EA043129B1 - IMPROVED POLYPEPTIDE MOLECULE WITH DOUBLE SPECIFICITY - Google Patents

Description

The present invention relates to a bispecific polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain providing a binding site derived from a T cell receptor (TCR) specific for a peptide epitope associated with a major histocompatibility complex (MHC) and a binding site derived from from an antibody capable of attracting human immune effector cells by specific binding to the surface antigen of said cells, as well as to methods for producing and using a bispecific polypeptide molecule.

State of the art

With the development of molecular cloning technology and deep knowledge of antibody engineering, there have been various formats of bispecific antibodies (bispecifics) that can be selected for optimal biological activity and clinical goals. In cancer therapy, bispecific antibodies have been developed to redirect the activity of immune effector cells to the tumor site through a first binding domain specific to the tumor cell epitope and a second binding domain specific to the immune effector cell epitope. Bispecific antibodies for targeting immune effector cells have been developed in a variety of formats, including formats without a crystallizable fragment (Fc) region and IgG-derived formats with symmetrical or asymmetric designs. In addition to redirecting effector cells to the tumor site, new uses have been established for bispecific antibodies. Bispecifics capable of inhibiting two related signaling molecules simultaneously could be developed to overcome inherited or acquired resistance and be more effective inhibitors of angiogenesis. In addition, bispecific antibodies can be used as promising immunostimulatory substances for the treatment of various diseases such as cancer. Bispecific antibodies can also be used to treat hemophilia type A by mimicking factor VIII function. Bispecific antibodies also have a wide range of potential applications in disorders and infectious diseases of the bones and diseases of the central nervous system (for a review, see 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)).

T cells express T cell receptor (TCR) complexes that are capable of inducing antigen-specific immune responses. Attraction of an antigenic peptide/molecule of the major histocompatibility complex (MHC) class I to a target cell with TCR causes the formation of an immune synapse and leads to signaling through CD3 co-receptors, which are components of the Treceptor (TCR) signal transduction complex. This signaling cascade controls T cell-mediated killing of an antigen-expressing cell by releasing and transferring granzymes and perforin from the T cell to the target cell.

From a historical point of view, the discovery and production of antibodies with single-chain variable domains (scFvs, described in Bird et al., 1988) served as the main impetus for the development of bispecific antibodies. This concept eventually led to the creation of BiTE format molecules and their clinical acceptance as an effective drug for the treatment of leukemia (Baeuerle, P.A.; Reinhardt, C. Bispecific T-cell antibodies for cancer therapy. Cancer Res. 2009, 69, 4941 -4944). In cancer, bispecific antibodies that co-recruit the CD3 epsilon subunit and the surface antigen on the tumor cell induce T cell-mediated killing of the tumor cell while bypassing the need for direct interaction between TCR and MHC class I molecules complexed with the antigen. This expands the T-cell repertoire in terms of tumor recognition and effector cell function (Baeuerle, P.A.; Reinhardt, C. Bispecific T-cell grasping 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 T-cell based cancer immunotherapy approaches are currently being investigated. cells, including BiTE® (bispecific T-cell activator) antibody constructs, which have a unique design and mechanism of action. They are constructed by genetic binding on a single polypeptide chain of minimal binding domains of monoclonal antibodies to tumor-associated surface antigens and to the T-cell receptor-associated CD3 molecule. Simultaneous recruitment of the target cell antigen and the CD3 molecule leads to the activation of polyclonal cytotoxic T cells, resulting in lysis of the target cell. BiTE®-derived blinatumomab that targets CD19 is under investigation for a wide range of B-cell malignancies and has recently been approved in the US by the FDA for the treatment of Philadelphia chromosome-negative B-cell relapsed/refractory acute B-linear lymphoblastic leukemia under trademark BLINCYTO™ (BLINCYTO™). The BiTE® platform is one of the most clinically advanced

- 1 043129 variants of T-cell immunotherapy.

However, the disadvantages of small bispecific molecules such as BiTE® have been found to be low production yield, difficult purification processes, tendency to aggregation, and a very short serum half-life. To overcome the inherent limitations of this class of molecules, various human IgG-based bispecific formats have been developed, starting with the concept of recombinant bispecific prototypical immunoglobulin (Ig)-G-like antibodies developed over two decades ago when Morrison and colleagues fused flexible linker peptides with C-terminals of the heavy chains of the IgG molecule, and then single-chain variable domains, with different binding specificities (Coloma, M.J. and Morrison, S.L. (1997) Design and production of novel tetravalent bispecific antibodies. Nat. Biotechnol. 15, 159-163). These molecules could be distinguished from normal antibodies because they had dual functionality. Technical hurdles initially hindered further development, resulting in bispecific antibodies (bsAbs) remaining a topic of R&D, primarily in academia and biotechnology. However, rapidly advancing technologies that have opened up opportunities for the design, manufacture and development of recombinant protein derivatives, combined with renewed interest from the pharmaceutical industry, have given a decisive impetus to bsAb research. Many different bsAb formats are now available for the development of therapeutic proteins (for reviews, see Gramer, mAbs. 2013; 5(6):962-973, Weidle, Cancer Genomics Proteomics. 2013 Nov-Dec; 10(6):239 -50, Brinkmann, M.A. 2017 Feb/Mar;9(2):182-212.). Briefly, the incorporation of Fc moieties (crystallizable fragment) consisting of CH2 and CH3 domains resulted in increased productivity, simplified purification processes, and increased stability. In addition, the serum half-life of such IgG-based drugs was increased due to:

i) increase in size; and ii) interaction of the Fc fragment with the human Fc receptor FcRn.

The development of IgG-based bispecific formats was further fueled by the discovery and introduction of engineered mutations to facilitate heterodimerization of two distinct CH3 domains, thereby linking 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), who proposed the knobs-into-holes principle. holes) as a novel and efficient strategy for the design of antibody heavy chain homodimer constructs for heterodimerization. In this approach, the overhang variant was first generated by replacing a small amino acid with a larger one in the CH3 domain in the CD4-IgG immunoadhesin: T366Y. The protrusion was designed to fit into a trough in the CH3 domain of a humanized anti-CD3 antibody resulting from the expedient replacement of a large residue with a smaller one: Y407T. The chimeric anti-CD3/CD4-IgG antibody is a protein pool with up to 92% purified protein A obtained by co-expression of these two heavy chains together with the light chain of an anti-CD3 antibody. In contrast, only up to 57% of a chimeric anti-CD3/CD4-IgG antibody could be generated due to co-expression in which the heavy chains contained wild-type CH3 domains. Thus, the ridge-to-trough modification facilitates the construction of hybrids between antibody and immunoadhesins and most likely other Fc fragment-containing bifunctional therapeutics, including bispecific immunoadhesins 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) propose a ridge-to-trough mutation to improve heterodimerization (trough: T366W/trough: T366S+L368A+Y407V ) in the CH3 domain of the Fc domain. This concept was further improved by introducing additional cysteine residues to form a stabilizing disulfide bond between heterodimeric CH3 domains, as described by Merchant et al. 1998, Nature Biotechnology (An Efficient Route to Human Bispecific IgG).

Other concepts for obtaining heterodimeric molecules have been proposed

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 coengagement of distinct target antigens); Von Kreudenstein et al. 2013, MAbs (Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design.)

For an overview of these concepts, see Ha et al.

- 2 043129

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).

With the introduction of Fc fragments consisting of hinges, CH2 and CH3 domains, or parts thereof, into bispecific molecules, the problem of nonspecific immobilization of these molecules induced by the interaction of the Fc fragment and the Fc gamma receptor (FcgR) has arisen. FcgRs consist of various cell surface molecules (FcgRI, FcgRIIa, FcgRIIb, FcgRIII) that bind with different affinities to epitopes represented by Fc fragments of IgG molecules. As such, non-specific (i.e., not induced by either of the two binding domains of the bispecific molecule) immobilization is unfavorable due to the identified:

i) influencing the pharmacokinetics of the molecule; and ii) off-target activation of immune effector cells of various Fc variants and mutations preventing FcgR binding.

Morgan et al. 1995, Immunology (The N-terminal end of the CH2 domain of chimeric human IgGl anti-HLA-DR is necessary for Clq, FcyRI and FcyRIII binding) proposed the replacement of residues 233-236 of the human IgG1 molecule with the corresponding human IgG2 sequences, resulting in complete inhibition of FcgRI binding, complete inhibition of C1q binding, and reduced FcgRIII binding.

Patent E P1075496 proposes antibodies and other Fc fragment-containing molecules with variations in the Fc region (233P, 234V, 235A and no residue or with G at position 236 and 327G, 330S and 331S), where the recombinant antibody is able to bind to the target molecule without inducing significant complement-dependent lysis or cell-mediated destruction of the target.

Dual affinity retargeting molecules (DART) are used, for example, to obtain optimal retargeting killing of B-cell lymphoma cells by T cells. The DART technology was first described by Moore et al. (in: 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). As shown by comparison with a single-chain bispecific antibody carrying identical Fv sequences of antibodies to CD19 and CD3, DART molecules have a great potential to induce B-cell lysis. The next step in the development of DART technology was DART-Fc molecules, which are 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). This molecule combines the high potency of the DART construct and, among other positive characteristics, an extended serum half-life of Fc-based molecules.

αβΤΚΡ (TCR) recognizes antigenic peptides presented by MHC molecules and is responsible for T cell specificity. Both α- and β-chains of TCR have variable (V) and constant domains. V domains are involved in antigenic peptide binding, and constant domains penetrate the T cell membrane. Based on crystal structure analysis of TCR associated with the peptide-MHC complex, complementarity determining regions (CDRs) 3 of both _Va and Ve chains preferentially interact with the peptide, while CDRs 1 and 2 interact with the MHC molecule. However, peptide recognition by CDR 1 and recognition of an MHC molecule by CDR 3 have 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 αβ-TCR heterodimer is closely associated with the CD3, CD4 or CD8 proteins, and other adhesion and signal transduction proteins. Binding of the antigenic peptide via TCR V regions stimulates T cell activation by signal transduction via TCR constant domains via the cytoplasmic CD3 and CD4 or CD8 proteins.

In contrast to the full-length TCR format, single-chain TCRs (scTKPs) have significant advantages in terms of design, soluble protein expression, and clinical potential. With respect to the prospects for soluble protein expression (i.e. production), scTKP is produced as an individual polypeptide, avoiding the need to obtain each TCR strand as separate polypeptides and allowing for a large amount of well-designed scTKP that binds to its ligand of the peptide-MHC complex. . This characteristic allows you to obtain the production yield required for clinical use. Finally, from a clinical point of view, a V-region-only (scTv) scTKP can be in the format of therapeutic agents or reagents for diagnostic purposes, similar to scFv fragments.

US 2006-0166875 proposes a single chain T cell receptor (scTKP) comprising a segment consisting of a TKR alpha chain variable region sequence fused to the N-terminus of an extracellular TKR alpha chain constant region sequence, a beta segment consisting of a beta variable region TKR chain fused to the N-terminus of the extracellular con sequence

- 3 043129 of the constant region of the TCR beta chain, and a linker sequence connecting the C-terminus of the alpha segment to the N-terminus of the beta segment, or vice versa, and the extracellular sequences of the constant region of the alpha and beta segments are connected by a disulfide bond, the length of the linker sequence and the position disulfide bonds are such that the variable region sequences of the alpha and beta segments are mutually oriented, essentially as in natural alpha/beta T cell receptors. Also proposed are complexes of two or more of these scTKPs and the use of these scTKPs in therapy and various types of screening. In contrast to the scTKP described in US 2006-0166875, US 2012-0252742 proposes a soluble single-chain human TCR without constant domains, consisting only of variable TCR fragments (scTv), which is useful for many purposes, including in the treatment of cancer. , viral and autoimmune diseases.

McCormack E et al. (in: 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) disclose that NY -ESO-1 and LAGE-1 are cancer-testicular antigens with an ideal profile for antitumor immunotherapy, combining elevated levels in many cancers with highly restricted expression in normal tissues, and sharing the HLA-A*0201, 157-165 epitope. They provide data to describe the specificity and antitumor activity of a bifunctional ImmTAC construct containing a soluble, high affinity T cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, has been shown to kill HLA-A2, antigen positive tumor cell lines, and freshly isolated HLA-A2 and LAGE-1 positive NSCLC cells. Using in vivo optical imaging, the results demonstrate in vivo targeting of high affinity, fluorescently labeled, NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in transplant mice. ImmTAC-NYE activity in vivo was tested in a tumor model in which human lymphocytes were stably co-transplanted into immunodeficient NSG mice bearing tumor grafts; activity was observed in both tumor prevention and advanced tumor models using green fluorescent protein (GFP) fluorescent readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. In general, LAGE-1 RNA was expressed in tumor samples at a higher frequency and at a higher level than NY-ESO-1. ImmTACs comprise a single chain Fv fragment derived from the anti-CD3 antibody UCHT-1 covalently linked to the C- or N-terminus of the TCR alpha or beta chain.

Patent EP1868650 relates to diabody molecules and their use in the treatment of a spectrum of diseases and disorders, including immunological disorders, infectious disease, intoxication, and cancer. 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, antigens can be from the same or different molecules. The individual polypeptide chains of a diabody molecule can be covalently linked via, but not limited to, non-peptide covalent bonds, such as disulfide bonding of cysteine residues found in each polypeptide chain. In specific embodiments, diabody molecules further include the Fc region provided herein as this allows inherent antibody properties (eg, long half-life) to be incorporated into the molecule by design. EP 1868650 states the need for the presence of immunoglobulin light or heavy chain variable domain binding regions and discusses the subject of functional Fc receptor binding agents.

WO 2016/184592 A1 proposes bispecific molecules in which one specificity is provided by TCR and the other by an antibody that is directed against an antigen or epitope on the surface of lymphocytes, but it does not disclose the specific arrangement of the TCR elements and variable regions of the antibody proposed herein. document.

EP 2258720A1 relates to a functional T cell receptor (TCR) fusion protein (TFP) that recognizes and binds to at least one epitope presented by the MHC and contains at least one amino acid sequence that recognizes and binds to an antigen.

It is an object of the present invention to provide improved bispecific molecules capable of targeting peptide-MHC complexes that can be easily prepared, have high stability, and also provide high activity when binding to appropriate antigenic epitopes. Other objects and advantages of the present invention will become apparent upon examination of the following description and its preferred embodiments, as well as the corresponding examples.

In a first aspect of the invention, the above object is achieved by providing a dual specificity polypeptide molecule selected from the group of molecules comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first variable domain binding site (VD1) of an antibody specifically binding with the surface antigen of the human immune effector cell, and the first binding site of the variable domain (VR1) TCR, specifically binding to

MHC-associated peptide epitope, and the first linker (LINK1) connecting these domains;

the second polypeptide chain includes a second TCR variable domain binding site (VR2) that specifically binds to an MHC-associated peptide epitope, and a second antibody variable domain binding site (VD2) that specifically binds to the surface antigen of a human immune effector cell, and a second linker (LINK2) , connecting the specified domains;

wherein said first binding site (VD1) and said second binding site (VD2) are associated to form a first binding site (VD1)(VD2) that binds to an epitope of a cell surface molecule;

said first attachment site (VR1) and said second attachment site (VR2) associate to form a second attachment site (VR1)(VR2) that binds to an MHC-associated peptide epitope;

wherein said two polypeptide chains are fused to human IgG hinge domains and/or human IgG Fc domains or dimerizing regions thereof; and where these two polypeptide chains are connected by covalent and/or non-covalent bonds between the specified hinge domains and/or Fc domains; and wherein said dual specificity polypeptide molecule is capable of simultaneously binding to a cell surface molecule and an MHC associated peptide epitope, and dual specificity polypeptide molecules wherein the order of binding sites in the polypeptide chain is selected from VD1-VR1; VD1-VR2; VD2-VR1; VD2-VR2; VR1-VD1; VR1-VD2; VR2-VD1; VR2-VD2, and where the domains are connected using either LINK1 or LINK2.

Preferred is a dual specificity polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain, where:

the first polypeptide chain includes a first variable domain binding site (VD1) derived from an antibody capable of recruiting human immune effector cells by specific binding to the surface antigen of said cells, and a first variable domain binding site (VR1) derived from an MHC-specific TCR -associated peptide epitope, and the first linker site (LINK1), connecting the two domains;

the second polypeptide chain includes a second variable domain binding site (VR2) derived from a TCR specific for an MHC-associated peptide epitope and a second variable domain binding site (VD2) derived from an antibody capable of recruiting human immune effector cells through specific binding to the surface antigen of these cells, and the second linker site (LINK2), connecting the two domains;

wherein said first binding site (VD1) and said second binding site (VD2) associate to form a first binding site (VD1)(VD2) that binds to an epitope of a cell surface molecule;

said first attachment site (VR1) and said second attachment site (VR2) associate to form a second attachment site (VR1)(VR2) that binds to an MHC-associated peptide epitope; where at least one of these polypeptide chains is connected at its C-terminus to hinge regions, CH2 and/or CH3 domains or parts thereof derived from human IgG; and wherein said dual specificity polypeptide molecule is capable of simultaneously binding to an immune effector cell antigen and an MHC-associated peptide epitope.

Preferably, the dual specificity polypeptide molecule of the present invention binds with high specificity to both an immune effector cell antigen and a specific antigen epitope presented as a complex of the peptide to the MHC molecule, for example, 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, for example, as measured by biolayer interferometry as described in Example 6, or as determined by flow cytometry.

The dual specificity polypeptide molecules of the present invention are shown in the present context in terms of a dual specificity polypeptide molecule comprising a first polypeptide chain comprising the sequence of SEQ ID NO: 16 and a second polypeptide chain comprising the sequence of SEQ ID NO: 17.

In a second aspect of the invention, the above object is solved by providing a nucleic acid(s) encoding a first polypeptide chain and/or a second polypeptide chain as provided herein, or an expression vector(s) comprising(s) such nucleic acid. In a third aspect of the invention, the above problem is solved by providing a host cell comprising the vector(s) as defined herein.

In the fourth aspect of the invention, the above problem is solved by proposing a method for obtaining

- 5 043129 molecules of a polypeptide with dual specificity in accordance with the present invention, including the appropriate expression of the specified(s) expression vector(s), comprising(their) nucleic acid(s) according to the invention in a suitable host cell, and suitable purification molecules(molecules) from the cell and/or their environment.

In a fifth aspect of the invention, the above problem is solved by providing a pharmaceutical composition comprising a dual specificity polypeptide molecule according to the invention, a nucleic acid or an expression vector(s) according to the invention, or a cell according to the invention together with one or more pharmaceutically acceptable carriers. or excipients.

In a sixth aspect of the invention, the invention relates to a dual specificity polypeptide molecule according to the invention, nucleic acid(s) or expression vector(s) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention for applications in medicine.

In a seventh aspect, the invention relates to a dual specificity polypeptide molecule according to the invention, a nucleic acid(s) or an expression vector(s) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention. , for use in the treatment of a disease or disorder according to the present description, in particular, selected from cancer and infectious diseases.

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

In a ninth aspect of the invention, the invention relates to a method for inducing an immune response in a patient or subject comprising administering a therapeutically effective amount of a dual specificity polypeptide molecule according to the invention or a pharmaceutical composition according to the invention.

In a tenth aspect, the invention relates to a method for killing target cells in a patient or subject, comprising administering to the patient an effective amount of a dual specificity polypeptide molecule in accordance with the invention.

As mentioned above, the invention provides new improved polypeptide molecules with dual specificity. The molecules generally comprise a first polypeptide chain and a second polypeptide chain, wherein the chains together provide an antibody variable domain that is specific for an immune effector cell surface antigen epitope and a TCR variable domain that is specific for an MHC-associated peptide epitope, e.g., a cancer epitope or epitopes. presented in connection with an infection, such as a viral infection such as HIV. The antibody and TCR-derived variable domains are stabilized by covalent and non-covalent bonds formed between Fc fragments or regions thereof located on both polypeptide chains. A dual specificity polypeptide molecule is therefore capable of simultaneously binding to a cellular receptor and an MHC-associated peptide epitope.

In the context of the present invention, the variable domains (VD1) and (VD2) are formed from antibodies capable of recruiting human immune effector cells by specifically binding to the surface antigen of said effector cells. In one particular embodiment, said antibodies specifically bind to human TCR-CD3 complex epitopes, including the TKR-alpha, TKR-beta, CD3-gamma, CD3-delta, CD3-epsilon, and CD3zeta peptide chains.

A dual specificity polypeptide molecule according to the present invention comprises a first polypeptide and a second polypeptide chain, forming a first (VD1) and a second (VD2) binding site, respectively, of a variable domain derived from an antibody capable of recruiting human immune effector cells through specific binding with the surface antigen of these cells. This first binding site (VD1) and said second binding site (VD2) are associated to form a first binding site (VD1)(VD2) that binds to an immune effector cell surface antigen epitope; In addition, the first and second polypeptide chains of the polypeptide molecule comprise a first (VR1) and a second (VR2) binding site, respectively, of a variable domain derived from a TCR specific for an MHC-associated peptide epitope. Said first binding site (VR1) and said second binding site (VR2) associate to form a second binding site (VR1)(VR2) that binds to said MHC-associated peptide epitope. In one embodiment of the dual specificity polypeptide molecule according to the invention, the order/orientation of the regions in the first polypeptide chain is selected from VD1-LINK1-VR1, and VR1-LINK1VD1; in another embodiment, the order/orientation of the regions in the second polypeptide chain

- 6 043129 is selected from VD2-LINK2-VR2, and VR2-LINK-VD2, that is, the location of the binding sites can be changed to obtain a left or right molecule (see, for example, Fig. 5). In addition, the configuration of the alpha and beta chains of the TCR-related portion can be changed.

In the context of the present invention, a dual affinity polypeptide molecule according to the invention is exemplified by a structure that binds to a SLYNTVATL peptide (SEQ ID NO: 7) when presented as a complex of peptide and MHC. However, the inventive concept is, of course, not limited to this particular peptide, and includes essentially any disease or disorder-associated epitope presented with an MHC molecule. This presentation can be associated with MHC molecules of both class I and II. Molecules of the human major histocompatibility complex (MHC-I) are present on the surface of all nucleated cells and display a wide range of peptide epitopes for control by various CD8+ T cells. CD8+ T cell responses are essential for the control and suppression of viral infections, as well as for the destruction of transformed and oncogenic cells. Examples of preferred recognizable peptide epitopes are provided in the relevant literature and, in particular, include the peptides disclosed in table. 1-5 of WO 2016/170139; tab. 1-5 of WO 2016/102272; tab. 1 or 2 applications WO 2016/156202; tab. 1-4 applications WO 2016/146751; tab. 2 applications WO 2011/113819; tab. 1-4b of WO 2016/156230; tab. 1-4b of WO 2016/177784; tab. 1-4 applications WO 2016/202963; tab. 1 and 2 applications WO 2016/207164; tab. 1-4 applications WO 2017/001491; tab. 1-4 applications WO 2017/005733; tab. 1-8 of WO 2017/021527; tab. 1-3 applications WO 2017/036936; tab. 1-4 PCT/EP 2016/073416 for cancer treatment, US Patent Application 2016-0187351, US Patent Application 2017-0165335, US Patent Application 2017-0035807, US Patent Application 2016-0280759, US Patent Application US Patent Application 2016-0287687, US Patent Application 2016-0346371, US Patent Application 2016-0368965, US Patent Application 20170022251, US Patent Application 2017-0002055, US Patent Application 2017-0029486, in US Patent Application 2017- 0037089, US Patent Application 2017-0136108, US Patent Application 20170101473, US Patent Application 2017-0096461, US Patent Application 2017-0165337, US Patent Application 2017-0189505, US Patent Application 2 017-0173132, in patent US Application 20170296640, US Patent Application 2017-0253633 and US Patent Application 2017-0260249, the contents of which are incorporated herein in their entirety by reference. In another aspect, a dual affinity polypeptide molecule according to the invention recognizes a peptide consisting of any of the peptides described in the above patent applications.

In one aspect, a dual affinity polypeptide molecule according to the invention binds to or is able to be specifically recognized/able to bind to one or more peptides with a total length of 8 to 100 amino acids, 8 and 30 amino acids, 8 to 16 amino acids, preferably from 8 and 14 amino acids, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in the case of extended peptides binding to class II molecules, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. In yet another aspect, a dual affinity polypeptide molecule according to the invention binds to or is able to be specifically recognized/able to bind to one or more peptides with a total length of 8 to 12 amino acids, 8 to 10 amino acids, 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.

Other suitable epitopes may be identified from databases such as, for example, the Immune Epitope Database (accessed at: www.iedb.org).

The term human immune effector cell(s) refers to a cell in the context of the natural cell diversity of the human immune system that, when activated, is capable of causing a change in the viability of a target cell. The term target cell viability may refer within the scope of the invention to the ability of a target cell to survive, proliferate and/or interact with other cells. Such interaction can be either direct, for example, when the target cell comes into contact with another cell, or indirect, for example, when the target cell secretes substances that affect the functioning of another distant cell. The target cell in relation to a person can be either natural or foreign. In the case where the cell is native to a human, it is preferable that the target cell undergoes transformation to become a malignant cell. The natural cell may further be a pathologically modified natural cell, for example, a natural cell infected with an organism such as a virus, plasmodium, or bacterium. In case the cell is foreign to the human, it is preferable that the target cell is an invasive pathogen such as an invasive bacterium or Plasmodium.

Preferred is a dual specificity polypeptide molecule according to the invention, wherein said first and second polypeptide chains further comprise at least one hinge domain and/or an Fc domain or a fragment thereof. In antibodies, the hinge or hinge region or hinge domain refers to the flexible region of the heavy chain located between the CH1 domain and the CH2 domain. It is approximately 25 amino acids long and is subdivided into

- 7 043129 upper hinge region, middle hinge region or main hinge region and lower hinge region. The hinge subdomain refers to the upper hinge region, the middle (or main) hinge region, or the lower hinge region. The amino acid sequences of the hinges of IgG1, IgG2, IgG3 and IgG4 molecules are (EC numbering is given):

lgG1: E216PKSCDKTHTCPPCPAPELLG (SEQ ID No. 1) lgG2: E216RKCCVECPPCPAPPVAGP (SEQ ID No. 2) lgG3:

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE216PKSCDTPPPCPRCPAPEL

LG (SEQ ID No. 3) lgG4: E216SKYGPPCPSCPAPEFLG (SEQ ID No. 4)

The main hinge region usually contains at least one cysteine bridge connecting two heavy chains. In addition, in order to reduce unwanted antibody-dependent cell-mediated cytotoxicity (ADCC), mutations can be introduced into the lower hinge region.

Preferred is a polypeptide molecule with dual specificity in accordance with the present invention, comprising at least one crystallizing fragment (Fc) domain of IgG, i.e., a crystallizing fragment (Fc region), an antibody tail region that interacts with Fc receptors, and some proteins of the complement system. The Fc regions contain two or three heavy chain constant domains (CH domains 2, 3, and 4) in each polypeptide chain. The Fco regions of IgG molecules also host a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is required for Fc receptor mediated activity. Small size bispecific antibody formats such as BiTE® and DART (~50 kDa) can result in rapid clearance and short half-life. Therefore, to obtain improved pharmacokinetic properties, a dual specificity scTv cell receptor polypeptide molecule (eg, CD3) can be fused to an Fc domain (human IgG1), which increases the molecular weight. Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q/M428L and M252Y/S254T/T256E+H433K/N434F, have been shown to increase neonatal Fc receptor (FcRn) binding affinity and IgG1 in vivo half-life . Thus, the serum half-life of the Fc-containing molecule can be further increased.

In the dual specificity polypeptide molecules of the invention, said Fc domain may comprise a CH2 domain comprising at least one effector suppression mutation. Preferably, these mutations are introduced into the ELLGGP sequence (SEQ ID NO: 50) of the human IgG1 molecule (residues 233-238) or corresponding residues of other isotypes) known to be associated with effector functions. In principle, one or more mutations corresponding to residues derived from IgG2 and/or IgG4 are introduced into the IgG1 Fc fragment. Preferred are: E233P, L234V, L235A and no residue or G at position 236. Another mutation is P331S. Patent No. EP1075496 discloses a recombinant antibody comprising a chimeric domain that is derived from two or more heavy chain CH2 domains of a human immunoglobulin molecule, wherein the human immunoglobulin molecules are selected from IgG1, IgG2, and IgG4, and wherein the chimeric domain is the heavy chain CH2 domain of a human immunoglobulin molecule , having the following blocks of amino acids at the indicated positions: 233P, 234V, 235A and no residue or G at position 236 and 327G, 330S and 331S in accordance with the EU numbering system, and its sequence is at least 98% identical to the CH2 sequence (residues 231 -340) human IgG1, IgG2 or IgG4 molecules having the indicated amino acid modifications.

Examples of preferred partial CH2 sequences to use may be (in whole or in part) the following:

231APPVA-G R SVF LF R R KR KDTLMIS RTP EVTCVWDVS H E D R EVKFN WYVDGVE

VHNAKTKPREEQYQSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEK334 (SEQ ID No. 5); And

231APPVA-G R SVF LF R R KR KDTLM IS RTP EVTCVWDVS H E D R EVKFN WYVDGVE

VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEK334 (SEQ ID No. 6), where the changes are underlined that position 297 is N (glycosylated variant) or a residue selected from the group with A, G and Q (deglycosylated variant).

In the dual specificity polypeptide molecules of the invention, said Fc domain may comprise a CH3 domain comprising at least one heterodimer-forming mutation. In order to maximize the yield of the dual specificity heterodimeric Fc protein of interest and to simplify purification, socket and socket mutations can be inserted into the Fc domain. Due to the effect of this design with the addition of protruding massive hydrophobic residues (protrusion) in one strand and the creation of complementary hydrophobic pockets (cavities) on the other strand, the Fc domains form heterodimers instead of their usual homodimers. An overhang variant can be generated by replacing a small amino acid with a larger one for insertion into a recess in the opposite domain created by replacing a large residue with a smaller one (Ridgway, J.B.B.; Presta, L.G.; 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 dual specificity polypeptide molecule according to the invention wherein said tang-to-trough mutation is selected from T366W as the trough and T366'S, L368'A and Y407'V as the trough in the CHS domain (see e.g. , application WO 98/50431). This set of mutations can be further extended to include the K4O9A and F405'K mutations, as described by Wei et al. (Structural basis of a novel heterodimeric Fc for bispecific antibody production, Oncotarget. 2017). The other ridge might be T366Y and the trough might be Y407'T.

The dual specificity polypeptide molecules of the invention may additionally include 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 in order to improve the stability of the molecules, optimally not adversely affecting binding characteristics of the bivalent molecule, and/or improving heterodimerization. For additional stability, a disulfide bond can be introduced into the CH3 domain of both chains, the bump and the trough, by adding a single cysteine. Preferred is a dual specificity polypeptide molecule according to the invention, wherein the Fc domain comprises a CH3 domain comprising at least one additional cysteine residue, eg S354C and/or Y349C.

Preferred is a dual specificity polypeptide molecule according to the invention, wherein said CD molecule is selected from the group of CD molecules associated with the immune response, CD3, such as chains

CD3y, CD36, and CD3£, CD4, CD7, CD8, CD10,

CD11 b, 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, FceRI, TKR-alpha/beta, TKR -gamma/-delta and HLA-DR.

Depending on the combination of the two antigen-binding units of the dual specificity polypeptide molecule according to the invention, certain advantages can be achieved in terms of the function of the molecule, in particular increased activity.

Preferred is an exemplary dual-specific polypeptide molecule according to the invention wherein the regions in the first polypeptide chain of the sequence SEQ ID NO: 28 for VD1, SEQ ID NO: 29 for VR1, SEQ ID NO: 30 for LINK1; and regions in the second polypeptide chain include the sequence of SEQ ID NO: 31 for VD2, SEQ ID NO: 32 for VR2, and SEQ ID NO: 30 for LINK2.

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

Also preferred is an exemplary dual specificity polypeptide molecule according to the invention, comprising a first polypeptide chain comprising the sequence of SEQ ID NO: 16 (1st chain of the molecule as a whole) and a second polypeptide chain comprising the sequence of SEQ ID NO: 17 (2nd chain of the whole molecule).

Further, even more preferred is an exemplary dual specificity polypeptide molecule according to the invention, wherein said first binding site (VD1)(VD2) that binds to an epitope of a human immune cell surface antigen (eg, CD3) is humanized; and/or said second binding site (VR1)(VR2) that binds to said MHC-associated peptide epitope has affinity maturity.

Humanized antibodies are antibodies (or portions thereof) of a non-human species whose protein sequences have been modified to increase their similarity to antibody variants naturally produced in humans. The humanization process is typically applied to monoclonal antibodies designed for human administration (eg, antibodies developed as anti-cancer drugs). Suitable humanization methods are known from the literature and are reviewed, for example, by Olimpieri, Pier Paolo, Paolo Marcatili, and Anna Tramontano. Tabhu: Tools for Antibody Humanization. Bioinformatics 31.3 (2015): 434-435. PMC;

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Safdari Y, Farajnia S, Asgharzadeh M, Khalili M. Antibody humanization methods - a review and update. Biotechnol Genet Eng Rev. 2013;29:175-86; or Ahmadzadeh V, Farajnia S, Feizi MA, Nejad RA. Antibody humanization methods for development of therapeutic applications. Monoclon Antib Immunodiagn Immunother.

2014 Apr; 33(2):67-73.

In general, in vitro affinity maturity of TKP and antibodies can be obtained according to procedures described in the literature, in particular using yeast or phage-based surface display (based on, for example, the work of 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 more recent 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 invention preferably specifically bind to the human immune cell surface antigen and the HLA peptid-molecule complex, respectively. The term specific binding, as used in connection with the term binding sites as used herein, and its grammatical variants are used to refer to a site with a binding affinity (KD) for an HLA peptide-molecule complex and/or an antibody epitope with a value of 100 μM or less. The binding sites (VD1)(VD2) and (VR1)(VR2) of the present invention bind to a CD antibody epitope or peptide-HLA molecule complex, respectively, with a binding affinity (K _D ) of about 100 μM or less, about 50 μM or less , about 25 μM or less, or about 10 μM or less. More preferred are high affinity binding sites with a binding affinity of about 1 μM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 500 pM or less, about 200 pM or less, about 100 pM or less. Non-limiting examples of preferred binding affinity ranges for the binding sites of the present invention include values from about 10 to about 100 pM, from 100 pM to about 1 nM, from 1 to about 10 nM; about 10 to about 20 nM; about 20 to about 30 nM; about 30 to about 40 nM; about 40 to about 50 nM; about 50 to about 60 nM; about 60 to about 70 nM; about 70 to about 80 nM; from about 80 to about 90 nM; and from about 90 to about 100 nM, for example, measured using the biolayer interferometry method, as described in example 6.

In one aspect, the invention discloses a polypeptide 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%, at least 99% identical to the amino acid sequence described herein, for example, the amino acid sequences of SEQ ID NO: 1 to 58. In another aspect, the invention discloses a first or second polypeptide whose sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence described herein. In yet another aspect, the invention discloses a dual specificity polypeptide molecule, the sequence of which 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%, at least 99% identical to one or more amino acid sequences described herein. The invention further discloses aspects in which an identity percentage of 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% applicable to any of the structural region sequences described in FIG. 1, for example, VD1, VR1, Link1, VR2, VD2, Link2 or the hinge region, and corresponds to the description or is part of the sequences proposed in the present description.

In one aspect, the polypeptides or polypeptide molecules with dual specificity described in the present context, can be changed in various ways, including substitutions, deletions, truncations and insertions of one or more amino acids. In another aspect, polypeptides or polypeptide molecules with dual specificity described in the present context may include 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, polypeptides or polypeptide molecules with dual specificity described in the present context may include 1-5, 1-10, 1-20, 2-5, 2-10, 5-20, 5-50 or 10- 100 amino acid substitutions, deletions or insertions. 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 are applicable to any of structural regions described in FIG. 1, such as VD1, VR1, Link1, VR2, VD2, Link2, or hinge areas. The invention further discloses aspects in which 1-5, 1-10, 1-20, 2-5, 2-10, 5-20, 5-50 or 10-100 amino acid substitutions, deletions or insertions are applicable to the sequences of any of the structural regions described in FIG. 1, such as VD1, VR1, Link1, VR2, VD2, Link2, or the hinge region, and is as described or is part of the sequences provided herein.

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 may be added to the N-terminus or C - the end of the polypeptide or polypeptide molecule described herein, for example, amino acid sequences 1-58.

In one aspect, VD1 may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 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% identical to the amino acid sequence of SEQ ID NO: 28.

In one aspect, VR1 may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 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% identical to the amino acid sequence of SEQ ID NO: 29.

In one aspect, LINK1 or LINK2 may 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%, at least 99% identical to the amino acid sequence of SEQ ID NO: 30.

In one aspect, VD2 may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 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% identical to the amino acid sequence of c SEQ ID NO: 31.

In one aspect, VR2 may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 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% identical to the amino acid sequence of SEQ ID NO: 32.

In one aspect, the hinge may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 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% identical to the amino acid sequence c SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3 or SEQ ID NO: 4. In one aspect, CH2 may 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%, at least 99% identical to the amino acid sequence of SEQ ID NO: 5, or SEQ ID NO: 6.

In one aspect, the Fc region may 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%, at least 99% identical to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

In one aspect, the invention discloses a polypeptide 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%, at least 99% identical to the amino acid sequence of SEQ ID NO: 43, 44, 45 or 46.

In one aspect, the dual specificity polypeptides or polypeptide molecules disclosed herein may be modified by substitution of one or more residues at various, possibly selective, sites along the length of the polypeptide chain. Such substitutions may be conservative, for example, when one amino acid is replaced by an amino acid with similar structure and characteristics, as well as when a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be the replacement of amino acids of the same or similar size and chemical nature, as, for example, when replacing leucine with isoleucine. In studies of sequence variation within families of naturally occurring homologous proteins, certain amino acid substitutions are tolerated more often than others, and they are often associated with similarities in size, charge, polarity, and hydrophobicity between the parent amino acid and its substitution; and such is the basis for determining conservative substitutions.

In another preferred embodiment, a dual specificity polypeptide molecule according to the invention, said molecule carries an active substance or portion thereof which is linked or conjugated to it. Said active agent may be selected from the group consisting of a detectable label, an immunostimulatory molecule, and a therapeutic agent.

The detectable label may be selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, 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, radiotracers, enzymes, nucleic acid probes, and contrast agents.

Therapeutic agents that may be associated with the molecules of the invention include immunomodulators, radioactive compounds, enzymes (eg perforin), chemotherapeutic agents (eg cisplatin) or toxin. Other suitable therapeutic agents include low molecular weight cytotoxic agents, ie compounds with the ability to kill mammalian cells having a molecular weight below 700 Daltons. Such compounds may also contain toxic metals capable of exerting a cytotoxic effect. In addition, it should be understood that these small molecular weight cytotoxic substances also include prodrugs, ie, compounds that, when degraded or converted under the influence of physiological conditions, release cytotoxic substances. Examples of such agents include cisplatin, maytansine derivatives, rachelmicin rachelmicin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e. proteins or their fragments capable of destroying mammalian cells. For example, ricin, diphtheria toxin, bacterial exotoxin A pseudomonas, DNase and RNase; radionuclides, i.e. unstable isotopes of elements that decay with the simultaneous emission of one or more α- or β-particles or γ-rays. For example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents can be used to facilitate the association of these radionuclides with molecules, or their multimers; immunostimulants, i.e. immune effector molecules that stimulate an immune response. For example, cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet factor-4, melanoma growth stimulator protein, complement activators; or heterogeneous 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 can be DNA, cDNA, PNA, RNA, or combinations thereof. The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be naturally occurring or may be synthesized. In general, the DNA segments encoding the peptides, polypeptides and proteins of the present invention are assembled from cDNA fragments and short oligonucleotide linkers or from a series of oligonucleotides to obtain a synthetic gene that is capable of being expressed in a recombinant transcription unit containing regulatory elements derived from a microbial or viral operon. Expression product means a polypeptide or protein that is a natural translation product of a gene and any nucleic acid sequence encoding equivalents resulting from the degeneracy of the genetic code and thus encoding the same nucleic acid(s) . The term fragment, when referring to a coding sequence, means a stretch of DNA comprising less than the entire coding region, the expression product of which essentially retains the same biological function or activity as the expression product of the entire coding region. Depending on the intended use, the nucleic acid can be codon-optimized for expression in an appropriate (eg, microbiological) host cell. The redundancy of the genetic code allows some amino acids to be coded for by more than one codon, however some particular codons are less optimal than others due to the relative availability of suitable tRNAs as well as other factors (Gustafsson et al., 2004).

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The nucleic acid may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof, both single and/or double stranded; natural or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone, and may or may not contain introns, provided that it encodes polypeptide chains.

The nucleic acid (eg, DNA) can then be incorporated into and/or expressed in a suitable host to produce a polypeptide comprising the polypeptide chain of the invention. Thus, a nucleic acid (e.g., DNA) encoding a polypeptide chain of the invention can be used according to known techniques, modified as appropriate to reflect the ideas disclosed herein, to construct an expression vector which is then used to transform a suitable cell- a host for expression and production of a polypeptide of the invention, as is known in the art. The nucleic acid (e.g., DNA, or in the case of retroviral vectors, RNA) encoding the polypeptide(s) chain(s) constituting the compound of the invention can be attached to a wide variety of other nucleic acid sequences (e.g., DNA) for introduction into corresponding host. The companion nucleic acid will depend on the nature of the host, the mode of introduction of the DNA into the host, and whether maintenance in episomal or integrative form is desired. Typically, the nucleic acid is introduced into an expression vector, such as a plasmid, with the correct orientation and correct reading frame for expression. If necessary, the nucleic acid may be linked to appropriate transcriptional and translational coordination nucleotide sequences recognized by the desired host, although such control elements are usually present in the expression vector. The vector is then administered to the host using standard methods. Typically, not all hosts are transformed by the vector. Therefore, it will be necessary to isolate the transformed host cells. One selection technique involves introducing into the expression vector a nucleic acid sequence, with any necessary control elements, that encodes a selected trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such a selectable trait may be on another vector that is used to co-transform the desired host cell. Host cells that have been transformed with the recombinant nucleic acid of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art, in accordance with the teachings disclosed herein, leading to expression of the polypeptide, which can then be isolated.

Many expression systems are known, including bacteria (eg E. coli and Bacillus subtilis), yeast (eg Saccharomyces cerevisiae), filamentous fungi (eg Aspergillus spec), plant cells, animal and insect cells. Preferably the system is mammalian cells such as CHO cells available from the American Type Culture Collection ATCC.

In one embodiment, the disclosure provides a method for producing a molecule as described herein, the method comprising cultivating a host cell capable of expressing the polypeptide chain(s) under conditions suitable to promote expression of said chain(s).

In one aspect, to obtain cells expressing molecules as described herein, nucleic acids encoding polypeptide chains comprising TKRalpha and/or TKR-beta binding domains are cloned into expression vectors such as gamma retrovirus or lentivirus. In another aspect, to obtain cells expressing molecules according to the present description, RNA is synthesized using techniques known in the art, for example, in vitro transcription systems. The in vitro synthesized RNAs are then introduced into suitable cells by electroporation in order to express the polypeptide chains.

To increase expression levels, nucleic acids encoding chains as described herein can be operably linked to strong promoters such as long terminal repeats of retrovirus (LTR), cytomegalovirus (CMV), mouse stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK ), β-actin, ubiquitin and the combined simian virus 40 (SV40)/CD43 promoter, elongation factor (EF)-1a and the spleen necrosis virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the expressed nucleic acid. In addition to strong promoters, expression cassettes as described herein may contain additional elements that can enhance transgene expression, including the central polypurine tract (cPPT), which promotes nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and a post-transcriptional regulatory element woodchuck hepatitis virus (wPRE), which increases the level of transgene expression by increasing RNA stability (Zufferey et al., 1999) (Zufferey et al., 1999).

The alpha and beta chains of the binding domain of the 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.

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In one embodiment, the host cell is genetically modified to express a molecule as described herein. Host cells as described herein may be allogeneic or autologous for the patient to be treated.

In still another aspect, the invention relates to a pharmaceutical composition comprising a dual specificity polypeptide molecule according to the present invention, a nucleic acid(s) or an expression vector(s) according to the present invention, or a cell according to the present invention. together with one or more pharmaceutically acceptable carriers or excipients.

The compositions of the invention may include bulk drug compositions suitable for the manufacture of pharmaceutical compositions (i.e., crude or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unitary dosage forms. Such compositions include a prophylactically or therapeutically effective amount of a polypeptide (substance) molecule with dual specificity for prophylactic and/or therapeutic purposes disclosed herein, or a combination of a substance and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of one or more molecular species of the invention and a pharmaceutically acceptable carrier.

Pharmaceutical compositions preferably include molecules either in free form or in salt form. Preferably, the salts are pharmaceutically acceptable salts of molecules, such as, for example, chloride or acetate (trifluoroacetate). It should be noted that the salts of the molecules according to the present invention are substantially different from the molecules in their in vivo state(s), since the molecules are not salts in vivo.

One embodiment of the present invention thus relates to a non-naturally occurring molecule according to the invention which is synthetically produced (eg synthesized) in the form of 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 substantially from the molecules in their in vivo state(s), since the synthesized molecules are not salts in vivo. Preferably, the salts are pharmaceutically acceptable salts of the molecules. Salts according to the invention include alkali and alkaline earth salts, such as salts of the Hofmeister series, including as anions PO ₄ ^3- , SO4 ^2- , CH3COO ^- , Cl ^- , Br ^- , NO3 ^- , ClO ₄ ^- , I ^- , SCN ^- and as cations NH4 ⁺ , Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ , Li ⁺ , Zn ²⁺ , Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Cu ²⁺ and Ba ²⁺ . In particular, the salts are selected from (ΝΗ ₄ ) ₃ ΡΟ4, (ΝΗ ₄ ) ₂ ΗΡΟ4, (ΝΗ ₄ )Η ₂ ΡΟ4, (NH ₄ ) ₂ SO4, NH4CH3COO, NH4CI, NH ₄ Br, NH ₄ NO ₃ , NH4CIO4, nh ₄ i, NhkSCN, Rb ₃ PO ₄ , Rb ₂ HPO ₄ , RbH ₂ PO ₄ , Rb ₂ SO ₄ , Rb ₄ CH ₃ COO, Rb 4 CI, Rb ₄ Br, Rb _{4 NO 3} , Rb ₄ CIO ₄ , Rb ₄ l, Rb ₄ SCN, K ₃ PO ₄ , K ₂ HPO ₄ , KH ₂ PO ₄ , Yu8O4, KCHeCOO, KCl, KBr, KNO ₃ , KCIO4, 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 , NaCIO ₄ , Nai, NaSCN, ZnCI ₂ Cs ₃ PO ₄ , Cs ₂ HPO ₄ , CsH ₂ PO ₄ , Cs ₂ SO ₄ , CsCH ₃ COO, CsCI, CsBr, CsNO ₃ , CsCIO ₄ , Csl, CsSCN, Li ₃ PO ₄ , Li ₂ HPO ₄ , LiH ₂ PO ₄ , Li ₂ SO ₄ , LiCH ₃ COO, LiCI, LiBr, LiNO ₃ , LiCIO 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 , MgCI ₂ , MgBr ₂ , Mg(NO ₃ ) ₂ , Mg(CIO ₄ ) ₂ , Mgl ₂ , Mg(SCN) ₂ , MnCI ₂ , Ca ₃ (PO ₄ ),, Ca ₂ HPO ₄ , Ca(H ₂ PO ₄ ) ₂ , CaSO ₄ , Ca(CH ₃ COO) ₂ , CaCI ₂ , CaBr ₂ , Ca(NO ₃ ) ₂ , Ca(CIO ₄ ) ₂ , Cab, 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 , BaCI ₂ , BaBr ₂ , Ba(NO ₃ ) ₂ , Ba(CIO ₄ ) ₂ , Bal ₂ and Ba(SCN) ₂ .

Especially preferred are NH acetate, MgCl ₂ , KH ₂ PO ₄ , Na ₂ SO ₄ , KCl, NaCl and CaCl ₂ , such as, for example, the chloride or acetate (trifluoroacetate) salts.

In one aspect, a polypeptide described herein is in the form of a pharmaceutically acceptable salt. In another aspect, the polypeptide is in the form of a pharmaceutical salt in crystalline form.

In one aspect, a pharmaceutically acceptable salt as described herein refers to salts having toxicity profiles in the range that is acceptable for pharmaceutical use.

Used in the context of this invention, the term pharmaceutically acceptable salt refers to derivatives of the disclosed peptides, and the peptide is modified by obtaining acidic or basic salts of the substance. For example, acid salts are prepared from the free base (generally where the neutral form of the drug has a neutral -NH ₂ group) using a reaction with an appropriate acid. Suitable acids for making acid salts include both organic acids, e.g., 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 acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, ptoluenesulfonic acid, salicylic acid and the like, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Conversely, the preparation of basic salts of acidic components that may be present on the peptide is carried out using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

In one aspect, pharmaceutically acceptable salts can increase the solubility and/or stability of the peptides described in the present context. In another aspect, the pharmaceutical salts described herein may be prepared by conventional means from an appropriate carrier peptide or complex by reacting, for example, an appropriate acid or base with the peptides or complexes described herein. In another aspect, the pharmaceutically acceptable salts are in crystalline or semi-crystalline form. In yet another aspect, pharmaceutically acceptable salts may include, for example, those described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use by P. H. Stahl and C. G. Wermuth (Wiley-VCH 2002) and L. D. Bighley, S. M. Berge, D. C Monkhouse, in Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J. C Boylan, Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995, pp. 453-499, each of these references is incorporated herein by reference in its entirety.

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

In one particular embodiment, pharmaceutically acceptable means approved by a federal or state regulatory agency, or listed by the USP or other generally accepted pharmacopoeias, for use in animals, particularly humans. The term carrier refers to the diluent, adjuvant, excipient, or delivery vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those derived from hydrocarbons, animal or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Salt solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, in particular for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, sodium phosphate, sodium acetate, L-histidine, skimmed milk powder, glycerin, propylene, glycol, water, ethanol and the like. The composition may also optionally contain small amounts of wetting or emulsifying agents or pH adjusting agents. Such compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

Typically, the ingredients of the compositions of the invention are supplied either singly or mixed in unit dosage form, for example, as a dry lyophilized powder or dehydrated concentrate in a hermetically sealed container such as an ampoule or sachet, indicating the amount of active substance. If the composition is to be administered by infusion, it may be reconstituted in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

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

Preferred is a dual specificity polypeptide molecule according to the invention, a nucleic acid or an expression vector according to the invention, or a cell according to the invention, or a pharmaceutical composition according to the invention for use in the treatment or prevention of a disease or disorder selected from: immunological disorders, infectious disease, intoxication, and cancer, including the treatment or prevention of certain cancers or other abnormal proliferative conditions, including (but not limited to) the following: carcinoma, including carcinoma of the bladder, breast, colon, kidney, liver, lung, ovary , pancreas, stomach, prostate, cervix

- 15 043129 uterus, thyroid and skin; including squamous cell carcinoma; tumors of the hematopoietic system of lymphoid origin, including acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; tumors of the hematopoietic system of myeloid origin, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other neoplastic diseases including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; neoplastic diseases of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, schwannomas and osteosarcoma; and other neoplastic diseases including melanoma, xeroderma pigmentosa, keratoactantoma, seminoma, follicular thyroid cancer, and teratocarcinoma. Additional cancers may include, but are not limited to, follicular lymphomas, p53-mutated carcinomas, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific embodiments, the treatment or prevention of cancer or dysproliferative changes (such as metaplasia and dysplasia) or hyperproliferative disorders is carried out using the methods and compositions of the invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus . In other specific embodiments, the treatment or prevention of sarcoma, melanoma or leukemia is carried out using the methods and compositions according to the invention.

The invention also relates to methods for eliciting an immune response in a patient or subject comprising administering a therapeutically effective amount of a dual specificity polypeptide molecule according to the invention or a pharmaceutical composition according to the invention. In one aspect, a population of a dual specificity polypeptide molecule according to the invention or a pharmaceutical composition according to the invention is administered to a patient or subject in need thereof.

The invention further relates to a method for killing target cells in a patient or subject, comprising administering to the patient an effective amount of a dual specificity polypeptide molecule in accordance with the invention.

The invention also provides methods for preventing, treating, or controlling one or more symptoms associated with an inflammatory disorder in a subject, further comprising administering to said subject a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents of the invention. The invention also provides methods for preventing, treating, or controlling symptoms associated with an autoimmune disease, further comprising administering to said subject a therapeutically or prophylactically effective amount of one or more immunomodulatory substances of the invention. Infectious diseases that are treatable or preventable with the molecules of the invention are caused by infectious agents, including, but not limited to, viruses, bacteria, fungi, unicellular organisms, and viruses. Viral diseases that can be treated or prevented by the molecules of the invention in combination with the methods of the invention include those caused by hepatitis A, hepatitis B, hepatitis C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, Coxsackie enterovirus, mumps virus, measles virus, rubella virus, poliovirus, smallpox virus, Epstein-Barr virus, human immunodeficiency virus I (HIV-I), human immunodeficiency virus II (HIV-II), but not limited to, and pathogens of viral diseases such as viral meningitis, encephalitis, dengue or smallpox, but not limited to them.

Bacterial diseases that can be treated or prevented by the molecules of the invention in combination with the methods of the invention include those caused by bacteria, including mycobacteria, rickettsiae, mycoplasmas, bacteria of the Neisseria family, S. pneumonia, Borrelia burgdorferi (Lyme borreliosis), Bacillus anthracis (anthrax), tetanus bacilli, streptococcus, staphylococcus, mycobacteria, tetanus bacilli, bacteria that cause whooping cough, cholera, plague, diphtheria, chlamydia, bacteria of the S. aureus family and legionella.

Protozoal diseases that can be treated or prevented by the molecules of the invention in combination with the methods of the present invention that are caused by protozoa include, but are not limited to, leishmania, coccidiosis, trypanosomes, or malaria. Parasitic diseases that can be treated or prevented by the molecules of the invention in combination with the methods of the present invention that are caused by parasites include, but are not limited to, chlamydia and rickettsiosis.

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), pathogen (e.g.,

- 16 043129

B-lymphotropic papovavirus (LPV); whooping cough; Borna's disease virus (BDV); bovine coronavirus; choriomeningitis virus; dengue virus; virus, E. coli; ebola; echovirus 1; echovirus11 (EV); endotoxin (LPS); enterobacteria; enteroorphan virus; enteroviruses; viral feline leukemia; foot-and-mouth disease virus; gibbon leukemia virus (GALV); gram-negative bacteria; Helicobacter pylori; hepatitis B virus (HBV); herpes simplex virus; HIV-1; human cytomegalovirus; human coronovirus; influenza virus A, B and C; legionella; leishmania mexican; listeriosis; measles virus; meningococci; morbilliviruses; mouse hepatitis virus; murine leukemia virus; murine herpesvirus gamma; mouse retrovirus; murine coronavirus, murine hepatitis virus; mycobacterial complex M; gonococci; bird pseudoplague virus; parvovirus B 19; protozoa of the genus Plasmodium falciparum; poxvirus; Pseudomonas aeruginosa; rotavirus; Salmonella typhiurium; shigella; streptococci; lymphotropic T-cell virus 1; vaccinia virus).

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

The dual specificity polypeptide molecule of the invention can be used in a method for preventing or treating a disease or condition whose symptoms are ameliorated by administration of the dual specificity polypeptide molecule. Such drugs may be presented in the form of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. Dual specificity polypeptide molecules for therapeutic purposes are typically supplied as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. This pharmaceutical composition may be presented in any suitable form, (depending on the desired route of administration to the patient). It may be presented as a single dosage form, is usually provided in a sealed container, and may be presented as part of a kit. Such a kit usually (although not necessarily) contains instructions for use. It may contain a plurality of said unit dosage forms. The pharmaceutical composition may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular or intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

In one aspect, the peptides or other molecules described in the present context may be combined with an aqueous carrier. In one aspect, the aqueous vehicle is selected from: ion exchanger, alumina, aluminum stearate, magnesium stearate, lecithin, whey proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated partial glycerides vegetable fatty acids, salts or electrolytes such as protamine sulfate, dibasic sodium phosphate, dicalcium phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g. microcrystalline cellulose , hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate), starch, lactose monohydrate, mannitol, trehalose, sodium lauryl sulfate and sodium croscarmellose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polymethacrylate, waxes, polyethylene polyoxypropylene block copolymers, polyethylene glycol and lanolin.

In one aspect, the aqueous carrier comprises a plurality of components, such as water, along with a non-aqueous carrier component, such as those described herein. In another aspect, an aqueous carrier is capable of providing improved properties in combination with a peptide or other molecule described herein, such as improved solubility, activity, and/or improved immunotherapeutic properties. In addition, the composition may contain adjuvants such as buffers, binders, ballasts, diluents, fragrances, lubricants, etc. The pharmaceutically acceptable diluent may, for example, include solvents, bulking agents, stabilizers, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like having physiological compatibility. Examples of pharmaceutically acceptable diluents include one or more of saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases it will be preferable to include one or more isotonic substances, for example sugars such as trehalose and sucrose, polyhydric alcohols such as mannitol, sorbitol or sodium chloride. Pharmaceutically acceptable substances such as wetting agents or small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers are also within the scope of the present invention. In addition, the composition may contain adjuvants such as buffers, binders, ballasts, diluents, flavors and lubricants.

The dosages of the dual specificity polypeptide molecules of the present invention may vary widely depending on the disease or disorder being treated, the age and condition of the individual being treated, etc.; for example, a suitable dosage range for a dual specificity polypeptide molecule may be between 25 ng/kg and 50 μg/kg. The physician will ultimately determine the appropriate dosage to use.

Pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided in substantially pure form, e.g., at least 80% pure, at least 85% pure, at least 90% pure, at least 91% pure. 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%.

The preferred characteristics of each aspect of the invention apply to each of the other aspects, mutatis mutandis. The prior art documents referenced herein are incorporated in their entirety, to the extent permitted by law. While the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made without departing from the spirit and scope of the present invention as defined in the appended claims. The present invention will be further illustrated by means of the following examples, which are given for illustrative purposes only and are not intended to limit the scope of the invention in any way.

In FIG. 1 is a brief overview diagram of a preferred embodiment of the present invention, a dual specificity polypeptide molecule containing human IgG1 Fc. VD1, VD2=variable domains derived from an antibody; VR1, VR2=variable domains derived from TCR; Link1, Link2=connection linkers; Cys-Cys=cysteine bridges.

In FIG. 2 is a brief overview diagram of 4 different dual specificity polypeptide molecule designs containing human IgG Fc tested in the context of the present invention. Black=TCR-derived variable domains; light grey=antibody derived variable domains; white=constant domains derived from the human IgG molecule. Lip-to-hollow mutations are represented as a cylinder. IA-ID diabody molecules according to the invention.

In FIG. 3 shows the analysis by size exclusion HPLC of various bispecific TKP/mAb molecules with the molecular structure according to the constructs shown in FIG. 2 that were purified using a 2-column purification process. The monomer content of 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%; ID_4: 95.22%; ID_5: 92.76%; ID_4: 99.31%; ID_5: 99.44%.

In FIG. 4 shows activity assay results for various TKP/mAb bispecific constructs (as shown in FIG. 2) designed as IgG4 based molecules. Jurkat_NFATRE_luc2 cells were co-incubated with HIV-peptide-loaded SLYNTVATL (SEQ ID NO: 7) T2 cells in the presence of increasing concentrations of bispecific TCR molecules (bssTKP). The bispecific molecule TKP/mAb-diabody IA-IgG4 showed higher activity than the two alternative TKP/mAb molecules with dual specificity.

In FIG. 5 shows activity assay results for various TKP/mAb bispecific constructs (as shown in FIG. 2) designed as IgG1 based molecules. Jurkat_NFATRE_luc2 cells were co-incubated with HIV-peptide-loaded SLYNTVATL (SEQ ID NO: 7) T2 cells in the presence of increasing concentrations of bispecific TCR molecules (bssTKP).

The bispecific TKP/mAb diabody molecules ID_1, IA_3 and IA1 showed significantly higher activity than the three alternative dual specificity TKP/mAb diabody molecules.

In FIG. 6 shows the results of activity analyzes performed on various IgG1-based TKP/mAb bispecific constructs (as shown in FIG. 2) using various antibody variable domains, both of which target the TKP-CD3 complex. The ID_1 construct includes the variable domains of the CD3-targeted UCHT1(V9) antibody, where the ID_4 and ID_5 constructs include the alpha/beta TKR-specific antibody BMA031 variable domains. Jurkat_NFATRE_luc2 cells were co-incubated with HIV-peptide-loaded SLYNTVATL (SEQ ID NO: 7) T2 cells in the presence of increasing concentrations of bispecific TCR molecules (bssTKP).

In FIG. 7 is a brief overview diagram of the possible orientations of the VD and VR domains in the molecules of the present invention. VH: VH domain derived from an antibody, VL: VL domain derived from an antibody; Va: derived from TKR V-alpha; Ve: formed from TKR V-beta.

In FIG. 8 shows the results of size-exclusion HPLC analysis of aggregates (HMWS high molecular weight fractions) among various IgG1-based TKP/mAb bispecific molecules. The aggregates were analyzed after purification and after storing the molecules at 40°C for 1 week.

- 18 043129 and 2 weeks respectively.

In FIG. 9 shows the results of activity assays performed for various IgG1 TKP/mAb bispecific molecules. Activity was assayed after purification and after storage of the molecules at 40° C. for 1 and 2 weeks, respectively. Storage under stress at 40°C did not lead to a significant loss of activity of the molecules, but for molecules III and IV, a sharp increase in non-specific (i.e., target-independent) activation of Jurkat T-cells was found.

In FIG. 10 shows the results of lactate dehydrogenase release assays for various bispecific TKP/mAb constructs (as shown in FIG. 2) designed as IgG1-based molecules PBMCs isolated from a healthy donor co-incubated with HIV peptide SLYNTVATL (SEQ ID NO: 7) loaded T2 cells in the presence of increasing concentrations of bispecific TCR molecules (bssTKP). The bispecific TKP/mAb diabody molecules IA_3 and ID_1 caused significantly more lysis of target cells than the three alternative TKP/mAb diabody molecules with dual specificity. As shown in the right side graph, none of the TKP/mAb bispecific constructs analyzed caused detectable lysis of T2 cells loaded with the irrelevant peptide (SEQ ID NO: 49).

In FIG. 11 shows the results of the lactate dehydrogenase release assay for the bispecific TKP/mAb-IA_5 diabody construct targeting the tumor-associated peptide PRAME-004 (SEQ ID NO: 49) presented on HLA-A*02. CD8-positive T cells isolated from a healthy donor were co-incubated with UACC-257, SW982 and U2OS cancer cell lines presenting varying amounts of PRAME-004 and HLA-A*02-1 peptide complexes on the cell surface (approx. 1100, about 770 and about 240 copies per cell, respectively, as determined by MS method) at a 5:1 effector to target ratio in the presence of increasing concentrations of TKP/mAb-diabody molecules. After 48 hours of co-culture, target cell lysis was quantified using a lactate dehydrogenase release assay according to the manufacturer's instructions (Promega).

In FIG. 12 shows the results of the lactate dehydrogenase release assay for the TKP/mAb-diabody bispecific constructs IA_5 and IA_6 using stability/affinity matured TKP and their improved version, respectively, for the tumor-associated peptide PRAME-004 (SEQ ID NO: 49) presented on HLA- A*02. CD8-positive T cells isolated from a healthy donor were co-incubated with the U2OS cancer cell line presenting approx. 240 copies per cell of PRAME-004:HLA-A*02-1 complexes, or unloaded T2 cells (effector to target ratio 5:1) in the presence of increasing concentrations of TKP/mAb diabody molecules. After 48 hours of co-culture, target cell lysis was quantified using a lactate dehydrogenase release assay according to the manufacturer's instructions (Promega).

In FIG. 13 shows the results of a stability study under thermal stress conditions for the TKP/mAb-diabody constructs IA_5 and IA_6 using TKP with matured stability/affinity and their improved version, respectively, for the tumor-associated peptide PRAME-004 (SEQ ID NO: 49) presented on HLA-A*02. For this purpose, the proteins were formulated in PBS at a concentration of 1 mg/ml and then stored at 40°C for two weeks. Protein integrity and recovery was assessed by size exclusion HPLC. So the number of high molecular weight fractions was determined in accordance with the percentage of the area of the peak eluted to the main peak. The degree of extraction of the monomeric protein was calculated by comparing the area of the main peak of the samples, not exposed and subjected to stress.

Examples

Example 1 Construction of Fc-Containing Bispecific TKP/mAb Diabodies and Control Molecules

Fc-containing bispecific TKP/mAb diabodies and control molecules (as shown in Figure 2) were designed to specifically bind to the human TKP-CD3 complex and the peptide-MHC complex, comprising the HIV-derived peptide SLYNTVATL (SEQ ID NO: 7 ) associated with the HLA-A2*01 molecule. To target the TKP-CD3 complex, the VH and VL domains derived from the CD3-specific, humanized hUCHT1(V9) antibody 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 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 variant 10 used in the humanized version (own data). The V-alpha and V-beta domains of the previously described stability/affinity matured single chain 868Z11 human T cell receptor disclosed by Aggen et al. were used to target the complex of peptide and MHC molecule. (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 TKP/mAb diabodies, the DNA sequences encoding

- 19 043129 Various combinations of VH and VL (corresponding to VD1 and VD2, respectively) and Va and νβ (corresponding to VR1 and VR2, respectively), as well as the coding linkers Link1 and Link2, were obtained by genetic synthesis. The resulting DNA sequences were cloned in frame into expression vectors encoding the hinge, CH2 and CH3 domain derived from human IgG4 [access code no: K01316] and human IgG1 [access code no: P01857], respectively, and were further genetically modified. Genetic modifications have been made to introduce ridge-to-trough mutations into the CH3 domains with and without additional interstrand stabilization by disulfide bonding; in order to remove the N-glycosylation site in CH2 (eg N297Q mutation); for the purpose of introducing silencing mutations of the Fc gene; in order to introduce, for additional stabilization, a disulfide bond in VL and VH, respectively, according to the methods described in 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 the obtained bispecific TKP/mAb-diabodies, their variants, as well as the corresponding sequences are presented in table. 1.

Table 1. Overview of all derived and evaluated Fc-containing bispecific TKP/mAb diabodies:

KiH: ledge-to-trough;

K/O: Fc gene silencing;

KiH-ds: ledge-in-trough with artificial disulfide bond stabilization for CH3 and CH3' bonding;

ds-hUCHT1(V9): with stabilization due to the disulfide bond of the variable domains of hUCHT1(V9);

Link1: Linker connecting VR1 to nVD1.

Molecule TKR monoclonal antibody SEQ ID modifications IA-lgG4 868Z11 hUCHT1(V9) SEQ ID no. 8 SEQ ID no. 9 lgG4 (KiH) 1А_1 868Z11 hUCHT1(V9) SEQ ID no. 10 SEQ ID no. eleven lgG1 (K/O, KiH) 1А_2 868Z11 hUCHT1(V9) SEQ ID no. 12 lgG1 (K/O, KiH-ds)

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SEQ ID no. 13 1A_3 868Z11 ds-hUCHT1(V9) SEQ ID no. 14 SEQ ID no. 15 lgG1 (K/O, KiH-ds) Yu_1 868Z11 ds-hUCHT1(V9) SEQ ID no. 16 SEQ ID no. 17 lgG1 (K/O, KiH-ds) Yu_4 868Z11 hBMA031(var10) SEQ ID no. 18 SEQ ID no. 19 lgG1 (K/O, KiH-ds) Yu_5 868Z11 hBMA031(var10) SEQ ID no. 20 SEQ ID no. 21 extended Linkl lgG1 (K/O, KiH-ds) ID_4 868Z11 hBMA031(var10) SEQ ID no. 22 SEQ ID no. 23 lgG1 (K/O, KiH-ds) ID_5 868Z11 hBMA031(var10) SEQ ID no. 24 SEQ ID no. 25 extended Linkl lgG1 (K/O, KiH-ds) IA_5 R16P1C10I hUCHT1(Var17) SEQ ID no. 43 SEQ ID no. 44 lgG1 (K/O, KiH-ds) IA_6 R16P1 С101#6 hUCHT1(Var17) SEQJD no. 45 lgG1 (K/O, KiH-ds) SEQ ID no. 46

Were designed different control molecules with the same specificity, table. 2 using the indicated VH, VL, V-alpha and V-beta domains in combination with IgG1 or IgG4 derived constant domains having the genetically modified characteristics described above.

Table 2. Overview of all derived and evaluated Fc-containing bispecific control molecules:

KiH: ledge-to-trough;

K/O: Fc gene silencing.

- 21 043129

Molecule TKR monoclonal antibody SEQ ID modifications HI-lgG4 868Z11 hUCHT1(V9) SEQID No. 38 SEQID No. 39 lgG4 (KiH) IV-lgG4 868Z11 hUCHT1(V9) SEQID No. 40 SEQID No. 41 lgG4 II 868Z11 hUCHT1(V9) SEQID No. 33 SEQID No. 34 lgG1 (K/O, KiH) III 868Z11 hUCHT1(V9) SEQID No. 35 SEQID No. 36 lgG1 (K/O, KiH) IV 868Z11 hUCHT1(V9) SEQID No. 37 lgG1 (K/O) SEQID No. 42

Example 2 Preparation and Purification of Fc-Containing Bispecific TKP/mAb Diabodies

The recombinant protein expression vectors were designed as monocistronic, controlled by pUC19 derived promoter elements derived from human cytomegalovirus (HCMV). Plasmid DNA was amplified in E. coli according to standard culture procedures and then purified using commercially available kits (Macherey & Nagel). Purified plasmid DNA was used to transiently transfect CHO-S cells according to the manufacturer's instructions (ExpiCHO™ system; Thermo Fisher Scientific). Transfected CHO cells were cultured for 6-14 days at 32-37°C with one or two additions of ExpiCHO™ Feed.

The collection of conditioned cell supernatant was carried out by centrifugation (4000xg; 30 min) and clarified by filtration (0.22 μm). Bispecific molecules were purified using an Akta Pure 25 L FPLC (GE Lifesciences) rapid resolution liquid chromatography system equipped with simultaneous affinity and size exclusion chromatography equipment. Affinity chromatography was performed on Protein A columns (GE Lifesciences) following standard affinity chromatography protocols. Size exclusion chromatography was performed immediately after elution (pH 2.8) from the affinity column to obtain highly purified monomeric protein using the Superdex 200 pg system on 16/600 columns (GE Lifesciences) following standard protocols. Protein concentrations were determined on a NanoDrop system (Thermo Scientific) using calculated extinction coefficients according to predicted protein sequences. Concentration, if necessary, and buffer exchange were performed using Vivaspin devices (Sartorius). Finally, the purified molecules were stored in phosphate buffered saline at a concentration of about 1 mg/ml at 2-8°C.

Since proteins for therapeutic use must have acceptable stability when exposed to acid, the percentage of monomeric protein eluting from the Protein A absorption column was evaluated to ensure robust industrial purification processes (Table 3). It is obvious that the introduction of stabilizing mutations into the molecules, as well as the choice of certain orientations of the binding domains, significantly affect the stability upon exposure to acid.

- 22 043129

Table 3. Protein fraction after elution with acid from the absorption column

Molecule Monomer eluted from the absorption columns (% of total peak area) IA-lgG4 (VH-beta) n. O. 1A_1 (VH-beta) 49 1A_2 (VH-beta) 54 1A_3 (dsVH-beta) 63 ID_1 (alpha-dsVH) 46 IC_4 (VH-alpha) 62 1С_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 size exclusion HPLC on MabPac SEC-1 columns (5 µm, 7.8 x 300 mm) by passing 50 mM sodium phosphate at pH 6 ,8 containing 300 mM NaCl on an Agilent 1100 system (see FIG. 3). DNS-PAGE under non-reducing and reducing conditions confirmed the purity and expected size of various dual-specific TKP/mAb molecules (data not shown).

Example 3 Specific and Target Cell-Dependent T-Cell Activation Induced by Fc-Containing TCR/mAb Diabodies

The T-cell activation activity of Fc-containing TKP/mAb diabodies was assessed using the T-Cell Activation Bioassay (Promega). The assay consists of genetically engineered Jurkat T cells that express the luciferase reporter activated by the NFAT factor response element (NFAT-RE). Analyzes were performed according to the manufacturer. Briefly, T2 cells either loaded with HIV-specific SLYNTVATL peptide (SEQ ID NO: 7) or without loaded peptide (unloaded control) were then co-incubated with Promega's kit-modified Jurkat cells in the presence of increasing concentrations of TKP/mAb bispecific molecules. The activation of Jurkat reporter T cells was analyzed 16-20 hours later by measuring the luminescence intensity.

Representative activity assay results are selected for IgG4-based (FIG. 4) and IgG1-based (FIG. 5) TKP/mAb bispecific molecules, respectively. The data indicate that regardless of the IgG isotype of the constant domains used, the Fc-containing TKP/mAb-diabody IA and ID constructs exhibited increased T cell activation compared to the alternative TKP/mAb II, III and IV bispecific constructs, as follows: was measured by the degree of activation and/or the corresponding indicators of the EU ₅₀ . In addition, induced non-specific activation of T cells by Fc-containing TKP/mAb diabodies, compared to unloaded T2 cells, was reduced or at least was at the level of non-specific activation observed for alternative bispecific TKP/mAb constructs. In accordance with the results presented above, the dual specificity TKP/mAb-diabody molecules are preferred molecules for therapeutic intervention because they induce strong activation of effector T cells in a highly target dependent mechanism.

In addition, a lactate dehydrogenase release assay (Promega) was used to quantify PBMC-mediated lysis of SLYNTVATL peptide-loaded (SEQ ID NO: 7) T2 cells induced by various TKP/mAb bispecific molecules (FIG. 10). Consistent with the results of the T-Cell Activation Bioassay above, again, the Fc-containing constructs TKP/mAb-diabody IA and ID were superior to the alternative bispecific constructs TKP/mAb II, III and IV, as indicated by the increased absolute rate of cell lysis. -targets and a lower concentration of TCR in the bispecific molecule required to obtain half the maximum (EC ₅₀ ) level of destruction of target cells. With respect to the TKP/mAb II, III and IV constructs, the TKP/mAb-diabody IA and ID constructs did not cause lysis of T2 cells loaded with the irrelevant peptide (SEQ ID NO: 49), confirming target-specific lysis relative to T2 cells.

Example 4 Development of Fc-Containing Bispecific TKP/mAb Diabodies as a Molecular Platform

Fc-containing bispecific TKP/mAb diabody constructs have been designed to serve as a molecular platform providing framework regions for various variable

- 23 043129 domains derived from TCR and monoclonal antibodies that target various peptide complexes and MHC molecules and cell surface antigens, respectively. To validate suitability as a platform, the variable domains derived from monoclonal antibodies were replaced in the first set of molecules.

The variable domains of the anti-CD3 antibody hUCHT1(V9) (construct ID_1) were replaced with those of the hBMA031 (var10) anti-TKR antibody using the same domain orientation (constructs ID_4 and ID_5) or a different orientation (IC_4, IC_5) (for more details see Table 1 and Fig. 7). Expression, purification and characterization of these molecules were performed as described above. The degree of purification and integrity of the final preparations exceeded 92% according to the analysis by size exclusion HPLC.

Activity assay results revealed target-dependent activation of Jurkat reporter T cells and minimal non-specific activity against unloaded T2 cells for both hUCHT1 (construct ID_1) and hBMA031 (construct ID_4 and ID_5) antibody variable domains, confirming the suitability of the platform. constructs with dual specificity based on TKP/mAb-diabodies (Fig. 6). Notably, when the variable domains of TCR and constructs ID_4 and ID_5 monoclonal antibodies were exchanged on each of the polypeptide chains to give constructs IC_4 and IC_5, no T cell activation was observed (data not shown). The latter fact indicates that, despite the possibility of using bispecific TKP/mAb diabodies as a platform for constructs to introduce various variable domains of TKP and a monoclonal antibody, careful optimization of domain orientation is required to achieve optimal molecular activity.

Example 5 Stability of Fc-Containing Bispecific TKP/mAb Diabodies

The stability of bispecific TKP/mAb molecules was initially assessed using the Protein Thermal Shift Assay (Thermo Fisher Scientific) according to the manufacturer's instructions using the 7500 Real time PCR system (Applied Biosciences). Briefly, purified molecules were mixed with PTS buffer and PTS dye and subjected to an increasing temperature gradient while continuously monitoring the fluorescence of the samples. The recorded fluorescence signals were analyzed using the PTS program (Thermo Fisher Scientific) and the melting points (T _M ) were calculated using a modified technique.

Stress stability studies were performed by storing purified molecules after dissolution in PBS at 40° C. for up to two weeks. Samples were analyzed for protein integrity by size exclusion HPLC and activity by T-Cell Activation Assay (Promega) as described above.

As expected, storage at 40°C caused the formation of aggregates/high molecular weight fractions, as determined using size exclusion HPLC analyzes (see Fig. 8). The results of activity assays of IgG1 molecules after purification and incubation at 40°C are shown in FIG. 9. Although a significant decrease in activity after storage at 40°C was not observed for any of the tested molecules, it was observed that molecules III and IV under stress caused a significant proportion of non-specific (i.e., target-independent) activation of Jurkat T cells . In contrast, the bispecific TKP/mAb diabodies retained their target-dependent activity despite the presence of some aggregates, as detected by size exclusion HPLC.

Example 6 Preparation of cancer-targeting TKP/mAb-diabody bispecific molecules

To further validate the potential of the TKP/mAb-diabody bispecific construct platform, TKR-derived variable domains were replaced with TKR variable domains that were stability/affinity matured by yeast display as described previously (Smith et al., 2015, T-Cell Receptor Engineering and Analysis Using the Yeast Display Platform Methods Mol Biol 1319:95-141). TCR variable domains specifically binding to HIV-derived peptide SLYNTVATL (SEQ ID NO: 7) in association with HLAA*02 molecules were replaced with TCR variable domains specifically binding to tumor-associated peptide PRAME-004 (SEQ ID NO: 49), associated with the HLA-A*02 molecule. In addition, the variable domains of the humanized hUCHT1(V9) T cell recruiting antibody were replaced with the variable domains of hUCHT1(Var17), a recently humanized version of the UCHT1 antibody, to generate the TCR/mAb-diabody IA_5 molecule that targets the peptide PRAME-004 (comprising SEQ ID NO: 43 and SEQ ID NO: 44). Expression, purification and characterization of this molecule was carried out as described in example No. 2. The degree of purification and integrity of the final preparation exceeded 96% according to analysis by size exclusion HPLC.

The binding affinities of the bispecific constructs TKP/mAb-diabody to PRAME-004:HLAA*02 were determined using biolayer interferometry. The measurements were carried out on an Octet RED384 system using the settings recommended by the manufacturer. Briefly, purified TKP/mAb-diabody bispecific molecules were loaded onto biosensors (ANS) prior to analysis of serial dilutions of HLA-A*02/PRAME-004.

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The activity of this TKP/mAb-diabody construct targeting the PRAME004 peptide to induce tumor cell lysis was assessed by human CD8-positive T cell-mediated lysis assay of the UACC-257, SW982 and U2OS human cancer cell lines presenting various amounts of copies of the PRAME-004 peptide in association with HLA-A*02 molecules on the surface of tumor cells (UACC-257 - about 1100, SW982 - about 770, U2OS about 240 copies of PRAME-004 per cell, as determined by quantitative MS analysis) , as determined by the lactate dehydrogenase release assay.

As shown in FIG. 11, the TKP/mAb-IA_5 diabody construct targeting the PRAME-004 peptide caused concentration-dependent lysis of PRAME-004-positive tumor cell lines. Efficient lysis due to this TKP/mAb-diabody molecule was observed even for tumor cells of the U2OS line, expressing only 240 copies of PRAME-004 per tumor cell. These results further demonstrate that the TKP/mAb-diabody format is useful as a molecular platform to allow the introduction of the variable domains of various TKRs as well as the variable domains of various T cell recruiting antibodies.

Example 7 Possibility of Genetically Engineering TKP/mAb Diabody Constructs

The TKP variable domains used in the IA_5 construct were further improved in terms of affinity for the PRAME-004 peptide and TKR stability, and were used to genetically engineer the TKP/mAb diabody scaffold to produce the IA_6 construct (containing SEQ ID NO: 45 and SEQ ID NO: 46). Expression, purification and characterization of the TKP/mAb-diabodies IA_5 and IA_6 molecules were performed as described in Example No. 2. The purity and integrity of the final preparations exceeded 97% according to size exclusion HPLC analysis.

The activity of the variant of the TKP/mAb diabody IA_6, with improved stability and affinity, towards the PRAME-004 peptide was evaluated in an experimental cytotoxicity assay with U2OS tumor cells presenting low amounts of the PRAME-004 complex and HLAA*02 or unloaded T2 cells in as target cells and CD8-positive human T cells as effector cells.

As shown in FIG. 12, the inventors observed an increase in the cytotoxic activity of the TKP/mAb-diabody IA_6 molecule containing the variable domains of the TKP variant with improved stability/affinity compared to the precursor construct IA_5. For both constructs, IA_5 and IA_6, PRAME-004 dependent lysis could be confirmed as no cytolysis of T2 target negative cells was detected.

The protein construct was then thermally stressed at 40° C. for up to two weeks to analyze the stability of the PRAME-004-specific TKP/mAb diabody variants IA_5 and IA_6. SEC HPLC analysis showed significantly improved stability of the IA_6 variant compared to the IA_5 precursor construct (see FIG. 13). The temperature-induced increase in high molecular weight constructs (ie, eluted to the main peak) was less pronounced with IA_6 than with IA_5. Consistent with this result, the recovery of intact monomeric protein after thermal stress was 87% and 92% for IA_5 and IA_6, respectively.

These exemplary design data demonstrate that highly active and stable TKP/mAb-diabody constructs can be further improved by incorporating TKP variable domains with improved stability/affinity, resulting in improved therapeutic proteins with improved performance.

Example 8 Preferred Design Examples

In addition to the HIV-specific TCR-based bispecific construct as described in the present context (SEQ ID NO: 16 and SEQ ID NO: 17, with D orientation), the invention further provides several other exemplary HIV-specific constructs that have been tested . These constructs are based on improved humanized variants of the underlying anti-CD3 antibody (UCHT1) that have been fused to HIV-specific TCR 868 as disclosed herein in all four possible orientations (sequences SEQ ID NO: 51 through SEQ ID NO : 58, in orientations A-D).

Humanization of UCHT1 was performed with VH-1-46 and VK1-018 as acceptor scaffolds for the heavy and light chain CDRs, respectively. The selected J segments were JK1 and JH4, for the light and heavy chain, respectively.

The results obtained are summarized in the following table. 4:

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V9 (Zhu et al, 1995) The present invention DRB1 score 1232 -1190 Titer [mg/l] 0.75 3 Tm for F(ab) [°C] 83.0 86.4 EC50 activation effector cells [pM] 63 8

Data from the table. 4 demonstrate that humanization according to the invention is potentially less immunogenic (lower DRB1 scores); the molecules are more stable (increasing the melting point by about 3°C); and more active (~8-fold reduction in EC50) compared to the standard (V9) (for information on the analysis, see example 3).

Claims (15)

1. A polypeptide molecule with dual specificity, containing a first polypeptide chain and a second polypeptide chain, where: a) the first polypeptide chain contains ai) the first variable domain (VD1) of the antibody, aii) the first variable domain (VR1) of the T-cell receptor (TCR) and aiii) the first linker (LINK1) connecting these domains; b) the second polypeptide chain contains bi) a second variable domain (VR2) of TCR, bii) a second variable domain (VD2) of an antibody, and biii) a second linker (LINK2) connecting said domains; wherein said first variable domain (VD1) and said second variable domain (VD2) associate to form a first binding site (VD1)(VD2) that specifically binds to a cell surface antigen of a human immune effector cell; where the first variable domain (VR1) is one of a TCR Va domain and a TCR Ve domain, and the second variable domain (VR2) is the other of a TCR Va domain and a TCR Ve domain, said first variable domain (VR1) and said second variable domain (VR2 ) are associated with the formation of a second binding site (VR1)(VR2) that specifically binds to the MHC-associated peptide epitope; wherein said two polypeptide chains are fused to human IgG hinge domains and/or human IgG Fc domains or dimerizing regions thereof; where these two polypeptide chains are connected by covalent and/or non-covalent bonds between the specified hinge domains and/or Fc domains; wherein said dual specificity polypeptide molecule is capable of simultaneously binding to a cell surface molecule and a cell and an MHC-associated peptide epitope, and wherein the order of the variable domains in the two polypeptide chains is selected from VD1-VR1 and VR2VD2 or both VD2-VR2 and VR1-VD1. 2. A dual specificity polypeptide molecule according to claim 1, wherein the LINK1 and/or LINK2 linker sequences contain at least one sequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS and TVLSSAS. 3. A dual specificity polypeptide molecule according to any one of claims 1 or 2, wherein said first and second polypeptide chains further comprise at least one hinge domain and an Fc domain or fragments thereof derived from human IgG1, IgG2 or IgG4, wherein preferably said Fc domain contains at least one mutation that suppresses effector function in the sequence of SEQ ID NO: 50 (ELLGGP) of human IgG1, preferably wherein said mutation that suppresses effector function is derived from one or more E1P, L2V, L3A mutations , and the absence of a residue at position 4 of SEQ ID NO: 50. 4. A dual specificity polypeptide molecule according to claim 3, wherein said Fc domain contains a CH3 domain containing at least one mutation that facilitates the formation of heterodimers, preferably wherein said mutations include bump-to-bottom mutations. 5. A dual specificity polypeptide molecule according to any one of claims 3 or 4, wherein said Fc domain comprises CH2 and CH3 domains containing at least two additional cysteine residues. 6. A dual specificity polypeptide molecule according to any one of claims 1 to 5, wherein said antibody-derived VD1 and VD2 domains have an engineered disulfide bridge introducing a covalent bond between VD1 and VD2, and wherein said cysteines are introduced into the framework region (FR ) 4 in the case of VL and into frame region 2 in the case of VH. 7. A polypeptide molecule with dual specificity in accordance with any one of claims 1-6, where from - 26 043129 it is known that said cell surface molecule causes activation of immune cells or is at least one selected from the group consisting of CD3, 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, FcsRI, TCRa/β and TCRy/δ, HLA-DR, preferably wherein said cell surface molecule is CD3y, CD3δ or CD3e. 8. A polypeptide molecule with dual specificity in accordance with any one of paragraphs.1-7, where the first polypeptide chain contains SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30; and the second polypeptide chain contains SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 30. 9. A dual specificity polypeptide molecule according to any one of claims 3 to 8, wherein the Fc region in the first polypeptide chain comprises the sequence of SEQ ID NO: 26 or SEQ ID NO: 47 (Fc1) and the Fc region of the second polypeptide chain contains the sequence of SEQ ID NO: 27 or SEQ ID NO: 48 (Fc2). 10. A dual specificity polypeptide molecule according to claim 1, wherein the first polypeptide chain comprises SEQ ID NO: 16, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55 or SEQ ID NO: 57 and wherein the second polypeptide chain comprises SEQ ID NO: 17, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58. 11. A polypeptide molecule with dual specificity in accordance with any one of paragraphs.1-10, where the specified first binding site (VD1)(VD2), which binds the surface antigen of these immune cells, humanized; and/or said second binding site (VR1)(VR2) that binds said MHC-associated peptide epitope has affinity matured and/or stability matured. 12. A nucleic acid encoding a first polypeptide chain and/or a second polypeptide chain according to any one of claims 1 to 11, or an expression vector containing at least one of said nucleic acids. 13. A host cell containing a vector as defined in claim 12, preferably wherein the host cell expresses said vector. 14. A pharmaceutical composition comprising a dual specificity polypeptide molecule according to any one of claims 1 to 11, a nucleic acid or an expression vector according to claim 12, or a cell according to claim 13, together with one or more pharmaceutically acceptable carriers or excipients. 15. Use of a dual specificity polypeptide molecule according to any one of claims 1 to 11, a nucleic acid or an expression vector according to claim 12, a cell according to claim 13 or a pharmaceutical composition according to claim 14 in the treatment of diseases or disorders, preferably for the prevention or treatment of a disease or disorder selected from cancer, infectious diseases, and immunological disorders.