EP2111415A2 - Monoclonal agonistic anti-ctla-4 antibodies - Google Patents

Monoclonal agonistic anti-ctla-4 antibodies

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Publication number
EP2111415A2
EP2111415A2 EP05826461A EP05826461A EP2111415A2 EP 2111415 A2 EP2111415 A2 EP 2111415A2 EP 05826461 A EP05826461 A EP 05826461A EP 05826461 A EP05826461 A EP 05826461A EP 2111415 A2 EP2111415 A2 EP 2111415A2
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EP
European Patent Office
Prior art keywords
seq
ctla
antibody
monoclonal antibody
antibodies
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.)
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Application number
EP05826461A
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German (de)
French (fr)
Inventor
Thomas TeGenero AG HANKE
Frank TeGenero AG HORLING
Martin TeGenero AG TRISCHLER
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Theramab LLC
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Theramab GmbH
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Publication of EP2111415A2 publication Critical patent/EP2111415A2/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/06Antipsoriatics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565Complementarity determining region [CDR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75Agonist effect on antigen

Definitions

  • the invention relates to an antibody which is specific for CTLA-4, a pharmaceutical composition containing such an antibody, nucleic acids encoding such antibodies, vectors containing such antibodies, cells transfected with such vectors, uses of such antibodies, methods for producing such antibodies, and methods for Preparation of a pharmaceutical composition containing such antibodies.
  • T lymphocytes are the main agents of a highly efficient immune response that protects the human body against invading 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 perform important effector functions such as the destruction of virus-infected cells or tumor cells themselves. As a result, they occupy a key position in the initiation and coordination of an immune response.
  • TCRs T cell antigen receptors
  • HLA major histocompatibility complex
  • CD receptors control the extent and quality of the response of the T cell.
  • a signal combination of TCR and at least one other CD receptor is required for the complete activation of T cells, which is characterized in particular by proliferation and cytokine production. This process is called "costimulation.”
  • costimulation The most important costimulatory CD molecule on quiescent human T cells is the CD28 molecule.
  • T cells Activation of T cells are effectively shut down. This task is accomplished through the interaction of a number of immunological control mechanisms. A particularly important role is played by inhibitory cell surface receptors such as the "Cytotoxic T-Lymphocyte Antigen-4" (CTLA-4) molecule, which will be explained in detail later.
  • CTL-4 Cytotoxic T-Lymphocyte Antigen-4"
  • Organ transplants d. H . in transplantation among non-HLA-identical individuals, activation of the T cells of the recipient is undesirable because activation of T cells by recognition of allo-antigens is the major cause of a chronic rejection reaction.
  • CTLA-4 ' (CD152) is a member of the immunoglobulin superfamily and structurally the closest relative of CD28 (Lenschow DJ, Walunas TL, Bluestone JA, CD28 / B7 system of T cell costimulation., Annu Rev Immunol, 1996. 14: 233- 58).
  • CD28 the physiological function of CTLA-4 is not the promotion, but the inhibition of T-cell activation.
  • CTLA-4 is very weak on quiescent T cells and strongly expressed on the cell surface of activated and regulatory T cells.
  • CTLA-4 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 a shutdown of T cell proliferation and suppression of the Cytokine expression (Egen JG, Kuhns MS, Allison JP, CTLA-4: new entry into biological function and use in tumor immunotherapy, Nat Immunol, 2002. 3 (7): 611-8).
  • CTLA-4 The inhibitory function of CTLA-4 on the T cell surface was first determined using immobilized monoclonal antibodies specific for mouse CTLA-4 molecule (Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB, Bluestone JA _ ⁇ _ CTLA-4 can function as a negative regulator of cell activation, Immunity, 1994.1 (5): 405-13) and man (Blair PJ, Riley JL, Levine BL, Lee KP, Craighead N, Francomano T, Perfetto SJ, Gray GS, Carreno BM, June CH, CTLA-4 ligation dellvers a unique signal to resting human
  • CTLA-4 blockade enhances the activation of T cells in vivo. Accordingly, blocking, ie antagonistic, anti-CTLA-4 antibodies potentiated an anti-tumor response (Chambers CA, Allison JP, Co-stimulation in cell responses, Curr Opin Immunol, 1997. 9 (3): 396-404), to lead but also autoimmunity (Luhder F, Hoglund P, Allison JP, 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 were initially obtained in the mouse system, could be confirmed in humans in the first clinical trials.
  • a polymorphism in the CTLA-4 gene that results in decreased expression and functionality of the CTLA-4 protein correlates with an increased likelihood of human involvement in the autoimmune diseases rheumatoid arthritis (Seidl C, Donner H, Fischer B, Usadel KH, Seifried E , Coldwater JP, Badenhoop K, CTLA4 codon 11 dimorphism in patients with rheumatoid arthritis Tissue Antigens, 1998.
  • agonist anti-CTLA-4 antibodies should be immunosuppressive.
  • this has so far only been convincingly demonstrated for artificially immobilized antibodies.
  • the genetically engineered transmembrane expression of an anti-CTLA-4 "single chain" antibody on artificial APC reduced TCR mediated proliferation and interleukin-2 secretion of T cells (Griffin MD, Hong DK, Holman PO, Lee KM, Whitters MJ, Mammin SM, Fallarino F, Collins M, Segal DM, Gaj ewski TF, Wreath DM, Bluestone JA, Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152) .J Immunol, 2000.
  • CTLA-4 antibody specific for the C 'D loop of the extracellular domain of CTLA-4 (Gribben JG, Freeman GJ, Boussiotis VA, Rennert P, Jellis CL Greenfield E, Barber M, Restivo VA Jr, X Ke, Gray GS, Nadler LM, CTLA4 mediates antigen-specific apoptosis of human T cells, Proc Natl Acad Sci USA, 1995. 92 (3): 811-5).
  • Targeted and well-tolerated inactivation of T cells by stimulating the inhibitory function of CTLA-4 would be desirable in the above-mentioned indications.
  • the invention is therefore based on the technical problem of providing substances and pharmaceutical compositions capable of stimulating the inhibitory function of CTLA-4.
  • the invention teaches an isolated monoclonal antibody specific and agonistic to CTLA-4, the heavy chain of the antibody containing a sequence selected from the group consisting of (Seq. ID): "22 , 23, 24, 25, 26, 27, 28, 29, 30, and 32 ".
  • the light chain of the antibody may contain a sequence selected from the group consisting of (Seq. ID):" 33, 34 , 35, 36, 37, and 38 ".
  • An antibody according to the invention with a heavy chain containing a sequence according to Seq. ID 27, 28, or 29, preferably containing or consisting of Sequence according to Seq. ID 30 or 32, and with a light chain containing a sequence according to Seq. ID 36 or 37, preferably containing or consisting of a sequence according to Seq. ID 38.
  • a specific antibody having the above basic structure are the following antibodies TGN2122. H and TGN2422. H .
  • the invention teaches an isolated monoclonal antibody which is specific and agonistic to CTLA-4, the heavy chain of the antibody containing a sequence 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 may contain a sequence selected from the group consisting of (Seq. ID):" 54, 55, 56, 57, 58, and 59 ⁇ .
  • an antibody according to the invention with a heavy chain containing a sequence according to Seq. ID 48, 49, or 50, preferably containing or consisting of the sequence according to Seq. ID 51 or 53, as well as with a light chain containing a sequence according to Seq. ID 57 or 58, preferably containing or consisting of a sequence according to Seq. ID 59.
  • a specific antibody having the above basic structure are the following antibodies TGN2122. C and TGN2422. C.
  • the antibodies mentioned above are humanized antibodies. Since it is already one humanized antibody, as described below for other variants of antibodies according to the invention is not required.
  • the antibody may or may not bind to the C '' D loop of CTLA-4. Rather, it may also be an antibody that does not bind to this loop.
  • the invention teaches an isolated monoclonal antibody which is specific and agonistic to CTLA-4, wherein the antibody does not bind to a CTLA-4 partial sequence according to SEQ. ID 1 binds.
  • the sequence according to SEQ. ID 1 is the C 'D loop of the CTLA-4.
  • an antibody of the invention binds to other regions 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 inhibitory activity of CTLA-4 in vivo, constitutes a useful therapeutic concept for autoimmune diseases or transplantation, and provides suitable drugs in the form of the antibodies or fragments thereof.
  • an antibody according to the invention contains at least one of the sequences according to SEQ. ID 2 to SEQ. ID 7 or SEQ. ID 8 to SEQ. -ID 13. These sequences are the CDRs of the heavy and light chain variable regions, with additional reference to Table 2.
  • an antibody according to the invention is humanized. This can be done in a customary manner, for example by using a human CTLA-4-specific monoclonal mouse antibody is chimaerized in such a way that the constant regions are exchanged for human or human-compatible constant regions. It is essential that preferably all CDRs according to Table 2 are maintained and indeed in their spatial arrangement to each other.
  • monoclonal antibodies can be used which at least one, preferably all, sequences according to SEQ. ID 2 to SEQ. ID 7 or SEQ. ID 8 to SEQ. ID 13, for example one of the sequences according to SEQ. ID 14 to 17, included.
  • antibodies according to the invention can also be one of the sequences according to SEQ. ID 18 to 21 included.
  • Suitable embodiments of antibodies which form a basis for humanization are the antibodies 4.8H10H5 and 4.3F6B5 described in detail below. A preparation of humanized antibodies from this is possible in a conventional manner, for example by genetic engineering humanization strategies.
  • antibody encompasses, in addition to the structures explicitly disclosed, also functionally corresponding antibodies which have been modified, for example, by chimerization, humanization or de-immunization (excision of T-cell epitopes from the human antibody which trigger undesired immune reactions) and specific antibodies Fragments of the light and / or heavy chain variable region underlying antibody of the above type.
  • chimerization humanization or de-immunization (excision of T-cell epitopes from the human antibody which trigger undesired immune reactions) and specific antibodies Fragments of the light and / or heavy chain variable region underlying antibody of the above type.
  • specific antibodies Fragments of the light and / or heavy chain variable region underlying antibody of the above type The production resp. Obtaining such antibodies with given immunogens is the A person of ordinary skill in the art is familiar and need not be explained in more detail.
  • the invention also relates to 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.
  • An inventive antibody or. a protein or peptide according to the invention is preferably soluble in water, in particular in physiological saline solution, i. e. not artificially networked. Furthermore, an antibody according to the invention is superagonistic, i. e. stimulates the physiological action of the T-cell inhibitory receptor CTLA-4.
  • the invention further relates to a pharmaceutical composition containing a monoclonal antibody according to the invention and / or an inventive Protein or peptide, and optionally at least one physiologically acceptable carrier and / or excipient, which will be explained in detail later. It is obtainable by mixing these components, the active ingredient being used in a physiologically effective dose. This dose can be easily determined in in-vitro experiments with cells and in animal experiments in the usual way.
  • Such a pharmaceutical composition is for the prophylaxis and / or therapy of a disease or condition selected from the group consisting of "rheumatoid arthritis, type I diabetes, multiple sclerosis, systemic lupus erythematosus, psoriasis, ulcerative colitis, Crohn's disease, allergies, rejection of allograft organ transplant , in particular organ transplants of the following organs: heart, kidney, liver, pancreas, lung, bone marrow, and "graft-versus-host" disease suitable.
  • the invention also includes a method for the prophylaxis and / or treatment of a above disease or condition wherein a patient is given the pharmaceutical composition in suitable dosage.
  • Suitable counterions for ionic compounds are, for example, Na + , K + , Li + or cyclohexylammonium.
  • Suitable solid or liquid galenic preparations are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or solutions for injection (i.v., i.p., i .m., s.
  • adjuvants such as carriers, blasting, binding, coating, swelling, lubricants or lubricants, flavoring agents, sweeteners and solubilizers, are used.
  • adjuvants may be magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talcum,
  • the invention further relates to a method for producing a monoclonal antibody according to the invention, wherein a nucleic acid according to the invention is introduced into a vector, wherein a cell is transfected by means of the vector, wherein the transfected cell is cultured, wherein a supernatant is separated from the cultured cells or the cultured cell is lysed and the lysate is recovered, and the monoclonal antibodies are separated from the separated supernatant or lysate.
  • Example 1 Antibodies according to the invention
  • Table 1 shows the binding properties of 4 new anti-CTLA-4 antibodies, 2 of which do not bind to the C "D loop of CTLA-4 (4.3F6B5 and 4.8H10H5) and 2 however, bind to it (3.7F10A2 and 4.7A8H6).
  • the latter are reference antibodies and are not the subject of the present invention.
  • the specificity of the antibodies to human CTLA-4 was investigated, both on transfected Jurkat E6.1 cells and on ex vivo activated human PBMCs (peripheral blood mononuclear cells). Cross-reactivity against rat CTLA-4 was demonstrated with a transfected BW cell line carrying the extracellular domain of rat CTLA-4 on the surface. Likewise, the cross-reactivity against the closely related T-
  • Fig. 1 shows the most important examples
  • CTLA-4 The specificity of antibodies to CTLA-4 was confirmed with human PBMCs previously stimulated ex vivo with PHA / IL-2. In resting cells, CTLA-4 is predominant localizes intracellularly and is brought to the surface only after activation. The thick curve shows the binding of the antibodies, the thin curve the binding of the isotype control to activated human PBMCs.
  • C In view of a possible usability of the antibodies in
  • Fig. Figure 2 shows the nonexistent specificity of 2 of 4 anti-CTLA-4 antibodies from Table 1 for the C "" D loop.
  • the two antibodies according to the invention 4.3F6B5 and 4.8H10H5, which showed agonistic mode of action in functional assays (see Example 2), are not specific for the human C 'D loop.
  • Jurkat E6.1 was used, which expresses a chimeric extracellular domain of CTLA-4 on the surface: this chimera consists of the murine receptor, in which the C S D loop against the corresponding human sequence, represented by the amino acids Pos 68 - 83, was exchanged.
  • Fig. Figure 3 shows that binding of anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 to CTLA-4 by addition of recombinant CD80 can be competed.
  • the Ergbenis suggests that 4.8H10H5 and 4.3F6B5 are located near the binding site for CD80, d. H . bind the MYPPPY loop, but not to the C 'D loop.
  • the aim of this experiment was to further narrow the binding properties of the antibodies.
  • Jurkat E6.1 cells carrying the extracellular domain on their surface were incubated with increasing concentration of CD80Fc protein and 1 ⁇ g / ml of CTLA-4 specific antibody. The co-incubation of recombinant protein and antibody leads to a
  • Figure 10 shows the binding of the humanized antibodies to the extracellular domain of human CTLA-4. Shown is the binding of the humanized antibodies to transfected cells (thick curve) compared to binding of the isotype control (thin curve) to the same cells. The FACS analysis shows that in the course of humanization the specificity of the antibodies to human CTLA-4 is maintained.
  • Antibodies 4.8H10H5 and 4.3F6B5 again, divided into heavy and light chains, with the border between the variable regions and the constant regions labeled.
  • the sequences were determined by RT-PCR or. determined by protein sequencing (Edman degradation).
  • Table 3 shows heavy chain sequences of the antibody TGN2122.
  • H Table 4 shows the for the heavy chain coding nucleic acid.
  • Table 5 shows heavy chain sequences of the antibody TGN2422.
  • H Table 6 shows the heavy chain coding nucleic acid.
  • Table 7 shows light chain sequences for both antibodies TGN2122. H and TGN2422.
  • H Table 8 shows the light chain encoding nucleic acid.
  • Table 9 shows heavy chain sequences of the antibody TGN2122.
  • C. shows the heavy chain coding nucleic acid.
  • Table 11 shows heavy chain sequences of the antibody TGN2422.
  • C. shows the heavy chain coding nucleic acid.
  • Table 13 shows light chain sequences for both antibodies TGN2122.
  • C. Table 14 shows the light chain encoding nucleic acid.
  • sequences Seq. IDs 31, 39, 52, and 60 are leader peptides that are not included in the current mature chains. Therefore, those antibodies are preferred which do not contain these sequences.
  • TGN2122. C isotype IgG1
  • TGN2422 was purified from mouse antibody 4.3F6B5
  • Example 2 Effect of Antibodies According to the Invention on a Comparative Antibody Attached to C 'D Loop binds, as well as commercially available anti-CTLA-4 antibodies.
  • FIG. Figure 4 shows the inhibitory effect of anti-CTLA-4 antibody 4.8H10H5 on the proliferation of human PBMCs.
  • the aim of this proliferation inhibition assay was to identify a functionally novel antibody compared to previously known CTLA-4 specific antibodies.
  • a superagonistic antibody As an essential property of a superagonistic antibody has been defined the ability to reduce the proliferation of human PBMC. Another criterion was that this effect with soluble, not artificially networked! Antibody is observed. These antibodies were evaluated as positive, reducing anti-CD3 (or superagonistic anti-CD28, not shown) T cell proliferation by at least 25%. Readout system was the measurement of proliferation by means of 3 H thymidine incorporation. In this assay system, the CTLA-4 specific antibodies were given in time with the activating anti-CD3 antibody and the proliferation determined after 63-66 hours.
  • Antibody 4.8H10H5 was able to inhibit proliferation of T cells based on the above criteria. For comparison, an antibody not positively evaluated in this assay system (2.10B11A1) is listed. Shown is the relative proliferation in relation to the positive control (anti-CD3 induced proliferation). Further controls included the respective isotype control (IgGl or IgG2) and a commercially available antibody (BNI3, BD Pharmingen). The control commercial antibodies (14D3, 8H5, 3H1833, BNI3) with specificity for CTLA-4 had no effect. Fig.
  • FIG. 5 shows the stimulatory effect of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on the IL-2 production of Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 molecule.
  • Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 receptor on their surface were used for this cell-autonomous read-out system for the functional characterization of the CTLA-4-specific MAK (D).
  • D This consists of the extracellular domain of the CTLA-4 receptor and the transmembrane and intracellular domain of CD28.
  • Activation of the chimeric receptor by CTLA-4-specific MAK induces CD28-specific activation markers such as IL-2 or CD69, which are detected by means of ELISA or ELISA. FACS analysis can be measured.
  • Potentially superagonistic CTLA-4 specific antibodies can be identified in this system using CD28 specific
  • Activation markers are identified. Control antibodies and CTLA-4 specific antibodies were cross-linked (using sheep anti mouse Ig) and incubated with 1 * 10 5 transfected Jurkat E6.1 cells for 48h. As an activation marker, IL-2 production was measured by ELISA.
  • CTLA-4 specific antibodies (1 ⁇ g / ml) on the IL-2 production of transfected Jurkat cells expressing a chimeric CTLA-4 / CD28 receptor.
  • B Effect of commercially available CTLA-4 specific antibodies (1 ⁇ g / ml) on the IL-2 production of transfected Jurkat cells expressing a chimeric CTLA-4 / CD28 receptor.
  • C Effect of CTLA-4 specific antibodies (l ⁇ g / ml) on IL-2 production of untransfected Jurkat E6.1 cells lacking the chimeric receptor (representative experiments). Two of the antibodies tested (4.3F6B5, 4.8H10H5) induce the IL-2 production of transfected Jurkat cells by activation of the chimeric receptor, while none of the commercially available antibodies with specificity for CTLA-4 was able to do so.
  • Fig. Figure 6 shows the stimulatory effect of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on the CD69 induction of Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 molecule.
  • D Using the method shown in Fig. In addition to IL-2 production, the assay of CD69 expression as a further activation marker was measured by FACS analysis. In contrast to IL-2 production, CD69 is an early activation marker and can be detected after 4 h of incubation of the antibodies with the transfected cells.
  • A Effect of CTLA-4-specific antibodies (1 ⁇ g / ml) on transfected CD69 expression Jurkat cells expressing a chimeric CTLA-4 / CD28 receptor
  • B Effect of commercially available CTLA-4 specific antibodies (1 ⁇ g / ml) on the CD69 expression of transfected Jurkat cells carrying a chimeric CTLA-4 / CD28 receptor
  • C Effect of CTLA-4 specific antibodies (l ⁇ g / ml) on CD69 expression of untransfected Jurkat E ⁇ .l cells lacking the chimeric receptor (representative experiments) As shown for IL-2, as well Antibodies 4.3F6B5 and 4.8H10H5 are able to activate the chimeric receptor, a
  • FIG. Figure 7 shows that the stimulatory effect of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on the chimeric CTLA-4 / CD28 construct by CD80 Fc protein can be reduced.
  • Jurkat E. ⁇ . 1 cells expressing the chimeric CTLA-4 / CD28 receptor (see Fig. 5, 6) were incubated with increasing concentration of antibody and in each case 1 ⁇ g / ml CD80 Fc protein.
  • Antibody-mediated CD69 expression is then reduced by CD80Fc as the recombinant protein is excessively incubated compared to the antibody. Rectangular squares show the current CD69 induction without CD80 co-incubation, the triangular curves show the CD69 induction diminished by I ⁇ g / ml CD80 Fc
  • Antibodies In addition, the concentration-dependent binding of the antibodies and the Jurkat cells is shown.
  • FIG. Figure 8 shows the cross-reactivity of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 with the rat CTLA-4 molecule and the stimulatory functionality of the antibodies in a chimeric receptor assay.
  • A Binding of anti-CTLA-4 antibodies to rat CTLA-4 was demonstrated by BW cells expressing the extracellular domain of the rat receptor on its surface. Analogous to the chimeric receptor on Jurkat cells ( Figure 5), these cells express a chimeric CTLA-4 CD28 receptor consisting of rat CTLA-4 (extracellular domain) and mouse CD28 (transmembrane / intracellular domain) (bright line: anti-CTLA). 4 Akö, dark curve: isotype control).
  • FIG. Figure 9 shows the inhibitory in vivo effect of anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on CD28
  • lymph nodes and spleens were obtained and analyzed by FACS for the activation marker CD25.
  • a total of three experiments with varying antibody concentrations were performed.
  • Both tested CTLA-4 specific antibodies reduced the JJ316-induced CD25 expression on lymph node as well as on spleen cells in 3 independent experiments by approx. 30 - 40%. Shown is a representative result.
  • Antibodies of the invention from both IgG1 and IgG4 isotypes, effectively induce CD69 surface expression while the isotype control resp. the addition of cell culture medium without effect remained (representative result).
  • FIG. 12 shows the inhibitory effect of the antibody TGN2122.
  • a recall-response-assay 10 ⁇ 5 human PBMCs of healthy donors with 2, 5 ⁇ g / ml tetanus toxoid were activated and at the same time the corresponding CTLA-4 specific antibodies resp.
  • the isotype control given to the experimental approach.
  • the proliferation was measured by 3 H thymidine incorporation after 120 h incubation.
  • 3 H-thymidine was added to the experimental batch for the last 15-18 h of the test duration and the 3 H-Thymidineinbau determined.
  • the corresponding isotype control and an approach in which unactivated non-antibody cells were included were included. Both antibodies were able to effectively inhibit tetanus toxoid-induced proliferation. In contrast, the isotype control showed no such inhibition.
  • the superagonistic properties of antibodies according to the invention are shown since inhibition in soluble form, i. e. without artificial cross-linking, took place.
  • n is specified in sequences X, the specified n can vary by + 1.
  • MGWSCIILFLVATATGVHS Leader peptide, not part of mature HC
  • H HC nucleotide sequence (SEQ ID NO: 40)
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  • MGWSCIILFLVATATGVHS Leader peptide, not part of mature HC
  • MGWSCIILFLVATATGVHS Leader peptide, not part of mature HC
  • MGWSCIILFLVATATGVHS Leader peptide, not part of the mature LC

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Abstract

The invention relates to a CTLA-4-specific, agonistic, isolated monoclonal antibody which does not bond to the C''D loop of CTLA-4.

Description

Antikörper antibody
Gebiet der ErfindungField of the invention
Die Erfindung betrifft einen Antikörper, welcher für CTLA-4 spezifisch ist , eine pharmazeutische Zusammensetzung enthaltend einen solchen Antikörper, Nukleinsäuren codierend solche Antikörper, Vektoren enthaltend solche Antikörper, mit solchen Vektoren transfizierte Zellen, Verwendungen solcher Antikörper, Verfahren zur Herstellung solcher Antikörper sowie Verfahren zur Herstellung einer pharmazeutischen Zusammensetzung enthaltend solche Antikörper .The invention relates to an antibody which is specific for CTLA-4, a pharmaceutical composition containing such an antibody, nucleic acids encoding such antibodies, vectors containing such antibodies, cells transfected with such vectors, uses of such antibodies, methods for producing such antibodies, and methods for Preparation of a pharmaceutical composition containing such antibodies.
Hintergrund der Erfindung und Stand der TechnikBackground of the invention and prior art
T-Lymphozyten (T-Zellen) sind die Hauptakteure einer hoch effizienten Immunantwort , die den menschlichen Körper gegen eindringende Pathogene wie Bakterien und Viren schützt . Sie regulieren die molekulare Wechselwirkung zwischen verschiedenen zellulären Komponenten des Immunsystems wie dendritischen Zellen, B-Zellen, Makrophagen oder anderen T- Zellen und führen wichtige Effektor-Funktionen wie die Zerstörung von virusbefallenen Zellen oder Tumorzellen selbst durch . Dadurch nehmen sie eine Schlüsselposition bei der Initiierung und Koordinierung einer Immunantwort ein.T lymphocytes (T cells) are the main agents of a highly efficient immune response that protects the human body against invading 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 perform important effector functions such as the destruction of virus-infected cells or tumor cells themselves. As a result, they occupy a key position in the initiation and coordination of an immune response.
Hochselektive Moleküle namens T Zell-Antigen-Rezeptoren (TCR) , die sich auf der Zeil-Oberfläche befinden, geben j eder T-Zelle ihre Identität und statten sie mit der Fähigkeit zur spezifischen Erkennung von Antigenen, die von Molekülen des Haupthistokompatibilitätskomplexes (HLA) präsentiert werden, aus . Zusätzliche Zell-Oberflächen- Rezeptoren der „CD" Nomenklatur regulieren die Art und Weise der T-ZeIl Antwort , die durch antigenabhängige Stimulation des TCR eingeleitet wird . Somit diktiert der TCR dieHighly selective molecules called T cell antigen receptors (TCRs), located on the cell surface, give each T cell its identity and endow it with the ability to specifically recognize antigens derived from Molecules of the major histocompatibility complex (HLA) are out. Additional cell-surface receptors of the "CD" nomenclature regulate the manner of the T-cell response initiated by antigen-dependent stimulation of the TCR, thus dictating the TCR
Spezifität einer Immunantwort , wohingegen die CD Rezeptoren den Umfang und die Qualität der Antwort der T-Zelle kontrollieren . Unter physiologischen Bedingungen wird eine Signalkombination von TCR und mindestens einem weiteren CD Rezeptoren für die vollständige Aktivierung von T-Zellen benötigt, die insbesondere durch Proliferation und Zytokinproduktion gekennzeichnet ist . Dieser Prozess wird „Kostimulation" genannt . Das wichtigsten kostimulatorische CD Molekül auf ruhenden menschlichen T-Zellen ist das CD28 Molekül .Specificity of an immune response, whereas the CD receptors control the extent and quality of the response of the T cell. Under physiological conditions, a signal combination of TCR and at least one other CD receptor is required for the complete activation of T cells, which is characterized in particular by proliferation and cytokine production. This process is called "costimulation." The most important costimulatory CD molecule on quiescent human T cells is the CD28 molecule.
Um eine Überreaktion des Immunsystems zu vermeiden, die zu einer unkontrollierten und damit gefährlichen Vermehrung von Lymphozyten und einer massiven Ausschüttung von entzündungfördernden Zytokinen führen würde, muss dieIn order to avoid an overreaction of the immune system, which would lead to an uncontrolled and thus dangerous multiplication of lymphocytes and a massive release of inflammation - promoting cytokines, the
Aktivierung von T-Zellen effektiv abgeschaltet werden . Diese Aufgabe wird durch das Zusammenspiel einer Reihe immunologischer Kontroll-Mechanismen bewältigt . Eine besonders wichtige Rolle kommt dabei inhibitorischen Zelloberflächenrezeptoren wie dem „Cytotoxic T-Lymphocyte Antigen-4" (CTLA-4 ) Molekül zu, welches später im Detail erläutert werden wird .Activation of T cells are effectively shut down. This task is accomplished through the interaction of a number of immunological control mechanisms. A particularly important role is played by inhibitory cell surface receptors such as the "Cytotoxic T-Lymphocyte Antigen-4" (CTLA-4) molecule, which will be explained in detail later.
Bei der Entstehung von Autoimmunkrankheiten wie Rheumatoider Arthritis , Typ I Diabetis , Multipler Sklerose, Colitis , oder Psoriasis ebenso wie bei der Entstehung von Allergien spielt eine unkontrollierte Antwort von T-Lymphozyten auf körpereigene Strukturen bzw. auf externe Antigene eine wichtige Rolle . So kann eine initial überschießende Aktivierung von T-Zellen, eine fehlende Inhibition autoreaktiver T-Zellen oder auch eine mangelhafte Zahl und/oder Funktion regulatorischer T-Zellen kausal mit diesen Krankheiten zusammenhängen . Auch bei allogenenIn the development of autoimmune diseases such as rheumatoid arthritis, type I diabetes, multiple sclerosis, colitis, or psoriasis as well as in the development of allergies plays an uncontrolled response of T-lymphocytes to the body's own structures or external antigens important role . Thus, an initially excessive activation of T cells, a lack of inhibition of autoreactive T cells, or even a deficient number and / or function of regulatory T cells may be causally related to these diseases. Also in allogeneic
Organtransplantationen, d. h . bei Transplantationen unter nicht-HLA-identischen Individuen, ist eine Aktivierung der T-Zellen des Empfängers unerwünscht , da die Aktivierung der T-Zellen durch Erkennung von Allo-Antigenen die Hauptursache für eine chronische Abstoßungsreaktion ist .Organ transplants, d. H . in transplantation among non-HLA-identical individuals, activation of the T cells of the recipient is undesirable because activation of T cells by recognition of allo-antigens is the major cause of a chronic rejection reaction.
Derzeitige Therapiekonzepte zur Unterdrückung der T-ZeIl- Antwort zielen auf die antigenunspezifische Unterdrückung ' der Aktivität sowohl schädlicher als auch nützlicher T- Zellen durch „unspezifische" Immunsuppressiva ab . Dadurch sind therapeutische Effekte häufig von schwerwiegenden Nebenwirkungen begleitet .Current therapeutic approaches to suppress T-cell response targeting the antigen nonspecific suppression 'of the activity of both harmful and beneficial T cells by "non-specific" immunosuppressants from. This therapeutic effects are often accompanied by serious side effects.
CTLA-4 ' (CD152 ) ist ein Mitglied der Immunglobulin- Superfamile und strukturell der nächste Verwandte von CD28 (Lenschow DJ, Walunas TL, Bluestone JA, CD28/B7 system of T cell costimulation . Annu Rev Immunol, 1996. 14 : 233-58 ) . Im Gegensatz zu CD28 ist die physiologische Funktion von CTLA-4 aber nicht die Förderung, sondern die Hemmung der T-ZeIl- Aktivierung . CTLA-4 ist sehr schwach auf ruhenden T-Zellen und stark auf der Zelloberfläche von aktivierten und regulatorischen T-Zellen exprimiert . Die Bindung von CTLA-4 an seine natürlichen Liganden B7-1 (CD80 ) und B7-2 (CD86 ) , die von Antigen-Präsentierenden Zellen (APC) ausgeprägt werden, führt zu einer Abschaltung der T-Zell-Proliferation und zur Unterdrückung der Zytokinexpression (Egen JG, Kuhns MS, Allison JP, CTLA-4 : new inslghts into i ts biological function and use in tumor immunotherapy. Nat Immunol , 2002. 3 ( 7 ) : 611-8 ) . Die inhibitorische Funktion von CTLA-4 auf der T-Zell-Oberfläche wurde zunächst mit Hilfe immobilisierter monoklonaler Antikörper mit Spezifität für das CTLA-4 Molekül der Maus (Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB, Bluestone JA_^_ CTLA-4 can function as a nega tive regulator of T cell activation, Immunity, 1994.1 (5) : 405-13 ) und des Menschen (Blair PJ, Riley JL, Levine BL, Lee KP, Craighead N, Francomano T, Perfetto SJ, Gray GS, Carreno BM, June CH, CTLA-4 ligation dellvers a unique signal to resting humanCTLA-4 ' (CD152) is a member of the immunoglobulin superfamily and structurally the closest relative of CD28 (Lenschow DJ, Walunas TL, Bluestone JA, CD28 / B7 system of T cell costimulation., Annu Rev Immunol, 1996. 14: 233- 58). 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 weak on quiescent T cells and strongly expressed 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 a shutdown of T cell proliferation and suppression of the Cytokine expression (Egen JG, Kuhns MS, Allison JP, CTLA-4: new entry into biological function and use in tumor immunotherapy, Nat Immunol, 2002. 3 (7): 611-8). The inhibitory function of CTLA-4 on the T cell surface was first determined using immobilized monoclonal antibodies specific for mouse CTLA-4 molecule (Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB, Bluestone JA _ ^ _ CTLA-4 can function as a negative regulator of cell activation, Immunity, 1994.1 (5): 405-13) and man (Blair PJ, Riley JL, Levine BL, Lee KP, Craighead N, Francomano T, Perfetto SJ, Gray GS, Carreno BM, June CH, CTLA-4 ligation dellvers a unique signal to resting human
CD4 T cells that inhlbits interleukin-2 secretion but allows BcI-X (L) induction, J Immunol, 1998. 160 ( 1 ) : 12-5 ) gezeigt und konnte belegt werden durch den Phänotyp von Mäusen, bei denen das CTLA-4 Gen durch homologe Rekombination gezielt inaktiviert wurde . Diese Tiere sterben rasch an einer lymphoproliferativen Erkrankung, die durch eine unkontrollierte Aktivierung von T-Zellen gekennzeichnet ist (Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH, 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 , sowie Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW, Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science, 1995. 270 (5238 ) : 985-8 ) .CD4 T cells that interleukin-2 secretion but allows BCI-X (L) induction, J Immunol, 1998. 160 (1): 12-5), and could be demonstrated by the phenotype of mice receiving CTLA-4 Gene was specifically inactivated by homologous recombination. These animals rapidly die of lymphoproliferative disease characterized by uncontrolled activation of T cells (Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH, Loss of CTLA-4 leads to massive lymphoproliferation and fatal Immunity, 1995. 3 (5): 541-7, as well as Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW, Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4, Science, 1995. 270 (5238): 985-8).
Im Umkehrschluss legen diese Ergebnisse nahe, dass CTLA-4 Blockade die Aktivierung von T-Zellen in vivo verstärkt . Dementsprechend potenzierten blockierende, also antagonistische, anti-CTLA-4 Antikörper eine anti-Tumor Antwort (Chambers CA, Allison JP, Co-stimula tion in T cell responses . Curr Opin Immunol , 1997. 9 (3) : 396-404 ) , führen aber auch Autoimmunität herbei (Luhder F, Hoglund P, Allison JP, 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 ) . Diese zunächst im Maussystem gewonnenen Erkenntnisse ließen sich in ersten klinischen Versuchen auch beim Menschen bestätigen . So führte die Gabe eines blockierenden anti-human CTLA-4 Antikörpers in Einzelfällen zu einer (partiellen) Remission bei Patienten mit metastasierendem Melanom (Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, Davis T,Conversely, these results suggest that CTLA-4 blockade enhances the activation of T cells in vivo. Accordingly, blocking, ie antagonistic, anti-CTLA-4 antibodies potentiated an anti-tumor response (Chambers CA, Allison JP, Co-stimulation in cell responses, Curr Opin Immunol, 1997. 9 (3): 396-404), to lead but also autoimmunity (Luhder F, Hoglund P, Allison JP, 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 were initially obtained in the mouse system, could be confirmed in humans in the first clinical trials. In isolated cases the administration of a blocking anti-human CTLA-4 antibody led to a (partial) remission in patients with metastatic melanoma (Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, Davis T,
Henry-Spires R, MacRae S , Willman A, Padera R, Jaklitsch MT, Shankar S , Chen TC, Korman A, Allison JP, Dranoff G, Biologie activi ty of cytotoxic T lymphocyte-associa ted antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian Carcinoma pa tients, Proc Natl Acad Sei U S A, 2003. 100 ( 8 ) : 4712-7 ) . Gleichzeitig wurden aber bei einem hohen Anteil der behandelten Patienten klinische Anzeichen für Autoimmunität gefunden ( Phan GQ, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber DJ, Restifo NP, Haworth LR, Seipp CA, Freezer LJ, Morton KE, Mavroukakis SA, Duray PH, Steinberg SM, Allison JP, Davis TA, Rosenberg SA, Cancer regression and autoimmuni ty induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients wi th metasta tic melanoma , Proc Natl Acad Sei U S A, 2003. 100 ( 14 ) : 8372-7 ) .Henry Spiers R, MacRae S, Willman A, Padera R, Jaklitsch MT, Shankar S, Chen TC, Korman A, Allison JP, Dranoff G, Biology activi ty of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma equivalents, Proc Natl Acad. USA, 2003. 100 (8): 4712-7). At the same time, clinical signs of autoimmunity were found in a high proportion of treated patients (Phan GQ, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber DJ, Restifo NP, Haworth LR, Seipp CA, Freezer LJ, Morton KE, Mavroukakis SA, Duray PH, Steinberg SM, Allison JP, Davis TA, Rosenberg SA, Cancer regression and autoimmune induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients wi th metastatic melanoma, Proc Natl Acad Sei USA, 2003. 100 ( 14): 8372-7).
Ein Polymorphismus im CTLA-4 Gen, der zu einer verminderten Expression und Funktionalität des CTLA-4 Proteins führt , korreliert mit einer erhöhten Wahrscheinlichkeit von Menschen, an den Autoimmunerkrankungen Rheumatoide Arthritis ( Seidl C, Donner H, Fischer B, Usadel KH, Seifried E, Kaltwasser JP, Badenhoop K, CTLA4 codon 11 dimorphism in patients with rheumatoid arthri tis . Tissue Antigens, 1998. Jan; 51 ( 1) : 62-6) , Multiple Sklerose (Harbo HF, Celius EG, Vartdal F, Spurkland A, CTLA4 promoter and exon 1 dimorphisms in multiple sclerosis . Tissue Antigens , 1999 ; 53 ( 1 ) : 106-10 ) oder Typ I Diabetis ( Donner H, Rau H, Walfish PG, Braun J, Siegmund T, Finke R, Herwig J, Usadel KH,A polymorphism in the CTLA-4 gene that results in decreased expression and functionality of the CTLA-4 protein correlates with an increased likelihood of human involvement in the autoimmune diseases rheumatoid arthritis (Seidl C, Donner H, Fischer B, Usadel KH, Seifried E , Coldwater JP, Badenhoop K, CTLA4 codon 11 dimorphism in patients with rheumatoid arthritis Tissue Antigens, 1998. Jan; 51 (1): 62-6), multiple sclerosis (Harbo HF, Celius EG, Vartdal F, Spurkland A, CTLA4 promoter and exon 1 dimorphisms in multiple sclerosis, Tissue Antigens, 1999; 53 (1): 106-10) or Type I Diabetis (Donner H, Rau H, Walfish PG, Braun J, Siegmund T, Finke R, Herwig J, Usadel KH,
Badenhoop K, CTLA4 alanine-17 confers genetic susceptibility to Graves ' disease and to type 1 diabetes mellitus . J Clin Endocrinol Metab, 1997. 82 ( 1 ) : 143-6 ) zu erkranken .Badenhoop K, CTLA4 alanine-17 confers genetic susceptibility to Graves' disease and type 1 diabetes mellitus. J Clin Endocrinol Metab, 1997. 82 (1): 143-6).
Im Gegensatz zu der Verstärkung einer T-Zell-Antwort mit den oben beschriebene blockierenden/antagonistischen anti-CTLA-4 Antikörpern sollten agonistische anti-CTLA-4 Antikörper immunsuppressiv wirken . Dies konnte bislang aber nur für künstlich immobilisierte Antikörper überzeugend gezeigt werden . So reduzierte die gentechnologisch bewerkstelligte transmembrane Expression eines anti-CTLA-4 „Einzelketten"- Antikörpers auf künstlichen APC die TCR vermittelte Proliferation und Interleukin-2 Ausschüttung von T-Zellen (Griffin MD, Hong DK, Holman PO, Lee KM, Whitters MJ, O ' Herrin SM, Fallarino F, Collins M, Segal DM, Gaj ewski TF, Kranz DM, Bluestone JA, Blockade of T cell activa tion using a surface-linked single-chain antibody to CTLA-4 (CD152) . J Immunol, 2000. 164 ( 9) : 4433-42 ) . Die Tatsache, dass bei diesem experimentellen Ansatz nicht nur voraktivierte, sondern auch ruhende T-Zellen gehemmt wurden, zeigt, dass eine wichtige Funktion von CTLA-4 die frühe Unterdrückung des TCR Signals ist . Zu ähnlichen Ergebnissen kamen Brunner et al . (Brunner MC, Chambers CA, Chan FK, Hanke J, Winoto A, Allison JP, CTLA-4-Mediated Inhibition of early events of T cell proliferation . J Immunol, 1999.162 ( 10 ) : 5813-20 ) bei der Analyse der CTLA-4 Signalwege in naiven T Zellen . Die transmembrane Expression eines anti-CTLA-4 „Einzelketten" Antikörpers auf allogenen Tumorzellen führte zu einer Reduktion der T-ZeIl vermittelten Eliminierung dieser Tumorzellen in Mäusen (Hwang KW, Sweatt WB, Brown IE, Blank C, Gaj ewski TF, Bluestone JA, Alegre ML, 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 ) . Diese Ergebnisse zeigten, dass eine immunologische anti-Tumor Antwort oder die Abstoßung von allogenen Organtransplantaten durch effizienteIn contrast to enhancing a T cell response with the anti-CTLA-4 blocking / antagonistic antibodies described above, agonist anti-CTLA-4 antibodies should be immunosuppressive. However, this has so far only been convincingly demonstrated for artificially immobilized antibodies. Thus, the genetically engineered transmembrane expression of an anti-CTLA-4 "single chain" antibody on artificial APC reduced TCR mediated proliferation and interleukin-2 secretion of T cells (Griffin MD, Hong DK, Holman PO, Lee KM, Whitters MJ, Mammin SM, Fallarino F, Collins M, Segal DM, Gaj ewski TF, Wreath DM, Bluestone JA, 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 not only pre-activated but also quiescent T cells were inhibited in this experimental approach demonstrates that an important function of CTLA-4 is the early suppression of the TCR signal Similar results were reported by Brunner et al. (Brunner MC, Chambers CA, Chan FK, Hanke J, Winoto A, Allison JP, CTLA-4 mediated inhibition of early events of cell proliferation J Immunol, 1999, 162 (10): 5813-20) in the analysis of CTLA-4 signaling pathways in naive T cells. The transmembrane expression of an anti-CTLA-4 "single chain" antibody on allogeneic tumor cells resulted in a reduction of T cell mediated elimination of these tumor cells in mice (Hwang KW, Sweatt WB, Brown IE, Blank C, Gaj ewski TF, Bluestone JA, Alegre ML, Cutting edge: targeted ligation of CTLA-4 in vivo by membrane-bound anti-CTLA-4 antibody to prevent rejection of allogeneic cells. J Immunol, 2002.169 (2): 633-7.) These results indicated that an immunological anti-tumor response or rejection of allogeneic organ transplants through efficient
Kreuzvernetzung von CTLA-4 unterdrückt werden kann . Diese Art der gezielten Hemmung der T-Zell-Aktivierung durch CTLA- 4 Ligation in vivo konnte bislang allerdings nur mit membrangebundenem anti-CTLA-4 Antikörperkonstrukten oder mit den natürlichen membranständigen Liganden erzielt werden .Cross-linking of CTLA-4 can be suppressed. However, this type of targeted inhibition of T cell activation by CTLA-4 ligation in vivo has so far only been achieved with membrane-bound anti-CTLA-4 antibody constructs or with the natural membrane-bound ligands.
Eine entsprechende Unterdrückung der T-Zell-Antwort im Tier durch löslichen anti-CTLA-4 Antikörper ist bislang noch nicht beschrieben . Lediglich wurde die in vitro Induktion von Apoptose in voraktivierten T-Zellen durch einen CTLA-4 Antikörper mit Spezifität für die C ' D Schleife der extrazellulären Domäne von CTLA-4 beschrieben (Gribben JG , Freeman GJ, Boussiotis VA, Rennert P, Jellis CL, Greenfield E, Barber M, Restivo VA Jr, X Ke, Gray GS, Nadler LM, CTLA4 mediates antigen-specific apoptosis of human T cells . Proc Natl Acad Sei U S A, 1995. 92 ( 3 ) : 811-5) .A corresponding suppression of the T cell response in animals by soluble anti-CTLA-4 antibody is not yet described. Only the in vitro induction of apoptosis in preactivated T cells was described by a CTLA-4 antibody specific for the C 'D loop of the extracellular domain of CTLA-4 (Gribben JG, Freeman GJ, Boussiotis VA, Rennert P, Jellis CL Greenfield E, Barber M, Restivo VA Jr, X Ke, Gray GS, Nadler LM, CTLA4 mediates antigen-specific apoptosis of human T cells, Proc Natl Acad Sci USA, 1995. 92 (3): 811-5).
Zusammenfassend zeigen die bislang erhaltenen Erkenntnisse eine inhibitorische Funktion von CTLA-4 auf T-Zellen, deren Wirkmechanismus aber noch nicht vollständig verstanden ist . Es werden folgende Mechanismen, die sich nicht gegenseitig ausschließen, diskutiert : i) Unterdrückung des aktivierenden TCR und/oder CD28 Signalweges , ii ) Kompetition der CD28- vermittelten Kostimulation durch höhere Affinität zu CD80 und CD86, iii) Erhöhung des Schwellenwertes der T-ZeIl- Aktivierung, iv) Attenuierung der T-Zell-Expansion, und/oder v) Aktivierung von regulatorischen T-Zellen und damit verbunden indirekte Hemmung von konventionellen T Zellen .In summary, the findings so far show an inhibitory function of CTLA-4 on T cells, whose mechanism of action is not fully understood. The following mechanisms, which are not mutually exclusive, are discussed: i) suppression of the activating TCR and / or CD28 signaling pathway, ii) competition of CD28-mediated costimulation through higher affinity for CD80 and CD86, iii) increasing the threshold of T cell activation, iv) attenuating T cell expansion, and / or v) activating regulatory T cells and, thus, indirectly inhibiting conventional T cells.
In den vorstehend genannten Indikationen wäre eine gezielte und gut verträgliche Inaktivierung von T-Zellen durch die Stimulation der inhibitorischen Funktion von CTLA-4 wünschenswert .Targeted and well-tolerated inactivation of T cells by stimulating the inhibitory function of CTLA-4 would be desirable in the above-mentioned indications.
Technisches Problem der Erfindung .Technical problem of the invention.
Der Erfindung liegt daher das technische Problem zu Grunde, Substanzen und pharmazeutische Zusammensetzung anzugeben, die zur Stimulation der inhibitorischen Funktion von CTLA-4 in der Lage sind.The invention is therefore based on the technical problem of providing substances and pharmaceutical compositions capable of stimulating the inhibitory function of CTLA-4.
Grundzüge der Erfindung und bevorzugte Ausführungsformen .Broad features of the invention and preferred embodiments.
Zur Lösung dieses technischen Problems lehrt die Erfindung einen Isolierten monoklonalen Antikörper, welcher für CTLA-4 spezifisch und agonistisch ist, wobei die schwere Kette des Antikörpers eine Sequenz enthält, welche ausgewählt ist aus der Gruppe bestehend aus ( Seq. -ID) : „22 , 23 , 24 , 25 , 26, 27 , 28 , 29, 30 , und 32" . Die leichte Kette des Antikörpers kann eine Sequenz enthalten, welche ausgewählt ist aus der Gruppe bestehend aus ( Seq . -ID) : „33 , 34 , 35 , 36, 37 , und 38" .To solve this technical problem, the invention teaches an isolated monoclonal antibody specific and agonistic to CTLA-4, the heavy chain of the antibody containing a sequence selected from the group consisting of (Seq. ID): "22 , 23, 24, 25, 26, 27, 28, 29, 30, and 32 ". The light chain of the antibody may contain a sequence selected from the group consisting of (Seq. ID):" 33, 34 , 35, 36, 37, and 38 ".
Bevorzugt ist ein erfindungsgemäßer Antikörper mit einer schweren Kette enthaltend eine Sequenz gemäß Seq. -ID 27 , 28 , oder 29 , vorzugsweise enthaltend oder bestehend aus der Sequenz gemäß Seq . -ID 30 oder 32 , sowie mit einer leichten Kette enthaltend eine Sequenz gemäß Seq . -ID 36 oder 37 , vorzugsweise enthaltend oder bestehend aus einer Sequenz gemäß Seq . -ID 38.An antibody according to the invention with a heavy chain containing a sequence according to Seq. ID 27, 28, or 29, preferably containing or consisting of Sequence according to Seq. ID 30 or 32, and with a light chain containing a sequence according to Seq. ID 36 or 37, preferably containing or consisting of a sequence according to Seq. ID 38.
Ein spezieller Antikörper mit dem vorstehenden grundsätzlichen Aufbau sind die folgend beschriebenen Antikörper TGN2122. H und TGN2422. H .A specific antibody having the above basic structure are the following antibodies TGN2122. H and TGN2422. H .
Des Weiteren lehrt die Erfindung einen isolierten monoklonalen Antikörper, welcher für CTLA-4 spezifisch und agonistisch ist, wobei die schwere Kette des Antikörpers eine Sequenz enthält , welche ausgewählt ist aus der Gruppe bestehend aus ( Seq. -ID) : „43 , 44 , 45 , 46, 47 , 48 , 49, 50 , 51 , und 53" . Die leichte Kette des Antikörpers kann eine Sequenz enthalten, welche ausgewählt ist aus der Gruppe bestehend aus ( Seq . -ID) : „54 , 55 , 56, 57 , 58 , und 59λΛ .Furthermore, the invention teaches an isolated monoclonal antibody which is specific and agonistic to CTLA-4, the heavy chain of the antibody containing a sequence 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 may contain a sequence selected from the group consisting of (Seq. ID):" 54, 55, 56, 57, 58, and 59 λΛ .
Bevorzugt ist ein insofern auch erfindungsgemäßer Antikörper mit einer schweren Kette enthaltend eine Sequenz gemäß Seq . - ID 48 , 49, oder 50 , vorzugsweise enthaltend oder bestehend aus der Sequenz gemäß Seq. -ID 51 oder 53 , sowie mit einer leichten Kette enthaltend eine Sequenz gemäß Seq. -ID 57 oder 58 , vorzugsweise enthaltend oder bestehend aus einer Sequenz gemäß Seq. -ID 59.Preference is given to an antibody according to the invention with a heavy chain containing a sequence according to Seq. ID 48, 49, or 50, preferably containing or consisting of the sequence according to Seq. ID 51 or 53, as well as with a light chain containing a sequence according to Seq. ID 57 or 58, preferably containing or consisting of a sequence according to Seq. ID 59.
Ein spezieller Antikörper mit dem vorstehenden grundsätzlichen Aufbau sind die folgend beschriebenen Antikörper TGN2122. C und TGN2422. C .A specific antibody having the above basic structure are the following antibodies TGN2122. C and TGN2422. C.
Bei den vorstehend genannten Antikörpern handelt es sich um humanisierte Antikörper . Da es sich um einen bereits humanisierten Antikörper handelt, ist eine Humanisierung, wie folgend für weitere Varianten erfindungsgemäßer Antikörper, nicht erforderlich . Der Antikörper kann, muß aber nicht an das C ' ' D-Loop von CTLA-4 binden . Vielmehr kann es sich auch um einen Antikörper handeln, der nicht an dieses Loop bindet . In Hinblick auf alle weiteren Ausführungsformen sowie Verwendungen und sonstiger Details und Erläuterungen gelten die folgend für eine weitere Variante der Erfindung angebrachten Erläuterungen analog und vollumfanglich .The antibodies mentioned above are humanized antibodies. Since it is already one humanized antibody, as described below for other variants of antibodies according to the invention is not required. The antibody may or may not bind to the C '' D loop of CTLA-4. Rather, it may also be an antibody that does not bind to this loop. With regard to all other embodiments as well as uses and other details and explanations, the following attached to a further variant of the invention explanations apply analogously and fully.
Schließlich lehrt die Erfindung einen isolierten monoklonalen Antikörper, welcher für CTLA-4 spezifisch und agonistisch ist, wobei der Antikörper nicht an eine CTLA-4 Teilsequenz gemäß SEQ . -ID 1 bindet . Bei der Sequenz gemäß SEQ. _ID 1 handelt es sich um das C ' D-Loop des CTLA-4. Mit anderen Worten ausgedrückt , bindet ein erfindungsgemäßer Antikörper an andere Bereiche des CTLA-4 Moleküls als das C ' ' D-Loop . Die Erfindung beruht auf der Erkenntnis , dass eine agonistische Stimulation von CTLA-4 , i . e . die inhibitorische Aktivität von CTLA-4 in vivo induzierend, ein sinnvolles therapeutisches Konzept für Autoimmunerkrankungen oder Transplantationen darstellt, und stellt hierfür geeignete Wirkstoffe in Form der Antikörpern oder Fragmenten hiervon zur Verfügung .Finally, the invention teaches an isolated monoclonal antibody which is specific and agonistic to CTLA-4, wherein the antibody does not bind to a CTLA-4 partial sequence according to SEQ. ID 1 binds. In the sequence according to SEQ. ID 1 is the C 'D loop of the CTLA-4. In other words, an antibody of the invention binds to other regions 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 inhibitory activity of CTLA-4 in vivo, constitutes a useful therapeutic concept for autoimmune diseases or transplantation, and provides suitable drugs in the form of the antibodies or fragments thereof.
Vorzugsweise enthält ein erfindungsgemäßer Antikörper zumindest eine der Sequenzen gemäß SEQ . -ID 2 bis SEQ . -ID 7 oder SEQ . -ID 8 bis SEQ . -ID 13. Bei diesen Sequenzen handelt handelt es sich um die CDRs der variablen Bereiche einer schweren und einer leichten Kette, wobei ergänzend auf die Tabelle 2 verwiesen wird . Vorzugsweise ist ein erfindungsgemäßer Antikörper humanisiert . Dies kann in fachüblicher Weise erfolgen, bespielsweise indem ein gegen Human CTLA-4 spezifischer monoklonaler Maus Antikörper dahingehend chimärisiert wird, dass die konstanten Bereiche gegen humane oder humanverträgliche konstanten Bereiche ausgetauscht werden . Wesentlich ist, dass vorzugsweise alle CDRs gemäß Tabelle 2 erhalten bleiben und zwar auch in ihrer räumlichen Anordnung zueinander . Als Basis für die Humanisierung können beispielsweise monoklonale Antikörper dienen, die zumindest eine, vorzugsweise alle, Sequenzen gemäß SEQ . -ID 2 bis SEQ . - ID 7 oder SEQ . -ID 8 bis SEQ . -ID 13 enthalten, beispielsweise eine der Sequenzen gemäß SEQ . -ID 14 bis 17 , enthalten . Konkret können erfindungsgemäße Antikörper auch eine der Sequenzen gemäß SEQ . -ID 18 bis 21 enthalten . Geeignete Ausführungsbeispiele von Antikörpern, die eine Basis zur Humanisierung bilden, sind die folgend im Detail beschriebenen Antikörper 4.8H10H5 und 4.3F6B5. Eine Herstellung humanisierter Antikörper hieraus ist in fachüblicher Weise beispielsweise durch gentechnologische Humanisierungsstrategien möglich .Preferably, an antibody according to the invention contains at least one of the sequences according to SEQ. ID 2 to SEQ. ID 7 or SEQ. ID 8 to SEQ. -ID 13. These sequences are the CDRs of the heavy and light chain variable regions, with additional reference to Table 2. Preferably, an antibody according to the invention is humanized. This can be done in a customary manner, for example by using a human CTLA-4-specific monoclonal mouse antibody is chimaerized in such a way that the constant regions are exchanged for human or human-compatible constant regions. It is essential that preferably all CDRs according to Table 2 are maintained and indeed in their spatial arrangement to each other. As a basis for the humanization, for example, monoclonal antibodies can be used which at least one, preferably all, sequences according to SEQ. ID 2 to SEQ. ID 7 or SEQ. ID 8 to SEQ. ID 13, for example one of the sequences according to SEQ. ID 14 to 17, included. Specifically, antibodies according to the invention can also be one of the sequences according to SEQ. ID 18 to 21 included. Suitable embodiments of antibodies which form a basis for humanization are the antibodies 4.8H10H5 and 4.3F6B5 described in detail below. A preparation of humanized antibodies from this is possible in a conventional manner, for example by genetic engineering humanization strategies.
Der Begriff des Antikörpers umfasst im Rahmen der Erfindung neben den explizit offenbarten Strukturen auch funktional entsprechende Antikörper, welche beispielsweise durch Chimerisierung, Humanisierung oder De-Immunisierung (Ausschneiden von T-Zellepitopen aus dem humanen Antikörper, die unerwünschte Immunreaktionen auslösen) modifiziert sind, sowie spezifische Fragmente der leichten und/oder der schweren Kette des variablen Bereiches zu Grunde liegender Antikörper vorstehender Art . Die Herstellung bzw . Gewinnung solcher Antikörper mit vorgegebenen Immunogenen ist dem Durchschnittsfachmann wohl vertraut und braucht nicht näher erläutert zu werden .In the context of the invention, the term "antibody" encompasses, in addition to the structures explicitly disclosed, also functionally corresponding antibodies which have been modified, for example, by chimerization, humanization or de-immunization (excision of T-cell epitopes from the human antibody which trigger undesired immune reactions) and specific antibodies Fragments of the light and / or heavy chain variable region underlying antibody of the above type. The production resp. Obtaining such antibodies with given immunogens is the A person of ordinary skill in the art is familiar and need not be explained in more detail.
Die Erfindung betrifft auch ein isoliertes Protein oder Peptid enthaltend zumindest eine der Sequenzen SEQ . -ID 2 bis 13 , insbesondere eine der Sequenzen SEQ . -ID 14 bis 17 oder SEQ . -ID 18 bis 21 , oder eine der Sequenzen Seq. -ID 22 , 23 , 24 , 25 , 26, 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36, 37 , 38 , oder 39, insbesondere eine der Sequenzen Seq. -ID 27 , 28 , 29 , 30 , 32 , 36, 37 , oder 38 , oder eine der Sequenzen Seq. -ID 43 , 44 , 45 , 46, 47 , 48 , 49, 50 , 51 , 52 , 53 , 54 , 55 , 56, 57 , 58 , 59, oder 60 , insbesondere eine der Sequenzen Seq . -ID 48 , 49 , 50 , 51 , 53 57 , 58 , oder 59, oder bestehend aus einer der genannten Sequenzen, eine isolierte Nukleinsäure codierend für ein solches Protein oder Peptid oder für eine leichte Kette und/oder schwere Kette eines erfindungsgemäßen Antikörpers , einen isolierten Vektor enthaltend eine solche Nukleinsäure, und eine isolierte Zelle, welche mit einem solchen Vektor transfiziert ist . Die vorstehenden Gegenstände sind alle zur Herstellung bzw . Konstruktion erfindungsgemäßer Antikörper geeignet .The invention also relates to 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 said sequences, an isolated nucleic acid coding for such a protein or peptide or for a light chain and / or heavy chain of an antibody according to the invention, an isolated vector containing such a nucleic acid, and an isolated cell transfected with such a vector. The above objects are all for the production or. Construction of antibodies according to the invention suitable.
Ein erfindungsgemäßer Antikörper bzw . ein erfindungsgemäßes Protein oder Peptid ist vorzugsweise in Wasser, insbesondere in physiologischer Kochsalzlösung, löslich, i . e . nicht artifiziell vernetzt . Des Weiteren ist ein erfindungsgemäßer Antikörper superagonistisch, i . e . stimuliert die physiologische Wirkweise des T-ZeIl inhibitorischen Rezeptors CTLA-4.An inventive antibody or. a protein or peptide according to the invention is preferably soluble in water, in particular in physiological saline solution, i. e. not artificially networked. Furthermore, an antibody according to the invention is superagonistic, i. e. stimulates the physiological action of the T-cell inhibitory receptor CTLA-4.
Die Erfindung betrifft des weiteren eine pharmazeutische Zusammensetzung enthaltend einen erfindungsgemäßen monoklonalen Antikörper und/oder ein erfindungsgemäßes Protein oder Peptid, sowie optional zumindest einen physiologisch verträglichen Trägerstoff und/oder Hilfsstoff, welche später im Detail erläutert werden . Sie ist durch Mischung dieser Komponenten erhältlich, wobei der Wirkstoff in physiologisch wirksamer Dosis eingesetzt wird. Diese Dosis läßt sich leicht in in-vitro Versuchen mit Zellen sowie in Tierversuchen in fachüblicher Weise ermitteln . Eine solche pharmazeutischen Zusammensetzung ist zur Prophylaxe und/oder Therapie einer Erkrankung oder eines Zustandes aus der Gruppe bestehend aus „Rheumatoider Arthritis , Typ I Diabetis , Multipler Sklerose, Systemischer Lupus Erythematodes , Psoriasis , Colitis Ulerosa, Morbus Crohn, Allergien, Abstoßung von allogenen Organtransplantanten, insbesondere Organtransplantaten der folgenden Organe : Herz , Niere, Leber, Bauchspeicheldrüse, Lunge, Knochenmark, und „Graft-versus-Host"- Erkrankung" geeignet . Insofern umfasst die Erfindung auch ein Verfahren zur Prophylaxe und/oder Behandlung einer vorstehenden Erkrankung oder eines vorstehenden Zustandes , wobei einem Patienten diem pharmazeutische Zusammensetzung in geeigneter Dosierung dargereicht wird.The invention further relates to a pharmaceutical composition containing a monoclonal antibody according to the invention and / or an inventive Protein or peptide, and optionally at least one physiologically acceptable carrier and / or excipient, which will be explained in detail later. It is obtainable by mixing these components, the active ingredient being used in a physiologically effective dose. This dose can be easily determined in in-vitro experiments with cells and in animal experiments in the usual way. Such a pharmaceutical composition is for the prophylaxis and / or therapy of a disease or condition selected from the group consisting of "rheumatoid arthritis, type I diabetes, multiple sclerosis, systemic lupus erythematosus, psoriasis, ulcerative colitis, Crohn's disease, allergies, rejection of allograft organ transplant , in particular organ transplants of the following organs: heart, kidney, liver, pancreas, lung, bone marrow, and "graft-versus-host" disease suitable. Thus, the invention also includes a method for the prophylaxis and / or treatment of a above disease or condition wherein a patient is given the pharmaceutical composition in suitable dosage.
Die galenische Herrichtung einer erfindungsgemäßen pharmazeutischen Zusammensetzung kann in fachüblicher Weise erfolgen . Als Gegenionen für ionische Verbindungen kommen beispielsweise Na+, K+, Li+ oder Cyclohexylammonium in Frage . Geeignete feste oder flüssige galenische Zubereitungsformen sind beispielsweise Granulate, Pulver, Dragees , Tabletten, (Mikro-) Kapseln, Suppositorien, Sirupe, Säfte, Suspensionen, Emulsionen, Tropfen oder Lösungen zur Inj ektion ( i . V. , i . p . , i .m. , s . c . ) oder Vernebelung (Aerosole) , transdermale Systeme, sowie Präparate mit protrahierter Wirkstoff-Freigabe, bei deren Herstellung übliche Hilfsmittel wie Trägerstoffe, Spreng- , Binde-, Überzugs-, Quellungs-, Gleit- oder Schmiermittel, Geschmacksstoffe, Süßungsmittel und Lösungsvermittler, Verwendung finden . Als Hilfsstoffe sei Magnesiumcarbonat , Titandioxid, Lactose, Mannit und andere Zucker, Talcum,The galenic preparation of a pharmaceutical composition according to the invention can be carried out in the usual way. Suitable counterions for ionic compounds are, for example, Na + , K + , Li + or cyclohexylammonium. Suitable solid or liquid galenic preparations are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or solutions for injection (i.v., i.p., i .m., s. c.) or nebulization (aerosols), transdermal systems, as well as preparations with protracted release of active ingredient, in their preparation conventional adjuvants such as carriers, blasting, binding, coating, swelling, lubricants or lubricants, flavoring agents, sweeteners and solubilizers, are used. As adjuvants may be magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talcum,
Milcheiweiß , Gelatine, Stärke, Zellulose und ihre Derivate, tierische und pflanzliche Öle wie Lebertran, Sonnenblumen-, Erdnuss- oder Sesamöl , Polyethylenglycole und Lösungsmittel,, wie etwa steriles Wasser und ein- oder mehrwertige Alkohole, beispielsweise Glycerin, genannt .Milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycols and solvents, such as sterile water and monohydric or polyhydric alcohols, such as glycerol, called.
Die Erfindung betrifft des Weiteren ein Verfahren zur Herstellung eines erfindungsgemäßen monoklonalen Antikörpers , wobei eine erfindungsgemäße Nukleinsäure in einen Vektor eingebracht wird, wobei mittels des Vektors eine Zelle transfiziert wird, wobei die transfizierte Zelle kultiviert wird, wobei ein Überstand von der kultivierte Zellen abgetrennt oder wobei die kultivierte Zelle lysiert und das Lysat gewonnen wird, und wobei aus dem abgetrennten Überstand oder dem Lysat die monoklonalen Antikörper abgetrennt werden .The invention further relates to a method for producing a monoclonal antibody according to the invention, wherein a nucleic acid according to the invention is introduced into a vector, wherein a cell is transfected by means of the vector, wherein the transfected cell is cultured, wherein a supernatant is separated from the cultured cells or the cultured cell is lysed and the lysate is recovered, and the monoclonal antibodies are separated from the separated supernatant or lysate.
Im Folgenden wird die Erfindung anhand von lediglich Ausführungsformen darstellenden Beispielen näher erläutert ,In the following, the invention will be explained in more detail by means of examples which represent only embodiments,
Beispiel 1 : erfindungsgemäße Antikörper undExample 1: Antibodies according to the invention and
VergleichsantikörperComparison Antibodies
In Tabelle 1 sind die Bindungseigenschaften von 4 neuen anti-CTLA-4 Antikörpern dargestellt, wovon 2 nicht an das C " D-Loop von CTLA-4 binden ( 4.3F6B5 und 4.8H10H5 ) und 2 dagegen hieran binden ( 3.7F10A2 und 4.7A8H6) . Letztere sind Vergleichsantikörper und sind nicht Gegenstand der vorliegenden Erfindung . Untersucht wurde die Spezifität der Antikörper für humanes CTLA-4 , sowohl auf transfizierten Jurkat E6.1 Zellen wie auch auf ex vivo aktivierten humanen PBMCs ( Peripherer Blut-mononukleärer Zellen) . Die Kreuzreaktivität gegen Ratten CTLA-4 wurde mit einer transfizierten BW Zelllinie gezeigt , die die extrazelluläre Domäne von Ratten CTLA-4 auf der Oberfläche trägt . Ebenso wurde die Kreuzreaktivität gegen die nahe verwandten T-Table 1 shows the binding properties of 4 new anti-CTLA-4 antibodies, 2 of which do not bind to the C "D loop of CTLA-4 (4.3F6B5 and 4.8H10H5) and 2 however, bind to it (3.7F10A2 and 4.7A8H6). The latter are reference antibodies and are not the subject of the present invention. The specificity of the antibodies to human CTLA-4 was investigated, both on transfected Jurkat E6.1 cells and on ex vivo activated human PBMCs (peripheral blood mononuclear cells). Cross-reactivity against rat CTLA-4 was demonstrated with a transfected BW cell line carrying the extracellular domain of rat CTLA-4 on the surface. Likewise, the cross-reactivity against the closely related T-
Zellrezeptoren CD28 und ICOS auf transfizierten Jurkat E6.1 bzw . L929 Zellen ausgeschlossen . Die Bindung oder Nichtbindung an die laterale C V N D Loop Struktur ist ausführlich in Fig . 2 dargestellt . Die dicken Kurven stehen für CTLA-4 und die dünnen Kurven für die Isotypkontrolle .Cell receptors CD28 and ICOS on transfected Jurkat E6.1 resp. L929 cells excluded. The binding or non-binding to the lateral C VN D loop structure is described in detail in FIG. 2 shown. The thick curves represent CTLA-4 and the thin curves represent the isotype control.
Fig . 1 zeigt exemplarisch die wichtigstenFig. 1 shows the most important examples
Bindungscharakteristika des erfindungsgemäßen anti-CTLA-4 Antikörpers 4.8H10H5 sowie des Vergleichsantikörpers 3.7F10A2. (A) Zur Identifizierung CTLA-4 spezifischer Antikörper wurden transfizierte Jurkat E6.1 Zellen verwendet, die einen Chimären CTLA-4 / CD28 Rezeptor auf ihrer Oberfläche tragen . Dieser besteht aus der extrazellulären Domäne von humanem CTLA-4 , welche die Spezifität der Antikörper begründet, und der transmembranen und intrazellulären Domäne von Maus CD28. Der CD28 Teil des Rezeptor gewährleistet eine stabile Oberflächenexpression des Chimären Rezeptors . Gezeigt ist die Bindung der Antikörper an transfizierte Zellen (dicke Kurve) im Vergleich zur Bindung an nicht-transfizierte Zellen (dünne Kurve) . (B) Die Spezifität der Antikörper für CTLA-4 wurde mit humanen PBMCs bestätigt, die zuvor ex vivo mit PHA/ IL-2 stimuliert wurden . In ruhenden Zellen ist CTLA-4 vorwiegend intrazellulär lokalisert und wird erst nach Aktivierung an die Oberfläche gebracht . Die dicke Kurve zeigt die Bindung der Antikörper, die dünne Kurve die Bindung der Isotypkontrolle an aktivierte humane PBMCs . (C) In Hinblick auf eine mögliche Verwendbarkeit der Antikörper inBinding characteristics of the inventive anti-CTLA-4 antibody 4.8H10H5 and the reference antibody 3.7F10A2. (A) Transfected Jurkat E6.1 cells carrying a chimeric CTLA-4 / CD28 receptor on their surface were used to identify CTLA-4 specific antibodies. This consists of the extracellular domain of human CTLA-4, which establishes the specificity of the antibodies, and the transmembrane and intracellular domain of mouse CD28. The CD28 part of the receptor ensures stable surface expression of the chimeric receptor. Shown is the binding of antibodies to transfected cells (thick curve) compared to binding to non-transfected cells (thin curve). (B) The specificity of antibodies to CTLA-4 was confirmed with human PBMCs previously stimulated ex vivo with PHA / IL-2. In resting cells, CTLA-4 is predominant localizes intracellularly and is brought to the surface only after activation. The thick curve shows the binding of the antibodies, the thin curve the binding of the isotype control to activated human PBMCs. (C) In view of a possible usability of the antibodies in
Tiermodellen wurde die Kreuzreaktivität gegen Ratten- CTLA-4 gezeigt . Hierzu wurden transfizierte BW Zellen verwendet, die einen Chimären humanes CTLA-4 / Maus CD28 Rezeptor auf ihrer Oberfläche tragen . Gezeigt ist die Bindung des Antikörpers an transfizierte Zellen (dicke Kurve) bzw . an nicht transfizierte Zellen (dünne Kurve) .Animal models demonstrated cross-reactivity to rat CTLA-4. Transfected BW cells carrying a chimeric human CTLA-4 / mouse CD28 receptor on their surface were used for this purpose. Shown is the binding of the antibody to transfected cells (thick curve) or. to non-transfected cells (thin curve).
Fig . 2 zeigt die nicht vorhandene Spezifität von 2 von 4 anti-CTLA-4 Antikörpern aus Tabelle 1 für den C " " D Loop . Überraschender Weise zeigte sich, dass die beiden erfindungsgemäßen Antikörper 4.3F6B5 und 4.8H10H5 , welche in funktionellen Assays agonistische Wirkweise zeigten ( siehe Beispiel 2 ) , nicht spezifisch sind für den menschlichen C ' D Loop . Dazu wurden Jurkat E6.1 verwendet, die eine chimäre extrazelluläre Domäne von CTLA-4 auf der Oberfläche exprimeren : diese Chimäre besteht aus dem murinen Rezeptor, bei dem der C S D Loop gegen die entsprechende humane Sequenz , dargestellt durch die Aminosäuren Pos 68 - 83 , ausgetauscht wurde . Die Bindung der Antikörper 3.7F10A2 und 4.7A8H6 an den C ' D Loop zeigt , dass das Konstrukt effizient exprimiert wurde . Für die Antikörper 3.7F10A2 und 4.7A8H6 ist diese Aminosäuresequenz zur Bindung ausreichend (dicke Linie, dünne Kurve : Isotypkontrolle ) . Für die Antikörper 4.8H10H5 und 4.3F6B5 ist diese Sequenz nicht hinreichend zur Bindung .Fig. Figure 2 shows the nonexistent specificity of 2 of 4 anti-CTLA-4 antibodies from Table 1 for the C "" D loop. Surprisingly, it was found that the two antibodies according to the invention 4.3F6B5 and 4.8H10H5, which showed agonistic mode of action in functional assays (see Example 2), are not specific for the human C 'D loop. For this purpose Jurkat E6.1 was used, which expresses a chimeric extracellular domain of CTLA-4 on the surface: this chimera consists of the murine receptor, in which the C S D loop against the corresponding human sequence, represented by the amino acids Pos 68 - 83, was exchanged. The binding of the antibodies 3.7F10A2 and 4.7A8H6 to the C 'D loop shows that the construct was efficiently expressed. For the antibodies 3.7F10A2 and 4.7A8H6, this amino acid sequence is sufficient for binding (thick line, thin curve: isotype control). For antibodies 4.8H10H5 and 4.3F6B5 this sequence is not sufficient for binding.
Fig . 3 zeigt , dass die Bindung der anti-CTLA-4 Antikörper 4.8H10H5 und 4.3F6B5 an CTLA-4 durch Zugabe von rekombinantem CD80 kompetiert werden kann . Das Ergbenis legt nahe, dass 4.8H10H5 und 4.3F6B5 in der Nähe der Bindungsstelle für CD80 , d . h . die MYPPPY Schleife, binden, nicht aber an den C ' D Loop . Ziel dieses Experimentes war es , die Bindeeigenschaften der Antikörper weiter einzugrenzen . Jurkat E6.1 Zellen, die die extrazelluläre Domäne auf ihrer Oberfläche tragen wurden mit einer steigenden Konzentration CD80Fc Protein und j e lμg/ ml CTLA- 4 spezifischer Antikörper inkubiert . Die Ko-Inkubation von rekombinantem Protein und Antikörper führt zu einerFig. Figure 3 shows that binding of anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 to CTLA-4 by addition of recombinant CD80 can be competed. The Ergbenis suggests that 4.8H10H5 and 4.3F6B5 are located near the binding site for CD80, d. H . bind the MYPPPY loop, but not to the C 'D loop. The aim of this experiment was to further narrow the binding properties of the antibodies. Jurkat E6.1 cells carrying the extracellular domain on their surface were incubated with increasing concentration of CD80Fc protein and 1 μg / ml of CTLA-4 specific antibody. The co-incubation of recombinant protein and antibody leads to a
Verdrängung der Bindung der Antikörper 4.8H10H5 und 4.3F6B5 an die extrazelluläre Domäne von CTLA-4.Displacement of the binding of antibodies 4.8H10H5 and 4.3F6B5 to the extracellular domain of CTLA-4.
Experimente, wie zur Figur 1 beschrieben, wurden entsprechend für die Antikörper TGN2122. C, TGN2422. C,Experiments as described for FIG. 1 were done accordingly for the antibodies TGN2122. C, TGN2422. C.,
TGN2122. H und TGN2422. H durchgeführt . Figur 10 zeigt die Bindung der humanisierten Antikörper an die extrazelluläre Domäne von humanem CTLA-4. Dargestellt ist die Bindung der humanisierten Antikörper an transfizierte Zellen (dicke Kurve) im Vergleich zur Bindung der Isotypenkontrolle (dünne Kurve) an dieselben Zellen . Die FACS Analyse zeigt, dass im Verlauf der Humanisierung die Spezifität der Antikörper für humanes CTLA-4 erhalten bleibt .TGN2122. H and TGN2422. H performed. Figure 10 shows the binding of the humanized antibodies to the extracellular domain of human CTLA-4. Shown is the binding of the humanized antibodies to transfected cells (thick curve) compared to binding of the isotype control (thin curve) to the same cells. The FACS analysis shows that in the course of humanization the specificity of the antibodies to human CTLA-4 is maintained.
Die Tabelle 2 gibt die Sequenzen der erfindungsgemäßenTable 2 gives the sequences of the invention
Antikörper 4.8H10H5 und 4.3F6B5 wieder, und zwar unterteilt in schwere und leichte Ketten, wobei die Grenze zwischen den variablen Bereichen und den konstanten Bereichen markiert ist . Die Sequenzen wurden mittels RT- PCR bzw . mittels Protein- Sequenzierung (Edman Abbau) ermittelt .Antibodies 4.8H10H5 and 4.3F6B5 again, divided into heavy and light chains, with the border between the variable regions and the constant regions labeled. The sequences were determined by RT-PCR or. determined by protein sequencing (Edman degradation).
Die Tabelle 3 zeigt Sequenzen der schweren Kette des Antikörpers TGN2122. H . Die Tabelle 4 zeigt die für die schwere Kette codierende Nukleinsäure . Die Tabelle 5 zeigt Sequenzen der schweren Kette des Antikörpers TGN2422. H . Die Tabelle 6 zeigt die für die schwere Kette codierende Nukleinsäure . Die Tabelle 7 zeigt Sequenzen der leichten Kette für beide Antikörper TGN2122. H und TGN2422. H . Die Tabelle 8 zeigt die für die leichte Kette codierende Nukleinsäure .Table 3 shows heavy chain sequences of the antibody TGN2122. H . Table 4 shows the for the heavy chain coding nucleic acid. Table 5 shows heavy chain sequences of the antibody TGN2422. H . Table 6 shows the heavy chain coding nucleic acid. Table 7 shows light chain sequences for both antibodies TGN2122. H and TGN2422. H . Table 8 shows the light chain encoding nucleic acid.
Die Tabelle 9 zeigt Sequenzen der schweren Kette des Antikörpers TGN2122. C . Die Tabelle 10 zeigt die für die schwere Kette codierende Nukleinsäure . Die Tabelle 11 zeigt Sequenzen der schweren Kette des Antikörpers TGN2422. C . Die Tabelle 12 zeigt die für die schwere Kette codierende Nukleinsäure . Die Tabelle 13 zeigt Sequenzen der leichten Kette für beide Antikörper TGN2122. C und TGN2422. C . Die Tabelle 14 zeigt die für die leichte Kette codierende Nukleinsäure .Table 9 shows heavy chain sequences of the antibody TGN2122. C. Table 10 shows the heavy chain coding nucleic acid. Table 11 shows heavy chain sequences of the antibody TGN2422. C. Table 12 shows the heavy chain coding nucleic acid. Table 13 shows light chain sequences for both antibodies TGN2122. C and TGN2422. C. Table 14 shows the light chain encoding nucleic acid.
Bei den Sequenzen Seq . -ID 31 , 39 , 52 , und 60 handelt es sich um Leaderpeptide, die bei den j eweiligen reifen Ketten nicht enthalten sind . Daher sind solche Antikörper bevorzugt , die diese Sequenzen nicht enthalten .In the sequences Seq. IDs 31, 39, 52, and 60 are leader peptides that are not included in the current mature chains. Therefore, those antibodies are preferred which do not contain these sequences.
Die Antikörper TGN2122. C ( Isotyp IgGl ) und TGN2422. C ( Isotyp IgG4 ) wurden aus dem Maus Antikörper 4.3F6B5 durchThe antibodies TGN2122. C (isotype IgG1) and TGN2422. C (isotype IgG4) were purified from mouse antibody 4.3F6B5
Humanisierung erhalten . Die Antikörper TGN2122. H ( Isotyp IgGl ) und TGN2422. H ( Isotyp IgG4 ) wurden aus dem Maus Antikörper 4.8H10H5 erhalten .Humanization received. The antibodies TGN2122. H (isotype IgG1) and TGN2422. H (isotype IgG4) were obtained from mouse antibody 4.8H10H5.
Beispiel 2 : Wirkung erfindungsgemäßer Antikörper gegenüber einem Vergleichsantikörper, der an das C ' D Loop bindet, sowie kommerziell erhältlichen anti-CTLA-4 Antikörpern .Example 2: Effect of Antibodies According to the Invention on a Comparative Antibody Attached to C 'D Loop binds, as well as commercially available anti-CTLA-4 antibodies.
Fig . 4 zeigt den inhibitorischen Effekt des anti-CTLA-4 Antikörpers 4.8H10H5 auf die Proliferation humaner PBMCs .Fig. Figure 4 shows the inhibitory effect of anti-CTLA-4 antibody 4.8H10H5 on the proliferation of human PBMCs.
Ziel dieses Proliferations- Inhibitions Assays war es , einen im Vergleich zu bereits bekannten CTLA-4 spezifischen Antikörpern funktionell neuartigen Antikörper zu identifizieren . Als eine wesentliche Eigenschaft eines superagonistischen Antikörpers wurde die Fähigkeit definiert, die Proliferation humaner PBMC zu reduzieren . Weiteres Kriterium war, dass dieser Effekt mit löslichem, nicht artifiziell vernetzten! Antikörper zu beobachten ist . Diej enigen Antikörper wurden als positiv evaluiert , die eine anti- CD3 (bzw . superagonistische anti- CD28 ; nicht gezeigt ) induzierte Proliferation der T- Zellen um mindestens 25% reduzierten . Readout- System war die Messung der Proliferation mittels 3H Thymidineinbau . In diesem Assay- System wurden die CTLA-4 spezifischen Antikörper zeitglich mit dem aktivierenden anti CD3 Antikörper gegeben und die Proliferation nach 63- 66 Stunden bestimmt . Antikörper 4.8H10H5 war in der Lage, basierend auf den genannten Kriterien, die Proliferation von T- Zellen zu inhibieren . Zum Vergleich ist ein in diesem Assay System nicht positiv evaluierter Antikörper ( 2.10B11A1 ) aufgeführt . Dargestellt ist die relative Proliferation in Relation zur Positivkontrolle ( anti- CD3 induzierte Proliferation) . Als weitere Kontrollen wurde die j eweilige Isotypkontrolle ( IgGl oder IgG2 ) und ein kommerziell erhältlicher Antikörper (BNI3 , BD Pharmingen) mitgeführt . Die zur Kontrolle mitgeführten kommerziellen Antikörper ( 14D3 , 8H5 , 3H1833 , BNI3 ) mit Spezifität für CTLA-4 blieben ohne Wirkung . Fig . 5 zeigt den stimulatorischen Effekt der anti-CTLA-4 Antikörper 4.8H10H5 und 4.3F6B5 auf die IL-2 Produktion von Jurkat E6.1 Zellen, die ein chimäres CTLA-4 /CD28 Molekül exprimieren . Für dieses zellautonome Read-out System zur funktionellen Charakterisierung der CTLA-4 spezifischen MAK wurden Jurkat E6.1 Zellen verwendet, die einen Chimären CTLA-4/ CD28 Rezeptor auf ihrer Oberfläche exprimieren ( D) . Dieser besteht aus der extrazellulären Domäne des CTLA-4 Rezeptors und der transmembranen und intrazellulären Domäne von CD28. Die Aktivierung des Chimären Rezeptors durch CTLA- 4 spezifische MAK induziert CD28-spezifische Aktivierungsmarker wie IL-2 oder CD69 , die mittels ELISA bzw . FACS-Analyse gemessen werden können . Potentiell superagonistische CTLA-4 spezifische Antikörper können in diesem System anhand von CD28 spezifischenThe aim of this proliferation inhibition assay was to identify a functionally novel antibody compared to previously known CTLA-4 specific antibodies. As an essential property of a superagonistic antibody has been defined the ability to reduce the proliferation of human PBMC. Another criterion was that this effect with soluble, not artificially networked! Antibody is observed. These antibodies were evaluated as positive, reducing anti-CD3 (or superagonistic anti-CD28, not shown) T cell proliferation by at least 25%. Readout system was the measurement of proliferation by means of 3 H thymidine incorporation. In this assay system, the CTLA-4 specific antibodies were given in time with the activating anti-CD3 antibody and the proliferation determined after 63-66 hours. Antibody 4.8H10H5 was able to inhibit proliferation of T cells based on the above criteria. For comparison, an antibody not positively evaluated in this assay system (2.10B11A1) is listed. Shown is the relative proliferation in relation to the positive control (anti-CD3 induced proliferation). Further controls included the respective isotype control (IgGl or IgG2) and a commercially available antibody (BNI3, BD Pharmingen). The control commercial antibodies (14D3, 8H5, 3H1833, BNI3) with specificity for CTLA-4 had no effect. Fig. Figure 5 shows the stimulatory effect of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on the IL-2 production of Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 molecule. Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 receptor on their surface were used for this cell-autonomous read-out system for the functional characterization of the CTLA-4-specific MAK (D). This consists of the extracellular domain of the CTLA-4 receptor and the transmembrane and intracellular domain of CD28. Activation of the chimeric receptor by CTLA-4-specific MAK induces CD28-specific activation markers such as IL-2 or CD69, which are detected by means of ELISA or ELISA. FACS analysis can be measured. Potentially superagonistic CTLA-4 specific antibodies can be identified in this system using CD28 specific
Aktivierungsmarkern identifiziert werden . Kontrollantikörper und CTLA-4 spezifische Antikörper wurden kreuzvernetzt (mittels Schaf anti Maus Ig) und mit 1 * 105 transfizierten Jurkat E6.1 Zellen für 48h inkubiert . Als Aktivierungsmarker wurde IL-2 Produktion mittels ELISA gemessen .Activation markers are identified. Control antibodies and CTLA-4 specific antibodies were cross-linked (using sheep anti mouse Ig) and incubated with 1 * 10 5 transfected Jurkat E6.1 cells for 48h. As an activation marker, IL-2 production was measured by ELISA.
Isotypkontrollen und kommerziell erhältliche Antikörper mit Spezifität für CTLA-4 wurden als Kontrollen mitgeführt . (A) Effekt der CTLA-4 spezifischen Antikörper ( lμg/ml ) auf die IL-2 Produktion transfizierter Jurkat- Zellen, die einen Chimären CTLA-4/ CD28 Rezeptor exprimieren . (B) Effekt der kommerziell erhältlichen CTLA-4 spezifischen Antikörper ( lμg/ml) auf die IL-2 Produktion transfizierter Jurkat- Zellen, die einen Chimären CTLA-4 / CD28 Rezeptor exprimieren . (C) Effekt der CTLA-4 spezifischen Antikörper ( lμg/ml) auf die IL-2 Produktion nicht transfizierter Jurkat E6.1 Zellen, denen der Chimäre Rezeptor fehlt , (repräsentative Experimente) . Zwei der getesteten Antikörper ( 4.3F6B5, 4.8H10H5 ) induzieren die IL-2 Produktion der transfizierten Jurkat Zellen durch Aktivierung des Chimären Rezeptors , während keiner der kommerziell erhältlichen Antikörper mit Spezifität für CTLA-4 hierzu in der Lage war .Isotype controls and commercially available antibodies with specificity for CTLA-4 were included as controls. (A) Effect of CTLA-4 specific antibodies (1 μg / ml) on the IL-2 production of transfected Jurkat cells expressing a chimeric CTLA-4 / CD28 receptor. (B) Effect of commercially available CTLA-4 specific antibodies (1 μg / ml) on the IL-2 production of transfected Jurkat cells expressing a chimeric CTLA-4 / CD28 receptor. (C) Effect of CTLA-4 specific antibodies (lμg / ml) on IL-2 production of untransfected Jurkat E6.1 cells lacking the chimeric receptor (representative experiments). Two of the antibodies tested (4.3F6B5, 4.8H10H5) induce the IL-2 production of transfected Jurkat cells by activation of the chimeric receptor, while none of the commercially available antibodies with specificity for CTLA-4 was able to do so.
Fig . 6 zeigt den stimulatorischen Effekt der anti-CTLA-4 Antikörper 4.8H10H5 und 4.3F6B5 auf die CD69 Induktion von Jurkat E6.1 Zellen, die ein chimäres CTLA-4 /CD28 Molekül exprimieren . (D) Unter Verwendung des in Abb . 6 beschriebenen Assay Systems wurde neben der IL-2 Produktion als weiterer Aktivierungsmarker die CD69 Expression mittels FACS Analyse gemessen . Im Gegensatz zur IL-2 Produktion ist CD69 ein „early activation marker" und kann bereits nach 4h Inkubation der Antikörper mit den transfizierten Zellen nachgewiesen werden . (A) Effekt der CTLA-4 spezifischen Antikörper ( lμg/ml ) auf die CD69 Expression transfizierter Jurkat-Zellen, die einen Chimären CTLA-4/ CD28 Rezeptor exprimieren . (B) Effekt der kommerziell erhältlichen CTLA-4 spezifischen Antikörper ( lμg/ml ) auf die CD69 Expression transfizierter Jurkat- Zellen, die einen Chimären CTLA-4/ CD28 Rezeptor exprimieren . (C) Effekt der CTLA-4 spezifischen Antikörper ( lμg/ml ) auf die CD69 Expression nicht transfizierter Jurkat Eβ . l Zellen, denen der chimäre Rezeptor fehlt , (repräsentative Experimente ) . Ebenso wie für IL-2 gezeigt, sind die Antikörper 4.3F6B5 und 4.8H10H5 in der Lage, den Chimären Rezeptor zu aktivieren, eineFig. Figure 6 shows the stimulatory effect of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on the CD69 induction of Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 molecule. (D) Using the method shown in Fig. In addition to IL-2 production, the assay of CD69 expression as a further activation marker was measured by FACS analysis. In contrast to IL-2 production, CD69 is an early activation marker and can be detected after 4 h of incubation of the antibodies with the transfected cells. (A) Effect of CTLA-4-specific antibodies (1 μg / ml) on transfected CD69 expression Jurkat cells expressing a chimeric CTLA-4 / CD28 receptor (B) Effect of commercially available CTLA-4 specific antibodies (1 μg / ml) on the CD69 expression of transfected Jurkat cells carrying a chimeric CTLA-4 / CD28 receptor (C) Effect of CTLA-4 specific antibodies (lμg / ml) on CD69 expression of untransfected Jurkat Eβ.l cells lacking the chimeric receptor (representative experiments) As shown for IL-2, as well Antibodies 4.3F6B5 and 4.8H10H5 are able to activate the chimeric receptor, a
Signaltransduktion auszulösen und die CD69 Expression zu induzieren . Keiner der kommerziell erhältlichen Antikörper mit Spezifität für CTLA-4 war hierzu in der Lage (repräsentative Experimente) .Trigger signal transduction and induce CD69 expression. None of the commercially available antibodies with specificity for CTLA-4 was able to do this (representative experiments).
In Fig . 7 ist dargestellt, dass der stimulatorische Effekt der anti-CTLA-4 Antikörper 4.8H10H5 und 4.3F6B5 auf das chimäre CTLA-4 /CD28 Konstrukt durch CD80 Fc Protein vermindert werden kann . Jurkat E . β . l Zellen, die den Chimären CTLA-4 / CD28 Rezeptor exprimieren (vgl . Abb . 5 , 6 ) wurden mit steigender Konzentration Antikörper und j eweils 1 μg/ml CD80 Fc Protein inkubiert . Die Antikörper-vermittelte CD69 Expression wird dann durch CD80Fc verringert , sobald das rekombinante Protein im Überschuss im Vergleich zum Antikörper inkubiert wird. Rechteckige Quadrate zeigen die j eweilige CD69 Induktion ohne CD80 Ko-Inkubation, die durch Dreiecke dargestellten Kurven zeigen die durch Iμg/ ml CD80 Fc verminderte CD69 Induktion durch die j eweiligenIn Fig. Figure 7 shows that the stimulatory effect of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on the chimeric CTLA-4 / CD28 construct by CD80 Fc protein can be reduced. Jurkat E. β. 1 cells expressing the chimeric CTLA-4 / CD28 receptor (see Fig. 5, 6) were incubated with increasing concentration of antibody and in each case 1 μg / ml CD80 Fc protein. Antibody-mediated CD69 expression is then reduced by CD80Fc as the recombinant protein is excessively incubated compared to the antibody. Rectangular squares show the current CD69 induction without CD80 co-incubation, the triangular curves show the CD69 induction diminished by Iμg / ml CD80 Fc
Antikörper . Zusätzlich ist die konzentrationsabhängige Bindung der Antikörper and die Jurkat Zellen gezeigt .Antibodies. In addition, the concentration-dependent binding of the antibodies and the Jurkat cells is shown.
In Fig . 8 ist die Kreuzreaktivität der anti-CTLA-4 Antikörper 4.8H10H5 und 4.3F6B5 mit dem CTLA-4 Molekül der Ratte und die stimulatorische Funktionalität der Antikörper in einem Chimären Rezeptor-Assay gezeigt . (A) Die Bindung von anti-CTLA-4 Antikörpern an Ratten CTLA-4 wurde mittels BW Zellen gezeigt , die die extrazelluläre Domäne des Ratten- Rezeptors auf ihrer Oberfläche exprimieren . Analog zu dem Chimären Rezeptor auf Jurkat Zellen (Abb . 5 ) exprimieren diese Zellen einen Chimären CTLA-4 CD28 Rezeptor bestehend aus Ratte CTLA-4 (extrazelluläre Domäne) und Maus CD28 (transmembrane/ intrazelluläre Domäne ) (helle Linie : anti- CTLA-4 Akö, dunkle Kurve : Isotypkontrolle) . (B) Für die kreuzreaktiven Antikörper 4.3F6B5 und 4.8H10H5 konnte die Aktivierung des Chimären Rezeptors anhand der IL-2 Induktion gezeigt werden . Wie im in Fig . 5 beschriebenen Assay System können mit den transfizierten BW Zellen potentiell superagonistische CTLA-4 spezifische Antikörper können anhand von CD28 spezifischen Aktivierungsmarkern identifiziert werden ( IL-2 ) . In einem Kontrollexperiment konnten die Antikörper auf BW Zellen, denen der chimäre Rezeptor fehlt, keine IL-2 Produktion induzieren .In Fig. Figure 8 shows the cross-reactivity of the anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 with the rat CTLA-4 molecule and the stimulatory functionality of the antibodies in a chimeric receptor assay. (A) Binding of anti-CTLA-4 antibodies to rat CTLA-4 was demonstrated by BW cells expressing the extracellular domain of the rat receptor on its surface. Analogous to the chimeric receptor on Jurkat cells (Figure 5), these cells express a chimeric CTLA-4 CD28 receptor consisting of rat CTLA-4 (extracellular domain) and mouse CD28 (transmembrane / intracellular domain) (bright line: anti-CTLA). 4 Akö, dark curve: isotype control). (B) For the cross-reactive antibodies 4.3F6B5 and 4.8H10H5, activation of the chimeric receptor was demonstrated by IL-2 induction. As in FIG. 5 can be identified with the transfected BW cells potentially superagonistic CTLA-4 specific antibodies by means of CD28 specific activation markers (IL-2). In a control experiment the antibodies on BW cells lacking the chimeric receptor could not induce IL-2 production.
Fig . 9 zeigt den inhibitorischen in vivo Effekt der anti- CTLA-4 Antikörper 4.8H10H5 und 4.3F6B5 auf die CD28Fig. Figure 9 shows the inhibitory in vivo effect of anti-CTLA-4 antibodies 4.8H10H5 and 4.3F6B5 on CD28
SuperMAB-vermittelte Aktivierung von T-Zellen in der Ratte . Dazu wurden aktivierender superagonistischer Ratten spezifischer CD28 Antikörper (JJ316) zusammen mit CTLA-4 spezifischen Antikörper bzw . Isotypkontrolle Ratten i . v . appliziert . Nach drei Tagen wurden Zellsuspensionen ausSuperMAB-mediated activation of T cells in the rat. For this purpose, activating superagonistic rats of specific CD28 antibodies (JJ316) together with CTLA-4 specific antibodies resp. Isotype Control Rats i. v. applied. After three days, cell suspensions were eliminated
Lymphknoten und Milz gewonnen und mittels FACS hinsichtlich des Aktivierungsmarkers CD25 analysiert . Insgesamt wurden drei Experimente mit variierender Antikörperkonzentration durchgeführt . Beide getesteten CTLA-4 spezifischen Antikörper reduzierten die JJ316 induzierte CD25 Expression auf Lymphknoten- wie auch auf Milzzellen in 3 unabhängigen Experimenten um ca . 30 - 40% . Gezeigt ist ein repräsentatives Ergebnis .The lymph nodes and spleens were obtained and analyzed by FACS for the activation marker CD25. A total of three experiments with varying antibody concentrations were performed. Both tested CTLA-4 specific antibodies reduced the JJ316-induced CD25 expression on lymph node as well as on spleen cells in 3 independent experiments by approx. 30 - 40%. Shown is a representative result.
In der Figur 11 ist der stimulatorische Effekt der humanisierten anti-CTLA-4 Antikörper ( 1 μg/ml ) in vitro auf die CD69 Expression von Jurkat E6.1 Zellen, die einen Chimären CTLA-4 /CD28 Rezeptor exprimieren, dargestellt . Es wurde entsprechend der Beschreibung zur Figur 6 vorgegangen . Der Figur 11 ist zu entnehmen, dass alle humanisiertenIn Figure 11, the stimulatory effect of the humanized anti-CTLA-4 antibody (1 μg / ml) in vitro on the CD69 expression of Jurkat E6.1 cells expressing a chimeric CTLA-4 / CD28 receptor is shown. The procedure was as described for FIG. 6. From Figure 11 it can be seen that all humanized
Antikörper der Erfindung, Sowohl vom IgGl als auch vom IgG4 Isotyp, effektiv die CD69 Oberflächenexpression induzieren, während die Isotypkontrolle bzw . die Zugabe von Zellkulturmedium ohne Effekt blieb (repräsentatives Ergebnis ) . Damit ist gezeigt, dass dass die funktionell neuartigen Eigenschaften der Antikörper 4.3F6B5 und 4.8H10H5 im Verlauf der Humanisierung erhalten geblieben sind . In der Figur 12 ist der inhibitorische Effekt der Antikörper TGN2122. C und TGN2122. H auf die Proliferation ex vivo stimulierter humaner PBMCs dargestellt . In einem Recall- Response-Assay wurden 10Λ5 humane PBMCs gesunder Spender mit 2 , 5 μg/ml Tetanus Toxoid aktiviert und zeitgleich der entsprechende CTLA-4 spezifische Antikörper bzw . die Isotypenkontrolle zum Versuchsansatz gegeben . Gemessen wurde die Proliferation mittels 3H-Thymidineinbau nach 120 h Inkubation . Hierzu wurde zum Versuchsansatz 3H-Thymidin für die letzten 15-18 h der Versuchsdauer zugegeben und der 3H- Thymidineinbau bestimmt . Als Kontrollen wurden die entsprechende Isotypenkontrolle und ein Ansatz , in dem sich nicht-aktivierte Zellen ohne Antikörper befanden, mitgeführt . Beide Antikörper waren in der Lage, die Tetanus Toxoid induzierte Proliferation effektiv zu inhibieren . Die Isotypenkontrolle zeigte dagegen keine solche Inhibierung . Mit diesen Versuchen sind die superagonistischen Eigenschaften erfindungsgemäßer Antikörper gezeigt, da eine Inhibierung in löslicher Form, i . e . ohne artifizielle Kreuzvernetzung, erfolgte .Antibodies of the invention, from both IgG1 and IgG4 isotypes, effectively induce CD69 surface expression while the isotype control resp. the addition of cell culture medium without effect remained (representative result). This shows that the functionally novel properties of the antibodies 4.3F6B5 and 4.8H10H5 have been preserved in the course of humanization. FIG. 12 shows the inhibitory effect of the antibody TGN2122. C and TGN2122. H on the proliferation of ex vivo stimulated human PBMCs. In a recall-response-assay 10 Λ 5 human PBMCs of healthy donors with 2, 5 μg / ml tetanus toxoid were activated and at the same time the corresponding CTLA-4 specific antibodies resp. the isotype control given to the experimental approach. The proliferation was measured by 3 H thymidine incorporation after 120 h incubation. For this purpose, 3 H-thymidine was added to the experimental batch for the last 15-18 h of the test duration and the 3 H-Thymidineinbau determined. As controls, the corresponding isotype control and an approach in which unactivated non-antibody cells were included were included. Both antibodies were able to effectively inhibit tetanus toxoid-induced proliferation. In contrast, the isotype control showed no such inhibition. With these experiments, the superagonistic properties of antibodies according to the invention are shown since inhibition in soluble form, i. e. without artificial cross-linking, took place.
Sofern in Sequenzen Xn angegeben ist, kann das angegebene n um + 1 variieren . If n is specified in sequences X, the specified n can vary by + 1.
Tab. 1 Bindungscharakteristika neuer anti-CTLA-4 AntikörperTable 1 Binding characteristics of new anti-CTLA-4 antibodies
Tab . 2Tab. 2
4.3F6B5 Schwere Kette4.3F6B5 Heavy Chain
CDR-Hl CDR-H2CDR-HL CDR-H2
EVQLQPSGPVL VKPGÄSVKISCKASPYTFTDYKIPJWVKQSHGKSLEWIGtf IYPYSGSSDYNOKEVQLQPSGPVL VKPGÄSVKISCKASPYTFTDYKIPJWVKQSHGKSLEWIGtf IYPYSGSSDYNOK
FKsJKÄTLTVDNSSTTAYMELRSLTSEDSAVYYCAR^GbAMDYiiΛi'GQGTAVTVSSFKsJKÄTLTVDNSSTTAYMELRSLTSEDSAVYYCAR ^ GbAMDYiiΛi'GQGTAVTVSS
CDR-H3CDR-H3
Leichte KetteLight chain
CDR-Ll CDR-L2CDR-L1 CDR-L2
DILMTQSPASLSASVGETVTITClSASENIYGALΪWYQRKQGKSPQLLIYiSATNLAElGMSSRFSDILMTQSPASLSASVGETVTITClSASENIYGALΪWYQRKQGKSPQLLIYiSATNLAElGMSSRFS
4.8H10H5 Schwere Kette4.8H10H5 Heavy chain
CDR-Hl CDR-H2CDR-HL CDR-H2
DVKL VESGGGL VKLGGSLKLSCAASfSFT FNI YYMS}WVRQTPEKRLELV£|AINPDGGNTYYPDTDVKL VESGGGL VKLGGSLKLSCAASfSFT FNI YYMS} WVRQTPEKRLELV £ | AINPDGGNTYYPDT
VKG^FTISRDNAKNTLYLQMSSLKSEDSALYYCARlϊGGPGFDsiΛfGQGTTLTVSSVCG ^ FTISRDNAKNTLYLQMSSLKSEDSALYYCARlϊGGPGFDsiΛfGQGTTLTVSS
CDR-H3CDR-H3
Leichte KetteLight chain
CDR-Ll CDR-L2CDR-L1 CDR-L2
ENVLTQSPAIMSASPGERVTMTCßASSSVSYMHlWYQQKSNTSPKLWIYiDTSKLAsiGVPGRFSGENVLTQSPAIMSASPGERVTMTCßASSSVSYMHlWYQQKSNTSPKLWIYiDTSKLAsiGVPGRFSG
SGSRNS YSLTISSMEAEDVATYYCIFPGSGFPFMYTJFGGGTKLEIKRSGSRNS YSLTISSMEAEDVATYYCIFPGSGFPFMYTJFGGGTKLEIKR
CDR-L3 TAB 3CDR-L3 TAB 3
TGN2122. H HC AminosäuresequenzTGN2122. H HC amino acid sequence
Seq . 30 EVQLVE_SGGGL_V_QP^Seq. 30 EVQLVE_SGGGL_V_QP ^
GLELVAAINPpGGNTYYPDTVKGRFT SWGQGTLVTVSSASTKGPSVFP]-APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVGLELVAAINPpGGNTYYPDTVKGRFT SWGQGTLVTVSSASTKGPSVFP] -APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq. 31 :Seq. 31:
MGWSCIILFLVATATGVHS = Leaderpeptid, nicht Teil der reifen HCMGWSCIILFLVATATGVHS = Leader peptide, not part of mature HC
XXXX = Variables Segment XXXX = konstantes SegmentXXXX = variable segment XXXX = constant segment
CDRsCDRs
Kabat IYYMS - Seq . 22Kabat IYYMS - Seq. 22
AbM GFTFNIYYMS - Seq. 25AbM GFTFNYYMS - Seq. 25
EVQLVESGGGLVQPGGSLRLSCAASGFTFNIYYMSWVRQAPGKGLELVAEVQLVESGGGLVQPGGSLRLSCAASGFTFNIYYMSWVRQAPGKGLELVA
AINPDGGNTYYPDTVKG - Seq. 23AINPDGGNTYYPDTVKG - Seq. 23
AINPDGGNTY - Seq . 26AINPDGGNTY - Seq. 26
AINPDGGNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAINPDGGNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
YGGPGFDS - Seq. 24 YGGPGFDS - Seq. 24 YGGPGFDSWGQGTLVTVSS - Seq. 27YGGPGFDS - Seq. 24 YGGPGFDS - Seq. 24 YGGPGFDSWGQGTLVTVSS - Seq. 27
IYYMSX14AINPDGGNTYYPDTVKGX32YGGPGFDS - Seq. 28 GFTFNIYYMSX14AINPDGGNTYX39YGGPGFDS - Seq. 29 TAB 4IYYMSX 14 AINPDGGNTYYPDTVKGX 32 YGGPGFDS - Seq. 28 GFTFNIYYMSX 14 AINPDGGNTYX 39 YGGPGFDS - Seq. 29 TAB 4
TGN2122. H HC Nukleotidsequenz (Seq. 40)TGN2122. H HC nucleotide sequence (SEQ ID NO: 40)
1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA
51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT
101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG AGGTGCAGCT101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG AGGTGCAGCT
151 GGTGGAGAGC GGCGGCGGCC TGGTGCAGCC CGGCGGCAGC CTGAGGCTGA151 GGTGGAGAGC GGCGGCGGCC TGGTGCAGCC CGGCGGCAGC CTGAGGCTGA
201 GCTGCGCCGC CAGCGGCTTC ACCTTCAACA TCTACTACAT GAGCTGGGTG201 GCTGCGCCGC CAGCGGCTTC ACCTTCAACA TCTACTACAT GAGCTGGGTG
251 AGGCAGGCCC CCGGCAAGGG CCTGGAGCTG GTGGCCGCCA TCAACCCCGA251 AGGCAGGCCC CCGGCAAGGG CCTGGAGCTG GTGGCCGCCA TCAACCCCGA
301 CGGCGGCAAC ACCTACTACC CCGACACCGT GAAGGGCAGG TTCACCATCA301 CGGCGGCAAC ACCTACTACC CCGACACCGT GAAGGGCAGG TTCACCATCA
351 GCAGGGACAA CGCCAAGAAC AGCCTGTACC TGCAGATGAA CAGCCTGAGG351 GCAGGGACAA CGCCAAGAAC AGCCTGTACC TGCAGATGAA CAGCCTGAGG
401 GCCGAGGACA CCGCCGTGTA CTACTGCGCC AGGTACGGCG GCCCCGGCTT401 GCCGAGGACA CCGCCGTGTA CTACTGCGCC AGGTACGGCG GCCCCGGCTT
451 CGACAGCTGG GGCCAGGGCA CCCTGGTGAC CGTGAGCAGC GGTGAGTCGT451 CGACAGCTGG GGCCAGGGCA CCCTGGTGAC CGTGAGCAGC GGTGAGTCGT
501 ACGCTAGCAA GCTTTCTGGG GCAGGCCAGG CCTGACCTTG GCTTTGGGGC501 ACGCTAGCAA GCTTTCTGGG GCAGGCCAGG CCTGACCTTG GCTTTGGGGC
551 AGGGAGGGGG CTAAGGTGAG GCAGGTGGCG CCAGCCAGGT GCACACCCAA551 AGGGAGGGGG CTAAGGTGAG GCAGGTGGCG CCAGCCAGGT GCACACCCAA
601 TGCCCATGAG CCCAGACACT GGACGCTGAA CCTCGCGGAC AGTTAAGAAC601 TGCCCATGAG CCCAGACACT GGACGCTGAA CCTCGCGGAC AGTTAAGAAC
651 CCAGGGGCCT CTGCGCCCTG GGCCCAGCTC TGTCCCACAC CGCGGTCACA651 CCAGGGGCCT CTGCGCCCTG GGCCCAGCTC TGTCCCACAC CGCGGTCACA
701 TGGCACCACC TCTCTTGCAG CCTCCACCAA GGGCCCATCG GTCTTCCCCC701 TGGCACCACC TCTCTTGCAG CCTCCACCAA GGGCCCATCG GTCTTCCCCC
751 TGGCACCCTC CTCCAAGAGC ACCTCTGGGG GCACAGCGGC CCTGGGCTGC751 TGGCACCCTC CTCCAAGAGC ACCTCTGGGG GCACAGCGGC CCTGGGCTGC
801 CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT GGAACTCAGG801 CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT GGAACTCAGG
851 CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA CAGTCCTCAG851 CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA CAGTCCTCAG
901 GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG CAGCTTGGGC901 GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG CAGCTTGGGC
951 ACCCAGACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGT951 ACCCAGACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGT
1001 GGACAAGAAA GTTGGTGAGA GGCCAGCACA GGGAGGGAGG GTGTCTGCTG1001 GGACAAGAAA GTTGGTGAGA GGCCAGCACA GGGAGGGAGG GTGTCTGCTG
1051 GAAGCCAGGC TCAGCGCTCC TGCCTGGACG CATCCCGGCT ATGCAGCCCC1051 GAAGCCAGGC TCAGCGCTCC TGCCTGGACG CATCCCGGCT ATGCAGCCCC
1101 AGTCCAGGGC AGCAAGGCAG GCCCCGTCTG CCTCTTCACC CGGAGGCCTC1101 AGTCCAGGGC AGCAAGGCAG GCCCCGTCTG CCTCTTCACC CGGAGGCCTC
1151 TGCCCGCCCC ACTCATGCTC AGGGAGAGGG TCTTCTGGCT TTTTCCCAGG1151 TGCCCGCCCC ACTCATGCTC AGGGAGAGGG TCTTCTGGCT TTTTCCCAGG
1201 CTCTGGGCAG GCACAGGCTA GGTGCCCCTA ACCCAGGCCC TGCACACAAA1201 CTCTGGGCAG GCACAGGCTA GGTGCCCCTA ACCCAGGCCC TGCACACAAA
1251 GGGGCAGGTG CTGGGCTCAG ACCTGCCAAG AGCCATATCC GGGAGGACCC1251 GGGGCAGGTG CTGGGCTCAG ACCTGCCAAG AGCCATATCC GGGAGGACCC
1301 TGCCCCTGAC CTAAGCCCAC CCCAAAGGCC AAACTCTCCA CTCCCTCAGC1301 TGCCCCTGAC CTAAGCCCAC CCCAAAGGCC AAACTCTCCA CTCCCTCAGC
1351 TCGGACACCT TCTCTCCTCC CAGATTCCAG TAACTCCCAA TCTTCTCTCT1351 TCGGACACCT TCTCTCCTCC CAGATTCCAG TAACTCCCAA TCTTCTCTCT
1401 GCAGAGCCCA AATCTTGTGA CAAAACTCAC ACATGCCCAC CGTGCCCAGG1401 GCAGAGCCCA AATCTTGTGA CAAAACTCAC ACATGCCCAC CGTGCCCAGG
1451 TAAGCCAGCC CAGGCCTCGC CCTCCAGCTC AAGGCGGGAC AGGTGCCCTA1451 TAAGCCAGCC CAGGCCTCGC CCTCCAGCTC AAGGCGGGAC AGGTGCCCTA
1501 GAGTAGCCTG CATCCAGGGA CAGGCCCCAG CCGGGTGCTG ACACGTCCAC1501 GAGTAGCCTG CATCCAGGGA CAGGCCCCAG CCGGGTGCTG ACACGTCCAC
1551 CTCCATCTCT TCCTCAGCAC CTGAACTCCT GGGGGGACCG TCAGTCTTCC1551 CTCCATCTCT TCCTCAGCAC CTGAACTCCT GGGGGGACCG TCAGTCTTCC
1601 TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG1601 TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG
1651 GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT1651 GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT
1701 CAACTGGTAC GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC1701 CAACTGGTAC GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC
1751 GGGAGGAGCA GTACAACAGC ACGTACCGGG TGGTCAGCGT CCTCACCGTC1751 GGGAGGAGCA GTACAACAGC ACGTACCGGG TGGTCAGCGT CCTCACCGTC
1801 CTGCACCAGG ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA1801 CTGCACCAGG ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA
1851 CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCΆAAGGTG1851 CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCΆAAGGTG
1901 GGACCCGTGG GGTGCGAGGG CCACATGGAC AGAGGCCGGC TCGGCCCACC1901 GGACCCGTGG GGTGCGAGGG CCACATGGAC AGAGGCCGGC TCGGCCCACC
1951 CTCTGcccTG AGAGTGACCG CTGTACCAΆC CTCTGTCCCT ACAGGGCAGC1951 CTCTGcccTG AGAGTGACCG CTGTACCAΆC CTCTGTCCCT ACAGGGCAGC
2001 CCCGAGAACC ACAGGTGTAC ACCCTGCCCC CATCCCGGGA TGAGCTGACC 2051 AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA 2101 CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTÄCAAGA 2151 CCACGCCTCC CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG 2201 CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC 2251 CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC 2301 TGTCTCCGGG TAAATGA .^ ü- ßies2001 CCCGAGAACC ACAGGTGTAC ACCCTGCCCC CATCCCGGGA TGAGCTGACC 2051 AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA 2101 CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTÄCAAGA 2151 CCACGCCTCC CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG 2201 CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC 2251 CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC 2301 TGTCTCCGGG TAAATGA . ^ U - ßies
TGN2422. H HC AminosäuresequenzTGN2422. H HC amino acid sequence
Seq . 32 : EVQLVESGGGLVQ^Seq. 32: EV Q LVESGGGLV Q ^
GLEL V^INPDGGNTYYPDTVKG3_FTI SRDNAKNGLEL V ^ INPDGGNTYYPDTVKG3_FTI SRDNAKN
SWGGGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVSWGGGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAP
EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGKEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Seq. 31 :Seq. 31:
MGWSCIILFLVATÄTGVHS = Leaderpeptid, nicht Teil der reifen HCMGWSCIILFLVATÄTGVHS = Leader peptide, not part of the mature HC
XXXX = Variables Segment XXXX = konstantes SegmentXXXX = variable segment XXXX = constant segment
CDRsCDRs
Kabat IYYMS - Seq. 22Kabat IYYMS - Seq. 22
AbM GFTFNIYYMS - Seq. 25AbM GFTFNYYMS - Seq. 25
EVQLVESGGGLVQPGGSLRLSCAASGFTFNIYYMSWVRQAPGKGLELVAEVQLVESGGGLVQPGGSLRLSCAASGFTFNIYYMSWVRQAPGKGLELVA
AINPDGGNTYYPDTVKG - Seq. 23AINPDGGNTYYPDTVKG - Seq. 23
AINPDGGNTY - Seq . 26AINPDGGNTY - Seq. 26
AINPDGGNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAINPDGGNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
YGGPGFDS - Seq . 24 YGGPGFDS - Seq. 24 YGGPGFDSWGQGTLVTVSS - Seq . 27 TAB 6YGGPGFDS - Seq. 24 YGGPGFDS - Seq. 24 YGGPGFDSWGQGTLVTVSS - Seq. 27 TAB 6
TGN2422. H HC Nukleotidsequenz (Seq. 41)TGN2422. H HC nucleotide sequence (SEQ ID NO: 41)
1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG AGGTGCAGCT 151 GGTGGAGAGC GGCGGCGGCC TGGTGCAGCC CGGCGGCAGC CTGAGGCTGA 201 GCTGCGCCGC CAGCGGCTTC ACCTTCAACA TCTACTACAT GAGCTGGGTG 251 AGGCAGGCCC CCGGCAAGGG CCTGGAGCTG GTGGCCGCCA TCAACCCCGA 301 CGGCGGCAAC ACCTACTACC CCGACACCGT GAAGGGCAGG TTCACCATCA 351 GCAGGGACAA CGCCAAGAAC AGCCTGTACC TGCAGATGAA CAGCCTGAGG 401 GCCGAGGACA CCGCCGTGTA CTACTGCGCC AGGTACGGCG GCCCCGGCTT 451 CGACAGCTGG GGCCAGGGCA CCCTGGTGAC CGTGAGCAGC GGTGAGTCGT 501 ACGCTAGCAA GCTTTCTGGG GCAGGCCGGG CCTGACTTTG GCTGGGGGCA 551 GGGAGGGGGC TAAGGTGACG CAGGTGGCGC CAGCCAGGTG CACACCCAAT 601 GCCCATGAGC CCAGACACTG GACCCTGCAT GGACCATCGC GGATAGACAA 651 GAACCGAGGG GCCTCTGCGC CCTGGGCCCA GCTCTGTCCC ACACCGCGGT1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG AGGTGCAGCT 151 GGTGGAGAGC GGCGGCGGCC TGGTGCAGCC CGGCGGCAGC CTGAGGCTGA 201 GCTGCGCCGC CAGCGGCTTC ACCTTCAACA TCTACTACAT GAGCTGGGTG 251 AGGCAGGCCC CCGGCAAGGG CCTGGAGCTG GTGGCCGCCA TCAACCCCGA 301 CGGCGGCAAC ACCTACTACC CCGACACCGT GAAGGGCAGG TTCACCATCA 351 GCAGGGACAA CGCCAAGAAC AGCCTGTACC TGCAGATGAA CAGCCTGAGG 401 GCCGAGGACA CCGCCGTGTA CTACTGCGCC AGGTACGGCG GCCCCGGCTT 451 GGCCAGGGCA CCCTGGTGAC CGACAGCTGG CGTGAGCAGC GGTGAGTCGT 501 ACGCTAGCAA GCTTTCTGGG GCAGGCCGGG CCTGACTTTG GCTGGGGGCA 551 GGGAGGGGGC TAAGGTGACG CAGGTGGCGC CAGCCAGGTG CACACCCAAT 601 GCCCATGAGC CCAGACACTG GACCCTGCAT GGACCATCGC GGATAGACAA 651 GAACCGAGGG GCCTCTGCGC CCTGGGCCCA GCTCTGTCCC ACACCGCGGT
701 CACATGGCAC CACCTCTCTT GCAGCTTCCA CCAAGGGCCC ATCCGTCTTC 751 CCCCTGGCGC CCTGCTCCAG GAGCACCTCC GAGAGCACAG CCGCCCTGGG701 CACATGGCAC CACCTCTCTT GCAGCTTCCA CCAAGGGCCC ATCCGTCTTC 751 CCCCTGGCGC CCTGCTCCAG GAGCACCTCC GAGAGCACAG CCGCCCTGGG
801 CTGCCTGGTC AAGGACTACT TCCCCGAACC GGTGACGGTG TCGTGGAACT 851 CAGGCGCCCT GACCAGCGGC GTGCACACCT TCCCGGCTGT CCTACAGTCC 901 TCAGGACTCT ACTCCCTCAG CAGCGTGGTG ACCGTGCCCT CCAGCAGCTT 951 GGGCACGAAG ACCTACACCT GCAACGTAGA TCACAAGCCC AGCAACACCA801 CTGCCTGGTC AAGGACTACT TCCCCGAACC GGTGACGGTG TCGTGGAACT 851 CAGGCGCCCT GACCAGCGGC GTGCACACCT TCCCGGCTGT CCTACAGTCC 901 TCAGGACTCT ACTCCCTCAG CAGCGTGGTG ACCGTGCCCT CCAGCAGCTT 951 GGGCACGAAG ACCTACACCT GCAACGTAGA TCACAAGCCC AGCAACACCA
1001 AGGTGGACAA GAGAGTTGGT GAGAGGCCAG CACAGGGAGG GAGGGTGTCT 1051 GCTGGAAGCC AGGCTCÄGCC CTCCTGCCTG GACGCACCCC GGCTGTGCAG 1101 CCCCAGCCCA GGGCAGCAAG GCATGCCCCA TCTGTCTCCT CACCCGGAGG 1151 CCTCTGACCA CCCCACTCAT GCTCAGGGAG AGGGTCTTCT GGATTTTTCC 1201 ACCAGGCTCC GGGCAGCCAC AGGCTGGATG CCCCTACCCC AGGCCCTGCG 1251 CATACAGGGG CAGGTGCTGC GCTCAGACCT GCCAAGAGCC ATATCCGGGA 1301 GGACCCTGCC CCTGACCTAA GCCCACCCCA AAGGCCAAAC TCTCCACTCC 1351 CTCAGCTCAG ACACCTTCTC TCCTCCCAGA TCTGAGTAAC TCCCAATCTT 1401 CTCTCTGCAG AGTCCAAATA TGGTCCCCCA TGCCCATCAT GCCCAGGTAA 1451 GCCAACCCAG GCCTCGCCCT CCAGCTCAAG GCGGGACAGG TGCCCTAGAG 1501 TAGCCTGCAT CCAGGGACAG GCCCCAGCCG GGTGCTGACG CATCCACCTC 1551 CATCTCTTCC TCAGCACCTG AGTTCCTGGG GGGACCATCA GTCTTCCTGT 1601 TCCCCCCAAA ACCCAAGGAC ACTCTCATGA TCTCCCGGAC CCCTGAGGTC 1651 ACGTGCGTGG TGGTGGACGT GAGCCAGGAA GACCCCGAGG TCCAGTTCAA 1701 CTGGTACGTG GATGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG 1751 AGGAGCAGTT CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG 1801 CACCAGGACT GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA1001 AGGTGGACAA GAGAGTTGGT GAGAGGCCAG CACAGGGAGG GAGGGTGTCT 1051 GCTGGAAGCC AGGCTCÄGCC CTCCTGCCTG GACGCACCCC GGCTGTGCAG 1101 CCCCAGCCCA GGGCAGCAAG GCATGCCCCA TCTGTCTCCT CACCCGGAGG 1151 CCTCTGACCA CCCCACTCAT GCTCAGGGAG AGGGTCTTCT GGATTTTTCC 1201 ACCAGGCTCC GGGCAGCCAC AGGCTGGATG CCCCTACCCC AGGCCCTGCG 1251 CATACAGGGG CAGGTGCTGC GCTCAGACCT GCCAAGAGCC ATATCCGGGA 1301 GGACCCTGCC CCTGACCTAA GCCCACCCCA AAGGCCAAAC TCTCCACTCC 1351 CTCAGCTCAG ACACCTTCTC TCCTCCCAGA TCTGAGTAAC TCCCAATCTT 1401 CTCTCTGCAG AGTCCAAATA TGGTCCCCCA TGCCCATCAT GCCCAGGTAA 1451 GCCAACCCAG GCCTCGCCCT CCAGCTCAAG GCGGGACAGG TGCCCTAGAG 1501 TAGCCTGCAT CCAGGGACAG GCCCCAGCCG GGTGCTGACG CATCCACCTC 1551 CATCTCTTCC TCAGCACCTG AGTTCCTGGG GGGACCATCA GTCTTCCTGT 1601 TCCCCCCAAA ACCCAAGGAC ACTCTCATGA TCTCCCGGAC CCCTGAGGTC 1651 ACGTGCGTGG TGGTGGACGT GAGCCAGGAA GACCCCGAGG TCCAGTTCAA 1701 CTGGTACGTG GATGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG 1751 AGGAGCAGTT CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG 1801 CACCAGGACT GGCTGAACGG CAAGGAGTAC A AGTGCAAGG TCTCCAACAA
1851 AGGCCTCCCG TCCTCCATCG AGAΆAACCAT CTCCAAAGCC AΆAGGTGGGA1851 AGGCCTCCCG TCCTCCATCG AGAΆAACCAT CTCCAAAGCC AΆAGGTGGGA
1901 CCCACGGGGT GCGAGGGCCA CATGGACAGA GGTCAGCTCG GCCCACCCTC1901 CCCACGGGGT GCGAGGGCCA CATGGACAGA GGTCAGCTCG GCCCACCCTC
1951 TGCCCTGGGA GTGACCGCTG TGCCAACCTC TGTCCCTACA GGGCAGCCCC1951 TGCCCTGGGA GTGACCGCTG TGCCAACCTC TGTCCCTACA GGGCAGCCCC
2001 GAGAGCCACA GGTGTACACC CTGCCCCCAT CCCAGGAGGA GATGACCAAG2001 GAGAGCCACA GGTGTACACC CTGCCCCCAT CCCAGGAGGA GATGACCAAG
2051 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTACC CCAGCGACAT2051 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTACC CCAGCGACAT
2101 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA2101 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
2151 CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAGGCTA2151 CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAGGCTA
2201 ACCGTGGACA AGAGCAGGTG GCAGGAGGGG AATGTCTTCT CATGCTCCGT2201 ACCGTGGACA AGAGCAGGTG GCAGGAGGGG AATGTCTTCT CATGCTCCGT
2251 GATGCATGAG GCTCTGCACA ACCACTACAC ACAGAAGAGC CTCTCCCTGT 2301 CTCTGGGTAA ATGA TAB 72251 GATGCATGAG GCTCTGCACA ACCACTACAC ACAGAAGAGC CTCTCCCTGT 2301 CTCTGGGTAA ATGA TAB 7
TGN2122/TGN2422. H-kappa LC AminosäuresequenzTGN2122 / TGN2422. H-kappa LC amino acid sequence
SEQ. 38 : ENVLT_Q_SPAT_LS_LSPGERAT_LS_CSASSSEQ. 38: ENVLT_Q_SPAT_LS_LSPGERAT_LS_CSASS
RLWIYDTSKLASGIPARFSGSGSRNPXTLTRLWIYDTSKLASGIPARFSGSGSRNPXTLT
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Seq . 39 :Seq. 39:
MGWSCIILFLVATÄTGVHS = Leaderpeptid, nicht Teil der reifen LCMGWSCIILFLVATÄTGVHS = Leader peptide, not part of the mature LC
XXXX = Variables Segment XXXX = konstantes SegmentXXXX = variable segment XXXX = constant segment
CDRsCDRs
Hier : Kabat=AbMHere: Kabat = AbM
SASSSVSYMH - Seq . 33 DTSKLAS - Seq . 34 ENVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLWIYDTSKLASGIPARFSSASSSVSYMH - Seq. 33 DTS CLASS - Seq. 34 ENVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLWIYDTSKLASGIPARFS
FPGSGFPFMYT - Seq . 35 GSGSRNDYTLTISSLEPEDFAVYYCFPGSGFPFMYTFGGGTKVEIK - Seq. 37FPGSGFPFMYT - Seq. 35 GSGSRNDYTLTISSLEPEDFAVYYCFPGSGFPFMYTFGGGTKVEIK - Seq. 37
SASSSVSYMHXi5DTSKLASX32FPGSGFPFMYT - Seq . 36 SASSSVSYMHXi 5 DTSKLASX 32 FPGSGFPFMYT - Seq. 36
32 ,1 u. U, \ 32 , 1 u. U, \
TAB 8TAB 8
TGN2122/TGN2422. H-kappa LC Nukleotidsequenz (Seq. 42)TGN2122 / TGN2422. H-kappa LC nucleotide sequence (SEQ 42)
1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG AGAACGTGCT 151 GACCCAGAGC CCCGCCACCC TGAGCCTGAG CCCCGGCGAG AGGGCCACCC 201 TGAGCTGCAG CGCCAGCAGC AGCGTGAGCT ACATGCACTG GTACCAGCAG 251 AAGCCCGGCC AGGCCCCCAG GCTGTGGATC TACGACACCA GCAAGCTGGC 301 CAGCGGCATC CCCGCCAGGT TCAGCGGCAG CGGCAGCAGG AACGACTACA 351 CCCTGACCAT CAGCAGCCTG GAGCCCGAGG ACTTCGCCGT GTACTACTGC 401 TTCCCCGGCA GCGGCTTCCC CTTCATGTAC ACCTTCGGCG GCGGCACCAA 451 GGTGGAGATC AAGCGTGAGT CGTACGCTAG CAAGCTTGAT ATCGAATTCT1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG AGAACGTGCT 151 GACCCAGAGC CCCGCCACCC TGAGCCTGAG CCCCGGCGAG AGGGCCACCC 201 TGAGCTGCAG CGCCAGCAGC AGCGTGAGCT ACATGCACTG GTACCAGCAG 251 AAGCCCGGCC AGGCCCCCAG GCTGTGGATC TACGACACCA GCAAGCTGGC 301 CAGCGGCATC CCCGCCAGGT TCAGCGGCAG CGGCAGCAGG AACGACTACA 351 CCCTGACCAT CAGCAGCCTG GAGCCCGAGG ACTTCGCCGT GTACTACTGC 401 TTCCCCGGCA GCGGCTTCCC CTTCATGTAC ACCTTCGGCG GCGGCACCAA 451 GGTGGAGATC AAGCGTGAGT CGTACGCTAG CAAGCTTGAT ATCGAATTCT
501 AAACTCTGAG GGGGTCGGAT GACGTGGCCA TTCTTTGCCT AΆAGCΆTTGA501 AAACTCTGAG GGGGTCGGAT GACGTGGCCA TTCTTTGCCT AΆAGCΆTTGA
551 GTTTACTGCA AGGTCAGAAA AGCATGCAAA GCCCTCAGAA TGGCTGCAAA 601 GAGCTCCAAC AAAACAATTT AGAACTTTAT TAAGGAATAG GGGGAAGCTA 651 GGAAGAAACT CAAAACATCA AGATTTTAAA TACGCTTCTT GGTCTCCTTG551 GTTTACTGCA AGGTCAGAAA AGCATGCAAA GCCCTCAGAA TGGCTGCAAA 601 GAGCTCCAAC AAAACAATTT AGAACTTTAT TAAGGAATAG GGGGAAGCTA 651 GGAAGAAACT CAAAACATCA AGATTTTAAA TACGCTTCTT GGTCTCCTTG
701 CTATAATTAT CTGGGATAAG CATGCTGTTT TCTGTCTGTC CCTAACATGC 751 CCTGTGATTA TCCGCAAACA ACACACCCAA GGGCAGAACT TTGTTACTTA701 CTATAATTAT CTGGGATAAG CATGCTGTTT TCTGTCTGTC CCTAACATGC 751 CCTGTGATTA TCCGCAAACA ACACACCCAA GGGCAGAACT TTGTTACTTA
801 AACACCATCC TGTTTGCTTC TTTCCTCAGG AACTGTGGCT GCACCATCTG801 AACACCATCC TGTTTGCTTC TTTCCTCAGG AACTGTGGCT GCACCATCTG
851 TCTTCATCTT CCCGCCÄTCT GATGAGCAGT TGΆAATCTGG AACTGCCTCT851 TCTTCATCTT CCCGCCÄTCT GATGAGCAGT TGΆAATCTGG AACTGCCTCT
901 GTTGTGTGCC TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG901 GTTGTGTGCC TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG
951 GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG951 GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG
1001 AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG1001 AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG
1051 AGCAAAGCAG ACTACGAGAA ΆCACAAÄGTC TACGCCTGCG AAGTCACCCA1051 AGCAAAGCAG ACTACGAGAA ΆCACAAÄGTC TACGCCTGCG AAGTCACCCA
1101 TCAGGGCCTG AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT 1151 AG 1101 TCAGGGCCTG AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT 1151 AG
TAB 9TAB 9
TGN2122. C HC AminosäuresequenzTGN2122. C HC amino acid sequence
GLEWIGYIYPYSGSSDTOGLEWIGYIYPYSGSSDTO
WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq. 52Seq. 52
MGWSCIILFLVATATGVHS = Leaderpeptid, nicht Teil der reifen HCMGWSCIILFLVATATGVHS = Leader peptide, not part of mature HC
XXXX = Variables Segment XXXX = konstantes SegmentXXXX = variable segment XXXX = constant segment
CDRsCDRs
Kabat DYKIH - Seq . 43Kabat DYKIH - Seq. 43
AbM GYTFTDYKIH - Seq. 46ABM GYTFTDYKIH - Seq. 46
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYKIHWVRQAPGQGLEWIGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYKIHWVRQAPGQGLEWIG
YIYPYSGSSDYNQKFKS -Seq . 44YIYPYSGSSDYNQKFKS -Seq. 44
YIYPYSGSSD - Seq. 47YIYPYSGSSD - Seq. 47
YIYPYSGSSDYNQKFKSRATLTVDNSISTAYMELSRLRSDDTAVYYCARYIYPYSGSSDYNQKFKSRATLTVDNSISTAYMELSRLRSDDTAVYYCAR
GGDAMDY - Seq. 45 GGDAMDY - Seq . 45 GGDAMDYWGQGTLVTVSS - Seq . 48GGDAMDY - Seq. 45 GGDAMDY - Seq. 45 GGDAMDYWGQGTLVTVSS - Seq. 48
DYKIHX14YIYPYSGSSDYNQKFKSX32GGDAMDY - Seq . 49 GYTFTDYKIHX14YIYPYSGSSDX39GGDAMDY - Seq . 50 TAB 10DYKIHX14YIYPYSGSSDYNQKFKSX32GGDAMDY - Seq. 49 GYTFTDYKIHX 14 YIYPYSGSSDX 39 GGDAMDY - Seq. 50 TAB 10
TGN2122. C HC Nukleotidsequenz (Seq. 61)TGN2122. C HC nucleotide sequence (SEQ ID NO: 61)
1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGÄCAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCC AGGTGCAGCT 151 GGTGCAGAGC GGCGCCGAGG TGAAGAAGCC CGGCGCCAGC GTGAAGGTGA 201 GCTGCAAGGC CAGCGGCTAC ACCTTCACCG ACTACAAGAT CCACTGGGTG 251 AGGCAGGCCC CCGGCCAGGG CCTGGAGTGG ATCGGCTACA TCTACCCCTA 301 CAGCGGCAGC AGCGACTACA ACCAGAAGTT CAAGAGCAGG GCCACCCTGA 351 CCGTGGACAA CAGCATCAGC ACCGCCTACA TGGAGCTGAG CAGGCTGAGG 401 AGCGACGACA CCGCCGTGTA CTACTGCGCC AGGGGCGGCG ACGCCATGGA 451 CTACTGGGGC CAGGGCACCC TGGTGACCGT GAGCAGCGGT GAGTCGTACG 501 CTAGCAAGCT TTCTGGGGCA GGCCAGGCCT GACCTTGGCT TTGGGGCAGG 551 GAGGGGGCTA AGGTGAGGCA GGTGGCGCCA GCCAGGTGCA CACCCAATGC1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGÄCAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCC AGGTGCAGCT 151 GGTGCAGAGC GGCGCCGAGG TGAAGAAGCC CGGCGCCAGC GTGAAGGTGA 201 GCTGCAAGGC CAGCGGCTAC ACCTTCACCG ACTACAAGAT CCACTGGGTG 251 AGGCAGGCCC CCGGCCAGGG CCTGGAGTGG ATCGGCTACA TCTACCCCTA 301 CAGCGGCAGC AGCGACTACA ACCAGAAGTT CAAGAGCAGG GCCACCCTGA 351 CCGTGGACAA CAGCATCAGC ACCGCCTACA TGGAGCTGAG CAGGCTGAGG 401 AGCGACGACA CCGCCGTGTA CTACTGCGCC AGGGGCGGCG ACGCCATGGA 451 CTACTGGGGC CAGGGCACCC TGGTGACCGT GAGCAGCGGT GAGTCGTACG 501 CTAGCAAGCT TTCTGGGGCA GGCCAGGCCT GACCTTGGCT TTGGGGCAGG 551 GAGGGGGCTA AGGTGAGGCA GGTGGCGCCA GCCAGGTGCA CACCCAATGC
601 ccATGAGccc AGACACTGGA CGCTGAACCT CGCGGACAGT TAΆGAACCCA601 ccATGAGccc AGACACTGGA CGCTGAACCT CGCGGACAGT TAΆGAACCCA
651 GGGGCCTCTG CGCCCTGGGC CCAGCTCTGT CCCACACCGC GGTCACATGG651 GGGGCCTCTG CGCCCTGGGC CCAGCTCTGT CCCACACCGC GGTCACATGG
701 CACCACCTCT CTTGCAGCCT CCACCAAGGG CCCATCGGTC TTCCCCCTGG701 CACCACCTCT CTTGCAGCCT CCACCAAGGG CCCATCGGTC TTCCCCCTGG
751 CACCCTCCTC CAAGAGCACC TCTGGGGGCA CAGCGGCCCT GGGCTGCCTG751 CACCCTCCTC CAAGAGCACC TCTGGGGGCA CAGCGGCCCT GGGCTGCCTG
801 GTCAAGGACT ACTTCCCCGA ACCGGTGÄCG GTGTCGTGGA ACTCAGGCGC801 GTCAAGGACT ACTTCCCCGA ACCGGTGÄCG GTGTCGTGGA ACTCAGGCGC
851 CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG TCCTCAGGAC851 CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG TCCTCAGGAC
901 TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAG CTTGGGCACC901 TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAG CTTGGGCACC
951 CAGACCTACA TCTGCAACGT GAATCACAAG CCCAGCAACA CCAAGGTGGA951 CAGACCTACA TCTGCAACGT GAATCACAAG CCCAGCAACA CCAAGGTGGA
1001 CAAGAAAGTT GGTGAGAGGC CAGCACAGGG AGGGAGGGTG TCTGCTGGAA1001 CAAGAAAGTT GGTGAGAGGC CAGCACAGGG AGGGAGGGTG TCTGCTGGAA
1051 GCCAGGCTCA GCGCTCCTGC CTGGACGCAT CCCGGCTATG CAGCCCCAGT1051 GCCAGGCTCA GCGCTCCTGC CTGGACGCAT CCCGGCTATG CAGCCCCAGT
1101 CCAGGGCAGC AAGGCAGGCC CCGTCTGCCT CTTCACCCGG AGGCCTCTGC1101 CCAGGGCAGC AAGGCAGGCC CCGTCTGCCT CTTCACCCGG AGGCCTCTGC
1151 CCGCCCCACT CATGCTCAGG GAGAGGGTCT TCTGGCTTTT TCCCAGGCTC1151 CCGCCCCACT CATGCTCAGG GAGAGGGTCT TCTGGCTTTT TCCCAGGCTC
1201 TGGGCAGGCA CAGGCTAGGT GCCCCTAACC CAGGCCCTGC ACACAAAGGG1201 TGGGCAGGCA CAGGCTAGGT GCCCCTAACC CAGGCCCTGC ACACAAAGGG
1251 GCAGGTGCTG GGCTCAGACC TGCCAAGAGC CATATCCGGG AGGACCCTGC1251 GCAGGTGCTG GGCTCAGACC TGCCAAGAGC CATATCCGGG AGGACCCTGC
1301 CCCTGACCTA AGCCCACCCC AAAGGCCAAA CTCTCCACTC CCTCAGCTCG1301 CCCTGACCTA AGCCCACCCC AAAGGCCAAA CTCTCCACTC CCTCAGCTCG
1351 GACACCTTCT CTCCTCCCAG ATTCCAGTAA CTCCCAATCT TCTCTCTGCA1351 GACACCTTCT CTCCTCCCAG ATTCCAGTAA CTCCCAATCT TCTCTCTGCA
1401 GAGCCCAAAT CTTGTGACAA AACTCACACA TGCCCACCGT GCCCAGGTAA1401 GAGCCCAAAT CTTGTGACAA AACTCACACA TGCCCACCGT GCCCAGGTAA
1451 GCCAGCCCAG GCCTCGCCCT CCAGCTCAAG GCGGGACAGG TGCCCTAGAG1451 GCCAGCCCAG GCCTCGCCCT CCAGCTCAAG GCGGGACAGG TGCCCTAGAG
1501 TAGCCTGCAT CCAGGGACAG GCCCCAGCCG GGTGCTGACA CGTCCACCTC1501 TAGCCTGCAT CCAGGGACAG GCCCCAGCCG GGTGCTGACA CGTCCACCTC
1551 CATCTCTTCC TCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT1551 CATCTCTTCC TCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT
1601 TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC1601 TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC
1651 ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA1651 ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA
1701 CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AÄGCCGCGGG1701 CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
1751 AGGAGCÄGTA CAACAGCACG TACCGGGTGG TCAGCGTCCT CACCGTCCTG1751 AGGAGCÄGTA CAACAGCACG TACCGGGTGG TCAGCGTCCT CACCGTCCTG
1801 CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA1801 CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA
1851 AGcccTCccA GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGTGGGΆ1851 AGcccTCccA GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGTGGGΆ
1901 CCCGTGGGGT GCGAGGGCCA CATGGACAGA GGCCGGCTCG GCCCACCCTC1901 CCCGTGGGGT GCGAGGGCCA CATGGACAGA GGCCGGCTCG GCCCACCCTC
1951 TGCCCTGAGA GTGACCGCTG TACCAACCTC TGTCCCTACA GGGCAGCCCC1951 TGCCCTGAGA GTGACCGCTG TACCAACCTC TGTCCCTACA GGGCAGCCCC
2001 GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG2001 GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG
2051 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT2051 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT
2101 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA2101 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
2151 CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC2151 CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC
2201 ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT2201 ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT
2251 GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT 2301 CTCCGGGTAA ATGA TAB 11 ! 2251 GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT 2301 CTCCGGGTAA ATGA TAB 11 !
TGN2422. C HC AminosäuresequenzTGN2422. C HC amino acid sequence
Seq . 53 : QYQkYQSGJ^ VKKPGA^Seq. 53: QYQkYQSGJ ^ VKKPGA ^
WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPETFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPE
FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE
GNVFSCSVMHEALHNHYTQKSLSLSLGKGNVFSCSVMHEALHNHYTQKSLSLSLGK
Seq. 52 :Seq. 52:
MGWSCIILFLVATATGVHS = Leaderpeptid, nicht Teil der reifen HCMGWSCIILFLVATATGVHS = Leader peptide, not part of mature HC
XXXX = Variables Segment XXXX = konstantes SegmentXXXX = variable segment XXXX = constant segment
CDRsCDRs
Kabat DYKIH - Seq . 43Kabat DYKIH - Seq. 43
AbM GYTFTDYKIH - Seq . 46ABM GYTFTDYKIH - Seq. 46
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYKIHWVRQAPGQGLEWIGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYKIHWVRQAPGQGLEWIG
YIYPYSGSSDYNQKFKS - Seq. 44YIYPYSGSSDYNQKFKS - Seq. 44
YIYPYSGSSD - Seq . 47YIYPYSGSSD - Seq. 47
YIYPYSGSSDYNQKFKSRATLTVDNSISTAYMELSRLRSDDTAVYYCARYIYPYSGSSDYNQKFKSRATLTVDNSISTAYMELSRLRSDDTAVYYCAR
GGDAMDY - Seq. 45 GGDAMDY - Seq . 45 GGDAMDYWGQGTLVTVSS - Seq . 48 TAB 12GGDAMDY - Seq. 45 GGDAMDY - Seq. 45 GGDAMDYWGQGTLVTVSS - Seq. 48 TAB 12
TGN2422. C HC Nukleotidsequenz (Seq. 62)TGN2422. C HC nucleotide sequence (SEQ ID NO: 62)
1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCC AGGTGCAGCT 151 GGTGCAGAGC GGCGCCGAGG TGAAGAAGCC CGGCGCCAGC GTGAAGGTGA 201 GCTGCAAGGC CAGCGGCTAC ACCTTCACCG ACTACAAGAT CCACTGGGTG 251 AGGCAGGCCC CCGGCCAGGG CCTGGAGTGG ATCGGCTACA TCTACCCCTA 301 CAGCGGCAGC AGCGACTACA ACCAGAAGTT CAAGAGCAGG GCCACCCTGA 351 CCGTGGACAA CAGCATCAGC ACCGCCTACA TGGAGCTGAG CAGGCTGAGG 401 AGCGACGACA CCGCCGTGTA CTACTGCGCC AGGGGCGGCG ACGCCATGGA 451 CTACTGGGGC CAGGGCACCC TGGTGACCGT GAGCAGCGGT GAGTCGTACG 501 CTAGCAAGCT TTCTGGGGCA GGCCGGGCCT GACTTTGGCT GGGGGCAGGG 551 AGGGGGCTAA GGTGACGCAG GTGGCGCCAG CCAGGTGCAC ACCCAATGCC 601 CATGAGCCCA GACACTGGAC CCTGCATGGA CCATCGCGGA TAGACAAGAA 651 CCGAGGGGCC TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC 701 ATGGCACCAC CTCTCTTGCA GCTTCCACCA AGGGCCCATC CGTCTTCCCC 751 CTGGCGCCCT GCTCCAGGAG CACCTCCGAG AGCACAGCCG CCCTGGGCTG1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCC AGGTGCAGCT 151 GGTGCAGAGC GGCGCCGAGG TGAAGAAGCC CGGCGCCAGC GTGAAGGTGA 201 GCTGCAAGGC CAGCGGCTAC ACCTTCACCG ACTACAAGAT CCACTGGGTG 251 AGGCAGGCCC CCGGCCAGGG CCTGGAGTGG ATCGGCTACA TCTACCCCTA 301 CAGCGGCAGC AGCGACTACA ACCAGAAGTT CAAGAGCAGG GCCACCCTGA 351 CCGTGGACAA CAGCATCAGC ACCGCCTACA TGGAGCTGAG CAGGCTGAGG 401 AGCGACGACA CCGCCGTGTA CTACTGCGCC AGGGGCGGCG ACGCCATGGA 451 CTACTGGGGC CAGGGCACCC TGGTGACCGT GAGCAGCGGT GAGTCGTACG 501 CTAGCAAGCT TTCTGGGGCA GGCCGGGCCT GACTTTGGCT GGGGGCAGGG 551 AGGGGGCTAA GGTGACGCAG GTGGCGCCAG CCAGGTGCAC ACCCAATGCC 601 CATGAGCCCA GACACTGGAC CCTGCATGGA CCATCGCGGA TAGACAAGAA 651 CCGAGGGGCC TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC 701 ATGGCACCAC CTCTCTTGCA GCTTCCACCA AGGGCCCATC CGTCTTCCCC CTGGCGCCCT 751 GCTCCAGGAG CACCTCCGAG AGCACAGCCG CCCTGGGCTG
801 ccTGGTCAAG GACTACTTCC CCGAΆCCGGT GACGGTGTCG TGGAΆCTCAG801 ccTGGTCAAG GACTACTTCC CCGAΆCCGGT GACGGTGTCG TGGAΆCTCAG
851 GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA 901 GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG851 GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA 901 GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG
951 cACGAAGAcc TACACCTGCA ACGTΆGATCA CAAGCCCAGC AACACCAAGG951 cACGAAGAcc TACACCTGCA ACGTΆGATCA CAAGCCCAGC AACACCAAGG
1001 TGGACAAGAG AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT 1051 GGAAGCCAGG CTCAGCCCTC CTGCCTGGAC GCACCCCGGC TGTGCAGCCC 1101 CAGCCCAGGG CAGCAAGGCA TGCCCCATCT GTCTCCTCAC CCGGAGGCCT 1151 CTGACCACCC CACTCATGCT CAGGGAGAGG GTCTTCTGGA TTTTTCCACC 1201 AGGCTCCGGG CAGCCACAGG CTGGATGCCC CTACCCCAGG CCCTGCGCAT 1251 ACAGGGGCAG GTGCTGCGCT CAGACCTGCC AAGAGCCATA TCCGGGAGGA 1301 CCCTGCCCCT GACCTAAGCC CACCCCAAAG GCCAAACTCT CCACTCCCTC 1351 AGCTCAGACA CCTTCTCTCC TCCCAGATCT GAGTAACTCC CAATCTTCTC 1401 TCTGCAGAGT CCAAATATGG TCCCCCATGC CCATCATGCC CAGGTAAGCC 1451 AACCCAGGCC TCGCCCTCCA GCTCAAGGCG GGACAGGTGC CCTAGAGTAG 1501 CCTGCATCCA GGGACAGGCC CCAGCCGGGT GCTGACGCAT CCACCTCCAT 1551 CTCTTCCTCA GCACCTGAGT TCCTGGGGGG ACCATCAGTC TTCCTGTTCC 1601 CCCCAAAACC CAAGGACACT CTCATGATCT CCCGGACCCC TGAGGTCACG 1651 TGCGTGGTGG TGGACGTGAG CCAGGAAGAC CCCGAGGTCC AGTTCAACTG1001 TGGACAAGAG AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT 1051 GGAAGCCAGG CTCAGCCCTC CTGCCTGGAC GCACCCCGGC TGTGCAGCCC 1101 CAGCCCAGGG CAGCAAGGCA TGCCCCATCT GTCTCCTCAC CCGGAGGCCT 1151 CTGACCACCC CACTCATGCT CAGGGAGAGG GTCTTCTGGA TTTTTCCACC 1201 AGGCTCCGGG CAGCCACAGG CTGGATGCCC CTACCCCAGG CCCTGCGCAT 1251 ACAGGGGCAG GTGCTGCGCT CAGACCTGCC AAGAGCCATA TCCGGGAGGA 1301 CCCTGCCCCT GACCTAAGCC CACCCCAAAG GCCAAACTCT CCACTCCCTC 1351 AGCTCAGACA CCTTCTCTCC TCCCAGATCT GAGTAACTCC CAATCTTCTC 1401 TCTGCAGAGT CCAAATATGG TCCCCCATGC CCATCATGCC CAGGTAAGCC 1451 AACCCAGGCC TCGCCCTCCA GCTCAAGGCG GGACAGGTGC CCTAGAGTAG 1501 CCTGCATCCA GGGACAGGCC CCAGCCGGGT GCTGACGCAT CCACCTCCAT 1551 CTCTTCCTCA GCACCTGAGT TCCTGGGGGG ACCATCAGTC TTCCTGTTCC 1601 CCCCAAAACC CAAGGACACT CTCATGATCT CCCGGACCCC TGAGGTCACG 1651 TGCGTGGTGG TGGACGTGAG CCAGGAAGAC CCCGAGGTCC AGTTCAACTG
1701 GTACGTGGAT GGCGTGGAGG TGCATAATGC CAΆGACAAAG CCGCGGGAGG1701 GTACGTGGAT GGCGTGGAGG TGCATAATGC CAΆGACAAAG CCGCGGGAGG
1751 AGCAGTTCAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC1751 AGCAGTTCAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC
1801 CAGGACTGGC TGAACGGCAA GGAGTACAAG TGCAΆGGTCT CCAACAAAGG1801 CAGGACTGGC TGAACGGCAA GGAGTACAAG TGCAΆGGTCT CCAACAAAGG
1851 CCTCCCGTCC TCCATCGAGA AAACCATCTC CAAAGCCAAA GGTGGGACCC 1901 ACGGGGTGCG AGGGCCACAT GGACAGAGGT CAGCTCGGCC CACCCTCTGC 1951 CCTGGGAGTG ACCGCTGTGC CAACCTCTGT CCCTACAGGG CAGCCCCGAG1851 CCTCCCGTCC TCCATCGAGA AAACCATCTC CAAAGCCAAA GGTGGGACCC 1901 ACGGGGTGCG AGGGCCACAT GGACAGAGGT CAGCTCGGCC CACCCTCTGC 1951 CCTGGGAGTG ACCGCTGTGC CAACCTCTGT CCCTACAGGG CAGCCCCGAG
2001 AGCCACAGGT GTACACCCTG CCCCCATCCC AGGAGGAGAT GACCAAGAΆC2001 AGCCACAGGT GTACACCCTG CCCCCATCCC AGGAGGAGAT GACCAAGAΆC
2051 CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTACCCCA GCGACATCGC 2101 CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACGC 2151 CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCTACAG CAGGCTAACC 2201 GTGGACAAGA GCAGGTGGCA GGAGGGGAAT GTCTTCTCAT GCTCCGTGAT 2251 GCATGAGGCT CTGCACAACC ACTACACACA GAAGAGCCTC TCCCTGTCTC2051 CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTACCCCA GCGACATCGC 2101 CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACGC 2151 CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCTACAG CAGGCTAACC 2201 GTGGACAAGA GCAGGTGGCA GGAGGGGAAT GTCTTCTCAT GCTCCGTGAT 2251 GCATGAGGCT CTGCACAACC ACTACACACA GAAGAGCCTC TCCCTGTCTC
2301 TGGGTAAΆΓG A TAB 132301 TGGGTAAΆΓG A TAB 13
TGN2122/TGN2422. C-kappa LC AminosäuresequenzTGN2122 / TGN2422. C-kappa LC amino acid sequence
Seq . 59 : PiQMTQS PS SLSAS _VGDRVT ITCGASEN iχGAJLNWχQRKPGKASeq. 59: PiQMTQS PS SLSAS _VGDRVT ITCGASEN iχGAJLNWχQRKPGKA
PKLLIYGATNLADGVPSRFSGSGSGRDYTLTISSLQPEDFATYFCQNILGTWTFGGGTK_VEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECPKLLIYGATNLADGVPSRFSGSGSGRDYTLTISSLQPEDFATYFCQNILGTWTFGGGTK_VEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Seq . 60 :Seq. 60:
MGWSCIILFLVATATGVHS = Leaderpeptid, nicht Teil der reifen LCMGWSCIILFLVATATGVHS = Leader peptide, not part of the mature LC
XXXX = Variables Segment XXXX = konstantes SegmentXXXX = variable segment XXXX = constant segment
CDRsCDRs
Hier : Kabat=AbMHere: Kabat = AbM
GASENIYGALN - Seq . 54 GATNLAD - Seq . 55 DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQRKPGKAPKLLIYGATNLADGVPSRFGASENIYGALN - Seq. 54 GATNLAD - Seq. 55 DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQRKPGKAPKLLIYGATNLADGVPSRF
QNILGTWT - Seq . 56 SGSGSGRDYTLTISSLQPEDFATYFCQNILGTWTFGGGTKVEIK - Seq. 58QNILGTWT - Seq. 56 SGSGSGRDYTLTISSLQPEDFATYFCQNILGTWTFGGGTKVEIK - Seq. 58
GASENIYGALNX15GATNLADX32QNILGTWT - Seq . 57 GASENIYGALNX 15 GATNLADX 32 QNILGTWT - Seq. 57
TAB 14TAB 14
TGN2122/TGN2422. C-kappa LC Nukleotidsequenz (Seq. 63)TGN2122 / TGN2422. C-kappa LC nucleotide sequence (SEQ ID NO: 63)
1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG ACATCCAGAT 151 GACCCAGAGC CCCAGCAGCC TGAGCGCCAG CGTGGGCGAC AGGGTGACCA 201 TCACCTGCGG CGCCAGCGAG AACATCTACG GCGCCCTGAA CTGGTACCAG 251 AGGAÄGCCCG GCAAGGCCCC CAAGCTGCTG ATCTACGGCG CCACCAACCT 301 GGCCGACGGC GTGCCCAGCA GGTTCAGCGG CAGCGGCAGC GGCAGGGACT 351 ACACCCTGAC CATCAGCAGC CTGCAGCCCG AGGACTTCGC CACCTACTTC 401 TGCCAGAACA TCCTGGGCAC CTGGACCTTC GGCGGCGGCA CCAAGGTGGA 451 GATCAAGCGT GAGTCGTACG CTAGCAAGCT TGATATCGAA TTCTAAACTC 501 TGAGGGGGTC GGATGACGTG GCCATTCTTT GCCTAAAGCA TTGAGTTTAC 551 TGCAAGGTCA GAAAAGCATG CAAAGCCCTC AGAATGGCTG CAAAGAGCTC 601 CAACAAAACA ATTTAGAACT TTATTAAGGA ATAGGGGGAA GCTAGGAAGA 651 AACTCAAAAC ATCAAGATTT TAAATACGCT TCTTGGTCTC CTTGCTATAA1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTAA 51 GGGGCTCACA GTAGCAGGCT TGAGGTCTGG ACATATATAT GGGTGACAAT 101 GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG ACATCCAGAT 151 GACCCAGAGC CCCAGCAGCC TGAGCGCCAG CGTGGGCGAC AGGGTGACCA 201 TCACCTGCGG CGCCAGCGAG AACATCTACG GCGCCCTGAA CTGGTACCAG 251 AGGAÄGCCCG GCAAGGCCCC CAAGCTGCTG ATCTACGGCG CCACCAACCT 301 GGCCGACGGC GTGCCCAGCA GGTTCAGCGG CAGCGGCAGC GGCAGGGACT 351 ACACCCTGAC CATCAGCAGC CTGCAGCCCG AGGACTTCGC CACCTACTTC 401 TGCCAGAACA TCCTGGGCAC CTGGACCTTC GGCGGCGGCA CCAAGGTGGA 451 GATCAAGCGT GAGTCGTACG CTAGCAAGCT TGATATCGAA TTCTAAACTC 501 TGAGGGGGTC GGATGACGTG GCCATTCTTT GCCTAAAGCA TTGAGTTTAC 551 TGCAAGGTCA GAAAAGCATG CAAAGCCCTC AGAATGGCTG CAAAGAGCTC 601 CAACAAAACA ATTTAGAACT TTATTAAGGA ATAGGGGGAA GCTAGGAAGA 651 AACTCAAAAC ATCAAGATTT TAAATACGCT TCTTGGTCTC CTTGCTATAA
701 TTATCTGGGA TAAGCATGCT GTTTTCTGTC TGTCCCTAAC ATGCCCTGTG 751 ATTATCCGCA AACAACACAC CCAAGGGCAG AACTTTGTTA CTTAAACACC 801 ATCCTGTTTG CTTCTTTCCT CAGGAACTGT GGCTGCACCA TCTGTCTTCA701 TTATCTGGGA TAAGCATGCT GTTTTCTGTC TGTCCCTAAC ATGCCCTGTG 751 ATTATCCGCA AACAACACAC CCAAGGGCAG AACTTTGTTA CTTAAACACC 801 ATCCTGTTTG CTTCTTTCCT CAGGAACTGT GGCTGCACCA TCTGTCTTCA
851 TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG 901 TGCCTGCTGA ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT 951 GGATAACGCC CTCCAATCGG GTAACTCCCA GGAGAGTGTC ACAGAGCAGG851 TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG 901 TGCCTGCTGA ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT 951 GGATAACGCC CTCCAATCGG GTAACTCCCA GGAGAGTGTC ACAGAGCAGG
1001 ACAGCAAGGA CAGCACCTÄC AGCCTCAGCA GCACCCTGAC GCTGAGCAAA 1051 GCAGACTACG AGAAACACAA AGTCTACGCC TGCGAAGTCA CCCATCAGGG 1101 CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG TGTTAG 1001 ACAGCAAGGA CAGCACCTÄC AGCCTCAGCA GCACCCTGAC GCTGAGCAAA 1051 GCAGACTACG AGAAACACAA AGTCTACGCC TGCGAAGTCA CCCATCAGGG 1101 CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG TGTTAG

Claims

Patentansprüche : Claims:
1. Isolierter monoklonaler Antikörper, welcher für CTLA-4 spezifisch und agonistisch ist, wobei die schwere Kette des Antikörpers eine Sequenz enthält, welche ausgewählt ist aus der Gruppe bestehend aus ( Seq. -ID) : „22 , 23 , 24 , 25 , 26, 27 , 28 , 29, 30 , und 32" .An isolated monoclonal antibody specific for CTLA-4 and agonistic, wherein 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, 30, and 32 ".
2. Isolierter monoklonaler Antikörper nach Anspruch 1, wobei die leichte Kette des Antikörpers eine Sequenz enthält , welche ausgewählt ist aus der Gruppe bestehend aus ( Seq . -ID) : „33 , 34 , 35 , 36 , 37 , und 38" .The isolated monoclonal antibody of claim 1, wherein the antibody light chain contains a sequence selected from the group consisting of (Seq. ID): "33, 34, 35, 36, 37, and 38".
3. Isolierter monoklonaler Antikörper nach Anspruch 1 mit einer schweren Kette enthaltend eine Sequenz gemäß Seq . -ID 27 , 28 , oder 29 , vorzugsweise enthaltend oder bestehend aus der Sequenz gemäß Seq. -ID 30 oder 32 , sowie mit einer leichten Kette enthaltend eine Sequenz gemäß Seq. -ID 36 oder 37 , vorzugsweise enthaltend oder bestehend aus einer Sequenz gemäß Seq . -ID 38.3. An isolated monoclonal antibody according to claim 1 having a heavy chain containing a sequence according to Seq. ID 27, 28, or 29, preferably containing or consisting of the sequence according to Seq. ID 30 or 32, and with a light chain containing a sequence according to Seq. ID 36 or 37, preferably containing or consisting of a sequence according to Seq. ID 38.
4. Isolierter monoklonaler Antikörper, welcher für CTLA-4 spezifisch und agonistisch ist , wobei die schwere Kette des Antikörpers eine Sequenz enthält , welche ausgewählt ist aus der Gruppe bestehend aus (Seq. -ID) : „43, 44 , 45 , 46, 47 , 48 , 49, 50 , 51 , und 53" .4. An isolated monoclonal antibody specific for CTLA-4 and agonistic, the heavy chain of the antibody containing a sequence selected from the group consisting of (Seq. ID): '43, 44, 45, 46, 47, 48, 49, 50, 51, and 53 ".
5. Isolierter monoklonaler Antikörper nach Anspruch 4 , wobei die leichte Kette des Antikörpers eine Sequenz enthält, welche ausgewählt ist aus der Gruppe bestehend aus ( Seq . -ID) : „54 , 55 , 56, 57 , 58 , und 59" . The isolated monoclonal antibody of claim 4, wherein the antibody light chain contains a sequence selected from the group consisting of (Seq. ID): "54, 55, 56, 57, 58, and 59".
6. Isolierter monoklonaler Antikörper nach Anspruch 4 mit einer schweren Kette enthaltend eine Sequenz gemäß Seq . -ID 48 , 49 , oder 50 , vorzugsweise enthaltend oder bestehend aus der Sequenz gemäß Seq . -ID 51 oder 53 , sowie mit einer leichten Kette enthaltend eine Sequenz gemäß Seq . -ID 57 oder 58 , vorzugsweise enthaltend oder bestehend aus einer Sequenz gemäß Seq . -ID 59.6. An isolated monoclonal antibody according to claim 4 having a heavy chain containing a sequence according to Seq. ID 48, 49, or 50, preferably containing or consisting of the sequence according to Seq. ID 51 or 53, as well as with a light chain containing a sequence according to Seq. ID 57 or 58, preferably containing or consisting of a sequence according to Seq. ID 59.
7. Isolierter monoklonaler Antikörper, welcher für CTLA-4 spezifisch und agonistisch ist , wobei der Antikörper nicht an eine CTLA-4 Teilsequenz gemäß SEQ . -ID 1 bindet .7. An isolated monoclonal antibody which is specific for CTLA-4 and agonistic, wherein the antibody does not correspond to a CTLA-4 partial sequence according to SEQ. ID 1 binds.
8. Isolierter monoklonaler Antikörper nach Anspruch 7 , enthaltend 1 , 2 , 3 , 4 , 5, oder 6 der Sequenzen gemäß SEQ . -ID 2 bis SEQ . -ID 7 oder der Sequenzen gemäß SEQ . - ID 8 bis SEQ . -ID 13.8. An isolated monoclonal antibody according to claim 7, containing 1, 2, 3, 4, 5, or 6 of the sequences according to SEQ. ID 2 to SEQ. ID 7 or the sequences according to SEQ. ID 8 to SEQ. ID 13.
9. Isolierter monoklonaler Antikörper nach Anspruch 7 oder 8 , welcher humanisiert ist .The isolated monoclonal antibody of claim 7 or 8 which is humanized.
10. Isolierter monoklonaler Antikörper nach einem der Ansprüche 7 bis 9 , enthaltend eine oder beide der Sequenzen j eweils gemäß SEQ . -ID 14 und SEQ . -ID 15 oder SEQ . -ID 16 und SEQ . -ID 17 , oder enthalten eine oder beide der Sequenzen j eweils gemäß SEQ . -ID 18 und SEQ . - ID 19 oder SEQ . -ID 20 und SEQ . -ID 21.10. An isolated monoclonal antibody according to any one of claims 7 to 9, containing one or both of the sequences in each case according to SEQ. ID 14 and SEQ. ID 15 or SEQ. ID 16 and SEQ. ID 17, or contain one or both of the sequences in each case according to SEQ. ID 18 and SEQ. ID 19 or SEQ. ID 20 and SEQ. ID 21.
11. Isoliertes Protein oder Peptid enthaltend zumindest eine der Sequenzen SEQ . -ID 2 bis 13 , insbesondere eine der Sequenzen SEQ . -ID 14 bis 17 oder SEQ . -ID 18 bis 21 , oder eine der Sequenzen Seq . -ID 22 , 23 , 24 , 25 , 26, 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36, 37 , 38 , oder 39 , insbesondere eine der Sequenzen Seq. -ID 27 , 28 , 29 , 30 , 32 , 36, 37 , oder 38 , oder eine der Sequenzen Seq. -ID 43, 44 , 45, 46, 47 , 48 , 49, 50 , 51 , 52 , 53 , 54 , 55 , 56, 57 , 58 , 59, oder 60 , insbesondere eine der Sequenzen Seq . -ID 48 , 49, 50 , 51 , 53 57 , 58 , oder 59, oder bestehend aus einer der genannten Sequenzen .11. 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 said sequences.
12. Isolierte Nukleinsäure codierend für ein Protein oder Peptid gemäß Anspruch 11 oder für eine leichte Kette und/oder schwere Kette eines Antikörpers nach einem der Ansprüche 1 bis 10 , insbesondere enthaltend oder bestehend aus einer Sequenzen Seq . -ID 61 , 62 , oder 63.12. An isolated nucleic acid encoding a protein or peptide according to claim 11 or a light chain and / or heavy chain of an antibody according to any one of claims 1 to 10, in particular containing or consisting of a sequence Seq. ID 61, 62, or 63.
13. Isolierter Vektor enthaltend zumindest eine Nukleinsäure nach Anspruch 12.13. Isolated vector containing at least one nucleic acid according to claim 12.
14. Isolierte Zelle, welche mit einem Vektor gemäß Anspruch 13 transfiziert ist .14. An isolated cell which is transfected with a vector according to claim 13.
15. Pharmazeutischen Zusammensetzung enthaltend einen monoklonalen Antikörper nach einem der Ansprüche 1 bis 10 und/oder ein Protein oder Peptid nach Anspruch 11 , sowie optional zumindest einen physiologisch verträglichen Trägerstoff und/oder Hilfsstoff .15. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 1 to 10 and / or a protein or peptide according to claim 11, and optionally at least one physiologically acceptable carrier and / or excipient.
16. Verwendung eines monoklonalen Antikörpers nach einem der Ansprüche 1 bis 10 und/oder eines Proteins oder Peptids nach Anspruch 11 zur Herstellung einer pharmazeutischen Zusammensetzung zur Prophylaxe und/oder Therapie einer Erkrankung oder eines Zustandes aus der Gruppe bestehend aus „Rheumatoider Arthritis, Typ I Diabetis , Multipler Sklerose, Systemischer Lupus Erythematodes , Psoriasis , Colitis Ulerosa, Morbus Crohn, Allergien, Abstoßung von allogenen Organtransplantanten, insbesondere Organtransplantaten der folgenden Organe : Herz , Niere, Leber,16. Use of a monoclonal antibody according to any one of claims 1 to 10 and / or a protein or peptide according to claim 11 for the preparation of a pharmaceutical composition for the prophylaxis and / or therapy of a disease or condition selected from the group consisting of 'rheumatoid arthritis, Type I Diabetic, multiple sclerosis, systemic lupus erythematosus, psoriasis, ulcerative colitis, Crohn's disease, allergies, rejection of allograft organ transplant, in particular organ transplants of the following organs: heart, kidney, liver,
Bauchspeicheldrüse, Lunge, Knochenmark, und „Graft- versus-Host"- Erkrankung" .Pancreas, lung, bone marrow, and graft-versus-host disease.
17. Verfahren zur Herstellung eines monoklonalen Antikörpers nach einem der Ansprüche 1 bis 10 , wobei eine Nukleinsäure nach Anspruch 12 in einen Vektor eingebracht wird, wobei mittels des Vektors eine Zelle transfiziert wird, wobei die transfizierte Zelle kultiviert wird, wobei ein Überstand von der kultivierte Zellen abgetrennt oder wobei die kultivierte Zelle lysiert und das Lysat gewonnen wird, und wobei aus dem abgetrennten Überstand oder dem Lysat die monoklonalen Antikörper abgetrennt werden .17. A process for producing a monoclonal antibody according to any one of claims 1 to 10, wherein a nucleic acid according to claim 12 is introduced into a vector, wherein by means of the vector a cell is transfected, wherein the transfected cell is cultured, wherein a supernatant of the cultured Cells are separated or wherein the cultured cell is lysed and the lysate is recovered, and wherein the separated supernatant or the lysate, the monoclonal antibodies are separated.
18. Verfahren zur Herstellung einer pharmazeutischen Zusammensetzung nach Anspruch 15 , wobei die monoklonalen Antikörper und/oder das Protein oder Peptid in physiologisch wirksamer Dosis mit dem zumindest einen physiologisch verträglichen Trägerstoff und/oder Hilfsstoff gemischt und zu einer definierten Darreichungsform hergerichtet wird. 18. A process for the preparation of a pharmaceutical composition according to claim 15, wherein the monoclonal antibody and / or the protein or peptide is mixed in physiologically effective dose with the at least one physiologically acceptable carrier and / or excipient and prepared to a defined dosage form.
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