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Definitions

• the invention relates to a novel co-stimulatory protein selectively expressed on the surface of dendritic cells, to its use in a method for the identification of immunomodulatory substances, to functional derivatives thereof, to agents interfering with the respective costi- mulatory pathway, and to uses of said derivatives and interfering agents.
• T lymphocyte response is a complex process involving cell-cell interactions and production of soluble mediators (cytokines or lymphokines).
• cytokines or lymphokines soluble mediators
• Optimal activation of T lymphocytes is believed to require two cell-cell interaction signals: an antigen specific or clonal T cell receptor (TCR) signaling upon binding to the peptide-MHC on the antigen presenting cells (APCs), as well as a second, antigen non-specific "costimulatory" signal.
• TCR antigen specific or clonal T cell receptor
• T cell encounters an antigen alone, without co-stimulation by costimulatory molecules, no significant amplification of an immune response against a given antigen occurs. Moreover, without co-stimulation, TCR engagement not only results in a failure to induce an immune response but leads to functional T-cell inactivation by either T cell anergy or apopto- sis, resulting in tolerance. If the co-stimulatory signal is provided, the T cell will respond with clonal expansion specific for the stimulating antigen.
• APCs that process and present the .antigen to T cells
• density of the peptide antigen/MHC ligand available for engagement of the TCR and the provision of soluble and/or membrane-bound costimulatory signals by APCs at the time of T cell engagement and activation.
• APCs that provide the signals required for activation of T cells include rnonocytes/macrophages, B lymphocytes, aid dendritic cells (DCs).
• DCs are considered as the most potent initiators of antigen-specific T cell responses in vivo.
• DCs have a distinct phenotype from acti- vated macrophages and are classified into different subtypes capable of initiating distinct immune responses. In vitro they show an approximately 100-fold greater potency than macrophages to activate naive T cells.
• B7 A typical and the best characterized example for costimulatory molecules expressed on APCs such as DCs are the members of the so-called B7 family.
• This family includes B7, also known as B7-1 or CD80 and B7-2, also called CD86.
• B7 are members of the im- munoglobulin (Ig) superfamily and comprise two extracellular Ig domains, an N-terminal variable (V)-like domain followed by a constant (C)-like domain.
• the ligands or counter- receptors of B7, expressed on the surface of T cells, are CD28 and CTLA-4.
• CD28 is a homodimeric glycoprotein of the Ig superfamily found on most mature human T cells that functions in T cell activation, is constitutively expressed on resting T cells and increases after activation.
• CTLA4 is a T cell surface molecule highly homolo- gous to CD28 but is not expressed on resting T cells and appears following T cell activation.
• TNF tumor necrosis factor
• TNF receptor family mem- bers including CD40-CD154, CD30-CD30L, CD27-CD70, 4-1BB-4-1BBL, RANK-
• RANKL OPGL
• Ox40-Ox40L have also been demonstrated to be involved in T-cell costimulation through T cell-DC interactions.
• the framework of costimulatory molecules which determine the qualitative and quantitative T-cell responses have not yet been fully elucidated.
• T cell activation remains a highly complex field and, therefore, T cell function abnormalities can until now only be addressed very insufficiently by any therapeutic interventions.
• a central function of the immune system is to distinguish foreign antigens, such as infectious agents, from self components of body tissues.
• the immune system normally acquires self tolerance (unresponsiveness to self) by clonal deletion of autoreactive T cells in the thymus in the perinatal period and by functional suppression of autoreactive T and B cells at later stages of development. Nevertheless, sometimes there is a failure in the maintenance of self tolerance, a failure to discriminate between self and non-self antigens, and an autoimmune response, characterized by the activation and clonal expansion of autoreactive lymphocytes and the production of autoantibodies, is produced against autologous antigens of normal body tissues. Although many autoimmune diseases are associated with autoantibodies and thus with B cells, T-cells may play an important role also in these pathological conditions as they can act on B-cell development and function.
• Autoimmune diseases are multifactorial in origin .and can be classified into organ-specific and nonorgan-specific (or systemic) autoimmune diseases.
• Clinical examples include: autoimmune hemolytic anemia, Hashimoto's thyroiditis, myasthenia gravis, Grave's disease, Goodpasture's syndrome, Crohn's disease, Guillain-Barre syndrome, psoriasis, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, uveitis, type I (insulin-dependent) diabetes, rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus (SLE), and multiple sclerosis.
• T helper cells type 1 TH1 cells
• TH2 cell T helper cell type 2
• Immunodeficiencies may be primary or secondary. Primary immunodeficiency is classified into four main groups depending on which component of the immune system is deficient: B cells, T cells, phagocytic cells, or complement. Over 70 primary immunodeficiencies have been described. T cell deficiencies are e.g. DiGeorge anoma- ly, chronic mucocutaneous candidiasis, Nezelof syndrome, natural killer cell deficiency, and idiopathic CD4 lymphocytopenia. T cell as well as B cell reactions are hampered in severe combined immunodeficiency (SCID) which is probably the most important form of primary immunodeficiencies in terms of patient numbers.
• SCID severe combined immunodeficiency
• immunodeficiency The - considerably more common - secondary immunodeficiency is an impairment of the immune system resulting from an illness in a previously normal person.
• the of course most important disease of this class is AIDS and AIDS related diseases.
• Other infectious diseases that may implicate immunodeficiencies are cytomegalovirus infection, infectious mononucleosis, acute bacterial disease, and severe mycobacterial or fungal disease.
• immunosuppressive agents such as radiation and immunosuppressive drugs.
• a medicament useful for the treatment of immunosuppressed conditions might also serve to stimulate the T cell response of a healthy individual.
• APCs is an important target for novel therapeutical, especially pharmacological approaches in the treatment of autoimmune dieseases, primary and secondary immunodeficiencies, allergies and asthma.
• the object of the present invention is therefore to provide novel means and methods for the modulation of T cell activation.
• a first embodiment of the invention which provides an agent which modulates T cell - APC interaction, said agent being an isolated antibody or antibo- dy derivative that selectively recognizes and binds to mammalian Sema4A protein.
• the antibody or antibody derivative binds to human Sema4A protein.
• an agent which modulates T cell - APC interaction said agent being an isolated antibody or antibody derivative that selectively recognizes and binds to mammalian Tim-2 protein.
• the antibody or antibody derivative binds to human Tim-2 protein.
• the two aforementioned embodiments have in common that said antibodies or antibody derivatives inhibit the costimulatory effect of Sema4A protein on T cell activation.
• composition comprising an antibody or antibody derivative binding to Sema4A protein or Tim-2 as described above and an excipient, an adjuvant, a diluent, and/or a carrier.
• Also encompassed by the present invention are (i) the use of an antibody or antibody derivative binding to Sema4A protein or Tim-2 as described above for the preparation of a medicament for the treatment of a disease selected from autoimmune diseases, allergies and asthma, and (ii) methods for the treatment of a disease selected from autoimmune diseases, allergies and asthma comprising administering to a patient in need thereof a thera-plastically effective amount of an antibody or antibody derivative as described above.
• an isolated and purified protein the sequence of which consists of SEQ ID NO: 1. Furthermore, there are provided proteins having an amino acid sequence at least 95 % identical, preferably at least 98 % identical to SEQ TD NO:l, wherein said proteins have the biological function of acting as costimulatory molecules and wherein this biological function can be tested using the assays described in detail below.
• SEQ ID NO:l human Sema4A protein according to the invention
• a human protein named TANGO 265 and having an amino acid sequence (SEQ ID NO:2) similar to the above has previously been described (DERWENT ® geneseq database, acces- sion no. AAB66043).
• the degree of identity between SEQ ID NO: 1 and SEQ ID NO:2 is 94 % (Scoring matrix: BLOSUM62).
• No clear indication of the biological function of TANGO 265 protein has been given in the prior art. It may represent an allelic form or a splice variant of the now discovered human Sema4A protein.
• SEQ ID NO:2 (human TANGO 265 protein):
• an isolated soluble mammalian Sema4A protein derivative comprising at least the extracellular sema domain of Sema4A protein and lacking at least a portion of the transmembrane domain of Sema4A protein.
• a Sema 4 A fusion protein comprising all or parts of a mammalian Sema4A protein fused to .another protein or protein domain.
• a fusion protein having the sequence SEQ ID NO:3 or a fusion protein essentially as shown in SEQ ID NO:3 but comprising the human counterparts of the respective protein components.
• SEQ ID NO:3 (mSema4A-Fc): , MALPSLGQDS WSLLRVFFFQ LFLLPSLPPA SGTGGQGPMP RVKYHAGDGH RALSFFQQKG
• a pharmaceutical composition that, when administered to a subject, stimulates T cell mediated immune responses in said subject, wherein said pharmaceutical composition comprises (i) a pharmaceutically active component selected from the group consisting of the Sema4A protein according to SEQ ID NO:l, a functional fragment or derivative thereof, such as a soluble mammalian Sema4A protein derivative as outlined above or a mammalian Sema4A fusion protein as described above, and (ii) one or more components selected from the group consisting of excipients, adjuvants, diluents and carriers.
• the pharmaceutical composition shows the physiological effect of stimulating T cell mediated immune responses.
• This effect can be assessed e.g. by an assay comprising the steps (i) stimulating naive CD4+ T cells with immobilized anti-CD3 antibodies and anti-CD28 antibodies in the presence or absence of said pharmaceutically active component and (ii) measuring the activation of the thus treated T cells by measuring T cell proliferation or IL-2 secretion. If in the presence of the compound T cell proliferation or IL-2 secretion is increased as compared to assays in which said compound is not included, this compound is classified in the context of this invention as compound having the biological effect of stimulating T cell mediated immune responses.
• a pharmaceutically active substance selected from the group consisting of the Sema4A protein according to SEQ ID NO:l, a functional fragment or derivative thereof, especially a soluble mammalian Sema4A protein derivative as outlined above and a mammalian Sema4A fusion protein as described above, wherein said pharmaceutically active substance has the biological effect of stimulating T cell mediated immune responses, for the preparation of a medicament for the treatment of primary or secondary immunodeficiencies or for the stimulation of normal T cell responses, as well as (ii) a method for the treatment of a disease selected from primary or secon- dary immunodeficiencies or for the stimulation of T cell responses comprising administering to a patient in need thereof a therapeutically effective amount of a substance selected from the aforementioned group.
• a further embodiment of the invention is represented by a mammalian Sema4A protein derivative reactive with Tim-2 antigen present on the surface of T cells.
• a method of identifying a compound capable of modulating T cell mediated immune responses in a mammal including man comprising the steps (i) preparing a candidate compound, (ii) contacting a T cell expressing a Sema4A receptor on its surface with said candidate compound, (iii) contacting said T cell with a Sema4A agent under conditions suitable to activate said T cell and (iv) detemiining if said candidate compound modulates the activation of said T cell, wherein said Sema4A agent is selected from the group consisting of a mammalian Sema4A protein, the human Sema4A protein as given by SEQ ID NO:l, a functional fragment or derivative thereof, a soluble mammalian Sema4A protein derivative as outlined above, a mammalian Sema4A fusion protein as described above, and a cell expressing Sema4A protein or a Sema4A protein derivative on its surface, wherein
• the modulation of the activation of said T cell is preferably determined by measuring T cell proliferation or the secretion of a cytokine, e.g. interleukin-2, interferon- gamma, and interleukin-4, by T cells into the culture medium.
• a cytokine e.g. interleukin-2, interferon- gamma, and interleukin-4
• bone marrow-derived dendritic cells are used as the Sema4A agent.
• T cells are contacted with an anti-CD3 antibody and optionally an anti-CD28 antibody in order to create conditions suitable to activate T cells.
• the T cells expressing a Sema4A receptor on their surface are preferably CD4+ T cells prepared from splenocytes.
• a Sema4A fusion protein and more preferably the Sema4A-Fc fusion protein of SEQ ID NO:3 is used in the above method.
• a functional human Sema4A protein or protein derivative is especially pre- ferred.
• the active subst.ances modulate the T cell mediated immune response by interacting with the DC - T cell costimulatory pathway.
• substances selected from the group consisting of anti-Sema4A and anti-Tim-2 antibodies or antibody derivatives, mammalian Sema4A proteins, the human Sema4A protein as given by SEQ ID NO: 1 , a functional fragment or derivative thereof, a soluble mammalian Sema4A protein derivative as outlined above, and compound identified by a method as described in the aforementioned embodiments, are used for the investigation of T cell costimulatory pathways or for the preparation of a medicament for the treatment of diseases linked to T cell activation abnormalities.
• T cell responses can be modulated in vitro .and in vivo by administering to the T cells a substance selected from the above group.
• Figure 1 shows the sequence alignment of human (upper line; SEQ ID NO:l) and mouse (bottom line; SEQ ID NO:4) Sema4A.
• the middle line indicates identical amino acid residues.
• Predicted signal sequence small-dashed line
• Sema domain solid line
• Ig-like domain large-dashed line
• transmembrane region bold line
• Figure 2 shows the results of the experiment of Example 5.
• Treatment with anti-Sema4A .antibodies blocks the development of experimental autoimmune encephalomyelitis (EAE).
• A EAE clinical disease course in mice treated with anti-Sema4A (open circles) or control ratlgGs (closed circles).
• B In vitro responses (proliferation, IL-4 and IFN- ⁇ production) of CD4+ T cells stimulated with myelin oligodendrocyte glycoprotein (MOG)-peptide.
• EAE experimental autoimmune encephalomyelitis
• the semaphorin family includes a large number of phylogenetically conserved proteins and comprises secreted and transmembrane proteins carrying a large "sema domain" (approximately 500 amino acid residues) in their extracellular regions.
• Many semaphorins of the secreted-type have been shown to be involved in axon guidance acting as chemorepulsion factors or delivering guidance cues to migrating axons during neuronal development.
• semaphorins are also crucially involved in T-cell costimulation including pathogenic immune reactions.
• Sema 4A is preferentially expressed on the cell surface of DCs and has a potent costimulatory activity including in vitro T cell activation and in vivo generation of antigen-specific T cells. Furthermore, it could be demonstrated that Sema4A is an important target for the treatment of autoimmune diseases: Administration of anti- Sema4A antibodies effectively inhibits the development of experimental autoimmune en- cephalomyelitis (EAE).
• EAE experimental autoimmune en- cephalomyelitis
• Tim-2 T cell surface antigen
• Tim-1 has been identified to be implicated in TH2 cell responses as an airway hyperreacti- vity regulatory gene.
• Tim-3 has been demonstrated to be expressed exclusively on TH1 cells and to be crucially involved in TH1 cell responses and macrophage acti- vation.
• a method for the identification of modulators using Sema4A, Tim-2 or the interaction bet- ween Sema4A and Tim-2 as target To test compounds for a modulating effect on Sema4A-T cell interaction, naive CD4+ T cells can be incubated with respective test compounds and stimulated with immobilized anti-CD3 antibodies, anti-CD28 antibodies and e.g. Sema4A-Fc. As a control, the same assays are performed without a test compound being added. As a readout, T cell prolifera- tion and interleukin-2 (IL-2) production can be determined.
• IL-2 interleukin-2
• interferon-gamma IFN- ⁇
• IL-4 secretion can be measured.
• the skilled person will set the remaining parameters of the assay system in an appropriate manner.
• One example for a suitable assay system for the assessment of Sema4A-T cell interaction and the modulation thereof by test compounds is described in detail in Examples 6 and 7 and the Methods be- low.
• HTS high throughput screening
• a test compound which is known to show the desired modulating or inhibitory function will also be included in the assay as a positive control.
• HTS also comprises ultra high throughput screening formats (UHTS).
• said UHTS formats may be carried out using 384- or 1536-well microplates, sub-microliter or sub-nanoliter pipettors, improved plate readers and procedures to deal with evaporation.
• HTS methods are described e.g. in US 5,876,946 and US 5,902,732.
• the expert in the field can adapt the method described above to a HTS or UHTS format without the need of carrying out an inventive step.
• autoimmune diseases such as autoimmune hemolytic anemia, Hashimoto's thyroiditis, myasthenia gravis, Grave's disease, Goodpasture's syndrome, Crohn's disease, Guillain-Barre syndrome, psoriasis, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, uveitis, type I (insulin-dependent) diabetes, rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus (SLE), and multiple sclerosis.
• autoimmune diseases such as autoimmune hemolytic anemia, Hashimoto's thyroiditis, myasthenia gravis, Grave's disease, Goodpasture's syndrome, Crohn's disease, Guillain-Barre syndrome, psoriasis, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, uveitis, type I (insulin-dependent) diabetes,
• primary and secondary immunodeficiencies can be addressed by an activation of the T (helper) cell system.
• Primary T cell immunodeficiencies are e.g. DiGe- orge anomaly, chronic mucocutaneous candidiasis, Nezelof syndrome, natural killer cell deficiency, idiopathic CD4 lymphocytopenia and SCID.
• Secondary immunodeficiencies are AIDS and AIDS related diseases, cytomegalovirus infection, infectious mononucleosis, acute bacterial disease, and severe mycobacterial or fungal disease. Other causes for immunodeficiency are treatment with immunosuppressive agents such as radiation and immunosuppressive drugs.
• a general stimulation of the immune response of a healthy organism can be useful. The stimulation of the immune response might be achieved by an antigen unspecific T cell stimulation. In all these cases, a compound activating and/or enhancing the costimulatory pathway will be useful.
• agents useful for the treatment of autoimmune diseases including - but not limited to - autoimmune hemolytic anemia, Hashimoto's thyroiditis, myasthenia gravis, Grave's disease, Goodpasture's syndrome, Crohn's disease, Guillain-Barre syndrome, psoriasis, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, uveitis, type I (insulin-dependent) diabetes, rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus (SLE), and multiple sclerosis.
• autoimmune diseases including - but not limited to - autoimmune hemolytic anemia, Hashimoto's thyroiditis, myasthenia gravis, Grave's disease, Goodpasture's syndrome, Crohn's disease, Guillain-Barre syndrome, psoriasis, myasthenia gravis, glomerulonephritis, autoimmune he
• an important embodiment of the invention are anti-Sema4A .antibodies and antibody derivatives which recognize and bind to Sema4A, thereby blocking the Sema4A mediated costimulatory pathway.
• the antibodies are preferably directed against the human Sema4A protein, may be obtained from any species and maybe polyclonal or monoclonal antibodies. Especially prefered are humanized monoclonal antibody proteins. The binding of such antibodies or antibody derivatives and the inhibiting effect thereof on T cell activation can be assessed as described below in the Examples and Methods.
• Fab fragments may be formed by protease di-reading, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering. Also included in this term are F(ab')2 fragments, which may be prepared by proteolytic cleavage with pepsin.
• an antibody protein of this kind is known as a single-chain-Fv (scFv).
• scFv-antibody proteins of this kind known from the prior art are described in Huston et al. (1988, PNAS 16: 5879-5883).
• various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity.
• scFv were prepared as fusion proteins with multimerisation domains.
• the multimerisation domains maybe, e.g. the CH3 region of an IgG or a coiled coil structure (helix structures) such as leucin-zipper domains.
• Diabody means a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883).
• the shortening of the Linker in an scFv molecule to 5- 10 amino acids leads to the formation of ho- modimers in which an inter-chain VH/NL-superimposition takes place.
• Diabodies may additionally be stabilised by the incorporation of disulphide bridges. Examples of diabody- antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217- 1226).
• minibodies Another sort of antibody derivative is represented by so-called minibodies. These are bi- valent, homodimeric scFv derivatives consisting of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisa- tion region which is connected to the scFv via a hinge region (e.g. also from IgGl) and a linker region.
• the disulphide bridges in the hinge region are mostly formed in higher cells and not in prokaryotes. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).
• triabody By triabody the skilled person means a trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423- 433). ScFv derivatives wherein NH-NL are fused directly without a linker sequence lead to the formation of trimers.
• the skilled person will also be familiar with miniantibodies having a bi-, tri- or tetravalent structure wherein the multimerisation is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 11:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).
• the invention relates to pharmaceutical compositions comprising said antibody or antibody derivative as active substance and to the use of said antibody or antibody derivative for the preparation of a medicament for the treatment of diseases such as autoimmune diseases, allergic diseases and asthma as already outlined above in detail.
• a further aspect of the invention is the use of said antibody or antibody derivative in a method for the treatment of said autoimmune diseases, allergic diseases and asthma.
• Suitable excipients, adjuvants, diluents and carriers that may be used in the pharmaceutical compositions are known in the art. Examples can be taken e.g. from the handbook: Genna- ro, Alfonso R.: "Remington's Pharmaceutical Sciences", Mack Publishing Company, Eas- ton, Pennsylvania, 1990.
• the costimulatory pathway mediated by Se- ma4A can also be blocked by administering anti-Tim-2-antibodies, respective antibody derivatives as outlined above and small molecules blocking Sema4A-Tim-2-interaction.
• Sema4A protein such as human Sema4A or functional fragments or derivatives thereof.
• functional fragment or derivative means a protein having part or all of the primary structure of a mammalian, preferably human Sema4A and possessing at least the biological property of binding to the Sema4A receptor on T cells.
• said functional fragment or derivative is a soluble Sema4A protein, more prefe- rably a soluble human Sema4A protein.
• a recombinant soluble Sema4A protein can be produced by standard cloning techniques known in the art, e.g. by deleting all or parts of the transmembrane domain of natural Sema4A protein (functional fragment).
• a preferred example of a functional derivative is a fusion protein construct including at least a portion of the extracellular domain of Sema4A protein and another protein, e.g. human immunoglobulin C gamma 1, that alters the solubility, binding affinity and/or valency of Sema4A protein.
• Another protein e.g. human immunoglobulin C gamma 1, that alters the solubility, binding affinity and/or valency of Sema4A protein.
• An example of a soluble functional derivative is Sema4A-Fc having the amino acid sequence according to SEQ ID NO:3.
• a pharmaceutical composition that, when administered to a subject, stimulates T cell mediated immune responses in said subject, said pharmaceutical composition comprising (i) a pharmaceutically active component selected from the group consisting of the Sema4A protein according to SEQ ID NO:l, a functional fragment and a functional derivative of mammalian Sema4A protein as outlined above, and (ii) one or more components selected from the group consisting of excipients, adjuvants, diluents and carriers.
• the pharmaceutical composition shows the physiological effect of stimulating T cell mediated immune responses.
• This effect can be assessed e.g. by an assay comprising the steps (i) stimulating naive CD4+ T cells with immobilized anti-CD3 antibodies and anti-CD28 antibodies in the presence or absence of said pharmaceutically active component and (ii) measuring the activation of the thus treated T cells by measuring T cell proliferation or IL-2 secretion. If in the presence of the compound T cell proliferation or IL-2 secretion is increases as compared to assays in which said compound is not included, this compound is classified in the context of this invention as compound having the biological effect of stimulating T cell mediated immune responses.
• a functional Sema4A derivative as defined above causes a strong in vitro T cell activation when administered together with anti-CD3 .and/or anti-CD28 .antibodies.
• a cytokine may be added in order to optimize stimulation of T cells.
• a pharmaceutically active substance selected from the group consisting of the Sema4A protein according to SEQ ID NO:l, a functional fragment and a functional derivative of mammalian Sema4A protein as outlined above, wherein said pharmaceutically active substance has the biological effect of stimula- ting T cell mediated immune responses, for the preparation of a medicament for the treatment of primary or secondary immunodeficiencies or for the stimulation of normal T cell responses, as well as (ii) a method for the treatment of a disease selected from primary or secondary immunodeficiencies or for the stimulation of T cell responses comprising administering to a patient in need thereof a therapeutically effective amount of a substance selected from the aforementioned group.
• the functional mammalian Sema4A protein derivative encompassed by the present invention is expected to specifically bind to Tim-2 antigen present on the surface of T cells.
• the invention also provides a method for treating immune system diseases by administering Sema4A protein, functional fragments or derivatives, including soluble human Sema4A fusion proteins, to react with T cells by binding to the Tim-2 antigen.
• a method for inhibiting T cell proliferation in graft versus host disease wherein Tim-2 positive T cells are reacted with Sema4A, preferably with a soluble human Sema4A protein fragment or derivative, to bind to the Tim-2 receptor, and an immunosuppressant is administered.
• Example 1 Isolation of mouse and human Sema4A
• the amino-terminal signal sequence is followed by a sema domain, an Ig-like domain, a hydrophobic transmembrane region, and a cytoplasmic tail.
• cystein residues in the semaphorin domain are conserved between Sema4A and CD100, another member of the semaphorin family.
• Sema4A Although the expression of mouse Sema4A during embryonic development has been re- ported, its expression profiles in the adult tissues have not been reported. To exclude the possible cross hybridization among the semaphorin family in the case of northern blot analysis, RT-PCR for analysis of Sema4A-expression using Clontech's mouse multiple tissue cD ⁇ A panels was performed. The results were as follows: Sema4A was expressed in a broad range of tissues with prominent levels in the brain, spleen, lung, kidney .and testis. In addition, the expression of Sema4A was not detectable by embryo day 7 but it becomes detectable and gradually increased during embryonic development, of which embryonic expression profiles are consistent with those reported previously.
• Sema4A-Fc recombi- nant soluble mouse Sema4A protein consisting of the putative extracellular region of mouse Sema4A fused with human IgGl Fc.
• Identity of the product obtained was shown by SDS-PAGE.
• Two micrograms of purified Sema4A-Fc protein was separated by gradient PAGE (4%-20%) in the presence of 0.1% SDS under reducing conditions or nonreducing conditions and visualized by silver staining. A band of approximately 120 kDa was observed for Sema4A-Fc under reducing conditions, and di- mer formation was apparent under non-reducing conditions.
• anti-mouse Sema4A monoclonal antibodies were produced by immunizing rats with Sema4A-Fc and screening hybridomas with mouse Sema4A-expressing CHO cell tans- fectants (Sema4A-CHO) by flow cytometric analysis. It could be confirmed that anti- Sema4A (SK31, rat IgG2a) specifically bound to Sema4A-CHO but not to either control CHO cell transfectants with neomycin resistance plasmid alone (CHOneo) or CD 100- expressing CHO cells (CD100-CHO).
• anti-Sema4A monoclonal antibodies
• Sema4A was expressed abundantly on the surface of bone marrow derived and splenic DCs. Its expression was moderately detected on the surface of B cells. However, its ex- pression was not detected on the surface of T cells where CD 100 is abundantly expressed.
• Sema4A has an effect on T cell activation
• CD4+ T cells were stimulated with immobilized anti-CD3 plus anti-CD28 in the presence or absence of Sema4A-Fc.
• Sema4A-Fc enhanced anti-CD3 induced T cell proliferation and IL-2 production.
• Sema4A promotes the differentiation of T cells into Th-1 like or Th-2 like effector populations under the respective culture conditions.
• Naive T cells were cultured with anti-CD3 plus anti-CD28 in the presence of IL-12 plus anti-IL-4 (Th-1 conditions) or IL-4 (Th-2 conditions) for 6 days, and the resulting cells were restimulated with anti-CD3 plus anti-CD28 for 48 hr.
• the production of IFN- ⁇ or IL-4 was measured by ELISA.
• the presence of Sema4A-Fc the induction of either IFN- ⁇ or IL-4 producing cells was sigmficantly enhanced compared to that in the absence of Sema4A-Fc.
• Sema4A-Fc did not have any effects on Th-1 or Th-2 -like effector populations.
• Sema4A costimulates T cells in combination with other costimulatory molecules, in particular, B7 family members (CD80 and CD86), expressed on DCs.
• Sema4A-Fc has an effect on mixed lymphocyte reactions (MLR) between allogeneic T cells and DCs.
• MLR mixed lymphocyte reactions
• Bone marrow derived DCs on a C57BL/6 background were utilized as stimulators in MLR with CD4+ T cells isolated from the spleen on a BALB/c background as responders.
• Sema4A-Fc significantly enhanced T cell proliferation in the MLR.
• the production of IL-2 in the culture supernatants was also enhanced by Sema4A-Fc.
• a class IN semaphorin, CD 100 has previously been shown to be involved in the activation of B cells and DCs.
• Sema4A-Fc has an effect on B cells (proliferation) and DCs (maturation) as it is the case for CD 100.
• Small resting B cells purified from C57BL/6 mice were stimulated with or without anti-CD40 and IL-4 in the presence of either Sema4A-Fc or CDIOO-Fc for 72 hr. Cells were pulsed with [ 3 H]thymidine.
• CDIOO-Fc significantly enhanced CD40-induced proliferation of B cells and IL-12 pro- duction of DCs
• Sema4A-Fc did not show such effects on these cells.
• mice were immunized with keyhole limpet haemocyanin (KLH) in complete Freund's adjuvant subcutaneously in the hind foot pad and then treated with Sema4A-Fc for every 4 days intravenously.
• KLH keyhole limpet haemocyanin
• CD4+ T cells were prepared from the draining lymph nodes, and were tested in vitro for antigen-specific responses of T cells.
• a dramatic increase in the proliferation and the production of both IL-4 and IF ⁇ - ⁇ of CD4+ T cells from draining lymph nodes was observed in mice treated with Sema4A-Fc but not with control human IgGl.
• CD4+ T cells were prepared five days after immunization from the draining lymph nodes and stimulated for 72 hr with various concentrations of MOG-peptide in the presence of irradiated splenocytes of C57BL/6 mice. Proliferation was assessed during the final 12 hr of culture by pulsing with 2 ⁇ Ci [ 3 H] thymidine. IL-4 and IFN- ⁇ production in the culture supernatants were measured by ELISA. As shown in Fig.
• Example 6 Screening assay for the identification of compounds having a modulating effect on Sema4A - T cell - interaction
• naive CD4+ T cells are incubated with respective test compounds and are stimulated with immobilized anti-CD3 plus anti-CD28 .antibodies and Sema4A-Fc. Controls are performed without addition of the test compound. As a readout, T cell proliferation and IL-2 production are measured.
• T cell stimulation activators Compounds which result in an increased T cell proliferation or IL-2 production are classified as T cell stimulation activators, whereas compounds resulting in a decreased T cell proliferation or IL-2 production are classified as T cell stimulation inhibitors. They are useful as lead compounds for the development of small molecule pharmaceuticals for the treatment of e.g. autoimmune diseases, allergies, or asthma (inhibitors) or primary and sec- ondary immunodeficiencies (activators).
• the assay is performed essentially as described in Example 6 with the exeption that it is carried out under HTS conditions described above and sufficiently known in the art.
• Sema4A receptor various cells (splenic B cells, bone-marrow derived DCs, splenic T cells or EL-4 cells) were stained with biotinylated Sema4A-Fc.
• the binding of biotinylated Sema4A-Fc was not detected on primary T cells, B cells or DCs. Even after the B cells and the DCs were stimulated with anti-CD40, the binding of biotinylated Sema4A-Fc was not detected.
• a cDNA library from EL-4 cells was constructed. Plasmid DNA from the library was introduced into COS7 cells. The tr ⁇ msfec- ted COS7 cells were allowed to bind biotinylated Sema4A-Fc or biotinylated human im- munoglobulin Fc fractions followed by magnetic beads conjugated with strepavidin. Cells binding Sema4A-Fc were enriched by magnetic sorting. A discrete band corresponding to a 960 bp insert appeared after a third round of sorting, whereas no bands were apparent with cells binding human immunoglobulin Fc fractions. Upon sequencing of the 960 bp cDNA insert of these clones, the full-length of cDNAs encoding Tim-2 was identified.
• a BLAST search of a mouse EST database of the National Center for Biotechnology Information identified a cDNA of mouse Sema4A (X85991). Using this sequence, a full-length cDNA was cloned from a cDNA library generated from bone marrow derived DCs by PCR using primers containing a sense sequence including a Sail site 5'- AGGTCGACCCATCTGGTGACCATCTCAGGCTGACCATGGC-3' (SEQ ID NO:7) and an antisense sequence including a Notl site and FLAG (DYKDDDDK; SEQ ID NO: 8) sequence 5'- ATGCGGCCGCTTACTTGTCATCGTCGTCCTTGTAGTCAGCCACTTCGGCGCC- CAGATGGTTG-3* (SEQ ID NO:9). The resulting Sall-Notl fragments were cloned into pEFBos vector.
• RT-PCR analysis for expression of Sema4A The expression profiles of Sema4A in mouse tissues were analysed by RT-PCR using mouse multiple tissue cDNA panels (Clontech). Based on the sequence of Sema4A, RT- PCR was performed using a sense 5'-AGACTGGCCTCTTACCACTGGAGTCATG-3' (SEQ TD NO: 10) and an antisense 5'-TAGTTGTCGGCATCTACGTCACTG-3' (SEQ TD NO:ll) oligonucleotide primers (94°C for 30 sec; 60°C for 30 sec; 72°C for 30 sec; 30 cycles).
• Sema4A protein A truncated form of Sema4A cDNA was prepared from the full-length Sema4A cDNA by PCR using a pair of oligonucleotide primers containing a sense sequence including a Sail site 5'-AGGTCGACCCATCTGGTGACCATCTCAGGCTGACCATGGC-3' (SEQ ID NO:12) and an antisense sequence including a Bglll site 5'- ATAGATCTGTACTTACTTTGGGCAGCCATGGAAGCTCCGC-3' (SEQ ID NO: 13).
• the resulting Sall-Bglll fragments were used to replace the Sall-BamHI DNA fragments of the pEFBos human IgGl Fc cassette.
• Stable P3U1 plasmacytoma transfectants carrying the expression plasmid were established by electropo- ration. Briefly, aliquots of 10 7 cells were transfected with 50 ⁇ g of the plasmid DNA digested with Hindlll and 5 ⁇ g of pMClneo vector digested with BamHI by electroporation. After selection in RPMI medium containing 10% FCS and 0.3 mg/ml of G418 for 10 days, individual G418-resistant colonies were isolated and cloned.
• the Sema4A-Fc protein was purified from culture supernatants by protein A-Sepharose (Amersham Pharmacia).
• Sema4A-CHO Transfectants Sema4A-CHO were generated by introducing full-length FLAG-tagged Sema4A cDNAs in the pEFBos vector and the pMClneo vector using Lipofectamine Plus 2000 (Life Technologies). Sema4A-CHO were selected by anti-FLAG mAb (M2, Sigma) and cloned. As a control transfectant, CHOneo was generated by transfection of CHO cells with the pMClneo vector alone.
• Anti-Sema4A (SK31, rat IgG2a) was established as follows. Rats were immunized three times and boosted once with 100 mg of Sema4A-Fc protein. Rat splenocytes were fused with P3U1 cells, and 7 days later, hybridomas were tested for the production of specific antibodies using Sema4A-CHO by flow cytometry.
• Flow cytometric analysis for expression of Sema4A and its counter receptor Anti-Sema4A, Sema4A-Fc and isotype-matched control Igs were biotinylated using a bi- otinylation kit (Boehringer Mannheim).
• a bi- otinylation kit Boehringer Mannheim.
• For flow cytometric analysis for Sema4A or its counter receptor aliquots of 10 6 cells were incubated with biotinylated anti-Sema4A, Sema4A-Fc, or control Igs on ice for 1 hr containing 5 mg/ml of Fc block (PharMingen). After washing with staining buffer, the cells were stained for 20 min with FITC-conjugated streptavidin (PharMingen). Cells were then washed and analyzed by a flow cytometer.
• CD4+ T cells were prepared from splenocytes using Mag- netic Cell Sorting (MACS) (Miltenyi Biotech, Germany). Cells (lxlO 5 cells) were stimulated with 5 ⁇ g/ml of anti-CD3 (2C11; PharMingen) coated flat-bottomed 96-well plates in the absence or presence of anti-CD28 (10 ⁇ g/ml) for 48 hr.
• MCS Mag- netic Cell Sorting
• naive CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 in the absence or presence of Sema4A-Fc for 6 days, which is supplemented with IL-12 plus anti-IL-4 to generate Th-1 -like cells or with IL-4 to generate Th-2-like cells. Then, the harvested cells were restimulated with anti-CD3 plus anti-CD28 for 48 hr.
• DCs were generated from the bone marrow progenitors of C57BL/6 mice, using GM-CSF, as previously described.
• Irradiated (3000 rad) DCs from C57BL/6 mice were cultured with CD4+ T cells (5xl0 4 cells/well) derived from BALB/c mice with or without Sema4A-Fc or human IgGl (PharMingen) in flat-bottomed 96-well plates for 72 hr. Cells were pulsed with 2 mCi of [ 3 H]thymidine for the final 12 hr of incubation. For IL-2 production, the levels of IL-2 in the culture supernatants were measured using ELISA kit (Endogen). For the MLR using fixed fully maturated DCs, bone marrow-derived DCs of C57BL/6 mice were treated with anti-CD40 (5 mg/ml) for 24 hr, then fixed with 0.8% paraformaldehyde and used as stimulators.
• Nonadherent splenic B cells from C57BL/6 mice (6-8 weeks) were isolated with a combination of anti-Thyl.2 (F7D5, Seroteck Ltd, U.K.) and rabbit complement (Wako, Japan). The remaining B cells were further fractionated through a Percoll gradient of 50%, 60%, 66%, and 70%, and the cells at the interface between 66°/o and 70% layers were collected.
• the resulting small resting B cells (1 x 10 5 cells/well) were cultured with or without 1 mg/ml of anti-CD40 (HM40-3, PharMingen) and 10 U/ml of IL-4 (Genzyme, Cambridge, MA) in the presence of either Sema4A-Fc or CDIOO-Fc in flat-bottomed 96-well plates for 72 h. Cells were pulsed with 2 mCi of [ 3 H]thymidine for the last 16 hr.
• IL-12 was quantitated after culturing DCs (1 x 10 6 cells/well) in 24- well plates for 72 hr with or without anti-CD40 (5 mg/ml) plus either Sema4A-Fc or CDIOO-Fc.
• the mature IL- 12p70 heterodimer was detected using a mouse IL-12 ELISA kit (Amersham Pharmacia).
• EAE was induced in 8- to 12-week-old C57BL/6 mice by subcutaneous injections of 100 mg/ml of mouse/rat MOG-peptide (MEVGWYRSPFSRNVHLYRNGK (SEQ ID NO: 14) , Kurabo, Japan) in CFA including heat inactivated Mycobacterium tuberculosis into the femoral region on both sides.
• pertussis toxin 100 ng; List Biological Labs, Campbell, CA
• Either anti-Sema4A or rat IgGs ICN Pharmaceuticals, Inc
• MOG-specific T cells were determined as follows: seven days after the immunization with the same procedure, CD4+ T cells were purified from the draining lymph nodes by MACS and lxl 0 5 cells were stimulated with various concentrations of MOG-peptide in the presence of irra- diated (3000 rad) splenocytes (1 x 10 6 cells/well) of C57BL/6 mice in flat-bottomed 96- well plates for 72 hr. For proliferation assay, cells were pulsed with 2 mCi of [ 3 H]thymidine for the last 16 hr. Levels of IL-4 and IFN- ⁇ in the culture supernatants were measured using ELISA kit (R&D systems).
• coli DH10B cells (Invitrogen) were transformed with the ligated DNA by electroporation. Aliquots of 2.0xl0 7 independent clones were used to transfect COS7 cells. COS7 cells were transfected with plasmid DNAs using lipofectamine plus (Invitrogen). Three days after transfection, the cells were harvested, resuspended to a concentration of 5xl0 6 cells/ml in PBS containing 5% FCS, 2.5 ⁇ g/ml of Fc block (PharMingen) and 5 ⁇ g/ml of biotinylated Sema4A-Fc or biotinylated human immunoglobulin Fc, and incubated on ice for 1 hr.
• the cells were washed with ice-cold PBS and suspended to 5x10 6 cells/ml in PBS containing Dynabeads M-280 streptavidin (Dynal A.S). After incubation for 30 min, the cells were washed with ice-cold PBS ten times using a Magnetic Particle Concentrator (Dynal A.S).
• the extrachromosomal plasmid DNA was extracted from binding cells by the Hirt method.
• the plasmid DNA was introduced into E. coli DH10B cells by electroporation, then applied to the second and third transfection by protoplast fusion. Magnetic sorting was repeated three times as described above.

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