WO1992015326A1 - Immunogenicite a mediation par complexe de ciblage - Google Patents

Immunogenicite a mediation par complexe de ciblage Download PDF

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Publication number
WO1992015326A1
WO1992015326A1 PCT/US1992/001588 US9201588W WO9215326A1 WO 1992015326 A1 WO1992015326 A1 WO 1992015326A1 US 9201588 W US9201588 W US 9201588W WO 9215326 A1 WO9215326 A1 WO 9215326A1
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Prior art keywords
targeting
complex
targeting complex
group
recognition
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PCT/US1992/001588
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English (en)
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Peter Schultz
Kevan M. Shokat
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The Regents Of The University Of California
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Publication of WO1992015326A1 publication Critical patent/WO1992015326A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell

Definitions

  • This invention is related to the targeting of the body's immune system to pathological organisms and tissues, and other undesirable materials.
  • the devastation caused by other viral diseases can not be circumvented through treatment of secondary effects.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HIV human immunodeficiency virus
  • 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.
  • 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.]
  • CD4 soluble CD4, sCD4
  • CD4 soluble CD4, sCD4
  • CD4 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) .
  • 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.
  • the present inventive targeting complex takes a fundamentally different approach to the challenges of HIV than does Capon or Traunecker.
  • 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.
  • the CD4 protein of the targeting complex is termed the targeting element.
  • 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.
  • 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.
  • 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.
  • DNP dinitrobenzene
  • sCD4 dinitrobenzene
  • 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.
  • 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.
  • 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.
  • 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.
  • AIDS a number of opportunistic diseases are characteristic of the diminished immunologic capacity brought on by the HIV infection.
  • 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.
  • panoply of cancers characteristic of AIDS patients they are unusual in their poor response to chemotherapeutic efforts.
  • 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. conserveed 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the maternal antibodies to an antigen unrelated to the target bind the antigen component of the inventive immunologic complex.
  • 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.
  • inventive immune complex Treatment of allergic reactions by administration of the inventive immune complex avoids many of the complications and limitations of presently available treatments.
  • a targeting agent directed against the allergin serves to eliminate the offending material from the body prior to its provoking the immune response.
  • 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 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.
  • 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.
  • the humoral antibodies are sufficiently cleared to avoid specific sensitization, such effects may be unusual.
  • 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.
  • the targeting complex of the present invention comprises a targeting element attached to a recognition element.
  • 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.
  • 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.
  • the inventive targeting complex 1 attaches to or becomes associated with the target organism 9 through targeting site 7.
  • 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.
  • 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.
  • 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.
  • sialic acid which binds influenza viruses
  • 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.
  • CD4 14 Vasoactive Intestinal Peptide 7 , Peptide T 8 , Sialic
  • 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.
  • 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.
  • 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.
  • Example 1 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.
  • 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.
  • the recognition site on the host cell can be used as the targeting segment in the present invention.
  • 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.
  • the virus is screened using a 'library' of 10 6 - 10 8 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.
  • Influenza binding synthetic peptides US Patent #4,981,782 issued to Judd, et al., issued 1/1/91
  • 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.
  • 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.
  • 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.
  • the present invention provides a method of attaching recognition elements to such pathogens despite the above described evasion methods.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mode and degree of Immune response can be graduated to some extent by the choice of the recognition element.
  • 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.
  • 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.
  • 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.
  • 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.
  • recognition element 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.
  • 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.
  • inventive immunologic complex 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.
  • 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.
  • K D 10 "15 M
  • the antigen, DNP elicits antibodies with 10 3 - 10 4 fold higher K A 's(2 x lo't ⁇ ' 1 ) than do antigens of similar size.
  • Karush Adv. in Immunol.. Vol. 2 pp. 1-40, 1962.
  • 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 10 4 - lO'fr 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.
  • 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) .
  • BSA bovine serum albumin
  • 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 H 2 0 2 (HRP substrates at 4°C and quantitated by densitometry of a positive exposure of the filter (Scheme 1) .
  • DAB 3,3'-diaminobenzidine tetrahydrochloride
  • H 2 0 2 H 2 0 2
  • CD4-DNP gpl20 complex 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
  • 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 ⁇ L of 100 ⁇ g/mL) was substituted for gpl20. Biotin- DNP (100 ⁇ L of 10 ⁇ g/mL) was substituted for CD4-DNP.
  • GAM-Ig-HRP concentration was 2 ⁇ g/mL.
  • 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.
  • biotin-DNP 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.
  • 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.
  • N-2.4-dinitrophenyl-eth ⁇ leneglvcol 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 CHC1 3 , and 0.095 g (0.85 mmol) of l,4-diazabicyclo[2.2.2]octane. Forty eight ⁇ L (0.38 mmol) of 2,4 dinitro-1-fluorobenzene was added in 8 ⁇ L 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 CHC1 3 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 CHC1 3 :CH 3 0H:acetone containing 0.1% Et 3 N.
  • 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 CHC1 3 . Twenty-two ⁇ L (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.
  • 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 CH 2 C1 2 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.
  • CD4-DNP To a solution of 200 ⁇ g of CD4 in 1 mL (0.1 M NaHC0 3 , pH 9.0) was added 35 ⁇ L (2.5 mM in DMSO) of 2 in 5 ⁇ L 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 ⁇ g/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 ⁇ L, (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 ⁇ L of 3% BSA in PBS.

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Abstract

Procédé de ciblage du système immutaire du corps à la recherche d'organismes et de tissus pathologiques et d'autres matériaux indésirables à l'aide d'un complexe de ciblage nouveau. Ledit procédé s'applique particulièrement au traitement des infections par le HIV. Ledit complexe est constitué d'un élément de ciblage qui se lie à un matériau indésirable. L'élément de ciblage est fixé sur un élément de reconnaissance qui permet au corps d'identifier le matériau ciblé comme étant nuisible. Le corps neutralise ensuite ledit matériau. Lorsque cela est nécessaire pour éviter l'empêchement stérique, un élément d'écartement est utilisé entre l'élément de ciblage et l'élément de reconnaissance. Cette caractéristique permet aux immunoglobulines ambiantes de se fixer sur l'élément de reconnaissance.
PCT/US1992/001588 1991-02-27 1992-02-26 Immunogenicite a mediation par complexe de ciblage WO1992015326A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6984382B1 (en) 1994-05-02 2006-01-10 Bernd Groner Bifunctional protein, preparation and use
US20160082102A1 (en) * 2009-10-13 2016-03-24 Yale University Bifunctional molecules with antibody-recruiting and entry inhibitory activity against the human immunodeficiency virus

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003690A1 (fr) * 1987-10-30 1989-05-05 Institut Pasteur Application de dinitro-trinitrophenyle et de ses derives ou d'anticorps anti-nitrophenyles a la modification de l'interaction entre des cellules sensibles d'un hote et un agent pathogene

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1989003690A1 (fr) * 1987-10-30 1989-05-05 Institut Pasteur Application de dinitro-trinitrophenyle et de ses derives ou d'anticorps anti-nitrophenyles a la modification de l'interaction entre des cellules sensibles d'un hote et un agent pathogene

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Volume 113, No. 5, issued 27 February 1991, K.M. SHOKAT et al., "Redirecting the Immune Response: Ligand-Mediated Immunogenicity", pp. 1861-1862. *
NATURE, Volume 255, issued 26 June 1975, G. DENNART, "Cell-mediated Immunity to Hapten Modified Self- and Non-self Antigens", pp. 712-713. *
NATURE, Volume 337, issued 09 February 1989, D.J. CAPON et al., "Designing CD4 Immunoadhesins for AIDS Therapy", pp. 525-531. *
NATURE, Volume 339, issued 04 May 1989, A. TRAUNECKER et al., "Highly Efficient Neutralization of HIV with Recombinant CD4-Immunoglobulin Molecules", pp. 68-70. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6984382B1 (en) 1994-05-02 2006-01-10 Bernd Groner Bifunctional protein, preparation and use
US20160082102A1 (en) * 2009-10-13 2016-03-24 Yale University Bifunctional molecules with antibody-recruiting and entry inhibitory activity against the human immunodeficiency virus
US10188727B2 (en) * 2009-10-13 2019-01-29 Yale University Bifunctional molecules with antibody-recruiting and entry inhibitory activity against the human immunodeficiency virus

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