dual specificity antibody, polypeptide molecules, nucleic acid, host cell, pharmaceutical compositio

专利摘要:
The present invention relates to a bispecific polypeptide molecule, comprising a first polypeptide chain and a second polypeptide chain, which provide a binding region derived from a T cell receptor (TCR, T-cell receptor) specific for an epitope associated with the complex main histocompatibility and a binding region derived from an antibody capable of recruiting human effector immune cells by binding to a surface antigen of said cells, as well as to methods of producing the bispecific polypeptide molecule and applications thereof.
公开号:BR112020000762A2
申请号:R112020000762-5
申请日:2018-07-13
公开日:2020-07-21
发明作者:Martin Hofmann；Felix Unverdorben；Sebastian Bunk；Dominik Maurer
申请人:Immatics Biotechnologies Gmbh；
IPC主号:

专利说明:

[001] [001] The present invention relates to a bispecific polypeptide molecule, which comprises a first polypeptide chain and a second polypeptide chain, which provide a binding region derived from a T-cell receptor (TCR, "T-cell receptor") specific for an epitope associated with the major histocompatibility complex and a binding region derived from an antibody capable of recruiting human effector immune cells by binding to a surface antigen of said cells, as well as methods of producing the bispecific polypeptide molecule and applications thereof. GENERAL ASPECTS OF THE INVENTION
[002] [002] With the development of molecular cloning technologies and the improvement of antibody engineering, several types of bispecific antibodies have emerged, which can be used to optimize biological activity and clinical application. For anticancer therapy, bispecific antibodies designed to redirect the activity of immune effector cells to the tumor site have been developed, which specifically bind to an epitope on tumor cells and a second epitope-specific binding domain on immune effector cells. Bispecific antibodies that modify the specificity of effector immune cells have been developed in several formats. Some of them do not have the crystallizable region (Fc), and others are derived from IgG and have a symmetrical or asymmetric structure. In addition to redirecting effector cells to the cancer site, bispecific antibodies have other well-established applications. For example, bispecific antibodies capable of inhibiting two related signaling molecules can be developed to overcome innate or acquired resistance and to act as more effective inhibitors of angiogenesis. Bispecific antibodies can also be used as immunostimulants for the treatment of various diseases, including cancer - a therapeutic strategy that has been showing promise. Bispecific antibodies that mimic factor VIII function can also be used to treat hemophilia A. Other prospects for applying bispecific antibodies are bone diseases, infections and diseases of the central nervous system (see review in Yang F. et al., Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies. Int J Mol Sci. 2016 Dec 28; 18 (1)).
[003] [003] T cells express complexes of T cell receptors (TCR, "T-cell receptor") capable of inducing specific immune responses to antigens. The interaction of the complex formed by the antigenic peptide and the major histocompatibility complex (MHC, “major histocompatibility complex”) class I with the target cell carrying the TCR induces the formation of an immune synapse that facilitates signaling mediated by CD3 coreceptors, which are components of the TCR signaling complex. The signaling cascade directs cell death induced by T cells that express the antigen and act by releasing and transferring granzymes and perforin to the target cell.
[004] [004] In the past, the discovery and production of single-chain antibody variable domains (scFvs, described by Bird et al., 1988) has been important for the development of bispecific antibodies. This concept led to the development of BiTE molecules and their clinical validation as a powerful anti-leukemic drug (Baeuerle, P.A .; Reinhardt, C.
[005] [005] Stieglmaier J., et al. (Utilizing the BiTE (bispecific T-cell engager) platform for immunotherapy of cancer. Expert Opin Biol Ther.
[006] [006] It was found, however, that bispecific small molecules like those of the BiTE® type have deficiencies, such as low yield in production, need for difficult purification processes, propensity to aggregation and very low serum half-life. To circumvent the intrinsic limitations of this class of molecules, several bispecific structures based on human IgG were developed from a concept involving antibodies similar to immunoglobulin G (IgG) conceived more than two decades ago, when Morrison and collaborators fused flexible ligand peptides to the chains IgG heavy-chain C-terminals and then added several single-chain variable domains with different binding specificities (Coloma, MJ and Morrison, SL (1997) Design and production of novel tetravalent bispecific antibodies. Nat. Biotechnol. 15, 159– 163). The molecules can be differentiated from 'normal' antibodies because they have a dual function. Initially, bispecific antibodies (bsAb) had their development restricted to research in academic and biotechnology circles due to technical difficulties. However, the rapid technological development allowed the creation, production and development of recombinant protein derivatives, which renewed the interest of the pharmaceutical industry and boosted the development of research in bsAb.
[007] [007] Another factor that promoted the development of bispecific formats based on IgG was the possibility of including artificial mutations that facilitate the heterodimerization of two different CH3 domains, thus connecting two different polypeptide chains. The basic concept was introduced by Ridgway JB, et al. in “Knobs-into-holes” engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 Jul; 9 (7): 617-21 ”, which revealed the“ key and lock ”approach, a new and effective technique for creating homodimers capable of promoting the heterodimerization of heavy chains of antibodies. In this technique, the “key” is created by replacing a small amino acid with a large amino acid in the CH3 domain of T366Y, a CD4 and IgG immunoadhesin. The "key" is designed to be inserted into a "lock" formed by the CH3 domain in a humanized anti-CD3 antibody created by a specific Y407T substitution, where a larger residue is exchanged for a smaller one. The hybrid between anti-CD3 / CD4 and IgG corresponds to up to 92% of the total purified protein A after simultaneous expression of these two heavy chains together with the anti-CD3 light chain. In contrast, only 57% of anti-CD3 / CD4-IgG hybrids are recovered after simultaneous expression in which heavy chains contained wild-type CH3 domains. The key-and-lock technique allows the construction of a hybrid of antibody and immunoadhesin and, probably, other bifunctional therapeutic products containing Fc, including bispecific immunoadhesins and bispecific antibodies. In “Atwell et al., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol, 1997 ”a mutation was revealed that generates a key-and-lock combination, where the key is generated by T366W and the lock by T366S, L368A and Y407V in the CH3 domain of the Fc domain, which resulted in better heterodimerization. The concept was further refined by introducing cysteine residues to form stable disulfide bridges between the CH3 heterodimeric domains, as described in “Merchant et al. An Efficient Route to Human Bispecific IgG, Nature Biotechnology, 1998 ”.
[008] [008] Other concepts of production of heterodimeric molecules were revealed by Muda et al., 2011, PEDS (“Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies”), Gunasekaran et al., 2010 , J Biol Chem (“Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG”), Moore et al., 2011, MAbs (“A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens ”) and Von Kreudenstein et al., 2013, MAbs (“ Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design ”). These concepts were summarized and reviewed by Ha et al. 2016, Front Immunol (“Immunoglobulin Fc Heterodimer Platform Technology: From Design to Application in Therapeutic Antibodies and Proteins”) and Liu et al., 2017, Front Immunol (“Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds”).
[009] [009] When Fc components such as hinge domains, CH2 and CH3 or parts of them were introduced into bispecific molecules, problems of non-specific immobilization of these molecules arose induced by interactions between Fc and the Fc-gamma receptor (FcgR). FcgR is formed by several cell surface molecules (FcgRI, FcgRIIa, FcgRIIb and FcgRIII), which have different epitope binding affinities exhibited by Fc components of IgG molecules. Since this type of nonspecific immobilization (i.e., it is not induced by either of the two binding domains of a bispecific molecule) is unfavorable due to its influence on the molecule's pharmacokinetics and the activation of immune effector cells far from the target, several variants have been identified Fc and mutations that promote FcgR ablation.
[0010] [0010] Morgan et al., 1995, Immunology (“The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, FcyRI and FcyRIII binding”) revealed the exchange of residues at positions 233 to 236 of human IgG1 and the corresponding sequence derived from human IgG2, which eliminated binding to FcgRI and C1q and decreased binding to FcgRIII.
[0011] [0011] EP1075496 reveals antibodies and other molecules that contain Fc and variations in the Fc region (233P, 234V, 235A, absence of residue or G in 236, 327G, 330S and 331S), where the recombinant antibody is able to bind to the molecule target without significant induction of complement-dependent lysis or cell-mediated destruction of the target.
[0012] [0012] Dual activity redirecting molecules (DART, “dual affinity retargeting“) are used in order to create, for example, an optimized redirection of cell death induced by T cells from B cell lymphoma. The original DART technology was described by Moore et al. (Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma, Blood. 2011 Apr 28; 117 (17): 4542-51). When compared with a bispecific single chain antibody with Fv sequences identical to those of anti-CD19 and anti-CD3 antibodies, DART molecules proved to be more potent in directing the lysis of B cells. DART technology was improved with the introduction of molecules DART-Fc, described by Root et al., 2016 Antibodies (“Development of PF-06671008, a Highly Potent Anti-P-cadherin / Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer”). This molecule combines the high power of DARTs with the prolonged serum half-life of Fc-based molecules, among other positive characteristics.
[0013] [0013] The αβTCR (TCR) recognizes antigenic peptides presented by MHC and is responsible for the specificity of T cells. The α and β chains of the TCR both have variable (V) and constant domains. The V domains participate in the binding of antigenic peptides, and the constant domains cross the T cell membrane. From the analysis of the crystalline structure of the TCR linked to the MHC peptide complex, it was found that the complementarity determining regions (CDR) type 3 of the V α and Vβ chains preferentially interact with the peptide, whereas CDRs 1 and 2 interact with MHC. However, recognition of the peptide by CDR 1 and MHC by CDR 3 has also been described (Piepenbrink et al., The basis for limited specificity and MHC restriction in a T cell receptor interface, Nat Commun, 2013; 4, 1948). The heterodimeric αβ TCR is closely associated with CD3, CD4 or CD8 proteins and other adhesion proteins or signal transducers. The binding of an antigenic peptide to the TCR V region initiates the activation of T cells mediated by signal transduction through the cytoplasmic proteins CD3 and CD4 or CD8 of the TCR constant domains.
[0014] [0014] Single-chain TCRs (scTCRs, "single-chain TCRs") provide significant advantages over the complete TCR for engineering, expression of soluble proteins and clinical potential. From the perspective of the expression of soluble proteins (i.e., manufacture), scTCRs are produced in the form of a single polypeptide, which avoids the need for separate production of each of the TCR chains and allows to produce larger amounts of the correctly assembled scTCR , which binds to the appropriate ligand formed by peptide and MHC. This characteristic allows to obtain the necessary productivity for clinical applications. Finally, also from a clinical point of view, scTCRs formed only by V regions (scTv) can be formatted as therapeutic or diagnostic agents in the same way as scFv fragments.
[0015] [0015] US 2006-0166875 discloses a single-chain T cell receptor (scTCR) comprising a segment formed by a variable alpha chain region of TCR fused to the N-terminus of the extracellular sequence of the constant region of the alpha chain of the TCR, a beta segment formed by a variable region of the beta chain of TCR fused to the N-terminus of the extracellular sequence of a constant region of beta chain of TCR, a linker sequence that links the C-terminus of the alpha segment to the N-terminus of the beta segment or vice versa the extracellular sequences of the constant region of the alpha and beta segments are linked by a disulfide source, and the length of the linker sequence and the position of the disulfide bond are such that the sequences of the variable region of the alpha and beta segments are both oriented substantially in the as seen in native alpha and beta T cell receptors. Complexes of two or more of said scTCRs and the use of scTCRs in various therapeutic and screening applications are also disclosed. In contrast to the scTCRs described in US 2006-0166875, US 2012-0252742 discloses a soluble single-chain human TCR without constant domains, formed only by the variable fragments of the TCR (scTv), which can be used for various purposes, such as treatment cancer and viral or autoimmune diseases.
[0016] [0016] McCormack E, et al. ("Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors. Cancer Immunol Immunother. 2013 Apr; 62 (4): 773-85") revealed that NY- ESO-1 and LAGE-1 are two cancer and testicular antigens whose profile is ideal for anti-tumor immunotherapy, as they undergo overregulation in several types of cancer, present strongly restricted expression in normal tissues and share a common epitope with HLA-A * 0201 in its 157-165 portion. The authors presented data describing the specificity of the antitumor activity of a bifunctional ImmTAC formed by a soluble T cell receptor (TCR) with high affinity for NY-ESO-1157-165 fused to an anti-CD3 scFv. The reagent thus formed, called ImmTAC-NYE, was shown to be able to kill newly isolated HLA-A2 tumor cells and HLA-A2 and LAGE-1 positive CPCNP cells. In vivo optical image studies have shown the in vivo action of high-affinity, NYESO-specific TCRs with fluorescent labeling against HLA-A2 and NY-ESO-1157-165 positive tumors in mouse xenograft models. The in vivo efficacy of ImmTAC-NYE was tested in a tumor model in which human lymphocytes were simultaneously stably grafted onto immunodeficient NSG mice with xenografts. Efficacy was observed both for tumor prevention and in models with established tumors by means of a GFP fluorescence reading. Quantitative RT-PCR was used to analyze the expression of NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 samples of NSCLC and 10 samples of ovarian cancer. In general, tumor samples expressed LAGE-1 RNA more frequently and at higher levels than NY-ESO-1 RNA. ImmTACs comprise a single chain Fv derived from the anti-CD3 UCHT-1 antibody, which covalently binds to the C or N termination of the alpha or beta chain of the TCR.
[0017] [0017] EP1868650 refers to molecules of diabodies and their use in the treatment of various diseases and conditions, including immunological diseases, infectious diseases, intoxication and cancer. Diabetes molecules comprise two polypeptide chains that associate to form at least two epitope binding sites, which can recognize identical or different epitopes from the same or different antigens, and such antigens can be of the same molecule or different molecules. The polypeptide chains of a diabody body can be covalently linked via non-peptide covalent bonds, including, without limitation, disulfide bonds from cysteine residues located on both polypeptide chains. In some specific configurations, the body molecules also have an Fc region, which is revealed here because it allows the molecule to be given properties similar to those of antibodies (eg long half-life). EP1868650 requires the presence of ligand regions from immunoglobulin light or heavy chain variable domains and includes an extensive discussion of functional Fc receptor ligands.
[0018] [0018] WO 2016/184592 A1 reveals bispecific molecules in which one of the specificities is derived from a TCR and the other from an antibody directed against a lymphocyte antigen or surface epitope, but does not reveal the specific arrangement of the elements of the TCR and the regions antibody variables as disclosed herein.
[0019] [0019] EP2258720A1 refers to a functional T cell receptor (TCR) fusion protein (TFP) that recognizes and binds to at least one epitope presented by MHC and contains at least one amino acid sequence that recognizes an antigen and binds to it.
[0020] [0020] It is the object of the present invention to provide improved bispecific molecules capable of acting against peptide complexes with MHC and which are easy to produce, have high stability and have high binding power to the respective antigenic epitopes. Other objects and advantages of the present invention will become apparent after studying the description below and the specific configurations thereof, as well as the respective examples.
[0021] [0021] In a first aspect of the invention, the above object is solved by providing a double specificity antibody selected from the group of molecules formed by a first polypeptide chain and a second polypeptide chain, being that: the first polypeptide chain comprises a first variable domain binding region (VD1) of an antibody that specifically binds to an antigen on the cell surface of an immune effector cell; and a first binding region of a variable domain (VR1) of a TCR that specifically binds to an MHC-associated peptide epitope; and a first linker (LINK1) that connects said domains; the second polypeptide chain comprises a second binding domain of a variable domain (VR2) of a TCR that specifically binds to an MHC-associated peptide epitope; and a second binding domain of a variable domain (VD2) of an antibody that specifically binds to a cell surface antigen of a human immune effector cell; and a second linker (LINK2) that connects said domains; wherein the first binding region (VD1) and said second binding region (VD2) associate in order to form a first binding site (VD1) (VD2) that binds to the epitope of the cell surface molecule; said first linker region (VR1) and said second linker region (VR2) associate in order to form a second linker site (VR1) (VR2) that binds to said MHC-associated peptide epitope; where the two polypeptide chains mentioned above are fused to form hinge domains of human IgG and / or domains of human IgG Fc or dimerizing portions thereof; and where the two polypeptide chains mentioned above are connected to each other by covalent and / or non-covalent bonds between said hinge domains and / or the Fc domains; and where said double-specific polypeptide molecule is capable of binding simultaneously to the cell surface molecule and the MHC-associated peptide epitope; and polypeptide molecules of double specificity, where the order of the binding regions of the polypeptide chains is one among VD1-VR1, VD1- VR2, VD2-VR1, VD2-VR2, VR1-VD1, VR1-VD2, VR2-VD1, VR2-VD2 and where the domains are connected to each other by LINK1 or LINK2.
[0022] A double specificity polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain is preferred, where the first polypeptide chain comprises: a first binding region of a variable domain (VD1) derived from an antibody capable of recruiting cells human effector immunity by specifically binding to a surface antigen of said cells, a first binding domain of a variable domain (VR1) derived from a specific TCR for a peptide epitope associated with MHC and an initial binding portion (LINK1) that connects the two Domains; and the second polypeptide chain comprises a second variable domain binding region (VR2) derived from a TCR specific for an MHC-associated peptide epitope, a second variable domain binding region (VD2) derived from an antibody capable of recruiting immune cells human effectors by specifically binding to a surface antigen from such cells and a second ligand (LINK2) that connects the two domains; said first binding region (VD1) and said second binding region (VD2) joining together to form a first binding site (VD1) (VD2) that binds to the epitope of the cell surface molecule, and said first binding region (VR1) and said second binding region (VR2) associate in order to form a second binding site (VR1) (VR2) that binds to said MHC-associated epitope; where at least one of said polypeptide chains is connected at its C-terminal end to the hinge regions, to the CH2 and / or CH3 domains or parts thereof derived from human IgG and where said double specific polypeptide molecule is capable of bind simultaneously to the immune effector cell antigen and to the MHC-associated peptide epitope.
[0023] Preferably, the dual specificity polypeptide molecule according to the present invention binds with high specificity to both the cellular immune effector antigen and a specific antigenic epitope presented in the form of a MHC peptide complex, e.g. binding affinity (KD) of about 100 nM or less, about 30 nM or less, about 10 nM or less, about 3 nM or less or about 1 nM or less, e.g., measured by interferometry of bi-layer as described in Example 6 or as determined by flow cytometry.
[0024] The double-specific polypeptide molecules according to the present invention are exemplified herein by a double-specific polypeptide molecule comprising a first polypeptide chain comprising SEQ No. 16 and a second polypeptide chain comprising SEQ No. 17.
[0025] [0025] In a second aspect of the invention, the object presented above is solved by providing one or more nucleic acids encoding a first polypeptide chain and / or a second polypeptide chain as disclosed herein or one or more expression vectors that comprise such nucleic acid. In a third aspect of the invention, the object presented above is solved by providing a host cell that comprises the vectors as defined herein.
[0026] [0026] In a fourth aspect of the invention, the object presented above is solved by providing a peptide molecule of double specificity according to the present invention, comprising the appropriate expression of said expression vectors, comprising nucleic acids as revealed in a appropriate host cell and appropriate purification of the cell molecules and / or the respective medium.
[0027] [0027] In a fifth aspect of the invention, the object presented above is solved by providing a pharmaceutical composition comprising the peptide molecule of double specificity according to the present invention, the nucleic acid or the expression vectors according to the invention or the cell according to the invention, together with one or more pharmaceutically acceptable vehicles or excipients.
[0028] [0028] In a sixth aspect of the invention, the invention relates to the double-specific polypeptide molecule according to the invention, to the nucleic acids or expression vectors according to the invention, to the cell according to the invention or to the composition pharmaceutical product according to the invention for use in medicine.
[0029] [0029] In a seventh aspect of the invention, the invention relates to the double-specific polypeptide molecule according to the invention, to the nucleic acids or expression vectors according to the invention, to the cell according to the invention or to the composition pharmaceutical according to the invention for use in the treatment of a disease or disorder as disclosed herein, which may be a cancer or an infectious disease.
[0030] [0030] In an eighth aspect of the invention, the invention relates to a method of treating a disease or disorder that comprises administering a therapeutically effective amount of the dual specific polypeptide molecule according to the invention, nucleic acids or expression vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention.
[0031] [0031] In a ninth aspect of the invention, the invention relates to a method of producing an immune response in a patient or research subject which comprises administering a therapeutically effective amount of the double-specific polypeptide molecule according to the invention or the pharmaceutical composition according to the invention.
[0032] [0032] In a tenth aspect, the invention relates to a method of killing target cells in a patient or research subject, which comprises administering to the patient an effective amount of a double-specific polypeptide molecule in accordance with the present invention.
[0033] [0033] As mentioned earlier, the invention provides new and improved double-specific polypeptide molecules. The molecule generally comprises a first polypeptide chain and a second polypeptide chain, and these chains together provide a variable domain of an antibody specific for an epitope of a surface antigen of an immune effector cell and a variable domain of a TCR specific for a peptide epitope associated with MHC, such as, for example, a cancer epitope or epitopes presented due to infection (eg viral infections such as HIV). Variable domains derived from antibodies and TCR are stabilized by covalent bonds between Fc segments or portions thereof located on both polypeptide chains. Said double-specific polypeptide molecule is capable of binding simultaneously to a cell surface receptor and an MHC-associated peptide epitope.
[0034] [0034] In the context of the present invention, the variable domains (VD1) and (VD2) are derived from antibodies capable of recruiting human immune effector cells by specifically binding to a surface antigen of said effector cells. In a preferred configuration, said antibodies specifically bind to epitopes of the TCR complex with human T cell CD3, which comprises the TCRα, TCRβ, CD3γ, CD3δ, CD and CD3 peptide chains.
[0035] The double specificity polypeptide molecule according to the present invention comprises a first polypeptide chain and a second polypeptide chain, which create a first linker region (VD1) and a second linker region (VD2), respectively, of a variable domain derived from an antibody capable of recruiting human effector immune cells by specifically binding to a surface antigen of said cells. The first binding region (VD1) and said second binding region (VD2) associate in order to form a first binding site (VD1) (VD2) that binds to the surface antigen of the immune effector cell. Furthermore, the first and second polypeptide chains of the polypeptide molecule comprise a first linker region (VR1) and a second linker region (VR2), respectively, both originating from a variable domain derived from a specific TCR for an MHC-associated peptide epitope. Said first binding region (VR1) and said second binding region (VR2) associate in order to form a second binding site (VR1) (VR2) that binds to said peptide epitope associated with MHC. In a double-specific polypeptide molecule configuration according to the invention, the order and orientation of the regions of the first polypeptide chain is one among VD1- LINK1-VR1 and VR1-LINK1-VD1; in another configuration, the order and orientation of the regions of the first polypeptide chain is one among VD2-LINK2-VR2, and VR2-LINK-VD2, that is, the arrangement of the binding sites can be rearranged in order to form a molecule “Left-handed” or a “right-handed” molecule (see, eg, Figure 5). Furthermore, the configuration of the alpha and beta chains of the TCR-related portion can also be changed.
[0036] [0036] In the context of the present invention, the double affinity polypeptide molecule according to the invention is exemplified by a structure that binds to the SLYNTVATL peptide (SEQ No. 7) when presented as a MHC peptide complex. However, the concept of the invention is evidently not restricted to this specific peptide and basically covers any epitope related to a disease or disorder that presents itself in a context involving an MHC molecule. The presentation can be related to MHC class I or MHC class II. All nucleated cells have molecules of the main class I histocompatibility complex (MHC class I) on their surfaces, which also have a large amount of peptide epitopes exposed to surveillance of the CD8 + T cell repertoire. The responses of CD8 + T cells are essential for the control and elimination of viral infections and for the elimination of transformed or tumorigenic cells. Some examples of preferred peptide epitopes for recognition are described in the respective literature and specifically include the peptides disclosed in Tables 1 to 5 of 2016/170139. Tables 1 to 5 of WO 2016/102272; Tables 1 and 2 of WO 2016/156202; Tables 1 to 4 of WO 2016/146751; Table 2 of WO 2011/113819; Tables 1 to 4b of WO 2016/156230; Tables 1 to 4b of WO 2016/177784; Tables 1 to 4 of WO 2016/202963; Tables 1 and 2 of WO 2016/207164; Tables 1 to 4 of WO 2017/001491; Tables 1 to 4 of WO 2017/005733; Tables 1 to 8 of WO 2017/021527; Tables 1 to 3 of WO 2017/036936; Tables 1 to 4 of PCT / EP2016 / 073416 for cancer treatment, US Publication 2016-0187351, US Publication 2017-0165335, US Publication 2017-0035807, US Publication 2016-0280759, US Publication 2016-0287687, US Publication 2016-0346371, US Publication 2016-0368965, US Publication 2017-0022251, US Publication 2017-0002055, US Publication 2017-0029486, US Publication 2017-0037089, US Publication 2017-0136108, Publication US Publication 2017-0101473, US Publication 2017-0096461, US Publication 2017-0165337, US Publication 2017-0189505, US Publication 2017-0173132, US Publication 2017-0296640, US Publication 2017-0253633 and Publication of USA 2017-0260249, the contents of which are hereby incorporated in full by reference. In another aspect, the double-affinity polypeptide molecule according to the invention recognizes a peptide formed by any of the peptides described in the aforementioned patent applications.
[0037] [0037] In one aspect, the double-affinity polypeptide molecule according to the invention binds or is able to specifically recognize or bind to one or more peptides with a total length of 8 to 100 amino acids, from 8 to 30 amino acids, from 8 to 16 amino acids, preferably from 8 to 14 amino acids, specifically from 8, 9, 10, 11, 12, 13 or 14 amino acids, in the case of binding to elongated class II peptides, the length can also be 15, 16 , 17, 18, 19, 20, 21 or 22 amino acids. In another aspect of the invention, the double-affinity polypeptide molecule according to the invention binds or is able to specifically recognize or bind to one or more peptides with a total length of 8 to 12 amino acids, from 8 to 10 amino acids, from 9 to 15 amino acids, 9 to 14 amino acids, 9 to 13 amino acids, 9 to 12 amino acids, 9 to 11 amino acids, 10 to 15 amino acids, 10 to 14 amino acids, 10 to 13 amino acids or 10 to 12 amino acids.
[0038] [0038] Other appropriate epitopes can be identified in databases, such as, for example, the Immune Epitope Database, available at www.iedb.org.
[0039] [0039] The term "human effector immune cell" (singular or plural) refers to a cell in the natural repertoire of cells of the human immune system, which can alter the viability of a target cell when activated.
[0040] In the double-specific polypeptide molecule according to the invention, it is preferable that the first and second polypeptide chains mentioned above comprise at least one hinge-like domain and / or an Fc domain or part of an Fc domain. In antibodies, the “hinge” or
[0041] [0041] The central hinge region usually contains a cysteine bridge that connects two heavy chains. It is also possible to introduce mutations in the lower hinge to reduce antibody-dependent cell-mediated cytotoxicity (ADCC), which is an undesirable characteristic.
[0042] A double specificity polypeptide molecule according to the present invention is preferred which comprises at least one domain with IgG crystallizable fragment (Fc), i.e., a crystallizable fragment region (Fc region), which is the caudal region of the antibody that interacts with Fc receptors and some proteins of the complement system.
[0043] [0043] In the dual specific polypeptide molecules of the invention, said Fc domain can comprise a CH2 domain that comprises at least one mutant mutant of the effector function.
[0044] [0044] Examples of preferred CH2 partial sequences which may be used (in whole or in part) are listed below: 231- APPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK--334 (SEQ 5); and 231- APPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK- 334 (with a number of gears (selected under 6),
[0045] [0045] In the dual specific polypeptide molecules of the invention, said Fc domain can comprise a CH3 domain that comprises at least one mutation that facilitates the formation of heterodimers.
[0046] [0046] A double-specific polypeptide molecule according to the invention is preferred where said key-and-lock mutation in the CH3 domain (see, eg, WO 98/50431) consists of T366W as the "key" and T366'S, L368'A and Y407'V as “lock”. This set of mutations can also be expanded by including the K409A and F405'K mutations, as revealed by Wei et al. (“Structural basis of a novel heterodimeric Fc for bispecific antibody production, Oncotarget. 2017”). Another possibility is T366Y as a “key” and Y407’T as a “lock”.
[0047] [0047] The dual specificity polypeptide molecules of the invention can also comprise cysteine bridges artificially introduced between at least one cysteine residue in the first polypeptide chain and at least one cysteine residue in the second polypeptide chain in order to make the molecules more stable, ideally without interfering with the binding properties and / or the better heterodimerization capacity of the bivalent molecule. To improve stability, a disulfide bridge can be introduced by adding a cysteine to the CH3 domain of the "key" and "lock" chains. The double-specific polypeptide molecule according to the invention is preferred where the Fc domain comprises a CH3 domain that comprises at least one additional cysteine residue (e.g. S354C and / or Y349C).
[0048] [0048] A polypeptide molecule of double specificity according to the invention is preferred where said CD molecule is selected from the group of CD molecules related to the immune response, such as CD3 (CD3γ, CD3δ and Cd3ε chains), CD4, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193,
[0049] [0049] The exemplary double-specific polypeptide molecule according to the invention is preferred, where the regions of the first polypeptide chain comprise SEQ No. 28 in VD1, SEQ No. 29 in VR1 and SEQ No. 30 in LINK1; and the regions of the second polypeptide chain comprise SEQ No. 31 in VD2, SEQ No. 32 in VR2 and SEQ No. 30 in LINK2.
[0050] More preferred is the double-specific polypeptide molecule according to the invention, where the Fc region of the first polypeptide chain comprises SEQ No. 26 (Fc1) and the Fc region of the second polypeptide chain comprises or SEQ No. 27 (Fc2).
[0051] More preferred is the double-specific polypeptide molecule according to the invention, comprising a first polypeptide chain comprising SEQ No. 16 (chain 1 of the complete molecule) and a second polypeptide chain comprising SEQ No. 17 (chain 2 of the molecule) complete).
[0052] Even more preferred is the exemplary double-specific polypeptide molecule according to the invention, wherein said first binding site (VD1) (VD2) which binds to the surface antigen epitope of human immune cells (e.g. CD3) is humanized and / or said binding site (VR1) (VR2) that binds to said peptide epitope associated with MHC is affinity matured.
[0053] [0053] Humanized antibodies are antibodies or parts of antibodies of non-human species whose protein sequences are modified in order to make them more similar to antibody variants produced naturally in humans. The humanization process is usually applied to monoclonal antibodies developed for administration to humans (eg antibodies developed for use as anticancer drugs). Appropriate humanization methods are well known in the literature, as in, for example, Olimpieri, Pier Paolo, Paolo Marcatili and Anna Tramontano. Tabhu: Tools for Antibody Humanization. Bioinformatics 31.3 (2015): 434–435. PMC; Safdari Y, Farajnia S, Asgharzadeh M, Khalili M.
[0054] [0054] In general, affinity maturation of TCRs and antibodies can be performed in vitro according to methods described in the literature, especially those that employ yeast or phage display (based on, e.g., Holler PD, et al In vitro evolution of a T cell receptor with high affinity for peptide / MHC. Proc Natl Acad Sci USA. 2000 May 9; 97 (10): 5387-92; Boder ET et al., Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA. 2000 Sep 26; 97 (20): 10701-5; and, as a recent example, Zhao Q, et al. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential.
[0055] The (VD1) (VD2) and (VR1) (VR2) binding sites of the present invention preferentially and specifically bind to a human immune cell surface antigen and a HLA peptide complex, respectively.
[0056] [0056] In one aspect, the disclosure provides a peptide whose sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequences described herein, such as, for example, amino acid sequences 1 to 58.
[0057] [0057] In one aspect, the polypeptides or polypeptides of double specificity described herein can be modified in several ways, such as by substitutions, deletions, truncations and insertions of one or more amino acids. In another aspect, the polypeptides or polypeptides of double specificity described herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more amino acid substitutions, deletions or insertions. In yet another aspect, the double-specific polypeptides or polypeptides described herein may include 1 to 5, 1 to 10, 1 to 20, 2 to 5, 2 to 10, 5 to 20, 5 to 50 or 10 to 100 substitutions, deletions or amino acid inserts. In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more amino acid substitutions, deletions or insertions apply to any of the structural regions described in Figure 1 (eg VD1, VR1, Link1, VR2, VD2, Link2 or hinge regions). This disclosure also provides aspects in which 1 to 5, 1 to 10, 1 to 20, 2 to 5, 2 to 10, 5 to 20, 5 to 50 or 10 to 100 amino acid substitutions, deletions or insertions apply to the sequences of any of the structural regions described in Figure 1 (eg VD1, VR1, Link1, VR2, VD2, Link2 or hinge region), whether as described or forming part of the sequences disclosed herein.
[0058] [0058] In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more amino acids can be added to the portion N-terminal or C-terminal of a double-specific polypeptide or peptide described herein, such as, for example, amino acid sequences 1 to 58.
[0059] [0059] In one aspect, the VD1 region can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % identical to SEQ No. 28 amino acid sequences.
[0060] [0060] In one aspect, the VR1 region can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % identical to SEQ No. 29 amino acid sequences.
[0061] [0061] In one aspect, the LINK1 or LINK2 regions can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least at least 99% identical to SEQ No. 30 amino acid sequences.
[0062] [0062] In one aspect, the VD2 region can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % identical to SEQ No. 31 amino acid sequences.
[0063] [0063] In one aspect, the VR2 region can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % identical to SEQ No. 32 amino acid sequences.
[0064] [0064] In one aspect, the hinge can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequences SEQ No. 1, SEQ No. 2, SEQ No. 3 or SEQ No. 4. In one aspect, the CH2 domain can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequences SEQ No. 5 or SEQ No. 6.
[0065] [0065] In one aspect, the disclosure provides a peptide that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least at least 97%, at least 98% or at least 99% identical to SEQ No. 43, 44, 45 or
[0066] [0066] In one aspect, the polypeptides or peptide molecules of dual specificity disclosed herein can be modified by replacing one or more residues at different, possibly selective, sites in the polypeptide chain. Such substitutions can be of a conservative nature, for example, replacing one amino acid with another of similar structure and characteristics, such as, for example, replacing a hydrophobic amino acid with another hydrophobic amino acid. Even more conservative would be to replace amino acids of similar or identical chemical size and nature, such as replacing leucine with isoleucine. In studies of sequence variations in naturally occurring homologous protein families, certain amino acid substitutions are tolerated much more frequently than others, and these often show correlation with similarities in size, charge, polarity and hydrophobicity between the original amino acid and its replacement , this being the basis for the definition of “conservative substitutions”.
[0067] [0067] In another preferred configuration of the dual specificity peptide molecule according to the invention, said molecule has an active agent or a portion of such agent that is coupled or conjugated with the molecule. Said active agent can be selected from the group formed by a detectable marker, an immunostimulatory molecule and a therapeutic agent.
[0068] [0068] The detectable marker can be selected from the group formed by biotin, streptavidin, an enzyme or catalytic fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion or a fluorescent, phosphorescent or chemiluminescent molecule. Examples of detectable markers used for diagnostic purposes are fluorescent markers, radiolabels, enzymes, nucleic acid probes and contrast reagents.
[0069] [0069] Examples of therapeutic agents that can be associated with the molecules of the invention are immunomodulators, radioactive compounds, enzymes (eg perforin), chemotherapeutic agents (eg cisplatin) or a toxin. Some other suitable therapeutic agents are small molecule cytotoxic agents, i.e., compounds capable of killing mammalian cells and having a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals capable of producing cytotoxic effects. It is understood that such small molecule cytotoxic agents also include prodrugs, i.e., compounds that undergo decay or undergo conversion under physiological conditions in order to generate cytotoxic agents. Examples of such agents are cisplatin, maytansine derivatives, raquelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sodium porfimer (photophorin II), temozolomide, topotecan, glucuronate, trimetholine and trimetherine, trimethystone and trimetherine, trimethystone and trimetherine. doxorubicin and peptide cytotoxins (i.e. proteins or protein fragments capable of killing mammalian cells). Some other examples are ricin, diphtheria toxin, bacterial exotoxin A from pseudomonas, DNase and RNase, radionuclides (i.e.
[0070] [0070] Another aspect of the present invention which relates to a nucleic acid molecule encoding a first polypeptide chain and / or a second polypeptide chain as disclosed herein or an expression vector comprising one of such nucleic acids. The nucleic acid molecules can be DNA, cDNA, PNA, RNA or a combination of them. The nucleotide sequence that encodes a particular peptide, oligopeptide or polypeptide can occur naturally or be constructed by synthetic means. In general, the segments of DNA encoding peptides, polypeptides and proteins of this invention are constructed from cDNA fragments and short oligonucleotide ligands or a series of oligonucleotides in order to provide a synthetic gene capable of being expressed in a transcription unit recombinant that comprises regulatory elements derived from a microbial or viral operon. The term "expression product" means the polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequences that produce equivalent coding resulting from the degenerate nature of the genetic code producing the same amino acid (s) ( s). The term "fragment" means, when referring to a coding sequence, a portion of the DNA that comprises less than the complete coding region, whose expression product retains basically the same biological function or activity as the expression product of the complete coding region. Depending on the intended use, the nucleic acid can have its codons optimized for expression in an appropriate host cell (eg a microbial cell). The genetic code is redundant and allows some amino acids to be encoded by more than one codon, but some codons are less suitable than others due to factors such as, for example, the relative availability of corresponding tRNAs (Gustafsson et al., 2004).
[0071] [0071] The nucleic acid can be, for example, DNA, cDNA, PNA, RNA or combinations thereof, either single-stranded or double-stranded, or native or stabilized forms of polynucleotides such as, for example, polynucleotides with a basic chain phosphorothioate, and may or may not contain introns, as long as it encodes the polypeptide chains.
[0072] The nucleic acid (e.g. DNA) can then be terminated and / or expressed in an appropriate host in order to produce a peptide that comprises the polypeptide chain of the invention. Therefore, it is possible to use the nucleic acid (eg DNA) that encodes the polypeptide chain of the invention or variant thereof according to known techniques and appropriately modified according to the teachings presented here to build an expression vector, which is then used to transform an appropriate host cell and produce the polypeptide of the invention, as is known in the art.
[0073] [0073] Many expression systems are known, including bacteria (eg E. coli and Bacillus subtilis), yeasts (eg Saccharomyces cerevisiae), filamentous fungi (eg Aspergillus sp.), Plant cells, animal cells and insect cells. Preferably, the system may consist of mammalian cells such as CHO cells available from the ATCC Cell Biology Collection.
[0074] [0074] In one embodiment, the description provides a method of producing a molecule as described here, the method comprising cultivating host cells capable of expressing the polypeptide chains under appropriate conditions to promote their expression.
[0075] In one aspect, nucleic acids that encode polypeptide chains comprising alpha TCR and / or beta TCR binding domains of the present description are cloned, which are then cloned into expression vectors (e.g. gamma retrovirus) or lentivirus) to generate T cells that express molecules of the present description. In another aspect, RNAs are synthesized by procedures known in the art (eg in vitro transcription systems), to obtain molecules that express cells of the present description. Then, the RNAs synthesized in vitro are introduced into appropriate cells by electroporation in order to express polypeptide chains.
[0076] [0076] To increase expression, nucleic acids encoding strands of the present description can be operably linked to strong promoters, such as long terminal repeats (LTRs) in retroviruses, cytomegaloviruses (CMV), murine stem cell viruses (MSCV) U3 , phosphoglycerate kinase (PGK), β-actin, ubiquitin and a promoter composed of simian virus 40 (SV40) / CD43, elongation factor (EF) -1a and the promoter of the splenic focus-forming virus (SFFV). In a preferred configuration, the promoter is heterologous to the expressed nucleic acid. In addition to the strong promoters, the expression cassettes of the present description may contain other elements that can encourage the expression of transgenes, such as a central polipurine tract (cPPT), which promotes the central translocation of lentiviral structures (Follenzi et al., 2000 ), and the post-transcriptional regulatory element of the groundhog hepatitis virus (wPRE), which increases levels of transgene expression by improving RNA stability (Zufferey et al., 1999).
[0077] [0077] The alpha and beta chains of the binding domain of a molecule of the present invention can be encoded by nucleic acids located in separate vectors or by polynucleotides located in the same vector.
[0078] [0078] In one configuration, a host cell is modified to express a molecule of the present description. The host cells of the present description can be allogeneic or autologous in relation to the patient to be treated.
[0079] [0079] Yet another aspect of the invention relates to a pharmaceutical composition comprising the double-specific peptide molecule according to the present invention, the nucleic acids or the expression vectors according to the present invention or the cell according to the present invention, together with one or more pharmaceutically acceptable vehicles or excipients. The compositions of the invention include bulk drug compositions useful for the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e. compositions suitable for administration to a patient or research subject), which can be used in the preparation of forms suitable for individual doses. Such compositions comprise an effective amount for prophylaxis or treatment of the dual specificity polypeptide molecule (agent) disclosed herein or a combination of the agent and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise an effective amount for prophylaxis or treatment of one or more molecules of the invention and a pharmaceutically acceptable carrier.
[0080] [0080] The pharmaceutical compositions preferably comprise free-form or salt-form molecules. Preferably, such salts are pharmaceutically acceptable salts of molecules such as, for example, chloride or acetate (trifluoracetate) salts. It should be noted that the salts of the molecules according to the present invention differ substantially from the molecules in their in vivo state, since these molecules do not appear as salts in vivo.
[0081] [0081] One embodiment of the present invention therefore relates to a molecule according to the invention that does not occur naturally and is produced by synthetic means (synthesized) in the form of a pharmaceutically acceptable salt. Methods of synthetic production of peptides and / or polypeptides are well known in the art. The salts of the molecules according to the present invention differ substantially from molecules in their in vivo state, since these molecules do not present themselves as salts when generated in vivo. Preferably, the salts are pharmaceutically acceptable salts of the molecules. Said salts according to the invention include alkaline and alkaline earth salts, such as the salts of the Hofmeister series, which comprise the anions PO43-, SO42-, CH3COO-, Cl-, Br-, NO3-, ClO4-, I-, SCN- and the cations NH4 +, Rb +, K +, Na +, Cs +, Li +, Zn2 +, Mg2 +, Ca2 +, Mn2 +, Cu2 + and Ba2 +.
[0082] [0082] In one aspect, a polypeptide described herein is in the form of a pharmaceutically acceptable salt. In another aspect, a polypeptide in the form of a pharmaceutical salt has a crystalline form.
[0083] [0083] In one aspect, one of the pharmaceutically acceptable salts described herein refers to salts that have toxicity profiles within an acceptable range for pharmaceutical applications.
[0084] [0084] As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptides where the peptide is modified by making acidic or basic salts of the agent. For example, acid salts are prepared from the free base (usually where the neutral form of the drug has a neutral NH2 group) involving reaction with an appropriate acid. Among the acids suitable for preparing acidic salts are both organic acids (eg acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like) as inorganic acids (eg hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and similar). On the other hand, basic salts of acidic groups that may be present in a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.
[0085] [0085] In one aspect, pharmaceutically acceptable salts can increase the solubility and / or stability of the peptides described herein.
[0086] [0086] The invention also comprises pharmaceutical compositions comprising a double-specific polypeptide molecule of the invention, a therapeutic antibody (e.g., a tumor-specific monoclonal antibody) specific for a given cancer antigen and a pharmaceutically acceptable carrier.
[0087] [0087] In a specific configuration, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, included in the US Pharmacopoeia or other pharmacopoeias generally recognized for use in animals and, more particularly, in humans. The term "vehicle" refers to a diluent, adjuvant excipient or vehicle used to deliver a treatment. Such pharmaceutical carriers can be sterile liquids, including water and oils; they may be of animal, vegetable, synthetic or petroleum origin; and may include peanut oil, soy oil, mineral oil, sesame oil and the like.
[0088] [0088] Another aspect of the present invention relates to the polypeptide molecule of double specificity according to the invention, to nucleic acids or expression vectors according to the invention, to the cell according to the invention or to the pharmaceutical composition according to with the invention for use in medicine. In general, the use of the double-specific polypeptide molecule depends on the medical context of the peptide antigen recognized by said molecule, as is described in more detail below.
[0089] The double specificity polypeptide molecule according to the invention, the nucleic acid or the expression vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention are preferred for use in the treatment or prevention of a disease or disorder selected from the group consisting of immunological diseases, infectious diseases, intoxication and cancers, including treatment of various cancers or other diseases involving abnormal proliferation, including, but not limited to, carcinoma (including bladder carcinomas, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroid and skin), including squamous cell carcinoma, hematopoietic tumors of lymphoid lineage (including acute lymphocytic leukemia, acute lymphoblastic leukemia, lymphoma of B cells, T cell lymphoma and Burkitt's lymphoma), hematopoietic tumors of myeloid lineage (including acute or chronic myeloid leukemia and leukemia promyelocytic ucemia), tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma) and other tumors (including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma),
[0090] [0090] The invention relates to methods of producing an immune response in a patient or research subject that comprise the administration of a therapeutically effective amount of the dual specificity polypeptide molecule according to the invention or the pharmaceutical composition according to invention. In one aspect, a population of dual specificity polypeptide molecules according to the invention or the pharmaceutical composition according to the invention is administered to a patient or research subject who needs it.
[0091] [0091] The invention also relates to a method of killing target cells in a patient or research subject, which comprises administering to the patient an effective amount of a double-specific polypeptide molecule according to the present invention.
[0092] [0092] The invention also provides methods to prevent, treat or manage one or more symptoms associated with an inflammatory disease in an individual, employing for that purpose the administration to said individual of an amount effective for treatment or prophylaxis of one or more anti-inflammatory agents. - inflammatory according to the invention. The invention also provides methods for preventing, treating or managing one or more symptoms associated with an autoimmune disease, employing for that purpose the administration to said individual of an amount effective for treatment or prophylaxis of one or more immunomodulatory agents according to the invention. Infectious diseases that can be treated or prevented by molecules of the invention are caused by infectious agents such as viruses, bacteria, fungi, protozoa, viruses, among others. Viral diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention include, without limitation, those caused by the hepatitis A virus, the hepatitis B virus, the hepatitis C virus, influenza, chickenpox, adenovirus, herpes simplex type 2 (HSV 1), herpes simplex type 2 (HSV 2), swine fever, rhinovirus, ecovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, Coxsackie virus, virus, Coxsackie virus mumps, measles virus, rubella virus, poliovirus, smallpox virus, Epstein-Barr virus, human immunodeficiency virus type 1 (HIV 1), human immunodeficiency virus type 2 (HIV 2) and agents of viral diseases such as meningitis viral, encephalitis, dengue or smallpox.
[0093] [0093] Some bacterial diseases that can be treated or prevented by using the molecules of the invention in conjunction with the methods of the present invention include, without limitation, mycobacteria, rickettsiae, mycoplasmas, Neisseria, S. pneumoniae, Borrelia burgdorferi (Lyme disease ), Bacillus anthracis (anthrax), tetanus, streptococcosis, staphylococcus, mycobacteria, tetanus, whooping cough, cholera, plague, diphtheria, chlamydia, S. aureus and legionella.
[0094] [0094] Some diseases caused by protozoa that can be treated or prevented by using the molecules of the invention in conjunction with the methods of the present invention are leishmaniasis, coccidosis, trypanosomiasis and malaria. Some parasitic diseases that can be treated or prevented by using the molecules of the invention in conjunction with the methods of the present invention are chlamydiosis and rickettsiosis, without limitation.
[0095] [0095] Non-limiting examples of infectious agents and diseases are bacteria (eg Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Proteus vulgaris, Staphylococcus viridans and Pseudomonas aeruginosa), pathogens. eg B-lymphotropic papovavirus (LPV), Bordetella pertussis, Borna disease virus (BDV), bovine coronavirus, choriomeningitis virus, dengue virus, E. coli adenovirus, ebola, ecovirus 1, ecovirus 11 (EV), endotoxin (LPS), enteric bacteria, orphan enteric virus, enterovirus, feline leukemia virus, foot-and-mouth disease virus and gibbon leukemia virus (GALV)), Gram-negative bacteria, Helicobacter pylorii, hepatitis B virus (HBV), herpesvirus simplex, HIV 1, cytomegalovirus, human coronavirus, influenza A, B and C, legionella, Mexican Leishmania, Listeria monocytogenes, measles virus, meningococcus, morbilivirus, murine hepatitis virus, murine leukemia virus, herpesvirus gamma murine re murine trovirus, murine coronavirus, mouse hepatitis virus, Mycobacterium avium type M; Neisseria gonorrhoeae, Newcastle disease virus, parvovirus B 19, Plasmodium falciparum, poxvirus, pseudomonas, rotavirus, Salmonella typhiurium, Shigella, streptococci, type 1 T cell lymphotropic virus and vaccinia virus).
[0096] [0096] In yet another aspect of the invention, the invention relates to a method of treating a disease or disorder which comprises administering a therapeutically effective amount of the dual specific polypeptide molecule according to the invention, nucleic acids or expression vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention.
[0097] [0097] The dual specificity polypeptide molecule of the invention can be used in a method of preventing or treating a disease or disorder that can be alleviated by administering the dual specificity polypeptide molecule. Such treatments can be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable vehicles or excipients. Dual specificity polypeptide molecules are generally supplied as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The sterile pharmaceutical composition can be in any appropriate format, depending on the desired method of administration to the patient.
[0098] [0098] In one aspect, the peptides and other molecules described herein can be combined in an aqueous vehicle. In one aspect, the aqueous vehicle is selected from ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, sorbate potassium, partial glyceride mixtures of saturated vegetable fatty acids, salts or electrolytes (such as protamine sulfate), sodium acid phosphate, dicalcium phosphate, potassium acid phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate , polyvinyl pyrrolidone, polyvinyl pyrrolidone acetate, cellulose-based substances (eg microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetosuccinate, hydroxypropyl methylcellulose phthalate), starch, monohydrate lactose, mannitol, sodium chloride and lauryl sodium, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polymethacrylate, waxes, polyethylene-pol block polymers yoxypropylene, polyethylene glycol and wool fat.
[0099] [0099] In one aspect, the aqueous vehicle contains several components (eg water) together with a component that is a non-aqueous vehicle, such as the compounds described herein. In another aspect, the aqueous vehicle is capable of printing better properties when combined with a peptide or other molecule described herein, such as, for example, better solubility, efficacy and / or better immunotherapy. The composition can also contain excipients, such as buffers, binding agents, abrasive agents, thinners, flavors and lubricants, among others. "Pharmaceutically acceptable diluent" can include, for example, solvents, volumizing agents, stabilizing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents and the like that are physiologically compatible. Examples of pharmaceutically acceptable diluents are one or more among saline, saline with phosphate buffer, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it may be preferable to include in the composition one or more isotonic agents, such as, for example, sugars such as trehalose and sucrose, polyalcohols such as mannitol and sorbitol or sodium chloride. Also included in the scope of the present invention are pharmaceutically acceptable substances as wetting agents or small amounts of ancillary substances such as wetting or emulsifying agents, preservatives or buffers. The composition can also contain excipients such as buffers, binding agents, abrasive agents, thinners, flavors and lubricants.
[00100] The doses of the dual specific polypeptide molecules of the present invention vary within very wide limits, depending on the disease or condition to be treated, the age and the clinical condition of the individual to be treated, among other factors. For example, an appropriate dose of a double-specific polypeptide molecule can range from 25 ng / kg to 50 μg / kg. The appropriate final doses should be determined by a doctor.
[00101] [00101] The pharmaceutical compositions, vectors, nucleic acids and cells of the invention can be supplied in a substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.
[00102] [00102] The preferred characteristics of each aspect of the invention are the same for each of the other aspects, mutatis mutandis.
[00103] [00103] Figure 1 shows a schematic view of a preferred embodiment of the present invention: the double-specific polypeptide molecule containing human IgG1 Fc. VD1, VD2 = antibody-derived variable domains; VR1, VR2 = variable domains derived from TCR; Link1, Link2 = linkers; Cis-Cis = cysteine bridges.
[00104] [00104] Figure 2 shows a schematic view of four IgG Fc structures containing peptide molecules of double specificity, as tested in the context of the present invention. black = TCR-derived variable domains; light gray = antibody-derived variable domains; blank = variable domains derived from human IgG. Keyhole and hole mutations are indicated by cylinders. IA-ID body molecules are in accordance with the invention.
[00105] [00105] Figure 3 shows an HPLC-SEC analysis of several bispecific TCR / mAb molecules designed according to the structures shown in Figure 2, which were purified by means of a two column purification process. The monomeric content of the various molecules was determined as follows: II: 93.84%; III: 96.54%; IV: 98.49%; IA_1: 95.48%; IA_3: 98.45%; ID_1: 95.75%; CI_4: 95.22%; CI_5: 92.76%; ID_4: 99.31%; ID_5: 99.44%.
[00106] [00106] Figure 4 shows the results of potency assays with several bispecific TCR / mAb structures based on IgG4 as shown in Figure 2. Jurkat_NFATRE_luc2 cells were incubated together with T2 cells loaded with the HIV peptide SLYNTVATL (SEQ No. 7 ) in the presence of increasing concentrations of bispecific TCR molecules (bssTCR). The bispecific TCR / mAb IA-IgG4 diabody molecules proved to be more potent than two different molecules with specificity for TCR and mAb.
[00107] [00107] Figure 5 shows the results of potency assays with several bispecific TCR / mAb structures based on IgG4 as shown in Figure 2. Jurkat_NFATRE_luc2 cells were incubated together with T2 cells loaded with the HIV peptide SLYNTVATL (SEQ No. 7 ) in the presence of increasing concentrations of bispecific TCR molecules (bssTCR). Bispecific diabetic molecules ID_1, IA_3 and IA1 proved to be much more potent than three different molecules with specificity for TCR and mAb.
[00108] [00108] Figure 6 shows the results of the power tests carried out with various bispecific IgG1 structures for TCR and mAb (see Figure 2), in which several variable domains of antibodies acting on the TCR-CD3 complex were used. The ID_1 structure comprises the variable domains of the UCHT1 (V9) antibody that act against CD3, and the structures ID_4 and ID_5 comprise variable domains of the BMA031 antibody, which is specific for alpha and beta TCR. Jurkat_NFATRE_luc2 cells were incubated together with T2 cells loaded with the HIV peptide SLYNTVATL (SEQ No. 7) in the presence of increasing concentrations of bispecific TCR molecules (bssTCR).
[00109] [00109] Figure 7 shows schematically the possible orientations of the VD and VR domains of the molecules of the present invention. VH: antibody-derived VH domain, VL: antibody-derived VL domain; Vα: TCR-derived valfa; Vβ: TCR-derived vbeta.
[00110] [00110] Figure 8 shows the results of HPLC-SEC analysis of high molecular weight aggregates within bispecific IgG1-based TCR / mAb molecules. The aggregates were analyzed after purification and after the molecules were stored at 40 ° C for 1 week and 2 weeks, respectively.
[00111] [00111] Figure 9 shows the results of the potency test performed with several bispecific TCR / mAb molecules based on IgG1.
[00112] [00112] Figure 10 shows the results of LDH release assays with several bispecific TCR / mAb structures based on IgG1 as shown in Figure 2. PBMC isolated from a healthy donor were incubated together with T2 cells loaded with the peptide from HIV SLYNTVATL (SEQ No. 7) in the presence of increasing concentrations of bispecific TCR molecules (bssTCR). Bispecific diacor molecules based on TCR / mAb IA_3 and ID_1 induced much more marked lysis of target cells than the three TCR / mAb molecules with double specificity. As shown on the right side of the graph, none of the bispecific TCR / mAb structures tested induced detectable lysis of T2 cells loaded with an irrelevant peptide (SEQ No. 49).
[00113] [00113] Figure 11 shows the results of an LDH release assay with the bispecific TCR / mAb IA_5 structure, which acts against the tumor-associated peptide PRAME-004 (SEQ No. 49), presented in HLA-A * 02. CD8 positive T cells from healthy donors were incubated together with cancer cell lines UACC-257, SW982 and U2OS that had different amounts of PRAME-004: HLA-A * 02-1 complexes on their surfaces (about 1100, about 770 and about 240 copies per cell, respectively, as determined by M / S analysis) with a 5: 1 effector: target ratio in the presence of increasing concentrations of TCR / mAb body molecules. After 48 hours of combined culture, cell lysis was quantified using LDH release assays according to the manufacturer's instructions (Promega).
[00114] [00114] Figure 12 shows the results of an LDH release assay with bispecific TCR / mAb diabody structures IA_5 and IA_6, which use a stability and affinity matured TCR and an improved version of it, respectively, against the peptide tumor-associated PRAME-004 (SEQ No. 49) presented in HLA-A * 02. CD8 positive T cells isolated from healthy donors were incubated together with the U2OS cancer cell line, which had about 240 copies per cell of PRAME-004: HLA-A * 02-1 complexes or unloaded T2 cells (effector ratio: 5: 1 target) in the presence of increasing concentrations of TCR / mAb diabody bodies. After 48 hours of combined culture, cell lysis was quantified using LDH release assays according to the manufacturer's instructions (Promega).
[00115] [00115] Figure 13 shows the results of a thermal stress stability test of bispecific TCR / mAb diabody structures IA_5 and IA_6, which use a stability and affinity matured TCR and an improved version of it, respectively, against the peptide tumor-associated PRAME-004 (SEQ No. 49) presented in HLA-A * 02. For this, the proteins were formulated in PBS at a concentration of 1 mg / mL and then stored at 40ºC for two weeks. Protein integrity and recovery were assessed by HPLC-SEC. Therefore, the amount of high molecular weight components was determined according to the percentage of the peak area eluted before the main peak. The recovery of monomeric protein was calculated by comparing the areas of the highest peaks of samples subjected or not to stress.
[00116] [00116] Bispecific TCR / mAb bodies containing Fc and control molecules (shown in Figure 2) were designed to specifically bind to the human TCR-CD3 complex and the peptide: MHC complex comprising the HIV-derived peptide SLYNTVATL (SEQ No. 7 ) that binds to HLA-A2 * 01. For target selection of the TCR-CD3 complex, VH and VL domains derived from the humanized antibody specific for CD3 hUCHT1 (V9) described by Zhu et al. (“Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation. J Immunol, 1995, 155, 1903–1910”) or VH and VL domains derived from the BMA031 antibody specific for alpha TCR and beta described in Shearman et al. (“Construction, expression and characterization of humanized antibodies directed against the human alpha / beta T cell receptor. J Immunol, 1991, 147, 4366-73”) and employed in the humanized version of variant 10 (data generated internally). To create specificity against the target of the peptide: MHC complex, Valfa and Vbeta domains of 868Z11, a single-chain T cell receptor matured for stability and affinity revealed by Aggen et al. (“Identification and engineering of human variable regions that allow expression of stable single-chain T cell receptors. PEDS, 2011, 24, 361-372”).
[00117] [00117] In the case of bispecific TCR / mAb bodies containing Fc, DNA sequences encoding various combinations of VH and VL (corresponding to VD1 and VD2, respectively) and Va and Vb (corresponding to VR1 and VR2, respectively), as well as the coding of Link1 and Link2 ligands were obtained by gene synthesis. The resulting DNA sequences were cloned into their respective frames in order to create expression vectors that encode the hinge region and CH2 and CH3 domains derived from human IgG4 [sequence no. K01316] and IgG1 [sequence no. P01857], respectively, and were subjected to subsequent manipulations.
[00118] [00118] Table 1: Presentation of all bispecific TCR / mAb diabodies containing generated and evaluated Fc.
[00119] [00119] C&F: Key-and-lock; K / O: Fc silenced; C & F- ds: Key-and-lock stabilized by an artificially created disulfide bond that connects CH3: CH3 '; ds-hUCHT1 (V9): variable domains stabilized by hUCHT1 disulfide bond (V9); Link1: Link connecting VR1 and VD1. TCR mAb molecule SEQ No. modifications IA-IgG4 868Z11 hUCHT1 (V9) SEQ ID No. 8 IgG4 (KiH) SEQ ID No. 9 IA_1 868Z11 hUCHT1 (V9) SEQ ID No. 10 IgG1 (K / O, KiH) SEQ ID No 11 IA_2 868Z11 hUCHT1 (V9) SEQ ID No. 12 IgG1 (K / O, KiH-ds) SEQ ID No. 13 IA_3 868Z11 ds-hUCHT1 (V9) SEQ ID No. 14 IgG1 (K / O, KiH-ds ) SEQ ID No. 15 ID_1 868Z11 ds-hUCHT1 (V9) SEQ ID No. 16 IgG1 (K / O, KiH-ds) SEQ ID No. 17 IC_4 868Z11 hBMA031 (var10) SEQ ID No. 18 IgG1 (K / O , KiH-ds) SEQ ID No. 19 IC_5 868Z11 hBMA031 (var10) SEQ ID No. 20 IgG1 (K / O, KiH-ds) SEQ ID No. 21 extended Link1 ID_4 868Z11 hBMA031 (var10) SEQ ID No. 22 IgG1 (K / O, KiH-ds) SEQ ID No. 23 ID_5 868Z11 hBMA031 (var10) SEQ ID No. 24 IgG1 (K / O, KiH-ds) SEQ ID No. 25 extended Link1 IA_5 R16P1C10I hUCHT1 (Var17) SEQ ID No. 43 IgG1 (K / O, KiH-ds) SEQ ID No. 44 IA_6 R16P1C10I # 6 hUCHT1 (Var17) SEQ_ID No. 45 IgG1 (K / O, KiH-ds) SEQ ID No. 46
[00120] [00120] Several control molecules have been constructed that have the same specificities (Table 2) using the same VH, VL, Valfa and Vbeta domains in combination with constant domains derived from IgG1 or IgG4 comprising characteristics introduced by genetic engineering, as previously described.
[00121] [00121] Table 2: Presentation of all bispecific control molecules containing Fc generated and evaluated.
[00122] [00122] KiH: Key-and-lock; K / O: Fc silenced. TCR mAb molecule SEQ No. modifications III-IgG4 868Z11 hUCHT1 (V9) SEQ ID No. 38 IgG4 (KiH) SEQ ID No. 39 IV-IgG4 868Z11 hUCHT1 (V9) SEQ ID No. 40 IgG4 SEQ ID No. 41 II 868Z11 hUCHT1 (V9) SEQ ID No. 33 IgG1 (K / O, KiH) SEQ ID No. 34 III 868Z11 hUCHT1 (V9) SEQ ID No. 35 IgG1 (K / O, KiH) SEQ ID No. 36 IV 868Z11 hUCHT1 (V9 ) SEQ ID No. 37 IgG1 (K / O) SEQ ID No. 42 EXAMPLE 2 - PRODUCTION AND PURIFICATION OF BIESPECIFIC TCR / MAB DIACORPOS THAT CONTAINS FC
[00123] [00123] Monocistronic recombinant protein expression vectors controlled by HCMV-derived promoter elements (derived from pUC19) were designed. Plasmid DNA was amplified in E. coli according to standard culture methods and then purified by commercially available kits (Macherey and Nagel). The purified plasmid DNA was used for transient transfection of CHO-S cells according to the manufacturer's instructions (ExpiCHO ™ system; Thermo Fisher Scientific). The transfected CHO cells were cultured for 6 to 14 days at 32 ° C to 37 ° C and then exposed once or twice to the ExpiCHO ™ Feed solution.
[00124] [00124] The conditioned cell supernatant was collected by centrifugation (4000 x g; 30 minutes) and purified by ultrafiltration (0.22 µm).
[00125] [00125] As all therapeutic proteins need to present some stability when exposed to acid in order to be able to use robust processes for purification on an industrial scale, the percentage of monomeric protein eluted from the protein A capture column was measured (Table 3) .
[00126] [00126] Table 3: Fractionation of the monomeric protein in the capture column after acid elution: Monomer molecule eluted from the capture column (% of the total peak area) IA-IgG4 (VH-beta) nd IA_1 (VH-beta) 49 IA_2 (VH-beta) 54 IA_3 (dsVH-beta) 63
[00127] [00127] After size exclusion chromatography, the purified bispecific molecules showed high purity (> 93% monomeric protein), verified by HPLC-SEC in MabPac SEC-1 columns (5 µm, 7.8 × 300 mm) in phosphate 50 mM sodium at pH 6.8 with 300 mM NaCl in an Agilent 1100 system (see Figure 3). Reducing and non-reducing SDS ‐ PAGE gels confirmed the purity and expected size of the various TCR / mAb molecules of double specificity (data not shown).
[00128] [00128] The potency of the TCR / mAb bodies containing Fc for T cell activation was evaluated in a T cell activation bioassay (Promega). The trial used a Jurkat cell line with a modified genome to express a luciferase reporter gene triggered by an NFAT response element. The tests were performed according to the manufacturer's instructions. In summary, T2 cells were loaded with the HIV-specific peptide SLYNTVATL (SEQ No. 7) or left unloaded (unloaded control) and then cultured along with modified Jurkat cells provided by Promega in the presence of increasing concentrations of TCR / mAb molecules bispecific. The activation of Jurkat T cells with a reporter gene was analyzed after 16 to 20 hours by measuring the luminescence intensity.
[00129] [00129] Representative potency assays for TCR / mAb molecules based on IgG4 (Figure 4) and IgG1 (Figure 5) are shown,
[00130] [00130] Furthermore, the LDH release assay (Promega) was used to quantify the CMSP-mediated lysis of T2 cells loaded with the SLYNTVATL peptide (SEQ No. 7) induced by various bispecific TCR / mAb molecules (Figure 10). In line with the results previously described for the T cell activation bioassay, the structures of TCR / mAb bodies containing Fc IA and ID were superior to structures 2, 3 and 5 of bispecific TCR / mAb bodies, which was indicated by an increase in the absolute level of lysis of target cells and a decrease in the bispecific TCR concentration necessary to achieve half the maximum target cell death (EC50). As for the TCR / mAb structures 2, 3 and 4, the TCR / mAb bodies with structure IA and ID did not induce lysis of T2 cells loaded with a non-relevant peptide (SEQ No. 49), which demonstrates the specificity of cell lysis for T2 cells.
[00131] [00131] Bispecific TCR / mAb diaphragm structures containing Fc were designed to serve as molecular platforms and provide support for variable domains derived from TCR and mAb that act against various peptide / MHC complexes and effector cell surface antigens, respectively. To assess whether the platform was adequate, the variable domains derived from mAb were exchanged into a first set of molecules. The variable domains of the anti-CD3 antibody hUCHT1 (V9) (structure ID_1) have been replaced by the domains of the anti-TCR antibody hBMA031 (var10) with identical domain orientation (structures ID_4 and ID_5) or different (IC_4, IC_5) (see details) Table 1 and Figure 7). The expression, purification and characterization of these molecules were carried out as previously described. According to HPLC-SEC analyzes, the purity and integrity of the final preparations were greater than 92%.
[00132] [00132] The potency assay revealed activation of Jurkat reporter T cells and minimal nonspecific activity against unloaded T2 cells, both for variable domains of antibodies hUCHT1 (structure ID_1) and hBMA031 (structure ID_4 and ID_5), which underlies the platform stability of dual specificity TCR / mAb structures (Figure 6). It should be noted that, when the variable TCR and mAb domains of the structures ID_4 and ID_5 were exchanged in the two polypeptide chains, structures IC_4 and IC_5 were created that did not induce T cell activation (data not shown). This latter finding indicates that, although bispecific TCR / mAb diabody can be used as a platform structure to incorporate several TCR and mAb variable domains, it is necessary to carefully optimize the orientation of the domains to obtain the ideal molecular activity.
[00133] [00133] The stability of bispecific TCR / mAb molecules was initially assessed by the Protein Thermal Shift Assay (Thermo Fisher Scientific) according to the manufacturer's instructions and using a 7500 real-time PCR system (Applied Biosciences). Briefly, the purified molecules were mixed with PTS buffer and PTS dye and subjected to an increasing temperature gradient to constantly monitor the fluorescence of the samples. The recorded fluorescence signals were analyzed using the PTS software (Thermo Fisher Scientific), and the melting points (TF) were calculated using the derivative method.
[00134] [00134] Stress stability studies were performed by storing purified molecules dissolved in PBS at 40 ° C for up to 2 weeks. Samples were analyzed to assess the integrity of proteins using HPLC-SEC and potency assays using the T cell activation assay (Promega), as previously described.
[00135] [00135] As expected, storage at 40 ° C induced the formation of high molecular weight aggregates and components, as demonstrated by HPLC-SEC assays (see Figure 8). The results of the potency tests of IgG1-based molecules after purification and incubation at 40ºC are shown in Figure 9. Although none of the tested molecules showed a significant reduction in potency after storage at 40 ° C, it was observed that molecules 3 and 4 exposed to stress induced a significant amount of non-specific (independent of the target) activation of Jurkat T cells. In contrast, bispecific TCR / mAb diabodies maintained their potency dependent on the target, although HPLC-SEC has shown some aggregates.
[00136] [00136] To better validate the platform capabilities of bispecific TCR / mAb diabody structures, the variable domains derived from TCR have been exchanged for variable domains of a TCR, which has been matured to improve stability and affinity through exposure in yeasts of according to a method previously described (Smith et al, 2015, “T Cell Receptor Engineering and Analysis Using the Yeast Display Platform. Methods Mol Biol. 1319: 95-141”). The TCR variable domains, which specifically bind to the HIV-derived peptide SLYNTVATL (SEQ No. 7) in the context of HLA-A * 02, have been replaced by TCR variable domains that specifically bind to the tumor-associated peptide PRAME-004 ( SEQ No. 49) linked to HLA-A * 02. Furthermore, the variable domains of the humanized T-cell recruiting antibody hUCHT1 (V9) were exchanged for variable domains of hUCHT1 (Var17), a humanized version of the UCHT1 antibody, thus creating an anti-PRAME TCR / mAb diabody called IA_5, formed by SEQ No. 43 and SEQ No. 44. The expression, purification and characterization of this molecule were carried out as described in Example 2. According to the HPLC-SEC analysis, the purity and integrity of the final preparations were greater than 96%.
[00137] [00137] Bispecific TCR / mAb bispecific binding affinities for PRAME-004: HLA-A * 02 were determined by bi-layer interferometry. Measurements were performed on an Octet RED384 device following the settings recommended by the manufacturer. Briefly, bispecific TCR / mAb diabody bodies were purified and loaded on biosensors (AHC) before serial dilutions of HLA-A * 02 / PRAME-004 were analyzed.
[00138] [00138] The ability to induce tumor cell lysis of the TCR / mAb diabody structure with anti-PRAME-004 activity was assessed by measuring, through an LDH release assay, the lysis of human cancer cell lines UACC -257, SW982 and U2OS, whose cell surfaces have varying levels of copies of the PRLA-004 peptide associated with HLA-A * 02 (about 1100 in UACC-257, about 770 in SW982 and about 240 U2OS copies of PRAME- 004 per cell, as determined by quantitative M / S analysis).
[00139] [00139] As shown in Figure 11, the TCR / mAb anti-PRAME-004 IA_5 diabody structure induced lysis of PRAME-004 tumor cell lines in a concentration dependent manner. Even U2OS tumor cells, which expressed only 240 copies of PRAME-004 per cell, were efficiently lysed by the TCR / mAb diabody body molecule. These results corroborate the fact that the TCR / mAb diabody applies to the molecular platform, allowing the introduction of variable domains of several TCRs and variable domains of several T cell recruiting antibodies.
[00140] [00140] The variable domains of TCR used in structure IA_5 were further improved in relation to their affinity for PRAME-004 and stability of the TCR and then used to make a support for a TCR / mAb diabody, which led to the construction of the structure IA_6, comprising SEQ No. 45 and SEQ No. 46). The expression, purification and characterization of the TCR / mAb diacor molecules IA_5 and IA_6 were performed as described in Example 2. According to the HPLC-SEC analysis, the purity and integrity of the final preparations were greater than 97%.
[00141] [00141] The IA-6 variant of the TCR / mAb anti-PRAME-004 body had its potency, stability and affinity evaluated in cytotoxicity experiments in which the tumor cell line U2OS (which presents small amounts of PRAME-004: HLA-A * 02) or unloaded T2 cells were used as target cells and human CD8 T cells were used as effector cells.
[00142] [00142] As shown in Figure 12, the inventors observed an increase in the cytotoxic potency of the TCR / Ab IA_6 diabody body, which comprises the variable domains of the TCR variant with increased stability and affinity in relation to the precursor structure IA_5. PRAME-004-dependent lysis was confirmed in both structures (IA_5 and IA_6), since no cytolysis of T2 cells was detected, which were negative for the target.
[00143] [00143] Protein structures were again subjected to thermal stress at 40 ° C for up to two weeks to analyze the stability of variants IA_5 and IA_6 of TCR / mAb specific bodies for PRAME-004. HPLC-SEC analyzes after thermal stress revealed significant improvement of variant IA_6 compared to precursor structure IA_5 (see Figure 13). The increase in high molecular weight species induced by temperature (i.e. elution before the main peak) of the structures was less pronounced with IA_6 than with IA_5. In line with this result, the recovery of intact monomeric protein after heat stress was 87% and 92% for IA_5 and IA_6, respectively.
[00144] [00144] These exemplary engineering data demonstrate that stable and high potency TCR / mAb diabody structures can be improved by incorporating variable TCR domains that promote stability and affinity, which results in therapeutic proteins with better characteristics.
[00145] [00145] In addition to the bispecific and HIV-specific TCR structure described here (SEQ No. 16 and SEQ No. 17, in D orientation), the invention also provides several other exemplary HIV structures, which have been tested.
[00146] [00146] The humanization of UCHT1 was carried out using
[00147] [00147] The results obtained are shown below in Table 4: V9 (Zhu et al., 1995) Present invention DRB1 score 1232 ~ 1190 Title [mg / L] 0.75 3 Tm of F (ab) [° C] 83.0 86.4 EC50 of effector cell activation 63 8 [M]
[00148] [00148] The data in Table 4 shows that the humanization of the invention offers less immunogenic potential (lower DRB1 score) and that the molecules are more stable (melting point 3ºC higher) and more potent (EC50 about 8 times lower) than the standard (V9). (See test in Example 3).

权利要求:
Claims (21)
[1]
1. A DOUBLE-SPECIFICITY ANTIBODY selected from the group of molecules formed by a first polypeptide chain and a second polypeptide chain, with: the first polypeptide chain comprising a first variable domain binding region (VD1) of an antibody that specifically binds an antigen on the cell surface of an immune effector cell; and a first binding region of a variable domain (VR1) of a TCR that specifically binds to an MHC-associated peptide epitope; and a first linker (LINK1) that connects said domains; the second polypeptide chain comprises a second binding domain of a variable domain (VR2) of a TCR that specifically binds to an MHC-associated peptide epitope; and a second binding domain of a variable domain (VD2) of an antibody that specifically binds to a cell surface antigen of a human immune effector cell; and a second linker (LINK2) that connects said domains; the first binding region (VD1) and said second binding region (VD2) being associated in order to form a first binding site (VD1) (VD2) that binds to a cell surface antigen of a human effector immune cell ; said first linker region (VR1) and said second linker region (VR2) associate in order to form a second linker site (VR1) (VR2) that binds to said MHC-associated peptide epitope; where the two polypeptide chains mentioned above are fused to form hinge domains of human IgG and / or domains of human IgG Fc or dimerizing portions thereof; and where the two polypeptide chains mentioned above are connected to each other by covalent and / or non-covalent bonds between said hinge domains and / or the Fc domains; and where said double-specific polypeptide molecule is capable of binding simultaneously to the cell surface molecule and the MHC-associated peptide epitope; and double specificity polypeptide molecule, characterized by the order of the binding regions of the two polypeptide chains is one among VD1- VR1 and VR2-VD2 or VD1-VR2 and VR1-VD2, or VD2-VR1 and VR2-VD1 or VD2-VR2 and VR1 -VD1 and where the domains are connected to each other by LINK1 or LINK2.
[2]
2. The double-specific polypeptide molecule according to claim 1, characterized by the order of the binding regions of the polypeptide chains is one among VD1-VR1 and VD2-VR2; and where domains are connected by LINK1 or LINK2, respectively.
[3]
3. The double-specific polypeptide molecule according to claim 1, characterized by the linker sequences LINK1 and / or LINK2 contain at least one sequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS and TVLSSAS.
[4]
The double specificity POLYEPTIDE MOLECULE according to any one of claims 1 to 3, characterized by the first and second polypeptide chains mentioned also comprising at least one hinge-like domain and an Fc domain or portions of the same derivatives of IgG1, IgG2 or Human IgG4.
[5]
5. The double-specific polypeptide molecule according to claim 4, characterized by the Fc domain comprising at least one mutant mutation of the effector function in a residue selected from positions 233, 234, 235, 236, 297 and 331, where, preferably , said mutant mutation of the effector function is generated by replacing at least one residue at positions 233, 234, 235, 236 and 331 with the corresponding residue derived from IgG2 or IgG4.
[6]
6. The double-specific polypeptide molecule according to any one of claims 3 to 5, characterized in that the Fc domain comprises a CH3 domain that comprises at least one mutation that facilitates the formation of heterodimers.
[7]
7. The double-specific polypeptide molecule according to claim 6, characterized by the mutations being in any of the positions between 366, 368, 405 and 407, where, preferably, said mutations comprise the key-and-type mutations lock T366W and T366'S or L368A 'and Y407'V.
[8]
The double-specific polypeptide molecule according to any one of claims 3 to 7, characterized in that said Fc domain comprises one or more CH2 and CH3 domains comprising at least two more cysteine residues, such as, for example, S354C and Y349C or L242C and K334C.
[9]
The double-specific polypeptide molecules according to any one of claims 1 to 8, characterized by the domains derived from VD1 and VD2 antibodies described above have an artificial disulfide bridge that introduces a covalent bond between VD1 and VD2 and where said cysteines are introduced in the structural region (“framework”) (FR) 4 in the case of VL in the structural region (“framework”) in the case of VH.
[10]
10. The double-specific polypeptide molecule according to any one of claims 1 to 9, characterized by the cell surface molecule is known to induce activation of immune cells or, at least, has been selected from the group formed by molecules related to the response immune, such as CD3 (CD3γ, CD3δ and CD3ε chains), CD4, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI, TCRα / β, TCRγ / δ and HLA-DR.
[11]
The double specificity POLYEPTIDE molecule according to any one of claims 1 to 10, characterized by the regions of the first polypeptide chain comprising SEQ No. 28 in VD1, SEQ No. 29 in VR1 and SEQ No. 30 in LINK1; and the regions of the second polypeptide chain comprise SEQ No. 31 in VD2, SEQ No. 32 in VR2 and SEQ No. 30 in LINK2.
[12]
The double specificity POLYPEEPTIC MOLECULE according to any one of claims 3 to 11, characterized by the Fc region of the first polypeptide chain comprising SEQ No. 26 or SEQ No. 47 (Fc1) and the Fc region of the second polypeptide chain comprising SEQ No. 27 or SEQ No. 48 (Fc2).
[13]
13. A POLYPEPTIDIC MOLECULE characterized by double specificity, comprising SEQ No. 16 or SEQ No. 43 or SEQ No. 45 or SEQ No. 51, 53, 55 or 57, and a second polypeptide chain comprising SEQ No. 17 or SEQ No. 44 or SEQ No. 46 or SEQ No. 52, 54, 56 or 58.
[14]
14. The double-specific polypeptide molecule according to any one of claims 1 to 13, characterized in that said molecule carries a detectable marker.
[15]
The double specificity POLYEPTIDE MOLECULE according to any one of claims 1 to 14, characterized by said first binding site (VD1) (VD2) that binds to a cell surface antigen of said immune cells is humanized, and / or said second binding site (VR1) (VR2) that binds to said peptide epitope associated with
MHC is matured in order to provide more affinity and / or stability.
[16]
16. A NUCLEIC ACID characterized by encoding the first polypeptide chain and / or the second polypeptide chain according to claims 1 to 15 or an expression vector comprising at least one of said nucleic acids.
[17]
17. A HOSTING CELL characterized by comprising and, optionally, expressing a vector as defined in claim 16.
[18]
18. A PHARMACEUTICAL COMPOSITION characterized by being composed of a polypeptide molecule of double specificity according to any one of claims 1 to 15, the nucleic acid or the expression vector according to claim 16, or the cell according to claim 17, together with one or more pharmaceutically acceptable vectors or excipients.
[19]
19. THE POLYPEPTIDIC MOLECULE characterized by the double specificity according to any one of claims 1 to 15, the nucleic acid or the expression vector according to claim 16, the cell according to claim 17 or the pharmaceutical composition according to claim 18 for use in medicine.
[20]
20. THE POLYPEPTIDIC MOLECULE characterized by the double specificity according to any one of claims 1 to 15, the nucleic acid or the expression vector according to claim 16, the cell according to claim 17 or the pharmaceutical composition according to claim 18 for use in preventing or treating a disease or disorder selected from the group consisting of cancer, infectious diseases and immune disorders.
[21]
21. A METHOD OF TREATING A DISEASE OR CONDITION characterized by the administration of a therapeutically effective amount of the double-specific polypeptide molecule according to any one of claims 1 to 15, the nucleic acid or the expression vector according to claim 16, the cell according to claim 17 or the pharmaceutical composition according to claim
18.

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PT3428194T|2021-11-18|

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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