DE102017115966A1 - Polypeptide molecule with improved dual specificity - Google Patents

Abstract

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) that is responsible for a peptide epitope attached to a major histocompatibility complex (MHC). is bound to a binding region derived from an antibody capable of recruiting human immune effector cells by binding specifically to a surface antigen of these cells, as well as methods for producing the bispecific polypeptide molecule and their use.

Description

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 ) specific for a peptide epitope bound to a major histocompatibility complex (MHC), and to a binding region derived from an antibody capable of recruiting human immune effector cells by specifically targeting a surface antigen of those cells as well as methods for producing the bispecific polypeptide molecule and their use.

Background of the invention

Through the development of molecular cloning technology and the in-depth understanding of antibody production, a variety of bispecific antibody formats are available, from which one can choose to achieve optimal biological effects and achieve the medical goal. In cancer therapy, bispecific antibodies have been developed with the aim of realigning the activity of immune effector cells by a first binding domain specific for an epitope on tumor cells and a second binding domain specific for an epitope on the immune effector cells to the tumor site , Bispecific antibodies for retargeting of immune effector cells have been developed in various formats, including formats without a fragment-crystallizable (Fc) region and IgG-derived formats with a symmetric or asymmetric structure. In addition to the re-targeting of effector cells to the cancer site, new applications for bispecific antibodies have been introduced. Bispecific molecules, which can inhibit two correlating signaling molecules at the same time, can be designed to overcome congenital or acquired resistance and become more potent angiogenesis inhibitors. In addition, bispecific antibodies can be used as promising immunostimulating agents for the treatment of various diseases such as cancer. Bispecific antibodies can also be used to treat hemophilia A by mimicking the mode of action of factor VIII. Bispecific antibodies have prospects of widespread use in bone diseases and central nervous system infections and disorders (discussed in US Pat Yang F. et al. Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies. Int J Mol Sci. December 28, 206; 18 (1) ). T cells express T cell receptor (TCR) complexes that are capable of eliciting antigen-specific immune responses. The intervention of the antigen-peptide / major histocompatibility complex (MHC) class I on the target cell with the TCR triggers the formation of an immunological synapse and leads to signaling by CD3 co-receptors, which are components of the TCR signaling complex. This signaling cascade initiates T-cell mediated killing of the cell, which expresses the antigen by releasing and transferring granzymes and perforin from the T cell to the target cell.

Historically, the discovery and production of variable, single-chain antibody domains (scFvs, described by Bird et al., 1988) has served as a major driver for the development of bispecific antibodies. This concept eventually led to the generation of BiTE molecules and their clinical testing as an effective drug for the treatment of leukemia (Baeuerle et al. 2009). In the case of cancer, bispecific antibodies that interfere with the CD3 epsilon subunit and a surface antigen on the tumor cell, resolve T cell-mediated killing of the tumor cell, while eliminating the need for direct interaction of the tumor cell TCR and the MHC class I is bypassed in a complex with an antigen. This extends the repertoire of T cells capable of recognizing the tumor and acting as effector cells ( Baeuerle, PA; Reinhardt, C. Bispecific T-cell participating antibodies for cancer therapy. Cancer Res. 2009, 69, 4941-4944 ).

Stieglmaier J., et al. (In: Utilizing the BiTE (bispecific T-cell engager) platform for immunotherapy of cancer. Expert Opin Biol Ther. 2015; 15 (8): 1093-9 ) describe that various approaches to T cell-based cancer immunotherapy are currently being explored, including BiTE® (bispecific T-cell engager) antibody constructs, which have a unique structure and unique mechanisms of action. They are prepared by genetically linking the minimal binding domains of monoclonal antibodies to tumor-associated surface antigens and the T-cell receptor-associated molecule CD3 into a single polypeptide chain. The simultaneous intervention of the target cell antigen and CD3 leads to the activation of polyclonal cytotoxic T cells, which leads to the lysis of the target cells. Blinatumomab, a CD19 targeting BiTE, is being investigated for a wide range of B-cell malignancies and has recently been reviewed in the US by the US Food and Drug Administration for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia under the trade name BLINCYTO ™. The BiTE platform represents one of the clinically best-studied options for T-cell immunotherapy.

However, it has been found that small, bispecific molecules such as BiTEs have the disadvantage that they give only low production yields, require complicated purification procedures, tend to aggregate and also have a very short serum half-life. To overcome the inherent limitations of this class of molecules, various bispecific formats based on human IgG have been developed, starting from the concept of recombinant bispecific antibodies similar to the prototype immunoglobulin (Ig) G as it was more than two decades ago when Morrison and colleagues fused flexible linker peptides to the IgG heavy chain C-termini followed by 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 could be differentiated from 'normal' antibodies because they had two different functions. Technical hurdles initially hampered further development and led to biospecific antibodies (bsAbs) remaining a focus of research & development, particularly in the academic and biotechnology environment. However, the rapidly evolving technologies that enabled the creation, production and development of derivatives of recombinant proteins, in combination with a reawakened interest from the pharmaceutical industry, gave a tremendous boost to the field of bsAb research. Today, many different bsAb formats that are suitable for the development of therapeutic proteins are available (for review see Gramer 2007, Weidle 2013, Brinkmann 2017). In summary, incorporation of Fc (fragment crystallizable) parts out CH2 and CH3 domains, for increased productivity, simplified purification processes, and improved stability. In addition, the serum half-life of these IgG-based drugs was prolonged due to i) the increase in size and ii) the interaction of the Fc portion with the human Fc receptor FcRn.

The development of IgG-based bispecific formats was further promoted by the appearance and inclusion of generated mutations to facilitate heterodimerization of two distinct CH3 domains, thereby joining two distinct polypeptide chains. The basic concept was developed by Ridgway JB et al. (In: 'Knobs-into-holes' engineering of CH3 domains for heavy chain heterodimerization Protein Eng. 1996 Jul; 9 (7): 617-21 ), which disclosed the 'knobs-into-holes' approach as a new and effective manufacturing strategy for the generation of heavy chain antibody homodimers for heterodimerization. In this approach, a 'Knob' variant was first obtained by replacing a small amino acid in the CH3 domain of a CD4-IgG immunoadhesin with a larger one: T366Y. The 'Knob' is designed to be introduced into the 'hole' in the CH3 domain of a humanized anti-CD3 antibody generated by the expedient replacement of a large residue with a smaller one: Y407T. The anti-CD3 / CD4-IgG hybrid represents up to 92% of protein A purified protein pool after expressing these two different heavy chains together with the anti-CD3 light chain. In contrast, after co-expression, in which heavy chains contained wild-type CH3 domains, only up to 57% of the anti-CD3 / CD4-IgG hybrid was recovered. Thus, production by the 'knob-into-holes' technology facilitates the creation of an antibody / immunoadhesin hybrid and probably other Fc-containing bifunctional therapeutics, including bispecific immunoadhesin and bispecific antibodies. Atwell et al., 1997, J Mol Biol (Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library) discloses a 'knob-in-hole' mutation (Knob: T366W / hole: T366S + L368A + Y407V) in the CH3 domain of the Fc domain for improved heterodimerization. This concept was further enhanced by the additional introduction of cysteine residues to form a stabilizing disulfide bond between the heterodimeric CH3 domains as described by Merchant et al. 1998, Nature Biotechnology (An Efficient Route to Human Bispecific IgG).

Other concepts for producing heterodimeric molecules have been described by Muda et al. a. 2011 discloses PEDS (Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.); Gunasekaran u. a. 2010, J Biol Chem (Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific and monovalent IgG.); Moore u. a. 2011, MAbs (A novel bispecific antibody format enabling simultaneous bivalent and monovalent co-engagement of distinct target antigens.); From Kreudenstein u. a. 2013, MAbs (Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design.) These concepts are used by Ha u. a. 2016, Front Immunol (Immunoglobulin Fc Heterodimer Platform Technology: From Design to Application in Therapeutic Antibodies and Proteins) and Liu et al. a. 2017, Front Immunol (Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel scaffolds) reviewed and reviewed.

With the inclusion of Fc parts made of joints [Hinges], CH2 and CH3 Domains or parts thereof exist in bispecific molecules, the problem of nonspecific immobilization of these occurred Molecules elicited by Fc: Fc-gamma receptor ( FcgR ) - interactions. FcgRs consist of different cell surface molecules ( FcgRI . FcgRIIa . FcgRIIb . FcgRIII ) which bind with different affinities to epitopes carried by Fc portions of IgG molecules. Because this nonspecific immobilization (ie one not triggered by one of the two binding domains of a bispecific molecule) is unfavorable for the following reasons: i) its effect on the pharmacokinetic properties of a molecule and ii) the off-target activation of immune effector cells various Fc variants and mutations discovered to be one FcgR Bond dissolve.

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) reveals the replacement of the radicals 233 - 236 of human IgG1 with the corresponding sequence, that of human IgG2 stems, resulting in a dissolved FcgRI Bond, resolved C1q Bond and a diminished FcgRIII Binding leads.

EP1075496 discloses antibodies and other Fc-containing molecules that exhibit variations in the Fc region ( 233P . 234V . 235A and no rest or G at the position 236 and 327g . 330S and 331S ), wherein the recombinant antibody is capable of binding the target molecule without causing significant complement dependent lysis or cell mediated destruction of the target.

Dual Affinity Retargeting (DART) molecules are used, for example, to target the destruction of B cell lymphomas by optimally inverted T cells.

The original DART technology is described in Moore et al. (In: Application of dual affinity retargeting molecules to achieve optimally redirected T-cell killing of B-cell lymphoma, Blood. Apr. 28, 2011; 117 (17): 4542-51 ). Comparison with a single chain, bispecific, antibody-bearing, identical CD19 and CD3 Antibody Fv sequence showed that DART molecules are more effective in controlling B cell lysis. The further development of DART technology was achieved by the DART-Fc molecules as described in 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). Among other positive features, this molecule combined the high efficacy of the DART with the extended serum half-life of Fc based molecules.

The aßTCR ( TCR ) recognizes antigenic peptides presented by MHC and is responsible for the specificity of T cells. Both α- and β-chains of TCR have variable (V) and constant domains. The V domains are involved in the binding of the antigenic peptides and the constant domains extend through the membranes of the T cells. In the crystal structure analysis of a bound to the peptide-MHC complex TCR , interacting Complementarity Determining Regions (CDR) 3 from both the V _α - as well as the V _β chains preferably with peptides, while CDRs 1 and 2 interact with the MHC. However, the recognition of the peptides by CDR 1 and recognizing the MHC by CDR 3 also described ( Piepenbrink et al., The basis for limited specificity and MHC restriction in a T cell receptor interface, Nat Commun, 2013; 4, 1948 ). The TCR ate heterodimer is tight with CDR3 proteins, CD4 or CD8 and other adhesion and signal transduction proteins. The binding of antigenic peptides by the TCR V regions triggers T cell activation by signaling through the constant TCR Domains over CD3 and CD4 or CD8 cytoplasmic proteins.

Unlike the full-length TCR Format bring single-chain TCRs ( scTCRs ) have considerable advantages in terms of the creation, the expression of soluble proteins and the medical possibilities. From the perspective of the expression of soluble proteins (ie the preparation) are TCRs prepared as a single polypeptide, avoiding each TCR Chain as a separate polypeptide and to allow larger amounts of properly assembled scTCR which binds to its peptide-MHC ligand. This feature may allow to achieve manufacturing yields necessary for medical use. From a medical point of view, finally scTCRs only from the V regions ( SCTV ), as therapeutics or diagnostic agents similar to scFv fragments.

US2006-0166875 discloses a single-chain T cell receptor ( scTCR ) comprising a segment formed by a sequence of the TCR alpha chain variable region fused to the N-terminus of a TCR alpha chain constant region extracellular sequence, a beta segment consisting of a TCR beta chain variable region fused to the N-terminus of a TCR beta chain constant region extracellular sequence, and a linker sequence comprising the C terminus of the N-terminus segment the beta segment or vice versa linked to the extracellular Sequences of the alpha and beta segments of the constant region are linked by a disulfide bond, the length of the linker sequence and the position of the disulfide bond are designed so that the sequences of the variable region of the alpha and beta segments substantially as in native alpha / beta T cell receptors are aligned together. Complexes of two or more scTCRs and the use of scTCRs in therapy and in various examination applications are also disclosed. In contrast to that in the US 2006-0166875 described scTCR , discloses the US 2012-0252742 a soluble, human, single chain TCR without constant domains consisting only of the variable fragments of the TCR ( SCTV ), which is useful for many purposes, including the treatment of cancer, viral diseases and autoimmune diseases.

McCormack E, et al. (In: Bi-specific TCR-anti CD3 redirected T-cell targeting of NYESO-1 and LAGE-1-positive tumors. Cancer Immunol Immunother. Apr 2013; 62 (4): 773-85 ) disclose that NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy and associate the upregulation in many cancers with the highly restricted expression in normal tissues and a common HLA-A * 0201 epitope, 157-165, share. They present data to describe the specificity and antitumor activity of a bifunctional ImmTAC which is a soluble, high affinity T cell receptor ( TCR ) specific for a NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent has been shown to kill Imm TAC-NYE, HLA-A2, antigen-positive tumor cell lines and freshly isolated HLA-A2 and LAGE-1 positive NSCLC cells. The in vivo optical imaging results demonstrate the in vivo alignment of fluorescently labeled high affinity NYESO-specific TCRs on HLA-A2, NYESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE activity was tested in a tumor model in which human lymphocytes were stably implanted into immunodeficient NSG mice already containing tumor xenografts, thereby having an effect in both tumor prevention using a GFP fluorescence reading system as well as in established tumor models. Quantitative RT-PCR was used to assess expression of both NY-ESO-1 and LAGE-1 antigens 15 normal tissues, 5 To examine cancer cell lines, 10 NSCLCs and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed in the tumor samples at a higher frequency and in greater amounts than NY-ESO-1. ImmTACs include a single-chain Fv derived from anti-CD3 antibody UCHT-1, which is covalently linked to the C- or N-terminus of the alpha or beta chain of the TCR linked, stems.

It is an object of the present invention to provide improved bispecific molecules capable of targeting peptide-MHC complexes which are easily prepared, show high stability, and moreover exhibit high activity when attached bind the respective antigen epitopes. Other objects and advantages of the present invention will become apparent upon examining the following description and preferred embodiments thereof as well as the respective examples.

In a first aspect of the invention, the above object is achieved by providing a double specificity polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain is a variable domain first binding region ( VD1 derived from an antibody capable of recruiting human immune effector cells by specifically binding to a surface antigen of these cells, and a first variable domain binding region ( VR1 ), by one TCR which is specific for an MHC-associated peptide epitope and a first linker segment ( LINK1 ) connecting the two domains, wherein the second polypeptide chain comprises a second variable domain binding region ( VR2 ), that of a TCR which is specific for an MHC-associated peptide epitope and a second binding region of a variable domain ( VD2 ) derived from an antibody capable of recruiting human immune effector cells by binding specifically to a surface antigen of these cells, and a second linker section ( LINK2 ) connecting the two domains, this first binding region ( VD1 ) and this second binding region ( VD2 ) to form a first binding site ( VD1 ) ( VD2 ) which binds the epitopes on the cell surface molecule, this first binding region ( VR1 ) and this second binding region ( VR2 ) to form a second binding site ( VR1 ) ( VR2 ) to bind this MHC-associated peptide epitope, at least one of said polypeptide chains being linked at its C-terminus to hinge regions, CH 2 and / or CH3 domains or portions thereof derived from human IgG and wherein said dual specificity polypeptide molecule is capable of simultaneously binding to the immune effector cell antigen and the MHC-associated peptide epitope.

Preferably, the dual specificity polypeptide molecule of the present invention binds with high specificity to both the immune effector cell antigen and a specific antigenic epitope as a peptide-MHC complex e.g. With a binding affinity ( KD ) of about 100 nM or less, about 30 nM or less, about 10 nM or less, about 3 nM or less, about 1 nM or less.

The double specificity polypeptide molecule of the present invention according to the present invention is represented herein by a double specificity polypeptide molecule comprising a first polypeptide chain of SEQ ID NO. 16 and a second polypeptide chain with SEQ ID NO. 17, exemplified.

In a second aspect of the invention, the above object is achieved by providing a nucleic acid (s) encoding a first polypeptide chain and / or a second polypeptide chain, as disclosed herein, or an expression vector (s) containing that nucleic acid include, encode. In a third aspect of the invention, the object set forth above is achieved by providing a host cell comprising a vector / vectors as defined herein.

In a fourth aspect of the invention, the above object is achieved by providing a method of producing a dual specificity polypeptide molecule according to the present invention which comprises appropriate expression in a suitable host cell of said expression vector (s), such as which comprises nucleic acid (s), and comprises the appropriate purification of the molecule (s) from the cell and / or its medium.

In a fifth aspect of the invention, the above object is achieved by providing a pharmaceutical composition comprising the double specificity polypeptide molecule according to the invention, the nucleic acid or the expression vector (s) according to the invention or the cell according to the invention together with one or more pharmaceutically acceptable carrier (s) or excipient (s).

In a sixth aspect of the invention, the invention relates to the polypeptide molecule having dual specificity according to the invention, the nucleic acid (s) or the expression vector (s) according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention medical use.

In a seventh aspect of the invention, the invention relates to the polypeptide molecule of dual specificity according to the invention, the nucleic acid or the expression vector (s) according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in the Treatment of a disease or disorder as disclosed in this patent, especially selected from cancers and infectious diseases.

In an eighth aspect of the invention, the invention relates to a method of treating a disease or disorder comprising administering a therapeutically effective amount of the double specificity polypeptide molecule according to the invention, the nucleic acid or the expression vector (s) according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention.

As mentioned above, the invention provides novel and improved polypeptide molecules with dual specificity. The molecules usually comprise a first polypeptide chain and a second polypeptide chain, wherein the chains together provide a variable domain of an antibody specific for an epitope or an immune effector cell surface antigen, and a variable domain of an antibody TCR which is responsible for an MHC-associated peptide epitope, e.g. As a cancer epitope or epitopes resulting in an infection, eg. A viral infection such as HIV, is specific. Variable domains of antibodies and TCRs are stabilized by covalent and non-covalent bonds formed between Fc portions or portions thereof located on both polypeptide chains. The polypeptide molecule of dual specificity is then able to bind simultaneously to the cell receptor and the MHC-associated peptide epitope.

In the context of the present invention, variable domains ( VD1 ) and ( VD2 ) derived from antibodies capable of recruiting human immune effector cells by binding specifically to a surface antigen of these effector cells. In a particular embodiment, these antibodies bind specifically to epitopes of the TCR - CD3 Complex of human T cells comprising the peptide chains TCRalpha, TCRbeta, CD3gamma, CD3delta, CD3epsilon, and CD3zeta.

EP1868650 refers to diabody molecules and their use in the treatment of a variety of diseases and disorders, including immunological disorders, infectious diseases, intoxications and cancers. The diabody molecules comprise two polypeptide chains that associate to form at least two epitope binding sites that can recognize the same or different epitopes on the same or different antigens. In addition, the antigens may be from the same or different molecules. The individual polypeptide chains of the diabody molecule can be covalently linked by covalent bonds other than peptide bonds, such as, but not limited to, disulfide bonds of cysteine residues located within each polypeptide chain. In particular embodiments, the diabody molecules further include an Fc region, which is disclosed herein, as it enables the generation of antibody-like properties (eg, long half-life) in the molecule. EP 1868650 requires the presence of binding regions of variable light chain or heavy chain domains of an immunoglobulin and discusses extensively functional Fc receptor binding agents.

The double specificity polypeptide molecule according to the present invention comprises a first polypeptide and a second polypeptide chain comprising a first ( VD1 ) and a second ( VD2 ) Provides a variable domain binding region derived from an antibody capable of recruitment of human immune effector cells by specifically binding to a surface antigen of those cells. This first binding region ( VD1 ) and this second binding region ( VD2 ) to form a first binding site ( VD1 () VD2 ) which binds the epitope of the immune effector cell surface antigen. In addition, the first and second polypeptide chains of the polypeptide molecule comprise a first ( VR1 ) and a second ( VR2 ) Binding region, each of a variable domain, by a TCR which is specific for an MHC-associated peptide epitope. This first binding region ( VR1 ) and this second binding region ( VR2 ) to form a second binding site ( VR1 () VR2 ) which binds the epitope of this MHC-associated peptide. In one embodiment of the dual specificity polypeptide according to the invention, the order / orientation of the regions in the first polypeptide chain is selected from VD1-LINK1-VR1 and VR1 - LINK1 - VD1 in another embodiment, in the order / orientation of the regions in the second polypeptide chain is selected from VD2-LINK2-VR2 and VR2-LINK-VD2, ie the location of the binding sites can be re-arranged into a "left-handed" or "right-handed" molecule (please refer ). Also, the configuration of the alpha and beta chains of the with TCR linked part exchanged.

In the light of the present invention, the dual affinity polypeptide molecule of the invention is exemplified by a construct that binds the SLYNTVATL peptide (SEQ ID NO: 7) when presented as a peptide-MHC complex. Nonetheless, the concept of the invention is clearly not limited to this particular peptide and, in principle, includes any disease or disorder related epitope presented in the context of the MHC molecule. This presentation can be made both in terms of MHC class I or II. Major histocompatibility complex class I molecules (MHC-I) are present on the surface of all nucleated cells and show a wide variety of peptide epitopes for monitoring by the CD8 ⁺ T-cell repertoire. CD8 ⁺ T-cell responses are essential for the control and clearance of viral infections as well as the elimination of transformed and tumorigenic cells. Examples of preferred peptide epitopes to be recognized can be found in the corresponding literature and in particular include those in Tables 1 to 5 of WO 2016/170139 ; Tables 1 to 5 of WO 2016/102272 ; Tables 1 or 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 ; and Tables 1 to 4 of PCT / EP2016 / 073416 disclosed peptides for the treatment (s) of cancer, the contents of each of which applications have been fully incorporated by reference herein.

Other suitable epitopes can be identified by databases such as the Immunopitope Database (accessed at http://www.iedb.org/).

The term "human immune effector cell (s)" refers to a cell within the natural cell repertoire in the human immune system which, when activated, is capable of inducing a change in the viability of a target cell. The term "viability of a target cell" may, within the scope of the invention, refer to the ability of the target cell to survive, proliferate and / or interact with other cells. This interaction can occur either directly, for example when the target cell is in contact with another cell or indirectly, for example when the target cell secretes substances, which have an impact on the functioning of another, remote cell. The target cell may be either a human native cell or a nonhuman cell. In case the cell is a human native cell, the target cell is advantageously a cell that has changed and become a malignant cell. The native cell may additionally be a pathologically altered native cell, for example a native cell associated with an organism, such as an organism. B. a virus, a plasmodium or a bacterium is infected. In the case where the cell is not a human but a non-human cell, the target cell is advantageously an invaded pathogenic cell, for example an invaded bacterium or plasmodium.

 IgG3:
 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE216PKSCDTPPPCPRCPAPELLG
 (SEQ ID No. 3)

Preferred is the polypeptide molecule having dual specificity according to the invention, said first and second polypeptide chains further comprising at least one hinge domain (Hingdomäne) and / or an Fc domain or a part thereof. In antibodies, the "hinge" or "hinge region" or "hinge domain" refers to the flexible part of a heavy chain located between the CH1 domain and the CH2 domain. It is about 25 amino acids long and is divided into an "upper joint", a "middle joint" or a "core joint" and a "lower joint". An "articular subdomain" refers to the upper joint, the middle (or core) joint, or the lower joint. The amino acid sequences of the joints of a IgG1 . IgG2 . IgG3 and IgG4 Molecule read as follows (EU numbering is indicated):

 IgG3:
 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE216PKSCDTPPPCPRCPAPELLG
 (SEQ ID No. 3) 

The core joint region usually contains at least one cysteine bridge connecting the two heavy chains. In addition, lower hinge region mutations can be triggered to reduce unwanted antibody-dependent cell-mediated cytotoxicity ( ADCC ) to improve.

Preferred is a polypeptide molecule with dual specificity according to the present invention which comprises at least one IgG domain with a crystallizable fragment ( fc ), ie, a crystallizable fragment (Fc region), the end region of an antibody that interacts with Fc receptors and some proteins of the complement system. Fc regions comprise two or three heavy chain constant domains (CH domains 2 . 3 and 4 ) in each polypeptide chain. The Fc regions of IgG also carry a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is of essential importance for Fc receptor-mediated activity. The small size of bispecific antibody formats such as BiTEs and DARTs (~ 50 kD) can lead to rapid clearance and a short half-life. Therefore, the scTv cellular receptor polypeptide molecule having dual specificity can be linked to one (human IgG1 ) Fc domain can be fused to increase the molecular mass and thereby obtain improved pharmacokinetic properties. Several mutations at the interface between the CH2 and CH3 domains, such as T250Q / M428L and M252Y / S254T / T256E + H433K / N434F, have been shown to enhance binding affinity to the neonatal Fc receptor in vivo ( FcRn ) and the half-life of IgG1 increase. This could further extend the serum half-life of an Fc-containing molecule.

In the double specificity polypeptide molecules of the invention, this may be Fc Domain comprising a CH2 domain containing at least one mutation that shuts down the effector function. Preferably, these mutations are incorporated into the ELLGGP sequence of human IgG1 (residues 233 - 238 or corresponding residues from other isotypes) that are known to be relevant to effector functions. Basically, one or more mutations that are from the IgG2 correspond to corresponding residues and / or IgG4 in IgG1 Fc brought in. Preference is given to: E233P, L234V, L235A and no residues or G at position 236 , Another mutation is P331S. EP1075496 discloses a recombinant antibody comprising a chimaeric domain derived from two or more human immunoglobulin heavy chain CH2 domains, wherein the human immunoglobulins of IgG1 . IgG2 and IgG4 selected and the chimeric domain is an immunoglobulin heavy chain CH2 domain having the following amino acid blocks at the positions shown: 233P, 234V, 235A and no residue or G at position 236 and 327g . 330S and 331S in accordance with the EU numbering system, and is at least 98% CH2 Sequence (residues 231 - 340 ) of human IgG1 . IgG2 or IgG4 identical, which has these modified amino acids.

 231-
 APPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
 VHNAKTKPREEQYQSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEK-
 334 (SEQ ID No. 5);

und

 231-
 APPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEK-
 334 (SEQ ID No. 6)

mit unterstrichenen Änderungen, die an Position 297 ein N (glykosilierte Variante) oder einen aus der Gruppe von A, G und Q (deglykosilierte Variante) ausgewählten Rest enthalten.Examples of preferred CH 2 subsequences to be used may be (fully or partially) as follows:

 231-
 APPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
 VHNAKTKPREEQYQSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEK-
 334 (SEQ ID No. 5); 

and

 231-
 APPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
 VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEK-
 334 (SEQ ID No. 6) 

with underlined changes in position 297 an N (glycosylated variant) or one from the group of A . G and Q (deglycosylated variant) selected radical.

In the double specificity polypeptide molecules of the invention, this Fc domain may comprise a CH3 domain containing at least one mutation facilitating the formation of heterodimers. In order to maximize the yield of the desired heterodimeric Fc protein with dual specificity and to simplify the purification, "Knobs-into-holes" mutations can be generated in the Fc domain. Fc domains having this structure are moved to form heterodimers rather than their normal homodimers by protruding bulky, hydrophobic "knobs" on a chain and complementary hydrophobic "holes" on the chain other chain are generated. The 'knob' variant can be obtained by replacing a small amino acid with a larger one for insertion into a hole in the opposite domain, which has been generated by the expedient replacement of a large residue with a smaller one (Ridgway, JBB, Presta, LG Carter, P. "Knobs-into-holes" engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Eng., 1996, 9, 617-621; WO 2002/002781 ).

Preferred is a polypeptide molecule with dual specificity according to the invention, this "knob-in-hole" mutation being selected from T366W as knob and T366'S, L368'A and Y407'V as hole in the CH3 domain (see, e.g. B. WO 98/50431 ). This set of mutations can be extended by including mutations K409A and F405'K as described by Wei et al. (Structural basis of a novel heterodimeric Fc for bispecific antibody production, oncotarget. 2017. Another knob can be T366Y and the hole is Y407'T.

The dual specificity polypeptide molecules of the invention may further comprise artificially introduced cysteine bridges between at least one cysteine residue on the first polypeptide chain and at least one cysteine residue on the second polypeptide chain to improve the stability of the molecules, optimally without affecting the binding properties of the bivalent molecules, and / or to achieve improved heterodimerization. For additional stability, the addition of a single cysteine to the CH3 domain of both the knob and hole chains can introduce a disulfide bond. Preferred is a polypeptide molecule having dual specificity according to the invention, wherein the Fc domain comprises a CH3 domain comprising at least one additional cysteine residue, for example S354C and / or Y349C.

Preference is given to a polypeptide molecule with dual specificity according to the invention, this CD molecule being selected from the group of CD3 CD3 molecules related to the immune response, such as e.g. CD3y, CD3δ and CD3ε chains, CD4, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41b, CD41b, CD42a, CD42b, CD44, CD45a, CD49, CD55, CD56, CD61, CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD153, CD193, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI, TCRalpha / beta, TCRgamma / delta and HLA-DR. Depending on the combination of the two antigen-binding moieties of the dual specificity polypeptide molecule of the invention, specific advantages in terms of the functionality of the molecule, in particular increased efficacy, may be achieved.

Preferred is the exemplary polypeptide molecule having dual specificity according to the invention, wherein the regions in the first polypeptide chain SEQ ID NO. 28 for VD1 , SEQ ID no. 29 for VR1 , SEQ ID no. 30 for LINK1 and the regions in the second polypeptide chain SEQ ID NO. 31 for VD2 , SEQ ID no. 32 for VR2 , and SEQ ID no. 30 for LINK2 include.

Also preferred is the exemplary polypeptide molecule of dual specificity according to the invention, wherein the FC region in the first polypeptide chain SEQ ID NO. 26 ( Fc1 ) and the FC region in the second polypeptide chain SEQ ID NO. 27 ( Fc2 ).

Further preferred is the exemplary polypeptide molecule having dual specificity according to the invention, which comprises a first polypeptide chain with SEQ ID NO. 16 ( 1 , complete molecule) and a second polypeptide chain with SEQ ID no. 17 ( 2 , complete molecule).

Even more preferred is the exemplary polypeptide molecule of dual specificity according to the invention, wherein said first binding site ( VD1 ) ( VD2 ) containing the epitope of the surface antigen of human immune cells (e.g. CD3 ) binds, is humanized, and / or this second binding site ( VR1 ) ( VR2 ) which binds this MHC-associated peptide epitope has matured in terms of affinity.

Humanized antibodies are antibodies (or parts thereof) of a non-human species whose protein sequences have been modified to enhance their similarity to antibody variants produced in humans under natural conditions. The process of "humanization" is usually applied to monoclonal antibodies that have been developed for administration to humans (for example, antibodies that have been developed as anticancer drugs). Suitable methods for humanization are known from the literature, and are described, for example, in Olimpieri, Pier Paolo, Paolo Marcatili and Anna Tramontano checked. "Tabhu: Tools for Antibody Humanization." Bioinformatics 31.3 (2015): 434-435 , PMC; Safdari Y1, Farajnia S, Asgharzadeh M, Khalili M. Antibody humanization methods - a review and update. Biotechnol Genet Eng Rev. 2013; 29: 175-86 ; or Ahmadzadeh V1, Farajnia S, Feizi MA, Nejad RA. Antibody humanization methods for development of therapeutic applications. Monoclon Antib Immunodiagn Immunother. Apr. 2014; 33 (2): 67-73 ,

In general, the affinity maturation of TCRs according to the methods described in the literature, in particular by using the presentation on the surface of yeast or phage (for example based on Holler PD, including in vitro evolution of a T cell receptor with high affinity for peptide / MHC. Proc Natl Acad Sci USA. 9 May 2000; 97 (10): 5387-92 ; Boder et al., Directed evolution of antibody fragments with monovalent femtomolar antigen binding affinity. Proc Natl Acad Sci USA. 26 Sep 2000; 97 (20): 10701-5 ; and, as a younger example, Zhao Q, et al. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential. Leukemia. 2015; 29 (11): 2238-2247 ).

The binding sites ( VD1 ) ( VD2 ) and ( VR1 () VR2 ) of the present specification preferably bind specifically to a peptide HLA molecule complex or a surface antigen of human immune cells. As used herein in connection with binding sites of the present specification, the terms "specific binding" and grammatical variants thereof are used with the meaning to designate a site having a binding affinity ( KD ) for a peptide-HLA molecule complex and / or an antibody epitope of 100 μM or less. The binding sites ( VD1 ) ( VD2 ) and ( VR1 () VR2 ) of the present specification bind to a peptide HLA molecule complex or a CD antibody epitope having a binding affinity ( KD ) of about 100 μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM or less. Even more preferred are high affinity binding sites having binding affinities of about 1 μM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less. By way of example only, the preferred ranges of binding affinity binding sites of the present invention are from about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; approximately 40nM to about 50nM; about 50 nM to about 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

In another preferred embodiment of the dual specificity polypeptide molecule of the invention, said molecule carries an active agent or a portion thereof coupled to or conjugated to the molecule. This agent can be selected from the group consisting of a detectable label, an immunostimulating molecule and a therapeutic.

The detectable label may be selected from the group consisting of biotin, streptavidin, an enzyme or its catalytically active fragments, a radionuclide, a nanoparticle, a paramagnetic metal ion or a fluorescent, phosphorescent or chemiluminescent molecule. Detectable labels for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

Therapeutics which may be associated with the molecules of the invention include immunomodulators, radioactive compounds, enzymes (for example perforin), chemotherapeutic agents (for example cisplatin) or a toxin. Other suitable therapeutics include low molecular weight cytotoxic agents, ie, compounds capable of killing mammalian cells which have a molecular weight of less than 700 daltons. Such compounds may also contain toxic metals which may exhibit a cytotoxic effect. In addition, it is believed that these low molecular weight cytotoxic agents also contain precursors, that is, agents that degrade or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cisplatin, maytansin derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreat glucuronate, auristatin E vincristine and doxorubicin; Peptide cytotoxins, ie proteins or fragments thereof capable of killing mammalian cells. For example, ricin, diphtheritoxin, bacterial exotoxin A from Pseudomonas, DNase and RNase, radio nuclides, ie, unstable isotopes of elements which decompose with simultaneous emission of one or more α or β particles or gamma rays. For example, chelating agents for iodine 131 , Rhenium 186 , Indium 111 , Yttrium 90 , Bismuth 210 and 213 , Actinium 225 and Astatine 213 can be used to facilitate the attachment of these radio-nuclides to the molecules or their multimers, immune stimulators, ie immune-effect molecules that stimulate the immune response. Examples of such immune effect molecules are cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet factor 4 , Melanoma growth-stimulating protein, complement activators or xenogeneic protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

Another aspect of the present invention then 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 such a nucleic acid. The nucleic acid molecule may be a DNA . cDNA . PNA . RNA and combinations thereof. The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be naturally occurring or synthetically produced. In general, DNA segments encoding the peptides, polypeptides and proteins of this invention will be useful cDNA Fragments and short oligonucleotide linkers or from an oligonucleotide series to provide a synthetic gene capable of being expressed in a recombinant transcriptional unit consisting of regulatory elements derived from a microbial or viral operon. The term "expression product" refers to the polypeptide or protein which is the natural translation product of the gene and any nucleic acid sequences encoding equivalents that result from the degeneracy of the genetic code and therefore encodes the same amino acid (n ). When referring to a coding sequence, the term "fragment" refers to a segment of DNA that comprises less than the entire coding region whose expression product retains substantially the same biological function or effect as the expression product of the complete coding region. Depending on the intended use, the nucleic acid may be codon-optimized for expression in a suitable (e.g., microbial) host cell. Redundancy in the genetic code allows some amino acids to be encoded by more than one codon, but certain codons are appropriate because of the relative availability tRNAs and other factors less "optimal" than others (Gustafsson et al., 2004). The nucleic acid may be, for example DNA . cDNA . PNA . RNA or combinations thereof, either single and / or double stranded, or native or stabilized forms of polynucleotides, such as polynucleotides with a phosphorothioate backbone, and may contain introns or contain no introns as long as they encode polypeptide chains.

The nucleic acid (eg. DNA ) can then be contained and / or expressed in a suitable host cell to produce a polypeptide comprising the polypeptide chain of the invention. Therefore, the nucleic acid (e.g. DNA ) encoding the polypeptide chain of the invention are used in accordance with known techniques that have been appropriately modified in view of the teachings herein to construct an expression vector which is then used to generate a suitable host cell for expression and expression Production of the polypeptide of the invention as known in the art transform. The nucleic acid (eg a DNA , or in the case of retroviral vectors, a RNA ) encoding the polypeptide chain (s) of which the compound of the invention is made can be treated with a wide variety of other sequences of nucleic acids (e.g. DNA ) for introduction to a suitable host. The supplemental nucleic acid depends on the nature of the host and the mode of introduction of the host DNA into the host and whether an episomal maintenance or integration is desired. In general, the nucleic acid is introduced into an expression vector, such as a plasmid, having the correct orientation and reading frame for expression. If necessary, the nucleic acid can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host using standard techniques. In general, not all hosts are transformed by the vector. Therefore, it will be necessary to select the transformed host cells. A selection technique involves inserting a nucleic acid sequence into the expression vector with all necessary control elements encoding a selectable property of the transformed cell, such as antibiotic resistance. Alternatively, the gene for such a selectable property may be another vector used to simultaneously transform the desired host cell. Host cells transformed by the recombinant nucleic acid of the invention were then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to enable expression of the polypeptide which can subsequently be recovered ,

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus spec), plant cells, animal cells and insect cells. Preferably, the system may be of mammalian cells, such as e.g. For example, CHO cells available from the ATCC Cell Biology Collection.

In one embodiment, the specification provides a method of producing a molecule as described herein, the method comprising culturing a host cell capable of expressing the polypeptide chain (s) under conditions suitable for expressing them To promote chain (s).

In one aspect, nucleic acid encoding polypeptide chains comprising TCR alpha and / or TCR beta binding domains are cloned into expression vectors, such as gammaretrovirus or lentivirus, to obtain cells expressing molecules of the present specification. In another aspect RNAs synthesized by techniques known in the art, e.g. In vitro transcription systems to obtain cells expressing molecules of the present specification. The in vitro synthesized RNAs are then electroporated into suitable cells to express polypeptide chains.

In order to increase expression, nucleic acid coding chains of the present specification may be operatively linked to strong promoters, such as retroviral long terminal repeats ( LTRs ), Cytomegalovirus ( CMV ), Mouse strain cell virus ( MSCV ) U3 , Phosphoglycerate kinase (PGK), β-actin, ubiquitin, and a monkey virus composite promoter 40 (SV40) / CD43, elongation factor (EF) -1a and the promoter of spleen [spleen] Focus-Forming Virus (SFFV). In a preferred embodiment, the promoter is heterologous to the expressed nucleic acid. In addition to strong promoters, expression cassettes of the present invention can contain additional elements that can enhance the expression of transgenes, including a central polypurine tract (cPPT) that promotes nuclear translocation of lentiviral constructs (Follenzi et al., 2000) and the woodchuck posttranscriptional regulatory element Hepatitis virus (wPRE), which increases the level of transgenic expression by increasing RNA stability (Zufferey et al., 1999).

The alpha and beta binding domain chains of a molecule of the present invention may be encoded by nucleic acids located in separate vectors or may be encoded by polynucleotides located in the same vector.

In one embodiment, a host cell has been designed to express a molecule of the present specification. Host cells of the present description may be allogenic or autologous with respect to a patient to be treated.

Yet another aspect of the invention relates to a pharmaceutical composition comprising the double specificity polypeptide molecule according to the invention, the nucleic acid (s) or expression vector (s) according to the invention or the cell according to the present invention together with one or more a plurality of pharmaceutically acceptable carrier (s) or excipient (s). The compositions of the invention include compositions of the mass production of drugs useful in the preparation of pharmaceutical compositions (e.g., contaminated or non-sterile compositions) and pharmaceutical compositions (ie, compositions suitable for administration to a subject or a patient suitable) which can be used in the preparation of dosage unit forms. Such compositions comprise a prophylactically or therapeutically effective amount of the dual specificity prophylactic and / or therapeutic polypeptide molecules (active ingredient) disclosed herein or a combination of the active ingredient and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of one or more molecules of the invention and a pharmaceutically acceptable carrier.

The pharmaceutical compositions preferably comprise the molecules either in free form or as a salt. Preferably, the salts are pharmaceutically acceptable salts of the molecules, such as chloride or acetate (trifluoroacetate) salts. It must be noted that the salts of the molecules according to the present invention deviate significantly from the molecules in their state / conditions in vivo since the molecules are not present as salts in vivo.

One embodiment of the present invention therefore relates to a molecule according to the invention which does not occur in nature which has been synthetically produced (e.g., synthesized) as a pharmaceutically acceptable salt. Methods for the synthetic production of peptides and / or polypeptides are well known in the art. The salts of the molecules according to the present invention differ significantly from the molecules in their state / conditions in vivo, since the molecules that are generated are not present as salts in vivo. Preferably, the salts are pharmaceutically acceptable salts of the molecules. These salts of the invention include alkali and alkaline, such as salts of the Hofmeister series, which the following anions: PO ₄ ^3-, SO ₄ ^2-, CH ₃ COO ^-, Cl ^-, Br ^-, NO ₃ ^-, ClO ₄ ^-, I ^-, SCN ^- and the following cations NH ₄ ^+, Rb ^+, K ^+, Na ^+, Cs ^+, Li ^+, Zn ^2+, Mg ^2+, Ca ^2+, Mn ^2+, Cu ^2+, and Include Ba ²⁺ . In particular, salts of (NH ₄ ) ₃ PO ₄ , (NH ₄ ) ₂ HPO ₄ , (NH ₄ ) H ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , NH ₄ CH ₃ COO, NH ₄ Cl, NH ₄ Br , NH ₄ NO ₃ , NH ₄ ClO ₄ , NH ₄ I, NH ₄ SCN, Rb ₃ PO ₄ , Rb ₂ HPO ₄ , RbH ₂ PO ₄ , Rb ₂ SO ₄ , Rb ₄ CH ₃ COO, Rb ₄ Cl, Rb ₄ Br, Rb ₄ NO ₃ , Rb ₄ ClO ₄ , Rb ₄ I, Rb ₄ SCN, K ₃ PO ₄ , K ₂ HPO ₄ , KH ₂ PO ₄ , K ₂ SO ₄ , KCH ₃ COO, KCl, KBr, KNO ₃ , KClO ₄ , KI, KSCN, Na ₃ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , Na ₂ SO ₄ , NaCH ₃ COO, NaCl, NaBr, NaNO ₃ , NaClO ₄ , NaI, NaSCN, ZnCl ₂ Cs ₃ PO ₄ , Cs ₂ HPO ₄ , CsH ₂ PO ₄ , Cs ₂ SO ₄ , CsCH ₃ COO, CsCl, CsBr, CsNO ₃ , CsClO ₄ , CsI, CsSCN, Li ₃ PO ₄ , Li ₂ HPO ₄ , LiH ₂ PO ₄ , Li ₂ SO ₄ , LiCH ₃ COO, LiCl, LiBr, LiNO ₃ , LiClO ₄ , Lil, LiSCN, Cu ₂ SO ₄ , Mg ₃ (PO ₄ ) ₂ , Mg ₂ HPO ₄ , Mg (H ₂ PO ₄ ) ₂ , Mg ₂ SO ₄ , Mg (CH ₃ COO) ₂ , MgCl ₂ , MgBr ₂ , Mg (NO ₃ ) ₂ , Mg (ClO ₄ ) ₂ , MgI ₂ , Mg (SCN) ₂ , MnCl ₂ , Ca ₃ ( PO ₄ ), Ca ₂ HPO ₄ , Ca (H ₂ PO ₄ ) ₂ , CaSO ₄ , Ca (CH ₃ COO) ₂ , CaCl ₂ , CaBr ₂ , Ca (NO ₃ ) ₂ , Ca (ClO ₄ ) ₂ , CaI ₂ , Ca (SCN) ₂ , Ba ₃ (PO ₄ ) ₂ , Ba ₂ HPO ₄ , Ba (H ₂ PO ₄ ) ₂ , BaSO ₄ , Ba (CH ₃ COO) ₂ , BaCl ₂ , BaBr ₂ , Ba ( NO ₃ ) ₂ , Ba (ClO ₄ ) ₂ , BaI ₂ , and Ba (SCN) ₂ . Particularly preferred are NH acetate, MgCl ₂ , KH ₂ PO ₄ , Na ₂ SO ₄ , KCl, NaCl and CaCl ₂ , such as chloride or acetate (trifluoroacetate) salts.

The invention also encompasses pharmaceutical compositions comprising a polypeptide molecule of the invention having dual specificity and a therapeutic antibody (eg, a tumor-specific monoclonal antibody) specific for a particular cancer antigen and a pharmaceutically acceptable carrier.

In a specific embodiment, the term "pharmaceutically acceptable" means that the pharmaceutically acceptable article has been approved by a state or state government agency or is listed in the US Pharmacopoeia or other generally accepted pharmacopoeia for use in animals or specifically in humans. The term "carrier" refers to a diluent excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including oils of mineral, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, lime, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, sodium phosphate, sodium acetate, L-histidine, dried skimmed milk, glycerol, propylene, glycol, water , Ethanol and the like. If desired, the composition may also contain minor amounts of wetting or emulsifying or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Generally, the ingredients of compositions of the invention are either added separately or mixed together in dosage unit form, for example as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet, on which the amount of active ingredient is indicated. If the composition is to be infused, it may be dispensed with an infusion bottle containing sterile water or pharmaceutical grade sterile saline. When the composition is administered by injection, a vial of sterile water or saline may be provided for injection so that the components may be mixed prior to administration.

Another aspect of the present invention then relates to the polypeptide molecule of dual specificity according to the invention, the nucleic acid or expression vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for medical use. In general, the use of the double specificity polypeptide molecule will depend on the medical background of the peptide antigen (s) recognized by that molecule, as further described below.

Preferred is the polypeptide molecule of dual specificity according to the invention, the nucleic acid or expression vector according to the invention or the cell according to the invention or the pharmaceutical composition according to the invention for use in the treatment or prevention of a disease or disorder resulting from immunological disorders, infectious diseases , Intoxication and cancers, including the treatment or prevention of a variety of cancers or other abnormal proliferative disorders, including (but not limited to) the following diseases: carcinoma, including the bladder, breast, bowel, kidney, liver, lung, Ovaries, pancreas, stomach, prostate, cervix, thyroid and skin, including squamous cell carcinoma, hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkett's lymphoma, hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma, other tumors including melanoma, seminoma, teratocarcinoma, Neuroblastoma and glioma, tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, schwannomas and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosum, keratoacanthoma, seminoma, thyroid carcinoma and teratocarcinoma. Additional cancers include, but are not limited to, follicular lymphoma, p53 mutant carcinomas, breast hormone-dependent tumors, prostate and ovaries, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific embodiments, changes in malignancy or dysproliferative changes (such as metaplasia and dysplasia) or hyperproliferative diseases are presented by the methods and compositions of the invention in the ovaries, bladder, breast, intestine, lung, skin, pancreas or the uterus treated or prevented. In other specific embodiments, sarcoma, melanoma or leukemia are treated with the methods and compositions of the invention.

The invention also provides methods for preventing, treating or alleviating one or more symptoms associated with an inflammatory disease of a subject, further comprising administering a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents according to the invention the subject. The invention also provides methods of preventing, treating or ameliorating one or more symptoms associated with an autoimmune disease, further comprising administering to the subject a therapeutically or prophylactically effective amount of one or more immunomodulating agents according to the invention. Infectious diseases that can be treated or prevented with the molecules of the invention are caused by infectious agents, including, but not limited to, viruses, bacteria, fungi, protozoa and viruses. Viral diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention include, but are not limited to, viral diseases characterized by hepatitis type A, type B hepatitis, Type C hepatitis, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papovavirus, cytomegalovirus, echinovirus , Arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, smallpox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and viral pathogens such as viral Meningitis, encephalitis, dengue or smallpox.

Bacterial diseases that can be treated or prevented using the molecules of the invention in association with the methods of the present invention caused by bacteria include, but are not limited to, Mycobacteria rickettsia, Mycoplasma, Neisseria, Streptococcus pneumoniae, Borrelia burgdorferi (Lyme disease ), Bacillus antracis (anthrax), tetanus, streptococcus, staphylococcus, mycobacterium, tetanus, pertussis, cholera, plague, diphtheria, chlamydia, S. aureus and legionella.

Protozoal diseases which can be treated or prevented using the molecules of the invention together with the methods of the present invention caused by protozoa include, but are not limited to, leishmaniasis; Coccidioa, Trypanosoma or malaria. Parasitic diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention caused by parasites include, but are not limited to, chlamydia and rickettsia.

Examples of infectious agents and infectious diseases include, but are not limited to, bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Proteus vulgaris, Staphylococcus viridans and Pseudomonas aeruginosa), a pathogen (e.g. Blymphotrophic papovavirus (LPV); Bordetella pertussis; Borna disease virus (BDV); Bovine coronavirus; Choriomeningitis virus; Dengue virus; a Virus, E. coli; Ebola; Echovirus 1; Echovirus-11 (EV); Endotoxin (LPS); Enterobacteria; entero-orphan virus; enterovirus; feline leukemia virus; foot-and-mouth disease virus; gibbon monkey leukemia virus (GALV); gram-negative bacteria; helicobacter pylorii; hepatitis B virus (HBV); herpes simplex virus; HIV-I; human cytomegalovirus human coronavirus; influenza A, B and C; Legionella; Leishmania mexicana; Listeria monocytogenes; measles virus; meningococcus; morbillivirus; murine hepatitis virus; murine leukemia virus; murine G amma-herpesvirus; murine retrovirus; murine coronavirus murine hepatitis virus; Mycobacterium avium-M; Neisseria gonorrhoeae; Newcastle Disease Virus; Parvovirus B 19; Plasmodium falciparum; Smallpox virus; Pseudomonas; rotavirus; Salmonella typhiurium; Shigella; Streptococci; T cell lymphocyte virus 1; Vaccinia virus).

In yet another aspect of the present invention, the invention relates to a method of treating a disease or disorder comprising administering a therapeutically effective amount of the dual specificity molecule according to the invention, the nucleic acid or the expression vector according to the invention to the cell according to of the invention or the pharmaceutical composition according to the invention.

The dual specificity polypeptide molecule can be used in a method for preventing or treating a disease or condition that is enhanced by administering the polypeptide molecule having dual specificity. Such treatments may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. Therapeutic polypeptide molecules having dual specificity are usually provided as part of a sterile pharmaceutical composition which will normally comprise a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form (depending on the desired method of administration to a patient). It may be provided in unit dosage form and is generally provided in a sealed container and may be provided as part of a kit. This kit would normally (but not necessarily) include instructions for use. It may contain a variety of these dosage unit forms. The pharmaceutical composition may be adapted for administration in any suitable manner, such as parenteral (including subcutaneous, intramuscular or intravenous) routes. These compositions may be prepared by any method known in the art in the pharmaceutical arts by mixing the active ingredient with the carrier (s) or excipient (s) under sterile conditions.

Doses of the dual specificity polypeptide molecules of the present invention may vary within a wide range, depending on the disease or disorder to be treated, For example, the age and condition of the subject to be treated, etc., for example, a suitable dosage range for a polypeptide molecule with dual specificity may be between 25 ng / kg and 50 μg / kg. A doctor will eventually determine the appropriate dosages to administer.

Pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in a substantially pure form, for example having a purity of 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%.

Preferred features of each aspect of the invention apply mutatis mutandis to any other aspect. The documents mentioned herein are fully incorporated, to the extent permitted by law. Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. The present invention will be further described in the following examples, which are provided for the purpose of illustration only and are not intended to limit the invention in any way.

shows a schematic overview of a preferred embodiment of the present invention, the human IgG1 Fc -containing polypeptide molecule having dual specificity. VD1 . VD2 = variable domains derived from antibodies; VR1 . VR2 = variable domains of TCR come; link1 . Link2 = connecting linkers; Cys-Cys = Cysteine bridges.

shows a schematic overview of 4 different constructs of IgG Double-specificity Fc-containing polypeptide molecules that have been studied in the background of the present invention. Black = variable domains of TCR come; light gray = variable domains derived from antibodies; white = constant domains of human IgG come. Knob-hole mutations are indicated by a cylinder. The diabody molecules IA - ID agree with the invention.

shows the HPLC SEC analysis of various bispecific TCR / mAb Molecules with a molecular structure according to the in shown constructs, which were purified in a 2-column purification process. The monomers contained in the different molecules were determined as follows. II: 93.84%; III: 96.54%; IV: 98.49%; IA_1: 95.48%; IA_3: 98.45%; ID_1: 95.75%; IC_4: 95.22%; IC_5: 92.76%; ID_4: 99.31%; ID_5: 99.44%.

shows the results of the efficacy assay with different bispecific TCR / mAb constructs (as in shown) as on IgG4 based molecules were produced. Jurkat_NFATRE_Iuc2 cells were co-infected with HIV peptide SLYNTVATL (SEQ ID NO: 7) T2 cells in the presence of increasing concentrations of bispecific TCR (bssTCR) molecules incubated. The bispecific TCR / mAb diabody molecule IA-IgG4 showed greater efficacy than two alternative TCR / mAb molecules with dual specificity.

shows the results of the efficacy assay with different bispecific TCR / mAb constructs (as in shown) as on IgG1 based molecules were produced. Jurkat_NFATRE_Iuc2 cells were co-infected with HIV peptide SLYNTVATL (SEQ ID NO: 7) T2 cells in the presence of increasing concentrations of bispecific TCR (bssTCR) molecules incubated. The bispecific TCR / mAb Diabody molecules ID_1 . IA_3 and IA1 showed a much greater effectiveness than three alternative TCR / mAb molecules with dual specificity.

shows the results of the efficacy assay, with different, on IgG1 based molecules with bispecific TCR / mAb constructs (as in shown), using different variable antibody domains, both of which are based on the TCR - CD3 Target complex. construct ID_1 comprises variable domains from that CD3 targeted antibody UCHT1 (V9), while the constructs ID_4 and ID_5 variable domains of alpha / beta TCR specific antibody BMA031. Jurkat_NFATRE_Iuc2 cells were co-infected with HIV-peptide SLYNTVATL (SEQ ID NO 7) T2 cells in the presence of increasing concentrations of bispecific TCR (bssTCR) molecules incubated.

Figure 4 shows a schematic overview of the possible orientations of the VD and VR domains in the molecules of the present invention. VH: antibody-derived VH domain, VL: antibody-derived VL domain, Va: von TCR native Valpha; Vβ: from TCR derived Vbeta.

shows the results of HPLC-SEC analysis of aggregates (HMWS high molecular weight species) within different bispecific IgG1 based TCR / mAb molecules. Aggregates were analyzed after purification and storage of the molecules at 40 ° C for 1 week and 2 weeks, respectively.

shows the results of the efficacy assay involving different bispecific IgG1 based TCR / mAb molecules was performed. The efficacy was analyzed after purification and storage of the molecules at 40 ° C for 1 week and 2 weeks, respectively. Storage at 40 ° C under load did not result in significant loss of efficacy of the molecules, but a dramatic increase in nonspecific (ie, target-independent) activation of Jurkat T cells was detected for molecules III and IV.

EXAMPLES

EXAMPLE 1

Structure of Fc-containing bispecific TCR / mAb diabodies and control molecules.

Fc-containing, bispecific TCR / mAb Diabodies and Control Molecules (as in ) were prepared to be specific to the human TCR - CD3 Complex and to the peptide: to bind MHC complex, which is that of HIV derived peptide SLYNTVATL linked to HLA-A2 * 01. To be on the TCR - CD3 Complex, VH and VL domains derived from the CD3 HUCHT1 (V9) -specific humanized antibody described by Zhu et al. (Identification of heavy chain residues in a humanized antibody). 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 alpha / beta TCR-specific antibody BMA031 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 in the humanized version variant 10 (In-house generated data) used To address the peptide: MHC complex, Valpha and Vbeta domains of the stability described above and the affinity matured human single-chain T cell receptor 868Z11 disclosed 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 ).

In the case of Fc-containing, bispecific TCR / mAb Diabodies DNA sequences encoding various combinations of VH and VL (the respective VD1 and VD2 correspond) and Va and Vb (each VR1 and VR2 correspond), as well as the coding for the linker link1 and Link2 were obtained by gene synthesis. resulting DNA Sequences were cloned in expression vectors encoding the hinge region, the CH2 and CH3 domain are each from human IgG4 [Registration number: K01316] and IgG1 [Registration Number: P01857] and have been further manipulated. The manipulation was performed to insert knob-in-hole mutations in CH3 domains with and without additional stabilization by disulfide bonds between the chains to form an N-glycosylation site in CH2 (e.g., N297Q mutation) to introduce Fc cut-off mutations to introduce VL and VH stabilization by additional disulfide bonds, respectively, according to the methods described by Reiter et al. Stabilization of the Fv Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions. Biochemistry, 1994, 33, 5451-5459 ). An overview of manufactured bispecific TCR / mAb Diabodies, the variants and the corresponding sequences are listed in Table 1. Table 1 Overview of all generated and verified Fc-containing bispecific TCR / mAb diabodies: KiH: Knob-into-hole; K / O: Fc-off; KiH-ds: Knob-into-hole stabilized with synthetic disulfide bonds to connect CH3: CH3 '; ds-HUCHT1 (V9): disulfide-bond-stabilized hUCHT1 (V9) variable domains; Link1: Linker connecting VR1 and VD1. molecule TCR mAb SEQ IDs modifications IA-IgG4 868Z11 HUCHT1 (V9) SEQ ID no. 8th 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 (var1 0) SEQ ID no. 18 IgG1 (K / O, KiH-ds) SEQ ID no. 19 IC_5 868Z11 hBMA031 (var1 0) SEQ ID no. 20 IgG1 (K / O, KiH-ds) extended link1 SEQ ID no. 21 ID_4 868Z11 hBMA031 (var1 0) SEQ ID no. 22 IgG1 (K / O, KiH-ds) SEQ ID no. 23 ID_5 868Z11 hBMA031 (var1 0) SEQ ID no. 24 IgG1 (K / O, KiH-ds) extended link1 SEQ ID no. 25

Various control molecules exhibiting the same specificities were constructed, Table 2, wherein these VH, VL, Valpha, and Vbeta domains are in combinations with constant domains derived from IgG1 or IgG4 and include features such as the features described above. Table 2 Overview of all generated and verified Fc-containing bispecific control molecules: KiH: Knob-into-hole, K / O: Fc-switched off. molecule TCR mAb SEQ IDs 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

Preparation and purification of Fc-containing, bispecific TCR / mAb diabodies.

Vectors for the expression of recombinant proteins have been created as monocistronic pUC19 derivatives, which are controlled by promoter elements derived from HCMV come. plasmid DNA was amplified in E. coli according to standard culture techniques and subsequently purified using commercially available kits (Macherey & Nagel). Purified plasmid DNA was used for the transient transfection of CHO-S cells according to the manufacturer's instructions (ExpiCHO ™ system; Thermo Fisher Scientific). Transfected CHO cells were cultured for 6-14 days at 32 ° C to 37 ° C and received one to two feeds of ExpiCHO ™ Feed-Solution.

The conditioned cell supernatant was harvested by centrifugation (4000 x g, 30 minutes) and purified by filtration (0.22 μm). Bispecific molecules were purified using an Äkta Pure 25 L FPLC system (GE Lifesciences) equipped to perform parallel affinity and size exclusion chromatography. Affinity chromatography was performed on Protein A columns (GE Lifesciences) following standard protocols for affinity chromatography. Immediately after elution (pH 2.8) from the affinity column to obtain high purity monomeric proteins using standard protocols Superdex 200 pg 16/600 columns (GE Lifesciences), size exclusion chromatography was performed. Protein concentrations were determined on a NanoDrop System (Thermo Scientific) using calculated extinction coefficients according to the predicted protein sequences. If necessary, the concentration and exchange of the buffer was carried out using Vivaspin instruments (Sartorius). Finally, purified molecules were stored in phosphate buffered saline at concentrations of approximately 1 mg / ml at temperatures of 2-8 ° C.

Since therapeutic proteins are said to exhibit adequate stability upon exposure to acids to facilitate robust industrial purification processes, the percentage of monomeric protein eluting from the Protein A capture column was determined (Table 3). It is evident that the introduction of stabilizing mutations into molecules, as well as the selection of characteristic orientations of binding domains, significantly affects the stability in the action of acids. Table 3: Fraction of monomeric protein after acid elution from the scavenging column: molecule The monomer eluted from the scavenging column (% of the total peak area) IA-IgG4 (VH-beta) not defined IA_1 (VH-beta) 49 IA_2 (VH-beta) 54 IA_3 (dsVH-beta) 63 ID_1 (alpha-dsVH) 46 IC_4 (VH-alpha) 62 IC_5 (VH-alpha) 67 ID_4 (alpha-VH) 65 ID_5 (alpha VH) 69 II 39 III 51 IV 76

After size exclusion chromatography, the purified bispecific molecules showed high purity (> 93% monomeric protein) as determined by HPLC-SEC on MabPac SEC-1 columns (5 μm, 7.8x300 mm) prepared in 50 mM sodium phosphate, pH 6 , 8, 300 mM NaCl within an Agilent 1100 system, go through (see ). Non-reducing and reducing SDS-PAGE confirmed the purity and expected size of the various TCR / mAb molecules with dual specificity (data not shown).

EXAMPLE 3

Specific and target cell-dependent T cell activation triggered by Fc-containing TCR / mAb diabodies.

The effectiveness of Fc-containing TCR / mAb Diabodies was determined for T cell activation using the T cell activation bioassay (Promega). The assay consists of a genetically engineered Jurkat cell line expressing a luciferase reporter driven by a NFAT response element (NFAT-RE). The assays are performed according to the manufacturer's instructions. Briefly, T2 cells loaded with either the HIV-specific peptide SLYNTVATL or without peptide loading (uncharged control) were subsequently co-administered with Promegas modified Jurkat cells in the presence of increasing concentrations of bispecific TCR / mAb molecules cultured. Jurkat reporter T-cell activation was analyzed after 16-20 hours by measuring the luminescence intensity.

Representative efficacy assay results are given for IgG4-based ( ) and IgG1 -based bispecific TCR / mAb molecules ( ). The data show that regardless of the IgG isotype of the constant domains used, the Fc-containing TCR / mAb diabody constructs IA and ID showed superior T cell activation compared to the alternative bispecific TCR / mAb Constructs II, III and IV as measured by the magnitude of activation and / or EC50 values, respectively. In addition, non-specific T-cell activation was Fc -containing TCR / mAb Diabodies elicited against uncharged T2 cells decreased, or at least equal to the level of activation, for the alternative bispecific TCR / mAb constructs was observed. According to the above results, the TCR / mAb Double specificity diabody molecules around preferred molecules for therapeutic intervention, as they elicit strong effector T cell activation in a highly target-dependent manner.

EXAMPLE 4

The development of Fc-containing, bispecific TCR / mAb diabodies as a molecular platform

Fc -containing bispecific TCR / mAb Diabody constructs were created to serve as a molecular platform to set the framework for various of TCR and mAb-derived variable domains that target different peptide: MHC complexes and effector cell surface antigens. To validate eligibility as a platform, the mAb-derived domains were exchanged in a first set of molecules. The variable domains of hUCHT1 (V9) anti- CD3 Antibodies (construct ID_1) were antagonized by the domains of hBMA031 (var10). TCR Replaces antibodies that use the same domain orientation (constructs ID_4 and ID_5 ) or another orientation ( IC_4 . IC_5 ) (see Table 1 and for the details). The expression, purification and characterization of these molecules was performed as described above. The purity and integrity of the final preparations exceeded 92% according to HPLC-SEC analysis.

The results of the efficacy assay showed target-dependent Jurkat reporter T-cell activation and minimal nonspecific action against uncharged T2 cells for both antibody variable domains hUCHT1 (construct ID_1) and hBMA031 (constructs ID_4 and ID_5 ), which the suitability of the TCR / mAb Diabody constructs ( ) with dual specificity for the platform. Especially if the variable TCR and mAb domains of the construct ID_4 and ID_5 were switched to each Polypeptidkette, from which the constructs IC_4 and IC_5 no T cell activation was observed (data not shown). The latter findings show that nevertheless bispecific TCR / mAb Diabodies as a platform construct for the inclusion of various variable TCR - and mAb domains can be used, a careful optimization of the domain orientation is needed to achieve the optimal activity of the molecules.

EXAMPLE 5

Stability of Fc-containing bispecific TCR / mAb diabodies

The stability of bispecific TCR / mAb Molecules were originally determined using the Protein Thermal Exchange Assay (Thermo Fisher Scientific) according to the manufacturer's instructions using a 7500 real-time PCR system (Applied Biosciences). In a nutshell Purified molecules were mixed with PTS buffer and PTS color and exposed to an increasing temperature gradient, constantly monitoring the fluorescence of the samples. The recorded fluorescence signals were analyzed using PTS software (Thermo Fisher Scientific) and the melting temperatures (T _M ) were calculated by the derivative method.

Stress stability studies were performed by storing purified molecules that were dissolved in PBS at 40 ° C for up to two weeks. Samples were analyzed for integrity of the proteins using HPLC SEC and efficacy was assayed as described above using the T cell activation assay (Promega).

As expected, storage at 40 ° C triggered the formation of high molecular weight aggregates / species as determined by HPLC-SEC studies (see ). The results of the efficacy assay of IgG1 based molecules after purification and incubation at 40 ° C are in shown. Although none of the tested molecules exhibited a significant reduction in potency after storage at 40 ° C, it was observed that loaded molecules III and IV elicited a significant level of nonspecific (ie, target-independent) Jurkat T cell activation. In contrast, the bispecific TCR / mAb Diabodies their target-dependent efficacy, despite the presence of some aggregates, as shown in HPLC-SEC.

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• <120> Polypeptide molecule with improved dual specificity
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QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

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Claims (21)

The dual specificity polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises a first variable domain (VD1) binding region derived from an antibody capable of recruiting human immune effector cells by binding specifically to a surface antigen of these cells, and a first variable domain (VR1) binding region derived from a TCR specific for an MHC-associated peptide epitope, and a first linker part (LINK1) connecting the domains; the second polypeptide chain comprises a second variable domain (VR2) binding region derived from a TCR specific for an MHC-associated peptide epitope, and a second variable domain (VD2) binding region derived from an antibody capable of recruiting human immune effector cells by binding specifically to a surface antigen of these cells, and a second linker part (LINK2) connecting the domains; wherein said first binding region (VD1) and said second binding region (VD2) associate to form a first binding site (VD1) (VD2) which binds the epitope of the cell surface molecule; associate this first binding region (VR1) and this second binding region (VR2) to form a second binding site (VR1) (VR2) which binds to the epitope of this MHC-associated peptide; wherein these two polypeptide chains are fused to human IgG hinge domains and / or human IgG Fc domains or parts thereof; and wherein these two polypeptide chains are linked by covalent and / or non-covalent bonds between these hinge domains and / or Fc domains; and wherein said dual specificity polypeptide molecule is capable of simultaneously binding the cell surface molecule and the MHC-associated peptide epitope. Polypeptide molecule with dual specificity after Claim 1 wherein the order of the binding regions in the polypeptide chains under VD1-VR1; VD1-VR2; VD2-VR1; VD2 VR2; VR1 VD1; VR1 VD2; VR2-VD1; VR2-VD2 is selected and where the domains are connected by either LINK1 or LINK2. Polypeptide molecule with dual specificity after Claim 1 where the linker sequences LINK1 and / or LINK2 contain at least one sequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS and TVLSSAS. Polypeptide molecule with dual specificity according to one of Claims 1 to 3 wherein said first and second polypeptide chains further comprise at least one hinge domain and an Fc domain or portions thereof derived from human IgG1, IgG2 or IgG4. Polypeptide with dual specificity Claim 4 wherein said Fc domain comprises at least one mutation which, for a residue selected from positions 233, 234, 235, 236, 297 and 331, shuts down the effector function, preferably wherein said mutation which shuts down the effector function is generated in that at least one residue at position 233, 234, 235, 236 and 331 is replaced by the corresponding residue derived from IgG2 or IgG4. Polypeptide molecules with dual specificity according to one of Claims 3 to 5 wherein said Fc domain comprises a CH3 domain containing at least one mutation facilitating the formation of heterodimers. The polypeptide with dual specificity Claim 6 These mutations are located at any position selected from 366, 368, 405 and 407, preferably wherein these mutations include T366W and T366'S, L368A 'and Y407'V as knob-in-hole mutations. Polypeptide molecule with dual specificity according to one of Claims 3 to 7 wherein said Fc domain comprises CH2 and CH3 domain (s) comprising at least two additional cysteine residues, for example S354C and Y349C or L242C and K334C. Polypeptide molecules with dual specificity according to one of Claims 1 to 8th where these antibody-derived domains VD1 and VD2 show a manipulated disulfide bond that is a covalent one Introducing binding between VD1 and VD2 and introducing these cysteines into the frame region (FR) 4 in the case of VL and frame region 2 in the case of VH. Polypeptide molecule with dual specificity according to one of Claims 1 to 9 , wherein said cell surface molecule is known to induce activation of immune cells or at least selected from the group consisting of molecules related to the immune response, CD3 such as e.g. CD3y, CD3δ, and CD3ε chains, TCRa / b and TCRg / d and HLA-DR. Polypeptide molecule with dual specificity according to one of Claims 1 to 10 wherein the regions in the first polypeptide chain SEQ ID NO. 28 for VD1, SEQ ID NO. 29 for VR1, SEQ ID no. 30 for LINK1 and the regions in the second polypeptide chain SEQ ID NO. 31 for VD2, SEQ ID no. 32 for VR2 and SEQ ID no. 30 for LINK2. Polypeptide molecule with dual specificity according to one of Claims 3 to 11 wherein the FC region in the first polypeptide chain SEQ ID NO. 26 (Fc1) and the FC region in the second polypeptide chain SEQ ID NO. 27 (Fc2). Polypeptide molecule with dual specificity, which has a first polypeptide chain with SEQ ID NO. 16 and a second polypeptide chain with SEQ ID NO. 17 includes. Polypeptide molecule with dual specificity according to one of Claims 1 to 13 , which molecule carries a detectable label. Polypeptide molecule with dual specificity according to one of Claims 1 to 14 wherein said first binding site (VD1) (VD2) which binds the surface antigen of said immune cells is humanized; and / or this second binding site (VR1) (VR2), which binds to this MHC-associated peptide epitope, has matured to achieve higher affinity and / or stability. Nucleic acid encoding the first polypeptide chain and / or the second polypeptide chain according to any one of Claims 1 to 15 or an expression vector comprising at least one of these nucleic acids. Host cell, a in Claim 16 comprises defined vector and optionally expressed. A pharmaceutical composition comprising the double specificity polypeptide molecule of any one of Claims 1 to 15 , the nucleic acid or the expression vector Claim 16 or the cell after Claim 17 together with one or more pharmaceutically acceptable carrier (s) or excipient (s). Polypeptide molecule with dual specificity according to one of Claims 1 to 15 , the nucleic acid or the expression vector according to Claim 16 or the cell after Claim 17 or the pharmaceutical composition according to Claim 18 for use in medicine. Polypeptide molecule with dual specificity according to one of Claims 1 to 15 , the nucleic acid or the expression vector according to Claim 16 , the cell after Claim 17 or the pharmaceutical composition according to Claim 18 for use in the prevention or treatment of a disease or disorder selected from cancer, infectious diseases and immune disorders. A method of treating a disease or disorder comprising administering a therapeutically effective amount of a double specificity polypeptide molecule according to any one of Claims 1 to 15 , the nucleic acid or the expression vector Claim 16 or the cell after Claim 17 or the pharmaceutical composition Claim 18 includes.

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