN09) producing hybridoma cell line was a generous gift of Harden McConnell (Stanford) . Cells were cultured and ascites fluid obtained. (Preston, et al.. Science. Vol. 242, pp. 1168-1171, 1988.) Antibody AN09 was isolated by affinity chromatography on protein A coupled Sepharose 4B (Rossman, J. Biol. Chem. , Vol. 264, pp. 14587-14590, 1989) and determined to be >95% pure by SDS polyacrylamide gel electrophoresis (PAGE) . (Geyer, et al., J. Biol. Chem.. Vol. 263, pp. 11760-11767, 1988.) Streptavidin was obtained from Boehringer Mannheim. Human Clq, (+)-biotin, bovine serum, albumin, 3,3'-diamino benzidine hydrochloride (DAB) , and horse radish peroxidase (HRP) were purchased from Sigma. Tetraethylene glycol diamine was purchased from Texaco. Goat antimouse Ig-HRP was obtained from Pierce. All other reagents were of the highest purity commercially available.

N-2.4-dinitrophenyl-ethγleneglvcol diamine l: To a 10 mL round bottom flask were added at room temperature 0.18 g (0.85 mmol) of tetraethylene glycol diamine, 1.5 of CHC1₃, and 0.095 g (0.85 mmol) of l,4-diazabicyclo[2.2.2]octane. Forty eight μL (0.38 mmol) of 2,4 dinitro-1-fluorobenzene was added in 8 μL aliquots over a 10 minute period. This solution was stirred overnight and the volatiles were removed by rotary evaporation at water aspirator pressure. The yellow oil was dissolved in a minimum amount of CHC1₃ and purified by flash chromatography on a 1.5 cm x 8 cm silica gel (Merck 60 230-400 mesh size) column, eluting with 9:6:1 CHC1₃:CH₃0H:acetone containing 0.1% Et₃N. The fractions containing the product (R = 0.07 in Column eluent) were combined and the solvent removed by rotary evaporation to afford 0.094 g (68%) of a yellow oil. IR (thin film) 3360, 2932, 2871, 1621, 1588, 1524, 1426, 1336, cm^"1; ^JH NMR (CDC1₃) d 9.04 (d, 1, J=3) , 8.78 (s, 1), 8.21 (dd, 1, J=3, 10), 6.96 (d, 1, J=10) , 4.49 (S, 2) 3.83 (t, 2, J=5) , 3.61 (m, 12), 2.94 (t, 2 J-5) ¹³C NMR (CDC1₃) d 148.4, 135.9, 130.3, 130.2, 124.1, 114.3, 70.8, 70.5, 70.5, 70.4, 70.2, 70.0, 68.4, 53.4, 43.1, 40.8; mass spectrum (FAB+) 359 (M+l) . High resolution mass spectrum (FAB+) : 359.1564.

N-2.4-dinitrophenyl-isothiocyanate tetraethylene glvcol diamine 2: To a 25 mL round bottom flask were added, 0.093 g (0.20 mmol) of 1, 0.1 mL (0.57 mmol) of diisopropyl ethyl amine, and 15 mL of CHC1₃. Twenty-two μL (0.29 mmol) of thiophosgene was added in three equal aliquots at room temperature. The solution was stirred for five hours. The volatiles were removed by rotary evaporation leaving a yellow oil. This was dissolved in a minimum of CHC1₃ and purified by flash chromatography on a 1 cm x 6 silica gel column eluting with ethyl acetate. The fractions containing product (R = 03.6 in column eluent) were combined and the solvent was removed by rotary evaporation to afford 0.054 g (52%) of a yellow oil. IR (thin film) 3360, 3107, 2871, 2195, 2111, 1621, 1588, 1524, 1426, 1336 cm'¹; ^*H NMR (CDC1₃) d 9.2 (1, d, J=3), 8.8 (s, 1), 8.25 (dd, 1, J=3, 10), 6.96 (d, 1, J=10) , 3.85 (t, 2, J=5) , 3.73 (m, 4) 3.68 (m, 8), 3.61 (q, 2, J=5, 10), ³C(NMR) 148.3, 135.9, 130.3 130.2, 124.2, 114.1, 70.7, 70.7, 70.6, 70.6, 69.2, 68.5, 43.2; mass spectrum (FAB+) 401 (M+l) ; high resolution mass spectrum (FAB+) : 401.1126.

N-2.4-dinitrophenyl- (+)-biotin tetraethylene glycol diamine 3: To a 25 mL round bottom flask 0.6 g (2.46 mmol) of (+)-biotin, 10 mL of CH₂C1₂ and 0.51 g (2.46 mmol) of dicyclohexylcarbodiimide were added and the flask was cooled to 0°C. After stirring for 30 minutes, 0.42 g (1.12 mmol) of 1 was added. Stirring was continued overnight at room temperature. The white solid was filtered and the volatiles were removed from the filtrate by rotary evaporation. The remaining yellow oil was dissolved in a minimum of CHC1₃ and was purified by flash chromatography on a 4 cm x 25 silica gel column eluting with 9:1 CHC1₃:CH₃0H. The fractions containing product (R = 0.23 in column eluent) to afford 0.043 g (7%) of a yellow oil. IR (thin film) 3351, 3290, 2928, 2863, 1702, 1621, 1588, 1524, 1426, 1401, 1336 cm'¹; ^*H (CDC1₃) d 9.10

(d, 1 J=3), 8.79 (S, 1), 8.25 (dd, 1, J=3, 10), 6.97 (d, 1 J=10) , 6.8 (t, 1, J=5) , 6.68 (s, 1), 5.74 (s, 1), 4.49 (dd, 1, J=5, 8), 4.29 (dd, 1, J=5, 7), 3.82 (t, 2 , J=5) , 3.65 (m, 10), 3.55 (t, 2, J=5) , 3.4 (m, 2), 3.15 (m, 1), 2.87 (dd, 1, J=5, 13), 2.72 (d, 1, J=13) , 2.2 (t, 2, J=8) , 1.65 (m, 4), 1.4 (m, 2); ¹³C NMR (CDC1₃) d 173.3, 164.1 148.4, 136.0, 130.4, 130.3, 124.2, 114.2, 70.7, 70.5,

70.4, 70.0, 69.9, 68.5, 61.7, 60.2, 55.6, 45.8, 43.2,

40.5, 39.1, 35.9, 28.2, 28.0, 25.6, 8.6; mass spectrum (FAB+) 585 (M+l) ; high resolution mass spectrum (FAB+) :

585.2358. CD4-DNP: To a solution of 200 μg of CD4 in 1 mL (0.1 M NaHC0₃, pH 9.0) was added 35 μL (2.5 mM in DMSO) of 2 in 5 μL aliquots. The solution was stirred to 4°C for 24 hrs. The solution was then applied to a Fast-Desalting column (Pharmacia-LKB) at l mL/min (10 mM phosphate, 150 mM NaCl, pH 7.3) . The protein concentration in the excluded peak was determined by BioRad protein assay (Cat. #500-0006) to be 50 μg/mL. The number of DNP groups per CD4 molecule was determined by O. O._^^^ to be 1.

ELISA: Immuno-blots were performed on nitrocellulose membranes (BRL, Maryland) supported in a Hybri-Dot manifold (BRL) . The nitrocellulose filters were pre- wetted with 10 mM phosphate, 150 mM NaCl pH 7.4 buffer (PBS) and placed in the manifold. The first protein solution, 50 μL, (concentrations as indicated in Figure legends) was added to each well and incubated for 30 minutes. Blocking was accomplished by 2 hour incubation with 300 μL of 3% BSA in PBS. The next components of the sandwich were added sequentially in 100 μL aliquots followed by 30 minute incubations and washed after each new solution with three 300 μL aliquots of PBS (five minutes each) . After the last protein was added (HRP- labelled) the membrane was removed from the manifold and washed 5 5 minutes in PBS and DAB substrate solution was added at 4°C. The reaction was quenched by washing with ddH₂0 to remove the hydrogen peroxide substrate. The dried blots were photographed, positives made, and these were read with a Bio-Rad model 620, video densitometer densitometer.

Claims

CLAIMS:

1. A therapeutic targeting complex capable of directing the body's immunologic defense system against an undesirable object, comprising: a. a targeting element capable of attaching or associating with a target b. optionally, a spacer molecule, and c. a recognition element.

2. The targeting complex of claim 1, wherein said targeting element is directed against a virus.

3. The targeting complex of claim 2, wherein said targeting element is directed against HIV and HIV infected cells.

4. The targeting complex of claim 3, wherein said targeting element is selected from the group comprising CD4, sCD4, vasoactive intestinal peptide, peptide T, and sialic acid, and derivatives and analogues thereof capable of associating with HIV.

5. The targeting complex of claim 3, wherein said recognition element is selected from the group dinitrobenzene, fluorescein, galactosyl α (1 → 3) galactosyl (Gal α 1 → 3 Gal) , trinitrophenol.

6. The targeting complex of claim 3, wherein said spacer molecule is selected from the group teraethylene glycol, diethylene glycol, triethylene glycol, pentaethylene glycol, succinate, glutamate, adipate, and polypeptide.

7. The targeting complex of claim 2, wherein said virus is selected from the group comprising vaccinia, rabies, epstein barr, rheo, rhinovirus, polio viruses, and influenza.

8. The targeting complex of claim 2, wherein the targeting element is selected from the group comprising epidermal growth factor, acetylcholine receptor, complement receptor, beta-adrenergic receptor, ICAM-1, polio virus receptor, sialic acid, and derivatives and analogues thereof capable of associating with a relevant target.

9. The targeting complex of claim 2, wherein said virus is selected from the group comprising rubella, chicken pox, small pox, hepatitis and herpes simplex.

10. The targeting complex of claim 1, wherein said targeting element is directed against bacteria, parasites, blood factors, autoimmune factors, or allergens.

11. The targeting complex of claim 10, wherein said targeting element is directed against the pathogens responsible for sleeping sickness, river blindness, toxoplasmosis, tuberculoses, amebic dysentery, malaria, tuberculoses, salmonella, or streptococcus.

12. The targeting complex of claim 1, wherein said recognition element elicits a non-specific defense or will associate with natural antibodies.

13. The targeting complex of claim 12, wherein said recognition element is a bacterial toxin, bacteriophage, animal virus, starch particle, protozoa, fungi, metazoal parasites, erythrocytes, serum proteins, tissue cell, and derivatives and analogues thereof capable of eliciting a non-specific defense reaction or associating with natural antibodies.

14. The targeting complex of claim 1, wherein said recognition element elicits a specific defense reaction.

15. The targeting complex of claim 14, wherein said recognition element is selected from the group comprising rubella antigen, polio antigen, small pox antigen, pertussis antigen, diphtheria antigen, and mumps antigen.

16. A method of making the therapeutic agent of claim 1, comprising attaching the targeting element with the recognition element, and optionally interspacing said elements with a spacer molecule.

17. A method of making the therapeutic agent of claim 7, comprising, a. producing a targeting element selected from the group comprising CD4, sCD4, vasoactive intestinal peptide, peptide T, and sialic acid, and derivatives and analogues thereof capable of associating with HIV, b. binding said targeting element to one of the group comprising tetraethylene glycol, diethylene glycol, triethylene glycol, pentaethylene glycol, succinate, glutamate, adipate and polypeptides, c. binding the targeting element spacer molecule module to a recognition element selected from the group dinitrobenzen, fluorescein, galactosyl a (1 → 3) , galactosyl (Gal α 1 → 3 Gal) , trinitrophenol.

18. A method of treating a disease comprising, a. mixing the therapeutic targeting complex of claim 1 with a carrier material; b. administrating the mixture produced to a patient.

19. The method of claim 18, wherein said material is administered by a method from the group comprising, intravenously, intramuscularly, transdermally, intra peritoneally, orally, topically or by inhalation.