Patent Application
20090123477
The invention concerns an antibody, which is specific for CTLA-4, a pharmaceutical compound containing such an antibody, nucleic acid encoding such antibodies, vectors containing such antibodies, cells transfixed with such vectors, applications of such antibodies, methods for the production of such antibodies and methods for the production of a pharmaceutical compound containing such antibodies.
T lymphocytes (T cells) are the main agents of a highly efficient immune response that protects the human body against penetrating pathogens, such as bacteria and viruses. They regulate the molecular interaction between different cellular components of the immune system, such as dendritic cells, B cells, macrophages or other T cells, and carry out important effector functions themselves, such as the destruction of virus-infected cells or tumor cells. This means that they take up a key position in initiating and coordinating an immune response.
Highly active molecules called T cell antigen receptors (TCR) located on the cell surface give each T cell an identity and give them the ability to specifically recognise antigens presented by molecules of the major histocompatibility complex (MHC). Additional cell surface receptors of the ‘CD’ type regulate the method and type of T cell response, which is initiated by antigen-related stimulation of the TCR. Thus the TCR dictates the specific nature of an immune response, whilst the CD receptors control the scope and quality of the T cell response. Under physiological conditions, a combination of signals from TCR and at least one further CD receptor is required for the complete activation of T cells, which is particularly characterised by proliferation and cytokine production. This process is called ‘co-stimulation’. The most important co-stimulating CD molecule on resting human T cells is the CD28 molecule.
In order to avoid an overreaction of the immune system, which would lead to an uncontrolled and hence dangerous propagation of lymphocytes and a massive production of inflammatory cytokines, it is necessary to effectively switch off the activation of T cells. This task is achieved by the combination of a number of immunological control mechanisms. For this purpose, inhibiting cell surface receptors, such as the ‘cytotoxic T lymphocyte antigen-4’ (CTLA-4) molecule, which will be explained in greater detail later on, play a particularly important role.
In the development of autoimmune diseases, such as rheumatoid arthritis, type I diabetes, multiple sclerosis, colitis or psoriasis, as well as the development of allergies, an uncontrolled response of T lymphocytes to autologous structures and/or external antigens plays an important role. It is therefore quite possible that an initial overactivation of T cells, a missing inhibition of autoreactive T cells or a deficiency in the number and/or function of regulatory T cells has a causal connection with these diseases. Also in the case of allogenous organ transplantations, i.e. transplantations between individuals that are not HLA identical, an activation of the T cells of the recipient is not wanted since the activation of T cells is the main cause for chronic rejection reaction due to the recognition of alloantigen.
Current therapy concepts for suppressing the T cell response aim at the non-antigen-specific suppression of the activity of both harmful as well as useful T cells through the use of ‘nonspecific’ immune suppressants. This means that therapeutic effects are often accompanied by serious side effects.
CTLA-4 (CD152) is a member of the immunoglobulin superfamily and is structurally the nearest relative of CD28 (Lenschow D J, Walunas T L, Bluestone J A, CD28/B7 system of T cell costimulation. Annu Rev Immunol, 1996. 14:233-58). But in contrast to CD28, the physiological function of CTLA-4 is not the promotion but the inhibition of T cell activation. CTLA-4 is very weakly expressed on resting T cells and strongly on the cell surface of activated and regulatory T cells. The binding of CTLA-4 to its natural ligands B7-1 (CD80) and B7-2 (CD86), which are expressed by antigen-presenting cells (APC), leads to switching the T cell proliferation off and suppressing the cytokine expression (Egen J G, Kuhns M S, Allison J P, CTLA-4: new insights into its biological function and use in tumour immunotherapy. Nat Immunol, 2002. 3(7):611-8). The inhibiting function of CTLA-4 on the surface of T cells was initially demonstrated with the help of immobilised monoclonal antibodies specifically for the CTLA-4 molecule of the mouse (Walunas T L, Lenschow D J, Bakker C Y, Linsley P S, Freeman G J, Green J M, Thompson C B, Bluestone J A, CTLA-4 can function as a negative regulator of T cell activation, Immunity, 1994. 1(5):405-13) and humans (Blair P J, Riley J L, Levine B L, Lee K P, Craighead N, Francomano T, Perfetto S J, Gray G S, Carreno B M, June C H, CTLA-4 litigation delivers a unique signal to resting human CD4 T cells that inhibits interleukin-2 secretion but allows Bcl-X(L) induction, J Immunol, 1998. 160(1):12-5) and could be proved through the phenotype of mice in which the CTLA-4 gene was deliberately deactivated through homologous recombination. These animals died quickly from a lymphoproliferating disease, which is characterised by an uncontrolled activation of T cells (Tivol E A, Borriello F, Schweitzer A N, Lynch W P, Bluestone J A, Sharpe A H, Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity, 1995. 3(5): 541-7, as well as Waterhouse P, Penninger J M, Timms E, Wakeham A, Shahinian A, Lee K P, Thompson C B, Griesser H, Mak T W, Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science, 1995. 270(5238):985-8).
Conversely, these results suggest that CTLA-4 blockade reinforces the activation of T cells in vivo. In line with this, blocking, i.e. antagonistic, anti-CTLA-4 antibodies potentised an antitumour response (Chambers C A, Allison J P, Costimulation in T cell responses. Curr Opin Immunol, 1997. 9(3):396-404), but also induce autoimmunity (Luhder F, Hoglund P, Allison J P, Benoist C, Mathis D, Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates the unfolding of autoimmune diabetes. J Exp Med, 1998. 187(3):427-32). These findings, which had first been gained in the mouse system, could also be confirmed in humans in the first clinical trials. For example, after administering blocking antihuman CTLA-4 antibodies, individual cases of patients with metastasizing melanoma experienced a (partial) remission (Hodi F S, Mihm M C, Soiffer R J, Haluska F G, Butler M, Seiden M V, Davis T, Henry-Spires R, MacRae S, Willman A, Padera R, Jaklitsch M T, Shankar S, Chen T C, Korman A, Allison J P, Dranoff G, Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients, Proc Natl Acad Sci USA, 2003. 100(8):4712-7). At the same time, clinical indications for autoimmunity were found in a large proportion of treated patients (Phan G Q, Yang J C, Sherry R M, Hwu P, Topalian S L, Schwartzentruber D J, Restifo N P, Haworth L R, Seipp C A, Freezer L J, Morton K E, Mavroukakis S A, Duray P H, Steinberg S M, Allison J P, Davis T A, Rosenberg S A, Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma, Proc Natl Adad Sci USA, 2003. 100(14):8372-7).
A polymorphism in the CTLA-4 gene leading to a reduced expression and functionality of the CTLA-4 protein correlates with an increased probability of people falling ill with autoimmune diseases such as rheumatoid arthritis (Seidl C, Donner H, Fischer B, Usadel K H, Seifried E, Kaltwasser J P, Badenhoop K, CTLA4 codon 17 dimorphism in patients with rheumatoid arthritis. Tissue Antigens, 1998. Jan; 51(1):62-6), multiple sclerosis (Harbo H F, Celius E G, Vartdal F, Spurkland A, CTLA4 promoter and exon 1 dimorphisms in multiple sclerosis. Tissue Antigens, 1999; 53(1):106-10) or type I diabetes (Donner H, Rau H, Walfish P G, Braun J, Siegmund T, Finke R, Herwig J, Usadel K H, Badenhoop K, CTLA4 alanine-17 confers genetic susceptibility to Graves' disease and to type 1 diabetes mellitus. J Clin Endocrinal Metab, 1997. 82(1):143-6).
In contrast to the reinforcement of a T cell response with the blocking/antagonistic anti-CTLA-4 antibodies described above, agonistic anti-CTLA-4 antibodies should have an immunosuppressive effect. However, up to now it was only possible to demonstrate that convincingly for artificially immobilised antibodies. Thus the transmembrane expression of a ‘single-chain’ anti-CTLA-4 antibody on artificial APC created in gene technology reduced the TCR-induced proliferation and Interleukin-2 dissemination of T cells (Griffin M D, Hong D K, Holman P O, Lee K M, Whitters M J, O'Herrin S M, Fallarino F, Collins M, Segal D M, Gajewski T F, Kranz D M, Bluestone J A, Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152). J Immunol, 2000. 164(9):4433-42). The fact that, in this experimental approach, not only pre-activated but also resting T cells were inhibited shows that an important function of CTLA-4 is the early suppression of the TCR signal. Similar results were obtained by Brunner et al. (Brunner M C, Chambers C A, Chan F K, Hanke J, Winoto A, Allison J P, CTLA-4-Mediated inhibition of early events of T cell proliferation. J Immunol, 1999. 162(10):5813-20) in the analysis of CTLA-4 signal paths in naïve T cells.
The transmembrane expression of a single-chain anti-CTLA-4 antibody on allogenous tumour cells led to a reduction of the T-cell-conveyed elimination of these tumour cells in mice (Hwang K W, Sweatt W B, Brown I E, Blank C, Gajewski T F, Bluestone J A, Alegre M L, Cutting edge: targeted ligation of CTLA-4 in vivo by membrane-bound anti-CTLA-4 antibody prevents rejection of allogeneic cells. J Immunol, 2002. 169(2):633-7). These results showed that an immunological anti-tumour response or the rejection of allogeneic organ transplants can be suppressed through efficient crosslinking of CTLA-4. However, up to now this type of targeted suppression of T cell activation through CTLA-4 ligation in vivo could only be achieved with membrane-bound anti-CTLA-4 antibody constructs or with the natural membrane-based ligands. Up to now, a corresponding suppression of the T cell response in the animal through soluble anti-CTLA-4 antibodies has not been described. What has been described is the in vitro induction of apoptosis in pre-activated T cells by an CTLA-4 antibody with specificity for the C″D loop of the extracellular domain of CTLA-4 (Gribben J G, Freeman G J, Boussiotis V A, Rennert P, Jellis C L, Greenfield E, Barber M, Restivo V A Jr, X Ke, Gray G S, Nadler L M, CTLA-4 mediates antigen-specific apoptosis of human T cells. Proc Natl Acad Sci USA, 1995. 92(3):811-5).
In conclusion, the findings so far show an inhibiting function of CTLA-4 on T cells; however, it is not yet fully clear how this mechanism is affected. The following mechanism, which are not mutually exclusive, are discussed: i) suppression of the activating TCR and/or CD28 signal path, ii) competition of the CD28-induced costimulation through higher affinity to CD80 and CD86, iii) increasing the threshold value of T cell activation, iv) attenuation of the T cell expansion and/or v) activation of regulatory cells and connected with that indirect suppression of conventional T cells.
In the above-mentioned indications, a selective inactivation of T cells through the stimulation of the inhibiting function of CTLA-4, which is well tolerated by the organism, is desirable.
Therefore the invention is based on the technical problem of stating substances and pharmaceutical compounds that are capable of stimulating the inhibiting function of CTLA-4.
To solve this technical problem, the invention teaches an isolated monoclonal antibody, which is specific and agonistic for CTLA-4, whereby the heavy chain of the antibody contains a sequence selected from the group consisting of (Seq.-ID): “22, 23, 24, 25, 26, 27, 28, 29, and 32”. The light chain of the antibody can contain a sequence that has been selected from the group consisting of (Seq.-ID): “33, 34, 35, 36, 37 and 38”.
The preference is for an antibody in accordance with the invention with a heavy chain containing a sequence in accordance with Seq.-ID 27, 28 or 29, preferably containing or consisting of the sequence in accordance with Seq.-ID 30 or 32, as well as with a light chain containing a sequence in accordance with Seq.-ID 36 or 37, preferably containing or consisting of a sequence in accordance with Seq.-ID 38.
Special antibodies with the above general structure are the antibodies TGN2122.H and TGN2422.H described below.
In addition, the invention teaches an isolated monoclonal antibody, which is specific and agonistic for CTLA-4, whereby the heavy chain of the antibody contains a sequence which is selected from the group consisting of (Seq.-ID): “43, 44, 45, 46, 47, 48, 49, 50, 51 and 53”. The light chain of the antibody can contain a sequence which is selected from the group consisting of (Seq.-ID): “54, 55, 56, 57, 58 and 59”.
The preference is an antibody, which is also in accordance with the invention, with a heavy chain containing a sequence in accordance with Seq.-ID 48, 49 or 50, preferably containing or consisting of a sequence in accordance with Seq.-ID 51 or 53 and with a light chain containing a sequence in accordance with Seq.-ID 57 or 58, preferably containing or consisting of a sequence in accordance with Seq.-ID 59.
Special antibodies with the above general structure are the antibodies TGN2122.C and TGN2422.C described below.
The above-mentioned antibodies are humanised antibodies. Since the antibodies are already humanised, a humanisation, as described below for further variants of antibodies covered by the invention, is not required. The antibody may, but does not have to bind to the C″D loop of CTLA-4. It may also be an antibody that does not bind to this loop. With respect to all further forms and applications as well as other details and explanations, the subsequent explanations for a further variant of the invention apply analogously and in full.
Finally, the invention teaches an isolated monoclonal antibody, which is specific and agonistic for CTLA-4, whereby the antibody does not bind to a partial CTLA-4 sequence in accordance with Seq.-ID 1. The sequence in accordance with Seq.-ID 1 is the C″D loop of the CTLA-4. Put another way, the antibody covered by the invention binds to other areas of the CTLA-4 molecule than the C″D loop. The invention is based on the finding that an agonistic stimulation of CTLA-4, i.e. inducing the inhibiting activity of CTLA-4 in vivo, is a reasonable therapeutic concept for autoimmune diseases or transplants, and provides suitable substances in the form of antibodies or fragments thereof for this purpose.
Antibodies covered by the invention contain preferably at least one of the sequences in accordance with Seq.-ID 2 to Seq.-ID 7 or Seq.-ID 8 to Seq.-ID 13. These sequences are the CDRs of the variable areas of a heavy and a light chain; please also refer to table 2.
Preferably an antibody covered by the invention is humanised. This can be accomplished using the usual methods, for example by chimaerising a specific monoclonal mouse antibody against human CTLA-4 in such a way that the constant areas are replaced by human constant areas or constant areas tolerated by human organisms. What is important is that preferably all CDRs in accordance with table 2 are retained, including their spatial arrangement to each other. Possible bases for the humanisation can be, for example, monoclonal antibodies containing at least one, but preferably all, sequences in accordance with Seq.-ID 2 to Seq.-ID 7 or Seq.-ID 8 to Seq.-ID 13, for example one of the sequences in accordance with Seq.-ID 14 to 17. In concrete situations, antibodies covered by the invention can also contain one of the sequences in accordance with Seq.-ID 18 to 21. Suitable realised examples of antibodies forming a basis for humanisation are the antibodies 4.8H10H5 and 4.3F6B5 describe in detail below. It is possible to make humanised antibodies from these using the usual methods of gene technology, for example by applying gene technological humanisation strategies.
In the context of the invention, the term antibody comprises the explicitly revealed structures as well as functionally equivalent antibodies, which have been modified using e.g. chimaerisation, humanisation, or de-immunisation (cutting out T cell epitopes from the human antibody that causes undesirable immune reactions), as well as specific fragments of the light and/or the heavy chain of the variable area of the antibodies of the type described above. The average professional in this field should be familiar with the production/cultivation of such antibodies with specified immunogens; therefore this does not have to be explained in detail.
The invention also concerns an isolated protein or peptide containing at least one of the sequences Seq.-ID 2 to 13, in particular one of the sequences Seq.-ID 14 to 17 or Seq.-ID 18 to 21, or one of the sequences Seq.-ID 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, in particular one of the sequences Seq.-ID 27, 28, 29, 30, 32, 36, 37 or 38, or one of the sequences Seq.-ID 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, in particular one of the sequences Seq.-ID 48, 49, 50, 51, 53, 57, 58 or 59, or consisting of one of the named sequences an isolated nucleic acid encoding for one such protein or peptide or for a light chain and/or a heavy chain of an antibody covered by the invention, an isolated vector containing such a nucleic acid, and an isolated cell, which is transfixed with such a vector. All the above items are suitable for the production or construction of antibodies in accordance with the invention.
An antibody in accordance with the invention or a protein or peptide in accordance with the invention should preferably be soluble in water, in particular in physiological salt solution, i.e. not artificially cross-linked. Also, an antibody in accordance with the invention is superagonistic, i.e. it stimulates the physiological activity of the T cell inhibiting receptor CTLA-4.
In addition, the invention concerns a pharmaceutical compound containing a monoclonal antibody in accordance with the invention and/or a protein or peptide in accordance with the invention as well as optionally at least one physiologically compatible carrier substance and/or agent, which will be explained in detail later on. It can be obtained by mixing these components, whereby the active substance is used in a physiologically effective dosis. This dosis can easily be determined with cells in in-vitro trials as well as with animal trials in the usual way. Such a pharmaceutical compound is suitable for prophylactic or therapeutic treatment of a disease or a condition from the group comprising “rheumatoid arthritis, type I diabetes, multiple sclerosis, systemic lupus erythematodes, psoriasis, ulcerative colitis, morbus crohn, allergies, rejection of allogenous organ transplants, in particular organ transplants of the following organs: heart, kidney, liver, pancreas, lung, bone marrow, and ‘Graft-Versus-Host’ disease”. To that extent, the invention also comprises a process for prophylaxis and/or treatment of one of the above diseases, whereby the patient is administered the pharmaceutical compound in a suitable dosis.
The galenic preparation of a pharmaceutical compound in accordance with the invention can be made in the usual way. Possible counter-ions for ionic compounds are for example Na+, K+, Li+ or cyclohexylammonium. Suitable solid or liquid galenic forms of preparations are for example granules, powder, coated tablets, tablets, (Micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or solutions for injection (i.v., i.p., i.m., s.c.) or atomisation (aerosols), transdermal systems as well as preparations with protracted release of the active substance, for the production of which the usual auxiliary substances are used, such as carrier substances, blasting agents, binding agents, coating materials, swelling agents, lubricants, flavourings, sweeteners and solubilisers. Possible excipients are magnesium carbonate, titanium dioxide, lactose, mannite and other sugars, talcum, milk protein, gelatine, starch, cellulose and its derivatives, animal and vegetable oils such as cod liver oil, oil of sunflowers, peanuts or sesame, polyethyleneglycols and solvents such as sterile water and monohydric or poly hydric alcohols, for example glycerine.
In addition, the invention concerns a process for the production of a monoclonal antibody in accordance with the invention, in which a nucleic acid in accordance with the invention is entered into a vector, whereby a cell is transfixed with the help of the vector, whereby the transfixed cell is cultivated, whereby an excess of the cultivated cell is cut off or whereby the cultivated cell is lysed and the lysate is obtained, and whereby the monoclonal antibodies are separated from the cut off excess or the lysate.
Hereinafter, the invention is explained in greater detail, using examples presenting just different product forms.
Table 1 shows the binding characteristics of 4 new anti-CTLA-4 antibodies, of which 2 do not bind to the C″D loop of CTLA-4 (4.3F6B5 and 4.8H10H5) and 2 that bind to it (3.7F10A2 and 4.7A8H6). The latter are reference antibodies and are not subject to this invention. Subject of the investigation was the specificity of the antibodies for human CTLA-4, both on transfixed Jurkat E6.1 cells as well as ex vivo activated human PBMCs (peripheral blood mononuclear cells). The cross reactivity against rat CTLA-4 was demonstrated with a transfixed BW cell line carrying the extracellular domain of rat CTLA-4 on the surface. Likewise the cross reactivity against the closely related T cell receptors CD28 and ICOS on transfixed Jurkat E6.1/L929 cells was eliminated. The binding or non-binding to the lateral C″D loop structure is illustrated in detail in
Table 2 contains the sequences of the 4.8H10H5 and 4.3F6B5 antibodies covered by the invention, with a division into heavy and light chains, with the boundary between the variable areas and the constant areas being marked. The sequences were determined with the help of RT-PCR and/or protein sequencing (Edman Abbau).
Table 3 contains sequences of the heavy chain of antibody TGN2122.H. Table 4 contains the nucleic acid encoding for the heavy chain. Table 5 contains sequences of the heavy chain of the antibody TGN2422.H. Table 6 shows the nucleic acid encoding for the heavy chain. Table 7 shows sequences of the light chain for both antibodies TGN2122.H and TGN2422.H. Table 8 shows the nucleic acid encoding for the light chain.
Table 9 shows sequences of the heavy chain of the antibody TGN2122.C. Table 10 shows the nucleic acid encoding for the heavy chain. Table 11 shows sequences of the heavy chain of the antibody TGN2422.C. Table 12 shows the nucleic acid encoding for the heavy chain. Table 13 shows sequences of the light chain for both antibodies TGN2122.C and TGN2422.C. Table 14 shows the nucleic acid encoding for the light chain.
In the case of the sequences Seq.-ID 31, 39, 52 and 60 we are dealing with leader peptides, which are not included in the respective mature chains. Therefore antibodies are preferred that do not contain these sequences.
The antibodies TGN2122.C (isotype IgG1) and TGN2422.C (isotype IgG4) were obtained by humanisation from the mouse antibody 4.3F6B5. The antibodies TGN2122.H (isotype IgG1) and TGN2422.H (isotype IgG4) were obtained from the mouse antibody 4.8H10H5.
As controls, isotype controls and commercially available antibodies with specificity for CTLA-4 were also carried. (A) Effect of the CTLA-4-specific antibodies (1 μg/ml) on the IL-2 production of transfixed Jurkat cells that express a chimaerous CTLA-4/CD28 receptor. (B) Effect of the commercially available CTLA-4-specific antibodies (1 μg/ml) on the IL-2 production of transfixed Jurkat cells that express a chimaerous CTLA-4/CD28 receptor. (C) Effect of the CTLA-4-specific antibodies (1 μg/ml) on the IL-2 production of not-transfixed Jurkat E6.1 cells that are missing the chimaerous receptor.
(Representative experiments). Two of the tested antibodies (4.3F6B5, 4.8H10H5) induce the IL-2 production of the transfixed Jurkat cells through activation of the chimaerous receptor whilst none of the commercially available antibodies with CTLA-4 specificity were able to do that.
(Representative experiments). As was shown for IL-2, the antibodies 4.3F6B5 and 4.8H10H5 are able to activate the chimaerous receptor, to trigger a signal transduction and to induce the CD69 expression. None of the commercially available antibodies with CTLA-4 specificity were able to do that. (Representative experiments).
(A) The binding of anti-CTLA-4 antibodies to rat CTLA-4 was demonstrated with BW cells expressing the extracellular domain of the rat receptor on their surface. Analogous to the chimaerous receptor on Jurkat cells (
Where Xn is included in sequences, the ‘n’ may vary by ±1.
| Number | Date | Country | Kind |
|---|---|---|---|
| DE102004063494.7 | Dec 2004 | DE | national |
