double antigen-induced bipartite functional complementation

专利摘要:
bipartite functional complementation induced by double antigen the present invention relates to the set of polypeptides and their uses. in particular, the present invention relates to the set of polypeptides in which this set comprises two polypeptides in which each comprises a target "t" portion of binding to an "a" antigen and a "f" fragment of a functional domain, wherein said two polypeptides are not associated with each other in the absence of a substrate that has an "a" on (na) its surface and where, through the dimerization of "f", the resulting dimer becomes functional. furthermore, the diagnostic and medical uses of said sets are described. moreover, the present invention relates to the nucleic acid molecule (s) encoding said set of polypeptides. the present invention also relates to a vector comprising the nucleotide sequence of the nucleic acid molecule (s) encoding said set of polypeptides. moreover, the present invention relates to pharmaceutical compositions comprising said set of polypeptides. further, the present invention relates to the kit comprising comprising said set of polypeptides.
公开号:BR112014017182A2
申请号:R112014017182-3
申请日:2013-01-14
公开日:2020-10-27
发明作者:Gernot Stuhler
申请人:Julius-Maximilians-Universität Würzburg；
IPC主号:

专利说明:

[001] [001] The present invention relates to a set of polypeptides and their uses. In particular, the present invention relates to a set of polypeptides in which this set is composed of two polypeptides each of which comprises a target "T" portion of binding to an "A" antigen and a fragment of " F "of a functional domain, in which said two polypeptides are not associated with each other in the absence of a substrate that has" A "on (a) its surface and in which, by dimerizing" M ", the resulting dimer becomes functional. In addition, medical and diagnostic uses of this set are described. In addition, the present invention relates to the nucleic acid molecule (s) encoding said set of polypeptides. The present invention also relates to a vector comprising the nucleotide sequence of the nucleic acid molecule (s) encoding said set of polypeptides. In addition, the present invention relates to pharmaceutical compositions comprising said set of polypeptides. In addition, the present invention relates to a kit comprising said set of polypeptides.
[002] [002] In recent years a number of articles have been seen that report a remarkable milestone of effectiveness with respect to bi-specific antibody constructs for tumor therapy in vitro and in initial clinical and preclinical tests . Nowadays, a substantial number of different bispecific constructs is available, which differ in size, composition, pharmacokinetics and ability to directly eliminate neoplastic cells or to engage immune effector cells for lysis of tumor cells.
[003] [003] Antibody-based immune cancer strategies are highly promising therapeutic options due to their excellent sensitivity and specificity for target structures.
[004] [004] The structural and functional modular organization of antibodies allows for extensive manipulation through genetic engineering. The different immunoglobulin-like domains can be separated and / or joined without losing the functional characteristics associated with specific domains. In addition, they can be combined and linked with heterologous protein domains, but also with non-peptide moieties. Therefore, it is possible to develop the fusion constructs in a rational way without the natural limitations of conventional antibodies.
[005] [005] Antibody-based fusion proteins can be generated with new biological and / or pharmaceutical properties. There are promising efforts to modify the ability of the Fc domain to induce ADCC (antibody dependent on cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity), through mutagenesis, depends on the intended application, or to reduce side effects ( mutations) or inhibitors to increase therapeutic efficacy (activating mutations). New applications that become possible through genetic engineering are even more varied, when the antigen-binding domain of antibodies is considered.
[006] [006] The antigen recognizing the variable domains of the heavy chain (VH) and light chain (VL) of an antibody can be joined by means of a peptide ligand through genetic engineering, preserving the ability to bind to the antigen. Such single-stranded antigen-binding fragments (scFvs) can be used as substitutes for small antibodies capable of penetrating tissue with high and low serum retention times for procedures.
[007] [007] Recent reports indicate an enormous potential for recombinant bispecific antibodies in anti-tumor therapy. Such bispecific antibodies recognize two antigens, one of which is expressed through the tumor, while the other is normally found in an immune cell. Most bispecific antibodies in anti-tumor therapy target a tumor-associated lineage marker, on the one hand, and CD3e, an invariant molecule of the T / CD3 receptor complex, on the other hand, recruiting, des - the way, T cells to destroy the tumor [Muller and Kontermann, Bispecific antibodies for cancer immunotherapy: current perspectives. Biopharmaceuticals 2010, 24 (2): 89 to 98].
[008] [008] Despite the many options for manipulating the structure and function of antibodies, the therapeutic effectiveness of such antibody-based reagents is limited by the nature of the target antigen, the accessibility of the antigen in the tumor and tissues associated with tumors, and the antibody's ability to cause or mediate the function of inducing desired cell death.
[009] [009] For example, when patients are treated with bispecific constructs directed against antigens also expressed in tissues with vital functions, serious side effects are observed. This is a serious problem since, with the exception of an unknown number of surface molecules of individually mutated cells and the monoclonal antibody of the T or B cell receptor, in the case of lymphomas, tumor specific antigens that discriminate a cell transformed with its healthy parent is not available.
[0010] [0010] Since the therapeutic concepts based on the use of bispecific antibodies generally depend on the recruitment of effector cells, it turns out that the more effective the tool (bispecific construct), the more likely the side effects - rivers occur, and even the expression of antigen per minute in unprocessed tissue can cause uncontrollable off-target effects.
[0011] [0011] In 2008, Science published the first report on the effectiveness of the MT103 / blinatumomab antibody involving the single-stranded bio-specific T cell (BITE); it induces remissions in about 80% of patients with relapsed or refractory lymphoma for standard immune chemotherapy at serum levels of about 5 orders of magnitude lower than the reported serum levels for rituximab monoclonal antibody (Bargou, R. et al, Science 321, 974 to 977, 2008). This publication and subsequent reports on the phase trials | of confirmation in acute lymphatic leukemia (ALL) marked the beginning of a new era of bi-specific antibodies, until then in severe death for almost two decades, due to systemic toxicity and little or no therapeutic activity. Especially with regard to the role of science, bi-specific antibodies have become a flourishing field again, in which more than 35 different formats have been counted (Reichert, Drugs Discov Today. 17 (2012) 954 to 963). These formats differ in size and are optimized for antigen affinity, stability, the ability to recruit effector cells (mainly T cells) and pharmacokinetics. The affinity or the greed of the constructs are manipulated by means of the affinity maturation using the various techniques or simply by joining several scFv domains in line, in order to create a multivalent construct. Even specific antibodies are reported, which are designed to improve display-binding capabilities by targeting two instead of a target molecule. Format stability can be optimized by adding immunoglobulin-like domains to mimic naturally occurring antibodies that simultaneously improve pharmacokinetic properties such as prolonged serum half-life and protection against proteolytic digestion by means of proteases. In addition, the stability of the formats can be improved by optimizing production. Since binding sequences that are used to covalently join scFv domains often lead to aggregates, production lines have been established, where they first produce two or three polypeptides that can be easily assembled, in order to generate a functional drug. These techniques use targeted disulfide bridges or covalently cross-linked reagents to join the two different polypeptides. Other techniques for making use of hetero-domains or homo-dimerization such as leucine-zipper domains, Fc domains and other protrusion-type domains in bore technologies (see, for example, WO 2007/062466). In addition, the interactions of VH and VL, which can be stabilized by binding the antigen, have been used in so-called open sandwich immunoassays to detect the antigen (Ueda, Nature Biotechnology 14 (1996), 1714 to 1718; Ohmuro- Matsuyama (2012) Detection of Protein Phosphorylation by Open- Sandwich Immunoassay, Integrative Proteomics, Dr. Hon-Chiu Leung (Ed.), ISBN: 978-953-51-0070-6; WO 2004/016782 / EP-A1 1,536,005.)
[0012] [0012] However, the bi / tricespecific and bi- or polyvalent constructs described in the art have disadvantages. First, the absence of truly specific tumor antigens that can be addressed as a target molecule. In fact, the more potent the bispecific antibody format, the more serious the collateral damage, because the target antigens addressed so far are differentiation antigens shared through tumors and non-malignant cells.
[0013] [0013] Thus, there is a need in the art for more specific treatment options in the treatment of cancer, in particular, there is a need to improve ways of detecting and / or eliminating carcinoma cells with a higher specificity and reducing side effects.
[0014] [0014] Similar needs exist in the area of hematopoietic stem cell transplantation, that is, the transplantation of stem cells obtained from another person to a patient. A patient suffering from relapsing or refractory leukemia or another hematological disease can be treated by chemotherapy / radiation (to eliminate hematopoietic malignant cells) in combination with a hematopoietic cell transplant from a healthy donor. If the elimination of malignant cells is incomplete, the tumor can grow back from the recipient cells that survive malignant, despite the presence of healthy cells provided through transplantation. As a result, survival rates among patients undergoing tumor treatment and allogeneic transplantation are significantly reduced.
[0015] [0015] However, it is difficult to eliminate (and similarly identify) survivors of malignant cells, with high specificity and, therefore, despite several attempts, good solutions to this problem have not been found. Therefore, there is a need in the art to provide better ways to specifically identify and / or eliminate such malignant receptor cells with minimal side effects on other cells.
[0016] [0016] The graft (stem cells for allogeneic transplantation), given right after conditioning therapy (radiation / chemotherapy) can replace and reconstitute hematopoiesis. The graft is harvested from stimulated bone marrow or peripheral blood cells and contains about one percent of the hematopoietic stem cells, which are the source of newly constructed blood cells. In addition, the graft normally contains a large number of immune system cells, in particular T lymphocytes, which are part of the immune system and can be very beneficial in cases where these T cells mount an immune attack against leukemic cells. This situation is also described and known as a graft versus leukemia effect. On the other hand, an errant immune response that directs T cells against the patient, known as graft versus host disease, is also frequently seen.
[0017] [0017] To minimize graft versus host disease, grafts are usually selected based on HLA (human leukocyte antigens) or MHC (major histocompatibility complex). The closer the antigens between donor and recipient coincide, the lower the likelihood of graft versus severe host. However, for many patients, a complete combined graft cannot be found. In these cases, bone marrow or peripheral blood stem cells are used that differ in one or even more HLA molecules. These clinical situation requires a strict immunosuppressive regimen after transplantation to keep the T cell system strictly under control.
[0018] [0018] Therefore, it is an objective of the present invention to provide improved ways to identify and / or specifically eliminate specific types of cells. In addition, it is an object of the present invention to provide improved ways to identify and / or eliminate cells that have a specific combination of two specific antigens on their cell surface specifically. In addition, it is an object of the present invention to provide improved ways of identifying and / or specifically eliminating carcinoma cells. In addition, it is an objective of the present invention to provide improved forms for specific identification and / or to eliminate cells that (1) are of a certain origin (such as, in the situation of a tissue or cells, the cells from the recipient
[0019] [0019] The objectives of the present invention are solved by means of a set of polypeptides comprising:
[0020] [0020] a first polypeptide comprising P1
[0021] [0021] a target portion T1,
[0022] [0022] wherein said target portion T1 specifically binds to an A1 antigen, and
[0023] [0023] an F1 fragment of a functional domain F,
[0024] [0024] in which neither said F1 fragment per se nor said polypeptide P1 per se is functional with respect to the function of said F domain,
[0025] [0025] and
[0026] [0026] a second polypeptide comprising P2
[0027] [0027] a target portion T2,
[0028] [0028] wherein said target portion T2 specifically binds to an A2 antigen, and
[0029] [0029] an F2 fragment of said functional domain F,
[0030] [0030] in which neither said fragment F2 per se, nor said polypeptide P2 per se is functional with respect to the function of said domain F,
[0031] [0031] in which said A1 antigen is different from said A2 antigen,
[0032] [0032] wherein said P1 polypeptide and said P2 polypeptide are not associated with each other in the absence of a substrate that has both A1 and A2 antigens on or on its surface, more specifically a cell that carries both antigens A1 and A2 to or on their cell surface, and
[0033] [0033] wherein, by dimerizing said fragment of said F1 polypeptide P1 with said fragment F2 of said polypeptide P2, the resulting dimer is functional with respect to the function of said domain F.
[0034] [0034] The objectives of the present invention are also solved by means of a set of polypeptides comprising:
[0035] [0035] a first polypeptide comprising P1
[0036] [0036] a target portion T1,
[0037] [0037] wherein said target portion T1 specifically binds to an A1 antigen, and
[0038] [0038] an F1 fragment of a functional domain F,
[0039] [0039] in which neither said F1 fragment per se, nor said polypeptide P1 per se is functional with respect to the function of said F domain,
[0040] [0040] and
[0041] [0041] a second polypeptide comprising P2
[0042] [0042] a target portion T2,
[0043] [0043] wherein said target portion T2 specifically binds to an A2 antigen, and
[0044] [0044] an F2 fragment of said functional domain F,
[0045] [0045] in which neither said F2 fragment per se nor said polypeptide P2 per se is functional with respect to the function of said F domain,
[0046] [0046] in which said A1 antigen is different from said A2 antigen,
[0047] [0047] where
[0048] Said fragment F1 comprises a VL domain of an antibody and said fragment F2 comprises a VH domain of the same antibody; or wherein said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody; or
[0049] Said F1 fragment comprises a VL domain of an antibody and said F2 fragment comprises a VH domain of the same antibody; or wherein said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody; and
[0050] [0050] wherein said P1 polypeptide and said P2 polypeptide are not associated with each other in the absence of a substrate that has both A1 and A2 antigens on or on its surface, more specifically a cell that carries both antigens A1 and A2 to or on their cell surface, and
[0051] [0051] wherein, by dimerizing said fragment of said F1 polypeptide P1 with said fragment F2 of said polypeptide P2, the resulting dimer is functional with respect to the function of said domain F.
[0052] [0052] The objectives of the present invention are also solved by a set of polypeptides comprising:
[0053] [0053] a first polypeptide comprising P1
[0054] [0054] a target portion T1,
[0055] [0055] wherein said target portion T1 specifically binds to an A1 antigen, and
[0056] [0056] an F1 fragment of a functional domain F,
[0057] [0057] in which neither said F1 fragment per se, nor said polypeptide P1 per se is functional with respect to the function of said F domain,
[0058] [0058] and
[0059] [0059] a second polypeptide comprising P2
[0060] [0060] a target portion T2,
[0061] [0061] wherein said target portion T2 specifically binds to an A2 antigen, and
[0062] [0062] an F2 fragment of said functional domain F,
[0063] [0063] in which neither said fragment F2 per se, nor the reference
[0064] [0064] in which said A1 antigen is different from said A2 antigen,
[0065] [0065] where
[0066] [0066] (c) said F1 fragment comprises a VL domain of an antibody and said F2 fragment comprises a VH domain of the same antibody; or wherein said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody;
[0067] [0067] (d) said polypeptide P1 and said polypeptide P2 are not associated with each other in the absence of a substrate that has both A1 and A2 antigens on its surface, more specifically a cell that carries both the A1 and A2 antigens, on their cell surface; or
[0068] [0068] (e) said F1 fragment comprises a VL domain of an antibody and said F2 fragment comprises a VH domain of the same antibody; or wherein said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody; and
[0069] [0069] wherein said polypeptide P1 and said polypeptide P2 are not associated with each other in the absence of a substrate that has both A1 and A2 antigens on its surface, more specifically a cell that carries both A1 and A2 antigens, on their cell surface, and
[0070] [0070] in which, by dimerizing said fragment of said F1 polypeptide P1 with said fragment F2 of said polypeptide P2, the resulting dimer is functional with respect to the function of said domain F, and wherein said polypeptides P1 and P2, in particular, said fragments F1 and F2, have, in the absence of a substrate or cells, with each other a constant KD dissociation in the range of 10º to 10º M.
[0071] [0071] The present invention also relates to the following items:
[0072] [0072] 1. The set of polypeptides comprising:
[0073] [0073] a first polypeptide comprising P1
[0074] [0074] a target portion T1,
[0075] [0075] wherein said target portion T1 specifically binds to an A1 antigen, and
[0076] [0076] an F1 fragment of a functional domain F,
[0077] [0077] in which neither said F1 fragment per se, nor said polypeptide P1 per se is functional with respect to the function of said F domain,
[0078] [0078] and
[0079] [0079] a second polypeptide comprising P2
[0080] [0080] a target portion T2,
[0081] [0081] wherein said target portion T2 specifically binds to an A2 antigen, and
[0082] [0082] an F2 fragment of said functional domain F,
[0083] [0083] in which neither said fragment F2 per se, nor said polypeptide P2 per se is functional with respect to the function of said domain F,
[0084] [0084] in which said A1 antigen is different from said A2 antigen,
[0085] [0085] wherein said P1 polypeptide and said P2 polypeptide are not associated with each other in the absence of a substrate that has both A1 and A2 antigens on its surface, more specifically a cell that carries both A1 and A2 antigens, on their cell surface, and
[0086] [0086] wherein, by dimerizing said fragment of said F1 polypeptide P1 with said fragment F2 of said polypeptide P2, the resulting dimer is functional with respect to the function of said domain F.
[0087] [0087] 2. The set of polypeptides according to item 1, in which a cell having both antigens A1 and A2, on its cell surface induces the dimerization of Fragment F1 of said polypeptide P1 with Fragment F2 of said P2 polypeptide, whereas a cell that does not carry both A1 and A2 antigens on its cell surface does not induce dimerization of Fragment F1 of said polypeptide P1 with Fragment F2 of said polypeptide P2.
[0088] [0088] 3. The set of polypeptides according to item 1 or 2, wherein said target portion T1 comprises an immunoglobulin module, preferably an immunoglobulin module which comprises a VL domain linked to a VH domain, more preferably, an immunoglobulin module of 11 comprising an scFv (single chain variant fragment) of an antibody, or an immunoglobulin module comprising a Vhh region of the variable domain of an mud antibody, camel antibody or shark antibodies,
[0089] [0089] and / or said target portion T2 comprises an immunoglobulin module, preferably an immunoglobulin | 2 module comprising a VL domain linked to a VH domain, more preferably, a 12 module of immunoglobulin comprising an scFv (single chain variant fragment) of an antibody, or an immunoglobulin module comprising a Vhh region of the variable domain of a mud antibody, the shark camel antibody or antibodies,
[0090] [0090] or wherein said target portion T1 and / or said target portion T2 comprises an aptamer or a natural ligand of said A1 antigen or A2 antigen, respectively
[0091] [0091] 4. The set of polypeptides according to any of the preceding items, in which said A1 antigen and / or Referenced antigen A it is an antigen expressed on the surface of cells of a tumor or on the surface of progenitor / precursor cells of a tumor, preferably an antigen expressed on the surface of cells of a hematological tumor or an antigen expressed on the surface of a tumor cell surface of a non-hematological tumor.
[0092] [0092] 5. The set of polypeptides according to any of the preceding items, in which the combination of antigen A1 and Antigen A2 is only found in carcinoma cells, and not in cells that are not cancerous, and in which , preferably, the combination of antigen and A1 Antigen A2 is specific for cancer cells of a certain type of cancer.
[0093] [0093] 6. The set of polypeptides according to any of the preceding items, wherein said antigen is an MHC A1 antigen, preferably an allele variant of any of the HLA-A, HLA-B, HLA-C alleles , HLA-DO, HLA-DR, or HLA-DM, more preferably, an allele variant of a class MHC molecule, more preferably, an allele variant selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A25, HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3, HLA-Cw4, and HLA-Cw7 and / or said A2 antigen is an antigen which is specific to a particular cell type or cell line.
[0094] [0094] 7. The set of polypeptides according to any of the preceding items, wherein said functional domain F is an immunoglobulin module, preferably an scFv (single chain variant fragment) of an antibody, or an fluorescent molecule, GFP or preferably a variant of GFP, or a molecule capable of mediating bioluminescence, preferably Gaussia luciferase.
[0095] [0095] 8. The set of polypeptides according to any of the preceding items, wherein said functional domain F is a domain that specifically binds to a carrier molecule, preferably a carrier molecule, which is a peptide or a carbohydrate molecule, or an affinity marker, preferably an affinity marker selected from the group consisting of a FLAG marker, a myc marker, a glutathione-S-transferase (GST) marker , a hemagglutinin marker (HA), a polyhistidine marker (His) and a maltose binding protein (PAM) marker.
[0096] [0096] 9. The set of polypeptides according to any of the preceding items, wherein said functional domain F is a domain that specifically binds to a radioactive compound, a domain that specifically binds to a toxin molecule that by itself is not able to penetrate through the cell membrane of a human cell and that is internalized in a human cell through association with the cell membrane of that cell, a domain that specifically binds to a fluorescent molecule, or a domain that specifically binds to a molecule capable of mediating bioluminescence.
[0097] [0097] 10. The set of polypeptides according to any of the preceding items, wherein said F1 fragment comprises a VL domain of an antibody and said fragment F2 comprises a VH domain of the same antibody, wherein, preferably, the said antibody is an anti-CD3 antibody, or wherein said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody, wherein, preferably, said antibody is an anti-CD3 antibody.
[0098] [0098] 11. The set of polypeptides according to any of the preceding items, for use in the treatment of a patient suffering from a tumor or for use in diagnosis in a patient suffering from a tumor, preferably for use in treatment of a patient suffering from a tumor and allogeneic tissue or having undergone cell transplantation or intended to undergo such transplantation or for use in the diagnosis of a patient suffering from a tumor undergoing or intended for allogeneic tissue or transplantation cells, where, preferably, said set of polypeptides is administered to said patient.
[0099] [0099] 12. A nucleic acid molecule or a set of nucleic acid molecules that encode the set of polypeptides or one of the polypeptides in the set of polypeptides according to any of the preceding items.
[00100] [00100] 13. Vector comprising the nucleotide sequence of the nucleic acid molecule according to item 12, or the sequence of one of the nucleic acid molecules of a set of nucleic acid molecules according to item 12.
[00101] [00101] 14. A pharmaceutical composition comprising either the set of polypeptides according to any items from 11 to 11 or the nucleic acid molecule / set of nucleic acid molecules according to item 12, or the vector according to the item 13, wherein, preferably, said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
[00102] [00102] 15. A kit comprising the set of polypeptides according to any of items 1-11.
[00103] [00103] Preferably, said A1 antigen is a cell surface molecule. Preferably, said A2 antigen is a cell surface molecule. Preferably, said A1 antigen is specific for the malignant state of a cell. Preferably, said A2 antigen is specific for a particular cell type or cell line or for the malignant state of a cell. Preferably, said A1 antigen is specific to a type of malignant cell. Preferably, said A2 antigen is specific to a type of malignant cell.
[00104] [00104] In one aspect, the present invention relates to the set of polypeptides as defined and described in the present invention, wherein, however, the A1 antigen is the same as the A2 antigen. In this way, in such a set of polypeptides P1 and P2, fragment F1 can be linked to the target portion and T1 fragment F2 can be linked to the target portion T2, while both T1 and T2 specifically bind the same antigen. In this context, the epitope on antigen A1, to which the target portion binds T1, can be the same or a different epitope like the epitope on antigen A2, to which the target portion binds T2. In the case of the epitope on antigen A1 it is the same as the epitope on antigen A2, Polypeptide P1 can comprise a target portion, which is identical to the target portion composed in P2. In addition, this aspect of the present invention is based on the advantage that the set of polypeptides of P1 and P2, with the interrupted F domain, does not exhibit off-target effects (for example, without pre-activation of T cells. Displaying CD3 and, consequently, in the less toxic properties and / or side effects, for example, compared to conventional bispecific antibodies).
[00105] [00105] In one embodiment, said fragment F1 and said fragment F2 together are said functional domain F.
[00106] [00106] In one embodiment, said polypeptide P1 and said polypeptide P2 are not covalently linked to each other, in the absence of a substrate that has both A1 and A2 antigens on their surface, more specifically, a cell that carries both A1 and A2 antigens, on its cell surface.
[00107] [00107] In one embodiment, said polypeptide P1 and Reference
[00108] [00108] Said polypeptide P1 and polypeptide P2 and / or, in particular, said fragment F1 and fragment F2, as understood there, more in particular the VH and VL, which can be understood there, are not associated with each other, especially when administered to an individual in need of medical intervention, that is, in need of therapy and / or diagnosis. Accordingly, the pharmaceutical or diagnostic means provided in the present invention comprise two polypeptides of P1 and P2 as included in the "set of polypeptides" defined in the present invention, in an unassociated manner. The association of the two said polypeptides takes place in vivo, under the presence of said substrate or cell. According to the presence of said substrate or cell, the association of the two said polypeptides can be (further) stabilized by means of a stabilizing agent (for example, an antigen, such as, for example, CD3 , HIS or DIG, as described in the present invention). Preferably, they are not associated with each other in the absence of said substrate or cells and / or do not dimer in the absence of said substrate or cell. More preferably, they are not associated with each other in the absence of said substrate or cells and / or do not dimerize in the absence of said substrate or cell, even if an agent is present, which stabilizes the association and / or dimerization of the polypeptide. - P1 peptide and P2 polypeptide and / or, in particular, F1 fragment and F2 fragment, that is, even if said P1 polypeptide and P2 polypeptide and / or, in particular, said F1 fragment and F2 fragment is present in a stabilizing agent / trimeric complex / P1 (F1) / P2 (F2) (for example, in a VH / VL antigen / trimeric complex).
[00109] [00109] In a specific embodiment, said polypeptide P1 and Polypeptide P2 and / or, in particular, said fragment F1 and fragment F2, as understood there, more in particular VH and VL, which can be understood there, they are associated with each other and / or dimerize in three parts of the formation complex, preferably through an interaction mediated by an agent that stabilizes the association and / or dimerization of polypeptide P1 and Polypeptide P2 and / or, in particular , Fragment F1 and Fragment F2 (for example, an antigen mediated interaction). More preferably, however, this association and / or dimerization occurs only in the presence of said substrate or cell.
[00110] [00110] The affinity force with which, for example, leucine zippers and / or immunoglobulin constant domains, such as CH3 or Fc fragments, hetero and homodimerization is estimated and be in constant KD dissociation in the range of 10 to 101 - M (see, for example, Zhu (1997) Protein Sci. 6, 781-8, .. Pluckthun (1997) Immunotech 3, 83-105). This KD interval is clearly smaller than the KD with which, in the absence of said substrate or cell, association and / or dimerization of said P1 and P2 polypeptides, in particular such F1 and F2 fragments, of the present invention can occur - laugh. Thus, in one embodiment, Polypeptide P1 and polypeptide P2 and / or, in particular, Fragment F1 and Fragment F2 as understood there, more particularly the VH and VL, which can be understood there, associated with the other and / or in the absence of dimerization of said substrate or cell only with a KD that is superior to Kd, for example, hetero- and homodimerization of leucine zippers and / or immunoglobulin constant domains, such as CH3 or Fc fragments. In the presence of said substrate or cell, it is anticipated that polypeptide P1 and Polypeptide P2 and / or, in particular, Fragment F1 and Fragment F2 as comprised therein, more in particular VH and VL, which can be understood there, associated with another and / or dimerizes with a KD, which is in the KD range of, for example, hetero- and homodimerization of leucine zippers and / or constant domains, such as immunoglobulin CH3 or Fc fragments, or even below this range.
[00111] [00111] The interaction force, for example, isolated VH and VL domains, in general, is of low affinity. Using calorimetry, fluorometric or ultraviolet difference spectroscopy and / or circular dichroism techniques, the KD dissociation constants from 10º to 10º M were determined (see, for example, spend JMB (2001) 305, 989 to 1010; Pluckthun (1992) Immunological comments No. 130). Using plasmonic surface resonance techniques (SPR biosensor Bl-Acore or BlAcore 2000, Pharmacia) and a system of anti HEL antibodies (anti hyen lysozyme antibody HyYHEL-10), Ueda (loc. Cit.) And Ohmuro- Matsuyama (loc. Cit.) It was found that isolated VH and VL domains do not dimerize at all (Ka <105 / M, below the detection limit). However, the association of VH and VL peptides was significantly increased in the presence of cognate antigens (Ka - 109 / m), with a notable reduction in the dissociation rate of the antigen / VH / VL trimer complex with a Kd calculated - 2.73 x 10º + 1.43 x 10 $ / s to 1.4 µM of the antigen. Therefore, it is particularly envisaged within the scope of the present invention that KD with which, in the absence of said substrate or cell, association and / or dimerization of said polypeptides P1 and P2, in particular of such fragments F1 and F2, of the present invention can occur only in, or even above, the KD or KD range of isolated VH and VL domains for example, as assessed in the context of dress, Pluckthun (1992 (op. cit.) ;. loc cit.) , Ueda (loc. Cit.) And Ohmuro-Matsuyama (loc. Cit.), In particular above KD or KD range of the VH / VL antigen / trimeric complex as was estimated in the context of Worn (loc. Cit), Pluckthun (1992; loc cit), Ueda (loc. Cit) and Ohmuro-Matsuyama (loc. Cit). In the pre-
[00112] [00112] In one aspect, Polypeptide P1 and polypeptide P2 and / or, in particular, Fragment F1 and Fragment F2 as understood there, more in particular VH and VL, which can be understood there, are not associated in the absence of said substrate or cell and / or does not dimerize in the absence of said substrate or cell. If at all, they associate with each other and / or dimerize in the absence of said substrate or cell only with a KD of 10º M above, preferably above 10º M, more preferably above 10º M and more preferably above of 10 -4M. In another aspect, if at all, do they associate with each other and / or dimerize in the absence of that substrate or cell with only one KD in the range of 10º M to 10 M, preferably from 107 M to 10 3 M, more preferably from 10º M to 10 M and even more preferably 10º M to 10º M. | in another aspect, polypeptide P1 and Polypeptide P2 and / or, in particular, Fragment F1 and Fragment F2 as understood there, more in particular the VH and VL, which can be understood there, are associated, in the presence of said substrate or of cells and / or dimerize in the presence of said substrate or cell. In particular, they associate with each other and / or dimerize in the presence of said substrate or cell with a
[00113] [00113] In a preferred embodiment, the same applies in the case of an agent that is present, which stabilizes the association and / or dimerization of polypeptide P1 and Polypeptide P2 and / or, in particular, fragment F1 and fragment F2. For example, as a stabilizing agent according to the present invention, it can be an antigen, such as, for example, CD3, HIS or DIG, as described in the present invention, capable of binding to the F domain, which, for example, it can comprise an antibody VH and AVL (F1 and F2, respectively, or F2 and F3, respectively).
[00114] [00114] If "present", in the context of the present invention and, in particular, in the above scope (i.e., in relation to said agent and / or said substrate or cells and / or said antigens A1 and A2), in particular it means being present at a concentration in the range of 0.01 µM to 1 MM, in a range of 0.1 to 500 µM, in a range of 0.1 to 300 µm, in the range of 0.1 to 100 µM , in a range of 1 to 500 µm, in a range of 10 to 500 µM. Being "absent", in the context of the present invention and, in particular, in the above context (that is, in relation to said agent and / or said substrate or cells and / or said antigens A1 and A2), in particular means a concentration below the ranges above or below 1 mM, 500 CU, 300 µM, 100 µM, 10 CU, 1 CU, 0.1 CU, 0.01 CU, 0.001 CU or 1 AM, where preferred the lowest values.
[00115] [00115] The person who is versed in the technique is readily in a position to measure the KD of dimerization, in particular, of P1 and P2,
[00116] [00116] In a particular aspect, the KDs referred to in the present invention (i) apply to, (ii) are in or (iii) are to be measured at a temperature of 4-38C, preferably from 4 to 20 " C (for example 10T) or 20 and 38C (for example 30T), and / or a pH of 4.5 to 8 (for example, a pH of 7),
[00117] [00117] "Not associated", in the context of the present invention, particularly means not functionally associated with respect to the function of the F domain, that is, not allowing F1 and F2 to form a functional F. Thus, in one aspect of the present invention, P1 and P2 can be linked to each other (for example, covalently) insofar as no functional domain F is formed by F1 and F2. It is, however, preferred that P1 and P2 are separated.
[00118] [00118] In one embodiment, said A1 antigen and / or said A2 antigen is a molecule.
[00119] [00119] In one embodiment, said A1 antigen and / or said A antigen it's protein.
[00120] [00120] In one embodiment, said A1 antigen and / or said A2 antigen is non-protein.
[00121] [00121] In one embodiment, said target portion binds T1 non-covalently to said A1 antigen.
[00122] [00122] In one embodiment, said target portion binds to T2 not covalently to said A2 antigen.
[00123] [00123] In one embodiment, a substrate with both antigens A1 and A2, on its surface induces dimerization of the F1 fragment of said polypeptide P1 with Fragment F2 of said polypeptide P2, while a substrate, which does not have both Al and A2 antigens, on their cell surface does not induce dimerization of Fragment F1 of said polypeptide P1 with Fragment F2 of said polypeptide P2.
[00124] [00124] In one embodiment, a cell having both antigens A1 and A2, on its cell surface induces dimerization of Fragment F1 of said polypeptide P1 with Fragment F2 of said polypeptide P2, whereas a cell that it does not carry both antigens A1 and A2, in its surface cell it does not induce dimerization of Fragment F1 of said polypeptide P1 with Fragment F2 of said polypeptide P2. In this context, "induces dimerization" particularly means "allows juxtaposition and subsequent dimerization".
[00125] [00125] In one embodiment, said target portion T1 comprises an immunoglobulin module and / or said target portion T2 comprises an immunoglobulin module.
[00126] [00126] In one embodiment, said target portion T1 comprises an immunoglobulin Il module, which comprises a VL domain linked to a VH domain, preferably an immunoglobulin Il module comprising an scFv (fragment of variant of single chain) of an antibody, a Fab fragment or an F (ab ') 2 (e.g., with additional parts, e.g., an Fe domain) of an antibody or a complete antibody.
[00127] [00127] and / or said target portion T2 comprises an immunoglobulin 12 module, comprising a VL domain linked to a VH domain, preferably an immunoglobulin 12 module comprising an scFv (variant fragment) single-stranded) of an antibody to a Fab fragment or an F (ab), (for example, with additional parts, for example, an Fc domain) of an antibody or a complete antibody.
[00128] [00128] In one embodiment, said target portion T1 and / or said target portion T2 comprises an immunoglobulin module comprising a VhH region of the variable domain of a mud antibody, a camel antibody, or a shark antibody.
[00129] [00129] In one embodiment, said target portion T1 and / or said target portion T2 is an aptamer or a natural ligand of said antigen A1 or Antigen A2, respectively.
[00130] [00130] In one embodiment, said target portion T1 and / or said target portion T2 comprises an Fv or scFv (variant fragment (single chain)) of an antibody.
[00131] [00131] In one embodiment, the immunoglobulin module included in the target portion T1 and T2 comprises a domain V selected from the group consisting of:
[00132] [00132] a V domain of an anti-HLA-A2 antibody comprising a VL domain comprising SEQ ID NO: 78 and 79 (CDRs 1 and 3) and DAS (CDR 2) and / or a VH domain which comprises SEQ ID NO: 75-77 (CDRs 1-3);
[00133] [00133] a V domain of an anti-HLA-Cw6 antibody comprising a VL domain comprising SEQ ID NO: 83 and 84 (CDRs 1 and 3) and DDS (CDR 2) and / or a VH domain that comprises SEQ ID NO: 80-82 (CDRs 1-3);
[00134] [00134] a V domain of an antic = EpCAM antibody comprising a VL domain comprising SEQ ID NO: 88 and 89 (CDRs 1 and 3) and WAS (CDR 2) and / or a VH domain comprising SEQ ID NO: 85-87 (CDRs 1-3);
[00135] [00135] an V domain of an anti-Her2 antibody comprising a VL domain comprising SEQ ID NO: 93 and 94 (CDRs 1 and 3) and SAS (CDR 2) and / or a VH domain comprising SEQ ID No. 90-92 (CDRs 1-3);
[00136] [00136] a V domain of an anti-EGFR1 antibody comprising a VL domain comprising SEQ ID NO: 98 and 99 (CDRs 1 and 3) and DAS (CDR 2) and / or a VH domain comprising SEQ ID No. 95-97 (CDRs 1-3);
[00137] [00137] a V domain of an anti-CEA antibody comprising a VL domain comprising SEQ ID NO: 103 and 104 (CDRs 1 and 3) and SAS (CDR 2) and / or a VH domain comprising SEQ ID No. 100-102 (CDRs 1-3);
[00138] A V domain of an anti-CD45 antibody comprising a VL domain comprising SEQ ID NO: 107 and 108 (CDRs 1 and 3) and LAS (CDR 2) and / or a VH domain comprising SEQ ID No. 105 and 106 (CDRs 1 and 2) and CDR3 or SEQ ID NO: 132-134 (CDRs 1-3);
[00139] [00139] a V domain of an anti-CD138 antibody comprising a VL domain comprising SEQ ID NO: 112 and 113 (CDRs and
[00140] [00140] an V domain of an anti-CD19 antibody comprising a VL domain comprising SEQ ID NO: 158 and 159 (CDRs 1 and 3) and DAS (CDR 2) and / or a VH domain comprising SEQ ID No. 155-157 (CDRs 1-3).
[00141] [00141] In a preferred, additional embodiment, the immunoglobulin module comprised in the target portion T1 and / or T2 comprises a domain V selected from the group consisting of:
[00142] [00142] a V domain of an anti-HLA-A2 antibody comprising a VL domain comprising SEQ ID NO: 52 and / or a VH domain comprising SEQ ID NO: 51;
[00143] [00143] a V domain of an anti-HLA-Cw6 antibody comprising a VL domain comprising SEQ ID NO: 54 and / or a VH domain comprising SEQ ID NO: 53;
[00144] [00144] a V domain of an anticEpCAM antibody comprising a VL domain comprising SEQ ID NO: 56 and / or a VH domain comprising SEQ ID NO: 55;
[00145] [00145] a V domain of an anti-Her2 antibody comprising a VL domain comprising SEQ ID NO: 58 and / or a VH domain comprising SEQ ID NO: 57;
[00146] [00146] a V domain of an anti-EGFR1 antibody comprising a VL domain comprising SEQ ID NO: 60 and / or a VH domain comprising SEQ ID NO: 59;
[00147] [00147] a V domain of an anti-CEA antibody comprising a VL domain comprising SEQ ID NO: 62 and / or a VH domain comprising SEQ ID NO: 61;
[00148] [00148] an V domain of an anti-CD45 antibody comprising a VL domain comprising SEQ ID NO: 64 and / or a VH domain comprising SEQ ID NO: 63; and
[00149] [00149] an V domain of an anti-CD138 antibody comprising a VL domain comprising SEQ ID NO: 66 and / or a VH domain comprising SEQ ID NO: 65;
[00150] [00150] a V domain of an anti-CD19 antibody comprising a VL domain comprising SEQ ID NO: 153 and / or a VH domain comprising SEQ ID NO: 152.
[00151] [00151] In another, preferred, embodiment, the immunoglobulin module comprised in the target portion T1 and / or T2 comprises a domain V comprising any of SEQ ID NO: 67-74 and
[00152] [00152] In one embodiment, polypeptide P1 has the general structure F1-T1 and / or polypeptide P2 has the general structure F2-T2. Fragment F and T portions can be separated by means of a ligand (for example, F1-ligand-T1 and / or F2-ligand-T2) and / or flanked by (one) amino acid elongation (s) additional (s) 1 and / or 2 (elongation - -F1- (ligand) -T1-elongation2 and / or elongation1-F2- (ligand) -T2-elongation2). It is preferred that the previous general structure is from the N-terminus to the C-terminus of the polypeptides, that is, N-F1- T1-C and / or N-F2-T2-C, N-F1-ligand-T1-C and / or N-F2-ligand-T2-C and N-Elongation1-F1- (ligand) -T1-elongation2-C and / or N- Elongation1-F2- (ligand) -T2-elongation2-C. In case the target portion is or comprises an immunoglobulin module which, like an Fv or scFv, the P1 polypeptide can have the general structure F1-VH1-VL1 and / or Polypeptide P2 can have the general structure F2-VH2-VL2 or polypeptide P1 can have the general structure F1-VL1-VH1 and / or polypeptide P2 can have the general structure F2-VL2-VH2. Also in these cases, the F fragment and the T portions can be separated by means of a ligand (for example, F1-ligand-VH / VL1I-VL / IVH1 and / or F2-ligand-VH / VL2-VL / VH2 ) and / or flanked by means of (a) stretching (s) 1 and / or 2 (stretching1-F1- (linker) of additional amino acids -
[00153] The linker described above, in particular that between the V domains, may comprise from 1 to 25 amino acids, preferably from 12 to amino acids, preferably from 12 to 16 or from 15 to 20 amino acids. The ligand described above may comprise one or more motifs (G3S) and / or (G4S), in particular 1, 2, 3, 4, 5 or 6 (G3S) and / or motifs (G4S), preferably 3 or 4 ( G3S) and / or reasons (G4S), more preferably 3 or 4 reasons (G4S).
[00154] [00154] In one embodiment, said immunoglobulin 1 module and said F1 fragment are separated by means of a linker comprising 1 to 12, preferably 3 to 12, amino acids, and / or said immunoglobulin and said module 12 of the F2 fragment are separated by means of a linker comprising from 1 to 12, preferably 3 to 12, amino acids.
[00155] [00155] In one embodiment, the VL domain of 11 is linked to the VH domain of 11 by means of a linker comprising 12 to 25 amino acids, preferably a linker with the sequence (G3S) 3 or (G3S) 4 or (G4S) 3 or (G4S) 4 and / or the VL domain of | 2 is linked to the VH domain of 12 by means of a linker comprising 12 to 25 amino acids, preferably a sequence binding agent ( G3S) 3 or (G3S) 4 or (G4S) 3 or (G4S) 4.
[00156] [00156] As mentioned, the linker as described above can comprise the motif (G3S) and / or (G4S). Alternative binders may consist entirely of or comprise the GEGTSTGSGGSGGSGGAD motif. The person skilled in the art can, without further ado, find and use additional ligands (peptides) known in the art.
[00157] Said additional amino acid elongations 1 and / or 2, may comprise or comprise from 1 to 200, 1 to 100, 1 to 70, 65 to 1, 50, 1 to 25 or 1 to 20 amino acids.
[00158] [00158] In one embodiment, said A1 antigen and / or said A2 antigen is an antigen expressed on the surface of tumor cells or on the surface of progenitor / precursor cells of a tumor, preferably an antigen expressed on the surface of cells from a hematological tumor, more preferably, an antigen expressed on the surface of cells selected from the group consisting of acute myeloid leukemia cells, chronic myeloid leukemia cells, lymphatic leukemia cells acute, chronic lymphatic leukemia cells, cell lymphoma cells, myeloproliferative syndrome, myelodysplastic cells, more preferably myeloma cells, or said A1 antigen and / or Referred A2 antigen is an antigen expressed on cell surface of a non-hematological tumor, preferably a cell selected from the group consisting of renal cell carcinoma cells, bladder cancer cells, cell lung cancer cells, mesothelioma cells, prostate cancer cells, brain cancer cells, bone cancer cells, sarcoma cells, soft tissue cancer cells, ovarian cancer cells, cancer cells - cervical cancer, breast cancer cells, endometrial cancer cells, uterine carcinoma cells, germ cell tumor cells, anal carcinoma cells, rectal carcinoma cells, colon carcinoma cells, colon cells small intestine carcinoma
[00159] [00159] In one embodiment, the combination of A1 antigen and Antigen A2 is found only in blood cells or blood cell precursor cells, preferably only in one type of blood cell.
[00160] [00160] In one embodiment, the combination of antigen A1 and Antigen A2 is only found on the target, in particular, the carcinoma cells, and not (or only negligibly) in cells that are not target cells, in that are not carcinogenic. In a preferred embodiment, the combination of antigen A1 and Antigen A it is specific for carcinoma cells of a certain type of cancer.
[00161] [00161] In one embodiment, the combination of antigen A1 and Antigen A2 distinguishes a certain type of cells, preferably a certain type of carcinoma cells, from any other cells.
[00162] [00162] "Certain types of cancer", in this context, means type of cancer, characterized by the same organ, in which the cancer is formed or, preferably, the type of cancer, characterized by the same pair of antigens from A1 and A2 (aberrant).
[00163] [00163] In one embodiment, the combination of A1 antigen and Antigen A2 is found in progenitor / precursor cells that are progenitor / precursor cells in a tumor and not in progenitor / precursor cells that are not progenitor / precursor cells in a tumor. a tumor.
[00164] [00164] In one embodiment, said antigen is an A1 antigen that is specific to the malignant state of a cell and said A2 antigen is an antigen that is specific to the cell type or cell line of said cell.
[00165] [00165] In one mode,
[00166] [00166] A1l antigen is EpCAM (epithelial cell adhesion molecule) and A2 antigen is CD10 (differentiation cluster 10), HER2 / neu (human epidermal growth factor receptor 2), (vascular endothelial growth factor receptor , VEGF-R), EGFR (epidermal growth factor receptor, also called HER1 (human epidermal growth factor 1 receptor) or ErbB1) or MDR (multiple drug resistance protein), or
[00167] [00167] antigen A1 is MCSP (chondroitin sulfate proteoglycan sulphate associated with melanoma) and antigen A2 is melanoferrin or EDBCAM, or
[00168] [00168] antigen A1 is CA125 (cancer antigen 125 / antigen of carbohydrate 125) and antigen A2 is CD227 (PEM (polymorphic epithelial mucin) or MUC1 (mucin-1)), or
[00169] [00169] antigen A1 is CD56 and antigen A2 is CD140b (PDGFRRB (platelet receptor beta growth factor)) or GD3 ganglioside, or
[00170] [00170] antigenA1 isoEGFRe antigen 2 is HER2, or
[00171] [00171] antigen A1 is PSMA (prostate specific membrane antigen) and antigen 2 and HER2, or
[00172] [00172] antigen 1 is Sialila Lewis and antigen 2 is EGFR, or
[00173] [00173] antigen 1 is CD44 and antigen 2 is ESA (epithelial surface antigen) (CD326, EDpCAM), CD24, CD133, MDR (multidrug resistance protein) or CD117, or
[00174] [00174] antigen 1 is CD34 is antigen 2 is CD19, CD79a, CD 2, CD7, HLA-DR (human leukocyte antigen DR), CD13, CD117, CD33 or CD15, or
[00175] [00175] antigen 1 is CD33 is antigen 2 is CD19, CD79a, CD 2, CD7, HLA-DR (human leukocyte antigen DR), CD13, CD117 or CD15, or
[00176] [00176] antigen 1 is a MUC-1 antigen and antigen 2 is CD10, CEA or CD57, or
[00177] [00177] antigen 1 is CD38 and antigen 2 is CD138, or
[00178] [00178] antigen 1 is a CD2 and antigen 2 is CD29 or CD49f, or
[00179] [00179] antigen 1 is carbonic anhydrase IX and antigen 2 is aquaporin, preferably aquaporin-2.
[00180] [00180] In one embodiment, said A1 antigen and / or Referred A2 antigen is selected from the group consisting of HLA-A (HLA-A of the main histocompatibility complex, class |, À [Homo sapiens]; Gene ID : 3105 updated on 13-Jan-2013; DAQB- 90C11.16-002; Chromosome: 6; NC 000006.11 (29910247..29913661), for HLA-A2: 1. mRNA = LOCAL NM 001242758 = NM version 001242758.1 Gl: 337752169 = Gen- Bank: AY191309.1 PRI 13-JAN-2013; 2. Protein = P79495 [UniParc]. Last modified on May 1, 1997. Version 1 .; for HLA-Cw6: MRNA = LOCAL HUMMHCCWSB6A = GenBank: VERSION M28160.1 GI: 531197PRI (18-AUG-1994); Protein = 029963 [UniParc]. Last modified on August 22, 2003. Version 2.); EBDCAM (a-
[00181] [00181] In one embodiment, said A1 antigen and / or said A2 antigen is an MHC antigen, preferably an allelic variant of any of the HLA-A, HLA-B, HLA-C, HLA-DQ alleles , HLA-DR, or HLA-DM, more preferably, an allele variant of a class MHC molecule, more preferably, an allele variant selected from the group consisting of HLA-A1, HLA-A2 , HLA-A3, HLA-A25, HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3, HLA-Cw4, HLA-Cw6 and HLA-Cw7.
[00182] [00182] In one embodiment, said A1 antigen is HLA-A2.
[00183] [00183] In one embodiment, said A1 antigen and / or said A2 antigen is selected from the group consisting of CD45, aquaporin, preferably aquaporin-2, class B 1 member scavenger receptor (SCARB1), CD34, CD33, CD138, CD15, CD1a, CD2, CD3, CD4, CD5, CD8, CD20, CD23, CD31, CD43, CD56, CD57, CD68, CD79a, CD146, synaptophysin, CD56, CD57, nicotinic acetylcholine receptor, muscle kinase (MUSK), voltage-dependent calcium channels (/ Q-type P), voltage-dependent potassium channels (CPVD), N-methyl-D-aspartate (NMDA) receptor, TSH (thyroid stimulating hormone) receptor, amphiphysin, HepPar-1, GQ1b ganglioside, GD3 ganglioside, GM1 ganglioside and glycophorin-A.
[00184] [00184] In a preferred embodiment, said A1 antigen is an MHC antigen and said A2 antigen is an antigen that is specific to a particular cell type or cell line.
[00185] [00185] In one embodiment, said functional domain F is an immunoglobulin module, preferably an scFv (single chain variant fragment) of an antibody, most preferably an Fv (variant fragment) of an antibody, or a fluorescent molecule, preferably a bimolecular complementing fluorescence molecule, more preferably GFP or a variant of GFP, or a molecule capable of mediating bioluminescence, preferably a luciferase molecule, more preferably Gaussia lucifera.
[00186] [00186] In one embodiment, said functional domain F is an Fv (variant fragment) of an antibody.
[00187] [00187] In one embodiment, said functional domain F specifically binds or is able to bind specifically to an antigen. In a specific aspect, said antigen may be an antigen that is present in cells of the human immune system. In a preferred embodiment, said binding activates said cells of the human immune system.
[00188] [00188] In one embodiment, said functional domain F is a T cell envelope domain, preferably a T cell envelope domain specifically binds to CD2, CD3, CD5, T cell receptor or CD28, more preferably a T cell surrounding domain specifically binds to CD3e, an NK cell surrounding domain (natural killer cells), preferably an NK cell surrounding domain specifically binds to CD1a, CD16a or CD56, a domain involving macrophages, from preferably a domain involving macrophages binds specifically to CD16a, CD32a, CD32b, CD89 or CD64, a monocyte surrounding domain, preferably, a monocyte surrounding domain specifically binds to CD32a, CD32b, CD64 or CD89, a granulocyte envelope domain, preferably a granulocyte envelope domain specifically binds to CD16b, CD32a, CD32b, CD64, or CD89, a granulocyte envelope domain, preferably a granulocyte envelope domain neutrophils, which specifically binds to CD89 (FcaRI), or a granulocyte surrounding domain monocytes and / or activated macrophages, preferably a granulocyte, neutrophil, monocyte and / or activated macrophage surrounding domain, which specifically binds specifically CD64 (FcyRI).
[00189] [00189] In one embodiment, said functional domain F is a domain that specifically binds to an antigen linked to a diagnostic or therapeutic compound.
[00190] [00190] In one embodiment, said functional domain F is a domain that specifically binds to a carrier molecule, or an affinity marker. Preferably, said carrier molecule is linked to a diagnostic or therapeutic compound. Preferably, said affinity marker is linked to a diagnostic or therapeutic compound.
[00191] [00191] - Preferably, said affinity marker is selected from the group consisting of a FLAG marker, a Myc marker, a glutathione-S-transferase (GST) marker, a hemagglutinin (HA) marker - tag, a polyhistidine marker (His), a digoxigenin marker (DIG) and a maltose binding protein (MBP) marker.
[00192] [00192] Preferably, said carrier molecule is a peptide or a carbohydrate molecule. In a preferred embodiment, said functional domain F is a domain that specifically binds to a carrier molecule, preferably a vehicle molecule linked to a diagnostic or therapeutic compound, wherein said molecule vehicle is selected from the group consisting of gelatin, inulin, dextran and hydroxyethyl starch.
[00193] [00193] In one embodiment, said therapeutic compound is a radioactive compound, preferably a radioactive compound, which comprises 90Y, 177Lu, 1311, 32P, 10B, or 213Bi. In one embodiment, said therapeutic compound is a toxin. Preferably, said toxin is selected from the group consisting of edema factor B. anthracis, lethal factor B. anthracis, iota toxin C. perfringes, C2 toxin C. botulinum, ADP-ribosyltransferase C. difficile, toxin of diphtheria of fragment A C. diphtheriae, toxin shiga Burgholderia sp. (subunit D), Clostridium perfringens str. 13 PFOA toxin perfringo-lysine O, ricin A chain, plant RIP bouganine, human RNA-SE3 ribonuclease (RNase A family, 3) and lethal anthrax factor endopeptidase. Another non-limiting example of a toxin according to the present invention is a toxin being or comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 160-168.
[00194] [00194] In one embodiment, said diagnostic compound is a radioactive compound, preferably a radioactive compound comprising 99mTc, 111In, 82Rb and 201TI. In one embodiment, said diagnostic compound is a fluorescent compound, preferably GFP, a variant of GFP, or a small molecule fluorescent compound, such as FITC (fluorescein isothiocyanate), PE (phycoerythrin), an Alexa Fluor dye (such as AlexaFluor488 or related dyes) or a cyanine dye (such as Cy3 (Indo-carbocyanin) or Cy5 (Indodicarbocyanine) or related dyes). In one embodiment, said diagnostic compound is a molecule capable of mediating bioluminescence, preferably a luciferase molecule, more preferably Gaussia luciferase.
[00195] [00195] In one embodiment, said F1 fragment comprises a VL domain of an antibody and said F2 fragment comprises a VH domain of the same antibody, wherein, preferably, said antibody is an anti-CD3 antibody, more preferably an anti-CD3e antibody, or an anti-His or anti-DIG antibody or said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody, wherein preferably, said antibody is an anti-CD3 antibody, more preferably an anti-CD3e antibody, or an anti-His and anti-DIG antibody.
[00196] [00196] In another modality, the VL and VH domains as included in fragment F1 and F2, respectively, or in fragment F2 and F1, respectively, may also have two different antibodies, specific to the same antigen (and for the same or a different epitope) or for different antigens. This is, for example, expected to be used where the new specifications are to be created (for example, in phage display approaches).
[00197] [00197] In another embodiment, the immunoglobulin module comprised in domain F comprises a domain V selected from the group consisting of:
[00198] [00198] an V domain of an anti-CD3 antibody, comprising a VL domain comprising SEQ ID NO: 18-20 (CDR 1-3) and / or a VH domain comprising SEQ ID NO: 15-17 ( CDR 1-3);
[00199] [00199] a V domain of an anti-CD3 antibody, comprising a VL domain comprising SEQ ID NO: 24-26 (CDR 1-3) and / or a VH domain comprising SEQ ID NO: 21-23 ( CDR 1-3);
[00200] [00200] an V domain of an anti-CD3 antibody, comprising a VL domain comprising SEQ ID NO: 30-32 (CDR 1-3) and / or a VH domain comprising SEQ ID NO: 27-29 ( CDR 1-3);
[00201] [00201] an V domain of an anti-CD3 antibody, comprising a VL domain comprising SEQ ID NOS: 36 and 37 (CDR 1 and 3) and DTS (CDR 2) and / or a VH domain comprising SEQ ID NO: 33-35 (CDRs 1-3);
[00202] A V domain of an anti-CD3 antibody, comprising a VL domain comprising SEQ ID NO: 41 and 42 (CDR 1 and 3) and YTN (CDR 2) and / or a VH domain comprising SEQ ID NO: 38-40 (CDRs1-3); and
[00203] [00203] a V domain of an antibody anti-His antibody comprising a VL domain comprising SEQ ID NO: 46 and 47 (CDR 1 and 3) and KVS (CDR 2) and / or a VH domain comprising SEQ ID NO: 43-45 (CDRs 1-3);
[00204] [00204] a V domain of an anti-DIG antibody, comprising a VL domain comprising SEQ ID NO: 50 and 131 (CDRs 1 and 3) and YSS (CDR 2) and / or a VH domain comprising SEQ ID No. 48 and 49 (CDR 1 and 2) and A (3 CDR).
[00205] [00205] “In another preferred, additional modality, the immunoglobulin module comprised in domain F comprises a domain V selected from the group consisting of:
[00206] [00206] an V domain of an anti-CD3 antibody comprising a VL domain comprising SEQ ID NO: 2 and / or a VH domain comprising SEQ ID NO: 1;
[00207] [00207] a V domain of an anti-CD3 antibody comprising a VL domain comprising SEQ ID NO: 4 and / or a VH domain comprising SEQ ID NO: 3;
[00208] [00208] an V domain of an anti-CD3 antibody comprising a VL domain comprising SEQ ID NO: 6 and / or a VH domain comprising SEQ ID NO: 5;
[00209] [00209] an V domain of an anti-CD3 antibody comprising a VL domain comprising SEQ ID NO: 8 and / or a VH domain comprising SEQ ID NO: 7;
[00210] [00210] an V domain of an anti-CD3 antibody comprising a VL domain comprising SEQ ID NO: 10 and / or a VH domain comprising SEQ ID NO: 9; and
[00211] [00211] a V domain of an anti-His antibody comprising a VL domain comprising SEQ ID NO: 12 and / or a VH domain comprising SEQ ID NO: 11;
[00212] [00212] an V domain of an anti-DIG antibody comprising a VL domain comprising SEQ ID NO: 14 and / or a VH domain comprising SEQ ID NO: 30.
[00213] [00213] In one embodiment, said functional domain F is a domain that specifically binds to a toxin molecule, preferably a toxin molecule that alone is not able to penetrate through the cell membrane of a human cell, and which, preferably, is internalized in a human cell through association with the cell membrane of said cell, where, preferably, said association with the cell membrane of said cell is mediated by means of specific binding to a heterodimer formed from two molecules, preferably two molecules associated with said cell membrane, wherein, preferably, said two molecules are the polypeptides of P1 and P2, as described in the present invention. In one embodiment, the referred to functional domain F is a domain that specifically binds to component A (active component) of a bacterial infection of two components of the AB toxin. In one embodiment, said functional domain F is a domain that specifically binds to a toxin selected from the group consisting of edema factor B. anthracis, lethal factor B. anthracis, iota toxin C. perfringens, C2 toxin C. botulinum num, ADP-ribosyltransferase C. diffícile, diphtheria toxin from fragment A C. diphtheriae, toxin shiga Burgholderia sp. (subunit D), Clostridium perfringens str. 13 PFOA toxin perfringolysin O, chain A ricin, plant RIP bouganine, Human RNASE3 ribonuclease (RNase A family, 3) and lethal anthrax factor endopeptidase. Another non-limiting example of a toxin according to the present invention is a toxin being or comprising an amino acid sequence selected from the group consisting of SEQ ID NO 160-168.
[00214] [00214] In one embodiment, said functional domain F is a domain that specifically binds to a fluorescent molecule, preferably a fluorescent molecule, which alone is not able to penetrate through the cell membrane of a human cell. Preferably, said fluorescent molecule is GFP or a variant of GFP, or a molecule that is or comprises a small molecule fluorescent compound, such as FITC (fluorescein isothiocyanate), PE (phycoerythrin), a Alexa Fluor dye (such as AlexaFluor488 or related dyes) or a cyanine dye (such as Cy3 (indocarbocyanine) or Cy5 (Indodicarbocyanine) or related dyes).
[00215] [00215] In one embodiment, said functional domain F is a domain that specifically binds to a molecule capable of mediating bioluminescence, preferably to a luciferase molecule, with greater preference for Gaussia luciferase.
[00216] [00216] In one embodiment, said functional domain F is a fluorescent molecule, preferably a bimolecular complementing fluorescence molecule, more preferably GFP or a variant of GFP, such as YFP, PCP, Venus, or Cerulea.
[00217] [00217] Particular examples of P1 or P2 polypeptides comprised in the set of polypeptides according to the present invention are polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 114-129 and 197.
[00218] [00218] In general, the present invention relates to the treatment or elimination of a population of undesirable cells and the treatment or prevention of any disorder or disease that comes with this population of unwanted cells. For this purpose, the set of polypeptides of the present invention is to be used.
[00219] [00219] In one embodiment, said set of polypeptides is a set of polypeptides for use in treating a patient suffering from a tumor or cancer or for use in diagnosing a patient suffering from a tumor or cancer, preferably for use in the treatment of a patient suffering from a tumor or cancer and undergoing allogeneic tissue or cell transplantation or intended to undergo such transplantation, or for use in diagnosis in a patient suffering from a tumor or cancer and is submitted to or destined for allogeneic tissue or cell transplantation, in which, preferably, said set of polypeptides is administered to the referred patient.
[00220] [00220] Examples of tumors to be treated or diagnosed are those for which tumor or cancer cells are described in the present invention above in relation to antigens A1 and / or A2.
[00221] [00221] In one embodiment, said treatment involves the elimination of recipient tissue / cells of a certain type of cell,
[00222] [00222] In one embodiment, the set of polypeptides of the present invention is for use in an allogeneic fixation transplant for hematopoietic neoplasms, for example, with non-coincident HLA antigens, in particular for use in therapeutic exfoliation incompatibility situation. In this example situation, the double information of the HLA haplotype recipient (HLApatient) and origin of the hematopoietic lineage (CD45) is presented exclusively in the patient's leukemia blasts and other hematopoietic cells. All other cells of the original recipient express the recipient haplotype but not the CD45 hematopoietic cell antigen (for example, non-hematopoietic cell receptors are positive for HLA-A2 but negative for CD45). Likewise, all hematopoietic cells from the donor expressed HLA haplotype molecules from the expressed donor which means that they are CD45 positive, but negative for HLA-A2, in the situation of an incompatibility transplant, where the patient, but not the donor is positive for HLA-A2. Therefore, the present invention also relates to bimolecular and complementary single-chain antibody constructs directed against HLA-A2, in cases where the patient, but is not the HLA-A2 positive donor, and a second specific construct for the CD45 hematopoietic lineage marker to specifically target all the patient's hematopoietic cells, including all hematological neoplasms. In this manner, the first P1 polypeptide can comprise a single chain construct antibody fragment directed against the patient's HLA variable (target portion T1) fused to the anti-CD3 F1 VL fragment (e.g., the F1 fragment). The second P2 polypeptide can comprise a single-chain variable fragment specific to construct a hematopoietic lineage marker (eg, CD45; target portion T2), fused to the VH-divided F2 anti-CD3 Fv fragment (F2 fragment) .
[00223] [00223] In one embodiment, said elimination involves the destruction of said recipient tissue cells / or said recipient precursor cells by means of the cells of the immune system, by means of a toxin or a compound radioactive.
[00224] [00224] In one embodiment, said set of polypeptides is a set of polypeptides for use in diagnosis in a patient suffering from allogeneic tissue or cell transplantation, where, preferably, said patient is a patient suffering from a tumor.
[00225] [00225] In one embodiment, said diagnostic use involves the specific detection of recipient cells of a given cell type or cell line among recipient cells of different type cells or cell lines and donor cells of the same type or type different and cell line.
[00226] [00226] In one embodiment, said diagnostic use involves the specific detection of recipient cells that are malignant cells among the recipient cells that are not malignant and between the donor cells. In one embodiment, said set of polypeptides is administered to a patient.
[00227] [00227] Preferably, said patient is a mammal, more preferably a human being.
[00228] [00228] In one embodiment, said administration occurs through bolus administration or through continuous administration.
[00229] [00229] In one embodiment, polypeptides P1 and P2 of said set of polypeptides are administered in parallel. In another embodiment, polypeptides P1 and P2 of said set of polypeptides are administered sequentially.
[00230] [00230] In one embodiment, one of the P1 or P2 Polypeptides of said set of polypeptides is administered via bolus administration, while the other is administered through continuous administration.
[00231] [00231] In one embodiment, is the amount of polypeptide administered in the range from 0.5 ug / m per day at 500 pug / m per day for the P1 or P2 polypeptide for the polypeptide or for each of the P1 and P2 polypeptides, preferably in the range of from 5 vg / m per day to 200 ug / m per day for Polypeptide P1 or Polypeptide P2 or for each of the polypeptides of P1 and P2, more preferably in the range of from 10 µg / m a day at 80 ug / m per day for the P1 or P2 polypeptide for the polypeptide or for each of the P1 and P2 polypeptides.
[00232] [00232] In one embodiment, is the amount of polypeptide administered in the range of from 0.05 µg / m per day at 0.5 ug / m per day for the P1 or P2 polypeptide for the polypeptide or for each of the P1 and P2 polypeptides.
[00233] [00233] In one embodiment, the amount of P1 polypeptide administered is different from the amount of P2 polypeptide administered.
[00234] [00234] In one embodiment, the amount of polypeptide is administered in the range of from 0.5 ug / m per day at 50 ug / m per day for the P1 or P2 polypeptide for the polypeptide or for each of the P1 and P2 polypeptides. In one embodiment, the amount of polypeptide is administered in the range of from 50 pom a day at 100 ugm per day for the P1 or P2 polypeptide for the polypeptide or for each of the P1 and P2 polypeptides. In a fashion-
[00235] [00235] Additional reference points for deriving the amounts of the P1 and P2 polypeptides to be administered can also be obtained through consultative studies carried out with bi-specific antibody constructs (for example, Bargou R et al Tumor regression in cancer patients, with very low doses of an antibody-enveloping T cell. Science 2008; 321 (5891): 974-7, and Topp MS et al Target therapy with the cell's blinatumomab antibody T involving minimal residual disease from refractory chemotherapy in line B in patients with acute lymphoblastic leukemia results in a high response rate and prolonged leukemia-free survival. J Clin Oncol. 2,011.29: 2493 to 8).
[00236] [00236] In one embodiment, said administration occurs continuously for at least 12 hours, for at least 1 day, or at least 2 days, or for at least 3 days, or for at least 4 days, or at least 5 days, or at least 6 days, or for at least 7 days, or at least 8 days, or at least 9 days, or at least 10 days, or at least 11 days, or at least 12 days, or at least 13 days , or at least 14 days, or at least 15 days, or for at least 16 days, or for at least 17 days, or for at least 18 days, or for at least 19 days, or for at least days or at least 21 days, or at least 22 days, or at least 23 days, or for at least 24 days, or at least 25 days, or at least 26 days, or at least 27 days, or at least 28 days, or at least 29 days, or at least 30 days, or at least 5 weeks, or for at least 6 weeks.
[00237] [00237] In one embodiment, said administration of said set of polypeptides or one of the polypeptides of said set of polypeptides occurs intravenously, preferably by intravenous injection.
[00238] [00238] In one embodiment, said administration of said set of polypeptides or one of the polypeptides of said set of polypeptides occurs subcutaneously, preferably by subcutaneous injection.
[00239] [00239] In one embodiment, said set of polypeptides is administered in combination with one or more drugs selected from the group consisting of an immunomodulatory drug, and / or a steroid, preferably prednisolone or prednisone.
[00240] [00240] In one embodiment, said set of polypeptides is administered in combination with a radioactive compound, preferably, a radioactive compound bound to an antigen, a carrier molecule, or an affinity marker, wherein said radioactive compound , said antigen, said carrier molecule or reference
[00241] [00241] In one embodiment, said set of polypeptides is administered in combination with a toxin, preferably a toxin bound to an antigen, a carrier molecule, or an affinity marker, wherein said toxin, said antigen, said vehicle molecule or said affinity marker is specifically linked via said functional domain F.
[00242] [00242] In one embodiment, said set of polypeptides is administered in combination with a fluorescent molecule, preferably, a fluorescent molecule attached to an antigen, a carrier molecule, or an affinity marker, wherein said fluorophore, said antigen, said carrier molecule or said affinity marker is specifically linked via said functional domain F.
[00243] [00243] In one embodiment, said functional domain F is a domain that specifically binds to an antigen that is not recognized as foreign by the immune system of said patient to whom said set of polypeptides is administered.
[00244] [00244] In a modality of two sets of polypeptides as described above (a first set of polypeptides and a second set of polypeptides) are administered simultaneously or sequentially. In a preferred embodiment, said first set of polypeptides has different F1 and F2 fragments, than said second set of polypeptides. In a preferred embodiment, said first set of polypeptides has the same F1 and F2 fragments, as said second set of polypeptides. In a preferred embodiment, the targeting portions T1 and T2 of said first set of polypeptides are linked to the same antigens as the targeting portions T1 and
[00245] [00245] In one embodiment, said patient underwent cancer treatment, prior to treatment with said set of polypeptides, said cancer treatment, preferably being chemotherapy, radiotherapy or operative tumor removal, or undergoes cancer treatment in parallel with treatment with said set of polypeptides, said cancer treatment preferably, chemotherapy, radiotherapy or operative removal of the tumor.
[00246] [00246] In one embodiment, said set of polypeptides or one of the polypeptides of said set of polypeptides was produced by means of a prokaryotic or eukaryotic expression system or by the synthesis of new peptide.
[00247] [00247] In one embodiment, said set of polypeptides or one of the polypeptides of said set of polypeptides is generated within said patient by expressing the protein from a nucleic acid introduced into said patient.
[00248] [00248] Many patients suffer from allergic or autoimmune diseases. In many of these cases, a population of clonal B cells produces an antibody that reacts with errant antigens expressed through patients' tissues or complexes with an allergen, which causes anaphylactic reactions. In both cases, it is desirable to specifically eliminate the errant B cell clone.
[00249] [00249] For this purpose, the combinatorial system can be modified so that an arm (P1 or P2, in particular, T1 or T2), recognizes an associated B cell antigen (for example, CD19,
[00250] Thus, according to the present invention, any one of said A1 and A2 antigens can also be a clonotypic antibody on the surface of a B cell, in particular a B cell that causes an autoimmune disorder. -immune.
[00251] [00251] In this context, for example, one of said A1 and A2 antigens may be CD19 and the other may be a clonotypic antibody on the surface of a B cell, in particular a B cell that causes an autoimmune disorder. immune.
[00252] [00252] “According to this aspect of the present invention, any one of said target portions T1 and T2 may comprise an allergen or substrate that binds to the clonotypic antibody on the surface of the B cell and / or that is, after binding to the clonotypic antibody, it is capable of causing an autoimmune disease. Non-limiting examples of an allergen consisting of any of the aforementioned targets T1 and T2 are hair allergens, such as dog hair, cat head (for example, Fel d 1, Feld D1A, Feld D1B ) or guinea pig hair, or pollen allergens, such as, for example, birch, grass, pollen allergens. Other non-limiting examples are dust mite allergens (for example, Tyr p 2, Der P1, Der f 2), cat allergens (for example, Fel d 1, Feld D1A, Feld D1B), peanut allergens (for example conglutin-7), fungal allergens (for example,
[00253] [00253] In this context, the present invention also relates to the set of polypeptides as described in the present invention, and, in particular in the above aspect, for use in the treatment or prevention of a disorder selected from the group consisting of
[00254] [00254] an autoimmune disorder; and
[00255] [00255] a hypersensitivity disorder.
[00256] [00256] Non-limiting examples of an autoimmune disorder to be treated or prevented according to the present invention are selected from the group consisting of
[00257] [00257] allergic diseases;
[00258] [00258] multiple sclerosis;
[00259] [00259] Psoriasis;
[00260] [00260] Systemic lupus erythematosus;
[00261] [00261] Sjogren's syndrome;
[00262] [00262] arthritereumatoid;
[00263] [00263] idiopathic thrombocytopenic purpura;
[00264] [00264] Diabetes;
[00265] [00265] Vasculitis;
[00266] [00266] Crohn's disease; and
[00267] [00267] Amyloidosis.
[00268] [00268] Non-limiting examples of a hypersensitivity disorder to be treated or prevented according to the present invention are selected from the group consisting of allergies (type hypersensitivity reaction | according to Coombs and Gell classification) , an antibody of dependent cytotoxic reaction (type II hypersensitivity reaction), an immune complex disease (type II hypersensitivity reaction), delayed type hypersensitivity (type IV hypersensitivity reaction) and an autoimmune disease receptor immune mediated (type V hypersensitivity reaction).
[00269] [00269] In a preferred embodiment, said autoimmune disorder or hypersensitivity comes along with or is triggered by allogeneic stem cell transplantation (ie, any type | hypersensitivity disorder for type V according to the classification of Coombs and Gell).
[00270] [00270] Many cells that are infected by a pathogen (for example, a virus, such as, for example, HIV, EBV, CMV) proteins encoded by means of the pathogen expressed on their cell surface. Therefore, in accordance with the present invention, any of said A1 and A2 antigens can also be such a pathogen-encoded protein, such as, for example, an HIV, EBV or CMV protein on the surface of a cell. In this context, the present invention also relates to the set of polypeptides, as described in the present invention for use in the treatment or prevention of an infectious disease, for example, an infectious disease.
[00271] [00271] The objectives of the present invention are also solved by a nucleic acid molecule or a set of nucleic acid molecules that encode the set of polypeptides or one of the polypeptides of the set of polypeptides, as defined in the above modalities, in which preferably, said nucleic acid molecule or nucleic acid molecules of said set of nucleic acid molecules comprising an export signal that mediates the secretion of the polypeptide (s) encoded by a cell bacterial or eukaryotic.
[00272] [00272] A non-limiting example of a nucleic acid molecule or a set of nucleic acid molecules according to the present invention comprises one or more of the nucleotide sequences, as described in any of SEQ ID NO: 135-150 and 196 .
[00273] [00273] The objectives of the present invention are also solved by a vector comprising the nucleotide sequence of the nucleic acid molecule as defined above, or the sequence of one of the nucleic acid molecules of a set of acid molecules nucleic acids as defined above.
[00274] [00274] The objectives of the present invention are also solved by a cell, which comprises said nucleic acid / set of nucleic acids or said vector.
[00275] [00275] The objectives of the present invention are also solved by a pharmaceutical composition comprising either the set of polypeptides, as defined above, or the nucleic acid molecule
[00276] [00276] The objectives of the present invention are also solved by a kit comprising the set of polypeptides as defined above and / or the nucleic acid molecule or set of nucleic acid molecules according to the present invention and / or the vector according to the present invention.
[00277] [00277] In one embodiment, the polypeptides of said set of polypeptides composed by means of said kit are contained in a single bottle.
[00278] [00278] In a preferred embodiment, the polypeptides of said set of polypeptides composed by means of said kit are contained in separate bottles.
[00279] [00279] In one embodiment, one or more of the polypeptides of said set of polypeptides composed by means of said kit are freeze-dried.
[00280] [00280] In one embodiment, one or more of the polypeptides of said set of polypeptides composed by means of said kit are in solution.
[00281] [00281] The objectives of the present invention are also solved by a method for the treatment of a patient suffering from a
[00282] [00282] tumor or cancer and / or who is undergoing tissue transplantation or allogeneic cell;
[00283] [00283] an autoimmune disorder; or
[00284] [00284] a hypersensitivity disorder.
[00285] [00285] This method can comprise the steps of:
[00286] [00286] - Obtaining a set of polypeptides, said set of polypeptides comprising
[00287] [00287] a first polypeptide comprising P1
[00288] [00288] a target portion T1,
[00289] [00289] wherein said target portion T1 specifically binds to an A1 antigen, and
[00290] [00290] an F1 fragment of a functional domain F,
[00291] [00291] in which neither said F1 fragment per se nor said polypeptide P1 per se is functional with respect to the function of said F domain,
[00292] [00292] and
[00293] [00293] "a second polypeptide comprising P2
[00294] [00294] a target portion T2,
[00295] [00295] wherein said target portion T2 specifically binds to an A2 antigen, said A2 antigen being a cell surface molecule that is specific to a particular cell type or cell line, and
[00296] [00296] an F2 fragment of said functional domain F,
[00297] [00297] in which neither said F2 fragment per se, nor said polypeptide P2 per se is functional with respect to the function of said F domain,
[00298] [00298] in which said A1 antigen is different from said A2 antigen,
[00299] [00299] wherein said polypeptide P1 and said polypeptide P2 are not associated with each other in the absence of a substrate that has both antigens of A1 and A2 on its surface, more specifically a cell that carries both A1 and A2 antigens, on their cell surface, and
[00300] [00300] wherein, by dimerizing said F1 fragment of said P1 polypeptide with said F2 fragment of said P2 polypeptide, the resulting dimer is functional with respect to the function of said F domain,
[00301] [00301] - Administer said set of polypeptides to the referred patient.
[00302] [00302] In such a treatment method, said set of polypeptides is as defined in the above modalities.
[00303] [00303] The objectives of the present invention are also solved by a method of using the set of polypeptides as described above for the treatment of a patient undergoing cell or tissue transplantation.
[00304] [00304] The objectives of the present invention are also solved through the use of a set of proteins, as defined in the above modalities for the manufacture of a medicine for the treatment of a patient suffering from the diseases defined above and described in a disorder or, for example, a patient suffering from cancer and / or undergoing cell or tissue transplantation.
[00305] [00305] As used in the present invention, the term "polypeptide" refers to a linear amino acid molecular chain containing more than 30 amino acids. Optionally, a polypeptide can include one or more disulfide bonds, or be chemically modified. In addition, optionally, a non-protein element (such as a fluorophore, RNA-aptamer, DNA-aptamer, or small molecule) can be attached to said linear molecular chain of amino acids. Such polypeptides can be produced by any known method. The polypeptide can, for example, be generated by expressing a nucleic acid that codes for said polypeptide, or it can be synthesized using solid phase synthesis methods, or it can be produced through conjugation or ligation - tion of existing molecules, for example, through chemical bonding.
[00306] [00306] The term "P1 polypeptide" is used to refer to a polypeptide comprising (i) a target portion, wherein said target portion specifically binds to an antigen, and (ii) a fragment of a functional domain, in which neither said fragment per se nor said Polypeptide P1 per se is functional with respect to the function of said functional domain. The term "P2 polypeptide" is used to refer to a polypeptide comprising (i) a target portion, wherein said target portion specifically binds to an antigen, and (ii) a fragment of a functional domain , in which neither said fragment, per se, nor said P2 Polypeptide per se is functional with respect to the function of said functional domain.
[00307] [00307] The term "domain", as used in the present invention, refers to a linear molecular chain of amino acids that includes the amino acid sequence of an entire polypeptide or a portion of a polypeptide. Optionally, a domain can include one or more disulfide bonds, or it can be chemically modified. In addition, a domain can optionally comprise a non-protein element (such as a fluorophore). In one embodiment, however, the term "domain" does not include compounds that are chemically modified or comprise non-protein element (s).
[00308] [00308] A "functional domain", as used in the present invention, is a domain that is capable of carrying out a certain function, such as binding to a specific antigen or specific binding partner, activation specificity of a given receptor, the mediation of toxic or fluorescence effects after excitation, with light of an appropriate wavelength.
[00309] [00309] The term "functional domain F" is preferably also intended to include compounds that are non-protein. In a fashion, however, it refers to a protein compound or a functional part thereof.
[00310] [00310] The term "a fragment of a domain", as used in the present invention, refers to a linear molecular chain of amino acids that corresponds to a part of a domain, but not the totality of the domain. Optionally, a fragment of the domain can include one or more disulfide bonds, or can be chemically modified. In addition, a domain can optionally comprise a non-protein element or part of such a non-protein element.
[00311] [00311] The term "F1 fragment" is used to refer to a fragment of a functional domain. The term "F2 fragment" is used to refer to a fragment of a functional domain.
[00312] [00312] The pairs of abbreviations P1, P2; T1, T2; F1, F2; A, A2; and E1, E2, as used in the present invention, are intended to designate different polypeptides, targeting moieties, fragments, antigens, and immunoglobulin modules, respectively. They are synonymous with first polypeptide, second polypeptide; first target portion, second target portion; first fragment, second fragment; first antigen, second antigen; and first immunoglobulin module, second immunoglobulin module, respectively.
[00313] [00313] The term "portion", as used in the present invention, refers to a linear molecular chain of amino acids that includes the amino acid sequence of an entire polypeptide or a portion of a polypeptide. Optionally, a moiety may include one or more disulfide bonds, or it may be chemically modified. In addition, optionally, a portion may comprise a non-protein element (such as an oligonucleotide). In one embodiment, however, the term "portion" does not include compounds that are chemically modified or comprise non-protein element (s).
[00314] [00314] The term "target portion T1" is used to refer to a portion that specifically binds to an antigen, for example antigen A1. The term "T2 target portion" is used to refer to a portion that specifically binds to an antigen, for example A2 antigen.
[00315] [00315] As used in the present invention, a "linker" is a sequence of amino acids within a polypeptide that links two parts of said polypeptide or two domains constituted by said polypeptide.
[00316] [00316] The term "nucleic acid molecule", as used in the present invention, defines a linear molecular chain consisting of more than 30 nucleotides. The term includes DNA, such as cDNA or genomic DNA and RNA.
[00317] [00317] The term "construct", as used in the present invention, refers to a nucleic acid molecule that comprises one or more recombinant nucleotide sequences. The term also includes polypeptides that are expressed from a recombinant nucleotide sequence or that are made artificially or recombinant molecules that comprise two or more amino acid sequences that are not naturally found within the same protein.
[00318] [00318] The term "specifically binds" or "specifically binds", as used in the present invention in the context of a molecule or domain that specifically binds to an interacting partner or antigen, or that specifically binds to a partner of interaction or antigen, means that a molecule or domain binds to said antigen interaction partner or, preferably, through non-covalent binding, or is able to bind said antigen interaction partner or, preferably, by means of non-covalent bonding, and they do not, or essentially do not cross-react with any other interaction partner or antigen with a structure similar to that of the interaction partner or antigen.
[00319] [00319] In the context of a target portion (such as target portion T1 or T2), which specifically binds to an antigen (such as A1 or A2 antigen), the term "specifically binds" is intended to refer to a situation in which any of said target portion is able to specifically bind to said antigen, or where it actually binds to it.
[00320] [00320] In the context of a T cell envelope domain, an NK cell envelope domain, macrophage envelope cells, a monocyte envelope domain, a granulocyte envelope domain, a neutrophil granulocyte envelope domain, or a enveloping domain of activated neutrophilic granulocytes, monocytes and / or macrophages, the term "specifically binds to" an antigen or molecule or "specifically binds to" an antigen or molecule is intended to refer to a situation in which either the respective - a domain is able to specifically bind to that antigen or molecule, or where it actually binds to it.
[00321] [00321] In the context of a functional domain being a domain that "specifically binds to" an antigen, molecule, compound, carrier molecule, or affinity marker, the term "specifically binds to" is intended refer to a situation in which any of the aforementioned functional domain is able to specifically bind to said antigen, molecule, compound, vehicle or molecule affinity marker, or where it actually binds to it.
[00322] [00322] In the context of a toxin, fluorophore, the affinity marker antigen, vehicle or molecule being "specifically linked by" a functional domain, this is intended to refer to a situation in which both said functional domain is capable of binding specifically to said toxin, fluorophore antigen, vehicle or affinity marker molecule, or where it actually binds to it.
[00323] [00323] As used in the present application, a molecule or antigen is "specific for a particular cell type or cell line" if it is expressed by means of said cell / cell type of said cell line, but not (or only negligibly) for other cell types or cells of another cell line. In some embodiments, a molecule or antigen is "specific for a particular cell type or cell line" if it is expressed by said cell type / cells of that cell line, and no more than some other cell types or cells from another cell line in addition to said cell type / cells from said cell line that expresses said antigen, as well as, while most other cell types or cells from another cell line in addition to said cell type / cells of that cell line that do not express the referred antigen (or only insignificantly). The term "specific for a particular cell type or cell line" can also mean that said molecule or antigen is expressed by said cell type / cells of said cell strain at a higher rate, or at a higher rate than the value or by other types of cells / cells of other cell lines, in the sense that there may be a small but detectable expression of that molecule also in other types of cells / cells of other cell lines. The term "marker", as used in the present invention in the context of a marker for a particular cell type or lineage cell, can refer to a molecule or antigen that is specific to a type of cell. cell or cells of a cell line, respectively, as described above.
[00324] [00324] As used in the present invention, the term "aptamer" refers to a small compound, composed of oligonucleic acid (such as RNA or DNA) or peptide or non-peptide molecule that binds to a molecule specific target, with high affinity.
[00325] [00325] As used in the present invention, the term "carrier molecule" refers to a molecule or part of a molecule that is not recognized as foreign by the immune system of a patient to whom the set of polypeptides according to the present invention is administered, or that causes not only or a weak immune reaction by a patient to whom the set of polypeptides according to the present invention is administered. Preferably, such a "carrier molecule" is being bound by or likely to be bound by another molecule, such as an antibody. In some embodiments, a "carrier molecule" is a molecule or part of a molecule that, in certain embodiments, the carrier molecule is covalently or non-covalently linked to a second molecule or part of a second molecule, for example example, a fluorophore or toxin.
[00326] [00326] The term "MHC" refers to the main histocompatibility complex, which is a set of genes that code for a group of molecules that comprise the cell surface molecules, which are necessary for the presentation of the antigen to T-cells, which are also responsible for the rejection of rapid grafts. In humans, MHC includes the genes HLA-A, HLA-B, HLA-C, HLA-DP, HLA-HQ and HLA-DR. In the present patent application, the term is used to refer to the genes of the Principal Histocompatibility Complex, as well as to the gene products encoded by these genes. The term "HLA" refers to human antigens. As used in the present invention, "HLA" is the human form of "MHC".
[00327] [00327] The term "allelic variant", as used in the present invention, designates any one of two or more alternative forms of a gene that occupy the same chromosomal site. For example, HLA-A1, HLA-A2, and HLA-A3 are the three allelic variants of HLA-A. The term allelic variant is also used in the present invention to refer to a protein encoded by means of an allelic variant of a gene.
[00328] [00328] The term "antigen", as used in the present invention, refers to a molecule known to be specifically | linked to or capable of being specifically bound by an antibody or the antigen binding part of a antibody. In its broadest sense, "A1 antigen" refers to an antigen, as defined above. In its broadest sense "A2 antigen" refers to an antigen, as defined above. The designations "A1 antigen" and "Antigen A2" were chosen to allow the distinction between "A1 antigen" and "A2 antigen". An "MHC antigen" is an antigen that is also a molecule belonging to the major histocompatibility complex. MHC antigens include class antigens | CPH (in humans, HLA-A, B-, e-C expression) and MHC class II (antigens in humans, HLA-DP, -DQ and-DR antigens). The phrase that a cell "carries an antigen" or "carries an antigen on its cell surface" is intended to refer to a situation in which a cell expresses an antigen that is present on the surface of the cells of that cell and accessible to a antibody from the outside of said cell. The phrase that a substrate "has an antigen on its surface" is intended to refer to a situation in which said antigen is present on the surface of said substrate and accessible to an antibody applied to said substrate.
[00329] [00329] The term "an antigen that is specific for the malignant state of a cell", as used in the present invention, refers to an antigen that a malignant cell of a given cell type (such as a cell malignant tumor of B cells) takes, on its cell surface, but that the cell of the same type of cell that is not malignant (such as a non-malignant B cell) does not carry (or only insignificantly) on its cell surface.
[00330] [00330] The term "an antigen / molecule that is specific to a type of malignant cell", as used in the present invention, refers to an antigen / molecule that a malignant cell of a given type of cells (such as malignant cells of the B cell tumor) takes, on its surface cell, but that cell of the same cell type that is not malignant (such as a non-malignant B cell) or cells of other types of cells (such as T cells or hepatocytes) it does not carry (or only negligibly) on its cell surface.
[00331] [00331] The term "immunoglobulin domain", as used in the present invention, refers to a domain that essentially consists of a globular region of an antibody chain. Immunoglobulin domains are characterized in that they retain the immunoglobulin folding characteristic of antibody molecules. Immunoglobulins, such as IgG, IgE, or IgM, are composed of a variable number of light and heavy chains. Each heavy and light chain contains a constant region and a variable region. Each light chain variable region (VL) and each heavy chain variable region (VH) contain three hypervariable regions, also called "complementarity determining regions" or "CDR". CDRs are primarily responsible for binding immunoglobulin to an antigen.
[00332] [00332] The terms "VH" or "VH domain" are used interchangeably and refer to the variable region of an immunoglobulin heavy chain of an antibody. The terms "VL" or "VL domain" are used interchangeably and refer to the variable region of an immunoglobulin light chain of an antibody.
[00333] [00333] The term "immunoglobulin module", as used in the present invention, refers to a molecule, which is part of a molecule or molecular assembly comprising one or more, preferably two or more, domains of immunoglobulin and is able to bind to an antigen. Preferably, an "immunoglobulin module" comprises a linear molecular chain of amino acids that includes the amino acid sequence of one or more, preferably two or more immunoglobulin domains. Optionally, an "immunoglobulin module" comprises one or more, preferably two or more, disulfide bridges. Included in the term "immunoglobulin module" are molecules or parts of a molecule that comprise or consist of a "single chain variant fragment" of an anti-
[00334] [00334] The term "module 11 immunoglobulin" is used to refer to an immunoglobulin module comprising a VL domain linked to a VH domain. Preferably, said VL domain and said VH domain of said immunoglobulin module 11 are derived from the same antibody. Preferably, said VL domain and said VH domain of said immunoglobulin module 11 form a dimer. Preferably, said dimer is able to specifically bind to an antigen. Said antigen can be, for example, the A1 antigen. In one embodiment, said "immunoglobulin module 1" comprises a "single chain variant fragment" of an antibody that is capable of specifically binding to an antigen, for example, the A1 antigen.
[00335] [00335] The term "module 12 immunoglobulin" is used to refer to an immunoglobulin module comprising a VL domain linked to a VH domain. Preferably, said VL domain and said VH domain of said immunoglobulin module 12 are derived from the same antibody. Preferably, said VL domain and said VH domain of said immunoglobulin module 12 form a dimer. Preferably, said dimer is able to specifically bind to an antigen. Said antigen can be, for example, the A2 antigen. In one embodiment, said "immunoglobulin module 12" comprises a "single chain variant fragment" of an antibody that is capable of specifically binding to an antigen, for example, the A2 antigen.
[00336] [00336] Within an immunoglobulin module construct comprising a VL domain linked to a VH domain, the domain
[00337] [00337] The terms "Fv" and "variant fragment", as used in the present invention, refer to an antibody fragment which is the minimum antibody fragment that contains a complete recognition antigen and binding site. This region consists of a dimer from a heavy variable region and a light chain in close association, not covalent (VH-VL). In this modality, the VH and VL domain together define an antigen binding site with antigen binding specificity on the surface of the VH-VL dimer.
[00338] [00338] The terms "scFv", "single chain Fv", and "single chain variant fragment" are used interchangeably and serve to designate an antibody or portion of an antibody in which the variable region of the heavy chain ( VH) and light chain variable region (VL) of a traditional two-chain antibody were joined to form a chain. Typically, a binder is inserted between the two chains to allow correct folding and the creation of an active binding site.
[00339] [00339] The term "mud antibody", as used in the present invention, refers to an antibody or part of an antibody derived from mud. The term "camel antibody", as used in the present invention, refers to an antibody or part of a camel-derived antibody. The term "shark antibody", as used in the present invention, refers to an antibody or part of an antibody derived from shark. Mud, camel and shark antibodies have an antigen-binding portion that is constructed through a single domain, VHH, (instead of a VH and a VL chain).
[00340] [00340] The term "T cell surrounding domain", as used in the present invention, is intended to refer to a domain that specifically binds to an antigen that is present on the surface of T cell cells.
[00341] [00341] The term "molecule capable of mediating bioluminescence", as used in the present invention, refers to a molecule (or a functional part of a molecule) that has an enzymatic activity that, in the presence of (s) ) appropriate substrate (s) catalyzes a reaction that causes bioluminescence. The term includes luciferases, such as firefly or Gaussia luciferases.
[00342] [00342] The term "GFP variant", as used in the present invention, refers to a molecule that has an amino acid sequence derived from the amino acid sequence of the green fluorescent protein of Aequorea victoria, introducing changes that result in greater fluorescence or fluorescence with different colors. The term is intended to include, among others, YFP (green fluorescent protein), CFP (cyan fluorescent protein), Venus (Nagai T et al., A variant of the yellow fluorescent protein with fast and efficient maturation for biological cell applications. Nat. Biotechnol 2002 Jan; 20 (1): 87 to 90), Cerulean (PCP enhanced with S72A, Y145A and H148D substitutions).
[00343] [00343] "Improved GFP" (and, similarly, "Improved YFP", "Improved PCP") refers to a GFP (YFP, PCP), which has been "humanized", as reported in Kain et al. (1995) Biotechniques 19 (4): 650-55. "Humanized" refers to changes made to GFP (YFP, PCP), a nucleic acid sequence to optimize the codons for expressing the protein in human cells.
[00344] [00344] The term "bimolecular fluorescence complementing molecule", as used in the present invention, refers to a fluorescent molecule that can be supplied as two fragments that, by themselves, are not fluorescent, but that after heterodifferentiation. merization between the two fragments form a dimer that is capable of fluorescence.
[00345] [00345] The term "therapeutic compound", as used in the present invention, refers to a compound suitable for preventing, treating, alleviating or curing a disease or disease state. Preferably, a "therapeutic compound" is a compound that, after entering a cell, is capable of causing the cell to die. In some modalities, a therapeutic compound can be a chemical or radioactive compound that damages vital cell structures or disrupts vital cell processes.
[00346] [00346] The term "diagnostic compound", as used in the present invention, refers to a compound that can be detected using common detection methods, such as methods used in the clinic or in diagnostic laboratories biochemical or medical, for example, a fluorescent compound, a radioactive compound, or a bioluminescence mediator molecule.
[00347] [00347] The term "progenitor / precursor cells" is intended to refer to immature, undifferentiated or partially differentiated cells, which are typically found in animal / human postnatals and have the potential to differentiate into a specific type of cell or in specific cell types. The term "progenitor / precursor cells of a tumor" means progenitor / precursor cells with altered properties (for example, in relation to their proliferation behavior or standard expression gene) that give rise to tumor cells. Examples of such tumor progenitor / precursor cells are, for example, leukemic or progenitor precursor cells.
[00348] [00348] The term "cancer", as used in the present invention, refers to a malignant cell, a group of cells, and malignant neoplasms. The term is intended to understand carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas. A "cancer cell" is a cell that is part of or derived from cancer. The term "tumor" is used interchangeably with the term "cancer".
[00349] [00349] As used in the present invention, the term "hematological tumor" refers to cancer of the blood or blood system (such as bone marrow cells, blood cells, construct, and precursor cells of mature blood cells). In some embodiments, the term "hematological tumor" refers to a hematological neoplasm. As used in the present invention, the term "non-hematological tumor" refers to a tumor that is not a hematological tumor.
[00350] [00350] The term "a patient who is suffering from allogeneic tissue or cell transplantation", as used in the present invention, refers to a situation in which the patient receives or has received the transplanted cells or transplanted tissue , which was / were obtained from another person. One situation as preferred in this regard is the situation with incompatible HLA antigens. The "ug / m2" unit, as used in the present invention in the context of an amount of a polypeptide administered, refers to a certain amount of polypeptide per square meter of body surface of the patient to whom said polypeptide is administered (the peptide can be administered by any suitable route of administration, such as by intravenous or subcutaneous injection). For example, the expression "The amount of polypeptide is administered 50 µg / m2 per day for Polypeptide P1." it is intended to refer to a situation in which the amount of P1 polypeptide administered per day is 50 mg per square meter of body surface of the patient to whom P1 polypeptide is administered. In the case of a patient who has a body surface of 2 m2, this means that 100 µg of P1 polypeptide is administered per day.
[00351] [00351] The present inventors have surprisingly discovered that, with a set of polypeptides according to the present invention, the problems of the state of the art indicated above can be overcome and the objectives described above can be achieved. In addition, the present inventors have surprisingly discovered that, with a set of polypeptides according to the present invention, cells with a combination of two specific antigens can be identified and / or eliminated with high specificity and side effects reduced.
[00352] [00352] It is an advantage of the combinatorial strategy of the present invention that none of the preformed F units (for example, anti-CD3 units) are used. F1 and CD3 of VH and VL) do not heterodimerize by themselves, not even in the presence of an agent that stabilizes their dimerization (for example, an antigen capable of binding to the F domain, such as, for example, CD3, HIS or DIG), and therefore would not result in an F functional domain (for example, it does not stimulate T cells). Exclusively in situations where both complementary constructs P1 and P2 connect simultaneously on the surface of a given cell, the two components of F1 and F2 reconstitute the F domain (for example, CD3 binding site). In this way, the function of the F domain (for example, the activation of T cells) occurs precisely when necessary, but not systematically. Thus, it can be assumed that the combinatorial strategy of the present invention has less toxic effects, for example, in comparison with normal bispecific antibody strategies. This is also evidenced by the examples attached, in particular
[00353] [00353] In particular, to label cells that express a predefined signature antigen, two single chain polypeptides were designed as parts of the final bipartite (bi-molecular) construct (bi-molecule / tries specific antibody construct), each compound through a binding to the single chain variable fragment antigen (scFv) and either the variable light chain (VL) or variable heavy chain (VH) domain of an antibody. When these two hybrid fragments bind their respective antigens on the surface of a single cell, the VL and VH domains interact with each other to reconstitute the binding site to the original antigen and, in this way, satisfy the desired requirements.
[00354] [00354] “As mentioned, it is an advantage of the set of polypeptides of the present invention that the binding of both target antigens on the cell surface is necessary for functional heterodimerization. The self-assembly of the two complementary parts and subsequent stimulation of T cells after the attachment of only one arm to its antigen can be ruled out, corroborating the published data showing that the binding of VH or VL by itself is of low affinity and that VH / VL heterodimers tend to dissociate quickly in the absence of antigen (Colman, 1987, Nature 326, 358 to 363; Amit, 1986, Science 233, 747-753; Lei, 2002, Int. Immunol 14, 389 to 400; Ueda, 1996, Nat Biotechnol 14, 1714 to 1718).
[00355] [00355] In contrast to the homo- or hetero-dimerization domains well known in the art (leucine-zipper, Fc-domains, button in the hole, etc.), VH and HL interactions are of low affinity. However, it has been shown that the VH / VL interaction can be stabilized after binding with the specific antigen. Without being limited
[00356] [00356] In addition to an advantage of the constructs of the present invention, P1 and P2, for example, the combinatorial nature of the immune response, it was surprisingly discovered, in the context of the present invention that the bi-molecular construct, with the interrupted F domain, for example, scFv-anti-CD3, does not exhibit off-target effects.
[00357] [00357] The set of polypeptides according to the present invention, in particular the polypeptides P1 and P2 included therein, also have the advantage of being more stable and / or having an improvement in shelf life (in particular, the 40), in comparison with conventional constructs, such as bispecific BiTE constructs. These conventional bispecific constructs tend to add (in particular, € 4).
[00358] [00358] It is anticipated that the polypeptides of the present invention, P1 and P2, more in particular F1 and F2, as understood there, more particularly of VH and VL, which can be understood there, due to their interface hydrophobic, are able to bind albumin. This leads to an improved retention time; that is, more bioavailability in vivo, but also in vitro, as, for example, in serum or blood samples.
[00359] [00359] The set of polypeptides according to the present
[00360] [00360] In an exemplary embodiment, this inventive principle is applied for the specific elimination of tumor cells:
[00361] [00361] New histopathological analyzes and flow cytometry revealed that tumor cells can be detected and distinguished from their untransformed counterparts not by means of single surface markers, but by the expression of aberrant antigen combinations / profiles , as it is known through hematopoietic neoplasms and cancer and cancer stem cells from several other sources. Thus, while a single antigen may not be sufficient to specifically identify a particular tumor cell, a specific combination of two antigens may allow the tumor cell to be discriminated against from any other cell type.
[00362] [00362] For example, the set of polypeptides according to the present invention can be used to specifically eliminate carcinoma cells characterized by the simultaneous expression of CD33 and CD19 antigens on their cell surface. This combination of antigens is found in certain types of acute leukemia cells and distinguishes these cells from any other cells (such as non-malignant cells), which can carry either CD33 or CD19 on their cell surface, but do not exert so much CD33 and CD19 on their cell surface (Ossenkoppele et al., Review of the relevance of aberrant antigen expression by flow cytometry in myeloid neoplasms. Br J] Haematol 2011, 153 (4): 421- 36).
[00363] [00363] To specifically eliminate the leukemic cells that carry both CD33 and CD19 on their cell surface, the first target T1 portion of the first P1 polypeptide may be a CD33 specific single chain variable (scFv) fragment. As fragment F1 of the functional domain F, the variable domain of the VL light chain of an anti-CD3 antibody can be chosen. The second target T2 portion of the second P2 polypeptide may be of some scFv for CD19. Like the F2 fragment of the functional F domain of the heavy chain of the VH variable domain of the anti-CD3 antibody, which can be chosen. THE
[00364] [00364] In the presence of a leukemia cell having both CD33 and CD19 on its cell surface, both P1 and P2 polypeptides bind to the cell simultaneously. As a consequence, the F1 and F2 fragments of the functional group F (that is, the heavy and light chain of the anti-CD3 Fv variable domain of such anti-CD3 antibody) are brought together in close proximity, they hetero-dimerize and, in this way, they complete the desired biological function, allowing the dimer of P1 and P2 to bind specifically to CD3.
[00365] [00365] CD3 is a cell surface molecule that is present on the surface of T cells. The molecule is part of the complex T cell signaling, and the cross-linking of CD3 molecules on the T cell surface after binding of a specific antibody-CD3 leads to the activation of T cells. By involving CD3 antigens on the surface of T cells, the heterodimers of P1 and P2 polypeptides are able to recruit and activate T cells. As a result, the typical effector mechanisms of a cytotoxic T cell response are extracted, which leads to cell lysis: release of lytic granules that contain cytotoxic proteins perforin, granzymes, and granulisine. Perforin forms pores in the membrane of the target cell through which granzymes can enter and induce apoptosis. These effects lead to the specific destruction of leukemic cells that transport both CD33 and the CD19 antigen on their cell surface.
[00366] [00366] Cells other than leukemic cells do not have both CD33 and CD19 antigen on their cell surface. Therefore, they cannot recruit both P1 and P2 polypeptides, and do not complement CD3 binding capacity and the involvement of CD3 positive T lymphocytes is achieved. Consequently, cells other than leukemic cells are not affected, and the destruction of malignant cells, with excellent specificity, is achieved.
[00367] [00367] This is in stark contrast to conventional bispecific antibodies. A conventional bispecific construct that involves T cells and has specificity for cells expressing CD33, which is a mediator of the destruction of all CD33 positive cells. Since CD33 is the marker of myeloid lineage, which is expressed in several myeloid cells and myeloid progenitor cells, the destruction of these cells would result in long-lasting aplasia and, probably, the death of the patient. A conventional bispecific construct that involves T cells and has specificity for CD19 positive cells would lead to the elimination of all cells that carry the CD19 antigen on their cell surface. CD19 is expressed in a significant subset of B-lymphocytes. The destruction of these cells would lead to a serious defect in the immune system. Thus, in addition to eliminating leukemic cells that simultaneously express CD33 and CD19 on the surface, the application of conventional bi-specific antibodies, with specificity for CD33 and CD19, would lead to the elimination of myeloid cells and a subset significant number of B-lymphocytes.
[00368] [00368] Thus, while conventional bispecific antibodies recognize only one antigen in the cell to be eliminated, the effector activation according to the present invention requires the simultaneous recognition of two specific antigens on the cell surface to be identified / eliminated. As a result, the present invention achieves significantly improved specificities and reduced side effects.
[00369] [00369] It is clear to a person skilled in the art that, within the principle of the present invention, several variations for the exemplary modality described above are possible.
[00370] [00370] For example, the method described in the exemplary mode above can easily be adapted for the identification and / or elimination of other types of tumor cells, in addition to the positive acute leukemia cells CD33 and CD19 by simply choosing the appropriate ones. targeting portions T1 and T2, which specifically bind to A1 and A2 antigens, respectively, which are present at the same time that the cells are identified / eliminated, but not present simultaneously in other cell types. As mentioned above, many, if not all, carcinoma cells (but also carcinoma cell progenitor / precursor cells) express a number of cell surface molecules, which alone are largely expressed in normal tissues, but are indicative for the malignant phenotype expressed in a nonphysiological combination. For example, CD34 is a marker for hematopoietic stem cells and CD7 can be detected in a subset of lymphoid cells. The combination of CD34 and CD7, however, is strongly associated with malignancy, and aberrant co-expression of both antigens can be detected in a substantial proportion of acute myeloid leukemia (Ossenkoppele et al., Review of the relevance of aberrant antigen expression by flow cytometry in myeloid neoplasms. Br J Haematol 2011, 153 (4): 421-36.). Likewise, aberrant co-expression of CD44 and CD117 has been described for ovarian cancer stem cells, CD44 and CD24 for pancreatic cancer initiating cells and the combination of EDCAM and CD44 in colon stem cells and cancer of breast (Natasha Y. Frank, Tobias Schatton, Markus H. Frank ,. the therapeutic promise of the cancer stem cell concept. J Clin Invest 2010; 120: 41-50). The expression of CD24 and CD29, as well as CD24 and CD49f was found to be specific for breast carcinoma (Vassilapoulos A et al. Identification and characterization of BRCA1 breast tumor initiation cancer cells related to breast stem cell markers normal Int. J. Biol Sci 2008; 4: 133-142). In addition, combinations with highly expressed antigen levels are indicative of a number of malignant diseases, such as myeloma CD38 and CD138.
[00371] [00371] In addition to the combinations of cancer-specific antigens listed above and those known from the scientific literature, additional combinations of two antigens that are expressed simultaneously in specific tumor cells, but not in other cells can be derived from a linear form by those who are versed in the technique.
[00372] [00372] First, the person who is knowledgeable can come up with a combination of antigen that is specific to a particular cancer by combining an antigen that is specific to the malignant state of the respective cell type with a cell type marker appropriate cell line marker. For example, carbonic anhydrase IX is a marker strongly associated with renal cell carcinoma and metastasis of renal cell carcinoma and thus represents a marker for the malignant state of renal cells. This marker located membrane, however, is also expressed in normal cells of the intestinal tract. When selecting as the second renal antigen marker antigen as aquaporin, the resulting combination of two antigens is specific for renal cell carcinoma cells and cells that result from renal cell carcinoma metastasis, whereas neither non-renal kidney cells malignant cells (which do not express carbonic anhydrase IX) or intestinal tract cells (which do not express aquatic
[00373] [00373] Detailed information on the markers for the malignant status of various types of cells and on the markers for numerous types of cells or cell lines are available from the literature and web-based resources (see below for more details) them) or can be obtained through direct experimentation (see below).
[00374] [00374] Examples of markers for the malignant state of a cell are: E-cadherin in epithelial cells and ductal breast carcinoma cells; Ca-125 for epithelioid neoplasms and ovarian carcinoma cells, adenocarcinoma cells and breast cancer cells; Her-2 / neu for breast cancer cells; cystic crude protein fluid disease (BRST-2 protein) for breast cancer cells; BCA-225 (associated glycoprotein breast carcinoma) for lung and breast cancer cells; CA 19-9 (antigen carbohydrate 19-9) for pancreas, bile ducts and carcinoma cells of the intestinal tract; CEA for colorectal cancer cells; CD117 (c-kit) for GIST cells (gastrointestinal stromal tumor) (and myeloid cells and mast cells); CD30 for Reed-Sternberg cells (and Ki-1 activated T cells and B cells); Epithelial antigen (BER-EP4), epithelial membrane antigen, and related epithelial antigen (MOC-31) for epithelial carcinoma cells; epidermal growth factor (HER1) receptor for cells of various types of cancer; Platelet derivative of growth factor receptor (PDGFR) alpha for cells of various types of cancer; Melanoma associated with the marker / Mart 1 / Melan-A for melanoma cells; CD133 for cancer stem cell populations and others; marker 72 (tumor associated with gp 72) for adenocarcinoma cells.
[00375] [00375] Other examples of markers for a malignant state of a cell / cells include: EDBCAM, CD19, HER-2, HER-3, HER-4,
[00376] [00376] Examples of antigens, which are specific for a specific cell type / cell line or for some cell types (cell lines / cell type / cell line markers) include: CD45 for hematopoietic cells ; CD34 for endothelial cells, stem cells and stromal cells; CD33 for myeloid cells; CD138 for plasma cells and a subset of epithelial cells; CD15 for epithelial, myeloid and Reed-Sternberg cells; CD1a for cortical thymocyctes and Langerhans cells; CD2 for thymus cells, T cells and natural killer cells (NK); CD3 for T-cells, CD4 for T-helper cells; T-cell CD5, a subset of B cells, and thymus carcinoma cells; CD8 for cytotoxic T cells; CD20 for B cells; CD23 for activated B cells; CD31 for endothelial cells; CD43 for T cells, myeloid cells, a subset of B cells, histiocytes and plasma cells; CD56 in NK cells; CD57 for neuroendocrine cells, and NK cells; CD68 for macrophages; CD79a for B and plasma cells; CD146 for the endothelial cell line; protein
[00377] [00377] It should be noted that there are situations in which it may be advantageous to count for the purposes of the present invention, on an antigen with a specificity less than perfect for the cell type or cell line of interest. For example, in situations where an antigen is not known which is found exclusively in the cell line / cell of interest and not in any other types of cell line / or in situations where it is not possible to confirm the unique specificity of an antigen , also antigens that are present in one or more other types of cell T cell lines / in addition to the cell / cell line of interest can be considered. A similar consideration applies to markers for the malignant state of a cell, or even for the specificity of the combination of two antigens. Thus, there are, for example, situations where, for the purposes of the present invention, a combination of two antigens is selected that is specific, not only for the cells of interest, but also for one or more (some) cell lines of other types of T cells // types of malignant cells.
[00378] [00378] Secondly, the person who is knowledgeable in the technique can come up with a combination of antigen that is specific to a certain cancer by direct experimentation. This can comprise the steps of (1) determining the surface antigens on the tumor cells to be eliminated and (2) identifying between these tumor cell surface antigens of two antigens that are not present simultaneously in other cell types (or, in some modalities, present in just a few other types of cells).
[00379] [00379] Often, experimentation may not be necessary to determine the surface antigens of tumor cells to be eliminated, because this information may already be available for the respective type of cancer from the printed literature (see, for example, David J. Dabbs, Diagnostic Immunohistochemistry, Churchill Livingstone, 3rd edition (2010), or F and J Lin Prichard, Immunohistochemistry Practical Handbook: Frequently Asked Questions, Springer, New York, 1st edition (2011)). Even the most extensive information is available through web-based resources. For example, the Cancer Genome Anatomy Project (CGAP), of the National Cancer Institute of the USA (NCI) systematically determined the profiles of several pre-cancer cells, normal and cancerous (Strausberg RL gene expression The Cancer Genome Anatomy Project: Constructing new information and technology platform for cancer research: Early Cancer Molecular Pathology, 1999 (Srivastava, S., Henson, DE, Gazdar, A., eds IOS Press.), Pp 365 -370). The resources generated by the CGAP initiative are available free of charge (http: Ilcgap.nci.nih.gov/) and include access to all necessary CGAP data and analysis tools. Similarly, the Cancer Genome Characterization Initiative (CGCI) of the National Cancer Institute focuses on tools to characterize the genomic changes involved in different tumors, for example methods of genomic characterization, including exome and analysis of the transcript using second generation sequencing. The data generated by the CGCI are available through a publicly accessible database (http: //cgap.nci.nih.gov/cgci.html). In this way, in many cases, information about the presence or absence of various known cell surface proteins on the tumor cells of interest can be derived simply by checking these publicly accessible databases. If desired, this information can then be verified in a second step by means of immunochemochemical / immunohistochemical analysis of tumor cells / tissue according to the methods described below.
[00380] [00380] If there is no information available on the proteins expressed by the tumor cells / tissue of interest, the person skilled in the art can perform a characterization of the antigens on the tumor cells / tissue using the immunocytochemical / immuno- histochemistry with an antibody panel (see, for example, "Practical Immunohistochemistry Handbook: Frequently Asked Questions", by Lin F and J Prichard, Springer New York, 1st edition (2011), or "Using Antibodies: A Laboratory Manual "and by Harlow and D Lane, Cold Spring Harbor Laboratory Press (1998)). In summary, a histological preparation or cells isolated from a tumor were incubated with a first antibody directed to a potential surface antigen and, after a washing step, the incubation of a second antibody directed against the Fe domain of the first antibody . This second antibody is labeled with a fluorophore, or an enzyme such as HRP (horseradish peroxidase), in order to visualize the expression of the target antigen. Antibody panels that can be used for high-yield antigen profiles of cell surface antigen profiles are commercially available from several manufacturers.
[00381] [00381] In addition, tools specifically dedicated to the high rate of proteomic cell characterization to identify and analyze cell surface protein expression are commercially available.
[00382] [00382] The immunocytochemistry / proteomics / immunocytochemistry analysis described above can be preceded (or, in some cases, replaced) by the entire genome of the expression of the tumor cell profile gene or by means of mass spectrometry analysis of proteins expressed by the tumor cells / tissue of interest. For example, the entire expression of genes from the genome of tumor cell profiles can be carried out to verify the expression of various cell surface molecules, and the presence of such antigens on the cell surface of tumor cells. it can then be confirmed by antibody staining methods as described above.
[00383] [00383] Further information on approaches to characterize cell surface antigens (cancer) is available in the relevant scientific literature (eg, Zhou J, L Belov, Huang PY, Shin JS, Salomão MJ, Chapuis PH, Bokey L, C Chan, Clarke C ,. Clarke SJ, Christopherson IR, Colorectal cancer antigen surface profiles using fluorescence multiplexing antibody microassays J Methods Immunol 2010; 355: 40 to 51; or Carter P, L Smith, Ryan M. Identification and validation of cell surface antigens for antibodies targeting oncology (Endocr Relat Cancer 2004; 11: 659 to 87).
[00384] [00384] In a next step, the person who is skilled in the technique can identify among the antigens on the cell surface of tumor cells a combination of two antigens that is not expressed simultaneously in other types of cells.
[00385] [00385] Often, the literature or databases published
[00386] [00386] The expression of various cell surface molecules in different types of cells has been systematically studied by researchers in recent decades, through immunophenotyping and gene expression profiles of almost any type of cell in the body. For example, detailed information on the expression of more than 360 "differentiation clusters" of antigens (or CD antigens) is available in print (for example, "Leukocyte and stromal cell molecules: CD markers", from Zola H, Swart B, Nicholson |, Voss ee; John Wiley & Sons, 1st ed (2007)) and in online depositories (for example, www.hcedm.org/Moleculelnformation/tabid/54/Default.aspx ), and includes information on levels of tissue distribution and antigen expression., as well as information on reactive antibodies to antigens and the epitopes to which these antibodies bind.
[00387] [00387] In addition, there are publicly available databases that provide access to a large amount of genomic data generated by the scientific community. For example, Omnibus (GEO), the Gene Expression platform of the United States National Biotechnology Information Center (NCBI) (Barrett T et al, NCBI GEO: .. File for functional genomics of 10-year joint data. Nucleic Acids Res. 2011; 39 (database edition): D1005-10) present files and give access to a huge collection of microassays, next generation sequencing, and other forms of functional genomic data from high throughput, and even provides web-based interfaces and applications for easy access to this information (http: //www.ncbi.nlm.nih.gov/geo/).
[00388] [00388] Once a pair of two antigens has been identified through these resources it appears to be absent from other types of cells.
[00389] [00389] It should be noted that while, in many cases, the highest degree of specificity (absolute preference specificity) is, of course, desirable, there are situations in which a lower degree of specificity is acceptable. For example, if the set of polypeptides is used for diagnostic purposes, a certain degree of cross-reactivity with other types of cells or tissues may be acceptable (especially in the case of solid tumors, since the posi- additional information helps to distinguish tumor cells from cross-reactive cells). In addition, if the set of polypeptides is used for therapeutic purposes, a certain degree of cross-reactivity with other types of cells or tissues may also be acceptable, depending on the severity of the disease in the treated patient and the types of cells / tissues affected. through cross-reactivity. Other situations
[00390] [00390] In cases where no suggestion of a suitable antigen combination can be derived from the literature or from public databases, the presence / absence of cell surface antigens from tumor cells from other types of cells can be verified by simple experimentation. For this purpose, a variety of types and / or tissues can be obtained from the above sources. Cells can be subjected to proteome, immunocytochemical / immunohistochemical and / or gene expression profile characterization. (It should be noted that such analysis of non-tumor cells / tissues has to be performed only once, in order to obtain the data that can be used for the creation of various constructs according to the present invention, which can be adapted - to different therapeutic or diagnostic situations.) After comparing the results obtained with the information on the cell surface antigens of the tumor cells of interest, a combination of two antigens that is not present in any other cells besides the cells tumor of interest can be easily identified.
[00391] [00391] “A similar systematic approach to identifying a pair of two antigens, which is specific for tumor cells, is also described in a recent publication by Balagurunathan, which relies on the gene expression of the entire genome profiling followed by immunohistochemistry (Yoganand Balagurunathan, Expression of the identification gene based on target profiles of the cell surface for the development of multimeric ligands in pancreatic cancer Mol Ther 2008; 7 3071-3080). Using DNA microarrays, the authors of this manuscript generated the databases of mRNA gene expression profiles for a significant number of pancreatic cancer samples and samples of normal tissues. The expression data for the genes encoding the cell surface molecules were analyzed by a multivariate computational method based on rules, in order to identify the combinations of genes that are preferentially expressed in tumor cells, but not in normal tissues. Aberrant co-expression of antigens that constitute a tumor-specific antigen combination was then confirmed through standard immunohistochemistry techniques in pancreatic tumor tissue and normal tissue microassays.
[00392] [00392] Having identified and validated such a combination of antigens that are specific to the tumor cells of interest, the constructs of polypeptide P1 and Polypeptide P2 can be manipulated using protein engineering techniques and standard methods of molecular biology (see, for example, L and M Howard Kaser, making and using antibodies: a Practical Manual, CRC Press, 1st edition (2006); Sambrook et al, Molecular Cloning :. Laboratory Manual, Cold Spring Harbor Laboratory Press , New York (2001)).
[00393] [00393] For many cell surface molecules, specific monoclonal antibodies are characterized and therefore readily available. In this way, in many cases, the person skilled in the art may have access to the monoclonal antibody hybridoma cells that are specific to the antigens in the identified antigen combination. Having the option of choosing from a panel of antibodies specific to a given antigen, a person who is skilled in the art can choose a reactive antibody that binds to an epitope close to the membrane in order to minimize the distance from the cell which expresses the cell effector antigen (Bluemel C, Hausmann S, P Fluhr, Sriskandarajah M, Stallcup WB, Baeuerle PA, Kufer p Epitope distance to the target cell membrane size and the antigen to determine the lysis power T cell-mediated by BITE-specific antibodies a large melanoma surface antigen Cancer Immunol Immunother August 2010; 59 (8): 1197 to 209). If there is no such antibody that is available against one or both of the antigens in the identified antigen combination, monoclonal antibodies against the antigens can be generated using standard techniques (for example, L and M KASER Howard, Fazer and with Antibodies: The Pratical Handbook, CRC Press, 1st edition (2006)). In addition, several companies offer complete services for the generation of tailor-made monoclonal antibodies and hybridoma cells.
[00394] [00394] - DNA or mRNA that codes for the variable domains of monoclonal antibodies of interest can be obtained from hybridomas by PCR amplification or cloning (Orlandi R, Gussow PT, Jones: cloning of immunoglobulin variable domains for expression via the polymerase chain reaction, Proc Natl Acad Sci USA 1989, 86 (10): 3833-3837, Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, Ratner D: Amplification by POR Universal of variable regions of the mouse immunoglobulin gene: the design of degenerate primers and an evaluation of the effect of DNA polymerase on activity 3 5 'exonuclease J Immunol Metods 2000, 233 (1-2): 167-177; Essono S, Frobert Y, Grassi J, CREMINO C, Boquet D: A general method that allows the design of oligo-nucleotides to amplify the variable regions of immunoglobulin J cDNA Immunol Methods 2003, 279: 251 to 266; G Howard and M Kaser, making and using antibodies: A Practical Manual, CRC Press, 1st edition (2006)) or already established vectors that comprise the DNA sequence of the variable fragment of the respective antibody. Often, the sequence can be extracted from public databases, where many sequences are deposited, and then the construct can still be generated through the synthesis of the gene, since it is offered by several suppliers commercial services (for example, creative Biolabs, Shirley, USA).
[00395] [00395] To form the P1 polypeptide construct, the coding sequence for the variable Fv fragment of an antibody specific for the antigen first of the pair of identified antigens (or, optionally, the sequence of a fragment single chain variable derived from the sequence that) is used for the first target portion (T1) and linked via a suitable linker (encoding, for example, to less than 12 aa), to a coding sequence for the first F1 fragment of a functional domain (for example, the VL domain of an anti-CD3 antibody). Likewise, to form the P2 polypeptide construct, the coding sequence for the variable Fv fragment of an antibody specific for the antigen of the second pair of identified antigens (or, optionally, the sequence of a variable fragment of single strand derived from the sequence) is being used for the second target portion (T2) and ligated through a linker appropriate to a sequence encoding the second F2 fragment of that functional domain (for example, the VH domain of an anti-CD3 antibody).
[00396] [00396] For any construct of a P1 or P2 polypeptide according to the present invention, modifications of the construct or the sequences used for the formation of the construct are considered in order to adapt the construct to the specific needs. For example, a construct can be modified in a way that reduces or suppresses its immunogenicity in humans. In the case where a sequence is derived from a non-human parent antibody, such as a murine antibody, changes in the sequence can be carried out that result in reduced immunogenicity in humans, maintaining or retaining substantially the antigen-binding properties of the parent antibody
[00397] [00397] Several modifications of the procedure described above and adaptations, in order to accommodate the modalities and variations described in this application are evident to the person with knowledge in the technique.
[00398] [00398] In addition to variations in relation to antigens in which the targeting portions T1 and T2 specifically bind to, several other modifications are possible. For example, instead of fragments of single chain variants (scFv) as the T1 and / or T2 target moieties, other types of monovalent antibodies or antibody-like structures can be employed. For example, an antibody / antibody-like structure derived from a mud, camel or shark antibody can be used. Since llama, camel and shark antibodies have an antigen-binding portion that is constructed by a single domain (instead of a VH chain and a VL chain), the resulting P1 or P2 polypeptide is much smaller and it can, in this way, better penetrate the tumor tissues.
[00399] [00399] Furthermore, since many tumor antigens are relevant to cell surface-bound receptors, the target chain T1 and / or T2 single chain Fv can be replaced by the natural or artificial ligand of such a linked receptor to the cell surface. As antibodies, these natural or artificial ligands confer excellent specificity to the target receptor. Alternatively, the target portion T1 and / or T2 can be an aptamer.
[00400] [00400] In addition, in order to increase the binding affinity of a target moiety for the antigen, the target moiety can be multimerized and / or altered by glycosylation or other types of post-translation or chemical modification or optimized through targeted mutagenesis, or a phage display selection process.
[00401] [00401] In addition, the F1 and F2 fragments (that is, the VL and VH fragments of anti-CD3 Fv in the exemplary embodiment described above) can be replaced by fragments of a functional domain other than F, which results in a different biological effect by complementing the two fragments. Using fragments of anti CDB56, anti CD1a, or anti CD16a, natural killer cells can be recruited and activated. Using fragments of anti CD16, natural killer cells, polymorphonuclear neutrophil leukocytes, monocytes and macrophages can be recruited and activated. Using the fragments of anti CD32a, anti CD32b, anti CD89, anti CD16a, or anti CD64, macrophages can be recruited and activated. Using the anti CD32a, anti CD32b, anti CD64 or anti CD89 fragments, monocytes can be recruited and activated. Using fragments of anti CD16b, anti CD89, anti CD32a, anti CD32b or anti CD64, granulocytes can be recruited and activated. In addition, as an alternative to anti CD3, T cells can also be recruited and activated using anti CD2, anti CD5, anti CD28, or anti TCR (T cell receptor) fragments. More information or additional options regarding the recruitment and activation of effector cells via binding antibody are available in the literature, for example, "Bispecific Antibodies", by Roland E. Kontermann (ereferidor), Springer Berlin Heidelberg; 1 Edition. (2011).
[00402] [00402] “An additional option is the use of a set of polypeptides from P1 and P2, with fragments F1 and F2 from a functional domain F, which binds to an antigen in an effector cell after complementation of the two fragments, but in which the binding to this effector cell antigen does not cause activation of effector cells, he said. This set of polypeptides ("first set of polypeptides") is then used (for example, administered to a patient) in combination with a second set of polypeptides by fragments.
[00403] [00403] Similar effects can be achieved with two sets of polypeptides with different targeting moieties, but the same functional domain: These sets of polypeptides are designed to have a functional domain directed against an effector cell antigen, which normally allows each set of polypeptides to activate effector cells alone. However, both sets of polypeptides are used at a concentration that is too low to cause the activation of efficient effector cells. If both sets of polypeptides are present at the same time (for example, in the simultaneous administration of a patient) to each set of polypeptides, alone is not able to activate the effector cell (due to its low concentration).
[00404] [00404] As another alternative to the recruitment / activation of effector cells, a "pre-targeting" approach can be followed, since it is well established for the constructs of bi-specific antibodies (Image of Cancer and Bispecific antibodies pre-targeting therapy Goldenberg DM, Chatal JF, Barbet J, Boerman O, Sharkey RM Ther Cancer Update 2007 Mar; 2 (1): 19-31). To this end, F1 and F2 are replaced by fragments of VH and VL of an antibody specific for an antigen, a support molecule (ie, a piece / molecule of a molecule that is not recognized as foreign by the system immune system of the patient to whom the said set of polypeptides is administered or a molecule that does not or only causes a weak immune reaction by a patient to whom it is administered) or an affinity marker. Subsequently (or simultaneously) to administer polypeptides P1 and P2, a therapeutic or diagnostic compound coupled to said antigen, vehicle or affinity marker molecule is administered. Only cells that carry both A1 and A2 antigens on their surface are linked by both polypeptide P1 and Polypeptide P2. Consequently, only to these cells does functional complementation lead to the generation of a binding site capable of recruiting the therapeutic or diagnostic compound through said antigen, vehicle molecule or affinity tag. This approach allows the exclusive targeting of target cells as a whole, with the possibility of administering and dosing therapeutic compounds, such as toxins or radioactive substances or compounds with precise diagnosis, while cells that do not express antigens or that express only one antigens are not affected.
[00405] [00405] “An appropriate carrier molecule can be for example a peptide or a carbohydrate molecule. Preferably, the carrier molecule can be gelatin, dextran, or hydroxyethyl starch, which are common plasma expanders that are metabolically inert, remain in the blood and are eliminated via the kidney, if they are small enough. Alternatively, the vehicle molecule may be inulin, a metabolically inert molecule that is routinely used in the clinic to determine glomerular clearance (and, in addition, there are antibodies that specifically recognize inulin).
[00406] A suitable affinity marker can be, for example, a Flag marker, a myc marker, a glutathione-S-transferase (GST) marker, a hemagglutinin marker (HA), a polyhistidine marker (His) , or a maltose-binding protein (MBP) marker, a digoxigenin (DIG) marker.
[00407] [00407] The therapeutic compound bound to the antigen, vehicle or affinity marker molecule can, for example, be a radioactive compound or a toxin.
[00408] [00408] “Suitable radioactive compounds are for example compounds comprising 90Y, 177Lu, 1311, 32P, 10B, or 213Bi. Recruiting the antigen, vehicle or affinity marker molecule attached to the radioactive compound to cells that express both the first and the second antigen leads to the accumulation of radioactivity to the tumor site, resulting in the specific destruction of tumor cells / tissue.
[00409] [00409] Alternatively, the therapeutic compound bound to the antigen, vehicle or affinity marker molecule may for example be a toxic compound that is not able to cross the cell membrane without prior attachment to the cell surface.
[00410] [00410] This prerequisite is fulfilled by the A components of classic AB toxins derived from a number of pathogenic bacteria such as Clostridium perfringens, C. botulinum, C. difficile, B. anthracis and others. AB-toxins are complexes of two-component proteins that interfere with the functions of internal cells. Component A is the "active" component (that is, killing a cell on the penetration of the membrane), but it is not able to cross the cell membrane on its own. Component B is the "binding" component, which in itself is non-toxic, but is essential for the absorption and penetration of the component A membrane.
[00411] [00411] For example, the protective antigen Bacillus anthracis (PA) is a component of classical B toxin that mediates the uptake of the real edema factor exotoxins anthrax and lethal factor (LF). LF without the PA component is not toxic as long as LF alone does not penetrate the membranes and therefore cannot execute its pathogenic capabilities (Pezard C, Berche P, Mock M. "Contribution of individual toxin components to virulence of Bacillus anthracis "1991 Infect. Immun 59 (10): 3472). However, when bound to molecules on the cell surface, LF is internalized and highly toxic to the cell.
[00412] [00412] After the dimerization of the P1 and P2 polypeptides, the function of the functional domain F is reconstituted. Through the interaction of the reconstituted functional domain with the antigen, vehicle or affinity marker molecule coupled with the toxin, the toxin is recruited into the cell membrane of the target cells, incorporated into the cells and kills the cells.
[00413] [00413] This principle can be easily adapted for the purposes of the present invention by the person skilled in the art, since it is already widely used in so-called immunotoxins, in which a target portion, mainly an antibody or ligand-like domain natural, it is coupled to the toxin component (see, for example, Immunotoxins for targeted cancer therapy Kreitman RJ, AAPS J. 2006 Aug 18; 8 (3): E532-51). Examples include diphtheria toxin-based immunotoxins (such as Denileucine diptitox (U.S. trade name Ontak), which has been approved by the FDA for the treatment of some T-cell lymphomas) or based on the B. anthracis Lethal Factor (Pastan |, Hassan R, FitzGerald DJ Kreitman RJ (2007) "Treatment of Immunotoxin Cancer" Annu Rev. Med 58: 221-37).
[00414] [00414] Suitable components A of AB-toxins can be, for example edema factor B. anthracis, lethal factor B. anthracis, iota toxin C. perfringens, C2C toxin. botulinum, C. difficie ADP-ribosyltransferase, C. diphtheriae diphtheria toxin from fragment A.
[00415] [00415] Alternatively, the therapeutic compound can be, for example, a cytotoxic compound that is toxic after entering a cell and is able to cross the cell membrane by itself, without prior attachment to the cell surface. In this case, the antigen, vehicle or affinity-marker molecule that the therapeutic compound is coupled to is selected in order to prevent the resulting conjugate (i.e., the therapeutic compound bound to the antigen / vehicle labeling / affinity molecule) to from the crossing of cell membranes and entering cells without prior binding of the conjugate to the cell surface (a suitable carrier molecule may for example be a hydroxyethyl starch vehicle). In this way, such a conjugate does not enter cells without previous obligation to its cell surface; since such a conjugate binds to the cell surface, however, it is internalized in the cell and the toxic compound kills the cell. The conjugate does not bind to cells, unless it is recruited in the presence of the inventive set of cell polypeptides that simultaneously express both A1 and A2 antigens on their surface.
[00416] [00416] The diagnostic compound coupled to the antigen, vehicle or affinity marker molecule can, for example, be a radioactive compound, a fluorophore, or a compound capable of mediating bioluminescence.
[00417] [00417] Suitable radioactive compounds are, for example, compounds comprising 99mTc, 111In, 82Rb and 201T7I. These compounds are detected through medical imaging procedures known in the clinic.
[00418] [00418] Alternatively, a fluorescent compound can be used as a diagnostic compound, such as GFP (green fluorescent protein) or a variant of GFP (for example, BFP (blue fluorescent protein), CFP (cyan fluorescent protein) ), or YFP (green fluorescent protein)), or a small fluorescent molecule compound like FITC (fluorescein isothiocyanate) or PE (phycoerythrin), alexa fluor dyes (like AlexaFluor488 and related dyes sold by Molecular Probes, for example) or cyanine dyes (such as Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) or related dyes).
[00419] [00419] —Alternatively, a compound capable of mediating biolumination
[00420] [00420] In addition, the F1 and F2 fragments (ie, the VL and VH fragments of anti-CD3 Fv in the exemplary embodiment described above) can be replaced by means of the VL and VH fragments of an antibody that is specific for a therapeutic or diagnostic compound (for example, in this case, the functional domain F is able to directly bind to the therapeutic or diagnostic compound). In the present invention, the same therapeutic and diagnostic compounds, as described above in the context of the "pre-targeting" approach can be considered.
[00421] [00421] In addition, the F1 and F2 fragments (that is, the VL and VH fragments of anti-CD3 Fv in the exemplary embodiment described above) can be substituted by fragments of a fluorescent or bioluminescent compound, which are biologically inactive in themselves, but recover their function (that is, their ability to mediate fluorescence or bioluminescence) through the association of the two fragments and functional complementation, thus allowing the specific identification of cells that carry both antigens of Al and A2.
[00422] [00422] “A number of fluorescent molecules that can be used in this context are well known and characterized in the art, including, but not limited to, GFP (green fluorescent protein), GFP derivatives (such as YFP (green fluorescent protein) and PCP (cyan fluorescent protein), Venus (Nagai T et al, The fast and efficient maturation yellow fluorescent protein variant for biological cell applications Nat Biotechnol 2002 Jan; 20 (1): 87- 90) , or Cerulean (PCP enhanced with S72A, and Y145A H148D replacements)). For these molecules, the divided fragments are described that self-assemble in the proximity situation in a process called bimolecular complementation of fluorescence (BiIFC).
[00423] [00423] For example, GFP, PCP, Venus, with an M153T substitution, or Cerulea can be divided after amino acid 155 (that is, for example, the F1 fragment can comprise amino acids 1 to 155 of GFP, whereas fragment F2 can comprise GFP amino acids 156-245, or vice versa). Alternatively, YFP or Venus can be divided after the amino acid 173. Further details on the GFP division and the GFP division variants can be found in Kerppola TK, visualization of molecular interactions through fluorescence analysis and bimolecular complementation :. Characteristics of fragment complementing protein. Chem Soc Rev. 2009; 38: 2876-86.
[00424] [00424] An example of a molecule that mediates bioluminescence and that can be used in this context is the division luciferase. Particularly suitable is the Gaussia princeps luciferase, which does not require cofactors that are active and catalyze the oxidation of the celenterated luciferin substrate (coelenterazine) in a reaction that emits blue light, or derivatives of Gaussia luciferase (Remy and Michnick |
[00425] [00425] Intravital images of tumor lesions are of paramount importance in cases where carcinoma cells infiltrate tissues and the complete elimination of all transformed cells is a prerequisite for healing. A surgeon looking for disseminated carcinoma cells at the site of the operation can use split GFP or split GFP derivatives fused with targeting portions and a multispectral assisted laser fluorescence camera system to detect cells expressing aberrantly, a target antigen profile, similar to the intraoperative use of fluorescence or bioluminescence that is already explored in some clinical situations (van Dam GM et al, tumor-specific intraoperative fluorescence images in ovarian cancer by receptor-a target folate: results for the first time in humans Nat Med 2011 Sep 18; 17 (10): 1815 to 9; Luker et al, In vivo ligand receptor images with Gaussia luciferase complementation. Nature Medicine 2011, doi : 10.1038 / nm.2590).
[00426] [00426] For the detection of complete division luciferase, the application of a substrate for luciferase, which can be luciferin or coelenterazine, is mandatory. Coelenterazine is preferred because coelenterazine emits ATP-independent light and is well established for in vivo imaging and in vivo applications. A surgeon will be able to view the cancer cells after having labeled the tumor with polypeptide P1 and P2 and injected in a non-toxic amount of coenterolazine intravenously.
[00427] [00427] In another exemplary embodiment, the principle of the present invention is applied in the context of a patient suffering from a hematopoietic tumor and who received a transplant of healthy hematopoietic cells from another person (the donor). In the present invention, the set of polypeptides according to the present invention can be used for specific elimination (or detection) of the remaining malignant hematopoietic cells from the recipient after transplantation of healthy donor hematopoietic cells.
[00428] [00428] To destroy malignant hematopoietic cells in a patient suffering from a hematopoietic tumor, the patient may be subjected to chemotherapy and / or radiation therapy. Subsequently, the patient receives a hematopoietic cell transplant from a healthy donor.
[00429] [00429] To minimize the risk of transplant rejection or graft versus host disease, transplantation of tissues / cells (eg bone marrow) from a donor that has the same set of MHC molecules (main histocompatibility complex) ) is generally preferred. However, many times no donor with the same set of MHC molecules ("identical HLA donor") can be identified. Therefore, the transplantation of grafts with one or two mismatches in the set of MHC variants, umbilical cord not related to up to three mismatches, or haploidentical transplants are increasingly used. Consequently, it is common for there to be at least one distinct difference between the set of MHC molecules expressed by the recipient's cells and the donor cells.
[00430] [00430] In transplantation according to this exemplary embodiment of the present invention, donor cells are used, so that they are different from recipient cells with respect to at least one of its HLA variants. This means that there is at least one "distinguishing antigen", which is present on the cell surface of recipient cells, but not on the cell surface of donor cells. For example, the distinguishing antigen can be HLA-A2, if the patient (ie, the recipient) is HLA-A2 positive, while the donor is HLA-A2 negative.
[00431] [00431] Despite chemotherapy / radiotherapy, the recipient's individual malignant hematopoietic cells may have escaped eradication. Since the surviving malignant hematopoietic cells are recipient cells, they carry the distinctive antigen that differentiates recipient cells from donor cells. At the same time, they are cells of origin of the hematopoietic lineage and, therefore, have markers of this cell lineage, such as CDA45, on their cell surface. Leukemia blasts and other hematopoietic cells of the patient are the only cells that simultaneously exhibit the distinctive antigen (here HLA-A2) and hematopoietic cell lineage markers (here CD45). The set of polypeptides according to the present invention exploits this fact to specifically eliminate cells.
[00432] [00432] For this purpose, the first target portion T1 of the first polypeptide P1 may be a scFv specific for the antigen itself which is present only in cells of the receptor (here HLA-A2). As an F1 fragment of the functional domain F, the light chain variable region (VL) of a specific antibody CD3e-can be chosen. The second target portion T2 of the second P2 polypeptide can be a single fragment
[00433] [00433] However, if both the distinctive antigen (for example, HLA-A2) and the CD45 antigen are present in a single cell, binding to their respective antigens brings the two polypeptides P1 and P2 in close proximity. Consequently, the mismatched VH and VL domains result in heterodimerization of the P1 and P2 polypeptides and in the formation of a functional Fv variable antibody fragment from the VH and VL domains that is capable of binding to CD3e (see figure 2) .
[00434] [00434] “As a result, T cells are activated and recruited via CD3β, and the cell that carries both HLA-A2 and CD45 on their cell surface is specifically eliminated by a cytotoxic T cell response.
[00435] [00435] A person skilled in the art understands that, within the principle of the present invention, several variations to this exemplary mode are possible.
[00436] [00436] For example, in the P2 polypeptide the scFv fragment that recognizes the CD45 hematopoietic cell marker can be replaced by a scFv fragment that recognizes a cell marker of different lineage or cell type, that is, the target portion T2 may be a domain that specifically binds to an antigen that is specific to a cell line other than the hematopoietic cell line or to a particular cell type (for a detailed list of various cell line markers) cells and cell type markers that can be used in this context, see David J. Dabbs, immuno-diagnosis, Churchill Livingstone, 3rd edition (2010), or F and J Lin Prichard, Immunohistochemistry Practical Manual: Frequently Asked Questions , Springer, New York, 1st edition (2011)). To adapt the set of polypeptides of a cell line marker / alternative cell type marker, it is sufficient to replace the target portion of the T2 P2 polypeptide with a target portion that has binding specificity for a marker cell line / marker of the desired alternative cell type.
[00437] [00437] For example, in the case of metastatic renal cell carcinoma (RCC), a person who is knowledgeable in the technique can consult the aforementioned databases for information on cell surface proteins with cell-restricted expression kidney them. Among many other molecules, he will know that the expression of certain members of the aquaporin family is confined to kidney and erythrocyte cells. Having obtained this information, a person skilled in the art will construct a P2 polypeptide recognizing that a member of the aquaporin family is confined to erythrocyte kidney cells and fused to the heavy chain variable (VH) region of a specific CD3e antibody. . In the case where the patient suffering from renal cell carcinoma is A2 HLA positive and a kidney transplant from a healthy donor is HLA A2 negative, the doctor treating the patient can use both constructs (fused anti-aquaporin with anti-CD3 (VH) and anti HLA-A2 fused to the light chain
[00438] [00438] Another example is hepatocellular carcinoma (HCC). Hepatocytes are largely involved in a number of metabolic processes, including trafficking in lipoproteins. To this end, hepatocytes express receptors for high-density lipoproteins (HDL) on their surfaces (class B 1 member scavengers, SCARB1). The treatment of a patient with HLA A2 positive HCC suffering from HCC which expresses SCARB1 on the surface of tumor cells and metastases can be performed by a P2 polypeptide construct comprising a SCARB1 addressing Fvcs domain fused to the variable region the heavy chain (VH) of said CD3e specific antibody and a P1 polypeptide (anti-HLA A2 scFv fused to the light chain (VL) of said CD3 specific antibody: £) and transplantation of healthy liver cells from one HLA A2 negative donor. In this case, all hepatocytes and malignant cells
[00439] [00439] Several markers that are specific to certain types of cells or cell lines or some types of cells / lines are known (for a list of examples, see above). More information on lineage markers, differentiation antigens and tissue markers, as well as their distribution in tissues are easily accessible from published sources (see, for example, David J. Dabbs, Diagnostic immunohistochemistry, Churchill Livingstone, 3rd edition (2010), or F and J Lin Prichard, Handbook of practical immunohistochemistry: Frequently Asked Questions, Springer, New York, 1st edition (2011)) and public databases (such as the Gene Express Atlas of European Bioinformatics Institute (EBI), http: Ilhvww.ebi.ac.uk / GXA /, or the omnibus gene expression (GEO) of the platform, see above). In addition, these markers can be identified and / or verified in a simple way by a person who is skilled in the art using methods similar to those described above for the identification of specific combinations of tumor antigens.
[00440] [00440] In certain preferred embodiments, an antigen with less than the perfect specificity for a given cell type or cell line is used (that is, an antigen is used, which is present in more than one, but preferably only a few, cell types or cell lines). In some modalities,
[00441] [00441] The concept can be adapted to any other HLA haplotype besides HLA-A2 used in the exemplary modality above, while the recipient cells are positive for this HLA antigen and the donor cells are negative for it. Possible HLA antigens include, but are not limited to, HLA A1, HLA A2, HLA A3, HLA A25, HLA B7, HLA B8, HLA B35, HLA B44 and HLA Cw3, HLA Cw4, HLA Cw6, HLA Cw7. To adapt the set of polypeptides to an alternative HLA antigen, it is sufficient to replace the target portion of the T1 P1 polypeptide with a target portion that has binding specificity for the desired alternative HLA antigen. By an appropriate choice of target portion T1, it is clear that it is also possible to specifically eliminate cells from the donor.
[00442] [00442] In addition, instead of a VL domain and a VH domain, which, after assembly, forms a domain capable of binding to CD3e (ie fragment F1 and fragment F2 of P1 and P2 polypeptides, respectively) , the VL and VH domains can be replaced with domains / fragments that, during assembly, give a different function to the resulting dimer. In this regard, all the variations described above for the exemplary modality, concerning the e-limitation and / or detection of tumor cells identified by means of a specific combination of two cell surface antigens are equally applicable. For example, when the assembly of the complemented functional domain can mediate the binding / activation of other effector cells than T cells, it can be adapted to a "pre-targeting" approach, it can be linked to a therapeutic or diagnostic compound, or it can form a fluorescent molecule / molecule capable of mediating bioluminescence.
[00443] [00443] The various options for choosing fragments F1 and F2 and for choosing one of the targeting portions T1 or T2 above described in the exemplary modality, concerns the application of the principle of the present invention for the specific elimination of cells which can, of course, be considered.
[00444] [00444] From the exemplary modalities and variations described, it will be clear to a person skilled in the art that the principle of the present invention described above, can not only be used for the highly specific identification and / or elimination of cells tumor or the remaining malignant recipient cells after a cell transplant, but also for the identification and / or elimination of any other cell type having a specific combination of two antigens that distinguishes it from other cell types.
[00445] [00445] In the following, reference is made to the figures:
[00446] [00446] Figure 1 shows the principle of the present invention. Figure 1A: P1 and P2 antigen and polypeptide design. Figure 1B: If a cell expresses both antigens 1 and 2 on its cell surface, the simultaneous binding of polypeptide P1 and polypeptide P2 to the surface of the cell leads P1 and P2, in close proximity, to cause the association of fragments F1 and F2 and restore the biological function of the F domain through complementation. No restoration of biological function occurs only if the A1 antigen (Figure 1C) or the A2 antigen (Figure 1D) is present on the cell surface.
[00447] [00447] Figure 2 shows an exemplary embodiment of the present invention, in an allogeneic transplantation configuration of hematopoietic neoplasms with non-coincident HLA antigens. In this situation, the double information of recipient HLA haplotype (HLApatient) and origin of the hematopoietic lineage (CD45) is presented exclusively in leukemia blasts and other hematopoietic cells of the patient. The first P1 polypeptide comprises a construct of the single chain antibody fragment directed against the patient's HLA variable (target portion T1) fused to the anti-CD3 VL fragment (Fragment F1). The second P2 polypeptide comprises a single-chain variable fragment specific to construct the hematopoietic lineage marker CD45 (target portion T2), fused to the divided anti-CD3 FV VH fragment (F2 fragment).
[00448] [00448] CD45: specific antigen for hematopoietic cells. HLApatient: HLA-specific antigen for the patient's cells, that is, an allelic variant of human MHC that is present on the surface of the patient's cells (= cells of the cell transplant recipient), but absent on the cell surface of the patient. donor. aCD45 scFv: scFv with binding specificity for CD45. aHLA scFv patient: scFV with binding specificity for HLA patient. CD3 (VH): variable region of an immunoglobulin heavy chain of an antibody with binding specificity for CD3. CD3 (VL): variable region of an immunoglobulin light chain of an antibody with specificity for binding to CD3.
[00449] [00449] After the connection of the two constructs through their aCD45 scFv and aHLApatient scFv, respectively, to a cell that carries both the CD45 and the HLApatient antigen, assembly of CD3 (VH) with CD3 (VL) leads to functional complementation of the anti - body with the specificity of binding to CD3, thus allowing the recruitment and activation of T cells through CD3 molecules on their specific cell surface.
[00450] [00450] Figure 3 shows the constructs used in the experiments described in figures 4 to 9. (Construct 85 differs from construct 71 in that construct 85 has a Flag marker while construct 71 has a myc marker. Construct 75 differs from construct 82 in that construct 75 has a Flag marker while construct 82 has a myc marker.) VHCD3: variable region of an anti-CD3 antibody heavy chain; VLCD3: variable region of the light chain of an anti-CD3 antibody; VHA Z: heavy chain variable region of an anti-HLA-A2 antibody; VLAZ: variable region of the light chain of an anti-HLA-A2 antibody; VL45: variable region of the heavy chain of an anti-CD45 antibody; VH45: variable region of the light chain of an anti-CD45 antibody; L18, L7, L15, L6, L19: amino acid linker 18, 7, 15, 6, 19, respectively.
[00451] [00451] Figure 4 shows the conventional tandem bi-specific single-chain scFv constructs used to control the assay system. Briefly, bispecific antibody constructs with specificity for CD3 and HLA A2 were titrated as indicated for a U266 co-culture, a positive HLA A2 cell line, myeloma CD45 positive, and negative HLA A2 T cells (depleted monocytes of peripheral blood mononuclear cells), and the production of interleukin-2 by means of T cells was determined. The stimulation capacity of the substantial T cells was detected for the two constructs FvCD3-HLA-A 2 85 and 71, which differ in their respective markers Flag or Myc (For construct domain structure see Figure 3. ). The bispecific HLA-A2-CD3 tandem Fv constructs were less efficient and the single-stranded addressing or HLA-A2 constructs when CD3 did not stimulate T cells at all. Positive control is conducted using non-specific stimulation of PHA-L (phytohemagglutinin).
[00452] [00452] Figure 5 shows the stimulating capacity of T cells and highly and exclusively specific if a complementary pair of constructs according to the present invention is used, but not if only one of the two constructs of a pair is used individually.
[00453] [00453] Figure 6 shows a first of three competitive blocking experiences. The bispecific set of the construct FvCD3-HLA-A2 (construct 71) was given to co-cultures of U266 and T cells, as described and the stimulation function was determined by means of induced the production of IL-2 by T lymphocytes, . The function of stimulatory T cells was blocked by means of single chain constructs, which occupy the target epitope of the HLA A2 molecule (construct 4, the * 100 concentration). The intrinsic stimulation of T cells by specific HLA A2 or CD3 single chain constructs (construct 4 (concentration * 100) or construct 36 (concentration * 9)) has been discarded. PHA-L was used as a positive control.
[00454] [00454] Figure 7 shows that "tridominium constructs" (that is, constructs according to the present invention) must first bind to the surface of a single dimerization cell and complement the competitive epitope T cell fitting functions as blocking experiences. Briefly, constructs 42 and 45 were given to co-cultures of U266 cells and negative HLA-A2 T lymphocytes and stimulatory capacity was determined through the production of IL-2 T cells. In experimental situations where HLA epitopes A2 or CD45 molecules were competitively blocked by means of constructs 4 or 46 (both concentrations * 100), the T cell stimulation function was revoked. These results clearly indicate that the two respective "tridominium constructs" must connect at the same time on the surface of a cell, in order to restore or to complement the function of the surrounding T cells. The intrinsic stimulating activity of any construct (42, 45.4, 46 and 36) was discarded using the different concentrations.
[00455] [00455] Figure 8 shows the experiment analogous to Figure 7, for the combination of constructs 42 and 55. Once again, the capacity of stimulating T cells of the combination of the two "tri-domain constructs" was abolished through competitive blocking the antigenic epitopes on HLA A2 or the CD45 molecule. More importantly, these results again show that the segmentation module can be easily replaced by another module, with adequate specificity. More importantly, the VL-VH-VL configuration of construct 42 and the VH-VH-VL configuration of construct 55 prevent homo-or hetero-dimerization or self-assembly of the constructs without prior connection to a substrate that expresses both , HLA A2 and CDA45 antigens.
[00456] [00456] Figure 9 shows the lysis of U266 cells by negative HLA A2 T cells in a sample comprising the constructs (VH-VL) ("both constructs") VLCD3-scFVHLA-A 2 and VHCD3-scFvCD45. No significant lysis was observed in control samples that comprise only one of the two constructs.
[00457] [00457] Figure 10 shows the target restoration of the polypeptides. The binding of two separate polypeptides (P1 and P2) to their respective antigens in a target cell, each consisting of a specific variable single chain antibody fragment (scFv VH-VL) fused with variable light (VL) or the variable heavy chain (VH) domain of an antibody specific for CD3 (fragment F1 and F2), allows VH / VL heterodimerization and the formation of a functional CD3 binding site to exercise T cells.
[00458] [00458] Figure 11 shows that the VH / VL dimerization CD3 involves T cells and is dual-restricted antigen. U266 multiple myeloma, primary pro-lymphocytic T-cell leukemia (T-PLL), and acute myeloid leukemia THP-1 cells, all HLA-A2-positive and CD45-positive, were probed with blood mononuclear cell donors peripheral HLA-A2-negative (PBMC) and polypeptides as indicated. T cell involvement was assessed by interleukin-2 (IL-2) reactive production (A) and lysis of target cells (B). The bispecific set scFv (CD3 (VH-VL) -. HLA-A2 (VH-VL) of the antibody was used as a positive control (C), if the binding of polypeptides in THP-1 cells is blocked by an excess competitive analysis of scFvCD45 (left) and right) inhibitors ((blocking individual antigen epitopes in the target cell), as indicated, and reactive IL2 production by PBMC donor was investigated. (D), the antigen from individual or negative cell lines double RAJI and KMS scFvHLA-A 2 - 12-BM were probed with the polypeptides, PHA-L was used as a non-specific stimulus control of PBMC.
[00459] [00459] Figure 12 shows the targeted therapy through in vivo CD3VH / VL conditional complementation. (A), mouse survival (n = 6 per group) after intraperitoneal injection of 5 x 106 acute leukemia THP-1 cells together with 1.25 x 105, donor HLA-A2-negative T cells specific to CMV and polypeptides
[00460] [00460] Figure 13 shows that polypeptides directed by EGFR and EpCAM involve T cells for the destruction of carcinoma cells. The human colon cancer cell line EG-FR and double positive EpCAM COLO-206F and the melanoma cell line of FM-55 (EGFR-positive but EpCAM-negative) were probed with PBMC in the presence of specific polypeptides for the EGFR (CD3 (VH) -EGFR (VH-VL)) and EBCAM (CD3 (VL) -EpCAM (VH-VL)), as indicated. T cell involvement was assessed by reactive interferon-y production (IFN-y) (A) and the activation of caspase-3 in target cells (B).
[00461] [00461] Figure 14 shows that polypeptides directed by HLA-A 2 and CEA redirect T cells for the destruction of tumor cells. The human colon cancer cell line COLO-206F, melanoma cell line of FM-55 and the ovarian cancer cell line OVCAR were probed with PBMC in the presence of specific HLA-A2 (CD3 (VL) polypeptides) -HLA- A2 (VH-VL)) and CEA (CD3 (VH) -CEA (VH-VL)), as indicated. T cell involvement was assessed by producing reactive IFN-y. The samples were tested and analyzed as duplicates.
[00462] [00462] Figure 15 shows that the targeting polypeptides HLA-A2 and EGFR redirect T cells for the destruction of tumor cells. The human cell lines of COLO-206F, OVCAR and FM-55 were probed with PBMC in the presence of specific polypeptides
[00463] [00463] Figure 16 shows that the target polypeptides HLA-A2 and EGFR redirect T cells for tumor cell destruction. The human cell lines of COLO-206F, FM-55 and OVCAR were probed with PBMC in the presence of specific polypeptides for HLA-A2 (CD3 (VL) -HLA-A2 (VH-VL)) and Her2 (CD3 (VH) -Her2 (VH-VL)), as indicated. T cell involvement was assessed through the production of reactive IFN-y. The samples were tested and analyzed as duplicates.
[00464] [00464] Figure 17 shows that the target polypeptides HLA-A2 and EGFR redirect T cells for the destruction of tumor cells. In this experiment, the split antiCD3 fragments (CD3 (VH) and CD3 (VL)) for the anti-CD45 and anti-HLA-A2 target portions were exchanged, compared with the CD45 and HLA-A 2 polypeptides used in Figure 5.7-9, 11.12, 14-16. The human myeloma cell line U266 was probed with PBMC in the presence of polypeptides specific for CD45 (CD3 (VL) -CD45 (VH-VL)) and HLA-A2 (CD3 (VH) - HLA-A2 ( VH-VL)) as indicated. T cell involvement was assessed through the production of reactive IFN-y. The samples were tested and analyzed as duplicates.
[00465] [00465] Figure 18 shows that the targeted polypeptides EG-FR and EpCAM redirect T cells for the destruction of tumor cells. The human colon cancer cell lines COLO-206F and CX-1 and the ovarian cancer cell line OVCAR were probed with PBMC in the presence of specific polypeptides for EDpCAM (CD3 (VL) -EpCAM (VH-VL )) and EGFR (CD3 (VH) -EGFR (VH-VL)), as indicated. T cell involvement was assessed by producing reactive IFN-y. The samples were tested and analyzed as duplicates.
[00466] [00466] Figure 19 shows that the directed polypeptides Her2 and EpCAM redirect T cells for the destruction of tumor cells. The human ovarian cancer cell line OVCAR was probed with PBMC in the presence of specific polypeptides for EpCAM (CD3 (VL) -EpCAM (VH-VL)) and Her2 (CD3 (VH) -Her2 (VH-VL)), as indicated. T cell involvement was assessed by producing reactive IFN-y. The samples were tested and analyzed as duplicates.
[00467] [00467] Figure 20 shows that the directed polypeptides CD45 and CD138 redirect T cells for the destruction of tumor cells. The human myeloma cell line AMO-1 was probed with PBMC in the presence of CD45-specific polypeptides (CD3 (VL) -CD45 (VH-VL) from the upper panel, CD3 (VH) -CD45 (VH-VL) from the panel bottom) and CD138 (CD3 (VH), CD138 (VH-VL) top panel, CD3 (VL), CD138 (VH-VL) bottom panel), as indicated. T cell involvement was assessed by producing reactive IFN-y. The samples were tested and analyzed as duplicates.
[00468] [00468] Figure 21 shows that targeting a single antigen (CD138) with targeting CD138 polypeptides redirects T cells to the destruction of tumor cells. The human myeloma cell line AMO-1 was probed with PBMC in the presence of specific polypeptides for CD138 (CD3 (VL) -CD138 (VH-VL) and (CD3 (VH) -CD138 (VH-VL)), as indicated. T cell involvement was assessed by the production of reactive IFN-γ. Samples were assayed and analyzed as duplicates.
[00469] [00469] Figure 22 shows that targeting a single antigen (CD45) with targeting CD45 polypeptides redirects T cells to the destruction of tumor cells. Cell lines
[00470] [00470] Figure 23 shows the target restoration of two polypeptides directed against a single antigen on the surface of the target cell, two different epitopes (top) or the same epitope (bottom) of the antigen. The binding of two separate polypeptides (P1 and P2) to their respective epitope, on the same antigen, in a target cell. To target two different epitopes, the target portion of each polypeptide is composed of a specific single chain variable antibody (scFv) fragment. To target the same epitope, the target portion of each polypeptide is made up of the same variable single chain antibody (scFv) fragment. The targeting parts are fused via peptide ligands to the variable light chain domain (VL) or the variable heavy chain domain (VH) of an antibody specific for CD3 (fragment F1 and F2), allowing for VH heterodimerization / VL and the formation of a functional CD3 binding region (functional domain) to involve T cells.
[00471] [00471] Figure 24 shows the possibility of using different effector forms to kill a target cell with a kit of parts of polypeptides. For this purpose, the anti-CD3 module (F1 and F2) is replaced by an anti-HIS module (hexa-histidine), which, after simultaneous binding of polypeptide 1 and 2, complements a binding site hexa-histidine and therefore binds labeled histidine with payloads (for example a HIS toxin). The target portion T1 (VH-VL) of polypeptide P1 specifically binds to HLA-A2, the target portion T2 (VH-VL) of polypeptide P2 specifically binds to CD45. The F1 P1 polypeptide fragment comprises a VH domain of an antibody against a hexa-histidine tag and F2 fragment of the P2 polypeptide comprises a VL domain of the same antibody. The human myeloid leukemia cell line THP-1 was probed with a histidine marker (His) Clostridium perfringens from the iota toxin component in
[00472] [00472] Figure 25 shows that the targeted HLA-A2 and CD45 polypeptides, which comprise an antibody against a His identifier separation, kill tumor cells using a subunit A Shiga toxin histidine marker (His) at a concentration - tion of 0.01Jug / ml. The same experimental configuration was used as in figure F24.
[00473] [00473] Figure 26 shows that the targeted HLA-A2 and CD45 polypeptides, which comprise an antibody against a His identifier separation, kill tumor cells using a histidine (His) labeled Shiga toxin subunit A at a concentration of
[00474] [00474] Figure 27 shows that the target polypeptides EG-FR and EDCAM, which comprise an F functional domain with F1 and F2 are VH and HL of a specific antibody to digoxigenin (ADIG), the tumor cells, using a digoxigenin marked with the horseradish peroxidase (HRP) molecule. The T1 (VH-VL) target portion of the P1 polypeptide specifically binds to EGFR, the target portion
[00475] [00475] Figure 28 shows that the directed polypeptides CD45 and HLA-Cw6 redirect T cells for the destruction of the patient's cells. Cells from primary patients with known HLA-haplotypes were used. A51 = cells from a patient with MDS (myelodysplastic syndrome), homozygous for the HLA-Cw6 haplotype. A49 = cells of a patient after transplantation of anogenous bone marrow, heterozygous for the HLA-Cw6 haplotype. Patients' cells were incubated with healthy PBMCs for 30 hours, in the presence of specific polypeptides for CD45 (CD3 (VL) -CD45 (VH-VL) and HLA-Cw6 (CD3 (VH) -HLA-CWS6 (VH- VL)) as indicated. T cell involvement was assessed by producing reactive IFN-y. Samples were assayed and analyzed as duplicates.
[00476] [00476] Figure 29 shows that the directed polypeptides EG-FR and EpCAM redirect T cells for primary cancer and the destruction of the patient's cells. A44 tumor cells were removed from malignant ascites in a 48-year-old male patient with metastatic pancreatic cancer. Tumor cells from patients were incubated with PBMC from the patients themselves (collected by phlebotomy) for 30 hours, in the presence of specific polypeptides for EDCAM (CD3 (VL) -EpCAM (VH-VL) and EGFR (CD3 ( VH) -EGFR (VH VL)), as indicated.The involvement of T cells was assessed by the production of reactive IFN-y.The samples were assayed and analyzed as duplicates.
[00477] [00477] Figure 30 shows that the target polypeptides CD45 and HLA-A 2 redirect restricted CMV of CD8 + T cells in destruction of tumor cells. Human tumor cells THP-1 and U266 were incubated with restricted CMV from T cells from a healthy HLA-A2 negative donor for 30 hours in the presence of HLA-A2 (CD3 (VL) -HLA-A2 (VH VL) specific polypeptides) ) and CD45 (CD3 (VH) -CD45 (VH-VL)), as indicated. Tandem bispecific antibody scFv (CD3 (VH-VL) x HLA-A2 (VH-VL)) was used as a positive control The involvement of T cells was assessed by the production of reactive IFN-y The samples were streaked and analyzed as duplicates.
[00478] [00478] Figure 31 shows the main idea to eliminate autoimmune disorder or hypersensitivity causing B cell clones, with a polypeptide part kit, consisting of an allergen-specific polypeptide and a type-specific polypeptide. cell. The first polypeptide P1 has an allergen in its target group (for example BeTV-1A, Der-f2, conglutin-7, Can-f1, Feld-d1). The second P2 polypeptide has in its target group a specific variable single chain antibody fragment (scFv, VH-VL) targeting a cell surface protein (for example, CD19, CD138, CD38). Both targeting parts are fused to either the light chain variable domain (VL) or the variable heavy chain domain (VH) of a CD3 specific antibody (F1 and F2 fragments).
[00479] [00479] In the following, reference is made to certain genes (humans) or proteins also referred to in the specification, the examples and figures attached, as well as (partially) in the claims
[00480] [00480] CD45: Gene ID: 5788, updated on 13-Jan-2013,
[00481] [00481] CD34: Protein: P28906-1 / 2 Last modified on July 15, 1998. Version 2.
[00482] [00482] CD33: Gene ID: 945, updated on 30-Dec-2012: Protein: P20138 [UniParc]. Last modified on October 17, 2006. Version 2. Checksum: 1C73E588240FBAD8
[00483] [00483] CD138: Gene ID: 6382, updated on 6-Jan-2013,
[00484] [00484] CD15: Gene ID: 2526, updated on 5-Jan-2013
[00485] [00485] CDia: Gene ID: 909, updated on 30-Dec-2012, PO6126 [UniParc]. Last modified on February 9, 2010. Version
[00486] [00486] CD2: Gene ID: 914, updated on 5-Jan-2013; PO6729 [UniParc]. Last modified October 23, 2007. Version
[00487] [00487] CD3e: Gene ID: 916, updated on 5-Jan-2013, Po7766 [UniParc]. Last modified on February 1, 1996. Version
[00488] [00488] CD4: Gene ID: 920, updated on 13-Jan-2013; PO01730 [UniParc]. Last modified on November 1, 1988. Version 1. Checksum: 20ED893F9E56D236
[00489] [00489] CDB5: Gene ID: 921, updated on 30-Dec-2012; PO6127 [UniParc]. Last modified on November 30, 2010. Ver-
[00490] [00490] CDê8a: Gene ID: 925, updated on 30-Dec-2012; Isoform 1/2 (membrane) P01732-1 / 2 (mCD8alpha) [UniParc]. Last modified on July 21, 1986. Version 1. Checksum: FC- CA29BAA73726BB
[00491] [00491] CD20: Gene ID: 931, updated on 6-Jan-2013; P11836 [UniParc]. Last modified October 1, 1989. Version
[00492] [00492] CD23: Gene ID: 2208, updated on 4-Jan-2013; PO6734 [UniParc]. Last modified on January 1, 1988. Version
[00493] [00493] CD31: Gene ID: 5175, updated on 13-Jan-2013; Long isoform [UniParc].
[00494] [00494] Last modified on April 1, 1990. Version 1. Check-sum: C57BBFAZ00A407A6, P16284-1 / 2/3/4/5/6 = Isoforms 1-6
[00495] [00495] CD43: Gene ID: 6693, updated on 30-Dec-2012; P16150 [UniParc]. Last modified on April 1, 1990. Version 1. Checksum: C9C9AB8435D5E1FE
[00496] [00496] CD56: Gene ID: 4684, updated on 30-Dec-2012; Isoform 1 [UniParc]. Last modified on July 22, 2008. Version 3. Checksum: FD3B9DE80D802554, P13591-2 / 1/3/4/4/6, Isoforms 1-6
[00497] [00497] CD57: Gene ID: 27087, updated on 5-Jan-2013
[00498] [00498] CD68: Gene ID: 968, updated on 6-Jan-2013; Long isoform (CD68.1) [UniParc].
[00499] [00499] Last modified on May 15, 2007. Version 2. Check-sum: 69E68D69EDESEFBO, P34810 / 2, Isoform 1/2
[00500] [00500] CD79a: Gene | D: 973, updated on 5-Jan-2013; Isoform 1 (Long) [UniParc].
[00501] [00501] Last modified on June 1, 1994. Version 2 ,, Checksum: 6E5B837409969292, P11912-1 / 2, Isoform 1/2
[00502] [00502] CD146: Gene ID: 4162, updated on 30-Dec-2012; Isoform 1 [UniParc]. Last modified on January 10, 2006. Version 2. Checksum: ESGSCB8AC7BAO0738E, P43121-1 / 2, Isoform 1/2.
[00503] [00503] Gene ID: 6440, updated on 30-Dec-2012 and Gene | D: 6439, updated on 30-Dec-2012, P07988 [UniParc]. Last modified on May 1, 1992. Version 3. Checksum: 9FD7F66678A35153, and Isoforma 1 [UniParc]. Last modified on April 1, 1990. Version
[00504] [00504] Gene l | D: 6855, updated on 30-Dec-2012, PO8247 [Uni- Parc]. Last modified on August 1, 1991. Version 3. Checksum: 592289C43B12EFA7 Nicotinic acetylcholine receptors:
[00505] [00505] Gene ID: 1138, updated on 30-Dec-2012, Gene ID: 1136, updated on 6-Jan-2013, Gene ID: 1139, updated on 13-Jan-2013, Gene ID: 1137, updated on 30 -Dec-2012, Gene ID: 1141, updated on 5-Jan-2013 Musicus specific musk kinase:
[00506] [00506] Gene lD: 4593, updated on 8-Jan-2013, Isoform 1 [Uni- Parc]. Last modified on January 1, 1998. Version 1. Check-sum: 3DDC20E179FA010C, 015146-1 / 2, Isoform 1/2 Voltage-dependent calcium channel (type P / Q):
[00507] [00507] Gene lD: 773, updated on 5-Jan-2013; Isoform 1 (1A-1) (BI-1-GGCAG) [UniParc]. Last modified on July 15, 1999. Version 2. Checksum: 2F2F378ACEO02FD56, O00555-1 / 2/3/4/5/6/7, Isoforms 1-7, Gene ID: 25398, updated on 11-Jan-2013, J3KP41 [UniParc]. Last modified on October 3, 2012. Version 1. Checksum: AEDF4D2A5E49263F
[00508] [00508] Gene lD: 3737, updated on 30-Dec-2012, Gene ID: 3736, updated on 8-Jan-2013, Gene ID: 3742, updated on 8-Jan-2013 N-methyl-D-aspartate receptor (NMDA):
[00509] [00509] “Gene ID: 2904, updated on 5-Jan-2013, Q13224 [Uni- Parc]. Last modified on June 20, 2001. Version 3. Check-sum: 40AEB12BE6ESOCEF; Gene ID: 2902, updated on 30-Dec-2012, Isoform 3 (Long) (NR1-3) [UniParc]. Last modified on June 1, 1994. Version 1. Checksum: CDF5402769E530AB, Q05586- 1/2/3/4/5, Isoforms 1-5
[00510] [00510] TSHR: Gene ID: 7253, updated on 4-Jan-2013, Isoform Long [UniParc]. Last modified on March 29, 2005. Version 2. Checksum: D2EESCEBFD64A65F, P16473-1 / 2/3, Isoforms 1-3
[00511] [00511] Amrphiphysin:
[00512] [00512] Gene | D: 273, updated on 8-Jan-2013, Isoform 1 (128 kDa) [UniParc].
[00513] [00513] Last modified on February 1, 1996. Version 1., Checksum: 78B4F75AB75BA357, P49418-1 / 2, Isoform 1-2
[00514] [00514] GQIB ganglioside: Gene ID: 29906, updated on 30-Dec-2012
[00515] [00515] GD3: Gene ID: 117189, updated on 22-Jun-2012
[00516] [00516] Ca-125: Gene ID: 94025, updated on 30-Dec-2012, Q8WXI7 [UniParc]. Last modified on March 1, 2003. Version
[00517] [00517] Her-2 / neu: Gene ID: 2064, updated on 13-Jan-2013,
[00518] [00518] gross cystic disease fluid Protein 15; Gene ID: 5304, updated on 30-Dec-2012
[00519] [00519] CD11I7: GenelD: 3815, updated on 6-Jan-2013
[00520] [00520] CD30: Gene ID: 943, updated on 6-Jan-2013; Long isoform [UniParc]. Last modified on December 1, 1992. Version 1. Checksum: 7A407CC78A6GEOBC8, P28908-1 / 2, Isoform 1/2 Platelet-derived alpha PDGFR receptor growth factor:
[00521] [00521] Gene lD: 5159, updated on 13-Jan-2013, Gene ID: 5156, updated on 13-Jan-2013, Isoform 1 [UniParc]. Last modified on April 1, 1990. Version 1. Checksum: 5ESFB9940ACD1BE8, P16234-1 / 2/3, Isoforms 1-3; PO9619 [UniParc]. Last modified on July 1, 1989. Version 1. Checksum: 038C15E531D6E89D Marker associated with Melanoma VMart 1:
[00522] [00522] Gene ID: 2315, updated on 30-Dec-2012; Q16655 [Uni- Parc]. Last modified on November 1, 1996. Version 1. Check-sum: B755BFF39CFCB16E
[00523] [00523] CD133: Gene ID: 8842, updated on 13-Jan-2013; Isoform 1 (AC133-1) (S2) [UniParc].
[00524] [00524] Last modified on June 1, 1998. Version 1. Check-sum: D21CBCOSADB2DEDF, O43490-1 / 2/3/4/5/6/7, Isoforms 1-7
[00525] [00525] In the following, reference is made to the examples which are given to illustrate, not to limit the present invention. Examples Example 1 Cloning of recombinant antibody constructs
[00526] [00526] DNA sequences derived from hybridoma cells and encoding the variable domains of anti-CD3, anti-CD45 and anti-HLA A2 antibodies, respectively, were used to generate the antibody constructs described in Figure 3 by conventional methods of molecular biology (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001)). The constructs were designed to carry different affinity tags to facilitate identification and purification on the expression of recombinant proteins (Myc-, flag, His-Tag). For more details on domain arrangement, affinity markers and ligands of constructs, see Figure 3.
[00527] [00527] pelB Leader encodes a sequence of amino acids that directs a protein expressed in bacteria to the bacterial periplasm. The command sequence is cleaved by means of bacterial enzymes and the protein can be isolated. Example 2 Expression and purification of recombinant antibodies
[00528] [00528] Periplasmic Protein Expression:
[00529] [00529] The recombinant antibody constructs were expressed in the E. coli periplasm, strain TG1 using a suitable prokaryotic expression vector. Two liters of 2 x TY medium, including 0.1% glucose and 100 µg / ml ampicillin, was inoculated with 20 ml overnight culture of transformed TG1 and grown to the exponential stage (DO600 0.8- 0.9) to 37 (TC. Since the antibody fragments are under the control of the lactose promoter, protein expression was induced by adding 1 mM IPTG, followed by incubation at room temperature (room temperature) with agitation for an additional 3 hours The cells were harvested by centrifugation for 10 min at 2,750 xg and € 4 and resuspended in 100 ml or an appropriate buffer solution. performed by adding 50 μg / ml of freshly dissolved lysozyme [Roche Diagnostics] and incubated for 25 min on ice, then 10 mM MASO4 was added to stabilize the spheroblasts, and the cells were centrifuged for 10 min at 6,200 x g and 4% .Finally, the obtained supernatant, which contains the periplasma protein optically, was dialyzed against PBS overnight at 4 ° C and spun again for 15 min, as indicated above. Then, the recombinant proteins were purified by Ni-NTA-IMAC (immobilized metal affinity chromatography of nickel nitrile-triacetic acid).
[00530] [00530] For the purification of recombinant proteins with a His6 marker, an IMAC was performed by means of immobilized nickel nitrilatriacetic acid (NTA) from agarose beads [Qiagen]. First, a 1 ml NiI-NTA agarose column needed to be equilibrated with about 10 ml of sterile PBS, or a sodium phosphate buffered solution with 20 mM imidazole. Then, the crude protein, either precipitated from cytoplasmic expression, or dialyzed from periplasmic expression, was gradually applied to the column. After washing with about 20 ml of an appropriate IMAC wash buffer (sodium phosphate in buffered solution containing 20 - 35 mM imidazole) until no more protein is detected in the flow, the bound protein it was eluted from the column in 500 µl fractions with a sodium phosphate buffered solution including 250 mM imidazole.
[00531] [00531] All wash and elution fractions collected were tested for protein by a qualitative Bradford assay by adding 10 µl of each 90 µl sample of 1 x Bradford solution. The verification of the purification process was carried out by means of an SDS-PAGE analysis. To this end, the eluted fractions were run in parallel with crude protein, flow, and the washing fraction under reduced conditions. Finally, the positive fractions determined by the colorimetric reaction were pooled in peak and minor fractions and dialyzed against PBS overnight at 4 º C. For use in stimulation assays, the purified proteins that needed to be sterile filtered, and its concentration were determined
[00532] [00532] As an alternative to Example 2, the DNA encoding (VH) CD3-EGFR (VH-VL), (VH) CD3-CEA (VH-VL), (VH) CD3-Her2 (VH-VL), (VH) CD3 -HLA-A2 (VH-VL), (VH) CD3-HLA-CW6 (VH-VL) (VH) CD3-CD138 (VH-VL), (VH) antiDig-EGFR (VH-VL) , (VH) antiHis-HLA-A2 (VH-VL), (VL) CD3-CEA (VH-VL), (VL) CD3-EpCAM (VH-VL), (VL) antiDig-EpCAM (VH-VL) , (VL) antiHis-CD45 (VH-VL), (VL) CD3-CD45 (VH-VL) were synthesized and proteins were produced and isolated by GenScript (Piscataway, NJ, USA). The DNA was codon optimized for the expression of E. coli (vector E3), the optimized expression, grown in 2 liters standard LB medium, the protein was obtained from inclusion bodies or periplasm (pelB leader) in a Ni-HiTrap column step. Bacterial endotoxins were removed by dialysis against 5 liters of 1x phosphate buffered saline (PBS). Concentration was measured by Bradford protein assay, using bovine serum albumin (BSA) as standard. Purity was estimated by densitometric analysis of an SDS-PAGE gel stained with Coomassie blue. The aliquots were stored at -80 * C or € 4. The storage buffer was used 1xPBS, 5% glycerol, 0.5% sodium sarcosine lauroyl, pH 7.4. Example 3 Cell culture techniques
[00533] [00533] Cell Culture:
[00534] [00534] Mammalian cells were grown in T75 tissue culture flasks, in 20 ml of the appropriate culture medium, at 37 ° C with 5% CO 2. The cells were divided every 2 - 3 days. Adherent cells first needed to be isolated with 1 x trypsin-EDTA. The cells were counted using a vital dye, eosin or trypan blue. For storage, 60 to 80% confluence cells were harvested by centrifugation for 5 min at 450 xg, resuspended in FCS with 10% DMSO, aliquoted in cryovials and gradually frozen at -80 (C. The cells were quickly thawed at 37ºC in a water bath and carefully added to 5 ml of medium. In order to remove the DM-SO, the cells were again centrifuged, resuspended in fresh medium and transferred to a flask of tissue culture.
[00535] [00535] —PBMC, which comprises lymphocytes and monocytes, has previously been isolated from the lining of a healthy human donor by means of density centrifugation using the LSM lymphocyte separation solution based on Ficoll 1077 (PAA Laboratories, Pasching, Austria). Since, during use, however, these PBMCs appeared as a population of non-homogeneous cells, the separation from erythrocytes, remaining granulocytes, and thrombocytes was repeated as follows. Thawed PBMCs, resuspended in 30 ml of RPMI 1640 medium containing 10% FCS and Pen-Strep, were carefully layered over 10 ml of LSM 1077 and centrifuged for 5 min at 800 x g without locking. After discarding the upper phase, PBMC concentrated in the interphase was transferred to a fresh tube, resuspended in 30 ml of medium and centrifuged for 5 min at 450 x gq. Monocytes were removed by culturing PBMC from a 10 cm tissue culture plate overnight, allowing monocytes to adhere to the plate. Finally, PBMC, which remains in the solution, was harvested.
[00536] [00536] As an alternative to Example 3, primary human carcinoma cells from a patient with metastatic pancreatic cancer were extracted from the patient's ascites sacs (Figure 29).
[00537] [00537] As an alternative to Example 3, the generation of human cytomegalievirus (CMV) specific T cells: Briefly, dendritic cells (DC) were generated from plastic adherent monocytes from HLA-A0201 PBMC negative, donor BO702 +. After 72 hours of cultivation in GM-CSF / DC medium containing IL-4 (Cellgeix), DC were matured in medium containing IL4 (100ng / ml), GM-CSF (800IU / ml), LPS (10ng / ml) and IFN-y (100U / ml) with 2.5ug / ml CMV pp65 derived from TPRVTGGG peptide. After 16h, DC were irradiated (30Gy) and co-incubated with-CD45RO, T cells CD57 + CD8-naive in the proportion 1: 4 in medium containing 5% of serum AB and | L21 (10ng / ml). Fresh medium, IL7 and IL15 was added on days 3, 5 and 7 of culture, before
[00538] [00538] Flow cytometry:
[00539] [00539] The binding of antibody fusion proteins with tumor cells that present the antigen and / or T lymphocytes was tested by means of flow cytometry. For this purpose, 2.5-5 x 105 cells were incubated with 10 µg / ml scFv or 0.004-4 µg / ml of fusion proteins titrated in 100 µl of a suitable buffer solution (such as PBS albumin + bovine serum, or other acceptable buffer solution) per well of a 96-well V-shaped plate at 4 ° C for two hours. After washing three times with 150 µl of a suitable buffer solution, cells were incubated with FITC-conjugated anti-His6 marker or anti-Flag marker or anti-myc marker antibody at RT for 30 min and washed again twice . For propagation and background staining tests, two samples of each cell type were prepared in addition, one of the cells not stained and stained with an anti-FITC-conjugated His6 antibody, without any protein. Finally, the cells were resuspended in 500 µl of a suitable buffer solution, transferred to FACS tubes and analyzed using flow cytometry.
[00540] [00540] The stimulating properties of recombinant proteins were tested in a cell-based stimulation assay. In this way, T cell activation mediated by bispecific antibodies and "tridominium constructs" was determined by measuring PBMC stimulation in terms of induced IL-2 release.
[00541] [00541] CDA45 pos / HLA A2 from the U266 myeloma cell line were seeded in a 96-well flat-bottomed cell culture plate at a density of 105 cells per well in 100 µl of culture medium. The titrated stimulator proteins were added as indicated in 100 µl of medium per well and were preincubated for one hour at 37 ° C and 5% CO 2 to ensure sufficient binding. Unstimulated PBMC, thawed and isolated the day before, was then added to an indicated density and incubated for 24 hours at 37 ° C (5% CO 2 CO.). The plates were then centrifuged for 5 min. at 450 xg to collect cell-free supernatants for IL-2 quantification by ELISA.
[00542] [00542] “As an indicator for stimulatory activity, T cell activation induced by bispecific antibodies was measured in terms of IL-2 release. After PBMC stimulation, the concentration of secreted IL-2 in the supernatant was determined by sandwich IL-2 ELI-SA.
[00543] [00543] First, a 96 well ELISA plate was coated with 400 ng / 100 µl per mouse antibody well.
[00544] [00544] The seven-point standard curve was created by graphically representing the absorbance signals of each standard sample against the IL-2 concentration. In this way, the amount of IL-2 in each supernatant can be determined by interpolating the standard curve equipped with the nonlinear regression equation for an exponential association phase using GraphPad Prismº.
[00545] [00545] In cell culture of 100 µl of supernatant, the concentration
[00546] [00546] The HLA-A2 / CD45 of the U266 positive cell line or U266 myeloma cell line was labeled with 10 µM CF-SE (CFDA SE Vibrant Invitrogen Cell Tracer Kit) in 350 µl PBS for 10 min at room temperature (RT) in the dark. The labeling reaction was stopped by adding 5 ml of fetal calf serum (FCS), followed by a one hour incubation at RT. After two washes, CFSE-labeled target cells were resuspended in assay medium and co-incubated with peripheral blood mononuclear cells (PBMC) from a healthy HLA-A2 negative donor in a ratio of 1: 10 (5 * 105 and 5 * U266 106 PBMC in 2 ml) and 27 nM of antibody constructs, as indicated. A sample treated with Triton was used as a positive control (100% lysis) and a sample without construct antibody as a negative control (0% lysis). After 24h, apoptotic cells were visualized by spot 7AAD (Biozol, 10 min at room temperature) and the% specific lysis of labeled U266 CFSE cells was calculated using flow cytometry techniques. Caspase-3 assay (alternative to Example 4):
[00547] [00547] Staining was performed after co-incubating the target cells with T-cells (tumor cells: T cells ratio 2: 1), with or without the specific polypeptides for 4h. Surface staining for HLA-A2 and CD45 was performed first, followed by fixation and permeabilization (Fix + Perm, BD Biosciences). The activated caspase-3 antibody was then added over 30 min. (BD Biosciences). The cells were washed with 1xPBS 5% human serum (HS, PAA Laboratories) and analyzed on a BD-FACS Canto-ll. % of specific Apoptosis was calculated as (% experimental value -% spontaneous release) / (100% -% spontaneous release) * 100. Azul Alamar Assay (alternative from Example 4):
[00548] [00548] The Blue alamarº assay (Abd Serotec) was used to measure cell proliferation and viability, after exposure to toxins. Briefly, the cells were grown in 100 µl cell culture medium per well (96 well plate). For analysis, 10 µl of Blue alamar was added per well and incubated in the incubator for 30 to 120 minutes. The absorbance was read with a BioRad plate reader at 570 nm and 600 nMA, only blank media were used. The percentage difference in the reduction of cell proliferation between different groups of polypeptides was calculated as indicated by the manufacturer, using cells that grow in culture without toxin as a control. Digoxigenin assay (alternative to Example 4):
[00549] [00549] First, peroxidase from horseradish (HRP, Sigma-Aldrich Chemie GmbH) was labeled with digoxigenin NHS-ester (Sigma-Aldrich Chemie GmbH), in a 1/3 molar ratio. Dig-HRP was cleaned with Bio-Spin'Y micro chromatography columns (Bi- oRad) and stored at 4º in the dark. Colo -206F cells were first incubated with indicated polypeptides in various concentrations for 90 minutes. The cells were washed with PBS and resuspended in cell culture medium with Dig-HRP and incubated for 30 minutes. Then the cells were washed twice with PBS and resuspended in 50 µl of PBS. 50 ul of stabilized chromogen (Invitrogen 7 “) was added over 15-30 minutes at room temperature in the dark. 50 mL of stop solution was added and the absorbance was read with a BioRad plate reader at 450 nm. Mice (alternatives to Example 4):
[00550] [00550] HLA.A2transgenic, immunodeficient mice (NodScid IL-2rg - / - HLA.A2 / B2m tg; Bank number 14570, Jackson Lab, in Bar Harbor, Maine, USA) for the in vivo experiment (Figure 12A) were kept in certified animal facilities (ZEMM, Center for Experimental Molecular Medicine, University of Würzburg hospital), according to European guidelines. Female mice, from 6 to 10 weeks of age, were divided into five groups of six mice per group (n = 30). 5X106 THP-1 cells, 1.25 x105CMV specific CD8 + T-cells (tumor cells: proportion of T cells 40/1) and 0.5 µg of the polypeptides were injected intraperitoneally (ip), as indicated. After the injection, the mice were monitored by daily inspection. A second injection of 1.16x105-specific CD8 + CMV T cells / mouse was given on day 13 and injections of polypeptides were repeated every three days a week. The animals were sacrificed when the increase in body weight was greater than 80% or if they seemed to be dying according to institutional guidelines.
[00551] [00551] The domain structure, affinity markers and ligands of the constructs or polypeptides used in Examples 5-9 or Figures 4-11 are shown in Figure 3. These constructs and all constructs or polypeptides used in Figures 4-30 were prepared as described in Examples 1 and 2. Cell culture and the functional assays of Examples 5-9 and culture, functional assays and in vivo work for Figures 4-30 were performed as described in Examples 3 and 4. Example 5
[00552] [00552] The myeloma line of the positive target cell CD45 and HLA A2 U266 was co-incubated with negative HLA A2 T cells (monocytes spread PBMC (peripheral blood mononuclear cells) from a healthy donor and varying amounts of HLA A2 and Bi-specific antibodies CD3 constructs, as indicated (Numbers. 85, 82, 75 and 71) of PHA-L (phytohemagglutinin, a lectin that causes non-specific stimulation of T cells, 1 µg / ml of concentration final), it was used as a positive control and single-chain scFv constructs with specificity for HLA A2 (Number 4) or CD3 (number 36) were investigated.The production of IL2 (interleukin-2) by means of T cells was measured using ELISA techniques No IL2 production was found in experimental situations without any constructs The data obtained are shown in Figure 4. Example 6
[00553] [00553] The myeloma lineage of the positive target cell CD45 and HLA A2 U266 was co-incubated with negative HLA A2 T cells (monocytes depleted PBMC) from a healthy donor and varying amounts of tridominium "constructs" added separately (Numbers 42, 45, 55; numbers referring to the constructs, as shown in Figure 3) or in combinations of (42 + 45 or 42 55). PHA-L and single-chain scFv constructs with specificity for CD45 (Numbers 46 and 17) were given as controls. IL2 production using T cells was measured using ELISA techniques. No IL production was found in experimental situations, without any constructs. The data obtained are represented in Figure 5. Example 7
[00554] [00554] Alignment of myeloma of CD45 positive and HLA A2 target cells U266 was co-incubated with HLA A2 negative T cells (monocyte-streaked PBMC) from a healthy donor and the HLA A2 bispecific antibody and CD3 construct c alone (number 71, 27 nM) or in combination with single-chain scFv constructs that block HLA A2 antigenic epitopes (Number 4, one hundred times higher compared to construct 71, that is, 2700 nM) or CD3 (Number 36, nine times the excess compared to construct concentration 71, that is, 2438 nM). IL2 production by means of T cells was measured using ELISA techniques and PHA-L is given as a control. The data obtained are shown in Figure 6. Example 8
[00555] [00555] Alignment of myeloma of CD45 positive and HLA A2 target cells U266 was co-incubated with negative HLA A2 T cells (monocyte-streaked PBMC) from a healthy donor and the combination of constructs 42 and 45. The stimulating function T cells were blocked using single chain constructs specific for HLA A2 (number 4) or CD45 (number 46). The complementation of the T cell stimulating function was tested by testing the constructs
[00556] [00556] Alignment of myeloma of CD45 positive and HLA A2 U266 target cells was co-incubated with negative HLA A2 T cells (monocyte-streaked PBMC) from a healthy donor and the combination of constructs 42 and 55. The stimulating function T cells were blocked using single chain constructs specific for HLA A2 (number 4) or CD45 (number 46). Complementing the T cell stimulating function was tested by testing constructs 42 and 55 separately or single-chain scFv constructs directed against CD3 (number 36). IL2 production by means of T cells was measured using ELISA techniques and PHA-L is given as a control. The concentration of constructs was 27 nM, unless otherwise indicated. ("9 x" indicates a concentration of 243 NM, "100X" indicates a concentration of 2,700 nM.) The data obtained are shown in Figure 8.
[00557] [00557] The results of the previous examples clearly demonstrate that the two constructs of (42 45) or (42 55) must first bind to their ligands on the surface of a single cell in order to, subsequently, complete the T cell envelope function. Example 10
[00558] [00558] AlisedoCD45 and U266 positive HLA A2 myeloma target cell line by HLA A2 negative T cells (monocyte depleted PBMC), in the presence of VLCD3 scFvHLA-A2 (27 nMol) or VH-scFvCD45 (27 nMol) or a The combination of both of these constructs (27 nmol each) was determined using flow based cytometry techniques. The percentage of lysis was calculated by means of apoptotic U266 cells divided by total U266 cells and the background apoptosis was subtracted. The data obtained are shown in Figure
[00559] [00559] As part of the final bipartite construct, two polypeptides were designed, each consisting of a single antigen-binding variable fragment chain (scFv) and the variable light chain (VL) or variable heavy chain ( VH) domain of an anti-CD3 antibody activated by the T cell (Figure 10). When these two polypeptides bind their respective antigens on the surface of a single cell, the VL and VH domains interact with each other to restore the original anti-CD3 binding site. The thus specific heterodimer in the formed target engages and stimulates T cells to destroy tumor cells.
[00560] [00560] This scenario is fully validated in vitro when T lymphocytes are confronted with target cells that have been incubated with the two different polypeptides. As proof of principle, the major histocompatibility antigen HLA-A2 and the hematopoietic lineage marker CD45 were targeted as first and second antigens, both of which are expressed in U266 myeloma cells, the primary cells from a patient with pro-lymphocytic leukemia of the T cell lineage (T-PLL), and THP-1 of acute myeloid leukemia of blasts (Figure 11). Due to the VL / VH interaction described, the triespecific heterodimer now potently stimulates T cells to secrete interleukin-2 (IL-2) (Figure 11a) and to lysis the marked tumor cells in nanomolar concentration (Figu -
[00561] [00561] To demonstrate that the two molecules must first bind their antigens to the surface of the target cell for VH / VL hetero-dimerization to occur, single-chain variable fragments specific for HLA-A2 and CD45 were used to block the respective epitopes on the target. As shown in Figure 11c, when present in large excess, these inhibitors prevent the two polypeptides from triggering T cells in a dope-dependent manner. In addition, T cells were not stimulated when the target cells were omitted (data not shown) or when the target cells were probed which express CD45 only (Raji cells, figure 11D) or no target molecule (KMS-12 -BM, figure 11D). Example 12
[00562] [00562] For the proof of concept in vivo, a model of transplantation of stem cells of allogeneic incompatibility was used in which the residual leukemic and hematopoietic cells of a patient, all HLA-A2 and CD45-positive, must be eliminated to obtain stem cells from allogeneic donors (HLA-A2-negative, CD45- positive) the opportunity to graft and reconstitute hematopoiesis (see Figure 2). To place the specificity of the bipartite construct for the test, immunodeficient mice expressing the human HLA-A2 transgene in practically all nucleated cells were used, the question being whether HLA-A2-positive murine tissues, but CDA45 negative would suffer collateral damage. THP-1 cells were injected intraperitoneally with or without CD8 T lymphocytes from an HLA-A2 negative donor, which had been selected for specificity for cytomegalovirus (CMV) to prevent human anti-murine immune reactivity. Intraperitoneal tumors developed rapidly in mice that did not receive the polypeptides, and in mice treated with either types of individual molecules or a combination of both polypeptides, but without T cells. In all cases, the fatal disseminated disease developed within 3 to 4 weeks (Figure 12A). In contrast, all tumor-bearing mice treated with T cells and repeated injections of both polypeptides survived the end of the experiment on day 31, albeit with palpable tumors at the injection site. These results clearly show that the bipartite construct truly redefines T cells regardless of their specificity for tumor cells that simultaneously express both target molecules (HLA-A2) and CD45 in vivo. As an aside, a T cell recreating bispecific antibodies against HLA-A2 would cause damage, redirecting T cells against all HLA-A2positive murine tissues. Likewise, a bi-specific CD45-binding antibody would have mediated lysis of all hematopoietic cells, including the donor's leukemia and T-cell blast THP-1 cells. In the present flat set, however, injection of the specific HLA-A2 polypeptide into transgenic HLA-A2 animals did not cause apparent toxicity.
[00563] [00563] In order to examine the best possible toxicity for the
[00564] [00564] To venture into solid tumors, the present inventors targeted the combinatorial approach to epithelial cell adhesion molecule (EDPCAM) and epidermal growth factor receptor (EGFR) antigens. Both antigens are overexpressed in several carcinomas and have been extensively studied in the clinical phase of trials | le Ill. EGFR expression is closely associated with cell proliferation, while EpCAM is present on the basolateral surface of the epithelium virtually all simple and was recently found to act as a signaling protein in the Wnt pathway, Maetzel, 2009, Nat cell Biol 11, 162 to 171. As Figure 13a illustrates, the two polypeptides trigger the release of
[00565] [00565] - The more the validation of the bipartite functional complementation strategy was carried out in full in in vitro experiments, using a combination of different polypeptides, there was the target of several cell surface antigens from different human cell lines.
[00566] [00566] FM-55 positive human tumor HLA A2 cell lines (myeloma), Colo-206F (colon cancer) and OVCAR (ovarian cancer) were co-incubated with HLA-A2 PBMC negative from a healthy donor , polypeptide against HLA-A2 (CD3 (VL) - HLA-A2 (VH-VL)) and with a second target polypeptide or CEA (CD3 (VH) - CEA (VH-VL)), EGFR (CD3 (VH) ) - EGFR (VH-VL)) or Her2 (CD3 (VH) - Her2 (VH-VL)). IL2 or IFN-y production of lymphocytes was measured using ELISA techniques. These data demonstrate that (i) a specific combination of antigens, an antigen signature,
[00567] [00567] To demonstrate the interchangeability of the functional domain, the F1 and F2 fragments of a set of polypeptides were exchanged with each other, maintaining their specific complementation capacity for the restoration of their target domain from the original antibody to involve T cells. Therefore, the set of polypeptides against the target antigen, CD45 and HLA-A2 was used. The polypeptide against CD45 had CD3 (VL) as the F1 fragment and the polypeptide against HLA-A2 had CD3 (VH) as the F2 fragment. CD45 and HLA-A2 The U266 positive myeloma cell line was co-incubated with HLA-A2 negative T cells from a healthy donor and polypeptides against CD45 (CD3 (VL) - CD45 (VH-VL)) and HLA- A2 (CD3 (VH) - HLA-A2 (VH-VL)) in varying amounts. T cell involvement was assessed by the production of reactive IFN-y, measured using ELISA techniques. No IFN-y production was found in experimental situations, without any polypeptides. The data obtained are represented in Figure 17. Example 16
[00568] [00568] The bipartite functional complementation strategy was still tested, targeting a set of antigens, already used as targets for cancer antibody therapy (EGFR, EpCAM and HER2) (Her2 is a target for Trastuzumab in cancer of breast, EGFR is a target for cetuximab in colorectal cancer and EDCAM is a target for Catumazuma for the treatment of neoplastic ascites). Positive cells for
[00569] [00569] To test an antigen combination with accurate clinical correlation, the CD45 and CD138 combination was used to target human multiple myeloma (MM) cells. Most of these human cells are positive for CD45 and CD138. Bispecific T cell recruitment antibodies against CD45 would kill all a patient's hematopoietic cells and against CD138 would cause serious side effects due to their expression in various normal tissues (epithelial cells, endothelium, trophoblastic cells and glandular cells of the Gl tract , the Atlas human protein, Version:
[00570] [00570] Another application of the bipartite functional complementation strategy is the individual target antigens on the cell surface and killing the positive tumor cells of individual antigens. A major disadvantage for bispecific antibodies of T cell recruitment with functional anti-CD3 binding sides are severe side effects caused by the activation of nonspecific T cells and the release of cytokines (Linke, R. et al Catumaxomab :. Clinical development and future directions MAbs 2, 129-136 (2010)) The advantage of this bipartite functional complementation strategy is the fact that antibodies in which the activation of anti-CD3 T cells functional domain is restored exclusively in the target cell, no T cell domain activation is present. CD45 and CD138 AMO-1 and U266 positive human multiple myeloma cells were co-incubated with PBMC from a healthy donor and the combination of polypeptides against a single target antigen, or CD138 (CD3 (VH) - CD138 (VH + VL) + CD3 (VL) - CD138 (VH + VL)) or CD45 (CD3 (VH) - CD45 (VH + VL) + CD3 (VL) - CD45 (VH + VL)). Complementation of the lymphocyte stimulating function was assessed by the production of reactive IFN-y, measured using ELISA techniques. No IFN-y production was found in experimental situations with simple polypeptides or without any polypeptides. The data obtained are presented in Figure 21 and 22. In Figure 23, the approach of the single antigen is illustrated, by means of a set of polypeptides targeting two different epitopes (top) or the same epitope (bottom ) on the target A1 antigen. Example 19
[00571] [00571] This is an example to demonstrate that the functional complementation strategy can still be developed for the targeted delivery of cargo and that the different effector ways are possible to kill a target cell. By complementing the F1 and F2 fragments of a set of polypeptides linked to the target, the newly formed antibody binding site can bind any molecule that is specific. In order to direct a His-tagged charge precisely to a target cell, fragments of VH and VL from an anti-HIS antibody (hexahistidine) were used. After the simultaneous binding of a polypeptide (antiHis (VL) -CD45 (VH-VL) and polypeptide 2 (antiHis (VH) -HLA-A2 (VH-VL) to its target, specific CD45 and HLA-A2 antigens , a hexa-histidine at the binding site is completed on the target that binds histidine to high-affinity labeled charges, the charge being a His-labeled toxin, as given in the example in the present invention. and THP-1 HLA-A2 were co-incubated with Clostridium perfringes labeled with (His) histidine component of toxin la (Figure 24) or a Shiga toxin marked with histidine (His) subunit A (figures 25, 26) in combination with polypeptides against CD45 (antiHis (VL) -CD45 (VH-VL)) and HLA-A2 (antiHis (VH) -HLA-A2 (VH-VL)) The complementation of His-tagged toxin binding and the subsequent target cell death was assessed by measuring cell viability using a Blue wing test. In the highest concentration of polypeptides used (80nm), a difference the clear in target cell death, measured as a reduction in cell viability, was found in experimental situations with a combination of both polypeptides in relation to individual polypeptides.
[00572] [00572] To further demonstrate the versatility, flexibility and interchangeability of the bipartite functional complementation strategy, the VH and VL fragments of an anti-digoxigenin antibody were used to identify and mark the double antigen of positive cells with HRP labeled with Digoxigenin (horseradish peroxidase). EGFR and EpCAM positive Colo-206F cells were co-incubated with polypeptides against EGFR (antiDig (VH) - EGFR (VH + VL)) and EDCAM (antiDig (VL) - EBPCAM (VH + VL)). In the target complementation of the functional anti-digoxigenin domain, indicated by the HRP digoxigenin labeling of Colo-206F cells, it was evaluated by measuring peroxidase activity, using a standard ELISA kit (Invitrogen'Y). A clear difference in the target cells marked with Dig - HRP was found in an experimental situation, with a combination of both polypeptides in relation to individual polypeptides. The data obtained are shown in Figure 27. Example 21
[00573] [00573] Using human leukocyte antigens (HLA) as a double antigen arm restricted to two-way functional complementation, this haplotype strategy was further validated by exchanging the functional domains of the polypeptides with the VH and VL fragments of an antibody anti-HLA-Cw6. HLA-Cw6 CMSPs from positive primary patients were co-incubated with HLA-Cw6 negative PBMC from a healthy donor, polypeptide against CD45 (CD3 (VL) - CD45 (VH-VL)) and HLA-Cw6 (CD3 (VH) - HLA-Cw6 (VH-VL)). IFN-y production through lymphocytes was measured using ELISA techniques. These data demonstrate that hematopoietic cells from patients with other haplotypes than HLA-A2 can be targeted simply by exchanging a target domain (anti HLA-A2, Figure 5, 7-9, 11-12) on the other (anti HLA-Cw6). The data obtained are shown in Figures 28. Example 22
[00574] [00574] The bipartite functional complementation strategy induced by double antigen was further validated in an in vitro patient trial, using the primary carcinoma cells of the newly isolated patient and the target antigens already used for cancer therapy in the clinic. - single or clinical trials (EGFR, EDCAM, CEA and HER2). Malignant cells from a 48-year-old male patient with metastatic pancreatic cancer were co-incubated with patients who have peripheral blood lymphocytes and the combination of polypeptides against EGFR (CD3 (VH) - EGFR (VH + VL )), EBDCAM (CD3 (VL) - EpCAM (VH + VL)), Her2 (CD3 (VH) - Her2 (VH + VL)), CEA (CD3 (VH) - CEA (VH-VL)) and HLA- A2 (CD3 (VL) - HLA-A2 (VH-VL)). The complementation of the lymphocyte stimulating function was assessed through the production of reactive IFN-y, measured using ELISA techniques. No IFN-y production was found in experimental situations, without any polypeptides. These data demonstrate the potential of this strategy in using the patient's own immune system cells to attack and kill their transformed malignant cells. The data obtained are presented in Figures 29.
[00575] [00575] A highly enriched CD3 / CD8 CMV restricted T cell population was used to show that any T cell, regardless of its specificity, can serve as an effector cell in killing the double antigen positive tumor cells by this complementation strategy. U266 positive for CD45 and HLA-A2 and THP-1 cells were co-incubated with specific T cell cytomegalievirus (CMV) from a healthy donor HLA-A2 negative and polypeptides against CD45 (CD3 (VH) - CD45 (VH-VL)) and HLA-A2 (CD3 (VL) - HLA-A2 (VH-VL)) in varying amounts. The bispecific scFv antibody set (CD3 (VH-VL) x HLA-A2 (VH-VL)) was used as a positive control. T cell involvement was assessed through the production of reactive IFN-y, measured using ELISA techniques. No IFN-y production was found in experimental situations with simple polypeptides or without any polypeptides. The data obtained are shown in Figure 30. Cells from the same frozen aliquot batch, CMV-specific T cells and THP-1 cells, were used for the in vivo murine model (Figure 12A). Example 24
[00576] [00576] This illustration represents the potential to target specific autoimmune B cell allergens / clones, with the bipartite functional complementation strategy. When using a synthetic allergen as a target portion, the bound polypeptide allergen will specifically bind to its clonotypic B cell receptor expressed on the surface of the anti-allergen specific B cell clone. The second arm of the bipartite strategy will use a specific B-cell polypeptide (CD19, CD20, CD38, CD138), restricting complementation followed by the effector domain with subsequent death of target cells for the anti-specific B cell clone. -allergic. The ultimate goal of this strategy is to eliminate the B cell clone it causes and allergic or autoimmune disease (top of Figure 31), while sparing B cells with other specificities or other than B cell cells ( for example mast cells or basophilic cells) that bind the antibody responsible for the disease via Fc-receptors (bottom of Figure 31).
[00577] [00577] The characteristics of the present invention described in the specification, the claims and / or the attached drawings can, both separately and in any combination thereof, constitute a material for carrying out the present invention in its various forms .

权利要求:
Claims (25)
[1]
1. Set of polypeptides, characterized by the fact that it comprises: a first P1 polypeptide comprising (i) a target portion T1, in which said target portion T1 specifically binds to an A1 antigen, and (ii) an F1 fragment of a functional domain F, in which neither said fragment F1 alone, nor said polypeptide P1 alone is functional with respect to the function of said domain F, and a second polypeptide P2 which comprises (i) a target portion T2, wherein said target portion T2 specifically binds to an A2 antigen, and (ii) an F2 fragment of said functional domain F, in which neither said fragment F2 per se, nor said polypeptide P2 itself is functional with respect to the function of said F domain, in which said A1 antigen is different from said A2 antigen, in which said P1 polypeptide and said P2 polypeptide are not associated with each other. another in the absence of a substrate that has both A1 and A2 antigens, in its surface, and where, by dimerizing said F1 fragment of said P1 polypeptide with said F2 fragment of said P2 polypeptide, the resulting dimer is functional with respect to the function of said F domain.
[2]
2. Set of polypeptides according to claim
1, characterized by the fact that said P1 polypeptide and said P2 polypeptide are not associated with each other in the absence of a cell that carries both A1 and A2 antigens on their cell surface.
[3]
3. Set of polypeptides according to claim 1 or 2, characterized by the fact that a cell having both antigens A1 and A2, on its cell surface induces the dimerization of the F1 fragment of said polypeptide P1 with the F2 fragment of said polypeptide P2, whereas a cell that does not carry both antigens A1 and A2, on its cell surface, does not induce the dimerization of the F1 fragment of said polypeptide P1 with Fragment F2 of said polypeptide P2.
[4]
4. Set of polypeptides according to any one of the claims | to 3, characterized by the fact that said polypeptides P1 and P2 have, in the absence of said substrate or cells, with each other a constant KD dissociation in the range of 10º M to 10 M, in the range of 107 M to 10º M or in the range of 10º M to 10º M; and / or said polypeptides P1 and P2 have, in the presence of said substrate or cells, with each other a constant dissociation KD less than 10º M, less than 107 M less than 10º M or less than 10º M.
[5]
A set of polypeptides according to any one of claims 1 to 4, characterized by the fact that said A1 antigen and / or said A2 antigen is an antigen expressed on the surface of cells of a tumor or in surface of tumor progenitor / precursor cells.
[6]
A set of polypeptides according to any one of claims 1 to 5, characterized by the fact that the combination of antigen A1 and Antigen A2 is found only in cancer cells, and not in cells that are not cancerous.
[7]
7. Set of polypeptides according to claim 6, characterized by the fact that said antigen A1 and Antigen A2 is specific for cancer cells of a certain type of cancer.
[8]
8. Set of polypeptides according to any one of claims 1 to 7, characterized by the fact that said antigen A1 is an MHC antigen being an allelic variant selected from the group consisting of: HLA-A2, HLA-Cw6, HLA-A1, HLA-A3, HLA-A25, HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3, HLA-Cw4, HLA-Cw7; and / or said A2 antigen is an antigen that is specific to a particular cell type or cell line selected from the group consisting of: CD45; CD34; CD33; CD138; CD15; CD1a; CD2; CD3; CDA4; CD5; CD8; CD20; CD23; CD31; CD43; CD56; CD57; CD68; CD79a; CD146; pulmonary surfactant; synaptophysin; CD56; CD57; nicotinic acetylcholine receptor ;, muscle specific MUSK kinase; voltage-dependent calcium channels (type P / Q); voltage-dependent potassium channel (CPVD); N-methyl-D-aspartate (NM-DA) receptor; TSH; amphiphysin; HepPar-1; ganglioside GQ1b, GD3 or GM1; and glycophorin-A.
[9]
A set of polypeptides according to any one of claims 1 to 8, characterized by the fact that any one of said antigens A1 and A2 is selected from the group consisting of: HLA-A2; HLA-Cw6; EpCAM; CD45; Her2; EGFR; CD138; CEA; CD19; E-cadherin; Ca-125; Her-2 / neu; severe cystic disease fluid protein; BCA-225; CA 19-9; CD117; CD30; Epithelial BER-EPA4 antigen, epithelial membrane antigen and related epithelial MOC-31 antigen; epidermal growth factor receptor HER1;
platelet-derived growth factor alpha PDGFR receptor; marker associated with Melanoma / Mart 1 / Melan-A; CD133; TAG 72; and a clonotypic antibody on the surface of a B cell.
[10]
10. Set of polypeptides according to any one of claims 1 to 9, characterized by the fact that (i) one of said antigens A1 and A2 is EpCAM and the other is EGFR, HER2 / neu, CD10, VEGF-R or MDR ; (ii) one of said A1 and A2 antigens is MCSP and the other is melanoferrin or EDCAM; (ili) one of said antigens A1 and A2 is CA125 and the other is CD227 (iv) one of said antigens A1 and A2 is CD56 and the other is CD140b or ganglioside GD3; (v) one of said A1 and A2 antigens is EGFR and the other is HER 2; (vi) one of said A1 and A antigens is PSMA and the other is HER2; (vii) one of said A1 and A2 antigens is Sialil Lewis and the other is EGFR; (viii) one of said A1 and A2 antigens is CD44 and the other is ESA, CD24, CD133, CD117 or MDR; (ix) one of said antigens A1 and A2 is CD34 and the other is CD19, CD79a, CD2, CD7, HLA-DR, CD13, CD117, CD33 or CD15; (x) one of said A1 and A2 antigens is CD33 and the other is CD19, CD79a, CD2, CD7, HLA-DR, CD13, CD117 or CD15; (xi) one of said antigens A1 and A2 is MUC1 and the other is CD10, or CD57 CEA; (xii) one of said A1 and A2 antigens is CD38 and the other is CD138; (xiii) one of said antigens A1 and A2 is CD 24 and the other is CD29 or CD49f; (xiv) one of said antigens A1 and A2 is carbonic anhydrase IX and the other is aquaporin-2; (xv) one of said antigens Al and A is HA-A2 and the other is EDCAM; (xvi) one of said A1 and Aé antigens to HA-A2 and the other is CD45; (xvii) one of said A1 and AP antigens is HA-A2 and the other is EGFR; (xviii) one of said A1 and Aé antigens is HLA-A2 and the other is Her2; (xix) one of said antigens A1 and A is HA-A2 and the other is CEA; (xx) one of said A1 and A2 antigens is EpCAM and the other is CEA; (xxi) one of said antigens A1 and A2 is CD45 and the other is CD138; (xxii) one of said antigens A1 and A2 is EGFR and the other is CEA; (xxiii) one of said antigens A1 and A2 is Her2 and the other is CEA; or (xxiv) one of said A1 and A2 antigens is CD1I9 and the other is a clonotypic antibody on the surface of a B cell.
[11]
A set of polypeptides according to any one of claims 1 to 10, characterized in that said target portion T1 and / or T2 comprises an immunoglobulin module; or wherein said target portion T1 and / or T2 comprises an aptamer or a natural ligand of said antigen A1 or Antigen A2, respectively.
[12]
12. Set of polypeptides according to claim
tion 11, characterized by the fact that said target portion T1 comprises an immunoglobulin module 11 which comprises a VL domain linked to a VH domain or which comprises a variable domain of a VHH antibody of a llama antibody , a camel antibody or a shark antibody; and / or said target portion T2 comprises an immunoglobulin module 12 comprising a VL domain linked to a VH or VHH domain comprising a variable domain of a llama antibody, a camel antibody or a shark antibody.
[13]
13. Set of polypeptides according to claim 12, characterized in that said immunoglobulin module 11 comprises a scFv (single chain variant fragment), a Fab fragment or an F (ab ' ), an antibody or a complete antibody; and / or said immunoglobulin module 12 comprises a scFv (single chain variant fragment), a Fab fragment or an F (ab '), an antibody or a complete antibody.
[14]
14. Set of polypeptides according to any one of claims 1 to 4 and 7 to 13, characterized in that one of said target portion T1 and T2 comprises an allergen or substrate which binds to a clonotypic antibody on the surface of a B cell.
[15]
15. Set of polypeptides according to any one of claims 1 to 14, characterized in that said F1 fragment comprises an immunoglobulin module, or a fluorescent molecule or a molecule capable of mediating bioluminescence.
[16]
16. Set of polypeptides according to claims 15, characterized by the fact that said functional domain F is an Fv (variant fragment), or an scFv (single chain variant fragment) of an antibody.
7I9
[17]
17. Polypeptide pool according to any one of claims 1 to 16, characterized in that said F1 fragment comprises a VL domain of an antibody and said fragment F2 comprises a VH domain of the same antibody; or wherein said F1 fragment comprises a VH domain of an antibody and said F2 fragment comprises a VL domain of the same antibody.
[18]
A set of polypeptides according to any one of claims 1 to 17, characterized in that said F1 fragment comprises a VL domain of an anti-CD3, anti-His and anti-DIG antibody and said F2 fragment comprises a VH domain of the same antibody, or wherein said F1 fragment comprises a VH domain of an anti-CD3, anti-His and anti-DIG antibody and said F2 fragment comprises a VL domain of the same antibody.
[19]
19. Polypeptide set according to any one of claims 15 to 18, characterized in that said immunoglobulin module comprises a V domain selected from the group consisting of: (i) a V domain of an anti- CD3, which comprises a VL domain comprising SEQ ID NO: 2 and / or a VH domain comprising SEQ ID NO: 1; (ii) a V domain of an anti-CD3 antibody, which comprises a VL domain which comprises SEQ ID NO: 4 and / or a VH domain which comprises SEQ ID NO: 3; (li) a V domain of an anti-CD3 antibody, comprising a VL domain comprising SEQ ID NO: 6 and / or a VH domain comprising SEQ ID NO: 5; (iv) a V domain of an anti-CD3 antibody, which comprises a VL domain comprising SEQ ID NO: 8 and / or a domain
VH domain comprising SEQ ID NO: 7; (v) a V domain of an anti-CD3 antibody, which comprises a VL domain which comprises SEQ ID NO: 10 and / or a VH domain which comprises SEQ ID NO: 9; and (vi) a V domain of an antibody anti-His antibody comprising a VL domain comprising SEQ ID NO: 12 and / or a VH domain comprising SEQ ID NO: 11; (vii) a V domain of an anti-DIG antibody comprising a VL domain comprising SEQ ID NO: 14 and / or a VH domain comprising SEQ ID NO: 30.
[20]
20. Set of polypeptides according to any one of claims 1 to 19, characterized in that any of the polypeptides P1 and P2 is or comprises a sequence of amino acids selected from the group consisting of SEQ ID NO: 114 -129 and 197.
[21]
21. Set of polypeptides according to any one of claims 1 to 20, characterized in that it is for use in the treatment of a patient suffering from cancer and / or a tumor or for use in diagnosis in a patient suffering from cancer and / or a tumor.
[22]
22. Nucleic acid molecule or a set of nucleic acid molecules, characterized by the fact that they encode the set of polypeptides or one of the polypeptides in the set of polypeptides as defined in any one of claims 1 to
20.
[23]
23. Nucleic acid molecule or a set of nucleic acid molecules according to claim 22, characterized in that it comprises a nucleotide sequence as described in any one of SEQ ID NO *: 135-150 and 196.
[24]
24. A pharmaceutical composition comprising the set of polypeptides as defined in any one of claims 1 to 20 or the nucleic acid molecule / set of nucleic acid molecules as defined in claim 22 or 23, characterized in that, preferably, the said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
[25]
25. Kit characterized by the fact that it comprises the set of polypeptides as defined in any one of claims 1 to 20, or the nucleic acid molecule or set of nucleic acid molecules as defined in claim 22 or 23.

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法律状态:
2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-06-15| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-07-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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EP12151125|2012-01-13|

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EP12151125.7|2012-01-13|

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PCT/EP2013/050603|WO2013104804A2|2012-01-13|2013-01-14|Dual antigen-induced bipartite functional complementation|

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