CA2421842A1 - Antibody and/or chemokine constructs which bind to a chemokine receptor and their use in immunological disorders - Google Patents
Antibody and/or chemokine constructs which bind to a chemokine receptor and their use in immunological disorders Download PDFInfo
- Publication number
- CA2421842A1 CA2421842A1 CA002421842A CA2421842A CA2421842A1 CA 2421842 A1 CA2421842 A1 CA 2421842A1 CA 002421842 A CA002421842 A CA 002421842A CA 2421842 A CA2421842 A CA 2421842A CA 2421842 A1 CA2421842 A1 CA 2421842A1
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- Prior art keywords
- chemokine
- antibody
- cells
- construct
- ccr5
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Abstract
The present invention relates to the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the elimination of cells which are latently infected with a primate immunodeficiency virus. In addition, the present invention provides for the use of an antibody and/or chemokine construct whi ch binds to a chemokine receptor for the preparation of a pharmaceutical composition of the treatment, prevention and/or alleviation of inflammatory renal diseases, inflammatory bowel diseases, multiple sclerosis, skin diseases, diabetes or transplant rejection. Furthermore, the invention relat es to antibody constructs and/or chemokine constructs wherein said antibody construct comprises a binding site of chemokine receptor 5 and a binding sit e for CD3 and wherein said chemokine construct comprises RANTES and a toxin. T he invention also describes polynucleotides encoding said antibody- or chemokin e constructs, and vectors and hosts comprising said nucleic acid molecules. Additionally, the present invention relates to compositions comprising said antibody constructs, chemokine constructs, polynucleotides, vectors and/or hosts. Preferably said composition is a pharmaceutical composition. Describe d is also the use of antibody constructs, the chemokine constructs, the polynucleotides, the hosts and/or the vectors of the preparation of a pharmaceutical composition and methods for treating, preventing and/or alleviating an immunological disorder or for eliminating latently infected cells, wherein said cells are infected with a primate immunodeficiency virus , like HIV-1. Furthermore, the invention provides for a kit comprising the compounds of the invention.
Description
Antibody andlor chemokine constructs and their use in immunological disorders The present invention relates to the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composi-tion for the elimination of cells which are latently infected with a primate immunodefi-ciency virus. In addition, the present invention provides for the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the treatment, prevention and/or alleviation of in-flammatory renal diseases, infilammatory bowel diseases, multiple sclerosis, skin dis-eases, diabetes or transplant rejection. Furthermore, the invention relates to antibody constructs and/or chemokine constructs, in particular to constructs wherein said anti-body construct comprises a binding site for chemokine receptor 5 and a binding site for CD3 and wherein said chemokine construct comprises RANTES and a toxin. The in-vention also describes polynucleotides encoding said antibody- or chemokine con-structs, and vectors and hosts comprising said nucleic acid molecules.
Additionally, the present invention relates to compositions comprising said antibody constructs, chemo-kine constructs, polynucleotides, vectors and/or hosts. Preferably said composition is a pharmaceutical composition. Described is also the use of antibody constructs, the chemokine constructs, the polynucleotides, the hosts andlor the vectors for the prepa-ration of a pharmaceutical composition for treating, preventing and/or alleviating an immunological disorder or for eliminating latently infected cells, wherein said cells are infected with a primate immunodeficiency virus, like HIV-1. The present invention also relates to a method for treating, preventing and/or alleviating an immunological disorder or for the elimination cells which are latently infected with a primate immunodeficiency virus. Furthermore, the invention provides for a kit comprising the compounds of the invention.
Additionally, the present invention relates to compositions comprising said antibody constructs, chemo-kine constructs, polynucleotides, vectors and/or hosts. Preferably said composition is a pharmaceutical composition. Described is also the use of antibody constructs, the chemokine constructs, the polynucleotides, the hosts andlor the vectors for the prepa-ration of a pharmaceutical composition for treating, preventing and/or alleviating an immunological disorder or for eliminating latently infected cells, wherein said cells are infected with a primate immunodeficiency virus, like HIV-1. The present invention also relates to a method for treating, preventing and/or alleviating an immunological disorder or for the elimination cells which are latently infected with a primate immunodeficiency virus. Furthermore, the invention provides for a kit comprising the compounds of the invention.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturers specifications, instructions, etc.) are hereby incorporated by reference.
Immunological diseases/disorders, like autoimmune diseases, inflammation disorders as well as infections diseases are not only increasing but represent substantial threats to global health.
For example, in Germany, about 1 % of the population suffer from the autoimmune dis-ease rheumatoid arthritis. In addition, there is a number of other rheumatoid diseases also leading to arthritis. Currently, three groups of drugs - non-steroidal antirheumatics, cortisone preparations and second-line agents - and TNFa blocking agents are used for treating inflammatory joint diseases. Up to now, the therapy has focused on the lo-cal injection of cortisone preparations in combination with a systemic administration of antiphlogistics or second-line agents.
Non-steroidal antirheumatics have a mild analgetic and anti-inflammatory effect, but they have many side effects when applied frequently (e.g. gastric ulcers, nephroses). In high dosages, cortisone preparations have a strong decongestant and analgetic effect, however leading to a quick relapse after discontinuation of the therapy.
Moreover, cor-tisone preparations cannot stop the destruction process of the joint disease.
A long-term therapy with cortisone usually entails severe side effects (infections, Cushing's phenomenon, osteoporosis, parchment-like skin, metabolic and hormonal disorders).
The local injection of cortisone also has the essential disadvantage that the activity of the migrated white blood cells is only reduced. As the infiltrating cells are not de-stroyed, a quick relapse occurs after discontinuation of the therapy. As mentioned above, the same applies to the systemic application. Rarely is an inflammation due to the irritative effect of cortisone crystals aggravated after injection of cortisone. The du-ration of effect of a cortisone injection varies tremendously and ranges from primary ineffectiveness to a duration of effect of several weeks.
Immunological diseases/disorders, like autoimmune diseases, inflammation disorders as well as infections diseases are not only increasing but represent substantial threats to global health.
For example, in Germany, about 1 % of the population suffer from the autoimmune dis-ease rheumatoid arthritis. In addition, there is a number of other rheumatoid diseases also leading to arthritis. Currently, three groups of drugs - non-steroidal antirheumatics, cortisone preparations and second-line agents - and TNFa blocking agents are used for treating inflammatory joint diseases. Up to now, the therapy has focused on the lo-cal injection of cortisone preparations in combination with a systemic administration of antiphlogistics or second-line agents.
Non-steroidal antirheumatics have a mild analgetic and anti-inflammatory effect, but they have many side effects when applied frequently (e.g. gastric ulcers, nephroses). In high dosages, cortisone preparations have a strong decongestant and analgetic effect, however leading to a quick relapse after discontinuation of the therapy.
Moreover, cor-tisone preparations cannot stop the destruction process of the joint disease.
A long-term therapy with cortisone usually entails severe side effects (infections, Cushing's phenomenon, osteoporosis, parchment-like skin, metabolic and hormonal disorders).
The local injection of cortisone also has the essential disadvantage that the activity of the migrated white blood cells is only reduced. As the infiltrating cells are not de-stroyed, a quick relapse occurs after discontinuation of the therapy. As mentioned above, the same applies to the systemic application. Rarely is an inflammation due to the irritative effect of cortisone crystals aggravated after injection of cortisone. The du-ration of effect of a cortisone injection varies tremendously and ranges from primary ineffectiveness to a duration of effect of several weeks.
In rheumatology, second-line agents are used to achieve a long-term suppression of the inflammation and a reduction in cortisone preparations. Due to the considerable toxicity (allergies, infections, malignant diseases, renal insufficiency, blood pressure crises, pulmonary diseases) it is necessary for medical specialists to attend closely to the patients. After beginning treatment, no therapeutic effect may be apparent for the first three months. Currently, there are 4 or 5 of such second-line agents at disposal, which are used individually at first or are combined if the therapy is not effective.
Mostly, there is hardly anything known about the mode of action of second-line agents.
It is not yet entirely clear whether the application of second-line agents can diminish the destruction of the joint. In recent years, a new group of substances has been intro-duced into the treatment of rheumatoid arthritis, which is based on the blocking of cell signal substances (particularly TNFa) by means of monoclonal antibodies or soluble receptor constructs.
In addition, there are patients that do not respond to currently available therapies. In other cases, the conventional therapy has to be stopped due to intolerable side effects.
A similar situation exists for many other inflammatory and autoimmune diseases like inflammatory renal diseases, inflammatory bowel diseases, multiple sclerosis and transplant rejection, where current treatments have many limitations. For example, agents used in inflammatory and autoimmune diseases include anti-inflammatory and immunosuppressive agents like azathioprine, cyclophosphamide, glucocorticoids like prednisone and corticosteroids; immunosuppressants like cyclosporin A, Tacrolimus (FK506), Sirolimus (Rapamycin); and protein drugs like calcineurin, beta-interferon, anti-TNF alpha monoclonal antibodies (remicade). These agents show general immu-nomodulating effects and therefore efficacy and side effects profiles can pose severe limitations for the treatment options (Harrison's Principles of Internal Medicine, eds.
Fauci et al., 14t" edition, McGraw-Hill publisher).
Inflammatory bowel diseases (examples are Crohn's disease, ulcerative colitis) are treated with the anti-inflammatory agents sulfazsalazine (Azulfidine) and glucocorti-colds, like prednisone and, in selected cases, with TNF-a blocking agents. In ulcerative colitis immunosuppressive therapy with drugs such as azathioprine is well established, in severely ill patients the potent immunosuppressive agent cyclosporine is used (Harrison's Principles of Internal Medicine, eds. Fauci et al., 14t" edition, McGraw-Hill publisher).
In many cases no sufficient reduction of disease activity is achieved with current drugs, such that even surgical intervention is sometimes necessary.
Inflammatory renal diseases (nephritis) are treated with e.g. glucocorticoids, alkylating agents and/or plasmapheresis. Additional diseases with similar treatment options in-clude systemic lupus erythemtosus (SLE), Sjoegren's syndrome, polymyositis, derma-tomyositis, mixed connective tissue disease, antiphospholipidantibody syndrome.
For some of these diseases, few therapeutic options have been available up to now. All these diseases share an inflammatory component. However, the inflammatory compo-nent cannot be sufficiently suppressed by the currently available drugs. For some drugs, e.g. alkylating agents a maximal lifetime dose per patient cannot be exceeded.
Transplant rejection is treated using immunosuppressive agents including azathioprine, mycophenolate mofetil, glucocorticoids, cyclosporine, Tacrolimus (FK506), Sirolimus (Rapamycin). A combination of steroids and a low dose of mouse monoclonal antibody OKT3 binding to CD3 on T-cells is used to anergize and deplete T-cells, therapy is continued using immunosuppressants like cyclosporine. Human anti-mouse antibodies I(HAMAs) have common side effects and limit the use of OKT3 (Fauci et al. sic.
2381 ).
Approaches to treat multiple sclerosis include treatments which effect the overall im-mune system like anti-inflammatory agents including azathioprine, cyclophosphamide, prednisone, corticosteroids, cyclosporin A, calcineurin, Rapamycin, beta-interferon (Fauci et al, sic. 2415-2419; Wang (2000) j. Immunol. 165, 548-57). In addition, a num-ber of non-specific treatments are administered that may improve the quality of life in-cluding physical therapy and psycho-pharmacological agents. None of the treatment options mentioned above has a curative effect. Even the most promising compound, (3-interferon, leads only to a slower disease progression, while exhibiting significant side effects.
Furthermore, human immunodeficiency virus-type 1 (HIV-1 ), the most common cause of AIDS, has infected more than 50 million individuals (including those who have died), and the rate of new infections is estimated at nearly 6 million per year (AIDS
Epidemic Update: December 1999 (UNAIDS, Geneva, 1999), www.unaids.org). Equally disturb-ing are the uncertainties of the epidemic to come. Although sub-Saharan Africa re-mains the global epicenter, rates of infection have increased in recent times in the for-mer Soviet Union and parts of south and southeast Asia, including India and China, where literally hundreds of millions of individuals are potentially at risk.
In the United States, new waves of infection have been recognized in women, minorities, and younger generations of gay men. Combination antiretroviral therapy has afforded many people clinical relief, but the costs and toxicities of treatment are substantial, and HIV-1 infection remains a fatal disease. Moreover, the vast majority of infected people world-wide do not have access to these agents. Thus, although the demographics (and, in some instances, the natural history) of AIDS have changed, the epidemic is far from over; instead, it is evolving, expanding, and posing ever greater challenges.
Human immunodeficiency virus (HIV) cannot enter human cells unless it first binds to two key molecules on the cell surface, CD4 and a co-receptor. The co-receptor that is initially recognized is CCRS, later in the life cycle of the virus another chemokine re-ceptor CXCR4 becomes the co-receptor for HIV-1 (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). The HIV-strains that cause most transmissions of viruses by sexual contact are called M-tropic viruses. These HIV-1 strains (also known as NSI primary viruses) can replicate in pri-mary CD4+ T-cells and macrophages and use the chemokine receptor CCR5 (and, less often, CCR3) as their coreceptor. The T-tropic viruses (sometimes called SI pri-mary) can also replicate in primary CD4+ T-cells but can in addition infect established CD4+ T-cell lines in vitro, which they do via the chemokine receptor CXCR4 (fusin).
Many of these T-tropic strains can use CCR5 in addition to CXCR4, and some can en-ter macrophages via CCRS, at least under certain in vitro conditions (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)).
Whether other coreceptors contribute to HIV-1 pathogenesis is unresolved, but the ex-istence of another coreceptor for some T-tropic strains can be inferred from in vitro studies. Because M-tropic HIV-1 strains are implicated in about 90% of sexual trans-missions of HIV, CCR5 is the predominant coreceptor for the virus in patients;
trans-mission (or systemic establishment) of CXCR4-using (T-tropic) strains is rare (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, (1996), Paxton, Nature Med. 2, 412 (1996); Liu, Cell 86, 367 (1996); Samson, Nature 382, 722 (1996); Dean, Science 273, 1856 (1996); Huang, Nature Med. 2, 1240 (1996)). However, once SI viruses evolve in vivo (or if they are transmitted), they are especially virulent and cause faster disease progression (D'Souza, Nature Med.
2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996), Schuitemaker, J. Virol. 66, 1354 (1992); Connor, J. Virol. 67, 1772 (1993);
Richman, J.
Infect. Dis. 169, 968 (1994); R. I. Connor et al., J. Exp. Med. 185, 621 (1997); Trkola, Nature 384, 184 (1996)).
The numbers and identity of coreceptor molecules on target cells, and the ability of HIV-1 strains to likely enter cells via the different coreceptors, seem to be critical de-terminants of disease progression. These factors are major influences on both host-and virus-dependent aspects of HIV-1 infection. For example, a homozygous defect (delta 32) in CCR5 correlates strongly with resistance to HIV-1 infection in vivo and in vitro. Individuals who are heterozygous for a defective CCR5 allele are at best weakly protected against infection and have only a modestly slowed disease progression (Paxton, Nature Med. 2, 412 (1996); Liu, Cell 86, 367 (1996); Samson, Nature 382, 722 (1996); Dean, Science 273, 1856 (1996); Huang et al., Nature Med. 2, 1240 (1996)).
However, other factors can influence the level of CCR5 expression on activated CD4+
T-cells and thereby affect the efficiency of HIV-1 infection in vitro (Trkola, Nature 384, 184 (1996); Bleul, Proc. Natl. Acad. Sci. U.S.A. 94, 1925 (1997)). For reasons that are not yet clear, the amount of CCR5 expression on the cell surface (as measured by MIP-1 binding) varies by 20-fold on CD4+ T-cells from individuals with two wild-type CCR5 alleles (Trkola, Nature 384, 184 (1996)) (see figure). Staining with a CCRS-specific monoclonal antibody indicates a similar large variability (Wu, J. Exp. Med.
186:1373-81 (1997)). Such variation may far outweigh any effect of one defective allele for CCRS.
The causes of this variation should be the subject of intensive studies, as they point to controllable factors that could increase resistance to disease.
Most primary, clinical isolates of primate immunodeficiency viruses use the chemokine receptor CCR5 for entry (Feng, Science 272, 872 (1996); Choe, Cell 85, 1135 (1996);
Deng, Nature 381, 661 (1996); Dragic et al., Nature 381, p. 667; Doranz, Cell 85, 1149 (1996); Alkhatib, Science 272, 1955 (1996)). For most HIV-1 isolates that are transmit-ted and that predominate during the early years of infection, CCR5 is an obligate core-ceptor, and rare individuals that are genetically deficient in CCR5 expression are rela-tively resistant to HIV-1 infection (Connor, J. Exp. Med. 185, 621 (1997);
Zhang, Nature 383, 768 (1996); Bjorndal, J. Virol. 71, 7478 (1997); Dean, Science 273, 1856 (1996);
Liu, Cell 86, 367 (1996); Paxton, Nature Med. 2, 412 (1996); Samson, Nature 382, 722 (1996)). HIV-1 isolates arising later in the course of infection often use other chemokine receptors, frequently CXCR4, in addition to CCRS. Studies of chimeric envelope glyco-proteins demonstrated that the third variable (V3) loop of gp120 is a major determinant of which chemokine receptor is used (see references above and also Cocchi, Nature Med. 2, 1244 (1996); Bieniasz, EMBO J. 16, 2599 (1997); Speck, J. Virol. 71, (1997)). V3-deleted versions of gp120 do not bind CCRS, even though CD4 binding occurs at wild-type levels. Antibodies to the V3 loop interfere with gp120-CCR5 binding (Trkola, Nature 384, 184 (1996); Wu, Nature 384, 179 (1996); Lapham, Science 274, 602 (1996); Bandres, J. Virol. 72, 2500 (1998); Hill, Science 71, 6296 (1997)). These results support an involvement of the V3 loop in chemokine receptor binding.
Latency of HIV is established very early in the course of an infection, when M-tropic strains predominate. M-tropic strains depend on the presence of CCR5 on the target cell for infection. The importance of CCR5 as an essential co-receptor for M-tropic HIV-1 is emphasized by the fact that individuals lacking CCR5 due to a homozygous basepair deletion (delta32) are highly resistant to HIV-1 infection. In contrast to other markers like CD4, CD25, or CD45R0, CCR5 is only present on a subset of lympho-cytes and other cells that are prone to HIV-1 infection (Rottmann (1997) Am J
Pathol 151, 1341-1351; Naif (1998) J Virol 72, 830-836; Lee (1999) Proc. Natl Acad.
Sci. 96, 5315-5220).
Several approaches have been postulated to eliminate latent infected cells.
One strat-egy is to drive the latently infected to virus production and subsequent cell death. In this context, one approach is IL-2 (TNF-alpha, IL-6) administration in the presence of HAART until the viral reservoir is exhausted (Chun (1998) J. Exp. Med. 188, 83-91;
Chun (1999) Nat. Med. 5, 651-655; Stellbrink (1999) Abstracts of the 6th Conference on Retroviruses and Opportunistic Infections (Foundation for Retrovirology and Human Health, Alexandria, VA), abstr. 356. p. 135; Imamichi (1999) Abstracts of the 6th Con-ference on Retroviruses and Opportunistic Infections (Foundation for Retrovirology and Human Health, Alexandria, VA), abstr. 358, p. 135). These cells are believed to die af-ter activation. Whether the entire pool of latent infected cells can be exhausted is ques-tionable.
Another strategy tried was to specifically kill latently infected cells based on gp-120 ex-pression on the cell surface. Immunotoxins recognizing gp-120 have been proposed but failed for two reasons. The one construct tested in humans was a protein consisting of soluble CD4 linked to Pseudomonas aeroginosa exotoxin A (PE). The clinical results were disappointing due to dose-limiting hepatotoxicity without showing signs of efficacy and the program was terminated (Ashorn (1990) Proc. Natl Acad. Sci 87, 8889-8893;
Berger (1998) Proc. Natl Acad. Sci. 95, 11511-11513). The second reason for failure was that latent infected cells do not express viral surface glycoproteins, e.g. gp-120 and gp-41. Thus, approaches targeting gp-120 or gp-41 for the elimination of latently infected cells cannot work.
Other approaches to eliminate latent infected cells are based on eliminating the entire CD4+ T-cell compartment (Berger (1998) Proc. Natl Acad. Sci. 95, 11511-11513), or the CD25-positive compartment (Bell (1993) Proc. Natl Acad. Sci. 90, 1411-1415) the CD45R0 memory cell compartment (McCoig (1999) Proc. Natl. Acad. Sci 96, 11482-11485). However these markers do not adequately include all potentially infected cells.
Such cells include besides CD4-posifiive cells, or memory cells also macrophages, and non-hematopoietic cells.
In WO 98/18826 an antibody directed against the mammalian (e.g. human) chemokine receptor 5 is described and said antibody is proposed in a method of inhibiting the in-teraction of cell bearing CCR5 with a potential ligand, like HIV. It is proposed that said method inhibits an HIV infection. Furthermore, treatment options for inflammatory dis-eases, autoimmune diseases and graft rejection are proposed. Yet, all these treatment options are based on the assumption that specific antibodies like the immunoglobulin molecules themselves or functional portions thereof interfere with receptor-ligand inter-actions. However, whether these antibodies are capable of depleting the relevant cells is questionable. Furthermore, WO 98/18826 merely envisages the prevention of an in-teraction of HIV and the CCR5 receptor and thereby preventing an HIV
infection.
Leukocytes, in particular T-cells, are believed to be the key regulators of the immune response to infecfiive agenfis and are critical components for fihe initiation and mainte-nance of inflammatory processes, like inflammatory bowel disease inflammatory renal diseases, inflammatory joint disease, autoimmune disorders, like multiple sclerosis and arthritis, skin diseases, like psoriatic lesions, diabetes and in transplant rejection.
Therefore, the technical problem underlying the present invention is to provide for novel means and methods which can lead to the suppression of activated leukocytes involved in immunological pathologies, .like autoimmune diseases, inflammation process and/or viral infections of immune cells.
Accordingly, the present invention relates to the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the elimination of cells which are latently infected with a primate immu-nodeficiency virus, preferably a human immunodeficiency virus, most preferably HIV-1.
In context of the present invention the binding of said antibody and/or chemokine con-struct which binds to a chemokine receptor results in the depletion and/or destruction of the target cell, namely the cell latently infected with said primate immunodeficiency vi-rus.
In this invention it could surprisingly be shown that highly specific antibodies directed against an chemokine receptor were not able to destroy, lyse and/or deplete cells which express said chemokine receptor. However, antibody constructs or chemokine constructs as described and disclosed in the present invention were capable to specifi-cally interact with said chemokine-receptor positive cells and were able to deplete said cells. Said depletion/destruction may, e.g., be achieved by the attraction of specific ef fector cells, like monocytes, macrophages, T-cells (particularly preferred are cytotoxic T-cells) or dendritic cells. Even if monoclonal antibodies had been shown to be suc-cessful in the destruction/depletion of malignant cells (see, e.g., Maloney (1999), Sem Oncol. 26, 76-78), they appear to be ineffective against certain subtypes of leukocytes, (comprising lymphocytes, polynuclear leukocytes and monocytes) especially CCR5+
monocytes, T-cells and dendritic cells as documented herein and in the appended ex-amples.
In accordance with the present invenfiion, the term "antibody andlor chemokine con-struct" (i.e. antibody construct and/or chemokine construct) not only comprises the molecules and multifunctional constructs and compounds as described herein, but also comprises functional fragments thereof. Functional fragments of said constructs are meant to be fragments which are capable of binding. to/interacting with a chemokine receptor on a target cell and providing for means for depleting, lysing andlor destroying said target cell.
Specific chemokine receptors, in accordance with the present invention comprise, but are not limited to, CXCR3, CXCR4, CXCRS, CCR1, CCR2, CCR3, CCR4, CCRS, CCR6, CCR7, CCRB, CCR9, XCR1, CCR10 and CX3CR1. Chemokines and/or che-mokine ligands binding to said chemokine receptors are well known in the art and shown, inter alia, in Table 4. Furthermore, chemokines and corresponding receptors are disclosed in Murphy (2000), Pharm. Reviews 52, 145-176. The chemokines, che-mokirie ligands and/or receptors are preferably primate, more preferably human che-mokines/ligands/receptors.
The present invention also relates to the use of an antibody and/or chemokine con-struct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the treatment, prevention and/or alleviation of inflammatory renal dis-eases, inflammatory bowel diseases, multiple sclerosis, skin diseases, allergic reac-tions diabetes or transplant rejection.
Said skin diseases comprise, inter alia, psoriatic disorders, atopic dermatitis or chroni-cally inflamed skin. CCR6 .expression is upregulated in PBMCs derived from patients with psoriasis. In addition, CCR6 ligand (CCL20=MIP3alpha) and CCR6 are upregu-lated in psoriatic skin. Furthermore, CCL20 expressing keratinocytes colocalize with skin infiltrating T-cells (Homey (2000) J. Immunol. 164, 6621-6632).
Furthermore, CCR10 was detected on melanocytes, dermal fibroblasts, dermal endothelial cells, T-cells and skin-derived Langerhans cells but not keratinocytes. CCR10 ligand (CCL27) has a skin associated expression pattern (Homey (2000) J. Immunol. 164, 3465-3470;
Charbonnier (1999) J. Exp. Med 190, 1755-1768). In addition, CCR4 and its ligand (TARC, MDC) are upregulated in chronically inflamed skin. Moreover CCR4 is a hom-ing receptor for T-cells entering the skin. CCR4+ T-cells are only a small subpopulation of all T cells and therefore depletion of CCR4+ T-cells is indicated for various inflam-matory skin diseases (Campbell (1999) Nature 400, 776-780). CCR3 and exotoxin ex-pression is enhanced in atopic dermatitis and may contribute to the initiation and maintenance of inflammation (Yawalkar (1999) J. Invest. Dermatol. 113, 43-48).
Virtually all T-cells in rheumatoid arthritis, synovial fluid and in various inflamed tissues such as ulcerative colitis, chronic vaginitis and sarcoidosis express CXCR3.
Whereas fewer T-cells within normal lymph nodes are CXCR3 positive.
For multiple sclerosis it was shown that CCR5 and CXCR3 are predominantly ex-pressed on T-cells infiltrating demyelinating brain lesions, as well as in the peripheral blood of affected patients. The corresponding ligands MIP-1a and IP-10 were also de-tectable in the plaques (Balashov (1999) Proc. Natl. Acad. Sci. 96, 6873-6878). Elimi-nation of the T-cells would block the T-cell arm of this autoimmune disease.
Immunochemical analysis of the expression of the beta-chemokine receptors in post-mortem CNS tissue from patients with multiple sclerosis revealed that in chronic active MS lesions expression of CCR2, CCR3 and CCR5 was associated with foamy macro-phages and activated microglia while low levels of these chemokine receptors were expressed by microglial cells in control CNS tissue. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes and in 5/14 cases of MS CCR3 and were also expressed on astrocytes. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS (Simpson, J. Neuroimmunol., 2000, 108, 200).
High expression of CCR3 and CCR5 was also observed in T cells and B cells of lymph nodes derived from patients with Hodgkin disease. While CCR3 was equally distributed in CD4+ and CD8+ cells, CCR5 was mainly associated with CD4+ cells. These data suggest that chemokines are involved in the formation of the nonneoplastic leukocytic infiltrates in Hodgkin disease (Buri, Blood, 2001, 97, 1543-8).
Periodontal disease is a peripheral infection involving species of gram-negative organ-isms. In patients with moderate to advanced periodontal disease CCR5 chemokine re-ceptor expressing cells were found in the inflammatory infiltrates (Gamonal, J. Perio-dontal. Res., 2001, 36, 194-203 and Taubman, Crit. Rev. Oral. Biol. Med. 2001, 12, 125-35).
Diabetes type I is considered to be a T-cell mediated autoimmune disease. The ex-pression of CCR5 receptor in the pancreas was associated with the progression of type I diabetes in relevant animal models (Cameron (2000) J. Immunol. 165, 1102-1110). In particular, the CCR5 expression was associated with the development of insulinitis and spontaneous type I diabetes.
Specific chemokines are associated with T-cell migration in diabetes type I
relevant animal model: RANTES, MCP-1, MCP-3, MCP-5, IP10. These chemokines lead to a th1 immune response (Bradley (1999) J. Immunol. 162:2511-2520).
The above mentioned inflammatory bowel disease may comprise Morbus Crohn and colitis ulcerosa.
CCR9 is expressed on T-cells homing to the intestine and may be implied in Morbus Crohn and colitis ulcerosa. All intestinal lamina propria and intraepithelial lymphocytes express CCR9 (Zabel (1999) J. Exp. Med. 190, 1241-1256).
Additionally, the antibody- and/or chemokine construct as described in context of the present invention is also useful for avoiding complications during and/or after trans-plants, i.e. to avoid transplant rejections and graft versus host disease.
CCR7 is expressed on naive T-cells and dendritic cells and mediates cell migration to lymphatic organs. Elimination of CCR7+ cells would therefore prevent an immune re-sponse to novel antigens, e.g., following transplantation. Such a treatment would not be generally immune suppressing but selective for novel antigens and limited for the dura-tion of the adminisfiration of drugs of the invention depleting CCR7+ cells (Forster (1999) Cell 99, 23-33). CXCR5 is expressed on naive B cells in the peripheral blood and tonsils and memory T-cells. Elimination of CXCRS+ B-cells would prevent the es-tablishment of a humoral response. Furthermore, elimination of memory T-cells would reduce the cellular component of the immune response (Murphy (2000) Pharmacologi-cal Reviews 52, 145-176).
In order to provide pharmaceutical compositions for the treatment of allergies and/or allergic reactions, the antibody- and/or chemokine constructs as described herein may be employed. It was shown that CCR3 which binds exotoxin and RANTES, is ex-pressed on eosinophils, Th2 cells, mast cells, basophils, which are involved in allergic reactions (Romangnani (1999) Am. J. Pathol. 155, 1195-1204).
As far as the above mentioned renal or kidney diseases are concerned, it has been shown that CCR5 positive T-cells may play a role in interstitial processes leading to fibrosis. CCR5 positive cells have been identified in the interstitial infiltrate of various glomevular and interstitial diseases, as well as transplant rejection. Said disease com-prises acute and chronic nephritis, IgA nephropathy, and others (Segerer (1999), Kid-ney Int. 56, 52-64).
In a model of transient immune complex glomerulonephritis (IC-GN), CCR1, CCR2, and CCR5 were expressed early and were already downregulated at the peak of proteinuria and leukocyte infiltration. Expression of CCR5 was located to the glomerulus by in situ hybridization and quantitative reverse transcription-PCR of isolated glomeruli (Anders, J. Am. Soc. Nephrol., 2001, 12, 919-31 ). In kidneys of 38 patients with several renal diseases, CCR1- and CCRS-positive macrophages and T cells were detected in both glomeruli and interstitium as shown by immunohistochemistry. Renal CCRS-positive cells were dramatically decreased during convalescence induced by glucocorticoids (Furuichi, Am. J. Nephrol., 2000, 20, 291-9).
In a preferred embodiment of the present invention, the invention provides for the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition as described hereinabove, wherein said chemokine receptor is the chemokine receptor 5 (CCRS). It is preferred that said CCR5 is the human CCRS.
The chemokine receptor CCRS is a member of a large family of G protein coupled seven transmembrane domain receptors that binds the proinflammatory chemokines RANTES, MIP1-a, MIP1-f3 and MCP-2. Chemokines act in concert with adhesion molecules to induce the extravasation of leukocytes and to direct their migration to sites of tissue injury.
The CCR5 is expressed on a minority of T-cells and monocytes and is further the major co-receptor for M-trophic HIV-1 strains that predominate early in the course of an HIV-infection.
The pharmaceutical composition as described hereinabove is, therefore, particularly useful in the depletion of CCRS+ leukocytes and would be useful in the elimination of cells latently infected with HIV-1. Depletion of CCR5+ cells should therefore reduce the number of cells latently infected with HIV and should be particularly useful in combina-tion with active anti-viral, preferably anti-retroviral therapy.
In a particularly preferred embodiment said antibody construct is a bispecific antibody which binds to the chemokine receptor, preferably the CCRS, as a first antigen and a CD3 antigen of an effector cell as a second antigen. Preferably said CD3 antigen is on the surface of a T-cell, preferably a cytotoxic T-cell. Said CD3 therefore, denotes an antigen that is expressed on the above mentioned cells and may be part of the multi-molecular (T-) cell receptor complex.
Bispecific antibodies may be constructed by hybrid-hybridoma techniques, by cova-lently linking specific antibodies or by other approaches, like the diabody approach (Ki-priyanow, Int. J. Cancer 77 (1998), 763-773).
It is preferred that said bispecific antibody is a single chain antibody construct.
As is well known, Fv, the minimum antibody fragment which contains a complete anti-gen recognition and binding site, consists of a dimer of one heavy and one light chain variable domain (VF., and V~) in non-covalent association. In this configuration that cor-responds to the one found in native antibodies the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-V~ dimer. Collectively, the six CDRs confer antigen binding speci-ficity to the antibody. Frameworks (FRs) flanking the CDRs have a tertiary structure which is essentially conserved in native immunoglobulins of species as diverse as hu-man and mouse. These FRs serve to hold the CDRs in their appropriate orientation.
The constant domains are not required for binding function, but may aid in stabilizing VH-V~ interaction. Even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than an entire binding site (Painter, Biochem. 11 (1972), 1327-1337). Hence, said domain of the binding site of the antibody construct as defined and described in the present invention can be a pair of VH-V~, VH-VH or V~-V~
domains of different immunoglobulins. The order of VH and V~ domains within the polypeptide chain is not decisive for the present invention, the order of domains given hereinabove may be reversed usually without any loss of function. It is important, however, that the Vt., and V~ domains are arranged so that the antigen binding site can properly fold.
Different parts of the antibodies/immunoglobulins can be joined by means of conven-tional methods or constructed as a contiguous protein by means of recombinant DNA
techniques, e.g. in such a way that a nucleic acid molecule coding for a chimeric or humanized antibody chain is expressed in order to construct a contiguous protein (cf.
for example Mack et al. (1995) Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 7021-7025).
A single-chain antibody with the following Fv fragments is preferred: sc-Fv fragment of a monoclonal antibody against the chemokine receptor, preferably against CCRS, and an sc-Fv fragment of a monoclonal antibody against CD3. In this case, both the Fv fragment directed against the chemokine receptor and the Fv fragment against may be located in N-terminal position. The Fv fragment against CCR5 is preferred to be in N-terminal position. The order of the VL and VH antibody domains can be variable in both constructs, preferably, the order of the Fv fragment against CCR5 is VL-VH and the one of the Fv fragment against CD3 is VH-VL. The linkers between the variable domains as well between the two Fv fragments may consist of peptide linkers, prefera-bly of a hydrophilic flexible glycine- and serine-containing linker of 1 to 25 amino acids.
An additional histidine chain of ,e.g., 6 x His, in C- or N-terminal position can be used to simplify purification and detection.
Compared to conventional bispecific antibodies, bispecific single-chain antibodies have the advantage that they consist of only one protein chain and thus their composition is exactly defined. They have a low molecular weight of normally < 60 kD and can be pro-duced easily and on a large scale in suitable cell lines, e.g. in CHO cells, using recom-binant techniques. The most essential advantage, however, is that they have no con-stant antibody domains and thus only activate T-lymphocytes to lysis when these are bound to their target cells, i.e. to the chemokine-receptor expressing cells.
Therefore, single-chain antibodies are often superior to conventional bispecific antibodies as their clinical use entails fewer or.less severe side effects.
MC-1 was shown to bind specifically to the first part of the second extracellular loop of human CCR5 and did not crossreact with CCR5 derived from rhesus macaques as shown in the appended examples. Therefore, it is preferred that said single chain anti-body construct comprises V~ and VH domains of a antibody specific for the chemokine receptor, preferably the human CCRS, and VH and V~ domains of an antibody specific for a CD3 antigen. Said antibody specific for the human CCR5 is the murine anti-human CCR5 antibody MC-1, described, inter alia, in Mack (1998), J. Exp. Med.
187, 1215-1224 and in the appended examples. Yet, it is envisaged that other a-CCR5 anti-bodies, like MC-5 (as characterized in the appended examples and disclosed in Segerer (1999), loc. cit.) may be employed in the context of this invention.
The antibody specific for a CD3 antigen may be selected from the group consisting of antibodies rec-ognizing the gamma, delta, epsilon, zeta chains, particularly preferred are antibodies recognizing the epsilon chain and the CD3 zeta chain (Jakobs (1997) Cancer Immunol Immunother. 44, 257-264; Mezzanzanica (1991 ) Cancer Res 51, 5716-5721 ). Exam-ples of anti-epsilon chain antibodies are OKT3 (WO 91/09968, Kung et al., Science 206, 347-349 (1979); Van Wauwe, J. Immunol. 124, 2708-2713 (1980); Transy, Eur. J.
Immunol. 19, 947-950 (1989); Woodle, J. Immunol. 148, 2756-2763 (1992); Ada, Hu-man. Antibod. Hybridomas, 41-47 (1994)) and TR66 (Traunecker (1991 ) EMBO J.
10, 3655-3659). Examples of monoclonal antibodies against the CD3 zeta chain are H2D9, TIA2 (both Becton Dickinson), G3 (Serotec Ltd.).
In a particularly preferred embodiment of the use of the present invention, the V~ and VH domains of the single chain antibody as described above are arranged in the order V~(MC-1 )-VH(MC-1 )-VH(CD3)-V~(CD3), whereby it is particularly preferred that V~(MC-1) comprises the amino acid sequence as depicted in SEQ ID NO: 12, wherein said VH(MC-1) comprises the amino acid sequence as depicted in SEQ ID NO: 16, wherein said VH(CD3) comprises the amino acid sequence as depicted in SEQ ID NO: 26 and/or wherein said V~(CD3) comprises in SEQ ID NO: 28. Specific CDR parts of the MC-1 antibody are shown in SEQ ID NO: 29 to 34, wherein SEQ ID NO: 29 shows the CDR1 of V~ MC-1, SEQ ID NO: 30 shows the CDR2 of V~ MC-1, SEQ ID NO: 31 shows the CDR3 of V~-MC-1, SEQ ID NO: 32 shows the CDR1 of VH MC-1, SEQ ID NO: 33 shows the CDR2 of VH MC-1 and SEQ ID NO: 34 depicts the CDR 3 of VH MC-1.
Said bispecific antibody may, inter alia, comprise an amino acid sequence encoded by the nucleic acid sequence as depicted in SEQ ID NO: 17 or comprises the amino acid sequence as depicted in SEQ ID NO: 18.
In another embodiment of the use of the present invention, the antibody construct is a bispecific antibody which binds to said chemokine receptor as a first antigen and a toxin as a second antigen. The antibody may be covalently bound to said toxin, and said antibody-toxin construct may be constructed by chemical coupling, producing a fusion protein or a mosaic protein from said antibody and from a modified or unmodified prokaryotic or eukaryotic toxin. Furthermore, said antibody may be joined to said toxin via additional multimerization domains.
In a further embodiment of the use of the present invention said antibody construct can, via a multimerization domain, be bound in vitro and/or in vivo to a second antibody construct which binds to a CD3 antigen and/or a toxin. Said multimerization may, inter alia, be obtained via hetero(di)merization. For example, the hetero(di)merization region of constant immunoglobulin domains may be employed. Other multi- and/or heterodi-merization domains are known in the art and are based on leucine zippers, a-and f3-chains of T-cell receptors or MHC-class II molecules. Furthermore, jun- and fos-based domains may be employed (de Kuif (1996) J. Biol. Chem. 271, 7630-7634;
Kostelny (1992), J. Immunol. 148, 1547-1553). Additional examples of multimerization domains are p53- and MNT-domains as described in Sakamoto (1994) Proc. Natl. Acad.
Sci.
USA 91, 8974-8978; Lee (1994) Nat. Struct. Biol. 1, 877-890; Jeffrey (1995) Science 267, 1498-5102 or Nooren (1999) Nat. Struct. Biol. 6, 755-759).
In another embodiment of the invention, the above mentioned chemokine construct is a fusion construct of a modified or an unmodified chemokine with a modified or an un-modified toxin. Said construct may, inter alia via a multimerization domain, be bound in vitro and/or in vivo to an antibody construct which binds to a CD3 antigen and/or to a toxin. Suitable multimerization domains have been described in the art and are men-tioned hereinabove. The chemokine-toxin constructs may, inter alia, result from chemi-cal coupling, may be recombinantly produced (as shown in the appended examples), or may be produced as a fusion protein from a chemokine and a modified or unmodified prokaryofiic or eukaryotic toxin. It is particularly preferred that said chemokine binds to the human chemokine receptor CCR5 and comprises, inter alia, RANTES, MIP-1a, MIP-1 f3, MCP-2, MCP-3 or (a) fragments) thereof which are capable of binding to said receptor. A preferred toxin may be a truncated version of pseudomonas exotoxin, like PE38, PE40 or PE37. Most preferred, in context of this invention, is PE38.
Furthermore, and in accordance with the present invention, said chemokine construct may comprise the chemokine covalently bound to an antibody construct which binds to an antibody construct capable of binding to a CD3 antigen and/or which is a covalently bound to a toxin.
In a particularly preferred embodiment of the use of the present invention, the antibody and/or chemokine construct is a heterominibody construcfi comprising at least an anti-body and/or a chemokine which binds to a chemokine receptor, preferably to the receptor, most preferably to the human CCRS receptor. Said heterominibody construct may comprise at least one toxin and it is particularly preferred that said heterominibody construct binds to the chemokine receptor as defined hereinabove and/or to a CD3 an-tigen of an effector cell. Preferred chemokines are the chemokines mentioned herei-nabove and preferred toxins are the toxins described hereinabove, which may be modified or unmodified. Chemokines are well known in the art and described, inter alia, in Murphy (1999), loc. cit. Therefore, it is preferred that the chemokine is selected from the group consisting of RANTES, MIP-1f3, MIP-1a, MCP-2, and MCP-3 or a functional fragment thereof. The most preferred chemokine, in context of this invention is RAN-TES. Functional fragments of said chemokines are fragments which are capable of binding to or interacting with said chemokine receptor, preferably the human CCRS.
Heterominibodies are known in the art and their production is, inter alia, described in 5. Said heterominibody may be a multifunctional compound comprising at least one antibody and/or chemokine binding to or interacting with a chemokine recep-tor, preferably human CCRS, may (additionally) comprise a toxin as defined hereinbe-low and/or a binding site for the CD3 antigen.
In a preferred embodiment, the antibody- or chemokine construct to be used in the pre-sent invention is a fusion (poly)peptide or a mosaic (poly)peptide. Said fusion (poly)peptide may comprise merely the domains of the constructs as described herein above as well as (a) functional fragments) thereof. However, it is also envisaged that said fusion (poly)peptide comprises further domains and/or functional stretches.
Therefore, said fusion (poly)peptide can comprise at least one further domain, said domain being linked by covalent or non-covalent bonds. The linkage as well as the construction of such constructs, can be based on genetic fusion according to the meth-ods known in the art (Sambrook et al., loc. cit., Ausubel, "Current Protocols in Molecu-lar Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO
94/04686. The additional domain present in the construct may preferably be linked by a flexible linker, advantageously a (poly)peptide linker, wherein said (poly)peptide linker preferably comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the peptide, (poly)peptide or antibody or vice versa. Said linker may, inter alia, be a Glycine, a Serine and/or a Glycine/Serine linker. Additional linkers comprise oligomeri-zation domains. Oligomerization domains facilitate the combination of two or several autoantigens or fragments thereof in one functional molecule. Non-limiting examples of oligomerization domains comprise leucine zippers (like jun-fos, GCN4, E/EBP;
Kos-telny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sci. USA 94 (1997), 3673-3678, Williams, Genes Dev. 5 (1991 ), 1553-1563;Suter, "Phage Display of Pep-tides and Proteins", Chapter 11, (1996), Academic Press), antibody-derived oligomeri-zation domains, like constant domains CH1 and CL (Mueller, FEBS Letters 422 (1998), 259-264) and/or tetramerization domains like GCN4-LI (Zerangue, Proc. Natl.
Acad.
Sci. USA 97 (2000), 3591-3595).
Furthermore, the antibody- or chemokine construct to be used in the present invention or as described hereinbelow may comprise at least one further domain, inter alia, do-mains which provide for purification means, like, e.g. histidine stretches.
Said further domains) may be linked by covalent or non-covalent bonds.
The linkage can be based on genetic fusion according to the methods known in the art and described herein or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686. The additional domain present in the construct as described and disclosed in the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker to one of the binding site domains wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of one of said domains and the N-terminal end of the other of said domains when said polypeptide assumes a conformation suit-able for binding when disposed in aqueous solution. Preferably, said polypeptide linker is a polypeptide linker as described in the embodiments hereinbefore. The polypeptide of the invention may further comprise a cleavable linker or cleavage site for protein-ases, such as enterokinase It is also envisaged that said constructs disclosed for uses, compositions and methods of the present invention comprises (a) further domains) which may function as immu-nomodulators. Said immunomodulators comprise, but are not limited to cytokines, lym-phokines, T cell co-stimulatory ligands, etc.
Adequate activation resulting in priming of naive T-cells is critical to primary immunore-sponses and depends on two signals derived from professional APCs (antigen-presenting cells) like dendritic cells. The first signal is antigen-specific and normally mediated by stimulation of the clonotypic T-cell antigen receptor that is induced by processed antigen presented in the context of MHC class-I or MHC class-II
molecules.
However, this primary stimulus is insufficient to induce priming responses of naive T-cells, and the second signal is required which is provided by an interaction of specific T-cell surface molecules binding to co-stimulatory ligand molecules on antigen pre-senting cells, further supporting the proliferation of primed T-cells. The term "T-cell co-stimulatory ligand" therefore denotes in the light of the present invention molecules, which are able to support' priming of naive T-cells in combination with the primary stimulus and include, but are not limited to, members of the B7 family of proteins, in-cluding B7-1 (CD80) and 137-2 (CD86).
The antibody- and/or chemokine construct defined herein above or described herein-below may comprise further receptor or ligand function(s), and may comprise immuno-modulating effector molecule or a fragment thereof. An immuno-modulating effector molecule positively and/or negatively influences the humoral and/or cellular immune system, particularly its cellular and/or non-cellular components, its functions, and/or its interactions with other physiological systems. Said immuno-modulating effector mole-cule may be selected from the group consisting of cytokines, chemokines, macrophage migration inhibitory factor (MIF; as described, inter alia, in Bernhagen (1998), Mol Med 76(3-4); 151-61 or Metz (1997), Adv Immunol 66, 197-223), T-cell receptors and solu-ble MHC molecules. Such immuno-modulating effector molecules are well known in the art and are described, inter alia, in Paul, "Fundamental immunology", Raven Press, New York (1989). In particular, known cytokines and chemokines are described in Meager, "The Molecular Biology of Cytokines" (1998), John Wiley & Sons, Ltd., Chich-ester, West Sussex, England; (Bacon (1998). Cytokine Growth Factor Rev 9(2):167-73;
Oppenheim (1997). Clin Cancer Res 12, 2682-6; Taub, (1994) Ther. Immunol. 1 (4), 229-46 or Michiel, (1992). Semin Cancer Biol 3(1 ), 3-15).
Antibody and/or chemokine constructs as disclosed and described in the present in-vention and comprising (an) additional functional domains) may, inter alia, be multi-functional compounds, like heterominibodies, as described herein below.
The constructs to be used in the present invention or described herein may be con-structs which comprise domains originating from one species, preferably from mam-mals, more preferably from human. However, chimeric and/or humanized constructs are also envisaged and within the scope of the present invention.
In a particular preferred embodiment, the composition of the invention comprises a constructs to be used in the present invention or described herein is a cross-linked (poly)peptide construct. As mentioned herein, said cross-linking may be based on methods known in the art which comprise recombinant as well as biochemical methods.
In a yet further embodiment of the use of the present invention, the antibody construct or the chemokine construct to be used comprises at least one toxin. Said toxin may be Pseudomonas exotoxin A, diphtheria toxin and similar toxins. It is envisaged that trun-cated toxins are employed, like the PE38 or the PE40 of Pseudomonas toxin described in the appended examples.
Said toxin may be bound to said antibody or chemokine by means as described herei-nabove. It is also envisaged that said toxin is bound to the antibody/chemokine by means of a short peptide linker. The linker preferably consists of a flexible and hydro-philic amino acid sequence, in particular of glycines and serines. Preferably said linker has a length of 1 to 20 amino acids.
Several fusion proteins with a truncated version of Pseudomonas exotoxin A
have been designed so far. Most of them have been used to target and destroy malignant cells.
This toxin becomes activated upon proteolytic cleavage. A truncated version of the toxin (PE38) may be employed for the constructs of the present invention, as the full-length protein binds with its fist domain to the ubiquitous a2-macroglobulin receptor and is therefore toxic to most eukaryotic cells. Yet, this problem may be overcome by re-placing the first domain of Pseudomonas exotoxin A by a specific sequence in order to alter the binding specificity of the toxin.
Furthermore, the present invention relates to the use of a chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the elimination of cells which are latently infected with a primate immunodeficiency vi-rus wherein said chemokine construct comprises a amino acid sequence as depicted in SEQ ID NO: 24 or as encoded by the nucleotide sequence as depicted in SEQ ID
NO: 23.
As mentioned hereinabove, and in a preferred embodiment, the antibody and/or che-mokine constructs to be used within the scope of the present invention bind to or inter-act with the CD3 antigen. Preferably said CD3 antigen is on the surFace of an effector cell, namely a T-cell, preferably a cytotoxic T-cell.
It is particularly preferred that an antibody construct be used wherein said construct comprises a binding site for CCR5 and a binding site for CD3 and that a chemokine construct be used, wherein said chemokine construct comprises RANTES and said toxin is a truncated Pseudomonas exotoxin A (PE38).
The present invention, therefore also relates to antibody constructs comprising a bind-ing site for CCR5 and a binding site for CD3 as well as to chemokine constructs com-prising RANTES and the truncated Pseudomonas exotoxin A (PE38).
The present invention also relates to a polynucleotide encoding an antibody-construct as defined hereinabove or a polynucleotide encoding a chemokine construct as defined herein, wherein said polynucleotide is a polynucleotide comprising the nucleic acid molecule in particular encoding the polypeptide as depicted in SEQ ID NO: 18 or SEQ
ID NO: 24; a polynucleotide comprising the nucleic acid molecule as depicted in SEQ
ID NO: 17 or SEQ ID NO: 23; or (c) a polynucleotide hybridizing under stringent condi-tions to the complementary strand of a polynucleotide of (a) or (b).
With respect to the polynucleotides/nucleotide sequences characterized under (c) above, the term "hybridizing" in this context is understood as referring to conventional hybridization conditions, preferably such as hybridization in 50%formamide/6xSSC/0.1 %SDS and 1 OOpg/ml ssDNA, in which temperatures for hy-bridization are above 37°C and temperatures for washing in 0.1xSSC/0.1%SDS are above 55°C. Most preferably, the term "hybridizing" refers to stringent hybridization conditions, for example such as described in Sambrook., "Molecular Cloning: A
Labo-ratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. It is envisaged that the polynucleotides characterized under (c) above are highly homologous to the polynucleotides as defined in (a) and/or (b) and comprise a homol-ogy of at least 95%, more preferably of at least 97%, and most preferably 99%
with the polynucleotides of (a) and/or (b).
Polynucleotides as defined and characterized under (c), therefore may encode for polypeptides being highly homologous to the polypeptides as defined in (a) and (b).
The person skilled in the art can easily test the capacity of such homologous polypep-tides to bind to chemokine receptors, in particular to the human CCR5 receptor and/or to eliminate, deplete and/or destroy cells, for example, cells which are infected by a primate immunodeficiency virus, like HIV-1, or eliminate, deplete and/or destroy target cells involved in immunological disorders or disclosed herein. The person skilled in the art can easily adopt the in vitro, in vivo and ex vivo experiments of the appended ex-amples to verify the binding and/or depletion properties of such constructs.
Furthermore, said polynucleotide/nucleic acid molecule may contain, for example, thio-ester bonds and/or nucleotide analogues. Said modifications may be useful for the sta-bilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
The polynucleotide/nucleic acid molecule of the composition of the present invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination Said polynucleotide may be, e.g., DNA, cDNA, RNA or synthetically produced DNA
or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. Preferably said polynucleotide is part of a vector. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable condi-tions. Preferably, the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mam-malian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory ele-ments may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E.
coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of tran-scription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art; see also, e.g., the appended examples. The leader sequences) is (are) assembled in appropriate phase with translation, initiation and termination se-quences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Op-tionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product; see supra. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA
expres-sion vector pcDV1 (Pharmacia), pCDMB, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), or pSPORT1 (GIBCO BRL).
Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming of transfecting eukaryotic host cells, but control se-quences for prokaryotic hosts may also be used. Once the vector has been incorpo-rated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purifi-cation of the polypeptide of the invention may follow; see, e.g., the appended exam-ples.
As described above, the polynucleotide of the invention can be used alone or as part of a vector to express the antibody- and/or chemokine constructs to be used in the inven-tion or in cells, for, e.g., the treatment of immunological disorders or in anti-viral ther-apy. The polynucleotides or vectors containing the DNA sequences) encoding any one of the above described polypeptides is introduced into the cells which in turn produce the polypeptide of interest. Therefore, said polynucleotides and vectors may be used for gene therapy. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivery systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art;
see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239;
Is-ner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086;
Ono-dera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann.
N.Y.
Acad. Sci. 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51;
Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, US 5,580,859; US
5,589,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and refer-ences cited therein. The polynucleotides and vectors of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g., adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell. An example for an embryonic stem cell can be, inter alia, a stem cell as described in, Nagy, Proc. Natl.
Acad. Sci. USA 90 (1993), 8424-8428.
In accordance with the above, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engi-neering that comprise a polynucleotide encoding a polypeptide of the invention. Pref-erably, said vector is an expression vector and/or a gene transfer or targeting vector.
Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell populations.
Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the polynucleotides and vectors of the invention can be re-constituted into liposomes for delivery to target cells. The vectors containing the poly-nucleotides of the invention can be transferred into the host cell by well-known meth-ods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, su-pra. Once expressed, the polypeptides of the present invention can be purified accord-ing to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Pro-tein Purification", Springer-Verlag, N.Y. (1982). Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically (including extracorpore-ally)~or in developing and performing assay procedures.
In a still further embodiment, the present invention relates to a cell containing the poly-nucleotide or vector described above or to a host transformed with the vector of the invention. Preferably, said host/cell is a eukaryotic, most preferably a mammalian cell if therapeutic uses of the polypeptide are envisaged. Of course, yeast and less preferred prokaryotic, e.g., bacterial cells may serve as well, in particular if the produced poly-peptide is used as a diagnostic means.
The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromoso-mally.
The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of a polypeptide of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacte-ria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is meant to include yeast, higher plant, insect and pref erably mammalian cells. Depending upon the host employed in a recombinant produc-tion procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methio-nine amino acid residue. A polynucleotide coding for a polypeptide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a plasmid or a virus containing the coding sequence of the polypeptide of the invention and geneti-cally fused thereto an N-terminal FLAG-tag and/or C-terminal His-tag.
Preferably, the length of said FLAG-tag is about 4 to 8 amino acids, most preferably 8 amino acids.
Methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
The genetic constructs and methods described therein can be utilized for expression of the polypeptide of the invention in eukaryotic or prokaryotic hosts. In general, expres-sion vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as spe-cific genes which are capable of providing phenotypic selection of the transformed cells. Furthermore, transgenic animals, preferably mammals, comprising cells of the invention may be used for the large scale production of the antibody- andlor chemokine construct of the invention. It is most preferred that said transgenic animal produces the antibody construct of the invention.
In a further embodiment, the present invention thus relates to a process for the prepa-ration of a polypeptide described above comprising cultivating a (host) cell of the inven-tion under conditions suitable for the expression of the antibody- and/or chemokine construct and isolating the polypeptide from the cell or the culture medium.
The transformed hosts can be grown in fermentors and cultured according to tech-niques known in the, art to achieve optimal cell growth. The produced constructs of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., microbially expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against a tag of the polypeptide of the invention or as described in the appended examples.
Depending on the host cell, renaturation techniques may be required to attain proper conformation. If necessary, point substitutions seeking to optimize binding may be made in the DNA using conventional cassette mutagenesis or other protein engineering methodology such as is disclosed herein. Preparation of the polypeptides of the inven-tion may also be dependent on knowledge of the amino acid sequence (or corre-sponding DNA or RNA sequence) of bioactive proteins such as enzymes, toxins, growth factors, cell difFerentiation factors, receptors, anti-metabolites, hormones or various cytokines or lymphokines. Such sequences are reported in the literature and available through computerized data banks.
The present invention further relates to an antibody construct or the chemokine con-struct encoded by the polynucleotide as described hereinabove or produced by the method described hereinabove.
Furthermore, the constructs of the invention can be used in the management of immu-nological disorders, in particular autoimmune diseases, allergic diseases, inflammatory diseases and AIDS (HIV-infection), as documented in the appended examples.
Additionally, the present invention provides for compositions comprising the polynu-cleotide, the vector, the host, the antibody construct and/or the chemokine construct of the invention.
The term "composition", in context of this invention, comprises at least one polynucleo-tide, vector, host, antibody construct and/or chemokine construct as described herein.
Said composition, optionally, further comprises other molecules, either alone or in com-bination, like e.g. molecules which are capable of modulating and/or interfering with the immune system. The composition may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solutions) or (an) aerosol(s). In a preferred embodiment, said composition comprises at least two, preferably three, more preferably four, most preferably compounds as described in the invention.
Preferably, said composition is a pharmaceutical composition further comprising, op-tionally, a pharmaceutically acceptable carrier, diluent and/or excipient.
Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.
Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, in-traperitoneal, subcutaneous, intramuscular, topical or intradermal administration. Intra-venous administration is particularly preferred. The dosage regiment will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concur-rently. Generally, the regimen as a regular administration of the pharmaceutical com-position should be in the range of 1 pg to 10 mg units per day. If the regimen is a con-tinuous infusion, it should also be in the range of 1 pg to 10 mg units per kilogram of body weight per minute, respectively. However, a more preferred dosage for continu-ous infusion might be in the range of 0.01 p,g to 10 rng units per kilogram of body weight per hour. Particularly preferred dosages are recited herein below.
Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 102 copies of the DNA
molecule. The compositions of the invention may be administered locally or systemati-cally. Administration will generally be parenterally, e.g., intravenously; yet external ad-ministration is also envisaged. DNA may also be administered directed to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chlo-ride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient re-plenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention might comprise proteinaceous car-riers, like, e.g., serum albumin or immunoglobuline, preferably of human origin. Fur-thermore, it is envisaged that the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. Such agents might be drugs acting on the immunological system, drugs used in anti-viral treatment, in particular in HIV-treatment (for example, HAART) and AIDS management and/or anti-inflammatory drugs. It is, for example, en-visaged that patients are treated as early as possible with HAART until viral load is be-low detection level for several weeks to months. Early treatment of infected patients with HAART prevents the transition of viral strains from usage of CCR5 to other che-mokine receptors, like CXCR4 (Connor (1997) J. Exp. Med. 185, 621-628).
Constructs as disclosed in the present invention, for example, the CCR5xCD3 construct is admin-istered in addition to HAART to eliminate latently infected cells as well as cells that are prone to reinfection by HIV-1. The depletion of CCR5+ cells is repeated 1 to 10 times.
Doses of CCR5xCD3 are in the range of 0.5~.g/m2 to 10mg/m2, preferably 10~,g/m2 to 100~.g/m2. Doses can be administered intravenously, subcutaneously and/or into the cerebra-spinal fluid. After several treatment cycles with the bispecific antibody HAART
is discontinued and viral load is closely monitored. If viral load increases above detec-tion level, a new cycle of HAART and the bispecific antibody is initiated as described above.
It is envisaged by the present invention that the various polynucleotides and vectors of the invention are administered either alone or in any combination using standard vec-tors and/or gene delivery systems, and optionally together with a pharmaceutically ac-ceptable carrier or excipient. Subsequent to administration, said polynucleotides or vectors may be stably integrated into the genome of the subject. Preferably said sub-ject is a human.
On the other hand, viral vectors may be used which are specific for certain cells or tis-sues and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art. The pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delaying of different kinds of immuno-logical diseases, which may be related to inflammation, in particular inflammatory bowel diseases, inflammatory renal diseases, inflammatory joint diseases like (chronic) arthri-tis. Furthermore, the pharmaceutical composition of the present invention may be em-ployed to eliminate cells which are latently infected with a virus, preferably a primate immunodeficiency virus, more preferably with HIV(-1 ).
Furthermore, it is possible to use a pharmaceutical composition of the invention which comprises polynucleotide or vector of the invention in gene therapy. Suitable gene de-livery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991 ), 2726-2729).
Further methods for the delivery of nucleic acids comprise particle-mediated gene transfer as, e.g., described in Verma, Gene Ther.15 (1998), 692-699.
It is to be understood that the introduced polynucleotides and vectors express the gene product after introduction into said cell and preferably remain in this status during the lifetime of said cell. For example, cell lines which stably express the polynucleotide un-der the control of appropriate regulatory sequences may be engineered according to methods well known to those skilled in the art. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the polynu-cleotide of the invention and a selectable marker, either on the same or separate plas-mids. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
The selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl. Acad.
Sci. USA
77 (1980), 3567; O'Hare, Proc. Natl. Acad. Sci. USA 78 (1981 ), 1527), gpt, which con-fers resistance to mycophenolic acid (Mulligan, Proc. Natl. Acad. Sci. USA 78 (1981 ), 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, J. Mol. Biol. 150 (1981 ), 1 ); hygro, which confers resistance to hygromycin (Santerre, Gene 30 (1984), 147); or puromycin (pat, puromycin N-acetyl transferase).
Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, hisD, which allows cells to utilize histinol in place of his-tidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); and ODC
(ornithine de-carboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McCologue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).
In a further embodiment, the present invention relates to a composition, preferably a pharmaceutical composition, as described hereinabove, which further comprises a me-dicament for the treatment of an immunological disorder or a medicament for anti-HIV
treatment.
Said anti-HIV treatment may comprise HAART. HAART therapy consists of a cocktail of three classes anti-viral drugs. The classes are nucleosidal reverse transcriptase inhibi-tors (NRTI), non-nucleosidal reverse transcriptase inhibitors (NNRTI) and protease in-hibitors (PI). Usually 2 to 4 drugs from preferentially more than one class are combined to reduce viral load to almost non-detectable levels. Products, dosing schedules and common side effects are given in appended Tables I-III.
Said treatment of an immunological disorder may comprise anti-inflammatory agents and immunosuppressive agents. Anti-inflammatory agents may be selected from the group consisting of azathioprine, cyclophophamide, glucocorticoids like prednisone and corticosteroids. Immunosuppressive agents may comprise cyclosporin A, Tacrolimus (FK506), Sirolimus (Rapamycin). Protein drugs may comprise calcineurin, beta-interferon, anti-TNF alpha monoclonal antibodies (remicade). Dosing and use of anti-inflammatory agents and immunosuppressive agents is described, inter alia, in Fauci et al., sic. Further treatment options are known to the skilled artisan and, inter alia, de-scribed hereinabove.
In a particularly preferred embodiment, the present invention relates to a method for treating, preventing and/or alleviating an immunological disorder or for the elimination cells which are latently infected with a primate immunodeficiency virus comprising ad-ministering to a subject in need of such a treatment, prevention and/or alleviation an effective amount of the compounds and/or compositions. preferably the pharmaceutical compositions of the present invention.
The constructs described herein are particularly useful in specifically destroying che-mokine receptor positive cells. For example, a bispecific antibody, binding simultane-ously to CCR5 on target cells and to CD3 on T-cells, redirects cytotoxic T-cells to the CCR5 positive target cells. As shown in the appended examples the antibody construct specifically depletes CCR5 positive T-cells and monocytes, but is inactive against cells that do not express CCR5 such as CCRS deficient X32/032 PBMC. Furthermore, in vitro/ex vivo experiments the bispecific antibody construct eliminated more than 95% of CCR5 positive monocytes and T-cells from the synovial fluid of patients with arthritis.
Other constructs, like chemokine constructs, for example, a fusion protein of the che-mokine RANTES and a truncated version of the Pseudomonas exotoxin A (PE38) are able to bind to CCR5 and to downmodulate the receptor from the cell surface as exem-plified in the appended examples. Within 48 h RANTES-PE38 completely destroyed CCR5 positive CHO cells at a concentration of 2 nM. No cytotoxic effect was detectable against CCR5 negative CHO cells.
Based on the predominance of CCRS positive T-cells and monocytes in the infiltrate of chronically inflamed tissue, the specific depletion of CCR5 positive cells represents a new concept in the treatment of immunological disorders.
As described hereinabove, due to the fact that specific chemokine receptors are pres-ent on HIV-infected cells, namely CCRS, the compounds and compositions of the in-vention are particularly useful for the depletion/elimination of cells latently infected with primate immunodeficiency virus.
The present invention also relates to the use of the polynucleotide, the vector, the host, the antibody construct and/or the chemokine construct of the present invention for the preparation of a pharmaceutical composition for treating, preventing and/or alleviating an immunological disorder or for the preparation of a pharmaceutical composition for eliminating latently infected cells, wherein said cells are infected with a primate immu-nodeficiency virus, line a human immunodeficiency virus, in particular HIV-1.
Said immunological disorders may be autoimmune diseases, skin diseases, allergic diseases, inflammatory diseases, diabetes and transplant rejections, wherein said autoimmune disease is selected from the group consisting of multiple sclerosis, type I
diabetes, rheumatoid arthritis. Said skin diseases, may comprise psoriatic lesions, pso-riasis, atrophic dermatitis and the like. Inflammatory disease are mentioned herei-nabove is selected from the group consisting of inflammatory joint diseases, inflamma-tory renal diseases, inflammatory bowel diseases. In particular, said inflammatory bowel disease may comprise Morbus Crohn, sarcoidosis, systemic sclerosis, colla-genosis, myositis, neuritis. Inflammatory renal diseases may comprise nephritis, glo-merulonephritis, lupus nephritis, or IgA nephropathy.
In a variety of chronic inflammatory diseases an impressive accumulation of positive T-cells and macrophages is found at the site of inflammation. An accumulation of CCR5+ cells has been demonstrated in several types of inflammatory diseases, like arthritis, inflammatory renal diseases, transplant rejection, auto-immune diseases like multiple sclerosis and inflammatory bowel diseases. In contrast, in the peripheral blood of these patients only a minority of T-cells and monocytes express CCRS.
Therefore, CCR5 appears to be an excellent marker to identify leukocytes that are involved in chronic inflammation. The occurrence of a 32 by deletion in the CCR5 gene which pre-vents expression of CCRS, allows to study the pathophysiological role of CCR5 in chronic inflammatory diseases. In patients with rheumatoid arthritis the frequency of CCR5 deficient (032/32) individuals is significantly reduced. Moreover, the mean sur-vival of the kidney transplants is significantly longer in CCRS-X32/032 patients. These results make CCR5 a target for therapeutic intervention. Furthermore, the prevalence of CCR5 positive leukocytes in the diseased tissue in contrast to the rare expression of CCR5 on the peripheral blood leukocytes means that a specific elimination of positive leukocytes may be therapeutically useful by reducing the number of infiltrating cells in chronic inflammation, transplant rejection and autoimmune disease, like multiple sclerosis without significantly depleting peripheral blood leukocytes.
Eliminating CCR5 positive leukocytes from the inflammatory infiltrate will be of greater therapeutic benefit than simply blocking chemokine receptors of these cells, as they have already infil-trated the tissue.
As documented in the appended examples the antibody and/or chemokine constructs are particularly useful in the treatment, prevention and/or alleviation of inflammatory joint diseases. Therefore, the compositions of the present invention are particularly useful for the treatment of inflammatory joint diseases, like arthritis, in particular chronic arthritis.
The present invention furthermore, provides for medical methods and uses, wherein the composition, preferably the pharmaceutical composition, is to be administered in combination with antiviral agents and/or in combination with drugs to be employed in AIDS management.
As mentioned hereinabove, the main problem in AIDS management in the occurrence of latently HIV-infected cells. The current treatment options are based on anti-viral agents interfering with two enzymes of the HIV-1 virus, its protease and reverse tran-scriptase. The protease is essential to cleave the inactive viral pre-proteins to form the active products, while the reverse transcriptase is required to generate a DNA
interme-diate of the viral RNA genome. The DNA intermediate can then integrate into the host genome and remain there in a silent - latent form. The most efficient treatment option consists of highly active antiretroviral therapy (HAART) - a treatment regimen consist-ing of a combination of at least three anti-retroviral drugs and usually including at least one drug of the protease inhibitor class. The advent of highly active antiretroviral ther-apy (HAART) has had a significant impact on HIV-1-infected individuals, lowering cir-culating virus to undetectable levels (Oxenius (2000) Proc. Natl Acad. Sci.
97, 3383-3387; Perelson (1997) Nature (London) 387, 188-191; Hammer (1997) N. Engl. J.
Med.
337, 725-733; Gulick (1997) N. Engl. J. Med. 337, 734-739). Despite this, latently in-fected cells can remain in these individuals for significant periods of time (Chun (1997) Nature (London) 387, 183-188; Chun (1998) Proc. Natl. Acad. Sci. USA 95, 8869-8873;
Zhang (1999) N. Engl. J. Med. 340, 1605-1613); if HAART is withdrawn, these cells can produce virus (Harrigan (1999) AIDS 13, F59-F62). A pool of latently infected cells is generated early during primary HIV-1 infection (Chun (1998) Proc. Natl.
Acd. Sci. 95, 8869-8873). Considering the postulated long half life of latent viral reservoirs (Zhang (1999) N. Engl. J. Med. 340, 1605-1613, Finzi (1999) Nat. Med 5, 512-517) and the side effects and cost of chronic HAART (Flexner (1998) N. Engl. J. Med. 338, 1292; Carr (1998) Lancet 351, 1881-1883), it is important to develop new strategies to eliminate the latent reservoir. While HAART treatment has been highly successful in suppressing plasma viremia in HIV-infected individuals, there are still persistent reser-voirs of HIV including in latently infected CD4+ T-cells and other cells in the brain, gut associated lymphoid tissue and the genital tract (Chun (1999) Proc. Natl.
Acad. Sci. 96, 10958-10961 ). Re-emergence of plasma viremia after discontinuation of HAART
is due to those pre-existing viral reservoirs and HAART cannot eliminate those reservoirs (Chun (2000) Nature Med. 6, 757-761 ). Therefore, even HAART merely suppresses viral replication and reduces the viral load but does not prevent the occurrence of latent infected cells or eliminates such cells. Transmission of HIV-1 depends on the presence of CCRS, as individuals with a homozygous X32 deletion of the CCR5 allele are highly resistant against infection with HIV-1. Although highly active antiretroviral therapy can efficiently suppress replication of HIV-1, complete eradication of HIV has not been achieved to date. The main obstacle appears to be the inactivity of antiretroviral ther-apy against latently infected cells that can survive for several years and function as en-dogenous source for HIV-1. Many of these cells fail to express viral proteins and can evade the immune response. However, the majority of latently infected cells may still express CCRS, as this receptor is necessary for their initial infection. The compounds of the present invention are particularly useful in the depletion of CCR5+
cells and should significantly reduce the number of latently infected HIV+-cells. Other strategies to eliminate HIV-1 infected cells that depend on a specific recognition of viral proteins, e.g., surface expressed gp120, would be less effective against latently infected cells, as the virus is dormant in these cells.
Therefore, the compositions of the present invention are particularly useful in co-therapy approaches, which lead to a depletion of HIV-infected cells, preferably of CCR5 positive cells. It is preferred that the composition of the present invention is employed in combination with HAART. Therefore the construct of the invention may be used in HIV-therapy in combination with HAART as shown in the appended examples. Products, dosing schedules and common side effects of HAART are known and illustrated, inter alia, in Tables I, II and III.
Said combination may comprise the co-administration as well as an administration be-fore or after treatment with other anti-viral, preferably anti-retroviral, most preferably anti-HIV medication.
The present invention also provides for a kit comprising the polynucleotide, the vector, the host, the antibody construct and/or the chemokine construct of the present inven-tion.
Advantageously, the kit of the present invention further comprises, optionally (a) stor-age solutions) and/or remaining reagents or materials required for the conduct of sci-entific or therapeutic methods. Said kit may, inter alia, comprise drugs and/or medica-ments employed in the treatment of immunological disorders as defined herein and/or in AIDS management. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
The Figures show:
Figure 1.
Expression of various chemokine receptors (indicated on the x-axis) on T cells (first and second panel), monocytes (third panel) and neutrophils (forth panel) in the peripheral blood (white columns) and the synovial fluid (gray columns) of patients with arthritis other than gout. Each dot represents one patient and mean values are given as bars.
Expression of CXCR1 and CXCR2 on neutrophils is given as fluorescence intensity on the y-axis, while in all other cases the percentage of receptor positive cells is depicted.
Figure 2.
FRCS dot plots showing the expression of CCRS, CCR2 and CXCR4 on leukocytes in the peripheral blood (left) and synovial fluid (right) of one patient with rheumatoid arthri-tis. The cut-offs were set according to the isotype controls and are shown as vertical lines. In the synovial fluid the majority of T-cells and monocytes show a high level of CCRS expression, while in the peripheral blood only a minority of these cells express low levels of CCRS.
Figure 3.
Scheme of the bispecific single-chain antibody. The aCCR5 single-chain fragment (CCR5 VL / CCR5 VH) derived from the hybridoma MC-1 is fused to the N-terminus of a single-chain fragment directed against CD3 (CD3 VH / CD3 VL). Binding of the bispecific antibody to CD3~ T cells and CCR5 positive target cells results in crosslink-age of CD3, activation of effector T cells and lysis of CCR5 positive target cells.
Figure 4.
SDS page of the purified bispecific single-chain antibody aCCRS-aCD3. A single band of approx. 60 kD is visible under reducing (left) and non-reducing (right) conditions. No degradation or proteolysis of the bispecific antibody is detectable.
Figure 5.
Scheme of the chemokine-toxin RANTES-PE38. The chemokine RANTES is fused to the N-terminus of a truncated version of the Pseudomonas exotoxin A (PE38).
While the truncated toxin is unable to bind to eukaryotic cells, the fusion protein binds with the RANTES moiety to CCR5 and becomes internalized into the cell. Thereby the toxin in-hibits protein synthesis and. induces cell death.
Figure 6.
SDS-PAGE (left) and Westernblot (right) of the purified protein RANTES-PE38. A
dis-tinct band with the expected size of approx. 46 kD is visible in the coomassie stained SDS-PAGE and Westernblot.
Figure 7.
Binding of the aCCRS-aCD3 bispecific antibody to CD3 on CCR5 deficient lympho-cytes. Costaining with CD4 and CD8 demonstrated that the bispecific antibody binds to the subpopulation of CD4+ / CD8+ T cells. Multicolor analysis showed that no binding to other cell populations occurred.
Figure 8.
Binding of the aCCRS-aCD3 bispecific antibody to CCR5 on transfected CHO
cells.
CHO cells transfected with CCR5 are shown in black, while CXCR4 positive CHO
cells served as negative control and are shown in white.
Figure 9.
Binding of the aCCRS-aCD3 bispecific antibody to CCR5 on cultured monocytes.
Monocytes from a CCR5 positive donor are shown in black, while monocytes from a CCR5 deficient (~32/~32) donor served as negative control and are shown in white.
Figure 10.
CCR5 specific monoclonal antibodies were compared in their ability to induce down-modulation of CCR5 as analyzed by FACS. MAb MC-1 (squares), the parental antibody of the aCCRS-aCD3 bispecific antibody, showed significant internalization, while MC-4 (triangle) showed no induction of CCR5 internalization. CHO-CCR5 cells were incu-bated with various concentrations for 30 min at 37°C.
Figure 11.
Downmodulation of CCRS from the surface of PBMC with RANTES-PE38 (open sym-bols) and RANTES (closed symbols). Surface expression of CCR5 was determined on lymphocytes (squares) and monocytes (circles) and is given as % of the medium con-trol. The fusion protein RANTES-PE38 is able to downmodulate CCR5 from the cell surface with a somewhat lower efficiency than unmodified RANTES.
Figure 12.
Depletion of CCR5 positive monocytes by the bispecific antibody. CCR5 deficient PBMC 032/032) or wildtype PBMC (WT-PBMC) were cultured overnight and incubated with the bispecific antibody (100 ng/ml) or medium as control for 20 h.
Remaining monocytes (Mo) and lymphocytes (Ly) were identified by their light scatter properties in FACS. The CCR5 positive wildtype monocytes were completely depleted by the bispecific antibody, while the CCRS deficient monocytes survived.
Figure 13.
Depletion of CCR5 positive monocytes by the bispecific antibody. Dose response showing depletion of cultured monocytes with various concentrations of the aCCRS-aCD3 bispecific antibody. More than 90 % of monocytes were depleted at a concentra-tion of 33 ng/ml.
Figure 14.
The bispecific aCCRS-aCD3 antibody depletes lymphocytes and monocytes from the synovial fluid of a patient with chronic arthritis. Freshly draw synovial fluid was incu-bated with various concentrations of the bispecific antibody or medium as control for 20 h and analyzed by FACS. More than 95 % of both cell types were depleted at a con-centration of 31 ng/ml.
Figure 15.
The bispecific aCCRS-aCD3 antibody depletes lymphocytes and monocytes from the synovial fluid of a patient with chronic arthritis. Freshly draw synovial fluid was incu-bated with the bispecific antibody (500 ng/ml) or medium as control for 20 h and ana-lyzed by FACS (forward and sideward light scatter analysis). The bispecific antibody completely depleted the CCR5 positive monocytes and lymphocytes, while the negative granulocytes (PMN) survived. Consistent with our previous data all monocytes and lymphocytes in this synovial fluid expressed CCRS, while no expression of was found on granulocytes (PMN).
Figure 16.
The efficacy of the aCCRS-aCD3 bispecific single-chain antibody in depleting positive monocytes was compared with the efficacy of two unmodified monoclonal anti-bodies MC-1 and MC-5. PBMC from two different donors (F and N) were cultured over-night and then incubated for 24 h with medium in the presence or absence of antibody construct and antibody. Concentrations were as indicated. The cells were completely recovered and analyzed by FACS to quantify surviving monocytes and lymphocytes.
Shown are the results of two experiments per PBMC donor. Surprisingly only the bispecific antibody was able to considerably deplete CCR5 positive monocytes, while the unmodified monoclonal antibodies were largely ineffective.
Figure 17.
Example of the forward and sideward light scatter analysis of a representative experi-ment as shown in Fig. 16, indicating that only the aCCRS-aCD3 bispecific single-chain antibody was capable of depleting the monocytes in the left lower quadrants.
For com-parison of the localization of different cell types also see Figure 12 left panel.
Figure 18.
Destruction of CCR5 positive CHO cells with the chemokine-toxin RANTES-PE38.
CCR5 positive CHO cells and CXCR4 positive CHO cells were incubated for 40 h with the chemokine-toxin (10 nM) and analyzed by FACS. Dead cells appear in the left up-per region of the forward and sideward light scatter plot. RANTES-PE38 completely destroyed the CCR5 positive CHO cells while it had no effect on the CXCR4 positive CHO cells.
Figure 19.
Examples of antibody and / or chemokine constructs binding to chemokine receptor (CCR) expressing cells that are combined by peptide linkage or by multimerization do-mains: (A) shows various examples of antibody and chemokine constructs that interact with an effector cell by binding to an effector cell surface antigen, (B) shows examples of antibody and chemokine constructs that are linked to a toxin, (C) shows examples of antibody and chemokine constructs, that contain an antibody binding site for a toxin.
Figure 20.
4x105 CCR5+CHO cells were incubated with 19,5 ng/ml scFV CCR5xCD3 for 30 min-utes at 4°Cafter washing cells were incubated for 45 minutes at 4°C with 20 pg/ml anti-His-Tag monoclonal antibody. Binding of scFV CCR5xCD3 was detected with a mono-clonal goat anti-mouse IgG F(ab')2-PE conjugated antibody and analysed in a flow cy-tometer using the CeIIQuest software. Nonlinear regression analysis was performed with GraphPad Prizm.
Figure 21.
The cytotoxic activity of scFv CCR5xCD3 was tested in a FACS based assay with CCR5+CHO as target and CD3+ T-lymphocytes as effector cells. CD3+ T-cells were isolated from peripheral blood. CCR5+CHO cells were labeled with 12 pM PKH26.
Ef-fectoraarget cells in a ratio of 5:1 were incubated with dilutions of scFv CCR5xCD3 ranging from 320 ng/ml to 0.3 pg/ml for 16 hours at 37°C and 5% C02.
After staining with 1 pg/ml propidium iodine (PI), cells were analyzed by flow cytometry.
In order to verify the specificity of scFv CCR5xCD3 mediated lysis, stably transfected CHO cells were used as negative control target cells. The cytotoxicity assay was performed under identical conditions as described for CCR5+CHO cells.
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and nonlinear regression analysis was performed with GraphPad Prizm. The sigmoidal dose response curve revealed an EC50 value of 912 pg/ml. No cytotoxic ef fect of scFv CCR5xCD3 was observed using CXCR4+ CHO cells as target cells.
Figure 22.
The cytotoxic activity of scFv CCR5xCD3 on CCRS-positive cells was also tested using the CD3 positive T-cell line CB15 as effector cells in a FACS based assay.
CCR5+CHO
target cells labeled with 10 pM PKH26 were used in a effectoraarget ratio of 10:1 and incubated with 100 p1 of scFv CCR5xCD3 in different dilutions (40 pg/ml to 0.15 ng/ml) for 6 hours at 37°C at 5% C02. Cells were stained with 1 pg/ml propidium iodine (PI) and analyzed in duplicate in a flow cytometer.
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and nonlinear regression analysis was performed with GraphPad Prizm. The sigmoidal dose response curve revealed an EC50 value of 12.8 ng/ml.
Figure 23.
Reactivity of MC-1 with A) human PBMC and B) rhesus PBMC. PBMC (solid line), PBMC with PE conjugated goat anti-mouse antibody (dotted line) and PBMC with and PE conjugated goat anti-mouse antibody (solid bold line). Binding of MC-1 to hu-man PBMC (A), but not to rhesus PBMC (B) is indicated by the M1 marker line.
The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.
Example 1: Cell lines, PBMC preparation, synovial fluid 1.1 Generation of a CHO cell line expressing human CCR5 The cDNA sequence of CCR5 was amplified from genomic DNA of human PBMC by PCR with Pfu-polymerase (Stratagene), oligonucleotide primers were:
SEQ ID NO. 1 : 5' GGA ACA AGA TGG ATT ATC AAG TGT C 3' SEQ ID NO. 2: 5' CTG TGT ATG AAA ACT AAG CCA TGT G 3' The amplified fragment was gel purified, ligated into the PCR-Script Amp Sk(+) script vector (Stratagene) and sequenced. After subcloning into the PEF-DHFR vector, DHFR-deficient CHO cells were transfected by electroporation and selected for stable expression in nucleoside free MEM medium with 10% dialyzed FCS as described.
The CHO/CCR5 transfected cells were shown to be homogeneous by FACS-analysis.
1.2 PBMC purification PBMC were isolated from buffy coats or full blood of healthy donors by ficoll density gradient centrifugation. Where indicated PBMC were used from donors with a homozy-gous 32 basepair deletion in the CCR5 allele (~32/~32) preventing surface expression of CCRS. Specifically, buffy coats were diluted 1:2 in 0.9% NaCI, and 35 ml were lay-ered onto 15 ml of Ficoll Paque and centrifuged for 25 min at 400 g. The white inter-phase was harvested and thrombocytes depleted by three subsequent washing and centrifugation steps at 100 g for 6 min in RPMI with 10% FCS. Freshly isolated mono-cytes expressed a very low level of CCRS, but expression was strongly induced after culture of PBMC in RPMI with 10% FCS for 24 to 48 h at 37°C. The amount of FCS did not influence this induction. The expression of CCR5 on lymphocytes was not altered during culture.
1.3 Synovial fluid Synovial fluid of patients with arthritis was obtained from diagnostic or therapeutic ar-throcentesis and used for the experiments without further preparation.
Informed con-sent was obtained from all patients. Synovial fluid and blood samples were simultane-ously obtained from 23 patients who presented with gonarthritis for diagnostic or thera-peutic arthrocentesis. Diagnoses included rheumatoid arthritis (7), reactive arthritis (3), undifferentiated gonarthritis (4), psoriatic arthritis (3), osteoarthritis (2), ancylosing spondylitis (1 ) and gout (3) according to ACR criteria, where applicable.
Written in-formed consent was obtained from all patients. Synovial fluid was analyzed by light mi-croscopy. Crystals were identified by polarized light microscopy. Student's t-test and paired t-test was applied for statistical analysis.
1.4 Analysis of chemokine receptor expression in whole blood samples and synovial fluid Immediately after arthrocentesis SF (synovial fluid) leukocytes were isolated by two washing steps with 5% PBS in NaCI 0.9%. Synovial fluid cells and whole blood (con-taining 1 mM EDTA) were incubated on ice with monoclonal antibodies against chemo-kine receptors and the appropriate isotype controls at a concentration of 10pg/ml. The antibodies were for CCRS: MC-1 (Mack (1998) J. Exp. Med. 187, 1215-1224), for CCR2: DOC-3, which specifically binds to CCR2 (9), for CCR1: Clone 53504 (R&D-Systems), for CXCR1: 5A12 (Pharmingen), for CXCR2: 6C6 (Pharmingen), and for CXCR4 1265 (Pharmingen), IgG1-, IgG2a- and IgG2b-isotype controls (Sigma).
After two washing steps cells were incubated for 30 min on ice with a PE-conjugated rabbit-anti-mouse F(ab)2 fragment (R439, DAKO). Cells were washed twice and incubated with 10% mouse serum followed by a combination of CD4-FITC, CD8-PECy5 and CD14-APC (Immunotech). After lysis of erythrocytes, cells were immediately analyzed by flow cytometry (Becton-Dickinson). Calculations were performed with Cell Quest analysis software. Helper T cells, cytotoxic T cells, monocytes and neutrophils were identified by their light scatter properties and the expression or absence of CD4, CD8 and CD14. Chemokine receptor expression was calculated after defining a cutoff ac-cording to the isotype control.
In both acute and chronic joint effusions we found a consistently increased percentage of CD4+ and CD8+ T cells that expressed the chemokine receptor CCRS compared to the peripheral blood. These data are in good agreement with previous reports (Mack (1999) Arthritis Rheum. 42, 981-988; Qin (1998) J. Clin. Invest. 101, 746-754).
Chemokine receptor expression on T cells in non-crystal induced arthritis:
Approxi-mately 88% of CD4+ T cells and 93% of the CD8+ T cells from the synovial fluid stained positive for the chemokine receptor CCRS. Similarly, a major proportion of CD8+ and CD4+ T cells in the SF expressed CCR2 (66% and 48%) (Fig. 1 ). In con-trast, in the peripheral blood only a minority of T cells expressed the chemokine recep-tors CCR5 or CCR2. The enrichment in the synovial fluid was most pronounced for the CCRS+ helper-T cells (blood: SF ratio = 1:4). The majority of T lymphocytes stained positive for CXCR4 in both compartments. CXCR1, CXCR2 and CCR1 were only ex-pressed by a minor and variable percentage of T cells (Fig. 1 ). A typical example of one patient is shown in Fig. 2, showing the expression of CCRS, CCR2 and CXCR4 on leu-kocytes in the peripheral blood and synovial fluid.
Chemokine receptor expression on monocytes in non-crystal induced arthritis:
Consis-tent with previous data, the majority of monocytes in the SF expressed CCRS.
In addi-tion, a reduced expression of CXCR1, CXCR2, CXCR4 and CCR1 is here reported on monocytes in the synovial fluid compared to the peripheral blood (Fig. 1, 2).
Not only was a lower frequency of receptor positive cells found, but also a lower density of che-mokine receptors on the cell surface (data not shown). No differences could be de-tected in relation to the underlying diagnoses, duration of joint effusion or pretreatment.
CCR2 was equally expressed by all monocytes in both compartments (Fig.'s 1, 2).
Chemokine receptor expression on neutrophils in non-crystal induced arthritis:
Acute arthritis is characterized by a rapid influx of neutrophils into the inflamed joint. There-fore, the chemokine receptor expression on neutrophils from inflamed joint efFusions was analyzed. For the first. time a high expression of CXCR4 is described on a large fraction of neutrophils (60%) from the synovial fluid of patients with acute and chronic arthritis, while a much lower expression was found in the peripheral blood (24 %) (Fig.
1, 2). In arthritis other than gout CXCR1 and CXCR2 was reduced on neutrophils from the synovial fluid by approximately 50% compared to the peripheral blood. CCR1 was expressed only by a minority of neutrophils in both compartments.
1.5 Determination of CCR5 genotype Genomic DNA was prepared from frozen blood samples by affinity chromatography (Ruche Diagnostics). Subsequently a fragment of the CCR5 gene containing the po-tential 32 basepair deletion was amplified by a 40 cycle PCR with Taq polymerase. The primers were .
SEQ ID NO. 3: 5' TTT ACC AGA TCT CAA AAA GAA G 3' SEQ ID NO. 4: 5' GGA GAA GGA CAA TGT TGT AGG 3' Differences in the length of the PCR fragments (274 or 242 bp) allowed to identify CCRS-wildtype and CCRS-032 alleles.
Example 2: Construction of a bispecific antibody 2.1 Generation of monoclonal antibodies against human CCR5 To generate monoclonal antibodies against human CCRS, five BALB/c mice were im-munized intraperitoneally (i.p.) at four week intervals, first with 1x10' PBMC
cultured for days in IL-2 (100 U/ml) and six subsequent i.p. injections of 1x107 CHO cells ex-pressing high levels of CCRS. For this purpose, CCR5 transfected CHO cells were grown in the presence of 20 nM methotrexate to amplify expression of CCR5 and one clone expressing high levels of CCR5 was chosen. Four days after the last i.p.
injection of CHO/CCR5 cells, the spleens were removed and the cells fused with the P3X63-Ag8 cell line. Supernatants from approximately 6000 hybridomas were screened per fusion by flow cytometry on stable CHO/CCR5 cells and monoclonal antibodies against (MC-1, MC-4, MC-5) were detected after the third fusion. The specificity of MC-(IgG1), MC-4, MC-5 were tested on CHO cells stably transfected with CCR1-3 and CXCR4. In all cases no binding was detected. In addition the antibodies did not react with freshly isolated PBMGs and cultured PBMCs from a donor homozygous for the d32 deletion in the CCR5 gene.
2.2 Cloning of the variable domains of MAb MC-1 against CCR5 The light (VL) and heavy (VH) variable domains from the aCCR5 hybridoma MC-1 were cloned using PCR amplification (Orlandi (1989) Proc. Natl. Acad. Sci. 86, 3833).
Reverse transcription was carried out with random hexamer nucleotides and Super-Script reverse transcriptase (Gibco). The variable domains were amplified by PCR with Pfu-polymerase, subcloned into the vector PCR-script Amp SK+ (Stratagene) and se-quenced.
For PCR amplification of VL(1 ) the following primers were used:
SEQ ID NO. 5: 5' GACATTCAGC TGACCCAGTC TCCA 3' SEQ ID NO. 6: 5' GTTTTATTTC CAGCTTGGTC CC 3' For PCR amplification of VM(1 ) the following primers were used:
SEQ ID NO. 7: 5' ACCATGGGAT GGAGCTGTGT CATGCTCTT
SEQ ID NO. 8: 5' TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG
The nucleotide sequence of VL(1 ) obtained by RT PCR is SEQ ID NO. 9:
The corresponding translated protein sequence to VL(1 ) is SEQ ID NO. 10:
The nucleotide sequence of VL(1 ) without the primer sequences used for amplification, SEQ ID NO. 11:
l21 TATAATGCAA AAACCTTAAC AGAAGGTGTG CCATCAAGGT TCAGTGGCAG TGGATCAGGC
The corresponding translated protein sequence to SEQ ID NO. 11: of VL(1 ) is SEQ ID
NO. 12:
The sequence of VH(1 ) including the leader sequence obtained by RT PCR is SEQ
ID
NO. 13:
The corresponding translated protein sequence to VH(1) is SEQ ID NO. 14 The nucleotide sequence of VH(1 ) without the leader sequence and primer sequences used for amplification, SEQ ID NO. 15:
24l GACTGTTGAC AAATATTCCA GCACAGCCTA TATACAACTC AGCAGCCCGA CATCTGAGGA
The corresponding translated protein sequence to SEQ ID NO. 15 of VH(1 ) is SEQ ID
NO. 16:
2.3 Construction and expression of the bispecific single chain antibody CCR5xCD3 A schematic depiction of structure and mode of action of the CCR5xCD3 bispecific sin-gle chain antibody is shown in Fig 3. As described previously, the light and heavy vari-able domains were joined to a single-chain fragment using a (GIy4Ser1 )3 linker and expressed in the periplasmic space of E. coli to test binding of the recombinant protein to CCRS.
Subsequently, the DNA sequence of the aCCR5 single-chain fragment was subcloned with BsrG1 and BspE1 into an eukaryotic expression vector (pEF-DHFR) that already contained a single-chain fragment directed against CD3 with a C-terminally attached tail of 6 histidine residues (Mack (1995) Proc. Natl. Acad. Sci. 92, 7021 ).
The aCCR5 and aCD3 single-chain fragments were joined by a linker coding for GIy4Ser1 (see Fig 3).
The following order of the domains is chosen: VL(1 )-VH(1 )-VH(2)-VL(2), with (1 ) being the specificity against CCRS and (2) the specificity against CD3.
The bispecific CCR5xCD3 antibody has the following nucleotide sequence, SEQ ID
NO. 17:
The bispecific CCR5xCD3 antibody has the following protein sequence, SEQ ID
NO.
18:
A
The bispecific antibody was expressed in DHFR-deficient CHO cells and purified from the culture supernatant by affinity chromatography on immobilized Ni2+ ions (Hochuli (1988) Biotechnology 6, 1321-1325; Ni-NTA, Qiagen).
In summary, for the construction of bispecific antibodies, for example the single-chain technique may be used (Mack et al. (1995) Proc. Natl. Acad. Sci. U S A, 92:7021-7025;
Mack et al. (1997) J. Immunol. 158:3965-3970). In this case, as shown schematically in Figures 3 (top), the variable domains of the light (VL) and the heavy (VH) immuno-globulin chains of two different antibodies are fused in a particular order, optionally a histidine chain of 6 x His is attached in addition. The fusion is effected on a DNA basis so that a protein chain with four different variable domains is formed after expression (cf. Figures 3 (top)). The attached histidine chain enables a simple and efficient purifi-cation via immobilized Ni ions in one step. Figure 3 (top) shows a preferred embodi-ment of the bispecific antibody binding to the CD3 antigen on the surface of the effector cell and the human CCR5 on the surface of leukocytes as target cells.
Subsequently, a single-chain antibody with a specificity is generated by means of fu-sion PCR by inserting a linker of (GIy4Ser~)3 between the two variable antibody do-mains. In a further fusion PCR, the antibody fragment against CCRS is fused to the al-ready published antibody fragment against CD3, with a linker consisting of GIy4Ser~ is inserted (cf. Mack et al. supra).
In order to express the bispecific antibody, the corresponding DNA sequence is sub-cloned in a eukaryotic expression vector (e.g. PEF-DHFR, Mack et al. (1995) PNAS, supra) and transfected in DHFR-deficient CHO cells by means of electroporation. The bispecific antibody is purified from the supernatant of stably transfected CHO
cells by means of affinity chromatography at Ni-NTA, with elution taking place by lowering the pH value. Subsequently, the pH is adjusted and the protein is adjusted to a suitable concentration. Overall purification yield was approx. 900 pg/I culture supernatant. SDS-PAGE showed a single band of approx. 60 kD under reducing and non-reducing condi-tions without any detectable proteolysis or degradation of the protein (Fig.
4).
Example 3: Expression and purification of a chemokine-toxin fusion protein A schematic depiction of structure and mode of action of the RANTES-PE38 chemo-kine-toxin fusion protein is shown in Fig 5. A PCR fragment of RANTES, generated with the primers P1 and P2, was subcloned with Stul and Sall into a vector for periplasmic expression in E. coli (Mack (1995) Proc. Natl. Acad. Sci. 92, 7021 ). The restriction site Stul had previously been introduced at the 3' terminus of the OmpA signal sequence.
The DNA of a truncated version of Pseudomonas exotoxin A (PE38; Theuer (1993) Cancer Res. 53, 340), was amplified by PCR with Pfu-polymerase using the primers P3 and P4 and subcloned with BspE1 and Hind III into the vector that already contained the cDNA of RANTES. Primer P4 also added a tail of 6 histidine residues at the 3' ter-minus of PE38. During the periplasmic expression the OmpA signal sequence is cleaved off such that the recombinant protein starts with the first aminoacid of RAN-TES. The C-terminally attached tail of 6 histidine residues allowed purification by affinity chromatography on Ni-NTA (Qiagen).
List of primers:
SEQ ID NO. 19: 5' AAAGGCCTCCCCATATTCCTCGGA
SEQ ID NO. 20: 5' AAAGTCGACTCCGGACATCTCCAAAGAGTTGATGTAC
SEQ ID NO. 21: 5' AATCCGGAGGCGGCAGCCTGGCCGC
SEQID NO:22: 5' GGGAAGCTTAGTGATGGTGATGGTGATGCTTCAGGTCCTCGCGCGG
As described in the above the DNA sequence of RANTES was fused with the se-quence of a truncated version of the Pseudomonas exotoxin A (PE38) (Theuer (1993) Cancer Res. 53, 340). In a first version of the construct a Gly-Ser linker was spaced between RANTES and PE 38. However this resulted in a considerable proteolytic deg-radation of the fusion protein during expression in E. coli (data not shown).
In an at-tempt to stabilize the construct the linker and the fist three aminoacids of PE38 were removed. The new fusion protein showed no proteolysis during expression in the periplasmic space of E. coli as demonstrated by SDS-PAGE (Fig. 6 left panel) and Western-blot (Fig. 6 right panel). The corresponding constructs are depicted in SEQ ID
NOs: 23 and 24, respectively.
Example 4: Binding of the bispecific antibody to the target antigens CCR5 and CD3 Binding of the bispecific single-chain antibody to CHO cells or PBMC was determined by FACS-analysis (Fig. 7 to 9). The cells were incubated with the bispecific antibody for 60 min on ice followed by an antibody against 6xHis (Dianova, Hamburg, Germany) and a PE-conjugated polyclonal rabbit-anti mouse F(ab)2 fragment (R439, Dako, Ham-burg, Germany). As the bispecific antibody would also bind to CCRS, we performed the analysis with PBMC that lack expression of CCR5 due to a homozygous 32 basepair deletion in the CCR5 alleles. The antibody showed good binding to a subpopulation of lymphocytes. Co-staining with antibodies against CD4 and CD8 identified this sub-population as CD4 and CD8 positive T lymphocytes (Fig. 7). In addition, the bispecific antibody competed with the monoclonal CD3 antibody OKT-3 for binding to T
cells (data not shown).
Binding of the bispecific antibody to CCR5 was demonstrated on CCR5 overexpressing CHO cells and human monocytes (Fig. 8 and 9). The antibody showed excellent bind-ing to CCR5 transfected CHO cells (Fig. 8) and cultured monocytes (Fig 9), while no binding was detectable on CHO cells transfected with CXCR4 or on cultured mono-cytes from a donor with a homozygous CCRS-432/32 deletion. Overnight cultivation of monocytes induces expression of CCR5 on wild-type monocytes, while monocytes from donors with a homozygous CCRS-X32/032 deletion fail to express CCRS. Moreover we have shown that the CCR5 signal detectable with the bispecific antibody on cultured monocytes could be reduced to values below 15 % by preincubation of monocytes for 30 min at 37°C with AOP-RANTES (data not shown) that is known to efficiently induce internalization of CCR5 and reduce binding of CCR5 antibodies (25).
Example 5: Downmodulation of chemokine receptors 5.1 Downmodulation of CCR5 with mAb MC-1 against CCR5 The effect of MC-1 one on the surface expression of human CCR5 was measured.
For comparison a different monoclonal antibody MC-4 against CCR5 was used. CHO-CCRS cells were incubated with various concentrations of antibody MC-1 and MC-4 for 30 min. at 37°C. Cells were placed on ice and stained with MC-1 and MC-4 respec-tively at a concentration of 15 ug/ml for one hour on ice, followed by detection with a secondary antibody (rabbit anti-mouse FITC, F313 from DAKO). Analysis was per-formed on a FACSCalibur. Incubation with MC-1 at 37oC for 30 min resulted in a downmodulation of human CCR5 by 40% at a concentration of 10 ug/ml (Fig. 10).
5.2 Downmodulation of CCR5 by chemokine-toxin The fusion of RANTES to the N-terminus of a truncated version of the Pseudomonas exotoxin A is supposed to result in specific binding of the construct to cells expressing RANTES receptors such as CCRS, CCR1 and CCR3. Internalization of the chemokine receptors upon binding of the modified toxin would enhance the cellular uptake and cytotoxic activity of the construct (Fig. 5 lower panel). We therefore analyzed whether RANTES-PE38 is able to internalize CCR5 from the surface of primary monocytes and T cells (Fig. 11, open symbols). Internalization of CCR5 would indicate that the con-struct is able to bind to CCR5 and that RANTES remains functionally active after fusion to PE38. As shown in Fig. 11 the construct is able to internalize CCR5 from the surface of monocytes and lymphocytes. Unmodified RANTES served as positive control and was somewhat more efficient than RANTES-PE38 (Fig 11, closed symbols).
PBMC were incubated for 30 min at 37°C with various concentrations of RANTES or RANTES-PE38 diluted in RPMI with 10% FCS in a volume of 100 p1. Medium alone was used as control. The cells were then stained on ice for surface CCRS
expression using the monoclonal antibody MC-1 or medium as negative control followed by the PE-conjugated anti-mouse antibody 8439. The FACS-analysis was performed on a FACSCalibur (Becton Dickinson) and CeIIQuest software. Lymphocytes and monocytes were distinguished by their forward and sideward light scatter properties and expres-sion of CD14, CD4 and CDB. Relative surface CCR5 expression was calculated as [mean channel fluorescence (exp.) - mean channel fluorescence (negative control)] /
[mean channel fluorescence (medium) - mean channel fluorescence (negative control)].
Example 6: Depletion of cells with CCR5xCD3 antibody and RANTES-PE38 6.1 CCR5 specific depletion of monocytes from cultured PBMCs PBMC from CCRS-wildtype (WT) or CCR5 deficient (0321032) donors were incubated over night to induce expression of CCR5 on monocytes. Cultured PBMC were incu-bated with different concentrations of purified aCCRS-aCD3 bispecific antibodies or medium as control for 20 h. Surviving cells were analyzed on a FACSCalibur and counted.
In order to test the ability of the aCCRS-aCD3 bispecific single-chain antibody to de-plete CCR5 positive primary cells, we incubated human PBMC with the antibody (Fig.
12). Prior to incubation the PBMC were cultured overnight to upregulate CCR5 expres-sion on monocytes. By retargeting cytotoxic T cells the bispecific antibody depleted the majority of monocytes within 20 h in a concentration dependent manner (Fig.
13) with an almost complete elimination of CCR5 positive cells at concentration of 10 ng/ml. To verify that the depletion of.monocytes was due to their induced expression of CCRS, the same experiment was performed with PBMC from a donor with a homozygous 32 by deletion in the CCR5 allele that prevents surFace expression of CCR5. No depletion of CCR5 deficient monocytes was detectable after 20 h indicating that the depletion of cells with the bispecific antibody is restricted to monocytes that express CCR5 (Fig.
12), compare lower right panel to upper right panel, left panels serve as negative con-trols. Monocytes (Mo) and lymphocytes (Ly) were identified by their forward and side-wards light scatter properties. Monocytes appear in the lower left quadrant see arrows.
6.2 Depletion of monocytes and T lymphocytes from the synovial fluid of patients with arthritis Freshly drawn synovial fluid of patients with arthritis were incubated with different con-centrations of purified aCCRS-aCD3 bispecific antibodies or medium as control for 20 h. Surviving cells were analyzed on a FACSCalibur and counted.
The bispecific single-chain antibody could potentially be applied to deplete CCRS posi-tive T cells and monocytes from the inflamed joints of patients with arthritis. Therefore determined the depletion of CCR5 positive cells from the synovial fluid of patients with various types of arthritis was determined. It was shown previously that the majority of T
cells and monocytes in the inflamed synovial fluid express CCR5 (Mack (1999) loc.
cit.). In synovial samples obtained before depletion experiments it was confirmed by FACS analysis that the majority of lymphocytes and monocytes express CCRS, while no expression of CCR5 was detectable on granulocytes (data not shown). For the de-pletion experiments the synovial fluid was incubated ex vivo with different concentra-tions of the bispecific antibody for 20 h (Fig. 14). The synovial fluid was incubated im-mediately after puncture without any preparation to ensure that the conditions in vitro resemble most closely the situation in vivo when the antibody would be present within inflamed joints. As shown in Fig. 14 the bispecific antibody induced a depletion of the majority of lymphocytes and monocytes from the synovial fluid, while granulocytes that do not express CCR5 remained unaffected. A representative FACS analysis of the de-pletion of monocytes and lymphocytes in synovial fluid at a concentration of 0.5 uglml CCR5xCD3 is shown in Fig 15. Only the CCR5 negative neutrophils (PMN: polymor-pho-nuclear cells) are unaffected by the bispecific antibody.
Antibodies were incubated with synovial fluid for one or several days. After 24 hours, the CCR5 positive lymphocytes and monocytes have already almost disappeared.
When the medium is controlled after longer incubation, the monocytes have difFerenti-ated into macrophages which are visible at the bottom of the culture flask.
After an ap-propriate incubation with the bispecific antibody, no macrophages are visible.
A corresponding result can be obtained when cultivated PBMC are incubated with the bispecific antibody as described above. In this case, there is an almost complete de-pletion of CCR5 positive monocytes and an almost complete depletion of CCR5 posi-tive T-lymphocytes. The depletion of CCR5 positive T-cells and monocytes is shown.
The results show that the construct of the present invention is capable of destroying CCR5 positive monocytes. This applies to both monocytes from the joint aspirate and blood monocytes which express CCR5 when being differentiated into macrophages.
Depletion of the monocytes/macrophages takes place within a few hours (< 24 hrs). In particular the depletion of monocytes/macrophages in the joint is of great advantage in therapy since it is these cells that are mainly responsible for the joint destruction.
Moreover, for the activation of T-lymphocytes an interaction with macrophages is also required so that, at the same time, the function of the T-lymphocytes is suppressed.
In addition to the depletion of monocytes/macrophages, a considerable reduction in the number of CCR5 positive T-lymphocytes could be observed.
6.3 Comparison of the efficacy of the bispecific antibody CCR5xCD3 versus mono-clonal antibodies The efficacy of the aCCRS-aCD3 bispecific single-chain antibody in depleting positive monocytes was compared with the efficacy of two unmodified monoclonal anti-bodies. PBMC from two different donors (F and N) were cultured overnight and then incubated for 24 h with medium, the bispecific single-chain antibody (125 ng/ml), MC-1 (5pg/ml) and MC-5 (5pg/ml). The monoclonal antibody MC-1, the parental antibody for the bispecific single-chain antibody has the isotype mouse IgG-1 and the antibody MC-has the isotype IgG-2a. The cells were completely recovered and analyzed by FACS
to quantify surviving monocytes and lymphocytes.
Fig. 16 shows that surprisingly only the bispecific antibody was able to considerably deplete CCR5 positive monocytes, while the unmodified monoclonal antibodies were largely ineffective even when used in a 40 fold excess over the bispecific antibody CCR5xCD3. By FACS analysis using forward and sideward light scatter properties of lymphocytes and monocytes demonstrates that only the CCR5xCD3 bispecific antibody but not the monoclonal antibodies are capable of depleting cultured monocytes (Fig 17 compare right upper panel to lower panels).
6.4 Depletion of chemokine receptor expressing cells with RANTES-PE38 CHO cells expressing CCR5 or CXCR4 were grown to subconfluence on 24 well cul-ture plates and incubated with different concentrations of purified RANTES-PE38 or medium as control. After 40 hours the adherent and non-adherent cells were recovered and analyzed by FACS to measure the percentage of dead cells. It was previously es-tablished that dead (propidium iodide positive) CHO cells can be identified by their light scatter properties.
It was further analyzed the cytotoxic activity of RANTES-PE38. For that purpose we incubated CHO cells expressing human CCRS, murine CCR5 and human CXCR4 with various concentrations of the chemokine-toxin or medium. No surviving (adherent) hu-man or murine CCR5 positive CHO cells were detectable by light microscopy after 40 h incubation with as little as 10 nM RANTES-PE38. In contrast regular growth and sur-vival was observed when the CCR5 positive cells were incubated with medium or when CXCR4 positive CHO cells were incubated with equal concentrations of the chemokine-toxin (data not shown). To quantify the percentage of dead cells the adherent and non-adherent cells were analyzed by FACS. It was previously established that living and dead CHO cells can be identified by their light scatter properties, the position of dead and alive cells is indicated by arrows (Fig 18). As shown in Fig. 18 no cytotoxic effect of RANTES-PE38 was seen on CHO cells expressing CXCR4, while CHO cells express-ing human CCR5 were completely killed by 10 nM RANTES-PE38.
These experiments show that RANTES-PE 38 is able to internalize CCRS from the surface of cells and induces depletion of cells expressing the RANTES
receptors hCCR5 or mCCRS. The inactivity of the construct against CXCR4 positive CHO
cells demonstrates that the cytotoxic activity of the construct is restricted to cells that ex-press specific chemokine receptors.
Example 7: Virus infection assay with stable transfected cells GHOST 34 CCR5 cells are derived from HOS/CD4 cells stably expressing CCR5 and were provided by Dan Littman (Skirball Institute, New York). 2.5 x 104 cells in 48-well trays were exposed to 100 ~,I of chemokine at appropriate dilution for 30 min at 37°C.
100 ~,I of the NSI, CCRS-dependent HIV-1 strain, SF162 was added at 1000 focus forming units/ml (FFU/ml) and the cells incubated for a further 3 h. The cells were then washed and incubated in medium containing the appropriate chemokine for 4 days before fixing, staining in situ for p24 production and estimating foci of infection as pre-viously described.
EXAMPLE 8: Concentration dependent binding of CCR5xCD3 to CCRS express-ing CHO cells Chinese hamster ovary cells stably transfected with CCR5 (CCR5+CHO) were used as target cells for binding studies of bispecific scFv CCR5xCD3 (as described in Example 2 and Fig. 3). These cells were negative for CD3 and >95% positive for CCR5 as evaluated by binding assays with the parental antibody MC-1 (as described in Example 5 and Fig. 10). Binding was evaluated by a flow cytometry based binding assay.
4x105 CCR5+CHO cells were resuspended in 50 p1 FACS buffer (PBS with 1 % fetal calf serum (FCS) and 0,05% sodium azide) containing different dilutions of scFV
CCR5xCD3 ranging from 20 pg/ml to 19.5 ng/ml. Cells were incubated in a 96 well mi-crotiter plate for 30 minutes at 4°C. Cells were washed twice with FACS
buffer and in-cubated for 45 minutes at 4°C with 20 pg/ml anti-His-Tag monoclonal antibody (Di-anova). Specifically bound scFV CCR5xCD3 was detected with a monoclonal goat anti-mouse IgG F(ab')2 -PE conjugated antibody (Dianova). After washing, the cells were analysed in a flow cytometer (FACSCalibur, Becton Dickinson) using the CeIIQuest software (Becton Dickinson) to calculate the median values of the fluorescence intensi-ties of the different concentration samples. Nonlinear regression analysis was per-formed with GraphPad Prizm (Version 3.02). Concentration dependent binding of scFv CCR5xCD3 to CCRS expressing CHO cells was observed with a Kp value of 0,86 pg/ml (Fig. 20).
EXAMPLE 9: Cytotoxic activity of CCR5xCD3 with primary T lymphocytes as ef-fpctor cells The capacity of scFv CCR5xCD3 (as described in Example 2 and Fig. 3) to mediate cytotoxicity to CCRS-positive cells was tested using stably transfected CCR5+CHO as target cells and CD3 positive T-lymphocytes derived from peripheral blood as effector cells. For detection of cytotoxicity, a FACS based assay was performed.
CD3+ T-cells (include CD4+ and CD8+ cells) were isolated from peripheral blood by negative selection using a human T cell enrichment column (R&D Systems). For this purpose, PBMC were prepared by standard Ficoll-Hypaque density gradient separation and applied to the column. B cells and monocytes were bound to the column matrix, while T cells were eluated. The enriched T cells were washed in medium and used as effector cells.
For discrimination of target cells from effector cells by flow cytometry, CCR5+CHO cells were labeled with the aliphatic membrane dye PKH26 (Sigma) in a final concentration of 12 pM. 0.5x105 labeled CCRS+CHO cells and 2.5x105 CD3+ T-cells were seeded in a 96-well microtiter plate in a effectoraarget ratio of 5:1. 100 ~l dilutions of scFv CCR5xCD3 ranging from 320 ng/ml to 0.3 pg/ml were incubated with the cells for hours at 37°C in a humified atmosphere at 5% C02. Subsequently cells were centri-fuged for 3 minutes at 600xg, and the cell pellets were resuspended in 200 p1 FACS
buffer (PBS, 1 %FCS, 0.05% sodium azide). After staining with 1 pg/ml propidium iodine (PI), cells were analyzed in duplicate in a flow cytometer (FACSCalibur, Becton Dickin-son).
In order to verify the specificity of scFv CCR5xCD3 mediated lysis, stably transfected CHO cells were used as negative control target cells. The cytotoxicity assay was performed under identical conditions as described for CCR5+CHO cells.
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and a nonlinear regression analysis was performed with GraphPad Prizm. A
sigmoidal dose response curve was obtained (Fig. 21 ) revealing an EC50 value of 912 pg/ml. No cytotoxic effect of scFv CCR5xCD3 was observed using CXCR4+ CHO
cells as target cells.
EXAMPLE 10: Cytotoxic activity of CCR5xCD3 with T cell clone CB15 as efFector cells The cytotoxic activity of scFv CCR5xCD3 (as described in Example 2 and Fig. 3) on CCRS-positive cells was also tested using the CD3 positive T-cell line CB15 (CD4+) as effector cells. For detection of cytotoxicity, a FACS based assay was performed with CCR5 transfected CHO cells (CCR5+CHO) as target cells.
CCR5+CHO cells were labeled with the aliphatic membrane dye PKH26 (Sigma) in a final concentration of 10 pM. Effector and target cells were incubated in a microtiter plate in a ratio of 10:1 with 100 p1 of scFv CCR5xCD3 in dilutions ranging from 40 pg/ml to 0.15 ng/ml for 6 hours at 37°C in a humified atmosphere with 5% C02.
Cells were centrifuged for 3 minutes at 600xg and the cell pellets were resuspended in 200 p1 FACS buffer (PBS, 1 %FCS, 0.05% sodium azide). Cells were stained with 1 pg/ml propidium iodine (PI) and analyzed in duplicate in a flow cytometer (FACSCalibur, Becton Dickinson).
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and a nonlinear regression analysis was performed with GraphPad Prizm. A
sigmoidal dose response curve was obtained (Fig. 22) revealing an EC50 value of 12.8 ng/ml.
The results obtained with T cell clone CB15 as effector cells in bioactivity assay dem-onstrate that specific lysis mediated by scFv CCR5xCD3 is not restricted to the cyto-toxic activity of CD8+ CTL but that CD4+ T cells are also involved in this process.
EXAMPLE 11: Epitope mapping of parental CCR5 specific monoclonal antibody MC-1 used for construction of scFv CCR5xCD3 Epitope of parental CCR5 specific monoclonal antibody MC-1 used for construction of scFv CCR5xCD3 (as described in Example 2 and Fig. 3) was mapped by flow cytome-try using a panel of about 70 CHO-K1 cell lines stably expressing chimeric and point mutant receptors (Samson, J. Biol. Chem., 1997, 272, 24934-24941; Lee, J.
Biol.
Chem., 1999, 274, 9617-9626; Blanpain, J. Biol. Chem., 1999, 274, 34719-34727;
Blanpain, Blood, 2000, 96, 1638-1645). Cells were incubated for 30 min on ice with mab MC-1, washed and stained with PE-conjugated anti-mouse Ig antibody (Sigma).
CHO-K1 cells expressing CCR2b were used as negative control. MC-1 was shown to recognize the first part of the second extracellular loop (ECL2) of the CCR5 molecule (data not shown). ECL2 ranges from as 168-199 (RSQ KEGLHYTCSS HFPYSQYQFW
KNFQTLKIV) and is located between the transmembrane regions 4 and 5 of CCR5 as described by Chen, J. Virol., 1997, 71, 2705-2714.
The amino acid sequences of human and rhesus macaque CCR5 differ in eight amino acids with two amino acid changes are situated at position as 171 (K-->R) and as 198 (I-->M) in the ECL2 (Chen, J. Virol., 1997, 71, 2705-2714). Due to these amino acid changes potential crossreactivity of MC-1 with the ECL2 of rhesus macaque CCR5 was analyzed with human and rhesus PBMCs in a FACS based assay. PBMC of both spe-cies were isolated by standard ficoll gradient centrifugation. 5x105 cells were sus-pended in 50 p1 FACS buffer and 50 pg/ml of MC-1 was added. After 30 min incubation at 4°C, the cells were washed and stained with goat anti-mouse IgG
F(ab')2-PE conju-gated monoclonal antibody (Dianova) for 30 min at 4°C in the dark.
Cells were washed and analyzed in a flow cytometer (FACSCalibur, Becton Dickinson).
As shown in Fig. 23 MC-1 exclusively bound to human CCR5 but did not react with CCR5 derived from rhesus macaques. These data show that the epitope recognized by MC-1 is specific for human CCR5 and that lysin at position as 171 and isoleucin at po-sition as 198 in human CCR5 sequence are essential for this specificity.
Especially ly-sin at position as 171 which is located in the first part of ECL2 contributes to the spe-cific recognition of the human epitope of CCR5 by mab MC-1.
EXAMPLE 12: scFv CCR5xCD3 mediated reduction of virus production in HIV-1 infected monocytes PBMC were prepared from fresh buffy coats of healthy donors by Ficoll density cen-trifugation and monocytes were isolated by over night adherence to culture flasks. Re-maining PBL were removed and cultured separately at 37°C in a humidified atmos-phere at 5% C02.
Monocytes were seeded into a 48 well microtiter plate at a density of 5x104 cells/well and infected with the M-tropic HIV-1 strain BaL (moi=1 ) overnight at 37°C in a humidi-fied atmosphere at 5% C02. The virus was removed by washing and the monocytes were further cultured with unstimulated PBL (15 x 104 per well) + scFv CCR5xCD3 (1 pg/ml) + AZT (75 pM) or with unstimulated PBL (15 x 104 per well) alone as negative control. 5 days post infection (p.i.) monocytes were washed and cultured in the ab-sence of AZT or antibody. Supernatant was harvested on day 15 p.i. and HIV-1 replica-tion was quantified by measurement of p24 in an ELISA. This experimental approach led to a reduction of virus replication of 75% in samples containing scFV
CCR5xCD3 (75 ng/ml p24) compared to the control without scFv CCR5xCD3 (300 ng/ml p24).
Table I: Nucleosidal Reverse Transcriptase Inhibitors (nucleoside analogs, NRTI) Substance Trade- Dosing Sched- Common Side Effects and General name ule Remarks Zidovudine Retrovir300 mg, 2x dailyInitial gastrointestinal (GI) side effects, (AZT) anemia, neutropenia, myopathy Lamivudine Epivir 150 mg, 2x dailyGenerally well tolerated. Effective against (3TC) hepatitis Zidovudine,Com- 1 tablet 2x Combination-tablet containing daily 300 mg Lamiduvine bivir AZT and 150 mg 3TC
(AZT + 3TC) Didanosine Videx 200 mg, 2x daily15% peripheral neuropathy, pancreatitis;
(ddl) or avoid alcohol.
400 mg, 1x dailyContents alcohol: could be given simulta-on an empty neously with all NRTIs, Adefovir, Nevira-stomach (> 60 pine, and Efavirence; Delavirdine kg and weight) Indinavir should be given at least 1 hour before ddl; Nelfinavir to be given 1 hour after dd I .
ZalcitabineHivid 0,375-0,75 mg, 17-31 % peripheral neuropathy in difFerent (ddC) 3x daily studies; aphteous ulcerations Stavudine Zerit 20-40 mg, 2x Peripheral neuropathy (1-4% in earlier (d4T) daily studies; 24% in 'expanded access' pa-tients with CD4 > 50) Abacavir Ziagen 300 mg, 2x dailyAbout 3% reaction for hypersensitivity:
(ABA) fever, indisposition, possibly transient rash, gastrointestinal side effects.
Table II: Protease Inhibitors Substance Trade- Dosing Schedule Common Side Effects and name General Remarks Saquinavir Invirase 600 mg, 3x daily, Well tolerated. Limited take efficacy (hard gela- with high-fat meal due to poor resorption.
tine cap-sule, SQV-H) Saquinavir Forto- 1200 mg, 3x daily, Improved resorption compared take (soft gela-vase with high-fat meal to Invirase.
(>28g) tine cap-sule, SQV-S) Ritonavir Norvir 600 mg, (6 cap./7.5 Nausea and numb lips for ml) up to (RTV) 2x daily. Start with5 weeks. Occasionally hepatitis.
mg, 2x daily, then dot tolerated by up to in- 50% of crease within 10 the patients.
days to 600 mg, 2x daily;
Indinavir Crixivan 800 mg, every 8 hoursNeural calculus with 6-8%;
on re-(IDV) an empty stomach quires large liquid intake.
or with Occa-snack (<2g fat) sionally nausea and gastroin-testinal side effects.
Nelfinavir Viracept 750 mg, 3x daily, Often diarrhea, sometimes or 1250 nau-(NFV) mg, 2x daily with sea.
meals Table III: Non-Nucleosidal Reverse Transcriptase Inhibitors (NNRTI) Substance Trade- Dosing Sched- Common Side Effects and General name ule Remarks Nevirapine Viramune 200 mg, 1x Transient skin, hepatitis, induced daily liver (NVP) enzymes P450 3A4 DelavirdineRescriptor400 mg, 3x Transient skin, suppresses P450 daily 3A4 (DLV) Efavirence Sustiva 600 mg, 1x Initially dizziness, insomnia, daily momen-(EFV) in the eveningtart' transient skin: Induces P450 3A4;
avoid Claritithromycin.
Table IV: Chemokine receptors and chemokine ligands Chemokine Chemokine Ligands Receptors CXCR3 I-TAC (CXCL11), IP-10 (CXCL-10), Mig (CXCL9) CXCR4 SDF-1 (CXCL12) CXCR5 BCA1 (CXCL13) CCR1 MIP1alpha (CCL3), RANTES (CCLS), MCP-3 (CCL7), MCP-4 (CCL13), HCC1 (CCL14), LKN1 (CCL15) CCR2 MIP1alpha (CCL3), RANTES (CCLS), MCP-1 (CCL2), MCP-2 (CCLB), MCP-3 (CCL7), MCP-4 (CCL13) CCR3 RANTES (CCLS), MCP-2 (CCLB), MCP-3 (CCL7), MCP-4 (CCL13), eotaxin (CCL11 ), LKN1 (CCL15), MPIF-2 (CCL24) , eo-taxin-3 (CCL26) CCR4 TARC (CCL17), MDC (CCL22) CCR5 MIPlalpha (CCL3), MIP1 beta (CCL4), RANTES (CCLS), (CCLB), MCP-3 (CCL7), MCP-4 (CCL13), eotaxin (CCL11 ) CCR6 LARC~ (CCL20) CCR7 ELC (CCL19), SLC (CCL21 ) CCRB I-309 (CCL1 ), MIP1 beta (CCL4), TARC (CCL17) CCR9 TECK (CCL25) XCR1 XCL1, XCL2 CCR10 CCL27, CCL28 (Wang (2000) J. Biol. Chem. 275, 22313-22323) CX3CR1: fractalkine (CXC3CL1 ) 1, SEQUENCE LISTING
<110> MACK, Matthias <120> Antibody and/or chemokine constructs and their use in immunological disorders <130> E 2411 PCT
<140>
<141>
<160> 34 <170> PatentIn Ver. 2.1 <210> 1 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 1 ggaacaagat ggattatcaa gtgtc 25 <210> 2 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 2 ctgtgtatga aaactaagcc atgtg 25 <210> 3 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 3 tttaccagat ctcaaaaaga ag 22 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 4 ggagaaggac aatgttgtag g 21 <210> 5 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 5 gacattcagc tgacccagtc tcca 24 <210> 6 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 6 gttttatttc cagcttggtc cc 22 <210> 7 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 7 accatgggat ggagctgtgt catgctctt 29 <210> 8 <211> 34 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 8 tgaggagacg gtgaccgtgg tcccttggcc ccag 34 <210> 9 <211> 322 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(321) 3, <400>
gacattcagctg acccagtct ccagcctcc ctatctgca tctgtggga 48 AspIleGlnLeu ThrG1nSer ProAlaSer LeuSerAla SerValGly gaaactgtcacc atcacatgt cgagcaagt gagaatatt tacagttat 96 GluThrValThr IleThrCys ArgAlaSer GluAsnIle TyrSerTyr ttagcatggtat cagcagaaa cagggaaaa tctcctcaa ctcctggtc 144 LeuAlaTrpTyr GlnGlnLys GlnGlyLys SerProG1n LeuLeuVal tataatgcaaaa accttaaca gaaggtgtg ccatcaagg ttcagtggc 192 TyrAsnAlaLys ThrLeuThr GluGlyVal ProSerArg PheSerGly agtggatcaggc acacagttt tctctgaag atcaacagc ctgcagcct 240 SerG1ySerGly ThrGlnPhe SerLeuLys IleAsnSer LeuGlnPro gaagattttggg aattatttc tgtcaacat cattatgat actcctcgg 288 GluAspPheGly AsnTyrPhe CysGlnHis HisTyrAsp ThrProArg acgttcggtgga gggaccaag ctggaaata aaac 322 ThrPheGlyGly GlyThrLys LeuGluIle Lys <210> ZO
<211> 107 <212> PRT
<213> Mus sp.
<400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala 5er Val Gly l 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Asn Tyr Phe Cys Gln His His Tyr Asp Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 11 <211> 276 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(276) <400> 11 gcc tcc cta tct gca tct gtg gga gaa act gtc acc atc aca tgt cga 48 A1a Ser Leu Ser Ala Ser Val Gly Glu Thr Va1 Thr Ile Thr Cys Arg gcaagtgagaat atttac agttatttagca tggtatcag cagaaa cag 96 A1aSerGluAsn IleTyr SerTyrLeuAla TrpTyrGln GlnLys Gln ggaaaatctcct caactc ctggtctataat gcaaaaacc ttaaca gaa 144 GlyLysSerPro GlnLeu LeuValTyrAsn AlaLysThr LeuThr Glu ggtgtgccatca aggttc agtggcagtgga tcaggcaca cagttt tct 192 GlyValProSer ArgPhe SerGlySerGly SerGlyThr GlnPhe Ser ctgaagatcaac agcctg cagcctgaagat tttgggaat tatttc tgt 240 LeuLysIleAsn SexLeu GlnProGluAsp PheGlyAsn TyrPhe Cys caacatcattat gatact cctcggacgttc ggtgga 276 GlnHisHisTyr AspThr ProArgThrPhe GlyGly <210> 12 <211> 92 <212> PRT
<213> Mus sp.
<400> 12 Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu A1a Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Asn Tyr Phe Cys Gln His His Tyr Asp Thr Pro Arg Thr Phe Gly Gly 5~
<210> 13 <211> 414 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(414) <400> 13 atg gga tgg agc tgt gtc atg ctc ttc ttg gta gca aca get aca ggt 48 Met Gly Trp Ser Cys Val Met Leu Phe Leu Va1 A1a Thr A1a Thr Gly gtccactcccag gtccaactg cagcagcct ggggetggg agggtgagg 96 Va1HisSerGln ValGlnLeu GlnGlnPro GlyAlaGly ArgValArg cctggagettca gtgaagctg tcctgcaag gettctggc tactccttc 144 Pro.GlyAlaSer ValLysLeu SerCysLys A1aSerGly TyrSerPhe accagttactgg atgaactgg gtgaagcag aggcctgga caaggcctt 192 ThrSerTyrTrp MetAsnTrp ValLysGln ArgProGly GlnGlyLeu gagtggattggc atgattcat ccttccgat agtgaaact aggttaaat 240 GluTrpIleGly MetIleHis ProSerAsp SerGluThr ArgLeuAsn cagaagttcaac gacagggcc acattgact gttgacaaa tattccagc 288 G1nLysPheAsn AspArgAla ThrLeuThr ValAspLys TyrSerSer acagcctatata caactcagc agcccgaca tctgaggac tctgcggtc 336 ThrAlaTyrIle GlnLeuSer SerProThr SerGluAsp SerAlaVal tattactgtgca agaggagaa tattactac ggtatattt gactactgg 384 TyrTyrCysAla ArgGlyGlu TyrTyrTyr G1yIlePhe AspTyrTrp 115 120 125 .
ggccaagggacc acggtcacc gtctcctca 414 GlyGlnGlyThr ThrValThr ValSerSer <210> 14 <211> 138 <212> PRT
<213> Mus sp.
<400> 14 Met Gly Trp Ser Cys Val Met Leu Phe Leu Va1 Ala Thr Ala Thr Gly Val His Ser Gln Va1 Gln Leu Gln Gln Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg A1a Thr Leu Thr Val Asp Lys Tyr Sex Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro ThreSer Glu Asp Ser Ala Val Tyr Tyr Cys A1a Arg Gly G1u Tyr Tyr Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser <210> 15 <211> 354 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (2)..(352) <400> 15 c ttg gta gca aca get aca ggt gtc cac tcc cag gtc caa ctg cag cag 49 Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Val Gln Leu Gln Gln cct ggg get ggg agg gtg agg cct gga get tca gtg aag ctg tcc tgc 97 Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys aag get tct ggc tac tcc ttc acc agt tac tgg atg aac tgg gtg aag 145 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Va1 Lys cag agg cct gga caa ggc ctt gag tgg att ggc atg att cat cct tcc 193 Gln Arg Pro G1y G1n Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser gat agt gaa act agg tta aat cag aag ttc aac gac agg gcc aca ttg 241 Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu act gtt gac aaa tat tcc agc aca gcc tat ata caa ctc agc agc ccg 289 Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro aca tct gag gac tct gcg gtc tat tac tgt gca aga gga gaa tat tac 337 Thr Ser Glu Asp Ser Ala Va1 Tyr Tyr Cys Ala Arg Gly Glu Tyr Tyr 7, tac ggt ata ttt gac to 354 Tyr Gly Ile Phe Asp <210> 16 <211> 117 <212> PRT
<213> Mus sp.
<400> 16 Leu Val Ala Thr Ala Thr Gly Val 'His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Va1 Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Glu Tyr Tyr Tyr Gly Tle Phe Asp <210> 17 <211> 1545 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (58)..(1545) <400> 17 atgggatgga ctacaggtgt acactcc 57 gctgtatcat cctcttcttg gtagcaacag gat atc gtg acccag tctccagcctcc ctatctgca tctgtg gga 105 ctg Asp Ile Val ThrGln SerProAlaSer LeuSerA1a SerVal Gly Leu gaa act gtc atcaca tgtcgagcaagt gagaatatt tacagt tat 153 acc Glu Thr Val IleThr CysArgAlaSer GluAsnIle TyrSer Tyr Thr tta gca tgg cagcag aaacagggaaaa tctcctcaa ctcctg gtc 201 tat Leu Ala Trp GlnGln LysGlnGlyLys SerPr-oG1n LeuLeu Val Tyr $, tat aat gca aaa acc tta aca gaa ggt gtg cca tca agg ttc agt ggc 249 Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly agt gga tca ggc aca cag ttt tct ctg aag atc aac agc ctg cag cct 297 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro gaa gat ttt ggg aat tat ttc tgt caa cat cat tat gat act cct cgg 345 Glu Asp Phe Gly Asn Tyr Phe Cys Gln His His Tyr Asp Thr Pro Arg acg ttc ggt gga ggg acc aag ctc gag atc aaa ggt ggt ggt ggt tct 393 Thr Phe Gly G1y Gly Thr Lys Leu Glu Ile Lys Gly Gly G1y Gly Ser ggc ggc ggc ggc tcc ggt ggt ggt ggt tct cag gtc caa ctg cag cag 441 Gly G1y Gly Gly Ser Gly Gly Gly Gly Ser Gln Va1 Gln Leu Gln Gln cct ggg get ggg agg gtg agg cct gga get tca gtg aag ctg tcc tgc 489 Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys aag get tct ggc tac tcc ttc acc agt tac tgg atg aac tgg gtg aag 537 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Lys cag agg cct gga caa ggc ctt gag tgg att ggc atg att cat cct tcc 585 Gln Arg Pro Gly G1n Gly Leu Glu Trp I1e Gly Met Ile His Pro Ser gat agt gaa act agg tta aat cag aag ttc aac gac agg gcc aca ttg 633 Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu act gtt gac aaa tat tcc agc aca gcc tat ata caa ctc agc agc ccg 681 Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro acatctgaggac tctgcggtc tattactgt gcaagagga gaatattac 729 ThrSerGluAsp SerAlaVal TyrTyrCys AlaArgGly GluTyrTyr tacggtatattt gactactgg ggccaaggg accacggtc accgtctcc 777 TyrGlyIlePhe AspTyrTrp GlyGlnGly ThrThrVal ThrValSer tccggaggtggt ggatccgat atcaaactg ca.gcagtca ggggetgaa 825 SerGlyGlyGly G1ySerAsp IleLysLeu GlnGlnSer GlyAlaGlu ctggcaagacct ggggcctca gtgaagatg tcctgcaag acttctggc 873 LeuAlaArgPro GlyAlaSer ValLysMet SerCysLys ThrSerGly tac acc ttt act agg tac acg atg cac tgg gta aaa cag agg cct gga 921 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys G1n Arg Pro Gly 9' cagggtctggaatgg attgga tacattaatcct agccgtggt tatact 969 GlnGlyLeuGluTrp TleGly TyrIleAsnPro SerArgGly TyrThr aattacaatcagaag ttcaag gacaaggccaca ttgactaca gacaaa 1017 AsnTyrAsnGlnLys PheLys AspLysAlaThr LeuThrThr AspLys tcctccagcacagcc tacatg caactgagcagc ctgacatct gaggac 1065 SerSerSerThrAla TyrMet Gln,LeuSerSer LeuThrSer GluAsp tctgcagtctattac tgtgca agatattatgat gatcattac tgcctt 1113 SerAlaValTyrTyr CysAla ArgTyrTyrAsp AspHisTyr CysLeu gactactggcgccaa ggcacc actctcacagtc tcctcagtc gaaggt 1161 AspTyrTrpArgGln GlyThr ThrLeuThrVal SerSerVal GluGly ggaagtggaggttct ggtgga agtggaggttca ggtggagtc gacgac 1209 GlySerGlyGlySer GlyGly SerGlyGlySer GlyGlyVal AspAsp att cag ctg acc cag tct cca gca atc atg tct gca tct cca ggg gag 1257 Ile Gln Leu Thr G1n Ser Pro Ala Ile Met Ser A1a Ser Pro G1y Glu aag gtc acc atg acc tgc aga gcc agt tca agt gta agt tac atg aac 1305 Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn tgg tac cag cag aag tca ggc acc tcc ccc aaa aga tgg att tat gac 1353 Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp aca tcc aaa gtg get tct gga gtc cct tat cgc ttc agt ggc agt ggg 1401 Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly tct ggg acc tca tac tct ctc aca atc agc agc atg gag get gaa gat 1449 Ser Gly Thr Ser Tyr Ser Leu Thr I1e Ser Ser Met Glu Ala Glu Asp get gcc act tat tac tgc caa cag tgg agt agt aac ccg ctc acg ttc 1497 Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe gga get ggg acc aag ctg gag ctg aaa cat cat cac cat cat cat tag 1545 Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His His <210> 18 <211> 495 <212> PRT
<213> Mus sp.
<400> 18 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln G1y Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu G1n Pro Glu Asp Phe Gly Asn Tyr Phe Cys G1n His His Tyr Asp Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala G1y Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser .Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr I1e Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys A1a Arg Gly Glu Tyr Tyr 2l0 215 220 Tyr Gly Ile Phe Asp Tyr Trp Gly Gln G1y Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala G1u Leu A1a Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Va1 Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Arg Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly G1y Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Tle Gln Leu Thr Gln Ser Pro Ala Ile Met 5er Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly 435 . 440 445 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe 11' Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His His <210> 19 <211> 24 <212> DNA
<213> Mus sp.
<400> 19 aaaggcctcc ccatattcct cgga 24 <210> 20 <211> 37 <212> DNA
<213> Mus sp.
<400> 20 aaagtcgact ccggacatct ccaaagagtt gatgtac 37 <210> 21 <211> 25 <212> DNA
<213> Mus sp.
<400> 21 aatccggagg cggcagcctg gccgc 25 <210> 22 <211> 46 <212> DNA
<213> Mus sp.
<400> 22 gggaagctta gtgatggtga tggtgatgct tcaggtcctc gcgcgg 46 <210> 23 <211> 1245 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(1245) <400> 23 tcc cca tat tcc tcg gac acc aca ccc tgc tgc ttt gcc tac att gcc 48 Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala cgc cca ctg ccc cgt gcc cac atc aag gag tat ttc tac acc agt ggc 96 Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly 12.
aagtgctcc aaccca gcagtcgtcttt gtcacccga aagaaccgc caa 144 LysCysSer AsnPro AlaValValPhe ValThrArg LysAsnArg Gln gtgtgtgcc aaccca gagaagaaatgg gttcgggag tacatcaac tct 192 ValCysAla AsnPro GluLysLysTrp ValArgGlu TyrIleAsn Ser ttggagatg tccgga ggcggcagcctg gccgcgctg accgcgcac cag 240 LeuGluMet SerG1y GlyGlySerLeu A1aAlaLeu ThrAlaHis Gln gettgccac ctgccg ctggagactttc acccgtcat cgccagccg cgc 288 AlaCysHis LeuPro LeuGluThrPhe ThrArgHis ArgGlnPro Arg ggctgggaa caactg gagcagtgcggc tatccggtg cagcggctg gtc 336 GlyTrpG1u GlnLeu GluGlnCysGly TyrProVal GlnArgLeu Val gccctctac ctggcg gcgcggctgtcg tgg'aaccag gtcgaccag gtg 384 A1aLeuTyr LeuAla AlaArgLeuSer TrpAsnGln ValAspGln Val atccgcaac gccctg gccagccccggc agcggcggc gacctgggc gaa 432 IleArgAsn AlaLeu AlaSerProGly SerGlyGly AspLeuGly Glu gcgatccgc gagcag ccggagcaggcc cgtctggcc ctgaccctg gcc 480 AlaIleArg GluGln ProGluG1nAla ArgLeuAla LeuThrLeu Ala gccgccgag agcgag cgcttcgtccgg cagggcacc ggcaacgac gag 528 AlaAlaGlu SerGlu ArgPheValArg GlnG1yThr GlyAsnAsp Glu gccggcgcg gccaac ggcccggcggac agcggcgac gccctgctg gag 576 AlaG1yAla AlaAsn GlyProAlaAsp SerGlyAsp AlaLeuLeu Glu cgcaactat cccact ggcgcggagttc ctcggcgac ggcggcgac gtc 624 ArgAsnTyr ProThr GlyAlaG1uPhe LeuGlyAsp GlyGlyAsp Va1 agcttcagc acccgc ggcacgcagaac tggacggtg gagcggctg ctc 672 SerPheSer ThrArg GlyThrGlnAsn TrpThrVal GluArgLeu Leu 210. 215 220 caggcgcac cgccaa ctggaggagcgc ggctatgtg ttcgtcggc tac 720 GlnAlaHis ArgGln LeuGluGluArg GlyTyrVal PheValGly Tyr cacggcacc ttcctc gaagcggcgcaa agcatcgtc ttcggcggg gtg 768 HisGlyThr PheLeu GluAlaAlaGln SerIleVal PheGlyGly Val cgcgcgcgc agccag gacctcgacgcg atctggcgc,ggtttctat atc 816 ArgAlaArg SerGln AspLeuAspAla IleTrpArg GlyPheTyr Ile 260 ~ 265 270 gccggc gatccggcg ctggcc tacggctacgcc caggaccag gaaccc 864 AlaGly AspProAla LeuAla TyrG1yTyrAla GlnAspGln GluPro gacgca cgcggccgg atccgc aacggtgccctg ctgcgggtc tatgtg 912 AspAla ArgGlyArg IleArg AsnGlyAlaLeu LeuArgVal TyrVal ccgcgc tcgagcctg ccgggc ttctaccgcacc agcctgacc ctggcc 960 ProArg SerSerLeu ProGly PheTyrArgThr SerLeuThr LeuAla gcgccg gaggcggcg ggcgag gtcgaacggctg atcggccat ccgctg 1008 AlaPro GluAlaAla GlyG1u Va1GluArgLeu IleGlyHis ProLeu ccgctg cgcctggac gccatc accggccccgag gaggaaggc gggcgc 1056 ProLeu ArgLeuAsp AlaIle ThrGlyProGlu GluGluGly GlyArg ctggag accattctc ggctgg ccgctggccgag cgcaccgtg gtgatt 1104 LeuGlu ThrIleLeu GlyTrp ProLeuAlaGlu ArgThrVal ValIle ccctcg gcgatcccc accgac ccgcgcaacgtc ggcggcgac ctcgac 1152 ProSer AlaIlePro ThrAsp ProArgAsnVal GlyGlyAsp LeuAsp CCgtCC agCatCCCC gacaag gaacaggcgatc agcgccctg ccggac 1200 ProSer SerIlePro AspLys GluG1nAlaIle SerAlaLeu ProAsp tacgcc agccagccc ggcaaa ccgccgcgcgag gacctgaag taa 1245 TyrAla SerGlnPro GlyLys ProProArgGlu AspLeuLys <210>
<21l> 4 <212>
PRT
<213>
Mus sp.
<400> 24 Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg G1n Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu Glu Met Ser G1y Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly G1-y Asp Leu Gly Glu 14' Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala A1a Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp G1u Ala Gly Ala A1a Asn Gly Pro Ala Asp 5er Gly Asp Ala Leu Leu Glu 180 l85 190 Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val G1y Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser G1n Asp Leu Asp Ala Ile Trp Arg G1y Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Tle Thr Gly Pro Glu Glu Glu G1y Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val I1e Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys <210>
<2l1>
<212>
DNA
<213> .
Mus sp.
<220>.
<221>
CDS
<222> (363) (1)..
<400>
gat atc ctg cagcagtca ggggetgaactg gcaagacct ggggcc 48 aaa Asp Ile Leu GlnGlnSer GlyAlaGluLeu AlaArgPro GlyAla Lys tca gtg atg tcctgcaag acttctggctac acctttact aggtac 96 aag Ser Val Met SerCysLys ThrSerGlyTyr ThrPheThr ArgTyr Lys acg atg tgg gtaaaacag aggcctggacag ggtctggaa tggatt 144 cac Thr Met Trp ValLysGln ArgProGlyGln GlyLeuGlu TrpIle His 15.
ggatacattaat cctagccgt ggttatact aattacaat cagaagttc 192 GlyTyrIleAsn ProSerArg GlyTyrThr AsnTyrAsn GlnLysPhe aaggacaaggcc acattgact acagacaaa tcctccagc acagcctac 240 LysAspLysAla ThrLeuThr ThrAspLys SerSerSer ThrAlaTyr atgcaactgagc agcctgaca tctgaggac tctgcagtc tattactgt 288 MetGlnLeuSer SerLeuThr 5erGluAsp SerAlaVal TyrTyrCys gcaagatattat gatgatcat tactgcctt gactactgg cgccaaggc 336 AlaArgTyrTyr AspAspHis TyrCysLeu AspTyrTrp ArgGlnGly accactctcaca gtctcctca gtcgaa 363 ThrThrLeuThr ValSerSer ValG1u <210>
<211>
<212>
PRT
<213>
Mus sp.
<400>
Asp Ile Leu GlnGlnSer G1yAlaGlu LeuAlaArg ProGlyAla Lys Ser Val Met SerCysLys ThrSerGly TyrThrPhe ThrArgTyr Lys Thr Met Trp ValLysGln ArgProGly GlnGlyLeu GluTrpIle His Gly Tyr Asn ProSerArg GlyTyrThr AsnTyrAsn GlnLysPhe Tle Lys Asp Ala .ThrLeuThr ThrAspLys SerSerSer ThrAlaTyr Lys Met Gln Ser SerLeuThr SerGluAsp SerAlaVa1 TyrTyrCys Leu Ala Arg Tyr AspAspHis TyrCysLeu AspTyrTrp ArgGlnGly Tyr Thr Thr Thr ValSerSer ValGlu Leu <210> 27 <211> 324 <212> DNA
<213> Mus sp.
<220>
<221> CDS
16~
<222> (1)..(324) <400> 27 gtc gac gac att cag ctg acc cag tct cca gca atc atg tct gca tct 48 Val Asp Asp Ile Gln Leu Thr Gln Ser Pro A1a Ile Met Ser Ala Ser cca ggg gag aag gtc acc atg acc tgc aga gcc agt tca agt gta agt 96 Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser tac atg aac tgg tac cag cag aag tca ggc acc tcc ccc aaa aga tgg 144 Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp att tat gac aca tcc aaa gtg get tct gga gtc cct tat cgc ttc agt 192 Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser ggc agt ggg tct ggg acc tca tac tct ctc aca atc agc agc atg gag 240 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu get gaa gat get gcc act tat tac tgc caa cag tgg agt agt aac ccg 288 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro ctc acg ttc gga get ggg acc aag ctg gag ctg aaa 324 Leu Thr Phe Gly Ala Gly Thr Lys Leu G1u Leu Lys <210> 28 <211> 108 <212> PRT
<213> Mus sp.
<400> 28 Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met.Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala G1y Thr Lys Leu Glu Leu Lys 17, <210> 29 <211> 11 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 29 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Zeu A1a <2l0> 30 <211> 7 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 30 Asn Ala Lys Thr veu Thr Glu <210> 31 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 31 Gln His His Tyr Asp Thr Pro Arg Thr <210> 32 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial. Sequence: peptide <400> 32 Tyr Trp Met Asn <210> 33 <211> l9 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 33 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg <210> 34 <2l1> 10 <212> PRT
<2l3> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 34 Gly Glu Tyr Tyr Tyr Gly Ile Phe Asp Tyr
Mostly, there is hardly anything known about the mode of action of second-line agents.
It is not yet entirely clear whether the application of second-line agents can diminish the destruction of the joint. In recent years, a new group of substances has been intro-duced into the treatment of rheumatoid arthritis, which is based on the blocking of cell signal substances (particularly TNFa) by means of monoclonal antibodies or soluble receptor constructs.
In addition, there are patients that do not respond to currently available therapies. In other cases, the conventional therapy has to be stopped due to intolerable side effects.
A similar situation exists for many other inflammatory and autoimmune diseases like inflammatory renal diseases, inflammatory bowel diseases, multiple sclerosis and transplant rejection, where current treatments have many limitations. For example, agents used in inflammatory and autoimmune diseases include anti-inflammatory and immunosuppressive agents like azathioprine, cyclophosphamide, glucocorticoids like prednisone and corticosteroids; immunosuppressants like cyclosporin A, Tacrolimus (FK506), Sirolimus (Rapamycin); and protein drugs like calcineurin, beta-interferon, anti-TNF alpha monoclonal antibodies (remicade). These agents show general immu-nomodulating effects and therefore efficacy and side effects profiles can pose severe limitations for the treatment options (Harrison's Principles of Internal Medicine, eds.
Fauci et al., 14t" edition, McGraw-Hill publisher).
Inflammatory bowel diseases (examples are Crohn's disease, ulcerative colitis) are treated with the anti-inflammatory agents sulfazsalazine (Azulfidine) and glucocorti-colds, like prednisone and, in selected cases, with TNF-a blocking agents. In ulcerative colitis immunosuppressive therapy with drugs such as azathioprine is well established, in severely ill patients the potent immunosuppressive agent cyclosporine is used (Harrison's Principles of Internal Medicine, eds. Fauci et al., 14t" edition, McGraw-Hill publisher).
In many cases no sufficient reduction of disease activity is achieved with current drugs, such that even surgical intervention is sometimes necessary.
Inflammatory renal diseases (nephritis) are treated with e.g. glucocorticoids, alkylating agents and/or plasmapheresis. Additional diseases with similar treatment options in-clude systemic lupus erythemtosus (SLE), Sjoegren's syndrome, polymyositis, derma-tomyositis, mixed connective tissue disease, antiphospholipidantibody syndrome.
For some of these diseases, few therapeutic options have been available up to now. All these diseases share an inflammatory component. However, the inflammatory compo-nent cannot be sufficiently suppressed by the currently available drugs. For some drugs, e.g. alkylating agents a maximal lifetime dose per patient cannot be exceeded.
Transplant rejection is treated using immunosuppressive agents including azathioprine, mycophenolate mofetil, glucocorticoids, cyclosporine, Tacrolimus (FK506), Sirolimus (Rapamycin). A combination of steroids and a low dose of mouse monoclonal antibody OKT3 binding to CD3 on T-cells is used to anergize and deplete T-cells, therapy is continued using immunosuppressants like cyclosporine. Human anti-mouse antibodies I(HAMAs) have common side effects and limit the use of OKT3 (Fauci et al. sic.
2381 ).
Approaches to treat multiple sclerosis include treatments which effect the overall im-mune system like anti-inflammatory agents including azathioprine, cyclophosphamide, prednisone, corticosteroids, cyclosporin A, calcineurin, Rapamycin, beta-interferon (Fauci et al, sic. 2415-2419; Wang (2000) j. Immunol. 165, 548-57). In addition, a num-ber of non-specific treatments are administered that may improve the quality of life in-cluding physical therapy and psycho-pharmacological agents. None of the treatment options mentioned above has a curative effect. Even the most promising compound, (3-interferon, leads only to a slower disease progression, while exhibiting significant side effects.
Furthermore, human immunodeficiency virus-type 1 (HIV-1 ), the most common cause of AIDS, has infected more than 50 million individuals (including those who have died), and the rate of new infections is estimated at nearly 6 million per year (AIDS
Epidemic Update: December 1999 (UNAIDS, Geneva, 1999), www.unaids.org). Equally disturb-ing are the uncertainties of the epidemic to come. Although sub-Saharan Africa re-mains the global epicenter, rates of infection have increased in recent times in the for-mer Soviet Union and parts of south and southeast Asia, including India and China, where literally hundreds of millions of individuals are potentially at risk.
In the United States, new waves of infection have been recognized in women, minorities, and younger generations of gay men. Combination antiretroviral therapy has afforded many people clinical relief, but the costs and toxicities of treatment are substantial, and HIV-1 infection remains a fatal disease. Moreover, the vast majority of infected people world-wide do not have access to these agents. Thus, although the demographics (and, in some instances, the natural history) of AIDS have changed, the epidemic is far from over; instead, it is evolving, expanding, and posing ever greater challenges.
Human immunodeficiency virus (HIV) cannot enter human cells unless it first binds to two key molecules on the cell surface, CD4 and a co-receptor. The co-receptor that is initially recognized is CCRS, later in the life cycle of the virus another chemokine re-ceptor CXCR4 becomes the co-receptor for HIV-1 (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)). The HIV-strains that cause most transmissions of viruses by sexual contact are called M-tropic viruses. These HIV-1 strains (also known as NSI primary viruses) can replicate in pri-mary CD4+ T-cells and macrophages and use the chemokine receptor CCR5 (and, less often, CCR3) as their coreceptor. The T-tropic viruses (sometimes called SI pri-mary) can also replicate in primary CD4+ T-cells but can in addition infect established CD4+ T-cell lines in vitro, which they do via the chemokine receptor CXCR4 (fusin).
Many of these T-tropic strains can use CCR5 in addition to CXCR4, and some can en-ter macrophages via CCRS, at least under certain in vitro conditions (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996)).
Whether other coreceptors contribute to HIV-1 pathogenesis is unresolved, but the ex-istence of another coreceptor for some T-tropic strains can be inferred from in vitro studies. Because M-tropic HIV-1 strains are implicated in about 90% of sexual trans-missions of HIV, CCR5 is the predominant coreceptor for the virus in patients;
trans-mission (or systemic establishment) of CXCR4-using (T-tropic) strains is rare (D'Souza, Nature Med. 2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, (1996), Paxton, Nature Med. 2, 412 (1996); Liu, Cell 86, 367 (1996); Samson, Nature 382, 722 (1996); Dean, Science 273, 1856 (1996); Huang, Nature Med. 2, 1240 (1996)). However, once SI viruses evolve in vivo (or if they are transmitted), they are especially virulent and cause faster disease progression (D'Souza, Nature Med.
2, 1293 (1996); Premack, Nature Med. 2, 1174; Fauci, Nature 384, 529 (1996), Schuitemaker, J. Virol. 66, 1354 (1992); Connor, J. Virol. 67, 1772 (1993);
Richman, J.
Infect. Dis. 169, 968 (1994); R. I. Connor et al., J. Exp. Med. 185, 621 (1997); Trkola, Nature 384, 184 (1996)).
The numbers and identity of coreceptor molecules on target cells, and the ability of HIV-1 strains to likely enter cells via the different coreceptors, seem to be critical de-terminants of disease progression. These factors are major influences on both host-and virus-dependent aspects of HIV-1 infection. For example, a homozygous defect (delta 32) in CCR5 correlates strongly with resistance to HIV-1 infection in vivo and in vitro. Individuals who are heterozygous for a defective CCR5 allele are at best weakly protected against infection and have only a modestly slowed disease progression (Paxton, Nature Med. 2, 412 (1996); Liu, Cell 86, 367 (1996); Samson, Nature 382, 722 (1996); Dean, Science 273, 1856 (1996); Huang et al., Nature Med. 2, 1240 (1996)).
However, other factors can influence the level of CCR5 expression on activated CD4+
T-cells and thereby affect the efficiency of HIV-1 infection in vitro (Trkola, Nature 384, 184 (1996); Bleul, Proc. Natl. Acad. Sci. U.S.A. 94, 1925 (1997)). For reasons that are not yet clear, the amount of CCR5 expression on the cell surface (as measured by MIP-1 binding) varies by 20-fold on CD4+ T-cells from individuals with two wild-type CCR5 alleles (Trkola, Nature 384, 184 (1996)) (see figure). Staining with a CCRS-specific monoclonal antibody indicates a similar large variability (Wu, J. Exp. Med.
186:1373-81 (1997)). Such variation may far outweigh any effect of one defective allele for CCRS.
The causes of this variation should be the subject of intensive studies, as they point to controllable factors that could increase resistance to disease.
Most primary, clinical isolates of primate immunodeficiency viruses use the chemokine receptor CCR5 for entry (Feng, Science 272, 872 (1996); Choe, Cell 85, 1135 (1996);
Deng, Nature 381, 661 (1996); Dragic et al., Nature 381, p. 667; Doranz, Cell 85, 1149 (1996); Alkhatib, Science 272, 1955 (1996)). For most HIV-1 isolates that are transmit-ted and that predominate during the early years of infection, CCR5 is an obligate core-ceptor, and rare individuals that are genetically deficient in CCR5 expression are rela-tively resistant to HIV-1 infection (Connor, J. Exp. Med. 185, 621 (1997);
Zhang, Nature 383, 768 (1996); Bjorndal, J. Virol. 71, 7478 (1997); Dean, Science 273, 1856 (1996);
Liu, Cell 86, 367 (1996); Paxton, Nature Med. 2, 412 (1996); Samson, Nature 382, 722 (1996)). HIV-1 isolates arising later in the course of infection often use other chemokine receptors, frequently CXCR4, in addition to CCRS. Studies of chimeric envelope glyco-proteins demonstrated that the third variable (V3) loop of gp120 is a major determinant of which chemokine receptor is used (see references above and also Cocchi, Nature Med. 2, 1244 (1996); Bieniasz, EMBO J. 16, 2599 (1997); Speck, J. Virol. 71, (1997)). V3-deleted versions of gp120 do not bind CCRS, even though CD4 binding occurs at wild-type levels. Antibodies to the V3 loop interfere with gp120-CCR5 binding (Trkola, Nature 384, 184 (1996); Wu, Nature 384, 179 (1996); Lapham, Science 274, 602 (1996); Bandres, J. Virol. 72, 2500 (1998); Hill, Science 71, 6296 (1997)). These results support an involvement of the V3 loop in chemokine receptor binding.
Latency of HIV is established very early in the course of an infection, when M-tropic strains predominate. M-tropic strains depend on the presence of CCR5 on the target cell for infection. The importance of CCR5 as an essential co-receptor for M-tropic HIV-1 is emphasized by the fact that individuals lacking CCR5 due to a homozygous basepair deletion (delta32) are highly resistant to HIV-1 infection. In contrast to other markers like CD4, CD25, or CD45R0, CCR5 is only present on a subset of lympho-cytes and other cells that are prone to HIV-1 infection (Rottmann (1997) Am J
Pathol 151, 1341-1351; Naif (1998) J Virol 72, 830-836; Lee (1999) Proc. Natl Acad.
Sci. 96, 5315-5220).
Several approaches have been postulated to eliminate latent infected cells.
One strat-egy is to drive the latently infected to virus production and subsequent cell death. In this context, one approach is IL-2 (TNF-alpha, IL-6) administration in the presence of HAART until the viral reservoir is exhausted (Chun (1998) J. Exp. Med. 188, 83-91;
Chun (1999) Nat. Med. 5, 651-655; Stellbrink (1999) Abstracts of the 6th Conference on Retroviruses and Opportunistic Infections (Foundation for Retrovirology and Human Health, Alexandria, VA), abstr. 356. p. 135; Imamichi (1999) Abstracts of the 6th Con-ference on Retroviruses and Opportunistic Infections (Foundation for Retrovirology and Human Health, Alexandria, VA), abstr. 358, p. 135). These cells are believed to die af-ter activation. Whether the entire pool of latent infected cells can be exhausted is ques-tionable.
Another strategy tried was to specifically kill latently infected cells based on gp-120 ex-pression on the cell surface. Immunotoxins recognizing gp-120 have been proposed but failed for two reasons. The one construct tested in humans was a protein consisting of soluble CD4 linked to Pseudomonas aeroginosa exotoxin A (PE). The clinical results were disappointing due to dose-limiting hepatotoxicity without showing signs of efficacy and the program was terminated (Ashorn (1990) Proc. Natl Acad. Sci 87, 8889-8893;
Berger (1998) Proc. Natl Acad. Sci. 95, 11511-11513). The second reason for failure was that latent infected cells do not express viral surface glycoproteins, e.g. gp-120 and gp-41. Thus, approaches targeting gp-120 or gp-41 for the elimination of latently infected cells cannot work.
Other approaches to eliminate latent infected cells are based on eliminating the entire CD4+ T-cell compartment (Berger (1998) Proc. Natl Acad. Sci. 95, 11511-11513), or the CD25-positive compartment (Bell (1993) Proc. Natl Acad. Sci. 90, 1411-1415) the CD45R0 memory cell compartment (McCoig (1999) Proc. Natl. Acad. Sci 96, 11482-11485). However these markers do not adequately include all potentially infected cells.
Such cells include besides CD4-posifiive cells, or memory cells also macrophages, and non-hematopoietic cells.
In WO 98/18826 an antibody directed against the mammalian (e.g. human) chemokine receptor 5 is described and said antibody is proposed in a method of inhibiting the in-teraction of cell bearing CCR5 with a potential ligand, like HIV. It is proposed that said method inhibits an HIV infection. Furthermore, treatment options for inflammatory dis-eases, autoimmune diseases and graft rejection are proposed. Yet, all these treatment options are based on the assumption that specific antibodies like the immunoglobulin molecules themselves or functional portions thereof interfere with receptor-ligand inter-actions. However, whether these antibodies are capable of depleting the relevant cells is questionable. Furthermore, WO 98/18826 merely envisages the prevention of an in-teraction of HIV and the CCR5 receptor and thereby preventing an HIV
infection.
Leukocytes, in particular T-cells, are believed to be the key regulators of the immune response to infecfiive agenfis and are critical components for fihe initiation and mainte-nance of inflammatory processes, like inflammatory bowel disease inflammatory renal diseases, inflammatory joint disease, autoimmune disorders, like multiple sclerosis and arthritis, skin diseases, like psoriatic lesions, diabetes and in transplant rejection.
Therefore, the technical problem underlying the present invention is to provide for novel means and methods which can lead to the suppression of activated leukocytes involved in immunological pathologies, .like autoimmune diseases, inflammation process and/or viral infections of immune cells.
Accordingly, the present invention relates to the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the elimination of cells which are latently infected with a primate immu-nodeficiency virus, preferably a human immunodeficiency virus, most preferably HIV-1.
In context of the present invention the binding of said antibody and/or chemokine con-struct which binds to a chemokine receptor results in the depletion and/or destruction of the target cell, namely the cell latently infected with said primate immunodeficiency vi-rus.
In this invention it could surprisingly be shown that highly specific antibodies directed against an chemokine receptor were not able to destroy, lyse and/or deplete cells which express said chemokine receptor. However, antibody constructs or chemokine constructs as described and disclosed in the present invention were capable to specifi-cally interact with said chemokine-receptor positive cells and were able to deplete said cells. Said depletion/destruction may, e.g., be achieved by the attraction of specific ef fector cells, like monocytes, macrophages, T-cells (particularly preferred are cytotoxic T-cells) or dendritic cells. Even if monoclonal antibodies had been shown to be suc-cessful in the destruction/depletion of malignant cells (see, e.g., Maloney (1999), Sem Oncol. 26, 76-78), they appear to be ineffective against certain subtypes of leukocytes, (comprising lymphocytes, polynuclear leukocytes and monocytes) especially CCR5+
monocytes, T-cells and dendritic cells as documented herein and in the appended ex-amples.
In accordance with the present invenfiion, the term "antibody andlor chemokine con-struct" (i.e. antibody construct and/or chemokine construct) not only comprises the molecules and multifunctional constructs and compounds as described herein, but also comprises functional fragments thereof. Functional fragments of said constructs are meant to be fragments which are capable of binding. to/interacting with a chemokine receptor on a target cell and providing for means for depleting, lysing andlor destroying said target cell.
Specific chemokine receptors, in accordance with the present invention comprise, but are not limited to, CXCR3, CXCR4, CXCRS, CCR1, CCR2, CCR3, CCR4, CCRS, CCR6, CCR7, CCRB, CCR9, XCR1, CCR10 and CX3CR1. Chemokines and/or che-mokine ligands binding to said chemokine receptors are well known in the art and shown, inter alia, in Table 4. Furthermore, chemokines and corresponding receptors are disclosed in Murphy (2000), Pharm. Reviews 52, 145-176. The chemokines, che-mokirie ligands and/or receptors are preferably primate, more preferably human che-mokines/ligands/receptors.
The present invention also relates to the use of an antibody and/or chemokine con-struct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the treatment, prevention and/or alleviation of inflammatory renal dis-eases, inflammatory bowel diseases, multiple sclerosis, skin diseases, allergic reac-tions diabetes or transplant rejection.
Said skin diseases comprise, inter alia, psoriatic disorders, atopic dermatitis or chroni-cally inflamed skin. CCR6 .expression is upregulated in PBMCs derived from patients with psoriasis. In addition, CCR6 ligand (CCL20=MIP3alpha) and CCR6 are upregu-lated in psoriatic skin. Furthermore, CCL20 expressing keratinocytes colocalize with skin infiltrating T-cells (Homey (2000) J. Immunol. 164, 6621-6632).
Furthermore, CCR10 was detected on melanocytes, dermal fibroblasts, dermal endothelial cells, T-cells and skin-derived Langerhans cells but not keratinocytes. CCR10 ligand (CCL27) has a skin associated expression pattern (Homey (2000) J. Immunol. 164, 3465-3470;
Charbonnier (1999) J. Exp. Med 190, 1755-1768). In addition, CCR4 and its ligand (TARC, MDC) are upregulated in chronically inflamed skin. Moreover CCR4 is a hom-ing receptor for T-cells entering the skin. CCR4+ T-cells are only a small subpopulation of all T cells and therefore depletion of CCR4+ T-cells is indicated for various inflam-matory skin diseases (Campbell (1999) Nature 400, 776-780). CCR3 and exotoxin ex-pression is enhanced in atopic dermatitis and may contribute to the initiation and maintenance of inflammation (Yawalkar (1999) J. Invest. Dermatol. 113, 43-48).
Virtually all T-cells in rheumatoid arthritis, synovial fluid and in various inflamed tissues such as ulcerative colitis, chronic vaginitis and sarcoidosis express CXCR3.
Whereas fewer T-cells within normal lymph nodes are CXCR3 positive.
For multiple sclerosis it was shown that CCR5 and CXCR3 are predominantly ex-pressed on T-cells infiltrating demyelinating brain lesions, as well as in the peripheral blood of affected patients. The corresponding ligands MIP-1a and IP-10 were also de-tectable in the plaques (Balashov (1999) Proc. Natl. Acad. Sci. 96, 6873-6878). Elimi-nation of the T-cells would block the T-cell arm of this autoimmune disease.
Immunochemical analysis of the expression of the beta-chemokine receptors in post-mortem CNS tissue from patients with multiple sclerosis revealed that in chronic active MS lesions expression of CCR2, CCR3 and CCR5 was associated with foamy macro-phages and activated microglia while low levels of these chemokine receptors were expressed by microglial cells in control CNS tissue. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes and in 5/14 cases of MS CCR3 and were also expressed on astrocytes. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS (Simpson, J. Neuroimmunol., 2000, 108, 200).
High expression of CCR3 and CCR5 was also observed in T cells and B cells of lymph nodes derived from patients with Hodgkin disease. While CCR3 was equally distributed in CD4+ and CD8+ cells, CCR5 was mainly associated with CD4+ cells. These data suggest that chemokines are involved in the formation of the nonneoplastic leukocytic infiltrates in Hodgkin disease (Buri, Blood, 2001, 97, 1543-8).
Periodontal disease is a peripheral infection involving species of gram-negative organ-isms. In patients with moderate to advanced periodontal disease CCR5 chemokine re-ceptor expressing cells were found in the inflammatory infiltrates (Gamonal, J. Perio-dontal. Res., 2001, 36, 194-203 and Taubman, Crit. Rev. Oral. Biol. Med. 2001, 12, 125-35).
Diabetes type I is considered to be a T-cell mediated autoimmune disease. The ex-pression of CCR5 receptor in the pancreas was associated with the progression of type I diabetes in relevant animal models (Cameron (2000) J. Immunol. 165, 1102-1110). In particular, the CCR5 expression was associated with the development of insulinitis and spontaneous type I diabetes.
Specific chemokines are associated with T-cell migration in diabetes type I
relevant animal model: RANTES, MCP-1, MCP-3, MCP-5, IP10. These chemokines lead to a th1 immune response (Bradley (1999) J. Immunol. 162:2511-2520).
The above mentioned inflammatory bowel disease may comprise Morbus Crohn and colitis ulcerosa.
CCR9 is expressed on T-cells homing to the intestine and may be implied in Morbus Crohn and colitis ulcerosa. All intestinal lamina propria and intraepithelial lymphocytes express CCR9 (Zabel (1999) J. Exp. Med. 190, 1241-1256).
Additionally, the antibody- and/or chemokine construct as described in context of the present invention is also useful for avoiding complications during and/or after trans-plants, i.e. to avoid transplant rejections and graft versus host disease.
CCR7 is expressed on naive T-cells and dendritic cells and mediates cell migration to lymphatic organs. Elimination of CCR7+ cells would therefore prevent an immune re-sponse to novel antigens, e.g., following transplantation. Such a treatment would not be generally immune suppressing but selective for novel antigens and limited for the dura-tion of the adminisfiration of drugs of the invention depleting CCR7+ cells (Forster (1999) Cell 99, 23-33). CXCR5 is expressed on naive B cells in the peripheral blood and tonsils and memory T-cells. Elimination of CXCRS+ B-cells would prevent the es-tablishment of a humoral response. Furthermore, elimination of memory T-cells would reduce the cellular component of the immune response (Murphy (2000) Pharmacologi-cal Reviews 52, 145-176).
In order to provide pharmaceutical compositions for the treatment of allergies and/or allergic reactions, the antibody- and/or chemokine constructs as described herein may be employed. It was shown that CCR3 which binds exotoxin and RANTES, is ex-pressed on eosinophils, Th2 cells, mast cells, basophils, which are involved in allergic reactions (Romangnani (1999) Am. J. Pathol. 155, 1195-1204).
As far as the above mentioned renal or kidney diseases are concerned, it has been shown that CCR5 positive T-cells may play a role in interstitial processes leading to fibrosis. CCR5 positive cells have been identified in the interstitial infiltrate of various glomevular and interstitial diseases, as well as transplant rejection. Said disease com-prises acute and chronic nephritis, IgA nephropathy, and others (Segerer (1999), Kid-ney Int. 56, 52-64).
In a model of transient immune complex glomerulonephritis (IC-GN), CCR1, CCR2, and CCR5 were expressed early and were already downregulated at the peak of proteinuria and leukocyte infiltration. Expression of CCR5 was located to the glomerulus by in situ hybridization and quantitative reverse transcription-PCR of isolated glomeruli (Anders, J. Am. Soc. Nephrol., 2001, 12, 919-31 ). In kidneys of 38 patients with several renal diseases, CCR1- and CCRS-positive macrophages and T cells were detected in both glomeruli and interstitium as shown by immunohistochemistry. Renal CCRS-positive cells were dramatically decreased during convalescence induced by glucocorticoids (Furuichi, Am. J. Nephrol., 2000, 20, 291-9).
In a preferred embodiment of the present invention, the invention provides for the use of an antibody and/or chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition as described hereinabove, wherein said chemokine receptor is the chemokine receptor 5 (CCRS). It is preferred that said CCR5 is the human CCRS.
The chemokine receptor CCRS is a member of a large family of G protein coupled seven transmembrane domain receptors that binds the proinflammatory chemokines RANTES, MIP1-a, MIP1-f3 and MCP-2. Chemokines act in concert with adhesion molecules to induce the extravasation of leukocytes and to direct their migration to sites of tissue injury.
The CCR5 is expressed on a minority of T-cells and monocytes and is further the major co-receptor for M-trophic HIV-1 strains that predominate early in the course of an HIV-infection.
The pharmaceutical composition as described hereinabove is, therefore, particularly useful in the depletion of CCRS+ leukocytes and would be useful in the elimination of cells latently infected with HIV-1. Depletion of CCR5+ cells should therefore reduce the number of cells latently infected with HIV and should be particularly useful in combina-tion with active anti-viral, preferably anti-retroviral therapy.
In a particularly preferred embodiment said antibody construct is a bispecific antibody which binds to the chemokine receptor, preferably the CCRS, as a first antigen and a CD3 antigen of an effector cell as a second antigen. Preferably said CD3 antigen is on the surface of a T-cell, preferably a cytotoxic T-cell. Said CD3 therefore, denotes an antigen that is expressed on the above mentioned cells and may be part of the multi-molecular (T-) cell receptor complex.
Bispecific antibodies may be constructed by hybrid-hybridoma techniques, by cova-lently linking specific antibodies or by other approaches, like the diabody approach (Ki-priyanow, Int. J. Cancer 77 (1998), 763-773).
It is preferred that said bispecific antibody is a single chain antibody construct.
As is well known, Fv, the minimum antibody fragment which contains a complete anti-gen recognition and binding site, consists of a dimer of one heavy and one light chain variable domain (VF., and V~) in non-covalent association. In this configuration that cor-responds to the one found in native antibodies the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-V~ dimer. Collectively, the six CDRs confer antigen binding speci-ficity to the antibody. Frameworks (FRs) flanking the CDRs have a tertiary structure which is essentially conserved in native immunoglobulins of species as diverse as hu-man and mouse. These FRs serve to hold the CDRs in their appropriate orientation.
The constant domains are not required for binding function, but may aid in stabilizing VH-V~ interaction. Even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than an entire binding site (Painter, Biochem. 11 (1972), 1327-1337). Hence, said domain of the binding site of the antibody construct as defined and described in the present invention can be a pair of VH-V~, VH-VH or V~-V~
domains of different immunoglobulins. The order of VH and V~ domains within the polypeptide chain is not decisive for the present invention, the order of domains given hereinabove may be reversed usually without any loss of function. It is important, however, that the Vt., and V~ domains are arranged so that the antigen binding site can properly fold.
Different parts of the antibodies/immunoglobulins can be joined by means of conven-tional methods or constructed as a contiguous protein by means of recombinant DNA
techniques, e.g. in such a way that a nucleic acid molecule coding for a chimeric or humanized antibody chain is expressed in order to construct a contiguous protein (cf.
for example Mack et al. (1995) Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 7021-7025).
A single-chain antibody with the following Fv fragments is preferred: sc-Fv fragment of a monoclonal antibody against the chemokine receptor, preferably against CCRS, and an sc-Fv fragment of a monoclonal antibody against CD3. In this case, both the Fv fragment directed against the chemokine receptor and the Fv fragment against may be located in N-terminal position. The Fv fragment against CCR5 is preferred to be in N-terminal position. The order of the VL and VH antibody domains can be variable in both constructs, preferably, the order of the Fv fragment against CCR5 is VL-VH and the one of the Fv fragment against CD3 is VH-VL. The linkers between the variable domains as well between the two Fv fragments may consist of peptide linkers, prefera-bly of a hydrophilic flexible glycine- and serine-containing linker of 1 to 25 amino acids.
An additional histidine chain of ,e.g., 6 x His, in C- or N-terminal position can be used to simplify purification and detection.
Compared to conventional bispecific antibodies, bispecific single-chain antibodies have the advantage that they consist of only one protein chain and thus their composition is exactly defined. They have a low molecular weight of normally < 60 kD and can be pro-duced easily and on a large scale in suitable cell lines, e.g. in CHO cells, using recom-binant techniques. The most essential advantage, however, is that they have no con-stant antibody domains and thus only activate T-lymphocytes to lysis when these are bound to their target cells, i.e. to the chemokine-receptor expressing cells.
Therefore, single-chain antibodies are often superior to conventional bispecific antibodies as their clinical use entails fewer or.less severe side effects.
MC-1 was shown to bind specifically to the first part of the second extracellular loop of human CCR5 and did not crossreact with CCR5 derived from rhesus macaques as shown in the appended examples. Therefore, it is preferred that said single chain anti-body construct comprises V~ and VH domains of a antibody specific for the chemokine receptor, preferably the human CCRS, and VH and V~ domains of an antibody specific for a CD3 antigen. Said antibody specific for the human CCR5 is the murine anti-human CCR5 antibody MC-1, described, inter alia, in Mack (1998), J. Exp. Med.
187, 1215-1224 and in the appended examples. Yet, it is envisaged that other a-CCR5 anti-bodies, like MC-5 (as characterized in the appended examples and disclosed in Segerer (1999), loc. cit.) may be employed in the context of this invention.
The antibody specific for a CD3 antigen may be selected from the group consisting of antibodies rec-ognizing the gamma, delta, epsilon, zeta chains, particularly preferred are antibodies recognizing the epsilon chain and the CD3 zeta chain (Jakobs (1997) Cancer Immunol Immunother. 44, 257-264; Mezzanzanica (1991 ) Cancer Res 51, 5716-5721 ). Exam-ples of anti-epsilon chain antibodies are OKT3 (WO 91/09968, Kung et al., Science 206, 347-349 (1979); Van Wauwe, J. Immunol. 124, 2708-2713 (1980); Transy, Eur. J.
Immunol. 19, 947-950 (1989); Woodle, J. Immunol. 148, 2756-2763 (1992); Ada, Hu-man. Antibod. Hybridomas, 41-47 (1994)) and TR66 (Traunecker (1991 ) EMBO J.
10, 3655-3659). Examples of monoclonal antibodies against the CD3 zeta chain are H2D9, TIA2 (both Becton Dickinson), G3 (Serotec Ltd.).
In a particularly preferred embodiment of the use of the present invention, the V~ and VH domains of the single chain antibody as described above are arranged in the order V~(MC-1 )-VH(MC-1 )-VH(CD3)-V~(CD3), whereby it is particularly preferred that V~(MC-1) comprises the amino acid sequence as depicted in SEQ ID NO: 12, wherein said VH(MC-1) comprises the amino acid sequence as depicted in SEQ ID NO: 16, wherein said VH(CD3) comprises the amino acid sequence as depicted in SEQ ID NO: 26 and/or wherein said V~(CD3) comprises in SEQ ID NO: 28. Specific CDR parts of the MC-1 antibody are shown in SEQ ID NO: 29 to 34, wherein SEQ ID NO: 29 shows the CDR1 of V~ MC-1, SEQ ID NO: 30 shows the CDR2 of V~ MC-1, SEQ ID NO: 31 shows the CDR3 of V~-MC-1, SEQ ID NO: 32 shows the CDR1 of VH MC-1, SEQ ID NO: 33 shows the CDR2 of VH MC-1 and SEQ ID NO: 34 depicts the CDR 3 of VH MC-1.
Said bispecific antibody may, inter alia, comprise an amino acid sequence encoded by the nucleic acid sequence as depicted in SEQ ID NO: 17 or comprises the amino acid sequence as depicted in SEQ ID NO: 18.
In another embodiment of the use of the present invention, the antibody construct is a bispecific antibody which binds to said chemokine receptor as a first antigen and a toxin as a second antigen. The antibody may be covalently bound to said toxin, and said antibody-toxin construct may be constructed by chemical coupling, producing a fusion protein or a mosaic protein from said antibody and from a modified or unmodified prokaryotic or eukaryotic toxin. Furthermore, said antibody may be joined to said toxin via additional multimerization domains.
In a further embodiment of the use of the present invention said antibody construct can, via a multimerization domain, be bound in vitro and/or in vivo to a second antibody construct which binds to a CD3 antigen and/or a toxin. Said multimerization may, inter alia, be obtained via hetero(di)merization. For example, the hetero(di)merization region of constant immunoglobulin domains may be employed. Other multi- and/or heterodi-merization domains are known in the art and are based on leucine zippers, a-and f3-chains of T-cell receptors or MHC-class II molecules. Furthermore, jun- and fos-based domains may be employed (de Kuif (1996) J. Biol. Chem. 271, 7630-7634;
Kostelny (1992), J. Immunol. 148, 1547-1553). Additional examples of multimerization domains are p53- and MNT-domains as described in Sakamoto (1994) Proc. Natl. Acad.
Sci.
USA 91, 8974-8978; Lee (1994) Nat. Struct. Biol. 1, 877-890; Jeffrey (1995) Science 267, 1498-5102 or Nooren (1999) Nat. Struct. Biol. 6, 755-759).
In another embodiment of the invention, the above mentioned chemokine construct is a fusion construct of a modified or an unmodified chemokine with a modified or an un-modified toxin. Said construct may, inter alia via a multimerization domain, be bound in vitro and/or in vivo to an antibody construct which binds to a CD3 antigen and/or to a toxin. Suitable multimerization domains have been described in the art and are men-tioned hereinabove. The chemokine-toxin constructs may, inter alia, result from chemi-cal coupling, may be recombinantly produced (as shown in the appended examples), or may be produced as a fusion protein from a chemokine and a modified or unmodified prokaryofiic or eukaryotic toxin. It is particularly preferred that said chemokine binds to the human chemokine receptor CCR5 and comprises, inter alia, RANTES, MIP-1a, MIP-1 f3, MCP-2, MCP-3 or (a) fragments) thereof which are capable of binding to said receptor. A preferred toxin may be a truncated version of pseudomonas exotoxin, like PE38, PE40 or PE37. Most preferred, in context of this invention, is PE38.
Furthermore, and in accordance with the present invention, said chemokine construct may comprise the chemokine covalently bound to an antibody construct which binds to an antibody construct capable of binding to a CD3 antigen and/or which is a covalently bound to a toxin.
In a particularly preferred embodiment of the use of the present invention, the antibody and/or chemokine construct is a heterominibody construcfi comprising at least an anti-body and/or a chemokine which binds to a chemokine receptor, preferably to the receptor, most preferably to the human CCRS receptor. Said heterominibody construct may comprise at least one toxin and it is particularly preferred that said heterominibody construct binds to the chemokine receptor as defined hereinabove and/or to a CD3 an-tigen of an effector cell. Preferred chemokines are the chemokines mentioned herei-nabove and preferred toxins are the toxins described hereinabove, which may be modified or unmodified. Chemokines are well known in the art and described, inter alia, in Murphy (1999), loc. cit. Therefore, it is preferred that the chemokine is selected from the group consisting of RANTES, MIP-1f3, MIP-1a, MCP-2, and MCP-3 or a functional fragment thereof. The most preferred chemokine, in context of this invention is RAN-TES. Functional fragments of said chemokines are fragments which are capable of binding to or interacting with said chemokine receptor, preferably the human CCRS.
Heterominibodies are known in the art and their production is, inter alia, described in 5. Said heterominibody may be a multifunctional compound comprising at least one antibody and/or chemokine binding to or interacting with a chemokine recep-tor, preferably human CCRS, may (additionally) comprise a toxin as defined hereinbe-low and/or a binding site for the CD3 antigen.
In a preferred embodiment, the antibody- or chemokine construct to be used in the pre-sent invention is a fusion (poly)peptide or a mosaic (poly)peptide. Said fusion (poly)peptide may comprise merely the domains of the constructs as described herein above as well as (a) functional fragments) thereof. However, it is also envisaged that said fusion (poly)peptide comprises further domains and/or functional stretches.
Therefore, said fusion (poly)peptide can comprise at least one further domain, said domain being linked by covalent or non-covalent bonds. The linkage as well as the construction of such constructs, can be based on genetic fusion according to the meth-ods known in the art (Sambrook et al., loc. cit., Ausubel, "Current Protocols in Molecu-lar Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO
94/04686. The additional domain present in the construct may preferably be linked by a flexible linker, advantageously a (poly)peptide linker, wherein said (poly)peptide linker preferably comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the peptide, (poly)peptide or antibody or vice versa. Said linker may, inter alia, be a Glycine, a Serine and/or a Glycine/Serine linker. Additional linkers comprise oligomeri-zation domains. Oligomerization domains facilitate the combination of two or several autoantigens or fragments thereof in one functional molecule. Non-limiting examples of oligomerization domains comprise leucine zippers (like jun-fos, GCN4, E/EBP;
Kos-telny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sci. USA 94 (1997), 3673-3678, Williams, Genes Dev. 5 (1991 ), 1553-1563;Suter, "Phage Display of Pep-tides and Proteins", Chapter 11, (1996), Academic Press), antibody-derived oligomeri-zation domains, like constant domains CH1 and CL (Mueller, FEBS Letters 422 (1998), 259-264) and/or tetramerization domains like GCN4-LI (Zerangue, Proc. Natl.
Acad.
Sci. USA 97 (2000), 3591-3595).
Furthermore, the antibody- or chemokine construct to be used in the present invention or as described hereinbelow may comprise at least one further domain, inter alia, do-mains which provide for purification means, like, e.g. histidine stretches.
Said further domains) may be linked by covalent or non-covalent bonds.
The linkage can be based on genetic fusion according to the methods known in the art and described herein or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686. The additional domain present in the construct as described and disclosed in the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker to one of the binding site domains wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of one of said domains and the N-terminal end of the other of said domains when said polypeptide assumes a conformation suit-able for binding when disposed in aqueous solution. Preferably, said polypeptide linker is a polypeptide linker as described in the embodiments hereinbefore. The polypeptide of the invention may further comprise a cleavable linker or cleavage site for protein-ases, such as enterokinase It is also envisaged that said constructs disclosed for uses, compositions and methods of the present invention comprises (a) further domains) which may function as immu-nomodulators. Said immunomodulators comprise, but are not limited to cytokines, lym-phokines, T cell co-stimulatory ligands, etc.
Adequate activation resulting in priming of naive T-cells is critical to primary immunore-sponses and depends on two signals derived from professional APCs (antigen-presenting cells) like dendritic cells. The first signal is antigen-specific and normally mediated by stimulation of the clonotypic T-cell antigen receptor that is induced by processed antigen presented in the context of MHC class-I or MHC class-II
molecules.
However, this primary stimulus is insufficient to induce priming responses of naive T-cells, and the second signal is required which is provided by an interaction of specific T-cell surface molecules binding to co-stimulatory ligand molecules on antigen pre-senting cells, further supporting the proliferation of primed T-cells. The term "T-cell co-stimulatory ligand" therefore denotes in the light of the present invention molecules, which are able to support' priming of naive T-cells in combination with the primary stimulus and include, but are not limited to, members of the B7 family of proteins, in-cluding B7-1 (CD80) and 137-2 (CD86).
The antibody- and/or chemokine construct defined herein above or described herein-below may comprise further receptor or ligand function(s), and may comprise immuno-modulating effector molecule or a fragment thereof. An immuno-modulating effector molecule positively and/or negatively influences the humoral and/or cellular immune system, particularly its cellular and/or non-cellular components, its functions, and/or its interactions with other physiological systems. Said immuno-modulating effector mole-cule may be selected from the group consisting of cytokines, chemokines, macrophage migration inhibitory factor (MIF; as described, inter alia, in Bernhagen (1998), Mol Med 76(3-4); 151-61 or Metz (1997), Adv Immunol 66, 197-223), T-cell receptors and solu-ble MHC molecules. Such immuno-modulating effector molecules are well known in the art and are described, inter alia, in Paul, "Fundamental immunology", Raven Press, New York (1989). In particular, known cytokines and chemokines are described in Meager, "The Molecular Biology of Cytokines" (1998), John Wiley & Sons, Ltd., Chich-ester, West Sussex, England; (Bacon (1998). Cytokine Growth Factor Rev 9(2):167-73;
Oppenheim (1997). Clin Cancer Res 12, 2682-6; Taub, (1994) Ther. Immunol. 1 (4), 229-46 or Michiel, (1992). Semin Cancer Biol 3(1 ), 3-15).
Antibody and/or chemokine constructs as disclosed and described in the present in-vention and comprising (an) additional functional domains) may, inter alia, be multi-functional compounds, like heterominibodies, as described herein below.
The constructs to be used in the present invention or described herein may be con-structs which comprise domains originating from one species, preferably from mam-mals, more preferably from human. However, chimeric and/or humanized constructs are also envisaged and within the scope of the present invention.
In a particular preferred embodiment, the composition of the invention comprises a constructs to be used in the present invention or described herein is a cross-linked (poly)peptide construct. As mentioned herein, said cross-linking may be based on methods known in the art which comprise recombinant as well as biochemical methods.
In a yet further embodiment of the use of the present invention, the antibody construct or the chemokine construct to be used comprises at least one toxin. Said toxin may be Pseudomonas exotoxin A, diphtheria toxin and similar toxins. It is envisaged that trun-cated toxins are employed, like the PE38 or the PE40 of Pseudomonas toxin described in the appended examples.
Said toxin may be bound to said antibody or chemokine by means as described herei-nabove. It is also envisaged that said toxin is bound to the antibody/chemokine by means of a short peptide linker. The linker preferably consists of a flexible and hydro-philic amino acid sequence, in particular of glycines and serines. Preferably said linker has a length of 1 to 20 amino acids.
Several fusion proteins with a truncated version of Pseudomonas exotoxin A
have been designed so far. Most of them have been used to target and destroy malignant cells.
This toxin becomes activated upon proteolytic cleavage. A truncated version of the toxin (PE38) may be employed for the constructs of the present invention, as the full-length protein binds with its fist domain to the ubiquitous a2-macroglobulin receptor and is therefore toxic to most eukaryotic cells. Yet, this problem may be overcome by re-placing the first domain of Pseudomonas exotoxin A by a specific sequence in order to alter the binding specificity of the toxin.
Furthermore, the present invention relates to the use of a chemokine construct which binds to a chemokine receptor for the preparation of a pharmaceutical composition for the elimination of cells which are latently infected with a primate immunodeficiency vi-rus wherein said chemokine construct comprises a amino acid sequence as depicted in SEQ ID NO: 24 or as encoded by the nucleotide sequence as depicted in SEQ ID
NO: 23.
As mentioned hereinabove, and in a preferred embodiment, the antibody and/or che-mokine constructs to be used within the scope of the present invention bind to or inter-act with the CD3 antigen. Preferably said CD3 antigen is on the surFace of an effector cell, namely a T-cell, preferably a cytotoxic T-cell.
It is particularly preferred that an antibody construct be used wherein said construct comprises a binding site for CCR5 and a binding site for CD3 and that a chemokine construct be used, wherein said chemokine construct comprises RANTES and said toxin is a truncated Pseudomonas exotoxin A (PE38).
The present invention, therefore also relates to antibody constructs comprising a bind-ing site for CCR5 and a binding site for CD3 as well as to chemokine constructs com-prising RANTES and the truncated Pseudomonas exotoxin A (PE38).
The present invention also relates to a polynucleotide encoding an antibody-construct as defined hereinabove or a polynucleotide encoding a chemokine construct as defined herein, wherein said polynucleotide is a polynucleotide comprising the nucleic acid molecule in particular encoding the polypeptide as depicted in SEQ ID NO: 18 or SEQ
ID NO: 24; a polynucleotide comprising the nucleic acid molecule as depicted in SEQ
ID NO: 17 or SEQ ID NO: 23; or (c) a polynucleotide hybridizing under stringent condi-tions to the complementary strand of a polynucleotide of (a) or (b).
With respect to the polynucleotides/nucleotide sequences characterized under (c) above, the term "hybridizing" in this context is understood as referring to conventional hybridization conditions, preferably such as hybridization in 50%formamide/6xSSC/0.1 %SDS and 1 OOpg/ml ssDNA, in which temperatures for hy-bridization are above 37°C and temperatures for washing in 0.1xSSC/0.1%SDS are above 55°C. Most preferably, the term "hybridizing" refers to stringent hybridization conditions, for example such as described in Sambrook., "Molecular Cloning: A
Labo-ratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. It is envisaged that the polynucleotides characterized under (c) above are highly homologous to the polynucleotides as defined in (a) and/or (b) and comprise a homol-ogy of at least 95%, more preferably of at least 97%, and most preferably 99%
with the polynucleotides of (a) and/or (b).
Polynucleotides as defined and characterized under (c), therefore may encode for polypeptides being highly homologous to the polypeptides as defined in (a) and (b).
The person skilled in the art can easily test the capacity of such homologous polypep-tides to bind to chemokine receptors, in particular to the human CCR5 receptor and/or to eliminate, deplete and/or destroy cells, for example, cells which are infected by a primate immunodeficiency virus, like HIV-1, or eliminate, deplete and/or destroy target cells involved in immunological disorders or disclosed herein. The person skilled in the art can easily adopt the in vitro, in vivo and ex vivo experiments of the appended ex-amples to verify the binding and/or depletion properties of such constructs.
Furthermore, said polynucleotide/nucleic acid molecule may contain, for example, thio-ester bonds and/or nucleotide analogues. Said modifications may be useful for the sta-bilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
The polynucleotide/nucleic acid molecule of the composition of the present invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination Said polynucleotide may be, e.g., DNA, cDNA, RNA or synthetically produced DNA
or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. Preferably said polynucleotide is part of a vector. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable condi-tions. Preferably, the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mam-malian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory ele-ments may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E.
coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of tran-scription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art; see also, e.g., the appended examples. The leader sequences) is (are) assembled in appropriate phase with translation, initiation and termination se-quences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Op-tionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product; see supra. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA
expres-sion vector pcDV1 (Pharmacia), pCDMB, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), or pSPORT1 (GIBCO BRL).
Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming of transfecting eukaryotic host cells, but control se-quences for prokaryotic hosts may also be used. Once the vector has been incorpo-rated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purifi-cation of the polypeptide of the invention may follow; see, e.g., the appended exam-ples.
As described above, the polynucleotide of the invention can be used alone or as part of a vector to express the antibody- and/or chemokine constructs to be used in the inven-tion or in cells, for, e.g., the treatment of immunological disorders or in anti-viral ther-apy. The polynucleotides or vectors containing the DNA sequences) encoding any one of the above described polypeptides is introduced into the cells which in turn produce the polypeptide of interest. Therefore, said polynucleotides and vectors may be used for gene therapy. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivery systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art;
see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239;
Is-ner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086;
Ono-dera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann.
N.Y.
Acad. Sci. 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51;
Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, US 5,580,859; US
5,589,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and refer-ences cited therein. The polynucleotides and vectors of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g., adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell. An example for an embryonic stem cell can be, inter alia, a stem cell as described in, Nagy, Proc. Natl.
Acad. Sci. USA 90 (1993), 8424-8428.
In accordance with the above, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engi-neering that comprise a polynucleotide encoding a polypeptide of the invention. Pref-erably, said vector is an expression vector and/or a gene transfer or targeting vector.
Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell populations.
Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the polynucleotides and vectors of the invention can be re-constituted into liposomes for delivery to target cells. The vectors containing the poly-nucleotides of the invention can be transferred into the host cell by well-known meth-ods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, su-pra. Once expressed, the polypeptides of the present invention can be purified accord-ing to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Pro-tein Purification", Springer-Verlag, N.Y. (1982). Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically (including extracorpore-ally)~or in developing and performing assay procedures.
In a still further embodiment, the present invention relates to a cell containing the poly-nucleotide or vector described above or to a host transformed with the vector of the invention. Preferably, said host/cell is a eukaryotic, most preferably a mammalian cell if therapeutic uses of the polypeptide are envisaged. Of course, yeast and less preferred prokaryotic, e.g., bacterial cells may serve as well, in particular if the produced poly-peptide is used as a diagnostic means.
The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromoso-mally.
The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of a polypeptide of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacte-ria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is meant to include yeast, higher plant, insect and pref erably mammalian cells. Depending upon the host employed in a recombinant produc-tion procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methio-nine amino acid residue. A polynucleotide coding for a polypeptide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a plasmid or a virus containing the coding sequence of the polypeptide of the invention and geneti-cally fused thereto an N-terminal FLAG-tag and/or C-terminal His-tag.
Preferably, the length of said FLAG-tag is about 4 to 8 amino acids, most preferably 8 amino acids.
Methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
The genetic constructs and methods described therein can be utilized for expression of the polypeptide of the invention in eukaryotic or prokaryotic hosts. In general, expres-sion vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as spe-cific genes which are capable of providing phenotypic selection of the transformed cells. Furthermore, transgenic animals, preferably mammals, comprising cells of the invention may be used for the large scale production of the antibody- andlor chemokine construct of the invention. It is most preferred that said transgenic animal produces the antibody construct of the invention.
In a further embodiment, the present invention thus relates to a process for the prepa-ration of a polypeptide described above comprising cultivating a (host) cell of the inven-tion under conditions suitable for the expression of the antibody- and/or chemokine construct and isolating the polypeptide from the cell or the culture medium.
The transformed hosts can be grown in fermentors and cultured according to tech-niques known in the, art to achieve optimal cell growth. The produced constructs of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., microbially expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against a tag of the polypeptide of the invention or as described in the appended examples.
Depending on the host cell, renaturation techniques may be required to attain proper conformation. If necessary, point substitutions seeking to optimize binding may be made in the DNA using conventional cassette mutagenesis or other protein engineering methodology such as is disclosed herein. Preparation of the polypeptides of the inven-tion may also be dependent on knowledge of the amino acid sequence (or corre-sponding DNA or RNA sequence) of bioactive proteins such as enzymes, toxins, growth factors, cell difFerentiation factors, receptors, anti-metabolites, hormones or various cytokines or lymphokines. Such sequences are reported in the literature and available through computerized data banks.
The present invention further relates to an antibody construct or the chemokine con-struct encoded by the polynucleotide as described hereinabove or produced by the method described hereinabove.
Furthermore, the constructs of the invention can be used in the management of immu-nological disorders, in particular autoimmune diseases, allergic diseases, inflammatory diseases and AIDS (HIV-infection), as documented in the appended examples.
Additionally, the present invention provides for compositions comprising the polynu-cleotide, the vector, the host, the antibody construct and/or the chemokine construct of the invention.
The term "composition", in context of this invention, comprises at least one polynucleo-tide, vector, host, antibody construct and/or chemokine construct as described herein.
Said composition, optionally, further comprises other molecules, either alone or in com-bination, like e.g. molecules which are capable of modulating and/or interfering with the immune system. The composition may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solutions) or (an) aerosol(s). In a preferred embodiment, said composition comprises at least two, preferably three, more preferably four, most preferably compounds as described in the invention.
Preferably, said composition is a pharmaceutical composition further comprising, op-tionally, a pharmaceutically acceptable carrier, diluent and/or excipient.
Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.
Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, in-traperitoneal, subcutaneous, intramuscular, topical or intradermal administration. Intra-venous administration is particularly preferred. The dosage regiment will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concur-rently. Generally, the regimen as a regular administration of the pharmaceutical com-position should be in the range of 1 pg to 10 mg units per day. If the regimen is a con-tinuous infusion, it should also be in the range of 1 pg to 10 mg units per kilogram of body weight per minute, respectively. However, a more preferred dosage for continu-ous infusion might be in the range of 0.01 p,g to 10 rng units per kilogram of body weight per hour. Particularly preferred dosages are recited herein below.
Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 102 copies of the DNA
molecule. The compositions of the invention may be administered locally or systemati-cally. Administration will generally be parenterally, e.g., intravenously; yet external ad-ministration is also envisaged. DNA may also be administered directed to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chlo-ride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient re-plenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention might comprise proteinaceous car-riers, like, e.g., serum albumin or immunoglobuline, preferably of human origin. Fur-thermore, it is envisaged that the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. Such agents might be drugs acting on the immunological system, drugs used in anti-viral treatment, in particular in HIV-treatment (for example, HAART) and AIDS management and/or anti-inflammatory drugs. It is, for example, en-visaged that patients are treated as early as possible with HAART until viral load is be-low detection level for several weeks to months. Early treatment of infected patients with HAART prevents the transition of viral strains from usage of CCR5 to other che-mokine receptors, like CXCR4 (Connor (1997) J. Exp. Med. 185, 621-628).
Constructs as disclosed in the present invention, for example, the CCR5xCD3 construct is admin-istered in addition to HAART to eliminate latently infected cells as well as cells that are prone to reinfection by HIV-1. The depletion of CCR5+ cells is repeated 1 to 10 times.
Doses of CCR5xCD3 are in the range of 0.5~.g/m2 to 10mg/m2, preferably 10~,g/m2 to 100~.g/m2. Doses can be administered intravenously, subcutaneously and/or into the cerebra-spinal fluid. After several treatment cycles with the bispecific antibody HAART
is discontinued and viral load is closely monitored. If viral load increases above detec-tion level, a new cycle of HAART and the bispecific antibody is initiated as described above.
It is envisaged by the present invention that the various polynucleotides and vectors of the invention are administered either alone or in any combination using standard vec-tors and/or gene delivery systems, and optionally together with a pharmaceutically ac-ceptable carrier or excipient. Subsequent to administration, said polynucleotides or vectors may be stably integrated into the genome of the subject. Preferably said sub-ject is a human.
On the other hand, viral vectors may be used which are specific for certain cells or tis-sues and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art. The pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delaying of different kinds of immuno-logical diseases, which may be related to inflammation, in particular inflammatory bowel diseases, inflammatory renal diseases, inflammatory joint diseases like (chronic) arthri-tis. Furthermore, the pharmaceutical composition of the present invention may be em-ployed to eliminate cells which are latently infected with a virus, preferably a primate immunodeficiency virus, more preferably with HIV(-1 ).
Furthermore, it is possible to use a pharmaceutical composition of the invention which comprises polynucleotide or vector of the invention in gene therapy. Suitable gene de-livery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991 ), 2726-2729).
Further methods for the delivery of nucleic acids comprise particle-mediated gene transfer as, e.g., described in Verma, Gene Ther.15 (1998), 692-699.
It is to be understood that the introduced polynucleotides and vectors express the gene product after introduction into said cell and preferably remain in this status during the lifetime of said cell. For example, cell lines which stably express the polynucleotide un-der the control of appropriate regulatory sequences may be engineered according to methods well known to those skilled in the art. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the polynu-cleotide of the invention and a selectable marker, either on the same or separate plas-mids. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
The selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl. Acad.
Sci. USA
77 (1980), 3567; O'Hare, Proc. Natl. Acad. Sci. USA 78 (1981 ), 1527), gpt, which con-fers resistance to mycophenolic acid (Mulligan, Proc. Natl. Acad. Sci. USA 78 (1981 ), 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, J. Mol. Biol. 150 (1981 ), 1 ); hygro, which confers resistance to hygromycin (Santerre, Gene 30 (1984), 147); or puromycin (pat, puromycin N-acetyl transferase).
Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, hisD, which allows cells to utilize histinol in place of his-tidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); and ODC
(ornithine de-carboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McCologue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).
In a further embodiment, the present invention relates to a composition, preferably a pharmaceutical composition, as described hereinabove, which further comprises a me-dicament for the treatment of an immunological disorder or a medicament for anti-HIV
treatment.
Said anti-HIV treatment may comprise HAART. HAART therapy consists of a cocktail of three classes anti-viral drugs. The classes are nucleosidal reverse transcriptase inhibi-tors (NRTI), non-nucleosidal reverse transcriptase inhibitors (NNRTI) and protease in-hibitors (PI). Usually 2 to 4 drugs from preferentially more than one class are combined to reduce viral load to almost non-detectable levels. Products, dosing schedules and common side effects are given in appended Tables I-III.
Said treatment of an immunological disorder may comprise anti-inflammatory agents and immunosuppressive agents. Anti-inflammatory agents may be selected from the group consisting of azathioprine, cyclophophamide, glucocorticoids like prednisone and corticosteroids. Immunosuppressive agents may comprise cyclosporin A, Tacrolimus (FK506), Sirolimus (Rapamycin). Protein drugs may comprise calcineurin, beta-interferon, anti-TNF alpha monoclonal antibodies (remicade). Dosing and use of anti-inflammatory agents and immunosuppressive agents is described, inter alia, in Fauci et al., sic. Further treatment options are known to the skilled artisan and, inter alia, de-scribed hereinabove.
In a particularly preferred embodiment, the present invention relates to a method for treating, preventing and/or alleviating an immunological disorder or for the elimination cells which are latently infected with a primate immunodeficiency virus comprising ad-ministering to a subject in need of such a treatment, prevention and/or alleviation an effective amount of the compounds and/or compositions. preferably the pharmaceutical compositions of the present invention.
The constructs described herein are particularly useful in specifically destroying che-mokine receptor positive cells. For example, a bispecific antibody, binding simultane-ously to CCR5 on target cells and to CD3 on T-cells, redirects cytotoxic T-cells to the CCR5 positive target cells. As shown in the appended examples the antibody construct specifically depletes CCR5 positive T-cells and monocytes, but is inactive against cells that do not express CCR5 such as CCRS deficient X32/032 PBMC. Furthermore, in vitro/ex vivo experiments the bispecific antibody construct eliminated more than 95% of CCR5 positive monocytes and T-cells from the synovial fluid of patients with arthritis.
Other constructs, like chemokine constructs, for example, a fusion protein of the che-mokine RANTES and a truncated version of the Pseudomonas exotoxin A (PE38) are able to bind to CCR5 and to downmodulate the receptor from the cell surface as exem-plified in the appended examples. Within 48 h RANTES-PE38 completely destroyed CCR5 positive CHO cells at a concentration of 2 nM. No cytotoxic effect was detectable against CCR5 negative CHO cells.
Based on the predominance of CCRS positive T-cells and monocytes in the infiltrate of chronically inflamed tissue, the specific depletion of CCR5 positive cells represents a new concept in the treatment of immunological disorders.
As described hereinabove, due to the fact that specific chemokine receptors are pres-ent on HIV-infected cells, namely CCRS, the compounds and compositions of the in-vention are particularly useful for the depletion/elimination of cells latently infected with primate immunodeficiency virus.
The present invention also relates to the use of the polynucleotide, the vector, the host, the antibody construct and/or the chemokine construct of the present invention for the preparation of a pharmaceutical composition for treating, preventing and/or alleviating an immunological disorder or for the preparation of a pharmaceutical composition for eliminating latently infected cells, wherein said cells are infected with a primate immu-nodeficiency virus, line a human immunodeficiency virus, in particular HIV-1.
Said immunological disorders may be autoimmune diseases, skin diseases, allergic diseases, inflammatory diseases, diabetes and transplant rejections, wherein said autoimmune disease is selected from the group consisting of multiple sclerosis, type I
diabetes, rheumatoid arthritis. Said skin diseases, may comprise psoriatic lesions, pso-riasis, atrophic dermatitis and the like. Inflammatory disease are mentioned herei-nabove is selected from the group consisting of inflammatory joint diseases, inflamma-tory renal diseases, inflammatory bowel diseases. In particular, said inflammatory bowel disease may comprise Morbus Crohn, sarcoidosis, systemic sclerosis, colla-genosis, myositis, neuritis. Inflammatory renal diseases may comprise nephritis, glo-merulonephritis, lupus nephritis, or IgA nephropathy.
In a variety of chronic inflammatory diseases an impressive accumulation of positive T-cells and macrophages is found at the site of inflammation. An accumulation of CCR5+ cells has been demonstrated in several types of inflammatory diseases, like arthritis, inflammatory renal diseases, transplant rejection, auto-immune diseases like multiple sclerosis and inflammatory bowel diseases. In contrast, in the peripheral blood of these patients only a minority of T-cells and monocytes express CCRS.
Therefore, CCR5 appears to be an excellent marker to identify leukocytes that are involved in chronic inflammation. The occurrence of a 32 by deletion in the CCR5 gene which pre-vents expression of CCRS, allows to study the pathophysiological role of CCR5 in chronic inflammatory diseases. In patients with rheumatoid arthritis the frequency of CCR5 deficient (032/32) individuals is significantly reduced. Moreover, the mean sur-vival of the kidney transplants is significantly longer in CCRS-X32/032 patients. These results make CCR5 a target for therapeutic intervention. Furthermore, the prevalence of CCR5 positive leukocytes in the diseased tissue in contrast to the rare expression of CCR5 on the peripheral blood leukocytes means that a specific elimination of positive leukocytes may be therapeutically useful by reducing the number of infiltrating cells in chronic inflammation, transplant rejection and autoimmune disease, like multiple sclerosis without significantly depleting peripheral blood leukocytes.
Eliminating CCR5 positive leukocytes from the inflammatory infiltrate will be of greater therapeutic benefit than simply blocking chemokine receptors of these cells, as they have already infil-trated the tissue.
As documented in the appended examples the antibody and/or chemokine constructs are particularly useful in the treatment, prevention and/or alleviation of inflammatory joint diseases. Therefore, the compositions of the present invention are particularly useful for the treatment of inflammatory joint diseases, like arthritis, in particular chronic arthritis.
The present invention furthermore, provides for medical methods and uses, wherein the composition, preferably the pharmaceutical composition, is to be administered in combination with antiviral agents and/or in combination with drugs to be employed in AIDS management.
As mentioned hereinabove, the main problem in AIDS management in the occurrence of latently HIV-infected cells. The current treatment options are based on anti-viral agents interfering with two enzymes of the HIV-1 virus, its protease and reverse tran-scriptase. The protease is essential to cleave the inactive viral pre-proteins to form the active products, while the reverse transcriptase is required to generate a DNA
interme-diate of the viral RNA genome. The DNA intermediate can then integrate into the host genome and remain there in a silent - latent form. The most efficient treatment option consists of highly active antiretroviral therapy (HAART) - a treatment regimen consist-ing of a combination of at least three anti-retroviral drugs and usually including at least one drug of the protease inhibitor class. The advent of highly active antiretroviral ther-apy (HAART) has had a significant impact on HIV-1-infected individuals, lowering cir-culating virus to undetectable levels (Oxenius (2000) Proc. Natl Acad. Sci.
97, 3383-3387; Perelson (1997) Nature (London) 387, 188-191; Hammer (1997) N. Engl. J.
Med.
337, 725-733; Gulick (1997) N. Engl. J. Med. 337, 734-739). Despite this, latently in-fected cells can remain in these individuals for significant periods of time (Chun (1997) Nature (London) 387, 183-188; Chun (1998) Proc. Natl. Acad. Sci. USA 95, 8869-8873;
Zhang (1999) N. Engl. J. Med. 340, 1605-1613); if HAART is withdrawn, these cells can produce virus (Harrigan (1999) AIDS 13, F59-F62). A pool of latently infected cells is generated early during primary HIV-1 infection (Chun (1998) Proc. Natl.
Acd. Sci. 95, 8869-8873). Considering the postulated long half life of latent viral reservoirs (Zhang (1999) N. Engl. J. Med. 340, 1605-1613, Finzi (1999) Nat. Med 5, 512-517) and the side effects and cost of chronic HAART (Flexner (1998) N. Engl. J. Med. 338, 1292; Carr (1998) Lancet 351, 1881-1883), it is important to develop new strategies to eliminate the latent reservoir. While HAART treatment has been highly successful in suppressing plasma viremia in HIV-infected individuals, there are still persistent reser-voirs of HIV including in latently infected CD4+ T-cells and other cells in the brain, gut associated lymphoid tissue and the genital tract (Chun (1999) Proc. Natl.
Acad. Sci. 96, 10958-10961 ). Re-emergence of plasma viremia after discontinuation of HAART
is due to those pre-existing viral reservoirs and HAART cannot eliminate those reservoirs (Chun (2000) Nature Med. 6, 757-761 ). Therefore, even HAART merely suppresses viral replication and reduces the viral load but does not prevent the occurrence of latent infected cells or eliminates such cells. Transmission of HIV-1 depends on the presence of CCRS, as individuals with a homozygous X32 deletion of the CCR5 allele are highly resistant against infection with HIV-1. Although highly active antiretroviral therapy can efficiently suppress replication of HIV-1, complete eradication of HIV has not been achieved to date. The main obstacle appears to be the inactivity of antiretroviral ther-apy against latently infected cells that can survive for several years and function as en-dogenous source for HIV-1. Many of these cells fail to express viral proteins and can evade the immune response. However, the majority of latently infected cells may still express CCRS, as this receptor is necessary for their initial infection. The compounds of the present invention are particularly useful in the depletion of CCR5+
cells and should significantly reduce the number of latently infected HIV+-cells. Other strategies to eliminate HIV-1 infected cells that depend on a specific recognition of viral proteins, e.g., surface expressed gp120, would be less effective against latently infected cells, as the virus is dormant in these cells.
Therefore, the compositions of the present invention are particularly useful in co-therapy approaches, which lead to a depletion of HIV-infected cells, preferably of CCR5 positive cells. It is preferred that the composition of the present invention is employed in combination with HAART. Therefore the construct of the invention may be used in HIV-therapy in combination with HAART as shown in the appended examples. Products, dosing schedules and common side effects of HAART are known and illustrated, inter alia, in Tables I, II and III.
Said combination may comprise the co-administration as well as an administration be-fore or after treatment with other anti-viral, preferably anti-retroviral, most preferably anti-HIV medication.
The present invention also provides for a kit comprising the polynucleotide, the vector, the host, the antibody construct and/or the chemokine construct of the present inven-tion.
Advantageously, the kit of the present invention further comprises, optionally (a) stor-age solutions) and/or remaining reagents or materials required for the conduct of sci-entific or therapeutic methods. Said kit may, inter alia, comprise drugs and/or medica-ments employed in the treatment of immunological disorders as defined herein and/or in AIDS management. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
The Figures show:
Figure 1.
Expression of various chemokine receptors (indicated on the x-axis) on T cells (first and second panel), monocytes (third panel) and neutrophils (forth panel) in the peripheral blood (white columns) and the synovial fluid (gray columns) of patients with arthritis other than gout. Each dot represents one patient and mean values are given as bars.
Expression of CXCR1 and CXCR2 on neutrophils is given as fluorescence intensity on the y-axis, while in all other cases the percentage of receptor positive cells is depicted.
Figure 2.
FRCS dot plots showing the expression of CCRS, CCR2 and CXCR4 on leukocytes in the peripheral blood (left) and synovial fluid (right) of one patient with rheumatoid arthri-tis. The cut-offs were set according to the isotype controls and are shown as vertical lines. In the synovial fluid the majority of T-cells and monocytes show a high level of CCRS expression, while in the peripheral blood only a minority of these cells express low levels of CCRS.
Figure 3.
Scheme of the bispecific single-chain antibody. The aCCR5 single-chain fragment (CCR5 VL / CCR5 VH) derived from the hybridoma MC-1 is fused to the N-terminus of a single-chain fragment directed against CD3 (CD3 VH / CD3 VL). Binding of the bispecific antibody to CD3~ T cells and CCR5 positive target cells results in crosslink-age of CD3, activation of effector T cells and lysis of CCR5 positive target cells.
Figure 4.
SDS page of the purified bispecific single-chain antibody aCCRS-aCD3. A single band of approx. 60 kD is visible under reducing (left) and non-reducing (right) conditions. No degradation or proteolysis of the bispecific antibody is detectable.
Figure 5.
Scheme of the chemokine-toxin RANTES-PE38. The chemokine RANTES is fused to the N-terminus of a truncated version of the Pseudomonas exotoxin A (PE38).
While the truncated toxin is unable to bind to eukaryotic cells, the fusion protein binds with the RANTES moiety to CCR5 and becomes internalized into the cell. Thereby the toxin in-hibits protein synthesis and. induces cell death.
Figure 6.
SDS-PAGE (left) and Westernblot (right) of the purified protein RANTES-PE38. A
dis-tinct band with the expected size of approx. 46 kD is visible in the coomassie stained SDS-PAGE and Westernblot.
Figure 7.
Binding of the aCCRS-aCD3 bispecific antibody to CD3 on CCR5 deficient lympho-cytes. Costaining with CD4 and CD8 demonstrated that the bispecific antibody binds to the subpopulation of CD4+ / CD8+ T cells. Multicolor analysis showed that no binding to other cell populations occurred.
Figure 8.
Binding of the aCCRS-aCD3 bispecific antibody to CCR5 on transfected CHO
cells.
CHO cells transfected with CCR5 are shown in black, while CXCR4 positive CHO
cells served as negative control and are shown in white.
Figure 9.
Binding of the aCCRS-aCD3 bispecific antibody to CCR5 on cultured monocytes.
Monocytes from a CCR5 positive donor are shown in black, while monocytes from a CCR5 deficient (~32/~32) donor served as negative control and are shown in white.
Figure 10.
CCR5 specific monoclonal antibodies were compared in their ability to induce down-modulation of CCR5 as analyzed by FACS. MAb MC-1 (squares), the parental antibody of the aCCRS-aCD3 bispecific antibody, showed significant internalization, while MC-4 (triangle) showed no induction of CCR5 internalization. CHO-CCR5 cells were incu-bated with various concentrations for 30 min at 37°C.
Figure 11.
Downmodulation of CCRS from the surface of PBMC with RANTES-PE38 (open sym-bols) and RANTES (closed symbols). Surface expression of CCR5 was determined on lymphocytes (squares) and monocytes (circles) and is given as % of the medium con-trol. The fusion protein RANTES-PE38 is able to downmodulate CCR5 from the cell surface with a somewhat lower efficiency than unmodified RANTES.
Figure 12.
Depletion of CCR5 positive monocytes by the bispecific antibody. CCR5 deficient PBMC 032/032) or wildtype PBMC (WT-PBMC) were cultured overnight and incubated with the bispecific antibody (100 ng/ml) or medium as control for 20 h.
Remaining monocytes (Mo) and lymphocytes (Ly) were identified by their light scatter properties in FACS. The CCR5 positive wildtype monocytes were completely depleted by the bispecific antibody, while the CCRS deficient monocytes survived.
Figure 13.
Depletion of CCR5 positive monocytes by the bispecific antibody. Dose response showing depletion of cultured monocytes with various concentrations of the aCCRS-aCD3 bispecific antibody. More than 90 % of monocytes were depleted at a concentra-tion of 33 ng/ml.
Figure 14.
The bispecific aCCRS-aCD3 antibody depletes lymphocytes and monocytes from the synovial fluid of a patient with chronic arthritis. Freshly draw synovial fluid was incu-bated with various concentrations of the bispecific antibody or medium as control for 20 h and analyzed by FACS. More than 95 % of both cell types were depleted at a con-centration of 31 ng/ml.
Figure 15.
The bispecific aCCRS-aCD3 antibody depletes lymphocytes and monocytes from the synovial fluid of a patient with chronic arthritis. Freshly draw synovial fluid was incu-bated with the bispecific antibody (500 ng/ml) or medium as control for 20 h and ana-lyzed by FACS (forward and sideward light scatter analysis). The bispecific antibody completely depleted the CCR5 positive monocytes and lymphocytes, while the negative granulocytes (PMN) survived. Consistent with our previous data all monocytes and lymphocytes in this synovial fluid expressed CCRS, while no expression of was found on granulocytes (PMN).
Figure 16.
The efficacy of the aCCRS-aCD3 bispecific single-chain antibody in depleting positive monocytes was compared with the efficacy of two unmodified monoclonal anti-bodies MC-1 and MC-5. PBMC from two different donors (F and N) were cultured over-night and then incubated for 24 h with medium in the presence or absence of antibody construct and antibody. Concentrations were as indicated. The cells were completely recovered and analyzed by FACS to quantify surviving monocytes and lymphocytes.
Shown are the results of two experiments per PBMC donor. Surprisingly only the bispecific antibody was able to considerably deplete CCR5 positive monocytes, while the unmodified monoclonal antibodies were largely ineffective.
Figure 17.
Example of the forward and sideward light scatter analysis of a representative experi-ment as shown in Fig. 16, indicating that only the aCCRS-aCD3 bispecific single-chain antibody was capable of depleting the monocytes in the left lower quadrants.
For com-parison of the localization of different cell types also see Figure 12 left panel.
Figure 18.
Destruction of CCR5 positive CHO cells with the chemokine-toxin RANTES-PE38.
CCR5 positive CHO cells and CXCR4 positive CHO cells were incubated for 40 h with the chemokine-toxin (10 nM) and analyzed by FACS. Dead cells appear in the left up-per region of the forward and sideward light scatter plot. RANTES-PE38 completely destroyed the CCR5 positive CHO cells while it had no effect on the CXCR4 positive CHO cells.
Figure 19.
Examples of antibody and / or chemokine constructs binding to chemokine receptor (CCR) expressing cells that are combined by peptide linkage or by multimerization do-mains: (A) shows various examples of antibody and chemokine constructs that interact with an effector cell by binding to an effector cell surface antigen, (B) shows examples of antibody and chemokine constructs that are linked to a toxin, (C) shows examples of antibody and chemokine constructs, that contain an antibody binding site for a toxin.
Figure 20.
4x105 CCR5+CHO cells were incubated with 19,5 ng/ml scFV CCR5xCD3 for 30 min-utes at 4°Cafter washing cells were incubated for 45 minutes at 4°C with 20 pg/ml anti-His-Tag monoclonal antibody. Binding of scFV CCR5xCD3 was detected with a mono-clonal goat anti-mouse IgG F(ab')2-PE conjugated antibody and analysed in a flow cy-tometer using the CeIIQuest software. Nonlinear regression analysis was performed with GraphPad Prizm.
Figure 21.
The cytotoxic activity of scFv CCR5xCD3 was tested in a FACS based assay with CCR5+CHO as target and CD3+ T-lymphocytes as effector cells. CD3+ T-cells were isolated from peripheral blood. CCR5+CHO cells were labeled with 12 pM PKH26.
Ef-fectoraarget cells in a ratio of 5:1 were incubated with dilutions of scFv CCR5xCD3 ranging from 320 ng/ml to 0.3 pg/ml for 16 hours at 37°C and 5% C02.
After staining with 1 pg/ml propidium iodine (PI), cells were analyzed by flow cytometry.
In order to verify the specificity of scFv CCR5xCD3 mediated lysis, stably transfected CHO cells were used as negative control target cells. The cytotoxicity assay was performed under identical conditions as described for CCR5+CHO cells.
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and nonlinear regression analysis was performed with GraphPad Prizm. The sigmoidal dose response curve revealed an EC50 value of 912 pg/ml. No cytotoxic ef fect of scFv CCR5xCD3 was observed using CXCR4+ CHO cells as target cells.
Figure 22.
The cytotoxic activity of scFv CCR5xCD3 on CCRS-positive cells was also tested using the CD3 positive T-cell line CB15 as effector cells in a FACS based assay.
CCR5+CHO
target cells labeled with 10 pM PKH26 were used in a effectoraarget ratio of 10:1 and incubated with 100 p1 of scFv CCR5xCD3 in different dilutions (40 pg/ml to 0.15 ng/ml) for 6 hours at 37°C at 5% C02. Cells were stained with 1 pg/ml propidium iodine (PI) and analyzed in duplicate in a flow cytometer.
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and nonlinear regression analysis was performed with GraphPad Prizm. The sigmoidal dose response curve revealed an EC50 value of 12.8 ng/ml.
Figure 23.
Reactivity of MC-1 with A) human PBMC and B) rhesus PBMC. PBMC (solid line), PBMC with PE conjugated goat anti-mouse antibody (dotted line) and PBMC with and PE conjugated goat anti-mouse antibody (solid bold line). Binding of MC-1 to hu-man PBMC (A), but not to rhesus PBMC (B) is indicated by the M1 marker line.
The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.
Example 1: Cell lines, PBMC preparation, synovial fluid 1.1 Generation of a CHO cell line expressing human CCR5 The cDNA sequence of CCR5 was amplified from genomic DNA of human PBMC by PCR with Pfu-polymerase (Stratagene), oligonucleotide primers were:
SEQ ID NO. 1 : 5' GGA ACA AGA TGG ATT ATC AAG TGT C 3' SEQ ID NO. 2: 5' CTG TGT ATG AAA ACT AAG CCA TGT G 3' The amplified fragment was gel purified, ligated into the PCR-Script Amp Sk(+) script vector (Stratagene) and sequenced. After subcloning into the PEF-DHFR vector, DHFR-deficient CHO cells were transfected by electroporation and selected for stable expression in nucleoside free MEM medium with 10% dialyzed FCS as described.
The CHO/CCR5 transfected cells were shown to be homogeneous by FACS-analysis.
1.2 PBMC purification PBMC were isolated from buffy coats or full blood of healthy donors by ficoll density gradient centrifugation. Where indicated PBMC were used from donors with a homozy-gous 32 basepair deletion in the CCR5 allele (~32/~32) preventing surface expression of CCRS. Specifically, buffy coats were diluted 1:2 in 0.9% NaCI, and 35 ml were lay-ered onto 15 ml of Ficoll Paque and centrifuged for 25 min at 400 g. The white inter-phase was harvested and thrombocytes depleted by three subsequent washing and centrifugation steps at 100 g for 6 min in RPMI with 10% FCS. Freshly isolated mono-cytes expressed a very low level of CCRS, but expression was strongly induced after culture of PBMC in RPMI with 10% FCS for 24 to 48 h at 37°C. The amount of FCS did not influence this induction. The expression of CCR5 on lymphocytes was not altered during culture.
1.3 Synovial fluid Synovial fluid of patients with arthritis was obtained from diagnostic or therapeutic ar-throcentesis and used for the experiments without further preparation.
Informed con-sent was obtained from all patients. Synovial fluid and blood samples were simultane-ously obtained from 23 patients who presented with gonarthritis for diagnostic or thera-peutic arthrocentesis. Diagnoses included rheumatoid arthritis (7), reactive arthritis (3), undifferentiated gonarthritis (4), psoriatic arthritis (3), osteoarthritis (2), ancylosing spondylitis (1 ) and gout (3) according to ACR criteria, where applicable.
Written in-formed consent was obtained from all patients. Synovial fluid was analyzed by light mi-croscopy. Crystals were identified by polarized light microscopy. Student's t-test and paired t-test was applied for statistical analysis.
1.4 Analysis of chemokine receptor expression in whole blood samples and synovial fluid Immediately after arthrocentesis SF (synovial fluid) leukocytes were isolated by two washing steps with 5% PBS in NaCI 0.9%. Synovial fluid cells and whole blood (con-taining 1 mM EDTA) were incubated on ice with monoclonal antibodies against chemo-kine receptors and the appropriate isotype controls at a concentration of 10pg/ml. The antibodies were for CCRS: MC-1 (Mack (1998) J. Exp. Med. 187, 1215-1224), for CCR2: DOC-3, which specifically binds to CCR2 (9), for CCR1: Clone 53504 (R&D-Systems), for CXCR1: 5A12 (Pharmingen), for CXCR2: 6C6 (Pharmingen), and for CXCR4 1265 (Pharmingen), IgG1-, IgG2a- and IgG2b-isotype controls (Sigma).
After two washing steps cells were incubated for 30 min on ice with a PE-conjugated rabbit-anti-mouse F(ab)2 fragment (R439, DAKO). Cells were washed twice and incubated with 10% mouse serum followed by a combination of CD4-FITC, CD8-PECy5 and CD14-APC (Immunotech). After lysis of erythrocytes, cells were immediately analyzed by flow cytometry (Becton-Dickinson). Calculations were performed with Cell Quest analysis software. Helper T cells, cytotoxic T cells, monocytes and neutrophils were identified by their light scatter properties and the expression or absence of CD4, CD8 and CD14. Chemokine receptor expression was calculated after defining a cutoff ac-cording to the isotype control.
In both acute and chronic joint effusions we found a consistently increased percentage of CD4+ and CD8+ T cells that expressed the chemokine receptor CCRS compared to the peripheral blood. These data are in good agreement with previous reports (Mack (1999) Arthritis Rheum. 42, 981-988; Qin (1998) J. Clin. Invest. 101, 746-754).
Chemokine receptor expression on T cells in non-crystal induced arthritis:
Approxi-mately 88% of CD4+ T cells and 93% of the CD8+ T cells from the synovial fluid stained positive for the chemokine receptor CCRS. Similarly, a major proportion of CD8+ and CD4+ T cells in the SF expressed CCR2 (66% and 48%) (Fig. 1 ). In con-trast, in the peripheral blood only a minority of T cells expressed the chemokine recep-tors CCR5 or CCR2. The enrichment in the synovial fluid was most pronounced for the CCRS+ helper-T cells (blood: SF ratio = 1:4). The majority of T lymphocytes stained positive for CXCR4 in both compartments. CXCR1, CXCR2 and CCR1 were only ex-pressed by a minor and variable percentage of T cells (Fig. 1 ). A typical example of one patient is shown in Fig. 2, showing the expression of CCRS, CCR2 and CXCR4 on leu-kocytes in the peripheral blood and synovial fluid.
Chemokine receptor expression on monocytes in non-crystal induced arthritis:
Consis-tent with previous data, the majority of monocytes in the SF expressed CCRS.
In addi-tion, a reduced expression of CXCR1, CXCR2, CXCR4 and CCR1 is here reported on monocytes in the synovial fluid compared to the peripheral blood (Fig. 1, 2).
Not only was a lower frequency of receptor positive cells found, but also a lower density of che-mokine receptors on the cell surface (data not shown). No differences could be de-tected in relation to the underlying diagnoses, duration of joint effusion or pretreatment.
CCR2 was equally expressed by all monocytes in both compartments (Fig.'s 1, 2).
Chemokine receptor expression on neutrophils in non-crystal induced arthritis:
Acute arthritis is characterized by a rapid influx of neutrophils into the inflamed joint. There-fore, the chemokine receptor expression on neutrophils from inflamed joint efFusions was analyzed. For the first. time a high expression of CXCR4 is described on a large fraction of neutrophils (60%) from the synovial fluid of patients with acute and chronic arthritis, while a much lower expression was found in the peripheral blood (24 %) (Fig.
1, 2). In arthritis other than gout CXCR1 and CXCR2 was reduced on neutrophils from the synovial fluid by approximately 50% compared to the peripheral blood. CCR1 was expressed only by a minority of neutrophils in both compartments.
1.5 Determination of CCR5 genotype Genomic DNA was prepared from frozen blood samples by affinity chromatography (Ruche Diagnostics). Subsequently a fragment of the CCR5 gene containing the po-tential 32 basepair deletion was amplified by a 40 cycle PCR with Taq polymerase. The primers were .
SEQ ID NO. 3: 5' TTT ACC AGA TCT CAA AAA GAA G 3' SEQ ID NO. 4: 5' GGA GAA GGA CAA TGT TGT AGG 3' Differences in the length of the PCR fragments (274 or 242 bp) allowed to identify CCRS-wildtype and CCRS-032 alleles.
Example 2: Construction of a bispecific antibody 2.1 Generation of monoclonal antibodies against human CCR5 To generate monoclonal antibodies against human CCRS, five BALB/c mice were im-munized intraperitoneally (i.p.) at four week intervals, first with 1x10' PBMC
cultured for days in IL-2 (100 U/ml) and six subsequent i.p. injections of 1x107 CHO cells ex-pressing high levels of CCRS. For this purpose, CCR5 transfected CHO cells were grown in the presence of 20 nM methotrexate to amplify expression of CCR5 and one clone expressing high levels of CCR5 was chosen. Four days after the last i.p.
injection of CHO/CCR5 cells, the spleens were removed and the cells fused with the P3X63-Ag8 cell line. Supernatants from approximately 6000 hybridomas were screened per fusion by flow cytometry on stable CHO/CCR5 cells and monoclonal antibodies against (MC-1, MC-4, MC-5) were detected after the third fusion. The specificity of MC-(IgG1), MC-4, MC-5 were tested on CHO cells stably transfected with CCR1-3 and CXCR4. In all cases no binding was detected. In addition the antibodies did not react with freshly isolated PBMGs and cultured PBMCs from a donor homozygous for the d32 deletion in the CCR5 gene.
2.2 Cloning of the variable domains of MAb MC-1 against CCR5 The light (VL) and heavy (VH) variable domains from the aCCR5 hybridoma MC-1 were cloned using PCR amplification (Orlandi (1989) Proc. Natl. Acad. Sci. 86, 3833).
Reverse transcription was carried out with random hexamer nucleotides and Super-Script reverse transcriptase (Gibco). The variable domains were amplified by PCR with Pfu-polymerase, subcloned into the vector PCR-script Amp SK+ (Stratagene) and se-quenced.
For PCR amplification of VL(1 ) the following primers were used:
SEQ ID NO. 5: 5' GACATTCAGC TGACCCAGTC TCCA 3' SEQ ID NO. 6: 5' GTTTTATTTC CAGCTTGGTC CC 3' For PCR amplification of VM(1 ) the following primers were used:
SEQ ID NO. 7: 5' ACCATGGGAT GGAGCTGTGT CATGCTCTT
SEQ ID NO. 8: 5' TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG
The nucleotide sequence of VL(1 ) obtained by RT PCR is SEQ ID NO. 9:
The corresponding translated protein sequence to VL(1 ) is SEQ ID NO. 10:
The nucleotide sequence of VL(1 ) without the primer sequences used for amplification, SEQ ID NO. 11:
l21 TATAATGCAA AAACCTTAAC AGAAGGTGTG CCATCAAGGT TCAGTGGCAG TGGATCAGGC
The corresponding translated protein sequence to SEQ ID NO. 11: of VL(1 ) is SEQ ID
NO. 12:
The sequence of VH(1 ) including the leader sequence obtained by RT PCR is SEQ
ID
NO. 13:
The corresponding translated protein sequence to VH(1) is SEQ ID NO. 14 The nucleotide sequence of VH(1 ) without the leader sequence and primer sequences used for amplification, SEQ ID NO. 15:
24l GACTGTTGAC AAATATTCCA GCACAGCCTA TATACAACTC AGCAGCCCGA CATCTGAGGA
The corresponding translated protein sequence to SEQ ID NO. 15 of VH(1 ) is SEQ ID
NO. 16:
2.3 Construction and expression of the bispecific single chain antibody CCR5xCD3 A schematic depiction of structure and mode of action of the CCR5xCD3 bispecific sin-gle chain antibody is shown in Fig 3. As described previously, the light and heavy vari-able domains were joined to a single-chain fragment using a (GIy4Ser1 )3 linker and expressed in the periplasmic space of E. coli to test binding of the recombinant protein to CCRS.
Subsequently, the DNA sequence of the aCCR5 single-chain fragment was subcloned with BsrG1 and BspE1 into an eukaryotic expression vector (pEF-DHFR) that already contained a single-chain fragment directed against CD3 with a C-terminally attached tail of 6 histidine residues (Mack (1995) Proc. Natl. Acad. Sci. 92, 7021 ).
The aCCR5 and aCD3 single-chain fragments were joined by a linker coding for GIy4Ser1 (see Fig 3).
The following order of the domains is chosen: VL(1 )-VH(1 )-VH(2)-VL(2), with (1 ) being the specificity against CCRS and (2) the specificity against CD3.
The bispecific CCR5xCD3 antibody has the following nucleotide sequence, SEQ ID
NO. 17:
The bispecific CCR5xCD3 antibody has the following protein sequence, SEQ ID
NO.
18:
A
The bispecific antibody was expressed in DHFR-deficient CHO cells and purified from the culture supernatant by affinity chromatography on immobilized Ni2+ ions (Hochuli (1988) Biotechnology 6, 1321-1325; Ni-NTA, Qiagen).
In summary, for the construction of bispecific antibodies, for example the single-chain technique may be used (Mack et al. (1995) Proc. Natl. Acad. Sci. U S A, 92:7021-7025;
Mack et al. (1997) J. Immunol. 158:3965-3970). In this case, as shown schematically in Figures 3 (top), the variable domains of the light (VL) and the heavy (VH) immuno-globulin chains of two different antibodies are fused in a particular order, optionally a histidine chain of 6 x His is attached in addition. The fusion is effected on a DNA basis so that a protein chain with four different variable domains is formed after expression (cf. Figures 3 (top)). The attached histidine chain enables a simple and efficient purifi-cation via immobilized Ni ions in one step. Figure 3 (top) shows a preferred embodi-ment of the bispecific antibody binding to the CD3 antigen on the surface of the effector cell and the human CCR5 on the surface of leukocytes as target cells.
Subsequently, a single-chain antibody with a specificity is generated by means of fu-sion PCR by inserting a linker of (GIy4Ser~)3 between the two variable antibody do-mains. In a further fusion PCR, the antibody fragment against CCRS is fused to the al-ready published antibody fragment against CD3, with a linker consisting of GIy4Ser~ is inserted (cf. Mack et al. supra).
In order to express the bispecific antibody, the corresponding DNA sequence is sub-cloned in a eukaryotic expression vector (e.g. PEF-DHFR, Mack et al. (1995) PNAS, supra) and transfected in DHFR-deficient CHO cells by means of electroporation. The bispecific antibody is purified from the supernatant of stably transfected CHO
cells by means of affinity chromatography at Ni-NTA, with elution taking place by lowering the pH value. Subsequently, the pH is adjusted and the protein is adjusted to a suitable concentration. Overall purification yield was approx. 900 pg/I culture supernatant. SDS-PAGE showed a single band of approx. 60 kD under reducing and non-reducing condi-tions without any detectable proteolysis or degradation of the protein (Fig.
4).
Example 3: Expression and purification of a chemokine-toxin fusion protein A schematic depiction of structure and mode of action of the RANTES-PE38 chemo-kine-toxin fusion protein is shown in Fig 5. A PCR fragment of RANTES, generated with the primers P1 and P2, was subcloned with Stul and Sall into a vector for periplasmic expression in E. coli (Mack (1995) Proc. Natl. Acad. Sci. 92, 7021 ). The restriction site Stul had previously been introduced at the 3' terminus of the OmpA signal sequence.
The DNA of a truncated version of Pseudomonas exotoxin A (PE38; Theuer (1993) Cancer Res. 53, 340), was amplified by PCR with Pfu-polymerase using the primers P3 and P4 and subcloned with BspE1 and Hind III into the vector that already contained the cDNA of RANTES. Primer P4 also added a tail of 6 histidine residues at the 3' ter-minus of PE38. During the periplasmic expression the OmpA signal sequence is cleaved off such that the recombinant protein starts with the first aminoacid of RAN-TES. The C-terminally attached tail of 6 histidine residues allowed purification by affinity chromatography on Ni-NTA (Qiagen).
List of primers:
SEQ ID NO. 19: 5' AAAGGCCTCCCCATATTCCTCGGA
SEQ ID NO. 20: 5' AAAGTCGACTCCGGACATCTCCAAAGAGTTGATGTAC
SEQ ID NO. 21: 5' AATCCGGAGGCGGCAGCCTGGCCGC
SEQID NO:22: 5' GGGAAGCTTAGTGATGGTGATGGTGATGCTTCAGGTCCTCGCGCGG
As described in the above the DNA sequence of RANTES was fused with the se-quence of a truncated version of the Pseudomonas exotoxin A (PE38) (Theuer (1993) Cancer Res. 53, 340). In a first version of the construct a Gly-Ser linker was spaced between RANTES and PE 38. However this resulted in a considerable proteolytic deg-radation of the fusion protein during expression in E. coli (data not shown).
In an at-tempt to stabilize the construct the linker and the fist three aminoacids of PE38 were removed. The new fusion protein showed no proteolysis during expression in the periplasmic space of E. coli as demonstrated by SDS-PAGE (Fig. 6 left panel) and Western-blot (Fig. 6 right panel). The corresponding constructs are depicted in SEQ ID
NOs: 23 and 24, respectively.
Example 4: Binding of the bispecific antibody to the target antigens CCR5 and CD3 Binding of the bispecific single-chain antibody to CHO cells or PBMC was determined by FACS-analysis (Fig. 7 to 9). The cells were incubated with the bispecific antibody for 60 min on ice followed by an antibody against 6xHis (Dianova, Hamburg, Germany) and a PE-conjugated polyclonal rabbit-anti mouse F(ab)2 fragment (R439, Dako, Ham-burg, Germany). As the bispecific antibody would also bind to CCRS, we performed the analysis with PBMC that lack expression of CCR5 due to a homozygous 32 basepair deletion in the CCR5 alleles. The antibody showed good binding to a subpopulation of lymphocytes. Co-staining with antibodies against CD4 and CD8 identified this sub-population as CD4 and CD8 positive T lymphocytes (Fig. 7). In addition, the bispecific antibody competed with the monoclonal CD3 antibody OKT-3 for binding to T
cells (data not shown).
Binding of the bispecific antibody to CCR5 was demonstrated on CCR5 overexpressing CHO cells and human monocytes (Fig. 8 and 9). The antibody showed excellent bind-ing to CCR5 transfected CHO cells (Fig. 8) and cultured monocytes (Fig 9), while no binding was detectable on CHO cells transfected with CXCR4 or on cultured mono-cytes from a donor with a homozygous CCRS-432/32 deletion. Overnight cultivation of monocytes induces expression of CCR5 on wild-type monocytes, while monocytes from donors with a homozygous CCRS-X32/032 deletion fail to express CCRS. Moreover we have shown that the CCR5 signal detectable with the bispecific antibody on cultured monocytes could be reduced to values below 15 % by preincubation of monocytes for 30 min at 37°C with AOP-RANTES (data not shown) that is known to efficiently induce internalization of CCR5 and reduce binding of CCR5 antibodies (25).
Example 5: Downmodulation of chemokine receptors 5.1 Downmodulation of CCR5 with mAb MC-1 against CCR5 The effect of MC-1 one on the surface expression of human CCR5 was measured.
For comparison a different monoclonal antibody MC-4 against CCR5 was used. CHO-CCRS cells were incubated with various concentrations of antibody MC-1 and MC-4 for 30 min. at 37°C. Cells were placed on ice and stained with MC-1 and MC-4 respec-tively at a concentration of 15 ug/ml for one hour on ice, followed by detection with a secondary antibody (rabbit anti-mouse FITC, F313 from DAKO). Analysis was per-formed on a FACSCalibur. Incubation with MC-1 at 37oC for 30 min resulted in a downmodulation of human CCR5 by 40% at a concentration of 10 ug/ml (Fig. 10).
5.2 Downmodulation of CCR5 by chemokine-toxin The fusion of RANTES to the N-terminus of a truncated version of the Pseudomonas exotoxin A is supposed to result in specific binding of the construct to cells expressing RANTES receptors such as CCRS, CCR1 and CCR3. Internalization of the chemokine receptors upon binding of the modified toxin would enhance the cellular uptake and cytotoxic activity of the construct (Fig. 5 lower panel). We therefore analyzed whether RANTES-PE38 is able to internalize CCR5 from the surface of primary monocytes and T cells (Fig. 11, open symbols). Internalization of CCR5 would indicate that the con-struct is able to bind to CCR5 and that RANTES remains functionally active after fusion to PE38. As shown in Fig. 11 the construct is able to internalize CCR5 from the surface of monocytes and lymphocytes. Unmodified RANTES served as positive control and was somewhat more efficient than RANTES-PE38 (Fig 11, closed symbols).
PBMC were incubated for 30 min at 37°C with various concentrations of RANTES or RANTES-PE38 diluted in RPMI with 10% FCS in a volume of 100 p1. Medium alone was used as control. The cells were then stained on ice for surface CCRS
expression using the monoclonal antibody MC-1 or medium as negative control followed by the PE-conjugated anti-mouse antibody 8439. The FACS-analysis was performed on a FACSCalibur (Becton Dickinson) and CeIIQuest software. Lymphocytes and monocytes were distinguished by their forward and sideward light scatter properties and expres-sion of CD14, CD4 and CDB. Relative surface CCR5 expression was calculated as [mean channel fluorescence (exp.) - mean channel fluorescence (negative control)] /
[mean channel fluorescence (medium) - mean channel fluorescence (negative control)].
Example 6: Depletion of cells with CCR5xCD3 antibody and RANTES-PE38 6.1 CCR5 specific depletion of monocytes from cultured PBMCs PBMC from CCRS-wildtype (WT) or CCR5 deficient (0321032) donors were incubated over night to induce expression of CCR5 on monocytes. Cultured PBMC were incu-bated with different concentrations of purified aCCRS-aCD3 bispecific antibodies or medium as control for 20 h. Surviving cells were analyzed on a FACSCalibur and counted.
In order to test the ability of the aCCRS-aCD3 bispecific single-chain antibody to de-plete CCR5 positive primary cells, we incubated human PBMC with the antibody (Fig.
12). Prior to incubation the PBMC were cultured overnight to upregulate CCR5 expres-sion on monocytes. By retargeting cytotoxic T cells the bispecific antibody depleted the majority of monocytes within 20 h in a concentration dependent manner (Fig.
13) with an almost complete elimination of CCR5 positive cells at concentration of 10 ng/ml. To verify that the depletion of.monocytes was due to their induced expression of CCRS, the same experiment was performed with PBMC from a donor with a homozygous 32 by deletion in the CCR5 allele that prevents surFace expression of CCR5. No depletion of CCR5 deficient monocytes was detectable after 20 h indicating that the depletion of cells with the bispecific antibody is restricted to monocytes that express CCR5 (Fig.
12), compare lower right panel to upper right panel, left panels serve as negative con-trols. Monocytes (Mo) and lymphocytes (Ly) were identified by their forward and side-wards light scatter properties. Monocytes appear in the lower left quadrant see arrows.
6.2 Depletion of monocytes and T lymphocytes from the synovial fluid of patients with arthritis Freshly drawn synovial fluid of patients with arthritis were incubated with different con-centrations of purified aCCRS-aCD3 bispecific antibodies or medium as control for 20 h. Surviving cells were analyzed on a FACSCalibur and counted.
The bispecific single-chain antibody could potentially be applied to deplete CCRS posi-tive T cells and monocytes from the inflamed joints of patients with arthritis. Therefore determined the depletion of CCR5 positive cells from the synovial fluid of patients with various types of arthritis was determined. It was shown previously that the majority of T
cells and monocytes in the inflamed synovial fluid express CCR5 (Mack (1999) loc.
cit.). In synovial samples obtained before depletion experiments it was confirmed by FACS analysis that the majority of lymphocytes and monocytes express CCRS, while no expression of CCR5 was detectable on granulocytes (data not shown). For the de-pletion experiments the synovial fluid was incubated ex vivo with different concentra-tions of the bispecific antibody for 20 h (Fig. 14). The synovial fluid was incubated im-mediately after puncture without any preparation to ensure that the conditions in vitro resemble most closely the situation in vivo when the antibody would be present within inflamed joints. As shown in Fig. 14 the bispecific antibody induced a depletion of the majority of lymphocytes and monocytes from the synovial fluid, while granulocytes that do not express CCR5 remained unaffected. A representative FACS analysis of the de-pletion of monocytes and lymphocytes in synovial fluid at a concentration of 0.5 uglml CCR5xCD3 is shown in Fig 15. Only the CCR5 negative neutrophils (PMN: polymor-pho-nuclear cells) are unaffected by the bispecific antibody.
Antibodies were incubated with synovial fluid for one or several days. After 24 hours, the CCR5 positive lymphocytes and monocytes have already almost disappeared.
When the medium is controlled after longer incubation, the monocytes have difFerenti-ated into macrophages which are visible at the bottom of the culture flask.
After an ap-propriate incubation with the bispecific antibody, no macrophages are visible.
A corresponding result can be obtained when cultivated PBMC are incubated with the bispecific antibody as described above. In this case, there is an almost complete de-pletion of CCR5 positive monocytes and an almost complete depletion of CCR5 posi-tive T-lymphocytes. The depletion of CCR5 positive T-cells and monocytes is shown.
The results show that the construct of the present invention is capable of destroying CCR5 positive monocytes. This applies to both monocytes from the joint aspirate and blood monocytes which express CCR5 when being differentiated into macrophages.
Depletion of the monocytes/macrophages takes place within a few hours (< 24 hrs). In particular the depletion of monocytes/macrophages in the joint is of great advantage in therapy since it is these cells that are mainly responsible for the joint destruction.
Moreover, for the activation of T-lymphocytes an interaction with macrophages is also required so that, at the same time, the function of the T-lymphocytes is suppressed.
In addition to the depletion of monocytes/macrophages, a considerable reduction in the number of CCR5 positive T-lymphocytes could be observed.
6.3 Comparison of the efficacy of the bispecific antibody CCR5xCD3 versus mono-clonal antibodies The efficacy of the aCCRS-aCD3 bispecific single-chain antibody in depleting positive monocytes was compared with the efficacy of two unmodified monoclonal anti-bodies. PBMC from two different donors (F and N) were cultured overnight and then incubated for 24 h with medium, the bispecific single-chain antibody (125 ng/ml), MC-1 (5pg/ml) and MC-5 (5pg/ml). The monoclonal antibody MC-1, the parental antibody for the bispecific single-chain antibody has the isotype mouse IgG-1 and the antibody MC-has the isotype IgG-2a. The cells were completely recovered and analyzed by FACS
to quantify surviving monocytes and lymphocytes.
Fig. 16 shows that surprisingly only the bispecific antibody was able to considerably deplete CCR5 positive monocytes, while the unmodified monoclonal antibodies were largely ineffective even when used in a 40 fold excess over the bispecific antibody CCR5xCD3. By FACS analysis using forward and sideward light scatter properties of lymphocytes and monocytes demonstrates that only the CCR5xCD3 bispecific antibody but not the monoclonal antibodies are capable of depleting cultured monocytes (Fig 17 compare right upper panel to lower panels).
6.4 Depletion of chemokine receptor expressing cells with RANTES-PE38 CHO cells expressing CCR5 or CXCR4 were grown to subconfluence on 24 well cul-ture plates and incubated with different concentrations of purified RANTES-PE38 or medium as control. After 40 hours the adherent and non-adherent cells were recovered and analyzed by FACS to measure the percentage of dead cells. It was previously es-tablished that dead (propidium iodide positive) CHO cells can be identified by their light scatter properties.
It was further analyzed the cytotoxic activity of RANTES-PE38. For that purpose we incubated CHO cells expressing human CCRS, murine CCR5 and human CXCR4 with various concentrations of the chemokine-toxin or medium. No surviving (adherent) hu-man or murine CCR5 positive CHO cells were detectable by light microscopy after 40 h incubation with as little as 10 nM RANTES-PE38. In contrast regular growth and sur-vival was observed when the CCR5 positive cells were incubated with medium or when CXCR4 positive CHO cells were incubated with equal concentrations of the chemokine-toxin (data not shown). To quantify the percentage of dead cells the adherent and non-adherent cells were analyzed by FACS. It was previously established that living and dead CHO cells can be identified by their light scatter properties, the position of dead and alive cells is indicated by arrows (Fig 18). As shown in Fig. 18 no cytotoxic effect of RANTES-PE38 was seen on CHO cells expressing CXCR4, while CHO cells express-ing human CCR5 were completely killed by 10 nM RANTES-PE38.
These experiments show that RANTES-PE 38 is able to internalize CCRS from the surface of cells and induces depletion of cells expressing the RANTES
receptors hCCR5 or mCCRS. The inactivity of the construct against CXCR4 positive CHO
cells demonstrates that the cytotoxic activity of the construct is restricted to cells that ex-press specific chemokine receptors.
Example 7: Virus infection assay with stable transfected cells GHOST 34 CCR5 cells are derived from HOS/CD4 cells stably expressing CCR5 and were provided by Dan Littman (Skirball Institute, New York). 2.5 x 104 cells in 48-well trays were exposed to 100 ~,I of chemokine at appropriate dilution for 30 min at 37°C.
100 ~,I of the NSI, CCRS-dependent HIV-1 strain, SF162 was added at 1000 focus forming units/ml (FFU/ml) and the cells incubated for a further 3 h. The cells were then washed and incubated in medium containing the appropriate chemokine for 4 days before fixing, staining in situ for p24 production and estimating foci of infection as pre-viously described.
EXAMPLE 8: Concentration dependent binding of CCR5xCD3 to CCRS express-ing CHO cells Chinese hamster ovary cells stably transfected with CCR5 (CCR5+CHO) were used as target cells for binding studies of bispecific scFv CCR5xCD3 (as described in Example 2 and Fig. 3). These cells were negative for CD3 and >95% positive for CCR5 as evaluated by binding assays with the parental antibody MC-1 (as described in Example 5 and Fig. 10). Binding was evaluated by a flow cytometry based binding assay.
4x105 CCR5+CHO cells were resuspended in 50 p1 FACS buffer (PBS with 1 % fetal calf serum (FCS) and 0,05% sodium azide) containing different dilutions of scFV
CCR5xCD3 ranging from 20 pg/ml to 19.5 ng/ml. Cells were incubated in a 96 well mi-crotiter plate for 30 minutes at 4°C. Cells were washed twice with FACS
buffer and in-cubated for 45 minutes at 4°C with 20 pg/ml anti-His-Tag monoclonal antibody (Di-anova). Specifically bound scFV CCR5xCD3 was detected with a monoclonal goat anti-mouse IgG F(ab')2 -PE conjugated antibody (Dianova). After washing, the cells were analysed in a flow cytometer (FACSCalibur, Becton Dickinson) using the CeIIQuest software (Becton Dickinson) to calculate the median values of the fluorescence intensi-ties of the different concentration samples. Nonlinear regression analysis was per-formed with GraphPad Prizm (Version 3.02). Concentration dependent binding of scFv CCR5xCD3 to CCRS expressing CHO cells was observed with a Kp value of 0,86 pg/ml (Fig. 20).
EXAMPLE 9: Cytotoxic activity of CCR5xCD3 with primary T lymphocytes as ef-fpctor cells The capacity of scFv CCR5xCD3 (as described in Example 2 and Fig. 3) to mediate cytotoxicity to CCRS-positive cells was tested using stably transfected CCR5+CHO as target cells and CD3 positive T-lymphocytes derived from peripheral blood as effector cells. For detection of cytotoxicity, a FACS based assay was performed.
CD3+ T-cells (include CD4+ and CD8+ cells) were isolated from peripheral blood by negative selection using a human T cell enrichment column (R&D Systems). For this purpose, PBMC were prepared by standard Ficoll-Hypaque density gradient separation and applied to the column. B cells and monocytes were bound to the column matrix, while T cells were eluated. The enriched T cells were washed in medium and used as effector cells.
For discrimination of target cells from effector cells by flow cytometry, CCR5+CHO cells were labeled with the aliphatic membrane dye PKH26 (Sigma) in a final concentration of 12 pM. 0.5x105 labeled CCRS+CHO cells and 2.5x105 CD3+ T-cells were seeded in a 96-well microtiter plate in a effectoraarget ratio of 5:1. 100 ~l dilutions of scFv CCR5xCD3 ranging from 320 ng/ml to 0.3 pg/ml were incubated with the cells for hours at 37°C in a humified atmosphere at 5% C02. Subsequently cells were centri-fuged for 3 minutes at 600xg, and the cell pellets were resuspended in 200 p1 FACS
buffer (PBS, 1 %FCS, 0.05% sodium azide). After staining with 1 pg/ml propidium iodine (PI), cells were analyzed in duplicate in a flow cytometer (FACSCalibur, Becton Dickin-son).
In order to verify the specificity of scFv CCR5xCD3 mediated lysis, stably transfected CHO cells were used as negative control target cells. The cytotoxicity assay was performed under identical conditions as described for CCR5+CHO cells.
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and a nonlinear regression analysis was performed with GraphPad Prizm. A
sigmoidal dose response curve was obtained (Fig. 21 ) revealing an EC50 value of 912 pg/ml. No cytotoxic effect of scFv CCR5xCD3 was observed using CXCR4+ CHO
cells as target cells.
EXAMPLE 10: Cytotoxic activity of CCR5xCD3 with T cell clone CB15 as efFector cells The cytotoxic activity of scFv CCR5xCD3 (as described in Example 2 and Fig. 3) on CCRS-positive cells was also tested using the CD3 positive T-cell line CB15 (CD4+) as effector cells. For detection of cytotoxicity, a FACS based assay was performed with CCR5 transfected CHO cells (CCR5+CHO) as target cells.
CCR5+CHO cells were labeled with the aliphatic membrane dye PKH26 (Sigma) in a final concentration of 10 pM. Effector and target cells were incubated in a microtiter plate in a ratio of 10:1 with 100 p1 of scFv CCR5xCD3 in dilutions ranging from 40 pg/ml to 0.15 ng/ml for 6 hours at 37°C in a humified atmosphere with 5% C02.
Cells were centrifuged for 3 minutes at 600xg and the cell pellets were resuspended in 200 p1 FACS buffer (PBS, 1 %FCS, 0.05% sodium azide). Cells were stained with 1 pg/ml propidium iodine (PI) and analyzed in duplicate in a flow cytometer (FACSCalibur, Becton Dickinson).
Specific lysis of CCR5+CHO cells was calculated using the CeIIQuest software (Becton Dickinson) and a nonlinear regression analysis was performed with GraphPad Prizm. A
sigmoidal dose response curve was obtained (Fig. 22) revealing an EC50 value of 12.8 ng/ml.
The results obtained with T cell clone CB15 as effector cells in bioactivity assay dem-onstrate that specific lysis mediated by scFv CCR5xCD3 is not restricted to the cyto-toxic activity of CD8+ CTL but that CD4+ T cells are also involved in this process.
EXAMPLE 11: Epitope mapping of parental CCR5 specific monoclonal antibody MC-1 used for construction of scFv CCR5xCD3 Epitope of parental CCR5 specific monoclonal antibody MC-1 used for construction of scFv CCR5xCD3 (as described in Example 2 and Fig. 3) was mapped by flow cytome-try using a panel of about 70 CHO-K1 cell lines stably expressing chimeric and point mutant receptors (Samson, J. Biol. Chem., 1997, 272, 24934-24941; Lee, J.
Biol.
Chem., 1999, 274, 9617-9626; Blanpain, J. Biol. Chem., 1999, 274, 34719-34727;
Blanpain, Blood, 2000, 96, 1638-1645). Cells were incubated for 30 min on ice with mab MC-1, washed and stained with PE-conjugated anti-mouse Ig antibody (Sigma).
CHO-K1 cells expressing CCR2b were used as negative control. MC-1 was shown to recognize the first part of the second extracellular loop (ECL2) of the CCR5 molecule (data not shown). ECL2 ranges from as 168-199 (RSQ KEGLHYTCSS HFPYSQYQFW
KNFQTLKIV) and is located between the transmembrane regions 4 and 5 of CCR5 as described by Chen, J. Virol., 1997, 71, 2705-2714.
The amino acid sequences of human and rhesus macaque CCR5 differ in eight amino acids with two amino acid changes are situated at position as 171 (K-->R) and as 198 (I-->M) in the ECL2 (Chen, J. Virol., 1997, 71, 2705-2714). Due to these amino acid changes potential crossreactivity of MC-1 with the ECL2 of rhesus macaque CCR5 was analyzed with human and rhesus PBMCs in a FACS based assay. PBMC of both spe-cies were isolated by standard ficoll gradient centrifugation. 5x105 cells were sus-pended in 50 p1 FACS buffer and 50 pg/ml of MC-1 was added. After 30 min incubation at 4°C, the cells were washed and stained with goat anti-mouse IgG
F(ab')2-PE conju-gated monoclonal antibody (Dianova) for 30 min at 4°C in the dark.
Cells were washed and analyzed in a flow cytometer (FACSCalibur, Becton Dickinson).
As shown in Fig. 23 MC-1 exclusively bound to human CCR5 but did not react with CCR5 derived from rhesus macaques. These data show that the epitope recognized by MC-1 is specific for human CCR5 and that lysin at position as 171 and isoleucin at po-sition as 198 in human CCR5 sequence are essential for this specificity.
Especially ly-sin at position as 171 which is located in the first part of ECL2 contributes to the spe-cific recognition of the human epitope of CCR5 by mab MC-1.
EXAMPLE 12: scFv CCR5xCD3 mediated reduction of virus production in HIV-1 infected monocytes PBMC were prepared from fresh buffy coats of healthy donors by Ficoll density cen-trifugation and monocytes were isolated by over night adherence to culture flasks. Re-maining PBL were removed and cultured separately at 37°C in a humidified atmos-phere at 5% C02.
Monocytes were seeded into a 48 well microtiter plate at a density of 5x104 cells/well and infected with the M-tropic HIV-1 strain BaL (moi=1 ) overnight at 37°C in a humidi-fied atmosphere at 5% C02. The virus was removed by washing and the monocytes were further cultured with unstimulated PBL (15 x 104 per well) + scFv CCR5xCD3 (1 pg/ml) + AZT (75 pM) or with unstimulated PBL (15 x 104 per well) alone as negative control. 5 days post infection (p.i.) monocytes were washed and cultured in the ab-sence of AZT or antibody. Supernatant was harvested on day 15 p.i. and HIV-1 replica-tion was quantified by measurement of p24 in an ELISA. This experimental approach led to a reduction of virus replication of 75% in samples containing scFV
CCR5xCD3 (75 ng/ml p24) compared to the control without scFv CCR5xCD3 (300 ng/ml p24).
Table I: Nucleosidal Reverse Transcriptase Inhibitors (nucleoside analogs, NRTI) Substance Trade- Dosing Sched- Common Side Effects and General name ule Remarks Zidovudine Retrovir300 mg, 2x dailyInitial gastrointestinal (GI) side effects, (AZT) anemia, neutropenia, myopathy Lamivudine Epivir 150 mg, 2x dailyGenerally well tolerated. Effective against (3TC) hepatitis Zidovudine,Com- 1 tablet 2x Combination-tablet containing daily 300 mg Lamiduvine bivir AZT and 150 mg 3TC
(AZT + 3TC) Didanosine Videx 200 mg, 2x daily15% peripheral neuropathy, pancreatitis;
(ddl) or avoid alcohol.
400 mg, 1x dailyContents alcohol: could be given simulta-on an empty neously with all NRTIs, Adefovir, Nevira-stomach (> 60 pine, and Efavirence; Delavirdine kg and weight) Indinavir should be given at least 1 hour before ddl; Nelfinavir to be given 1 hour after dd I .
ZalcitabineHivid 0,375-0,75 mg, 17-31 % peripheral neuropathy in difFerent (ddC) 3x daily studies; aphteous ulcerations Stavudine Zerit 20-40 mg, 2x Peripheral neuropathy (1-4% in earlier (d4T) daily studies; 24% in 'expanded access' pa-tients with CD4 > 50) Abacavir Ziagen 300 mg, 2x dailyAbout 3% reaction for hypersensitivity:
(ABA) fever, indisposition, possibly transient rash, gastrointestinal side effects.
Table II: Protease Inhibitors Substance Trade- Dosing Schedule Common Side Effects and name General Remarks Saquinavir Invirase 600 mg, 3x daily, Well tolerated. Limited take efficacy (hard gela- with high-fat meal due to poor resorption.
tine cap-sule, SQV-H) Saquinavir Forto- 1200 mg, 3x daily, Improved resorption compared take (soft gela-vase with high-fat meal to Invirase.
(>28g) tine cap-sule, SQV-S) Ritonavir Norvir 600 mg, (6 cap./7.5 Nausea and numb lips for ml) up to (RTV) 2x daily. Start with5 weeks. Occasionally hepatitis.
mg, 2x daily, then dot tolerated by up to in- 50% of crease within 10 the patients.
days to 600 mg, 2x daily;
Indinavir Crixivan 800 mg, every 8 hoursNeural calculus with 6-8%;
on re-(IDV) an empty stomach quires large liquid intake.
or with Occa-snack (<2g fat) sionally nausea and gastroin-testinal side effects.
Nelfinavir Viracept 750 mg, 3x daily, Often diarrhea, sometimes or 1250 nau-(NFV) mg, 2x daily with sea.
meals Table III: Non-Nucleosidal Reverse Transcriptase Inhibitors (NNRTI) Substance Trade- Dosing Sched- Common Side Effects and General name ule Remarks Nevirapine Viramune 200 mg, 1x Transient skin, hepatitis, induced daily liver (NVP) enzymes P450 3A4 DelavirdineRescriptor400 mg, 3x Transient skin, suppresses P450 daily 3A4 (DLV) Efavirence Sustiva 600 mg, 1x Initially dizziness, insomnia, daily momen-(EFV) in the eveningtart' transient skin: Induces P450 3A4;
avoid Claritithromycin.
Table IV: Chemokine receptors and chemokine ligands Chemokine Chemokine Ligands Receptors CXCR3 I-TAC (CXCL11), IP-10 (CXCL-10), Mig (CXCL9) CXCR4 SDF-1 (CXCL12) CXCR5 BCA1 (CXCL13) CCR1 MIP1alpha (CCL3), RANTES (CCLS), MCP-3 (CCL7), MCP-4 (CCL13), HCC1 (CCL14), LKN1 (CCL15) CCR2 MIP1alpha (CCL3), RANTES (CCLS), MCP-1 (CCL2), MCP-2 (CCLB), MCP-3 (CCL7), MCP-4 (CCL13) CCR3 RANTES (CCLS), MCP-2 (CCLB), MCP-3 (CCL7), MCP-4 (CCL13), eotaxin (CCL11 ), LKN1 (CCL15), MPIF-2 (CCL24) , eo-taxin-3 (CCL26) CCR4 TARC (CCL17), MDC (CCL22) CCR5 MIPlalpha (CCL3), MIP1 beta (CCL4), RANTES (CCLS), (CCLB), MCP-3 (CCL7), MCP-4 (CCL13), eotaxin (CCL11 ) CCR6 LARC~ (CCL20) CCR7 ELC (CCL19), SLC (CCL21 ) CCRB I-309 (CCL1 ), MIP1 beta (CCL4), TARC (CCL17) CCR9 TECK (CCL25) XCR1 XCL1, XCL2 CCR10 CCL27, CCL28 (Wang (2000) J. Biol. Chem. 275, 22313-22323) CX3CR1: fractalkine (CXC3CL1 ) 1, SEQUENCE LISTING
<110> MACK, Matthias <120> Antibody and/or chemokine constructs and their use in immunological disorders <130> E 2411 PCT
<140>
<141>
<160> 34 <170> PatentIn Ver. 2.1 <210> 1 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 1 ggaacaagat ggattatcaa gtgtc 25 <210> 2 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 2 ctgtgtatga aaactaagcc atgtg 25 <210> 3 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 3 tttaccagat ctcaaaaaga ag 22 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 4 ggagaaggac aatgttgtag g 21 <210> 5 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 5 gacattcagc tgacccagtc tcca 24 <210> 6 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 6 gttttatttc cagcttggtc cc 22 <210> 7 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 7 accatgggat ggagctgtgt catgctctt 29 <210> 8 <211> 34 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 8 tgaggagacg gtgaccgtgg tcccttggcc ccag 34 <210> 9 <211> 322 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(321) 3, <400>
gacattcagctg acccagtct ccagcctcc ctatctgca tctgtggga 48 AspIleGlnLeu ThrG1nSer ProAlaSer LeuSerAla SerValGly gaaactgtcacc atcacatgt cgagcaagt gagaatatt tacagttat 96 GluThrValThr IleThrCys ArgAlaSer GluAsnIle TyrSerTyr ttagcatggtat cagcagaaa cagggaaaa tctcctcaa ctcctggtc 144 LeuAlaTrpTyr GlnGlnLys GlnGlyLys SerProG1n LeuLeuVal tataatgcaaaa accttaaca gaaggtgtg ccatcaagg ttcagtggc 192 TyrAsnAlaLys ThrLeuThr GluGlyVal ProSerArg PheSerGly agtggatcaggc acacagttt tctctgaag atcaacagc ctgcagcct 240 SerG1ySerGly ThrGlnPhe SerLeuLys IleAsnSer LeuGlnPro gaagattttggg aattatttc tgtcaacat cattatgat actcctcgg 288 GluAspPheGly AsnTyrPhe CysGlnHis HisTyrAsp ThrProArg acgttcggtgga gggaccaag ctggaaata aaac 322 ThrPheGlyGly GlyThrLys LeuGluIle Lys <210> ZO
<211> 107 <212> PRT
<213> Mus sp.
<400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala 5er Val Gly l 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Asn Tyr Phe Cys Gln His His Tyr Asp Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys <210> 11 <211> 276 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(276) <400> 11 gcc tcc cta tct gca tct gtg gga gaa act gtc acc atc aca tgt cga 48 A1a Ser Leu Ser Ala Ser Val Gly Glu Thr Va1 Thr Ile Thr Cys Arg gcaagtgagaat atttac agttatttagca tggtatcag cagaaa cag 96 A1aSerGluAsn IleTyr SerTyrLeuAla TrpTyrGln GlnLys Gln ggaaaatctcct caactc ctggtctataat gcaaaaacc ttaaca gaa 144 GlyLysSerPro GlnLeu LeuValTyrAsn AlaLysThr LeuThr Glu ggtgtgccatca aggttc agtggcagtgga tcaggcaca cagttt tct 192 GlyValProSer ArgPhe SerGlySerGly SerGlyThr GlnPhe Ser ctgaagatcaac agcctg cagcctgaagat tttgggaat tatttc tgt 240 LeuLysIleAsn SexLeu GlnProGluAsp PheGlyAsn TyrPhe Cys caacatcattat gatact cctcggacgttc ggtgga 276 GlnHisHisTyr AspThr ProArgThrPhe GlyGly <210> 12 <211> 92 <212> PRT
<213> Mus sp.
<400> 12 Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu A1a Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Asn Tyr Phe Cys Gln His His Tyr Asp Thr Pro Arg Thr Phe Gly Gly 5~
<210> 13 <211> 414 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(414) <400> 13 atg gga tgg agc tgt gtc atg ctc ttc ttg gta gca aca get aca ggt 48 Met Gly Trp Ser Cys Val Met Leu Phe Leu Va1 A1a Thr A1a Thr Gly gtccactcccag gtccaactg cagcagcct ggggetggg agggtgagg 96 Va1HisSerGln ValGlnLeu GlnGlnPro GlyAlaGly ArgValArg cctggagettca gtgaagctg tcctgcaag gettctggc tactccttc 144 Pro.GlyAlaSer ValLysLeu SerCysLys A1aSerGly TyrSerPhe accagttactgg atgaactgg gtgaagcag aggcctgga caaggcctt 192 ThrSerTyrTrp MetAsnTrp ValLysGln ArgProGly GlnGlyLeu gagtggattggc atgattcat ccttccgat agtgaaact aggttaaat 240 GluTrpIleGly MetIleHis ProSerAsp SerGluThr ArgLeuAsn cagaagttcaac gacagggcc acattgact gttgacaaa tattccagc 288 G1nLysPheAsn AspArgAla ThrLeuThr ValAspLys TyrSerSer acagcctatata caactcagc agcccgaca tctgaggac tctgcggtc 336 ThrAlaTyrIle GlnLeuSer SerProThr SerGluAsp SerAlaVal tattactgtgca agaggagaa tattactac ggtatattt gactactgg 384 TyrTyrCysAla ArgGlyGlu TyrTyrTyr G1yIlePhe AspTyrTrp 115 120 125 .
ggccaagggacc acggtcacc gtctcctca 414 GlyGlnGlyThr ThrValThr ValSerSer <210> 14 <211> 138 <212> PRT
<213> Mus sp.
<400> 14 Met Gly Trp Ser Cys Val Met Leu Phe Leu Va1 Ala Thr Ala Thr Gly Val His Ser Gln Va1 Gln Leu Gln Gln Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg A1a Thr Leu Thr Val Asp Lys Tyr Sex Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro ThreSer Glu Asp Ser Ala Val Tyr Tyr Cys A1a Arg Gly G1u Tyr Tyr Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser <210> 15 <211> 354 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (2)..(352) <400> 15 c ttg gta gca aca get aca ggt gtc cac tcc cag gtc caa ctg cag cag 49 Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Val Gln Leu Gln Gln cct ggg get ggg agg gtg agg cct gga get tca gtg aag ctg tcc tgc 97 Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys aag get tct ggc tac tcc ttc acc agt tac tgg atg aac tgg gtg aag 145 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Va1 Lys cag agg cct gga caa ggc ctt gag tgg att ggc atg att cat cct tcc 193 Gln Arg Pro G1y G1n Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser gat agt gaa act agg tta aat cag aag ttc aac gac agg gcc aca ttg 241 Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu act gtt gac aaa tat tcc agc aca gcc tat ata caa ctc agc agc ccg 289 Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro aca tct gag gac tct gcg gtc tat tac tgt gca aga gga gaa tat tac 337 Thr Ser Glu Asp Ser Ala Va1 Tyr Tyr Cys Ala Arg Gly Glu Tyr Tyr 7, tac ggt ata ttt gac to 354 Tyr Gly Ile Phe Asp <210> 16 <211> 117 <212> PRT
<213> Mus sp.
<400> 16 Leu Val Ala Thr Ala Thr Gly Val 'His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Va1 Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Glu Tyr Tyr Tyr Gly Tle Phe Asp <210> 17 <211> 1545 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (58)..(1545) <400> 17 atgggatgga ctacaggtgt acactcc 57 gctgtatcat cctcttcttg gtagcaacag gat atc gtg acccag tctccagcctcc ctatctgca tctgtg gga 105 ctg Asp Ile Val ThrGln SerProAlaSer LeuSerA1a SerVal Gly Leu gaa act gtc atcaca tgtcgagcaagt gagaatatt tacagt tat 153 acc Glu Thr Val IleThr CysArgAlaSer GluAsnIle TyrSer Tyr Thr tta gca tgg cagcag aaacagggaaaa tctcctcaa ctcctg gtc 201 tat Leu Ala Trp GlnGln LysGlnGlyLys SerPr-oG1n LeuLeu Val Tyr $, tat aat gca aaa acc tta aca gaa ggt gtg cca tca agg ttc agt ggc 249 Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly agt gga tca ggc aca cag ttt tct ctg aag atc aac agc ctg cag cct 297 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro gaa gat ttt ggg aat tat ttc tgt caa cat cat tat gat act cct cgg 345 Glu Asp Phe Gly Asn Tyr Phe Cys Gln His His Tyr Asp Thr Pro Arg acg ttc ggt gga ggg acc aag ctc gag atc aaa ggt ggt ggt ggt tct 393 Thr Phe Gly G1y Gly Thr Lys Leu Glu Ile Lys Gly Gly G1y Gly Ser ggc ggc ggc ggc tcc ggt ggt ggt ggt tct cag gtc caa ctg cag cag 441 Gly G1y Gly Gly Ser Gly Gly Gly Gly Ser Gln Va1 Gln Leu Gln Gln cct ggg get ggg agg gtg agg cct gga get tca gtg aag ctg tcc tgc 489 Pro Gly Ala Gly Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys aag get tct ggc tac tcc ttc acc agt tac tgg atg aac tgg gtg aag 537 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Lys cag agg cct gga caa ggc ctt gag tgg att ggc atg att cat cct tcc 585 Gln Arg Pro Gly G1n Gly Leu Glu Trp I1e Gly Met Ile His Pro Ser gat agt gaa act agg tta aat cag aag ttc aac gac agg gcc aca ttg 633 Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu act gtt gac aaa tat tcc agc aca gcc tat ata caa ctc agc agc ccg 681 Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Pro acatctgaggac tctgcggtc tattactgt gcaagagga gaatattac 729 ThrSerGluAsp SerAlaVal TyrTyrCys AlaArgGly GluTyrTyr tacggtatattt gactactgg ggccaaggg accacggtc accgtctcc 777 TyrGlyIlePhe AspTyrTrp GlyGlnGly ThrThrVal ThrValSer tccggaggtggt ggatccgat atcaaactg ca.gcagtca ggggetgaa 825 SerGlyGlyGly G1ySerAsp IleLysLeu GlnGlnSer GlyAlaGlu ctggcaagacct ggggcctca gtgaagatg tcctgcaag acttctggc 873 LeuAlaArgPro GlyAlaSer ValLysMet SerCysLys ThrSerGly tac acc ttt act agg tac acg atg cac tgg gta aaa cag agg cct gga 921 Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys G1n Arg Pro Gly 9' cagggtctggaatgg attgga tacattaatcct agccgtggt tatact 969 GlnGlyLeuGluTrp TleGly TyrIleAsnPro SerArgGly TyrThr aattacaatcagaag ttcaag gacaaggccaca ttgactaca gacaaa 1017 AsnTyrAsnGlnLys PheLys AspLysAlaThr LeuThrThr AspLys tcctccagcacagcc tacatg caactgagcagc ctgacatct gaggac 1065 SerSerSerThrAla TyrMet Gln,LeuSerSer LeuThrSer GluAsp tctgcagtctattac tgtgca agatattatgat gatcattac tgcctt 1113 SerAlaValTyrTyr CysAla ArgTyrTyrAsp AspHisTyr CysLeu gactactggcgccaa ggcacc actctcacagtc tcctcagtc gaaggt 1161 AspTyrTrpArgGln GlyThr ThrLeuThrVal SerSerVal GluGly ggaagtggaggttct ggtgga agtggaggttca ggtggagtc gacgac 1209 GlySerGlyGlySer GlyGly SerGlyGlySer GlyGlyVal AspAsp att cag ctg acc cag tct cca gca atc atg tct gca tct cca ggg gag 1257 Ile Gln Leu Thr G1n Ser Pro Ala Ile Met Ser A1a Ser Pro G1y Glu aag gtc acc atg acc tgc aga gcc agt tca agt gta agt tac atg aac 1305 Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn tgg tac cag cag aag tca ggc acc tcc ccc aaa aga tgg att tat gac 1353 Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp aca tcc aaa gtg get tct gga gtc cct tat cgc ttc agt ggc agt ggg 1401 Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly tct ggg acc tca tac tct ctc aca atc agc agc atg gag get gaa gat 1449 Ser Gly Thr Ser Tyr Ser Leu Thr I1e Ser Ser Met Glu Ala Glu Asp get gcc act tat tac tgc caa cag tgg agt agt aac ccg ctc acg ttc 1497 Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe gga get ggg acc aag ctg gag ctg aaa cat cat cac cat cat cat tag 1545 Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His His <210> 18 <211> 495 <212> PRT
<213> Mus sp.
<400> 18 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln G1y Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu G1n Pro Glu Asp Phe Gly Asn Tyr Phe Cys G1n His His Tyr Asp Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala G1y Arg Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser .Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg Ala Thr Leu Thr Val Asp Lys Tyr Ser Ser Thr Ala Tyr I1e Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys A1a Arg Gly Glu Tyr Tyr 2l0 215 220 Tyr Gly Ile Phe Asp Tyr Trp Gly Gln G1y Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala G1u Leu A1a Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Va1 Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Arg Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly G1y Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Tle Gln Leu Thr Gln Ser Pro Ala Ile Met 5er Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly 435 . 440 445 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe 11' Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His His <210> 19 <211> 24 <212> DNA
<213> Mus sp.
<400> 19 aaaggcctcc ccatattcct cgga 24 <210> 20 <211> 37 <212> DNA
<213> Mus sp.
<400> 20 aaagtcgact ccggacatct ccaaagagtt gatgtac 37 <210> 21 <211> 25 <212> DNA
<213> Mus sp.
<400> 21 aatccggagg cggcagcctg gccgc 25 <210> 22 <211> 46 <212> DNA
<213> Mus sp.
<400> 22 gggaagctta gtgatggtga tggtgatgct tcaggtcctc gcgcgg 46 <210> 23 <211> 1245 <212> DNA
<213> Mus sp.
<220>
<221> CDS
<222> (1)..(1245) <400> 23 tcc cca tat tcc tcg gac acc aca ccc tgc tgc ttt gcc tac att gcc 48 Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala cgc cca ctg ccc cgt gcc cac atc aag gag tat ttc tac acc agt ggc 96 Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly 12.
aagtgctcc aaccca gcagtcgtcttt gtcacccga aagaaccgc caa 144 LysCysSer AsnPro AlaValValPhe ValThrArg LysAsnArg Gln gtgtgtgcc aaccca gagaagaaatgg gttcgggag tacatcaac tct 192 ValCysAla AsnPro GluLysLysTrp ValArgGlu TyrIleAsn Ser ttggagatg tccgga ggcggcagcctg gccgcgctg accgcgcac cag 240 LeuGluMet SerG1y GlyGlySerLeu A1aAlaLeu ThrAlaHis Gln gettgccac ctgccg ctggagactttc acccgtcat cgccagccg cgc 288 AlaCysHis LeuPro LeuGluThrPhe ThrArgHis ArgGlnPro Arg ggctgggaa caactg gagcagtgcggc tatccggtg cagcggctg gtc 336 GlyTrpG1u GlnLeu GluGlnCysGly TyrProVal GlnArgLeu Val gccctctac ctggcg gcgcggctgtcg tgg'aaccag gtcgaccag gtg 384 A1aLeuTyr LeuAla AlaArgLeuSer TrpAsnGln ValAspGln Val atccgcaac gccctg gccagccccggc agcggcggc gacctgggc gaa 432 IleArgAsn AlaLeu AlaSerProGly SerGlyGly AspLeuGly Glu gcgatccgc gagcag ccggagcaggcc cgtctggcc ctgaccctg gcc 480 AlaIleArg GluGln ProGluG1nAla ArgLeuAla LeuThrLeu Ala gccgccgag agcgag cgcttcgtccgg cagggcacc ggcaacgac gag 528 AlaAlaGlu SerGlu ArgPheValArg GlnG1yThr GlyAsnAsp Glu gccggcgcg gccaac ggcccggcggac agcggcgac gccctgctg gag 576 AlaG1yAla AlaAsn GlyProAlaAsp SerGlyAsp AlaLeuLeu Glu cgcaactat cccact ggcgcggagttc ctcggcgac ggcggcgac gtc 624 ArgAsnTyr ProThr GlyAlaG1uPhe LeuGlyAsp GlyGlyAsp Va1 agcttcagc acccgc ggcacgcagaac tggacggtg gagcggctg ctc 672 SerPheSer ThrArg GlyThrGlnAsn TrpThrVal GluArgLeu Leu 210. 215 220 caggcgcac cgccaa ctggaggagcgc ggctatgtg ttcgtcggc tac 720 GlnAlaHis ArgGln LeuGluGluArg GlyTyrVal PheValGly Tyr cacggcacc ttcctc gaagcggcgcaa agcatcgtc ttcggcggg gtg 768 HisGlyThr PheLeu GluAlaAlaGln SerIleVal PheGlyGly Val cgcgcgcgc agccag gacctcgacgcg atctggcgc,ggtttctat atc 816 ArgAlaArg SerGln AspLeuAspAla IleTrpArg GlyPheTyr Ile 260 ~ 265 270 gccggc gatccggcg ctggcc tacggctacgcc caggaccag gaaccc 864 AlaGly AspProAla LeuAla TyrG1yTyrAla GlnAspGln GluPro gacgca cgcggccgg atccgc aacggtgccctg ctgcgggtc tatgtg 912 AspAla ArgGlyArg IleArg AsnGlyAlaLeu LeuArgVal TyrVal ccgcgc tcgagcctg ccgggc ttctaccgcacc agcctgacc ctggcc 960 ProArg SerSerLeu ProGly PheTyrArgThr SerLeuThr LeuAla gcgccg gaggcggcg ggcgag gtcgaacggctg atcggccat ccgctg 1008 AlaPro GluAlaAla GlyG1u Va1GluArgLeu IleGlyHis ProLeu ccgctg cgcctggac gccatc accggccccgag gaggaaggc gggcgc 1056 ProLeu ArgLeuAsp AlaIle ThrGlyProGlu GluGluGly GlyArg ctggag accattctc ggctgg ccgctggccgag cgcaccgtg gtgatt 1104 LeuGlu ThrIleLeu GlyTrp ProLeuAlaGlu ArgThrVal ValIle ccctcg gcgatcccc accgac ccgcgcaacgtc ggcggcgac ctcgac 1152 ProSer AlaIlePro ThrAsp ProArgAsnVal GlyGlyAsp LeuAsp CCgtCC agCatCCCC gacaag gaacaggcgatc agcgccctg ccggac 1200 ProSer SerIlePro AspLys GluG1nAlaIle SerAlaLeu ProAsp tacgcc agccagccc ggcaaa ccgccgcgcgag gacctgaag taa 1245 TyrAla SerGlnPro GlyLys ProProArgGlu AspLeuLys <210>
<21l> 4 <212>
PRT
<213>
Mus sp.
<400> 24 Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg G1n Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu Glu Met Ser G1y Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly G1-y Asp Leu Gly Glu 14' Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala A1a Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp G1u Ala Gly Ala A1a Asn Gly Pro Ala Asp 5er Gly Asp Ala Leu Leu Glu 180 l85 190 Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val G1y Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser G1n Asp Leu Asp Ala Ile Trp Arg G1y Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Tle Thr Gly Pro Glu Glu Glu G1y Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val I1e Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys <210>
<2l1>
<212>
DNA
<213> .
Mus sp.
<220>.
<221>
CDS
<222> (363) (1)..
<400>
gat atc ctg cagcagtca ggggetgaactg gcaagacct ggggcc 48 aaa Asp Ile Leu GlnGlnSer GlyAlaGluLeu AlaArgPro GlyAla Lys tca gtg atg tcctgcaag acttctggctac acctttact aggtac 96 aag Ser Val Met SerCysLys ThrSerGlyTyr ThrPheThr ArgTyr Lys acg atg tgg gtaaaacag aggcctggacag ggtctggaa tggatt 144 cac Thr Met Trp ValLysGln ArgProGlyGln GlyLeuGlu TrpIle His 15.
ggatacattaat cctagccgt ggttatact aattacaat cagaagttc 192 GlyTyrIleAsn ProSerArg GlyTyrThr AsnTyrAsn GlnLysPhe aaggacaaggcc acattgact acagacaaa tcctccagc acagcctac 240 LysAspLysAla ThrLeuThr ThrAspLys SerSerSer ThrAlaTyr atgcaactgagc agcctgaca tctgaggac tctgcagtc tattactgt 288 MetGlnLeuSer SerLeuThr 5erGluAsp SerAlaVal TyrTyrCys gcaagatattat gatgatcat tactgcctt gactactgg cgccaaggc 336 AlaArgTyrTyr AspAspHis TyrCysLeu AspTyrTrp ArgGlnGly accactctcaca gtctcctca gtcgaa 363 ThrThrLeuThr ValSerSer ValG1u <210>
<211>
<212>
PRT
<213>
Mus sp.
<400>
Asp Ile Leu GlnGlnSer G1yAlaGlu LeuAlaArg ProGlyAla Lys Ser Val Met SerCysLys ThrSerGly TyrThrPhe ThrArgTyr Lys Thr Met Trp ValLysGln ArgProGly GlnGlyLeu GluTrpIle His Gly Tyr Asn ProSerArg GlyTyrThr AsnTyrAsn GlnLysPhe Tle Lys Asp Ala .ThrLeuThr ThrAspLys SerSerSer ThrAlaTyr Lys Met Gln Ser SerLeuThr SerGluAsp SerAlaVa1 TyrTyrCys Leu Ala Arg Tyr AspAspHis TyrCysLeu AspTyrTrp ArgGlnGly Tyr Thr Thr Thr ValSerSer ValGlu Leu <210> 27 <211> 324 <212> DNA
<213> Mus sp.
<220>
<221> CDS
16~
<222> (1)..(324) <400> 27 gtc gac gac att cag ctg acc cag tct cca gca atc atg tct gca tct 48 Val Asp Asp Ile Gln Leu Thr Gln Ser Pro A1a Ile Met Ser Ala Ser cca ggg gag aag gtc acc atg acc tgc aga gcc agt tca agt gta agt 96 Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser tac atg aac tgg tac cag cag aag tca ggc acc tcc ccc aaa aga tgg 144 Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp att tat gac aca tcc aaa gtg get tct gga gtc cct tat cgc ttc agt 192 Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser ggc agt ggg tct ggg acc tca tac tct ctc aca atc agc agc atg gag 240 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu get gaa gat get gcc act tat tac tgc caa cag tgg agt agt aac ccg 288 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro ctc acg ttc gga get ggg acc aag ctg gag ctg aaa 324 Leu Thr Phe Gly Ala Gly Thr Lys Leu G1u Leu Lys <210> 28 <211> 108 <212> PRT
<213> Mus sp.
<400> 28 Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met.Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala G1y Thr Lys Leu Glu Leu Lys 17, <210> 29 <211> 11 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 29 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Zeu A1a <2l0> 30 <211> 7 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 30 Asn Ala Lys Thr veu Thr Glu <210> 31 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 31 Gln His His Tyr Asp Thr Pro Arg Thr <210> 32 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial. Sequence: peptide <400> 32 Tyr Trp Met Asn <210> 33 <211> l9 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 33 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Asn Asp Arg <210> 34 <2l1> 10 <212> PRT
<2l3> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 34 Gly Glu Tyr Tyr Tyr Gly Ile Phe Asp Tyr
Claims (50)
1. Use of an antibody and/or chemokine construct which binds to a chemokine re-ceptor for the preparation of a pharmaceutical composition for the elimination of cells which are latently infected with a primate immunodeficiency virus.
2. The use of claim 1, wherein said primate immunodeficiency virus is a human immunodeficiency virus.
3. The use of claim 2, wherein said human immunodeficiency virus is HIV-1.
4. Use of an antibody and/or chemokine construct which binds to a chemokine re-ceptor for the preparation of a pharmaceutical composition for the treatment, prevention and/or alleviation of inflammatory renal diseases, allergic reactions, inflammatory bowel diseases, multiple sclerosis, skin diseases, diabetes or transplant rejection.
5. The use of any one of claims 1 to 4, wherein said chemokine receptor is the chemokine receptor 5 (CCR5).
6. The use of claim 5, wherein said chemokine receptor 5 is the human CCR5.
7. The use of any one of claims 1 to 6, wherein said antibody construct is a bispecific antibody which binds to the chemokine receptor as a first antigen and a CD3 antigen of an effector cell as a second antigen.
8. The use of claim 7, wherein said bispecific antibody is a single chain antibody construct.
9. The use of claim 8, wherein said single chain antibody construct comprises V L
and V H domains of a antibody specific for the chemokine receptor and V H and V L
domains of an antibody specific for a CD3 antigen.
and V H domains of a antibody specific for the chemokine receptor and V H and V L
domains of an antibody specific for a CD3 antigen.
10. The use of claim 9, wherein said antibody specific for the chemokine receptor is the murine anti-human CCR5 antibody MC-1.
11. The use of claim 9 or 10, wherein said V L and V H domains are arranged in the order V L(MC-1)-V H(MC-1)-V H(CD3)-V L(CD3).
12. The use of claim 11, wherein said V L(MC-1) comprises the amino acid sequence as depicted in SEQ ID NO: 12, wherein said V H(MC-1) comprises the amino acid sequence as depicted in SEQ ID NO: 16, wherein said V H(CD3) comprises the amino acid sequence as depicted in SEQ ID NO: 26 and/or wherein said V L(CD3) comprises in SEQ ID NO: 28.
13. The use of any one of claims 5 to 12, wherein said bispecific antibody comprises an amino acid sequence encoded by the nucleic acid sequence as depicted in SEQ ID NO: 17 or comprises the amino acid sequence as depicted in SEQ ID
NO: 18.
NO: 18.
14. The use of any one of claims 1 to 6, wherein said antibody construct is a bispecific antibody which binds to said chemokine receptor as a first antigen and a toxin as a second antigen.
15. The use of any one of claims 1 to 6, wherein said antibody construct is cova-lently bound to a toxin.
16. The use of any one of claims 1 to 6, wherein said antibody construct can, via a multimerization domain, be bound in vitro and/or in vivo to a second antibody construct which binds to a CD3 antigen and/or a toxin.
17. The use of any one of claims 1 to 6, wherein said chemokine construct is a fu-sion construct of a modified or an unmodified chemokine with a modified or an unmodified toxin.
18. The use of any one of claims 1 to 6, wherein said chemokine construct can, via a multimerization domain, be bound in vitro and/or in vivo to an antibody construct which binds to a CD3 antigen and/or to a toxin.
19. The use of any one of claims 1 to 6, wherein said chemokine construct com-prises a chemokine covalently bound to an antibody construct which binds to an antibody construct which binds to a CD3 antigen and/or which is a covalently bound to a toxin.
20. The use of any one of claims 1 to 6, wherein said antibody and/or chemokine construct is a heterominibody construct comprising at least an antibody and/or a chemokine which binds to a chemokine receptor.
21. The use of claim 20, wherein said heterominibody construct comprises at least one toxin.
22. The use of claim 20 or 21, wherein said heterominibody construct binds to the chemokine receptor and/or to a CD3 antigen of an effector cell.
23. The use of any one of claims 17 to 22, wherein said chemokine is selected from the group consisting of RANTES, MIP-1.beta., MIP-1.alpha., MCP-2 and MCP-3..
24. The use of any one of claims 15 to 19, 21 and 22, wherein said toxin is a trun-cated Pseudomonas exotoxin A.
25. The use of any one of claims 17 to 24 wherein said chemokine construct com-prises a amino acid sequence as depicted in SEQ ID NO: 24 or as encoded by the nucleotide sequence as depicted in SEQ ID NO: 23.
26. The use of any one of claims 7 to 13, 16, 18, 19 and 22, wherein said CD3 anti-gen is on the surface of an effector cell which is a T-cell.
27. An antibody construct as defined in any one of claims 5 to 16, 20, 21 and 22, wherein said construct comprises a binding site for CCR5 and a binding site for CD3.
28. A chemokine construct as defined in any one of claims 17 to 22, wherein said chemokine construct comprises RANTES and said toxin is a truncated Pseu-domonas exotoxin A (PE38).
29. A polynucleotide encoding an antibody-construct as defined in any one of claims to 12 or a chemokine construct as defined in claims any one of claims 17 to 19, 23 and 24, wherein said polynucleotide is (a) a polynucleotide comprising the nucleic acid molecule in particular en-coding the polypeptide as depicted in SEQ ID NO: 18 or SEQ ID NO: 24;
(b) a polynucleotide comprising the nucleic acid molecule as depicted in SEQ
ID NO: 17 or SEQ ID NO: 23; or (c) a polynucleotide hybridizing under stringent conditions to the comple-mentary strand of a polynucleotide of (a) or (b).
(b) a polynucleotide comprising the nucleic acid molecule as depicted in SEQ
ID NO: 17 or SEQ ID NO: 23; or (c) a polynucleotide hybridizing under stringent conditions to the comple-mentary strand of a polynucleotide of (a) or (b).
30. The polynucleotide of claim 29 which is DNA or RNA.
31. A vector comprising the polynucleotide of claim 29 or 30.
32. The vector of claim 31 which is an expression vector or a gene transfer vector.
33. A host transformed with the vector of claim 31 or 32.
34. A method of producing the antibody construct or the chemokine construct as de-fined in claim 29 comprising culturing the host of claim 33 and isolating the pro-duced antibody construct or chemokine construct.
35. The antibody construct or the chemokine construct encoded by the polynucleo-tide of claim 29 or 30 or produced by the method of claim 34.
36. A composition comprising the polynucleotide of claim 29, the vector of claims 31 or 32, the host of claim 33, the antibody construct of claim 27 or 35 and/or the chemokine construct of claim 28 or 35.
37. The composition of claim 36 which is a pharmaceutical composition further com-prising, optionally, a pharmaceutically acceptable carrier, diluent and/or excipi-ent.
38. The composition of claim 36 or 37 which further comprises a medicament for the treatment of an immunological disorder or a medicament for anti-HIV treatment.
39. A method for treating, preventing and/or alleviating an immunological disorder or for the elimination of cells which are latently infected with a primate immunodefi-ciency virus comprising administering to a subject in need of such a treatment, alleviation and/or prevention an effective amount of the composition of any one of claims 36 to 38.
40. Use of the polynucleotide of claim 29 or 30, the vector of claim 31 or 32, the host of claim 33, the antibody construct of claims 27 or 35 and/or the chemokine con-struct of claim 28 or 38 for the preparation of a pharmaceutical composition for treating, preventing and/or alleviating an immunological disorder.
41. Use of the polynucleotide of claim 29 or 30, the vector of claim 31 or 32, the host of claim 33, the antibody construct of claims 27 or 35 and/or the chemokine con-struct of claim 28 or 38 for the preparation of a pharmaceutical composition for eliminating latently infected cells, wherein said cells are infected with a primate immunodeficiency virus.
42. The method of claim 39 or the use of claim 40, wherein said immunological dis-order is selected from the group consisting of autoimmune diseases, allergic dis-eases, skin diseases, inflammatory diseases, diabetes, graft versus host disease and transplant rejections.
43. The method or the use of claim 42, wherein said autoimmune disease is se-lected from the group consisting of multiple sclerosis, type I diabetes, and rheu-matoid arthritis.
44. The method or the use of claim 42, wherein said skin disease is selected from the group consisting of skin inflammation, atopic dermatitis and psoriasis.
45. The method or the use of claim 42, wherein said inflammatory disease is se-lected from the group consisting of inflammatory joint diseases, inflammatory re-nal diseases, inflammatory bowel diseases.
46. The method or the use of claim 45, wherein said inflammatory joint disease is (chronic) arthritis.
47. The method of claim 39 or the use of claim 41, wherein said infection with a pri-mate immunodeficiency virus is a HIV-1 infection.
48. The method or the use of claim 47, wherein said composition is to be adminis-tered in combination with antiviral agents and/or in combination with drugs to be employed in AIDS management.
49. The method or the use of claims 47, wherein said drugs to be employed in AIDS
management comprises drugs employed in HAART.
management comprises drugs employed in HAART.
50. A kit comprising the polynucleotide of claim 29 or 30, the vector of claim 31 or 32, the host of claim 33, the antibody construct of claims 27 or 35 and/or the chemokine construct of claim 28 or 38.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00119694 | 2000-09-08 | ||
EP00119694.8 | 2000-09-08 | ||
US09/948,004 US6723538B2 (en) | 1999-03-11 | 2001-09-05 | Bispecific antibody and chemokine receptor constructs |
US09/948,004 | 2001-09-05 | ||
PCT/EP2001/010433 WO2002020615A2 (en) | 2000-09-08 | 2001-09-10 | Antibody and/or chemokine constructs which bind to a chemokine receptor and their use in immunological disorders |
Publications (1)
Publication Number | Publication Date |
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CA2421842A1 true CA2421842A1 (en) | 2002-03-14 |
Family
ID=32842663
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002421842A Abandoned CA2421842A1 (en) | 2000-09-08 | 2001-09-10 | Antibody and/or chemokine constructs which bind to a chemokine receptor and their use in immunological disorders |
Country Status (10)
Country | Link |
---|---|
EP (1) | EP1317284A2 (en) |
JP (1) | JP2004508036A (en) |
AU (1) | AU2002212225A1 (en) |
CA (1) | CA2421842A1 (en) |
HU (1) | HUP0300942A2 (en) |
NO (1) | NO20031070L (en) |
PL (1) | PL366062A1 (en) |
RU (1) | RU2252786C2 (en) |
WO (1) | WO2002020615A2 (en) |
ZA (1) | ZA200301890B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4090348A1 (en) * | 2020-01-13 | 2022-11-23 | The Regents of The University of California | Methods for treating viral infections |
Families Citing this family (15)
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AU2002360073A1 (en) * | 2001-12-21 | 2003-07-09 | Micromet Ag | Mono-and dual chemokine/cytokine constructs |
EP2390268B1 (en) | 2002-11-08 | 2017-11-01 | Ablynx N.V. | Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor |
US8795674B2 (en) | 2005-09-30 | 2014-08-05 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Methods and compositions for modulating immune tolerance |
PT2292663E (en) | 2006-08-28 | 2013-11-04 | Kyowa Hakko Kirin Co Ltd | Antagonistic human light-specific human monoclonal antibodies |
AR065369A1 (en) | 2007-02-19 | 2009-06-03 | Novartis Ag | CICLOHEXIL DERIVATIVES - AMIDA OF ARIL- CARBOXILIC ACID |
WO2009032661A1 (en) | 2007-08-29 | 2009-03-12 | Sanofi-Aventis | Humanized anti-cxcr5 antibodies, derivatives thereof and their uses |
EP2585089B1 (en) * | 2010-06-28 | 2016-10-12 | Universitätsklinikum Freiburg | Blockade of ccl18 signaling via ccr6 as a therapeutic option in fibrotic diseases and cancer |
TWI638833B (en) | 2010-11-30 | 2018-10-21 | 中外製藥股份有限公司 | Cell damage induction treatment |
US9592289B2 (en) | 2012-03-26 | 2017-03-14 | Sanofi | Stable IgG4 based binding agent formulations |
KR102568808B1 (en) | 2014-04-07 | 2023-08-18 | 추가이 세이야쿠 가부시키가이샤 | Immunoactivating antigen-binding molecule |
JP6894702B2 (en) * | 2014-05-13 | 2021-06-30 | 中外製薬株式会社 | T cell redirection antigen-binding molecule for cells with immunosuppressive function |
JP6931329B2 (en) | 2015-11-18 | 2021-09-01 | 中外製薬株式会社 | Combination therapy using T cell redirection antigen-binding molecule for cells with immunosuppressive function |
WO2017086419A1 (en) | 2015-11-18 | 2017-05-26 | 中外製薬株式会社 | Method for enhancing humoral immune response |
WO2017210749A1 (en) * | 2016-06-10 | 2017-12-14 | Adelaide Research & Innovation Pty Ltd | Methods and products for treating autoimmune diseases |
CN109970856B (en) | 2017-12-27 | 2022-08-23 | 信达生物制药(苏州)有限公司 | anti-LAG-3 antibodies and uses thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE69910216T2 (en) * | 1998-07-22 | 2004-02-19 | Osprey Pharmaceuticals Ltd., Calgary | CONJUGATES FOR TREATING INFLAMMATORY DISEASES AND ASSOCIATED TISSUE DAMAGE |
WO2000053633A2 (en) * | 1999-03-11 | 2000-09-14 | Micromet Ag | Antibody and chemokine constructs that are directed to ccr5, and their use for treating autoimmune diseases |
-
2001
- 2001-09-10 CA CA002421842A patent/CA2421842A1/en not_active Abandoned
- 2001-09-10 AU AU2002212225A patent/AU2002212225A1/en not_active Abandoned
- 2001-09-10 EP EP01980364A patent/EP1317284A2/en not_active Ceased
- 2001-09-10 JP JP2002525234A patent/JP2004508036A/en not_active Withdrawn
- 2001-09-10 WO PCT/EP2001/010433 patent/WO2002020615A2/en active Application Filing
- 2001-09-10 PL PL01366062A patent/PL366062A1/en not_active Application Discontinuation
- 2001-09-10 RU RU2003108885/15A patent/RU2252786C2/en not_active IP Right Cessation
- 2001-09-10 HU HU0300942A patent/HUP0300942A2/en unknown
-
2003
- 2003-03-07 ZA ZA200301890A patent/ZA200301890B/en unknown
- 2003-03-07 NO NO20031070A patent/NO20031070L/en not_active Application Discontinuation
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4090348A1 (en) * | 2020-01-13 | 2022-11-23 | The Regents of The University of California | Methods for treating viral infections |
EP4090348A4 (en) * | 2020-01-13 | 2024-05-15 | Univ California | Methods for treating viral infections |
Also Published As
Publication number | Publication date |
---|---|
WO2002020615A2 (en) | 2002-03-14 |
RU2252786C2 (en) | 2005-05-27 |
HUP0300942A2 (en) | 2003-09-29 |
EP1317284A2 (en) | 2003-06-11 |
PL366062A1 (en) | 2005-01-24 |
WO2002020615A3 (en) | 2002-07-25 |
ZA200301890B (en) | 2004-03-16 |
JP2004508036A (en) | 2004-03-18 |
NO20031070L (en) | 2003-05-06 |
AU2002212225A1 (en) | 2002-03-22 |
NO20031070D0 (en) | 2003-03-07 |
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