Multispecific molecules

Abstract

To provide multi-specific molecules which are capable of simultaneously binding at least two different target antigens or epitopes.SOLUTION: The molecules comprise at least one binding domain molecule (BDM) that binds to a first target antigen or epitope. The BDM is modified for selective binding to a heterologous target, and coupled to a pharmacologically active protein or peptide, which is an antibody or antigen-binding fragment thereof or a non-antibody protein or peptide, binding to a second target antigen or epitope. The BDMs are coupled to a C-terminus of a polypeptide present within the pharmacologically active protein or peptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to a multispecific molecule capable of simultaneously binding to at least two different target antigens or target epitopes. The molecule comprises at least one binding domain molecule (BDM) that binds to a first target antigen or target epitope, such BDM being modified to selectively bind to a heterologous target and conjugated to a pharmacologically active protein or peptide, which is an antibody or its antigen-binding fragment or a non-antibody protein or peptide, that binds to a second target antigen or target epitope, the BDM being conjugated to the C-terminus of a polypeptide present within the pharmacologically active protein or peptide.

[0002] Built-in by reference All materials cited or referenced herein, and all materials cited or referenced in materials cited herein, together with any manufacturer's instructions, statements, product specifications, and product sheets relating to any product mentioned herein or in any documents incorporated herein by reference, are incorporated herein by reference in their entirety.

[0003] This application claims priority to Australian Patent Applications No. 2016900708 and No. 2016900709, the entire contents of which are incorporated herein by reference.

[0004] Sequence listing reference The entire contents of the electronically submitted sequence listing are incorporated by reference for all purposes. [Background technology]

[0005] Numerous recombinant proteins have been developed as therapeutic agents. However, proteins in their unmodified form are known to be rapidly removed in the body by renal filtration, cell clearance mechanisms in the reticuloendothelial system, or proteolysis (Francis (1992) Focus on Growth Factors 3:4-11). Various modifications of proteins and peptides have been developed to enhance the stability, circulation time, and biological activity of therapeutic proteins (see Francis (1992) Focus on Growth Factors 3:4-10). However, mechanisms that allow such therapeutic proteins to remain present in the body for longer periods are needed in this field.

[0006] Therapeutic monoclonal antibodies and antibody-related products, such as antibody-fusion proteins, antibody fragments, and antibody-drug conjugates (hereinafter collectively referred to as antibody products), have grown to become a major product class within the biopharmaceutical market. Today, antibody products are approved for the treatment of various diseases, including, to name a few, certain cancers, multiple sclerosis, asthma, and rheumatoid arthritis.

[0007] Despite the unparalleled successes in antibody drug development in recent years, the lack of efficacy against specific target diseases necessitates the exploration of new strategies for developing more effective antibody drugs. One method used to improve antibody efficacy is to create bispecific antibody-like protein structures that bind to two targets simultaneously. Conventional antibodies can only bind to one specific target (i.e., they have one specificity). Bispecific antibodies are generally engineered proteins composed of two different antibodies or antibody-like fragments (known as antibody-like scaffolds) fused together so that such bispecific antibodies can simultaneously bind to two different targets (i.e., have two specificities). Most antibody-like scaffolds are generally constructed from antibody fragments or made from antibody-like proteins that can bind to specific targets like antibodies. Bispecific antibodies enable more potent antibody drugs that can be designed to induce and activate immune effector cells such as T cells to specifically kill tumors, bind to multiple targets to act on multiple pathogenic pathways, bind to multiple sites on a single target cell or target protein to enhance specificity or induce synergistic induction, and target tumors with heterogeneous properties.

[0008] Currently, a key distinguishing factor among existing technologies used to produce bispecific antibody products is the general method of fusing various antibody-like scaffolds together. This type of bispecific antibody product has several significant drawbacks. First, small bispecific antibody products created by fusing two or more antibody-like scaffolds generally fall well below the renal threshold and typically have very short circulating half-lives ranging from a few minutes to a few hours. Such short half-lives necessitate daily or continuous infusion, which can lead to exceeding the drug's toxicity threshold. Second, many antibody-like scaffolds are insufficient due to their monovalent nature, meaning they have only one antigen-binding site compared to antibodies with two. Complete antibodies can bind to either antigen-binding site, improving the overall binding strength (known as the avidity effect) and offering certain advantages compared to monovalent antibody-like scaffolds.

[0009] Currently, there are only a handful of methods for producing bispecific antibody products in whole IgG format. Generally, these methods artificially create an antibody skeleton that combines two different parent antibodies with different specificities to form a single IgG (i.e., catumaxomab) with arms that bind to different targets. While this whole antibody method has some advantages over methods that fuse smaller fragments, this type of whole IgG bispecific method avoids the half-life problem seen in small fragment bispecific antibodies, but it does not address avidity loss because only one arm of the bispecific antibody binds to each target.

[0010] Therefore, there is a need in the art for methods to improve existing therapeutic proteins and antigen-binding molecules that can help improve their therapeutic efficacy and / or half-life. Furthermore, there is a need in the art for methods to overcome the current bispecific antibody methods and the drawbacks associated with their use. [Overview of the project]

[0011] This disclosure is based on a method for improving one or more characteristics of a protein or peptide, such as an antibody or immunoglobulin antigen-binding fragment. More specifically, this disclosure is based on a method for improving a protein with insufficient therapeutic effect (e.g., a therapeutic antibody) by converting it into a multispecific format. By conjugating a protein or peptide with at least one binding domain molecule (BDM) described herein, such a molecule is provided that can conjugate to multiple different targets by taking advantage of the fact that the binding targets (i.e., antigens or epitopes) of the protein and BDM are different. Thus, one or more characteristics of a protein or peptide can be improved, such as therapeutic efficacy, half-life, immune association, avidity, intracellular delivery, and / or tolerability. Thus, such a molecule provides an alternative to conventional bispecific antibodies.

[0012] Preferably, the target antigen or target epitope bound by BDM is different from the target antigen or target epitope bound by protein or peptide. For example, protein may bind to target antigen or target epitope present on cells or tissues, and BDM may bind to target antigen or target epitope present on immunomodulatory cells such as cytotoxic T cells or proteins that promote cell death, or to target antigen on human serum albumin (HSA) so as to extend the half-life of protein or peptide.

[0013] By utilizing the fact that proteins or peptides and BDMs bind simultaneously to their respective targets, the therapeutic efficacy of proteins or peptides can be enhanced by leveraging the functionality of the target to which the BDMs bind. Further binding of BDMs to proteins or peptides can convert them into bispecific, triplicate, or even multispecific forms. In particular, due to their small size, binding affinity, and solubility, these BDMs are ideal agents for improving the efficacy of inadequately therapeutic proteins or peptides, for example, by promoting the body's innate immune mechanisms that destroy tumor cells.

[0014] Therefore, this disclosure provides a multispecific molecule capable of binding to two or more different target antigens or target epitopes, such molecule (i) a binding domain molecule (BDM) that binds to a first target antigen or target epitope, the BDM comprising or comprising a V-like domain (VLD) scaffold having three exposed binding loops (BLs) contained therein, wherein at least two of the three BLs are modified or substituted with respect to their corresponding native sequences within the scaffold to selectively bind to a heterologous target antigen or target epitope, and (ii) comprising a pharmacologically active protein or peptide which is an antibody or its antigen-binding fragment or a non-antibody protein or peptide that binds to a second target antigen or target epitope, Herein, at least one BDM is bound to the C-terminus of a polypeptide present within its pharmacologically active protein or peptide.

[0015] For example, a pharmacologically active protein binds to its original target antigen or target epitope.

[0016] Preferably, the epitopes are located on separate antigens.

[0017] In one example, the first and second target antigens are different. In another example, the first and second target antigens are the same, but the molecule binds to different epitopes on that target antigen. In yet another example, the first and second target epitopes are different.

[0018] In one example, the molecule is a bispecific molecule. In another example, the molecule is a triplicate molecule.

[0019] In one example, the molecule contains one, two, three, four, or five BDMs (or multiples thereof, e.g., BDM dimers). In another example, the molecule contains one or two pairs of BDMs, where the BDMs in such pairs are identical. In one example, one, two, or three BDMs (or multiples thereof, e.g., dimers) are conjugated to a non-antibody protein or peptide.

[0020] In one example, the molecule comprises at least two BDMs, or at least one pair of BDMs, where each BDM (or BDM pair) binds to a different target antigen or target epitope. In a further example, each BDM (or BDM pair) binds to a target antigen or target epitope different from the target antigen or target epitope to which the pharmacologically active protein or peptide binds.

[0021] In another example, two, four, six, or eight BDMs are conjugated to a full-length antibody, where the molecule binds to at least two different target antigens or target epitopes. In one example, the molecule binds to two different target antigens or target epitopes. In another example, the molecule binds to three different target antigens or target epitopes. In yet another example, the molecule binds to four different target antigens or target epitopes.

[0022] In one example, the pharmacologically active protein is a full-length antibody or its immunoglobulin antigen-binding fragment. In another example, the protein is a non-antibody protein or peptide.

[0023] For example, non-antibody proteins or peptides are selected from the group consisting of the extracellular domains of proteins selected from blood coagulation factors, antikalin, toxoids, collagen-binding proteins, human serum-binding proteins (e.g., human serum albumin, HSA), tumor necrosis factor (TNF)-α receptor-binding proteins, integrin-binding proteins, vascular endothelial growth factor (VEGF) or its pseudo-substances, erythropoietin (EPO) or its pseudo-substances, C4-binding proteins, urokinase receptor antagonists, lymphokines, cytokines, osteoprotegerin (OPG), or programmed cell death 1 protein (PD1), programmed cell death ligand 1 (PD-L1), NKG2D, MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), and UL16-binding protein (ULBP).

[0024] In one example, the blood clotting factor is factor VIII or factor IX.

[0025] For example, toxoid is botulinum toxoid.

[0026] For example, lymphokines are IL-2 or its pseudo-substance, or GM-CSF or its pseudo-substance.

[0027] For example, cytokines may be G-CSF or its pseudo-substance, or stem cell factor (SCF) or its pseudo-substance.

[0028] In one example, the molecule contains one BDM bound to a non-antibody protein. In this example, the ratio of BDM to non-antibody protein is 1:1.

[0029] In one example, at least one BDM is attached to the C-terminus of the antibody heavy chain polypeptide. In another example, at least one BDM is attached to the C-terminus of both antibody heavy chain polypeptides.

[0030] In one example, at least one BDM is attached to the C-terminus of the antibody light chain polypeptide. In another example, at least one BDM is attached to the C-terminus of both antibody light chain polypeptides.

[0031] In one example, at least one BDM is attached to the C-terminus of all antibody heavy chain polypeptides and antibody light chain polypeptides.

[0032] In one example, at least one BDM is conjugated to the C-terminus of the CH1, CH2, or CH3 domain of an antibody heavy chain polypeptide.

[0033] In one example, at least one BDM is conjugated to the C-terminus of the antibody Fc.

[0034] For example, at least one BDM, (i) C-terminus of antibody light chain polypeptide, (ii) The C-terminus of each antibody light chain polypeptide, (iii) C-terminus of antibody heavy chain polypeptide, (iv) C-terminus of each antibody heavy chain polypeptide Combine them.

[0035] The full-length antibodies or immunoglobulin antigen-binding fragments described herein may be monospecific or bispecific. In the case of monospecific antibodies, the antibody may have a heavy chain variable domain and a light chain variable domain (i.e., a pair of V) that share the same specificity for a single target antigen or target epitope. H / V L This means that the antibody binds to such a target or epitope via the complementarity of the two arms of the Y-shaped antibody molecule. H / V L Each pair contains one pair, where each V H / V L It binds to different target antigens or target epitopes.

[0036] A full-length antibody contains two heavy chains and two light chains, each forming a pair. Therefore, in one example, the ratio of antibody chain to BDM is 4:2. In another example, the molecule contains four BDMs bound to the antibody (i.e., one BDM on each light chain and one BDM on each heavy chain). In one example, the ratio of antibody chain to BDM is 4:4. In another example, the molecule contains six BDMs bound to the antibody. In one example, the ratio of antibody chain to BDM is 4:6. In yet another example, the molecule contains eight BDMs bound to the antibody. In yet another example, the ratio of antibody chain to BDM is 4:8. In yet another example, the ratio of antibody chain to BDM is 4:2. n Here, n is a number from 1 to 5, including 1 and 5.

[0037] In one example, the immunoglobulin antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scFv, di-scFv, or chemically bound F(ab')2.

[0038] In one example, the molecule contains a single BDM conjugated with an immunoglobulin antigen-binding fragment. In one example, the ratio of the immunoglobulin antigen-binding fragment chain to the BDM is 2:1. In another example, the ratio of the immunoglobulin antigen-binding fragment chain to the BDM is 2:2. In yet another example, the BDM chain may be conjugated with an antigen-binding fragment, in which case the ratio of the immunoglobulin antigen-binding fragment chain to the BDM is 2:n, where n is a number from 1 to 16. In yet another example, n is a number from 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 4, or 2, or 1.

[0039] This disclosure intends for a number of different configurations in which at least one BDM can bind to a full-length antibody, for example: (i) Conjugate at least one BDM to the C-terminus of the CH3 domain of the antibody heavy chain polypeptide. (ii) Conjugate at least one BDM to the C-terminus of the CH1 domain of the light chain polypeptide. (iii) Conjugate at least one BDM to the C-terminus of the CH3 domain of the heavy chain polypeptide and the C-terminus of the CH1 domain of the light chain polypeptide. (iv) Conjugate at least one BDM to the C-terminus of the CH3 domain of both heavy chain polypeptides, or (v) Attach at least one BDM to the C-terminus of both CH1 domains of the light chain polypeptide.

[0040] With respect to immunoglobulin antigen-binding fragments, BDM may be conjugated to either the C-terminus of the heavy or light chain of the immunoglobulin fragment. In another example, BDM may be conjugated to the C-terminus of both the heavy and light chains of the immunoglobulin fragment. In yet another example, BDM may be conjugated to either the light and / or heavy chain of the immunoglobulin fragment (e.g., CH1) (e.g., Fab).

[0041] for example, (i) Conjugate at least one BDM to the C-terminus of the constant region of the Fab light chain polypeptide. (ii) Conjugate at least one BDM to the C-terminus of CH1 of Fab's heavy chain polypeptide, or (iii) At least one BDM is attached to the C-terminus of the constant region of the Fab light chain polypeptide and to the C-terminus of CH1 of the heavy chain polypeptide.

[0042] The BDM according to this disclosure preferably comprises or comprises a scaffold having three exposed binding loops (BLs) contained therein. The scaffold may be selected from the group consisting of a superfamily member containing an immunoglobulin-like (Ig-like) domain, a V-like domain, an i-body, a VNAR, or a VHH.

[0043] In one example, the exposed BL sequence is modified or substituted from the natural BL sequence, providing a modified binding loop that selectively binds to a heterologous target antigen or target epitope. These binding loops are designated as BL1, BL2, and BL3, respectively, as illustrated in Figure 1A. BL is similar to the CDR1, CDR2, and CDR3 regions of the antibody variable region.

[0044] In one example, BL1 and BL3 are modified or substituted from the natural BL sequence. In another example, BL1, BL2, and BL3 are modified or substituted from the natural BL sequence.

[0045] In another example, the BDM scaffold has less than 20% sequence identity to the human immunoglobulin variable region domain, and the scaffold has two or more modified BLs and exhibits selective binding to heterologous target antigens or target epitopes.

[0046] Ig-like domain-containing superfamily members may be selected from the group consisting of V-like domains (VLDs), C-set domains, ThyOx family member polypeptides, T cell receptors, CD2, CD4, CD8, class I MHC, class II MHC, CD1, cytokine receptors, G-CSF receptors, GM-CSF receptors, hormone receptors, growth hormone receptors, erythropoietin receptors, interferon-gamma receptors, prolactin receptors, NCAM, VCAM, ICAM, N-cadherin, E-cadherin, fibronectin, tenascin, and polypeptides or functional fragments of I-set-containing domains.

[0047] The BDM scaffolds provided in this disclosure may be selected from the group consisting of V-like domains (VLDs), C1-set domains, or C2-set domains. Combinations of BDMs conjugated to proteins or peptides are also intended. For example, one BDM may be a VLD and another may be a C-set domain.

[0048] In one example, the BDM scaffold comprises or consists of the extracellular portion of a native VLD, or a VLD having a modified binding loop to a native VLD (i.e., modified BDM), where the VLD is ACAN, ADORA3, ALCAM, JAML, AMIGO1, AXL, basidine, BCAM, BTNL2, 3, 8, 9 or 10, butyrophyllin (BTN), cell adhesion molecules (CAM), CD2, CD4, CD7, CD8, CD28, CD33, CD48, CD79, CD80, CD83, CD86, CD101, CD112, CD226, CD274, CD276, CD300, carcinoembryonic antigen-associated cell adhesion molecule (CEACAM), CRTAM, CTLA4, CXADR, C10orf54, ERMAP, ESAM, FAM187A, FCAMR, F11R, It is derived from proteins selected from the group consisting of GPA33, hyaluronan and proteoglycan link protein (HAPLN), HAVCR1, HEPACAM, HHLA2, HSPG2, ICOS, IGHA, IGSF, JAM2, JAM3, KDR, KIRREL, LY6G6F, MCAM, MOG, MPZ, MXRA8, NCA, NCR2, NCR3, NPHS1, PD1, PDCD1, PIGR, PILR, PSG, PTGFRN, PVR, sodium channel subunit protein (SCN), SEMA3D, sialoadhesin protein (SIGLEC), signal regulatory protein (SIRP), SLAMF6, SLAMF7, TIGIT, TIMD4, TREM, TREML, VCAN, VPREB, VSIG, VSTM, and VTCN1.

[0049] For example, a BDM scaffold may include or be composed of the extracellular portion of a native C-set domain (C1-set domain or C2-set domain), or a C-set domain having a modified binding loop to a native C-set domain (i.e., a modified C-set domain), where the C-set domains are AZGP1; basidine, B2M; CEACAM1, 3, 4, 5, 6, 7, 8; CD1A; CD1B; CD1C; CD1D; CD 1E;DMA;DQB2;DRB1;ELK2P1;FCGRT;HFE;HHLA2;HLA-A;HLA-B;HLA-B35;HLA-B57;HLA-C;HLA-CW;HLA-Cw;HLA-D;HL A-DMA;HLA-DMB;HLA-DOA;HLA-DOB;HLA-DP;HLA-DPA1;HLA-DPB1;HLA-DQA1;HLA-DQA2;HLA-DQB1;HLA-DQB2;HLA-D RA;HLA-DRB1;HLA-DRB2;HLA-DRB3;HLA-DRB4;HLA-DRw12;HLA-Dw12;HLA-E;HLA-F;HLA-G;HLA-G2.2;HLA-H;HLAC; IGHA1;IGHA2;IGHD;IGHE;IGHG1;IGHG2;IGHG3;IGHG4;IGHM;IGHV4-31;IGKC;IGKV1-5;IGKV2-24;IGL;IGLC1;IGLC 3;IGLL1;IGLV2-14;IGLV3-21;IGLV3-25;IGLV4-3;MICA;MICB;MR1;SIRPA;SIRPB1;SIRPG;SNC73;TAPBP;TAPBPL;TRBC1;TRBV19;TRBV21-1;TRBV3-1;TRBV5-4;TRBV7-2;Derived from proteins selected from the group consisting of micB, CD2, CD4, CD80, VCAM, and ICAM.

[0050] For example, the BDM scaffold includes or comprises an Ig-like domain having all or part of a native Ig-like domain or a modified binding loop (i.e., a modified Ig-like domain) relating to a native Ig-like domain, where the Ig-like domain is selected from the group consisting of ThyOx family member polypeptides, T cell receptors, CD2, CD4, CD8, class I MHC, class II MHC, CD1, cytokine receptors, G-CSF receptors, GM-CSF receptors, hormone receptors, growth hormone receptors, erythropoietin receptors, interferon-gamma receptors, prolactin receptors, NCAM, VCAM, ICAM, N-cadherin, E-cadherin, fibronectin, tenascin, and I-set-containing domain polypeptides or their functional fragments.

[0051] In this specification, terms such as "modified VLD" and "modified C-set domain" refer to BDMs in which at least two, preferably all three, of the exposed binding loops have been modified to achieve binding to a heterologous target antigen or target epitope. Modification may be achieved by amino acid substitution or by replacing all or part of the individual binding loops. The modified amino acid sequence within the binding loops confers selective binding activity to target antigens other than those to which an unmodified Ig-like domain-containing scaffold would bind. Amino acid modifications can be carried out at the nucleic acid or polypeptide level using methods known in the art.

[0052] The VLD or C-set domain may contain a BDM that binds to a heterologous target antigen or target epitope. The modified VLD or C-set domain may also include one or more modifications that alter the BDM's affinity for its native target. This affinity may be increased or decreased compared to the native VLD or C-set domain.

[0053] For example, a modified BDM may contain or consist of sequences that are at least approximately 60%, 70%, 75%, 80%, 85%, 87%, 90%, or 95% identical to the sequence of the natural VLD or C-set domain.

[0054] Those skilled in the art will be familiar with selecting appropriate heterobinding loop sequences for binding BDM to a desired target antigen. Such examples include those described in US7,166,697, the entirety of which is incorporated herein by reference.

[0055] In another example, BDM contains or is a component of all or part of a VLD protein or C-set domain protein, including 5 to 30 amino acid substitutions, 5 to 20 amino acid substitutions, 5 to 15 amino acid substitutions, 5 to 10 amino acid substitutions, or up to 5 amino acid substitutions compared to the corresponding native VLD protein or C-set domain protein. In another example, BDM is not the CLTA-4 VLD mutant molecules L104EA29Y or L104E described in US7,094,874.

[0056] In one example, the modified BDM contains one heterologous BL sequence. In another example, the modified BDM contains two heterologous BL sequences. In yet another example, the BDM contains three heterologous BL sequences.

[0057] In certain cases, BDM is a VLD scaffold containing or comprising the extracellular components of CTLA4, CD28, or ICOS. In further cases, the VLD scaffold is the extracellular components of human CTLA4. In another case, the BDM VLD is [Table 1] (Sequence ID 1) The sequence described in [reference] or its constituent elements are included in [reference].

[0058] In one example, alanine (A) at position 31 is replaced with tyrosine (Y). In another example, methionine (M) at position 56 is replaced with threonine (T).

[0059] In another example, the BDM VLD scaffold consists of framework sequences corresponding to residues 1-25, 34-54, 60-96, and 106-126 of sequence number 1.

[0060] In another example, the BDM scaffold includes or comprises sequences with at least approximately 70% sequence identity to it, or sequences with at least 75%, 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to sequence number 1 or residues 1-25, 34-54, 60-107, and 116-136 of sequence number 1.

[0061] In another example, one, two, or all three exposed binding loops of a natural BDM scaffold may be modified by amino acid substitution, addition, or deletion, and / or by optionally altering one or more physical properties (e.g., size, shape, charge, hydrophobicity, etc.).

[0062] Further examples include modifying the exposed binding loop (BL1) sequence ASPGKATE (SEQ ID NO: 2) or ASPGKYTE (SEQ ID NO: 7) of the native human CTLA-4 VLD sequence, and / or the exposed loop (BL2) sequence MMGNE (SEQ ID NO: 3) and / or the exposed binding loop (BL3) sequence ELMYPPPYY (SEQ ID NO: 4) by amino acid substitution, addition, deletion, or replacement with heterologous sequences.

[0063] For example, modify or replace the amino acid residues at positions 26-33 and / or 55-59 and / or 98-105 of SEQ ID NO: 1.

[0064] In another example, amino acid residues at positions 27–33 and / or 54–62 and / or 98–106 of sequence number 1 are modified or replaced with heterologous sequences.

[0065] In another example, the effect of modifying the natural human CTLA-4 VLD is to eliminate the VLD's natural affinity for CD80 and CD86.

[0066] In one example, BDM VLD scaffolding is arranged [Table 2] (Sequence ID 5) Includes or constitutes Here, Xn1, Xn2, and Xn3 are arbitrary amino acid residues, n is a number between 5 and 15, and the numbers 1, 2, and 3 refer to binding loop regions. More specifically, 1, 2, and 3 correspond to BL-1, BL-2, and BL-3 of BDM, respectively.

[0067] In one example, BDM VLD scaffolding is arranged [Table 3] (Sequence ID 6) Includes or constitutes Here, Xn1, Xn2, and Xn3 are arbitrary amino acid residues, n is a number between 5 and 15, and the numbers 1, 2, and 3 refer to binding loop regions. More specifically, 1, 2, and 3 correspond to BL-1, BL-2, and BL-3 of BDM, respectively.

[0068] In one example, BL-1, BL-2, and BL-3 of BDM each contain or are components of ASPGKATE (sequence number 2) or ASPGKYTE (sequence number 7), MMGNE (sequence number 3), and ELMYPPPYYL (sequence number 9), respectively, where BDM is bound to B7-1.

[0069] In one example, BL-1, BL-2, and BL-3 of the molecule BDM contain or are composed of TVSWVDME (SEQ ID NO: 10), WNGRW (SEQ ID NO: 11), and QLDPSWGYYWQGYE (SEQ ID NO: 12), respectively, where BDM is bound to sclerostin.

[0070] For example, BDM VLD is a sequence [Table 4] (Sequence ID 13) is included or is a component thereof, where BDM is bound to B7-1.

[0071] In another example, BDM VLD is an array [Table 5] (Sequence ID 14) is included or constitutes a compound in which BDM is bound to sclerostin.

[0072] In a further example, BL-1, BL-2, and BL-3 of the BDM are replaced with the CDR1, CDR2, and CDR3 sequences of the antibody, respectively. The antibody from which the CDR sequences are derived may originate from any species. In one example, the antibody is derived from a human. In another example, the antibody is derived from a domesticated animal, such as a cat, dog, rabbit, guinea pig, or horse.

[0073] In one example, at least one BDM may exist in the molecule in monomeric or dimeric form. In another example, at least one BDM may consist of a series of BDM monomers linked together.

[0074] Therefore, the term "BDM" as used herein includes BDM in monomeric form. In one example, BDM is a dimer. The dimer consists of cysteine ​​residues (Cys) in the stem of each CTLA4 monomer. 120 The dimers may be formed via disulfide bonds between them (each stem corresponds to approximately 10 residues connecting the VLD and the transmembrane region). Alternatively, the dimers may be formed by linking monomer units. In one example, a series (or daisy-chain) of monomeric BDMs may be linked together. For example, 2 to 16 BDM monomers may be linked head-to-tail in a daisy-chain-like arrangement. In another example, 2 to 14, 2 to 12, 2 to 10, 2 to 8, or 2 to 4 BDMs may be linked head-to-tail.

[0075] The linkage of BDM monomers can be achieved, for example, by utilization of covalent or non-covalent bonds, or by use of short peptide linkers as detailed herein. BDM monomers can be linked to each other using any of the linkage methods referred to herein. In another method, adjacent BDM monomers may be fused directly to each other.

[0076] In one example, the BDM is soluble. The "solubility" of the BDM scaffold of the present disclosure correlates with the generation of correctly folded monomer domains. Solubility may be evaluated, for example, by high performance liquid chromatography (HPLC). For example, in the case of soluble monomeric BDM, a single peak will occur in the HPLC chromatogram, while in the case of insoluble (e.g., multimeric or aggregated) BDM, multiple peaks will occur.

[0077] The binding of at least one BDM to a pharmacologically active protein may be achieved by methods known to those skilled in the art. The binding may be achieved, for example, by use of a linker, direct fusion, complexation or by covalent or non-covalent bonds.

[0078] In one example, the binding of a pharmacologically active protein to at least one BDM is achieved using a peptide linker. Any suitable peptide linker known in the art can be utilized in the present disclosure. In one example, the linker comprises the sequence (SGGGG) n S, (SEQ ID NO: 15), where n is any number from 2 to 8, or from 3 to 6 or 3 to 4. In one example, the linker comprises or consists of the sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 16) or SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17).

[0079] In another example, the binding of a pharmacologically active protein to at least one BDM is achieved without using a linker.

[0080] In one example, the molecule can bind simultaneously to a first target antigen, a second target antigen and / or optionally a third target antigen.

[0081] In another example, the molecule can bind to both B7-1-Fc and sclerostin simultaneously.

[0082] In a further example, a pharmacologically active protein or peptide and at least one BDM specifically bind to each of these target antigens. In another example, a molecule selectively binds to cells expressing two or more different target antigens or target epitopes recognized by the individual BDMs and pharmacologically active protein portions of that molecule, but does not selectively bind to cells expressing only one of the target antigens or target epitopes.

[0083] This disclosure also provides polypeptides comprising a BDM scaffold conjugated to a pharmacologically active protein or peptide. In one example, the polypeptide further comprises a linker. In one example, the linker comprises the sequence (SGGGG)nS, where n is any number from 2 to 8, or 3 to 6, or 3 to 4. In one example, the linker comprises or comprises the sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 16) or SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17).

[0084] This disclosure also provides polypeptides selected from the group that include or are components of any one of the sequences SEQ ID NOs: 5, 6, 13, 14, 19, 21, 22, 23, 24, 25, 27, 28, or 29. Preferably, the polypeptides are isolated. For example, the polypeptides of this disclosure include polypeptide tags. Examples of preferred tags include, but are not limited to, the p97 molecule, myc, the hexa-his tag, flag, and E7.

[0085] In one embodiment, the molecule according to the Disclosure is a nucleic acid. The Disclosure also provides nucleic acids that encode the polypeptides of the Disclosure, in particular one of the polypeptides SEQ ID NOs: 5, 6, 13, 19, 21, 22, 23, 24, 25, 27, 28, or 29. The nucleic acid may include DNA, RNA, or both.

[0086] In one example, the molecule is [Table 6] (Sequence ID 30) The BDM VLD nucleic acid sequence described in [reference] or its constituent elements, where N1 is the length of the nucleotide encoding the first binding loop, N2 is the length of the nucleotide encoding the second binding loop, and N3 is the length of the nucleotide encoding the third binding loop. In one example, N1, N2, and N3 are 15 to 45 nucleotides. In another example, N1 is 15 to 24 nucleotides, N2 is 15 nucleotides, and N3 is 30 to 45 nucleotides. N is any nucleotide (A, C, T, G).

[0087] In one example, a nucleic acid is provided as an expression construct, in which case such nucleic acid is functionally linked to a promoter. Such an expression construct may be a vector, such as a plasmid. In one example, the expression construct may be a bisistronic expression construct. The disclosure also intends separate expression constructs for the heavy and light chains of an antibody or immunoglobulin antigen-binding fragment. For example, one vector may contain a nucleic acid encoding an immunoglobulin light chain and a BDM VLD, and the other may contain a nucleic acid encoding an immunoglobulin heavy chain, or vice versa.

[0088] Nucleic acids may further include portions, such as FLAGs, to facilitate purification and identification. Systems for the cloning and expression of polypeptides in various different host cells are known and are described in detail herein.

[0089] This disclosure also provides host cells transformed with the nucleic acids described herein. Suitable host cells include bacteria, mammalian cells, yeast, mosses (bryophytes), and baculoviruses.

[0090] This disclosure also provides a method for producing the polypeptide molecule of this disclosure, comprising culturing the host cells of this disclosure under conditions that enable polypeptide expression, and optionally recovering the polypeptide. Depending on the host cells selected, the polypeptide may be glycosylated or unglycosylated.

[0091] This disclosure also provides a method for producing a multispecific molecule containing at least one BDM VLD conjugated to a pharmacologically active protein, and such method is (i) Provide a nucleic acid that encodes a BDM VLD sequence, wherein at least two of the three BLs of the VLD are modified or replaced with heterologous sequences. (ii) To provide nucleic acids that encode pharmacologically active proteins or peptides, and (iii) Optionally, provide a nucleic acid that codes for a linker sequence. (iv) Expressing the above nucleic acid sequence in a suitable vector, (v) Recovering the expressed protein. Includes.

[0092] This disclosure also provides a method for producing a multispecific molecule containing at least one BDM VLD conjugated to an antibody, and such method is (i) To provide nucleic acids that encode an antibody light chain sequence or a portion thereof, (ii) To provide nucleic acids that encode an antibody heavy chain sequence or a portion thereof, (iii) A nucleic acid encoding a BDM VLD sequence, wherein at least two of the three BLs of the VLD are modified or replaced with heterologous sequences. (iv) Expressing the above nucleic acid sequence in a suitable vector (multiple vectors are acceptable), and (v) Recovering the expressed protein. Includes.

[0093] In one example following the method, the antibody heavy chain consists of a full-length sequence. In another example following the method, the antibody heavy chain consists of a variable region and at least a CH1 region. In yet another example, the antibody heavy chain consists of a variable region and at least CH1 and CH2 regions.

[0094] In one example following the method, no linker sequence is present, and the nucleic acid sequence of the pharmacologically active protein or peptide is contiguous with the nucleic acid sequence of the BDM VLD. In another example, no linker sequence is present, and the nucleic acid sequences encoding the heavy and / or light chains of the antibody are contiguous with the BDM sequence.

[0095] The nucleic acid sequence of the antibody may further include a hinge region.

[0096] This disclosure also provides vectors (or more) comprising one or more nucleic acid sequences described herein. For example, a vector comprises nucleic acid sequences encoding a pharmacologically active protein and BDM VLD as described herein, and optionally a nucleic acid sequence encoding a linker. For example, a vector (or more) comprises nucleic acid sequences encoding an antibody light chain or antibody heavy chain as described herein, a nucleic acid sequence encoding a BDM VLD, and optionally a nucleic acid sequence encoding a linker. For another example, a vector (or more) comprises nucleic acid sequences encoding both an antibody heavy chain and an antibody light chain as described herein, a nucleic acid sequence encoding a BDM VLD, and optionally a nucleic acid sequence encoding a linker.

[0097] This disclosure also provides host cells containing a vector, or host cells containing one or more nucleic acid sequences described herein.

[0098] This disclosure also provides multispecific molecules that can be prepared by the methods described herein.

[0099] The molecules of this disclosure may be provided in compositions. Therefore, in another embodiment, this disclosure provides a pharmaceutical composition comprising the multispecific molecules described herein, together with a pharmacodynamically acceptable carrier and / or additive. The composition may be provided as a pharmaceutical. In one example, the composition is for use in the treatment of a disorder. In another example, the composition is for anti-aging or cosmetic purposes.

[0100] In another example, the molecules may be labeled with a drug that facilitates detection.

[0101] The compositions of this disclosure may be provided in the form of a kit with instructions for use according to a specific therapeutic indication.

[0102] This disclosure also provides the use of the multispecific molecules described herein for detecting one or more target antigens to which one or more portions of the molecule bind. [Brief explanation of the drawing]

[0103] [Figure 1] Figure A shows a schematic diagram of the sequence of the native human CTLA4 VLD scaffold, indicating the locations of the framework sequence and the sequences of binding loops 1, 2, and 3 (represented as BL1, BL2, and BL3). Figure B shows the locations of binding loop substitutions in the CTLA4 VLD, where BL1 is represented by Xn1, BL2 by Xn2, and BL3 by Xn3, where X is any amino acid and n is a number between 5 and 15. Figure C shows the sequence of the sclerostin human VLD scaffold, where the sequence of the binding loop region is underlined. [Figure 2] A schematic diagram of an antibody-VLD bispecific molecule according to an example of this disclosure is shown. The antibody binds to target A via the V domains of its heavy and light chains. The VLD, which is bound to the C-terminus of the CH3 constant domain of the antibody heavy chain, binds to target B. The bispecific molecule can bind to both target A and target B individually or simultaneously. [Figure 3]A schematic diagram of an antibody-VLD bispecific molecule according to an example of this disclosure is shown. The antibody binds to target A via the V domains of its heavy and light chains. The VLD is bound to target B at the C-terminus of each of the constant domains (constant regions, CLs) of the light chain. The bispecific molecule can bind to both target A and target B individually or simultaneously. [Figure 4] A schematic diagram of an antibody-VLD trispecific molecule according to an example of this disclosure is shown. The antibody binds to target A via the V domains of its heavy and light chains. The VLD is bound to the C-terminus of the light chain constant domain (CL) and the heavy chain constant domain (CH3), respectively. The trispecific molecule can bind to targets A, B, and C individually, simultaneously, or in combination of the three targets (e.g., target A and B, or target A and C, or target B and C). [Figure 5] A schematic diagram of a Fab-VLD bispecific molecule according to an example of this disclosure is shown. Fab binds to target A via the V domains of its heavy and light chains. VLD is bound to the C-terminus of the heavy chain constant domain (CH1) and binds to target B. The bispecific molecule can bind to both target A and target B individually or simultaneously. [Figure 6] A schematic diagram of a Fab-VLD bispecific molecule according to an example of this disclosure is shown. Fab binds to target A via the V domains of its heavy and light chains. VLD is bound to target B via the C-terminus of the constant domain (CL) of the light chain. The bispecific molecule can bind to both target A and target B individually or simultaneously. [Figure 7] A schematic diagram of a Fab-VLD triplicate molecule according to an example of this disclosure is shown. Fab binds to target A via the V domains of its heavy and light chains. VLD binds to target B via the C-terminus of the light chain constant domain (CL), and VLD also binds to target C via the C-terminus of the heavy chain constant domain (CH1). The triplicate molecule can bind to targets A, B, and C individually, simultaneously, or in combination of the three targets (e.g., target A and B, or target A and C, or target B and C). [Figure 8]Expression of the parent D1.3 Fab, as well as the bispecific and tripspecific mutants, under non-reducing conditions (SDS PAGE) is shown. D1.3 Fab is an anti-lysozyme Fab; D1.3 Fab-VLD×1(HC) is an anti-lysozyme Fab having a VLD fused to the heavy chain's CH1 domain; D1.3 Fab-VLD×1(LC) is an anti-lysozyme Fab having a VLD fused to the light chain's CL domain; and D1.3 Fab-VLD×2(HC+LC) is an anti-lysozyme Fab having a VLD fused to the heavy chain's CH1 domain and a VLD fused to the light chain's CL domain. [Figure 9] The SDS-PAGE expression analysis of the parent D1.3 Fab, bispecific mutant, and tripspecific mutant under reducing conditions is shown. D1.3 Fab is an anti-lysozyme Fab; D1.3 Fab-VLD×1(HC) is an anti-lysozyme Fab having a VLD fused to the CH1 domain of the heavy chain; D1.3 VLD×1(LC) is an anti-lysozyme Fab having a VLD fused to the CL domain of the light chain; and D1.3 VLD×2(HC+LC) is an anti-lysozyme Fab having a VLD fused to the CH1 domain of the heavy chain and a VLD fused to the CL domain of the light chain. [Figure 10]This shows BLitz® analysis of the initial binding of a bispecific molecule [IgG VLD×2(HC)] to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to B7.1-Fc. The bispecific molecule has each B7-1 bound VLD fused to the heavy chain of the D1.3 antibody [D1.3 IgG]. Trace 1 is the D1.3 anti-lysozyme antibody bound to lysozyme immobilized on the biosensor surface, with buffer added at point 1. Trace 2 is the D1.3 anti-lysozyme antibody bound to lysozyme, with B7-1-Fc added at point 1. B7-1-Fc was replaced with buffer at point 2. Trace 3 is the bispecific molecule-D1.3 IgG-VLD×2(HC) bound to lysozyme immobilized on the biosensor surface, with buffer added at point 1. Trace 4 is the bispecific molecule-D1.3 IgG-VLD×2(HC), bound to lysozyme immobilized on the biosensor surface, with B7-1-Fc subsequently added at point 1. B7-1-Fc was replaced with buffer at point 2. The sensorgram shows simultaneous binding of the dual targets to lysozyme and B7-1-Fc. [Figure 11]This shows BLitz® analysis of the initial binding of a bispecific molecule [IgG VLD×2(LC)] to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to B7-1-Fc. The bispecific molecule has a B7-1 bound VLD fused to the light chain of the D1.3 antibody [D1.3 IgG]. Trace 1 is the D1.3 anti-lysozyme antibody bound to lysozyme immobilized on the biosensor surface, with buffer added at point 1. Trace 2 is the D1.3 anti-lysozyme antibody bound to lysozyme immobilized on the biosensor surface, with B7-1-Fc added at point 1. B7-1-Fc was replaced with buffer at point 2. Trace 3 is the bispecific molecule-D1.3 IgG-VLD×2(LC) bound to lysozyme immobilized on the biosensor surface, with buffer added at point 1. Trace 4 is the bispecific molecule-D1.3 IgG-VLD×2(LC), bound to lysozyme immobilized on the biosensor surface, with B7-1-Fc subsequently added at point 1. B7-1-Fc was replaced with buffer at point 2. The sensorgram shows simultaneous binding of the dual targets to lysozyme and B7-1-Fc. [Figure 12]This shows BLitz® analysis of the initial binding of a trispecific [IgG VLD×4(HCLC)] to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to B7-1-Fc. The trispecific molecule has each B7-1-binding VLD fused to both the heavy and light chains of the D1.3 antibody [D1.3 IgG]. Trace 1 is the D1.3 anti-lysozyme antibody bound to lysozyme immobilized on the biosensor surface, with buffer added at point 1. Trace 2 is the D1.3 anti-lysozyme antibody bound to lysozyme immobilized on the biosensor surface, with B7-1-Fc added at point 1. B7-1-Fc was replaced with buffer at point 2. Trace 3 is the trispecific D1.3 IgG-VLD×4(HCLC) bound to lysozyme immobilized on the biosensor surface, with buffer added at point 1. Trace 4 is the triple-specific molecule -D1.3 IgG-VLD×4(HCLC), bound to lysozyme immobilized on the biosensor surface, with B7-1-Fc subsequently added at point 1. At point 2, B7-1-Fc was replaced with buffer. The sensorgram shows dual-target simultaneous binding to lysozyme and B7-1-Fc. [Figure 13]BLitz® analysis shows that the triplicate molecule of B7-1-Fc exhibits higher binding than the bispecific molecule. When bispecific and triplicate molecules with equivalent antibody counts were captured on biotin-labeled lysozyme conjugated to the surface of a biosensor, and then buffer or B7-1-Fc was added (point 1), the triplicate molecule showed higher binding ability than the bispecific molecule. Trace 1 is the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific and triplicate molecules. Point 1 shows the antibody bound after injecting only buffer. Trace 2 is the D1.3 anti-lysozyme antibody [D1.3 IgG], showing the case when B7-1-Fc was injected at point 1. At point 2, B7-1-Fc was replaced with buffer. Trace 3 is the bispecific D1.3 anti-lysozyme antibody [bispecific molecule-D1.3 IgG-VLD×2(LC)] with each VLD fused to the antibody CL chain. The captured bispecific molecule is shown to have bound to the B7-1-Fc injected at point 1. At point 2, the B7-1-Fc was replaced with buffer. Trace 4 is a trispecific D1.3 anti-lysozyme antibody [Trispecific molecule-D1.3 IgG-VLD×4(HC+LC)] which has each VLD fused to the CH and CL chains of the antibody. The captured bispecific molecule is shown to have bound to the B7-1-Fc injected at point 1. At point 2, the B7-1-Fc was replaced with buffer. [Figure 14] This image shows a superimposed sensorgram of a series of SPR bindings, illustrating the initial binding of a bispecific molecule [IgG VLD×2(HC)] to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to a certain concentration series of B7-1-Fc (50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml, and 0 μg / ml). The bispecific molecule contains each B7-1-binding VLD fused to the heavy chain of the D1.3 antibody [D1.3 IgG]. [Figure 15]This image shows a superimposed sensorgram of a series of SPR bindings, illustrating the initial binding of a bispecific molecule [IgG VLD×2(LC)] to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to a certain concentration series of B7-1-Fc (50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml, and 0 μg / ml). The bispecific molecule contains a B7-1-binding VLD fused to the light chain of the D1.3 antibody [D1.3 IgG]. [Figure 16] This image shows a superimposed sensorgram of a series of SPR bindings, illustrating the initial binding of a trispecific [IgG VLD×4(HC+LC)] to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to a certain concentration series of B7-1-Fc (50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml, and 0 μg / ml). The trispecific molecule contains each B7-1-binding VLD fused to both the heavy and light chains of the D1.3 antibody [D1.3 IgG]. [Figure 17] The image shows an overlapping SPR sensor gram for binding, exhibiting bispecificity [Fab-VLD×1(HC)], where the molecule binds to lysozyme and then co-binds to B7-1-Fc at a certain concentration series (25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml). The bispecificity molecule has a B7-1 binding VLD fused to a D1.3 Fab[D1.3 Fab] heavy chain. [Figure 18] The image shows an overlapping SPR sensor gram for binding, exhibiting bispecificity [Fab-VLD×1(LC)] by binding to lysozyme and then co-binding to B7-1-Fc at a certain concentration series (25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml). The bispecificity molecule has a B7-1 binding VLD fused with a D1.3 Fab[D1.3 Fab] light chain. [Figure 19]The image shows an overlapping SPR sensor gram for binding, exhibiting triple specificity [Fab-VLD×2(HC+LC)], where the molecule binds to lysozyme and then simultaneously binds to B7-1-Fc at a certain concentration series (25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml). The triple specificity molecule has each B7-1-binding VLD fused to both the heavy and light chains of D1.3 Fab [D1.3 Fab]. [Figure 20] The binding stoichiometry determined by SPR analysis of bispecific and tripspecific antibody-VLD molecules that bind to B7-1-Fc is shown. [Figure 21] The binding stoichiometry determined by the RMAX ratio obtained from SPR analysis of bispecific and tripspecific Fab-VLD molecules that bind to B7-1-Fc is shown. [Figure 22]This image shows a superimposed sensorgram of a series of SPR bindings, demonstrating that the triple-specific [IgG VLD×4(Scl-HC)(B7-LC)] first binds to biotin-labeled lysozyme captured with streptavidin, and then subsequently binds to B7-1-Fc and sclerostin in sequence. The triple-specific molecule has a sclerostin (Scl)-binding VLD fused to the heavy chain of the D1.3 antibody [D1.3 IgG] and a B7-1-binding VLD fused to the light chain. The trace for B7-1-Fc only is a sensorgram showing the case where the triple-specific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc. The sensorgram demonstrates dual-target simultaneous binding to lysozyme and B7-1-Fc. The trace for sclerostin only is a sensorgram showing the case where the triple-specific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of sclerostin. The sensorgram shows dual target simultaneous binding to lysozyme and sclerostin. The traces of B7-1-Fc and sclerostin are sensorgrams of the tripspecific molecule, showing binding to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc, and then the addition of sclerostin. The sensorgram shows triple target simultaneous binding to lysozyme, B7-1-Fc, and sclerostin. Injection of the tripspecific molecule: Point where IgG VLD×4(Scl-HC)(B7-LC) is added to the sensor surface. The trace shows the binding of IgG VLD×4(Scl-HC)(B7-LC) to lysozyme immobilized on the biosensor surface. Buffer injection 1: Point where the injection of IgG VLD×4(Scl-HC)(B7-LC) is stopped and replaced with buffer injection. The trace shows the dissociation of IgG VLD×4(Scl-HC)(B7-LC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: This is the point where the second analyte, B7-1-Fc, is added. The trace shows the binding of B7-1-Fc to IgG VLD×4(Scl-HC)(B7-LC), which is still bound to the lysozyme immobilized on the biosensor surface.Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with a buffer injection. The trace shows the dissociation of B7-1-Fc from IgG VLD×4(Scl-HC)(B7-LC) that is still bound to the lysozyme immobilized on the biosensor surface. Sclerostin injection: Point where the third analyte, sclerostin, is added. The trace shows sclerostin binding to IgG VLD×4(Scl-HC)(B7-LC) that is still bound to the lysozyme immobilized on the biosensor surface and simultaneously still bound to B7-1-Fc. Buffer injection 3: Point where the injection of sclerostin is stopped and replaced with a buffer injection. The trace shows the dissociation of sclerostin from IgG VLD×4(Scl-HC)(B7-LC) that is still bound to the lysozyme immobilized on the biosensor surface. [Figure 23]This BLitz® binding analysis shows that the triple-specific [Fab VLD×2(B7-HC)(Scl-LC)] first binds to biotin-labeled lysozyme captured with streptavidin, and then sequentially binds to B7-1-Fc and sclerostin. The triple-specific molecule has a sclerostin-binding VLD fused to the light chain of D1.3 Fab [D1.3 Fab] and a B7-1-binding VLD fused to the heavy chain. The trace shows triple-specific binding to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc. The binding trace shows dual-target simultaneous binding to lysozyme and B7-1-Fc. Adding sclerostin shows triple-target simultaneous binding to lysozyme, B7-1-Fc, and sclerostin. Addition of the triple-specific molecule: Point where Fab VLD×2(B7-1-HC)(Scl-LC) is added to the sensor surface. The trace shows the binding of Fab VLD×2(B7-l-HC)(Scl-LC) to lysozyme immobilized on the biosensor surface. Addition of B7-1-Fc: Point where B7-1-Fc is added. The trace shows the binding of B7-1-Fc to Fab VLD×2(B7l-HC)(Scl-LC) that is still bound to lysozyme immobilized on the biosensor surface. Addition of sclerostin: Point where sclerostin is added. The trace shows sclerostin binding to Fab VLD×2(B7l-HC)(Scl-LC) that is still bound to lysozyme immobilized on the biosensor surface and simultaneously still bound to B7-1-Fc. Addition of buffer: Point where sclerostin is replaced with buffer. [Figure 24]This BLitz® binding analysis shows that the triple-specific [Fab VLD×2(Scl-HC)(B7-LC)] first binds to biotin-labeled lysozyme captured with streptavidin, and then sequentially binds to B7-1-Fc and sclerostin. The triple-specific molecule has a sclerostin-binding VLD fused to the heavy chain of D1.3 Fab [D1.3 Fab] and a B7-1-binding VLD fused to the light chain. The trace shows triple-specific binding to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc. The binding trace shows dual-target simultaneous binding to lysozyme and B7-1-Fc. Adding sclerostin shows triple-target simultaneous binding to lysozyme, B7-1-Fc, and sclerostin. Addition of the triple-specific molecule: Point where Fab VLD×2(Scl-HC)(B7-LC) is added to the sensor surface. The trace shows the binding of Fab VLD×2(Scl-HC)(B7-LC) to lysozyme immobilized on the biosensor surface. Addition of B7-1-Fc: Point where B7-1-Fc is added. The trace shows the binding of B7-1-Fc to Fab VLD×2(Scl-HC)(B7-LC) that is still bound to lysozyme immobilized on the biosensor surface. Addition of sclerostin: Point where sclerostin is added. The trace shows sclerostin binding to Fab VLD×2(Scl-HC)(B7-LC) that is still bound to lysozyme immobilized on the biosensor surface and simultaneously still bound to B7-1-Fc. Addition of buffer: Point where sclerostin is replaced with buffer. [Figure 25] A schematic diagram of a protein (general description) conjugated with a VLD according to an example of this disclosure is shown. The protein conjugates to target A. The VLD is conjugated to the C-terminus of the protein polypeptide and conjugates to target B. The bispecific molecule can conjugate to both target A and target B individually or simultaneously. [Figure 26]This shows the analysis and detection of purified human serum albumin (HAS)-VLD fusion proteins by Western blotting using anti-His horseradish peroxidase (HRP). Lane 1 represents HSA with a VLD fused to the C-terminus, Lane 2 represents HSA with a VLD fused to the N-terminus, and Lane 3 represents HSA with VLDs fused to both the N-terminus and C-terminus. [Figure 27] Analysis using the ForteBio Blitz biosensor demonstrates the binding of the HSA-VLD fusion protein to B7-2-Fc. Trace 1 matches the HSA-VLD fusion protein as a B7-2-binding VLD bound to the C-terminus of HSA. Trace 2 matches the VDL-HSA-VLD construct, which has B7-2-binding VLDs bound to both the N-terminus and C-terminus of HSA. Point 1 corresponds to the addition of buffer. [Figure 28] Analysis using the ForteBio Blitz biosensor is shown, demonstrating the binding of HSA-VLD to CD3. The illustrated trace coincides with the binding of the molecule to CD3de. Point 1 corresponds to the addition of buffer. [Figure 29]Analysis using the ForteBio Blitz biosensor is shown, demonstrating the binding of the HSA-VLD fusion protein to anti-HSA aphibodies and B7-2-Fc. The illustrated traces correspond to the binding of the molecule to the anti-HSA aphibodies. Points 1, 2, and 3 correspond to the addition of buffer. Sequence Listing Key Sequence Number 1 Sequence Number 2 of Human Natural CTLA4 Scaffold (Extracellular Domain) Sequence Number 3 of CTLA4 Exposed Binding Loop 1 Sequence Number 4 of CTLA4 Exposed Binding Loop 2 Sequence Number 5 of CTLA4 Exposed Binding Loop 3 Sequence Number 6 of BDM VLD Scaffold Sequence Number 7 of BDM VLD Scaffold Sequence Number 8 of Binding Loop 1 Signal Peptide Sequence Number 9 of Binding Loop 3 Sequence Number 10 of Binding Loop 1 Sequence Number 11 of Binding Loop 2 Sequence Number 12 of Binding Loop 3 Sequence Number 13 of B7-1 Binding VLD Scaffold Sequence Number 14 of Sclerostin Binding VLD Scaffold Sequence Number 15 of Linker Sequence Number 16 of Linker Sequence Number 17 of Linker Sequence Number 18 of Anti-Lysozyme IgG1 Heavy Chain Sequence Number 19 of Anti-Lysozyme IgG1 Heavy Chain Linked to B7-1 Binding VLD by Linker Sequence Number 20 of Anti-Lysozyme IgG Kappa Light Chain Sequence Number 21 Sequence ID 22 of the anti-lysozyme IgG kappa light chain linked to B7-1 linked VLD by a linker Sequence ID 23 of the anti-lysozyme Fab kappa light chain linked to B7-1 linked VLD by a linker Sequence ID 24 of the anti-lysozyme Fab heavy chain linked to B7-1 linked VLD by a linker Sequence ID 25 of the anti-lysozyme Fab heavy chain with a C-terminal histidine tag and myc tag Sequence ID 26 of the anti-lysozyme IgG1 heavy chain fused with anti-sclerostin VLD Sequence ID 27 of the B7-2 linked VLD fused to the C-terminus of human serum albumin Sequence ID 28 of the B7-2 linked VLD fused to the N-terminus of human serum albumin Sequence ID 29 of the B7-2 linked VLD fused to both the N-terminus and C-terminus of human serum albumin Sequence ID 30 of the CD3 linked VLD fused to the C-terminus of human serum albumin Base sequence of the BDM VLD scaffold. [Modes for carrying out the invention]

[0104] overview Throughout this specification, unless otherwise specifically stated or designated in the context, references to a single step, composition, group of steps, or group of compositions should be interpreted as encompassing one or more such steps, compositions, group of steps, or group of compositions.

[0105] Those skilled in the art will understand that this disclosure is subject to changes and modifications other than those specifically described herein. It should be understood that this disclosure includes all such changes and modifications. Furthermore, this disclosure includes all steps, features, compositions and compounds that are referred to or indicated individually or collectively herein, as well as any combination of any two or more of such steps or features.

[0106] Any example or embodiment of the disclosure herein should be understood to apply mutatis mutandis to any other example of the disclosure unless otherwise specifically indicated.

[0107] Unless otherwise specifically stated, all technical and scientific terms used herein should be understood to have the same meaning as those commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0108] Unless otherwise specified, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. Sufficient descriptions and explanations can be found in original literature such as "Current Protocols in Immunology" by John Wiley & Sons (including all updates to date).

[0109] The descriptions and definitions of variable regions and their parts, immunoglobulins, antibodies and their fragments in this specification can be further clarified by the considerations in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0110] The terms "and / or," for example, "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be understood to explicitly support both meanings or either of them.

[0111] As used herein, "a" or "an" means "at least one" or "one or more" unless specifically indicated by the context.

[0112] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to mean that they include the element, integer, or step, or group of elements, integers, or steps described, but do not exclude any other element, integer, or step, or group of elements, integers, or steps.

[0113] Selection Definition In this specification, the letter "x" used in amino acid sequences indicates that any of the 20 standard amino acids may be placed in this position, unless otherwise specified.

[0114] As used herein, “identity” means the proportion of identical nucleotides or amino acid residues at corresponding positions within two or more sequences when they are aligned to maximize sequence matching, i.e., taking gaps and insertions into account. Identity can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The methods for determining identity are designed to maximize the agreement between the tested sequences. Furthermore, these identity determination methods are systematically compiled in the form of publicly available computer programs.Computer programs for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BLASTP, BLASTN, and FASTA (Altschul, S. et al., J. Molec. Biol. 215:403-410(1990) and Altschul et al. Nuc. Acids Res. 25:3389-3402(1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410(1990)).

[0115] As used herein, the term "sequence identity" refers to the percentage of identical residues in pairs when the polypeptide sequences of the disclosed herein and the target sequence are aligned over the residue count of the longer sequences of the two sequences. Identity is determined by dividing the number of identical residues by the total number of residues and multiplying the result by 100. Therefore, the identity between two sequences is typically expressed as a percentage.

[0116] The term "immunoglobulin" refers to a family of polypeptides that retain the immunoglobulin folding properties of antibody molecules, containing two beta sheets and, typically, a conserved disulfide bond. Members of the Ig superfamily include antibodies, T cell receptor molecules, ICAM molecules (involved in cell adhesion), and receptor molecules such as PDGF receptors involved in intracellular signal transduction. Preferably, this invention relates to antibodies.

[0117] An "immunoglobulin-like domain" refers to a beta-sandwich structure motif found in various proteins with different functions, such as extracellular matrix proteins, muscle proteins, immunoproteins, cell surface receptors, and enzymes. Members of Ig-like domains are divided into various superfamilies, including, for example, immunoglobulins, fibronectin type III, and cadherins. Other superfamilies containing Ig-like domain structural motifs include, for example, members of the PKD domain, β-galactosidase / glucosidase domains, the C-terminal domain of transglutaminase 2, actinoxanthine-like, CuZn superoxide dismutase-like, CBD9-like, lamin A / C globular tail domain, clathrin adapter attachment domain, integrin domain, PapD-like, violet acid phosphatase N-terminal domain, superoxide reductase-like, the N-terminal domain of thiol:disulfide exchange protein DsbD, and the invasine / inchmin cell adhesion fragment superfamilies. The similarity of Ig-like domain structures is maintained between different superfamily members regardless of significant sequence identity. The term includes Ig-like domain members within and between each superfamily. Therefore, the term “immunoglobulin-like (Ig-like) domain-containing superfamily” refers to polypeptides of Ig-like domain-containing members that fall into these superfamilies and any other superfamilies known in the art. Descriptions of various Ig-like domain-containing superfamilies can be found, for example, in Clarke et al., Structure Fold. Des. 7:1145-53 (1999) and in structure databases such as pdb.weizmann.ac.il / scop / data / scop.bcbhtml.

[0118] The term "antibody" may include all classes, e.g., IgG, IgM, IgA, IgD, or IgE, or subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, whether derived from any naturally occurring antibody-producing species or created by recombinant DNA technology, and whether isolated from serum, B cells, hybridomas, transfectomas, yeast, or bacteria, or produced by synthesis. The term "antibody" encompasses monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, primate-derived antibodies, or syn-humanized antibodies. The term "human antibody" refers to an antibody containing human-derived sequences, excluding any possible non-human CDR regions, and exhibiting minimal immunogenicity in humans.

[0119] As used herein, the term "full-length antibody" refers to an antibody in a substantially intact form, distinct from the antigen-binding fragment of the antibody. Full-length antibodies may be isolated or recombinant. Specifically, the whole antibody includes heavy and light chains containing an Fc region. The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof. The antibody protein includes a variable region composed of multiple polypeptide chains, e.g., a polypeptide containing a light chain variable region (VL) and a polypeptide containing a heavy chain variable region (VH). The antibody also includes a constant domain, a portion of which may be located within the constant region, which may contain a constant fragment or, in the case of a heavy chain, a crystallizable fragment (Fc). The VH and VL interact to form an Fv containing an antigen-binding region that can specifically bind to one or a few closely related antigens. The antibody-antigen binding interaction may manifest as an intermolecular contact with one or more amino acid residues of the complementarity-determining region (CDR). Generally, mammalian light chains are either κ or λ light chains, and mammalian heavy chains are α, δ, ε, γ, or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies may be human antibodies, humanized antibodies, or chimeric antibodies. In yet another example, antibodies may be from sharks, camelids, felines, or canines.

[0120] As used herein, the term “immunoglobulin antigen-binding fragment” refers to a fragment of an antibody, which includes a light chain variable region and a heavy chain variable region having a complementarity-determining region (CDR). Such a term encompasses Fab, F(ab')2, Fab', scFv, di-scFv, or chemically bound F(ab')2.

[0121] The term "Fab" is understood to refer to the region of an antibody that binds to an antigen and consists of one constant domain each in the heavy and light chains, and one variable domain.

[0122] The term "complementary" refers to immunoglobulin domains that form a corresponding pair. For example, the VH domain and VL domain of an antibody are complementary, two VH domains are not complementary, and two VL domains are not complementary.

[0123] The term "domain" refers to a folded protein structure that maintains its own tertiary structure independently of other parts of the protein.

[0124] The term “CDR” or “complementarity-determining region” refers to discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides in an antibody or antibody fragment (bound to an antigen). These specific regions are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlaps or subsets of amino acid residues when comparing amino acid residues with each other. However, when referring to the CDR of an antibody or transplanted antibody or its variant using any of the above definitions, the application shall be within the scope of the terms defined and used herein.

[0125] A "binding domain molecule (BDM)" refers to a monomeric domain that has structural features similar to the heavy chain variable region (VH) or light chain variable region (VL) of an antibody. These similar structural features include BL (binding loop) sequences, which are loop-like structures or regions of a surface polypeptide that function in a similar manner to the complementarity-determining region (CDR) of an antibody variable domain that binds to a specific antigen. A BDM scaffold consists of a framework sequence and three BL sequences contained between them. The BDMs used herein are not antibody variable domains. BDM scaffolds are described herein and include, for example, CTLA-4, lipocalin, fibronectin, ICOS, and CD28.

[0126] A “BL sequence” is a loop-like structure or region of a surface polypeptide that functions in a similar manner to the complementarity-determining region (CDR) of an antibody variable domain that binds to a specific antigen. Three antigen-binding loop sequences (referred to herein as BL-1, BL-2, and BL-3, respectively) are present in the BDM and are located within a scaffold sequence that provides the desired three-dimensional conformation of such loop sequences. The native BL sequences can be replaced with one or more corresponding antibody CDRs that can be transplanted onto the scaffold. Diversity can be introduced into the BL sites of the BDM, which is possible by randomizing the amino acid sequence of a specific loop in the scaffold, for example, by introducing an NNK codon, and then making a selection for the desired binding properties, for example, using display techniques. This mechanism is analogous to natural selection by high-affinity antigen-specific antibodies.

[0127] In the context of proteins, polypeptides, or peptides, the term “binding specificity” refers to the ability of a protein, peptide, and / or BDM to bind to its respective target antigen or target epitope, depending on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the target antigen or target epitope. For example, proteins and / or BDMs do not bind to proteins in general, but rather recognize and bind to specific protein structures. For instance, if a protein binds to epitope “A”, in a reaction involving labeled “A” and the protein, the amount of labeled “A” bound to the protein will be reduced by the presence of molecules containing epitope “A” (i.e., free, unlabeled “A”). It should also be understood that such terminology includes the “specific binding” of pharmacologically active proteins, peptides, and / or BDMs to their target antigens. The terms “specifically binds” or “binds specifically” should be understood to mean that a pharmacologically active protein (or, in the case of an antibody, its antigen-binding domain) or peptide and / or BDM of this disclosure reacts or associates with a particular target antigen more frequently, for a shorter duration, and / or with a higher affinity than an alternative target antigen. The reference to “binding” clearly supports the terms “specific binding” or “binding specific.” Typically, such terms are used to describe the affinity of a portion (i.e., the protein or BDM of this specification) for a given target antigen. In some situations, a low-affinity binding may be desirable if toxicity is a concern. In other situations, a high-affinity binding may be desirable to minimize cross-reactivity with other target antigens. One example of binding is specific binding as defined herein.

[0128] The term "selective coupling" is described in detail elsewhere in this specification.

[0129] The "binding affinity" or "affinity" of a portion of a molecule (i.e., a protein or BDM) to a selected target can be measured. The term "affinity" refers to the equilibrium constant of the reversible bond between two substances, and is expressed as the dissociation constant (Kd) or equilibrium dissociation constant (KD).

[0130] As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation after dilution.

[0131] In this invention, the term "antigen" means a pharmacologically active protein or peptide or a substance to which a BDM (biological protein) binds. An antigen will typically consist of one or more antigenic epitopes recognized by a BDM or protein or peptide. Protein antigens may be soluble proteins or membrane-bound proteins. Examples of soluble proteins include, but are not limited to, transcription factors, antibodies, growth factors, blood proteins (e.g., albumin), or drugs (e.g., steroids, pharmaceuticals, etc.). Types of membrane-bound proteins include growth factor receptors, tumor markers, cell surface markers, or markers that mediate intracellular transport (e.g., transferrin), or Fc receptors. An antigen typically refers to a substance that can elicit an immune response in a living organism. An antigen may be a polypeptide, a protein, a nucleic acid (e.g., DNA, RNA, or a combination of DNA and RNA), or other molecule.

[0132] As used herein, the term “epitope” (synonym “antigenic determinant”) should be understood to mean the region to which a protein (or the antigen-binding domain of an antibody or an immunoglobulin antigen-binding fragment) binds, or the region to which the BDM of this disclosure binds. Conventionally, such a term refers to the structure bound by the VH / VL pair of an immunoglobulin. An epitope defines the minimum binding site of an antibody or antibody-like domain (e.g., a BDM). This term is not necessarily limited to the specific residues or structures with which the protein and / or BDM of a molecule make contact. For example, this term includes the region of amino acids that are contacted by the CDR of an antibody or the BL sequence of an immunoglobulin antigen-binding fragment or a BDM, respectively, and the 5-10 (or more), 2-5, or 1-3 amino acids outside this region. In some examples, an epitope includes a series of discontinuous amino acids, i.e., “structural epitopes,” that are located close to each other when a polypeptide is folded and associate, for example, with another polypeptide. Such terms include epitopes consisting of linear peptide sequences (i.e., “continuous”) or epitopes consisting of discontinuous amino acid sequences (i.e., “structurally distinct” or “discontinuous”).

[0133] As used herein, the term "target" refers to an antigen or epitope. In certain examples, the target refers to a cell surface protein, such as a receptor or viral coat protein. In other examples, the target is a secreted protein.

[0134] As used herein, the term “antigen-binding domain” in an antibody or immunoglobulin antigen-binding fragment should be understood to mean a region of the antibody or immunoglobulin antigen-binding fragment that can specifically bind to a given antigen, more specifically, to an epitope present on the antigen. In antibodies, the antigen-binding domain is V H and V L This corresponds to V. H and V L Within the region, there is a CDR in contact with the epitope.

[0135] As used herein, the terms “position” or “positions” refer to the positions of amino acids within the amino acid sequences shown herein, typically counted from the left or 5' end of the sequence. As used herein, the term “corresponding” in the context of amino acid sequences of BDM refers to the positions of amino acids with reference to the natural or “wild-type” BDM sequence. Preferably, such positions would correspond to the amino acids BLS1 and / or BLS2 and / or BLS3, respectively, in the natural BDM sequence.

[0136] As used herein, the term “natural sequence” refers to a sequence having the same amino acid sequence as the corresponding naturally occurring polypeptide. Therefore, “natural BDM” refers to a polypeptide having the same amino acid sequence as the corresponding naturally occurring polypeptide. Such natural sequence polypeptides can be prepared by recombinant or synthetic means.

[0137] The term "protein" should be understood to include a single continuous unbranched polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other by covalent or non-covalent bonds (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0138] As used herein, the term "peptide" is understood to refer to a short chain (typically about 50 or fewer amino acids) consisting of amino acid monomers linked by peptide (amide) bonds.

[0139] As used herein, the term “isolated” refers to polypeptides, antibodies, proteins, etc., that have been identified, isolated, and / or recovered from their natural environment or from components of the environment in which they were produced. Contaminants of the natural environment include substances that are considered to interfere with the therapeutic use of polypeptides, and may include, for example, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. For example, polypeptides may be purified to (1) a concentration of over 80%, 85%, 90%, 95%, or 99% by weight as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, and / or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. The term “isolated polypeptide” includes in-situ polypeptides within recombinant cells, because recombinant cells will not contain at least one component of the polypeptide’s natural environment. Generally, the isolation of polypeptides will involve at least one purification step.

[0140] The term "recombinant" should be understood to mean artificially modified genetic products. Recombinant polypeptides include polypeptides expressed by artificial recombination techniques, for example, when they are expressed internally within cells, tissues, or other objects.

[0141] As used herein, the terms “detect” or “detecting” are understood to refer to both quantitative and qualitative levels, as well as combined levels. Therefore, this includes quantitative, semi-quantitative, and qualitative measurements of the target molecule.

[0142] As used herein, the term "subject" should be understood to mean any animal, including humans, such as mammals. Examples of subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human. The term "subject" also includes non-human subjects such as hamsters, rats, rabbits, cats, dogs, and horses.

[0143] Binding domain molecule (BDM) The binding domain molecule (BDM) of this disclosure preferably contains a protein scaffold having three exposed binding loops (BLs). The BLs can be modified, i.e., replaced or modified by amino acid substitution, to confer binding specificity to a given target antigen to the BDM. The BDM scaffold of this disclosure may be selected from the group consisting of immunoglobulin-like (Ig-like) domain-containing superfamily members, i-bodies, VNARs, or VHHs.

[0144] Ig-like domain-containing superfamily members may be selected from the group consisting of V-like domains such as CTLA-4 (e.g., VLD), C-set domains, ThyOx family member polypeptides, T cell receptors, CD2, CD4, CD8, class I MHC, class II MHC, CD1, cytokine receptors, G-CSF receptors, GM-CSF receptors, hormone receptors, growth hormone receptors, erythropoietin receptors, interferon-gamma receptors, prolactin receptors, NCAM, VCAM, ICAM, N-cadherin, E-cadherin, fibronectin, tenascin, and I-set-containing domain polypeptides or their functional fragments.

[0145] Examples of BDMs in accordance with this disclosure include lipocalin, protein A-derived molecules such as the Z-domain of protein A (afibody, SpA), afibody, adonectin (e.g., fibronectin), or ankyrin repeat protein (DARPin).

[0146] The BDMs of this disclosure have the advantage of being stable and tunable in both their scaffold domain structure and their ability to accept a wide range of heterogeneous binding loop sequences. Furthermore, the BDM scaffolds are readily available from human materials so that they have very little immunogenicity when used as therapeutic agents for humans. The BDM scaffolds can also be readily constructed to include or exclude naturally occurring polysaccharides.

[0147] The linking of heterogeneous binding loop sequences into BDM scaffolding can be carried out, for example, by chemical, biochemical, or recombinant methods.

[0148] Preferably, BDM as used in this disclosure refers to molecules other than human antibodies, such as those produced by B cells. The BDM molecules of this disclosure also exclude antibody fragments larger than the complementarity-determining region (CDR). Therefore, human antibody variable region fragments larger than approximately 50, 75, 100, or 110 amino acids are not included in the term BDM. Furthermore, BDM does not include antibody variable regions such as dAb structures, VH-VH structures, or VL-VL structures.

[0149] The term “scaffold” means a polypeptide support framework used to organize, orient, and hold heterogeneous binding loops or modified amino acid sequences that confer binding specificity to a given target. The scaffold is structurally separable from the binding specificity-conferring amino acid sequence. The structurally separable portions of the scaffold may include a wide variety of different structural motifs, such as beta-sandwiches, beta-sheets, alpha-helices, beta-barrels, coiled-coils, and other polypeptide secondary and tertiary structures known in the art. The scaffolds of this disclosure will also include one or more regions that can alter the amino acid sequence without substantially reducing the stability of the support framework structure. Exemplary regions that can be altered include binding loop segments that connect two strands of a beta-sandwich or beta-sheet.

[0150] The BDM scaffolds of this disclosure preferably exhibit less than 50% amino acid identity to the variable heavy chain or variable light chain sequence of human immunoglobulin. Generally, the scaffolds will exhibit amino acid sequence identity of less than 45%, less than 40%, less than 30%, less than 20%, less than 15%, or less than 10% compared to, for example, the amino acid sequence of the variable heavy chain or variable light chain of human immunoglobulin.

[0151] Modifiable scaffold residues are referred to herein as the outer binding loop or binding loop sequence (referred to herein as BL-1, BL-2, and BL-3, respectively). Residues that confer secondary or tertiary structural characteristics can be retained, modified, or preserved, as long as the overall structure of the scaffold is maintained. Those skilled in the art are knowledgeable about or can determine which residues function in the structural stability of polypeptide scaffolds, and the possible extent of modification of such residues.

[0152] In one example, the BDM scaffold is a V-like domain (VLD) protein.

[0153] VLDs are typically distinguished from those of antibodies or T cell receptors because they do not tend to bind to each other to form Fv-type molecules. VLDs are discussed in The Leucocyte Antigen Facts Book 1993, Eds Barclay et al., Academic Press, London; and CD Antigens 1996 (1997) Immunology Today 18, 100-101; and Arlene H Sharpe and Gordon J Freeman, (2002) Nature Reviews Immunology 2, 116-126, the full contents of which are incorporated herein by reference.

[0154] A person skilled in the art covered by this disclosure can easily determine a suitable VLD-containing protein suitable for use in this disclosure, for example, by searching the Uniprot database (www.uniprot.org).

[0155] VLD-containing proteins such as CTLA-4 may provide an alternative framework for developing novel binding sites with high affinity to target molecules. Single-domain V-like binding molecules derived from these binding sites are desirable because they are soluble. Examples of suitable VLD-containing binding sites include CTLA-4, CD28, and ICOS (Hutloff A et al, (1999) Nature 397(6716):263-6).

[0156] Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), along with its homologous cell surface proteins CD28 and ICOS, are involved in T cell regulation during immune responses. CTLA-4 is a 44 kDa homodimer that is transiently expressed primarily on the surface of activated T cells, where it interacts with surface antigens CD80 and CD86 on antigen-presenting cells to regulate the immune response (Waterhouse et al. (1996) Immunol Rev 153:183-207, van der Merwe et al. (1997) J Exp Med 185(3):393-403).

[0157] CD28 is a 44kDa homodimer that, like CTLA-4, is primarily expressed on T cells and regulates the immune response by interacting with surface antigens CD80 and CD86 on antigen-presenting cells (Linsley et al. (1990) J Immunol 182(5):2559-63). Current theories suggest that the level of the immune response is regulated by competition for available ligands between CTLA-4 and CD28. For example, knockout mice lacking the CTLA-4 gene exhibit massive over-proliferation of activated T cells (Waterhouse et al. (1995) Science 270(5238):985-8).

[0158] Each CTLA-4 monomeric subunit consists of an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular domain. The extracellular domain contains an N-terminal V-like domain (VLD; predicted to have a molecular weight of approximately 14 kDa based on homology to the immunoglobulin superfamily) and a stem of approximately 10 residues connecting the VLD to the transmembrane domain. The VLD contains surface loops corresponding to BL-1, BL-2, and BL-3 respectively (Metzler WJ et al (1997) Nat Struct Biol 4(7):527-31) and binds to CD80 and / or CD86. The sequences of human CTLA-4 have been determined to date (US5,434,131; US5,844,095; US5,851,795).

[0159] Structural and mutational studies of CTLA-4 suggest that binding to CD80 and CD86 is formed by the V-like beta chain of A'GFCC', as well as via the VLD surface formed by the highly conserved MYPPPYY sequence in BL-3. Dimerization of CTLA-4 monomers occurs at the cysteine ​​residues (Cys) of the two stems. 120 Dimerization occurs via disulfide bonds between the two extracellular domains, thereby tethering them, but there is no clear direct association between the V-like domains (Metzler WJ et al (1997) Nat Struct Biol 4(7):527-31). Dimerization appears to contribute only to high avidity to the ligand.

[0160] CD278, or ICOS (Inducible T Cell Costimulatory Molecular), is an immune checkpoint protein encoded by the ICOS gene. It is expressed on activated T cells. This protein belongs to the CD28 and CTLA-4 cell surface receptor families. This protein forms homodimers and plays a role in signal transduction, immune responses, and cell proliferation regulation.

[0161] Human CTLA4, CD28, and ICOS sequences are available in publicly accessible databases.

[0162] The human CTLA-4 sequence is available as UniProt reference number P16410. The extracellular domain of CTLA-4 corresponds to positions 36-161 of this sequence (where CTLA-4 has a total length of 126 amino acids). Amino acid residues 1-35 correspond to the signal peptide.

[0163] The human CD28 sequence is available as UniProt reference number P10747. The extracellular domain corresponds to positions 19-152 of the sequence.

[0164] The human ICOS sequence is available as UniProt reference number Q9Y6W8. The Ig-like VLD corresponds to positions 30-132 of the sequence.

[0165] For example, BDM is an immunoglobulin C-set domain protein, more preferably a C1-set domain protein. The C-set domain is a classical Ig-like domain similar to the constant domain of an antibody and is found almost exclusively in molecules involved in the immune system, such as major histocompatibility complex (MHC) class I and II complex molecules and various T cell receptors.

[0166] Those skilled in the art in the field covered by this disclosure can easily determine C-set domain-containing proteins suitable for use herein. For example, a search of the uniprot database (www.uniprot.org) reveals more than 300 human proteins.

[0167] Proteins like basidine contain a C-set domain (Xiao-Ling Yu et al. (2008) JBC vol 283(26):18056-18065). Further examples of C-set domains include the ROR1 extracellular domain and CEA family members such as CEACAM1-CEACAM8.

[0168] BDM can be made to mature in affinity using known selection and / or mutagenesis methods. Affinity-matured BDM may have 2, 5, 10, 20, 30 or more times the affinity of initial BDM. Apparent affinity can be determined by methods such as ELISA or other techniques familiar to those skilled in the art, such as surface plasmon resonance.

[0169] i-body is a single-domain antibody-like molecule derived from humans. The i-body framework is similar to that of shark-derived single-domain antibodies, and therefore shares the desirable biophysical and target-directing properties of shark antibodies. These are described, for example, in US7,977,071.

[0170] VNARs (variable new antigen receptors) refer to single variable region domain fragments derived from shark immunoglobulin novel antigen receptor antibodies (IgNARs). These are described, for example, in Griffiths K et al (2013) Antibodies 2(1):66-81.

[0171] VHH (heavy chain variable domain) domains or nanobodies refer to single monomeric variable antibody domains derived from camelid antibodies. These are described, for example, in Harmsen MM and HJ De Haard (2007) Appl Microbiol Biotechnol. 77(1):13-22.

[0172] By utilizing the binding specificity of BDM and the pharmacologically active protein portion of the molecule, the molecule can be configured to bind to one or more different target antigens or target epitopes, preferably at least two different target antigens or target epitopes. For example, the protein has binding specificity to a first target antigen, and at least one BDM has binding specificity to a second target antigen.

[0173] In examples where multiple BDMs are bound to a protein, the protein may have binding specificity to a first target antigen, the BDMs may have binding specificity to a second target antigen, and further BDMs (if present) may have binding specificity to a third target antigen. In other examples where the protein is an antibody or immunoglobulin antigen-binding fragment, the antibody or antigen-binding fragment may have binding specificity to a first target antigen, the BDMs (or BDM pairs if BDMs are present, for example, on each heavy chain or each light chain) may have binding specificity to a second target antigen, and further BDMs (or pairs thereof) may have binding specificity to a third target antigen.

[0174] The molecules of this disclosure may bind to at least one target antigen, at least two different target antigens, at least three different target antigens, at least four different target antigens, or at least five different target antigens.

[0175] Preferably, the molecule binds to one target antigen, two different target antigens, or three different target antigens. A variety of non-limiting examples are intended. (i) The protein or peptide binds to a first target antigen that is identical to or different from the second target antigen to which the BDM is bound. (ii) The antibody or immunoglobulin antigen-binding fragment binds to the first target antigen which is the same as the second target antigen to which BDM is bound. (iii) The antibody or immunoglobulin antigen-binding fragment binds to a first target antigen that is different from the second target antigen to which BDM is bound. (iv) The antibody or immunoglobulin antigen-binding fragment binds to the first target antigen, the BDM binds to the second target antigen which is the same as the first target antigen, and further BDM binds to the third target antigen which is different from the first and second target antigens. (v) The antibody or immunoglobulin antigen-binding fragment binds to the first target antigen, and the BDM and further BDM bind to the second and third target antigens, respectively, where the second and third target antigens may be the same or different, but the second and third target antigens are different from the first target antigen. (vi) The antibody or immunoglobulin antigen-binding fragment binds to the first target antigen, the BDM binds to the second target antigen, and the BDM binds to the third target antigen, where the first, second, and third target antigens are different. This includes the following.

[0176] It should be recognized that when two or more BDMs are linked together, the number of potential targets to which the molecule can bind may increase.

[0177] BDM generation As described herein, by using a bacterial expression system to replace one or more binding loop structures of a BDM domain (e.g., CTLA-4) with heterologous binding loop sequences of sclerostin or CD3, soluble nonglycosylated monomer-binding molecules were generated. Thus, V-like domains provide a basic framework for constructing soluble single-domain molecules in which the binding loop structure can be modified to engineer the binding specificity of the molecule.

[0178] The framework residues of BDM may be modified according to the structural characteristics present in camelid antibodies. Camel heavy chain immunoglobulins differ from conventional antibody structures in that they consist of a single VH domain.

[0179] When several unconventional substitutions (mainly changing from hydrophobic to polar) are performed on exposed framework residues, the hydrophobic surface is reduced, but the internal beta-sheet framework structure is maintained (Desmyter et al. (1996) Nat Struct Biol 3:803-811). Within the three binding loops, the lack of an antigen-binding surface normally provided by the VL domain is compensated for by several structural features. The BL2 loop is not significantly different from the other VH domains, but BL1 and BL3 adopt highly heterogeneous, non-standard conformations. For example, while the Ig molecule typically has 5 residues, the H1 loop can contain any number of residues from 2 to 8. However, the greatest variation is observed in BL3, with the length of this region varying between 7 and 21 residues in 17 reported camel antibody sequences (Muyldermans et al. (1994) Protein Eng 7:1129-1135). Thirdly, while many camelid VH domains possess disulfide bonds, camels have a disulfide bond linking BL1 and BL3, while llamas have a disulfide bond linking CDR1 and CDR2 (Vu et al. 1997). The function of this structural feature is thought to be to maintain loop stability and to provide a more curvilinear loop conformation than a planar one. Both of these aspects enable binding to the pocket inside the antigen and provide a large surface area. However, since not all camelid antibodies possess this disulfide bond, it is suggested that this is not an absolute structural requirement.

[0180] These characteristics allowed us to present the camelid V domain as a soluble molecule in vivo with sufficiently high affinity to elicit an effective immune response against a wide variety of target antigens.

[0181] A method for constructing and selecting single VLD molecules with novel binding affinity to target molecules is described in US7,166,697, the entire description of which is incorporated by reference. Such a method applies well-known molecular evolution techniques to V-like domains derived from immunoglobulin superfamily members. The method may involve the construction of phage or ribosome display libraries for screening a large number of mutant V-like domains.

[0182] The genome of fibrous fd bacteriophages is engineered to display proteins such as Ig-like proteins (Fab) encoded by DNA contained within the phage on their surface (Smith, 1985; Huse et al., 1989; McCafferty et al., 1990; Hoogenboom et al., 1991). Protein molecules can be displayed on the surface of Fd bacteriophages and are covalently bound to phage coat proteins encoded by gene III, or less frequently, gene VIII. Insertion of gene III coat protein into an antibody gene results in the expression of 3-5 recombinant protein molecules located at the terminals per phage. In contrast, antibody gene insertion into gene VIII can result in the display of approximately 2000 copies of recombinant protein per phage particle, but this can lead to a polyvalent system where the affinity of a single displayed protein may be masked. Fd phagemide vectors are also used because they allow for easy switching from the presentation of a functional Ig-like fragment on the surface of an Fd bacteriophage to the secretion of a soluble Ig-like fragment to E. coli. The recombinant protein fusion with the N-terminus of the gene III coat protein presented on the phage is made possible by strategically positioning the amber codon between the two protein genes. In the amber suppressor strain of E. coli, the resulting Ig domain-gene III fusion is fixed to the phage coat.

[0183] The protein affinity-based selection method is applicable to any high-affinity binding reagent, including antibodies, antigens, receptors, and ligands (see, for example, Winter and Milstein, (1991) Nature 349:293-299, the entire content of which is incorporated herein by reference). Therefore, the selection of the most affinity binding protein presented to the bacteriophage is combined with the recovery of the gene encoding such protein. Ig-presenting phages can be affinity-selected by binding them to their corresponding binding partners, which are covalently bound to beads or adsorbed on a plastic surface, using methods similar to ELISA or solid-phase radioimmunoassay. While almost all plastic surfaces adsorb protein antigens, commercially available products specifically designed for this purpose, such as Nunc Immunotube, are also available.

[0184] Ribosome display libraries use polypeptides that are de novo synthesized in a cell-free translation system and presented on the ribosome surface for selection (Hanes and Pluckthun, (1997) Proc Natl Acad Sci USA 94:4937-4942; He and Taussig, (1997) Nucle Acids Res 25:5132-5134). The "cell-free translation system" includes ribosomes, soluble enzymes required for protein synthesis (usually derived from the same cell as the ribosomes), transfer RNA, adenosine triphosphate, guanosine triphosphate, ribonucleoside triphosphate regeneration systems (such as phosphoenolpyruvate and pyruvate kinase), and salts and buffers required for the synthesis of proteins encoded by foreign mRNA. Polypeptide translation can be carried out under conditions in which intact polysomes are maintained, i.e., ribosomes, mRNA molecules, and translated polypeptides associate to form a single complex. This allows for effective "ribosome display" of the translated polypeptide.

[0185] In the selection process, translated polypeptides associated with corresponding ribosome complexes are mixed with target molecules bound to a matrix (e.g., Dynabeads). The target molecule may be any target compound (or a portion thereof), such as a DNA molecule, protein, receptor, cell surface molecule, metabolite, antibody, hormone, or virus. Since the ribosomes presenting the translated polypeptides will bind to the target molecules, these complexes can be selected and the mRNA re-amplified using RT-PCR.

[0186] While there are several alternative methods for modifying binding molecules, the general approach for any presentation protein follows a specific pattern: individual binding reagents are selected from a display library based on their affinity for their corresponding receptors. The genes encoding these reagents are modified by one or a combination of numerous in vivo and in vitro mutation strategies, constructing a novel gene pool for selecting and presenting the binding molecules with the highest affinity.

[0187] BDM dimer In one example, the BDM portion of the molecule is a dimer of the BDM monomer. Dimerization may occur spontaneously or be facilitated using a linker. For example, if BDM is CTLA-4 VLD, dimerization occurs if the sequences of the two stems are preserved, by their cysteine ​​residues (Cys 120 ) may be formed via disulfide bonds between them.

[0188] Alternatively, BDM monomers (e.g., CTLA-4 monomers) can be linked together using linkers. Using the same principle, multiple BDM monomers can be linked in tandem to form a string. Linkers facilitate greater flexibility and / or reduce steric hindrance between any two monomers. Linkers may be naturally occurring.

[0189] Other types of linkers that can be used, including the (Gly-Gly-Gly-Gly-Ser)n linker, are detailed below.

[0190] In vitro expression of the soluble form of CTLA-4 Neither the extracellular domain nor the V-like domain (VLD) of the human CTLA-4 molecule has been successfully expressed as soluble monomers in bacterial cells, which is likely due to the aggregation of expressed proteins (Linsley PS et al, (1995) J. Biol. Chem 270:15417-24). When the extracellular N-terminal domain (including Met1~Asp124 and Cys120) is expressed in E. coli, a 28 kDa dimeric protein is purified, in which two CTLA-4 V-like domains are linked by a disulfide bond at Cys120. These cysteine ​​molecules are removed when cleaved at Val114, and this cleavage was intended to express the 14 kDa VLD in a soluble monomer form. However, the product aggregated, which was concluded to be caused by the exposure of hydrophobic sites that are normally hidden by glycosylation (Linsley PS et al, (1995) J. Biol. Chem 270:15417-24).

[0191] Several successful cases have been reported of expressing monomeric glycosylated CTLA-4 extracellular domains in eukaryotic expression systems (i.e., CHO cells and yeast Pichia pastoris; Linsley PS et al, (1995) J. Biol. Chem 270:15417-24; Metzler WJ et al, (1997) Nat Struct Biol 4(7):527-31; Gerstmayer B et al, (1997) FEBS Lett 407(1):63-8). It is presumed that glycosylation in these eukaryotic expression systems occurs at the two N-linked glycosylation sites of the VLD (Asn76 and Asn108). However, high yields were only reported for the expression of a gene encoding CTLA-4 VLD fused with an Ig-CH2 / CH3 domain, which generates a dimeric recombinant protein containing two CTLA-4 VLDs bound to the Fc subunit (WO95 / 01994 and AU16458 / 95). AU60590 / 96 describes a mutant of CTLA-4 VLD in which the first tyrosine (Y) of the MYPPPY surface loop is replaced with a single amino acid, which retains and modifies affinity for the native CD80 and CD86 ligands. AU60590 / 96 describes this preferred soluble form of CTLA-4 VLD as a recombinant CTLA-4 / Ig fusion protein expressed in eukaryotic cells, but the problem of aggregation in prokaryotic expression systems remains unresolved. EP0757099A2 describes the use of CTLA-4 mutant molecules, for example, the effects of various changes in ligand binding of mutations in the BL sequence.

[0192] B7-1 (CD80) protein and B7-2 (CD86) protein The B7 protein is a superficial membrane protein found on activated antigen-presenting cells (APCs). When paired with either the T cell surface protein CD28 or CD152 (CTLA-4), it can emit either a co-stimulatory or co-inhibitory signal, thereby enhancing or reducing the activity of MHC-TCR signaling between APCs and T cells. B7 is present on activated APCs as well as on T cells.

[0193] The B7 protein family includes numerous members, such as B7-1, B7-2, B7-DC, and B7-H1 to B7-H7. The B7-1 protein, also known as CD80, binds to CD28 and CTLA-4 (cytotoxic T lymphocyte-associated protein 4). Its human sequence Uniprot reference number is P33681.

[0194] In one example, BDM binds to the B7-1 human protein. In another example, BDM binds to the B7-2 protein.

[0195] Sclerostin Sclerostin is a secreted glycoprotein with a cysteine ​​knot-like domain at its C-terminus and sequence similarity to the DAN family (abnormal genes in neuroblastoma selected by differential screening) of bone morphogenetic protein (BMP) antagonists. Sclerostin is produced by osteocytes and has an antianabolic effect on bone formation. The Uniprot reference number for the human sequence is Q9BQB4.

[0196] In one example, BDM binds to the human sclerostin protein.

[0197] Pharmacologically active proteins The complete protein species disclosed herein are pharmacologically active. Such proteins include antibodies (e.g., full-length antibodies), immunoglobulin antigen-binding fragments, or non-antibody proteins as described herein.

[0198] The term "pharmacologically active" means a substance determined to have the activity to affect a medical parameter or disease state, or to cause activation of cells involved in an immune response. The protein may be an agonist protein, an antagonist protein, or a pseudoprotein. The protein may also be a therapeutic antibody.

[0199] The terms "pseudo" or "agonist" refer to proteins (or peptides) that have biological activity comparable to that of natural proteins (e.g., EPO or G-CSF).

[0200] Suitable exemplary proteins in accordance with the present disclosure include, but are not limited to, the extracellular domains of proteins selected from blood coagulation factors, anticarin, toxoids, human serum albumin, collagen-binding proteins, TNF-α receptor-binding proteins, integrin-binding proteins, VEGF or its pseudo-substances, EPO or its pseudo-substances, C4-binding proteins, urokinase receptor antagonists, lymphokines, cytokines, osteoprotegerin (OPG), or programmed cell death 1 protein (PD1), programmed cell death ligand 1 (PD-L1), NKG2D, MHC class I polypeptide-associated sequence A (MICA), MHC class I polypeptide-associated sequence B (MICB), and UL16-binding proteins (ULBP).

[0201] Blood coagulation factors are well known in the art, and examples include factor VIII and factor IX, which are associated with hemophilia.

[0202] For example, the toxoid is a botulinum toxoid. The toxoid may be type A botulinum toxoid or type B botulinum toxoid. Synthetic forms of botulinum toxoids are also intended.

[0203] For example, lymphokines are IL-2 or GM-CSF or their pseudo-substances.

[0204] In one example, the cytokine is G-CSF or a pseudo-substance thereof, or a stem cell factor or a pseudo-substance thereof.

[0205] In one example, a protein is bound to BDM, which then binds to human serum albumin, extending the half-life of the protein in vivo compared to a protein without BDM.

[0206] antibody In certain cases, the multispecific molecule includes a full-length antibody. In one case, the antibody is a humanized antibody. In another case, the antibody is a human antibody. In yet another case, the antibody is a chimeric antibody.

[0207] The term "humanized antibody" should be understood as referring to a subclass of chimeric antibodies that have an antigen-binding site or variable region derived from a non-human species antibody, and an antibody structure based on the structure and / or sequence of a human antibody as the remaining portion. In humanized antibodies, the antigen-binding site generally includes a complementation-determining region (CDR) derived from a non-human antibody transplanted into a suitable framework region (FR) in the variable region of a human antibody, and the remaining portion being a region derived from a human antibody. The antigen-binding site may be wild-type (i.e., identical to that of a non-human antibody) or modified by one or more amino acid substitutions. In some cases, FR residues of the human antibody are replaced with corresponding non-human residues. Generally, a humanized antibody will contain at least one variable domain, typically substantially all of two variable domains, where all or substantially all CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all FR regions are FR regions of the consensus sequence of a human immunoglobulin.

[0208] The humanized antibodies disclosed herein will likely contain the constant region (Fc) of immunoglobulins, and typically the constant region of human immunoglobulins (Jones et al., (1986) Nature, 321:522-525; Riechmann et al., (1988) Nature, 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol. 2:593-596).

[0209] Methods for humanizing non-human antibodies or portions thereof (e.g., variable regions) are known in the art. Humanization is essentially possible according to the methods of Winter and collaborators (Jones et al., Nature, 321:522 525 (1986); Riechmann et al., Nature, 332:323 327 (1988)); Verhoeyen et al., Science, 239:1534 1536 (1988)) or the methods described in US5225539 or US5585089. Other methods for humanizing antibodies are not excluded.

[0210] As used herein, the term “human antibody” refers to an antibody having a variable region (e.g., VH, VL) and, optionally, a constant region derived from or matching a sequence found in humans, for example, in human germline cells or somatic cells. “Human” antibodies may include amino acid residues not encoded by human sequences, e.g., mutations introduced in vitro by random or site-directed mutations (certain mutations may involve conservative substitutions or mutations in a small number of antibody residues, e.g., one, two, three, four, five, or six of the antibody residues, e.g., one, two, three, four, five, or six of the residues constituting one or more CDRs of the antibody). These “human antibodies” do not need to be actually produced by humans; rather, they can be produced using recombinant techniques and / or isolated from transgenic animals containing nucleic acids encoding the constant and / or variable regions (e.g., those described above) of human antibodies (e.g., mice in which the endogenous immunoglobulin gene is partially or completely inactivated). Human antibodies can be produced using a variety of techniques known in the art, such as phage display libraries (e.g., those described in US5885793).

[0211] Human antibodies that recognize a selective epitope can also be produced using a technique called "guided selection." In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to induce the selection of a fully human antibody that recognizes the same epitope (see, e.g., US5,565,332).

[0212] Humanized immunoglobulins, including humanized antibodies, have been constructed by genetic engineering. Most of the humanized immunoglobulins described above contained a framework identical to that of a specific human immunoglobulin chain (i.e., acceptor or recipient), and three CDRs from a non-human (donor) immunoglobulin chain. Humanization may also include criteria for identifying a limited number of amino acids within the framework of the humanized immunoglobulin chain and selecting them to be identical to the amino acids at the positions in the donor rather than the acceptor in order to enhance the affinity of the antibody containing the humanized immunoglobulin chain.

[0213] Humanized antibodies generally have at least three major advantages over mouse or chimeric antibodies for use in human therapy. Since the effector portion of the antibody is human, it is thought to interact better with other parts of the human immune system (e.g., more efficiently destroy target cells by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC)). Furthermore, since the human immune system should not recognize the framework or constant region of the humanized antibody as foreign, the antibody response to such injected antibodies should be less than that to fully foreign mouse antibodies or partially foreign chimeric antibodies. Finally, mouse antibodies are known to have a significantly shorter half-life in the human bloodstream than human antibodies. Humanized antibodies can presumably have a half-life more similar to that of naturally occurring human antibodies, allowing for administration in smaller amounts and less frequently.

[0214] <> It will be recognized that any full-length antibody that binds to the desired target can be used in the present disclosure. In one example, the full-length antibody is further affinity matured and then bound to BDM.

[0215] In another example, the full-length immunoglobulin is a non-human immunoglobulin. In one example, the immunoglobulin is a mouse, rat, hamster, cat, dog, horse or bovine immunoglobulin.

[0216] Antibodies can be prepared using conventional methods. For example, polyclonal antisera or monoclonal antibodies can be produced using standard methods by using peptides or full-length target proteins. Mammals (e.g., mice, hamsters, or rabbits) can be immunized with the immunogenic form of a peptide that elicits an antibody response in mammals. Techniques for conferring high immunogenicity to peptides include conjugation to carriers or other techniques well known in the art. For example, proteins or peptides can be administered in the presence of adjuvants. The progress of immunization can be monitored by detecting the antibody titer in plasma or serum. Antibody levels can be evaluated using standard ELISA or other immunoassay procedures with the immunogen as the antigen. After immunization, antiserum can be obtained and, if desired, polyclonal antibodies can be isolated from the serum.

[0217] Antibodies can be produced from a variety of animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes, through cell culture, phages, or other means. Therefore, the antibodies useful in this disclosure are typically mammalian antibodies. Phage methods can be used to isolate initial antibodies or to produce mutants with modified specificity or avidity properties. Such techniques are common and well-known in the art. For example, an antibody may be produced by a recombinant method known in the art. For instance, a recombinant antibody can be produced by transfecting a host cell with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect a host cell with a DNA sequence expressing at least one VL region and one VH region. Exemplary descriptions of recombinant techniques for antibody production and synthesis include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); and Goding, Monoclonal Antibodies: Principles and Practice (Academic Press, 1993).

[0218] To enhance the efficacy of antibodies in vivo, it is sometimes desirable to modify them for effector function. For example, cysteine ​​residues may be introduced into the Fc region to form interchain disulfide bonds in this region. Antibodies thus produced may have improved internal distribution and / or high complement-mediated cytotoxicity and high antibody-dependent cell-mediated cytotoxicity (ADCC). See Caron et al., (1992) J. Exp Med., 176:1191-1195 and Shopes, (1992) J. Immunol., 148:2918-2922.

[0219] antibody fragment An antibody fragment comprises a portion of an intact antibody, which may include the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments suitable for use in this disclosure include Fab, F(ab')2, Fab', scFv, di-scFv, or chemically bound F(ab')2. In a particular example, the antibody fragment is Fab.

[0220] Fv refers to an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are strongly linked by non-covalent bonds. It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In summary, the six CDRs confer antigen-binding specificity to the antibody fragment.

[0221] The Fab fragment contains Fv and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that a few residues are added from the antibody hinge region to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines. The F(ab')2 fragment is prepared as a pair of Fab' fragments with hinge cysteine ​​residues in between.

[0222] Chemically linked F(ab')2 is a bispecific molecule formed by pairing two different Fab' fragments, each possessing a different binding specificity. Techniques for producing bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al (1985) Science 229:81 describes a procedure for producing F(ab')2 fragments by cleaving intact antibodies by protein degradation. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent the formation of intermolecular disulfides. Subsequently, the prepared Fab' fragments are converted to thionitrobenzoic acid (TNB) derivatives. Then, one of the Fab'-TNB derivatives is converted back to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody.

[0223] Production of multispecific molecules This disclosure also provides methods for creating the multispecific molecules of this disclosure.

[0224] Molecular expression is possible in both prokaryotic and eukaryotic cells. Prokaryotes are most often represented by a wide variety of bacterial strains. Bacteria can be either Gram-positive or Gram-negative. Typically, Gram-negative bacteria such as E. coli are preferred. Other microbial strains may also be used.

[0225] The sequences encoding the molecules can be cloned into vectors designed to express exogenous sequences in prokaryotic cells such as E. coli. These vectors may contain commonly used prokaryotic regulatory sequences, which are defined herein as containing a transcription initiation promoter and optionally an operator, along with a ribosome binding site sequence. Such commonly used promoters may include the beta-lactamase (penicillinase) and lactose (lac) promoter system (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057), and the lambda-derived PL promoter and N-gene ribosome binding site (Shimatake, et al., (1981) Nature 292:128).

[0226] Such expression vectors may also include selection markers, such as genes for a beta-lactamase or neomycin phosphotransferase that confer antibiotic resistance, so that the vector can replicate within bacteria and that cells carrying the plasmid can be selected when grown in the presence of antibiotics such as ampicillin or kanamycin.

[0227] Antibody expression in single-celled organisms such as E. coli and yeast is well established in the art. For an overview, see, for example, Andre Frenzel et al (2013) Front Immunol 4:217. Expression in cultured eukaryotic cells is also known to those skilled in the art as an option for producing the bispecific molecules described herein. See recent reviews, for example, Raff, ME (1993) Curr. Opinion Biotech. 4:573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6:553-560.

[0228] Molecular cloning techniques for achieving these objectives are publicly known in the art and are described, for example, in Ausubel et al., (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also publicly known in the art; see, for example, US4816567 or US5530101.

[0229] Systems for cloning and expressing polypeptides in various host cells are well known. Suitable host cell lines include bacterial, mammalian, yeast, and baculovirus lines. Mammalian cell lines available in the art for heterologous polypeptide expression include Chinese hamster ovary cells, HeLa cells, human fetal kidney cells, baby hamster kidney cells, NSO mouse melanoma cells, and many other cells. A suitable host would be selected considering, for example, the host's compatibility with the selection vector, the host's secretory characteristics, the host's ability to accurately fold proteins, and the host's fermentation requirements, as well as the toxicity of the product encoded by the expressed DNA sequence to the host, and the ease of purifying the expression product.

[0230] After isolation, the nucleic acid is functionally linked to a promoter and inserted into an expression construct or expression vector, and then subjected to further cloning (DNA amplification) or expression in a cell-free system or cells. The vector may be a plasmid, viral vector, such as a "phage" or phagemid, as appropriate.

[0231] To prepare the vector constructs of this disclosure, the nucleic acid sequences encoding the light chain sequence, heavy chain sequence, linker sequence, and BDM sequence of the antibody described herein are cloned into a suitable vector by standard methods.

[0232] In some examples, BDM will be cloned to the C-terminus of the antibody light chain sequence. In some examples, BDM will be cloned to the C-terminus of the antibody heavy chain sequence. In some examples, BDM will be cloned to the N-terminus of the antibody light chain sequence. In some examples, BDM will be cloned to both the N-terminus and C-terminus of the antibody light chain sequence. In some examples, BDM will be cloned to both the N-terminus and C-terminus of the antibody heavy chain sequence. In some examples, BDM will be cloned to both the N-terminus and C-terminus of both the antibody light chain sequence and the antibody heavy chain sequence. In some examples, the nucleic acid sequences encoding the antibody heavy chain or light chain and BDM are provided in one vector, and the other chain of the antibody is provided in another vector. In other examples, the nucleic acid sequences encoding the antibody light chain sequence, linker sequence, and BDM sequence are cloned into one vector, and the nucleic acid sequences encoding the antibody heavy chain sequence, linker sequence, and BDM sequence are cloned into another vector. Alternatively, a bisistronic vector may be used in which both the light and heavy chains of the antibody are expressed on the same vector. In certain cases, the coding sequences for the heavy and light chains (one of which may include BDM) may be present on a single vector, for example, in two expression cassettes within the same vector.

[0233] Nucleic acid sequences encoding molecules can also be inserted into vectors designed to express exogenous sequences in eukaryotic hosts. The regulatory elements of the vector may vary depending on the specific eukaryotic host.

[0234] Useful expression vectors may consist of sequences of chromosomal DNA, non-chromosomal DNA, and synthetic DNA, for example. Suitable vectors include derivatives of SV40 and known bacterial plasmids, such as E. coli plasmids col El, Pcr1, Pbr322, Pmb9 and their derivatives, and RP4; phage DNA, such as phage λ, e.g., NM989, and many derivatives of other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as 2u plasmid or its derivatives; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmid and phage DNA, such as plasmids modified to utilize phage DNA or other expression regulatory sequences; and the like.

[0235] Nucleic acid sequences encoding molecules can be incorporated into the genome of a eukaryotic host cell and replicated as host genome replication samples. Alternatively, a vector carrying such a nucleic acid sequence may contain a replication origin that enables extrachromosomal replication.

[0236] As used herein, the term “promoter” should be interpreted in its broadest sense, including transcriptional regulatory sequences of genomic genes, such as TATA boxes or initiator elements, but promoters are necessary for accurate transcription initiation, whether or not there are further regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that modify nucleic acid expression, for example, in response to development and / or external stimuli, or in a tissue-specific manner. In the context of this application, the term “promoter” is also used to describe derivatives that give, activate, or enhance the expression of recombinant nucleic acids, synthetic nucleic acids, or fusion nucleic acids, or nucleic acids to which the promoter is functionally linked. An exemplary promoter may contain further copies of one or more specific regulatory elements that further enhance the expression of the nucleic acid and / or modify spatial and / or transient expression.

[0237] As used herein, the term "functionally linked" means that the promoter is positioned relative to the nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0238] Many vectors are available for expression in cells. Generally, components of a vector include, but are not limited to, one or more of a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, a polyadenylation sequence, and a transcription termination sequence. Those skilled in the art will recognize sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0239] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-α promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the α-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter; hybrid regulatory elements including the CMV enhancer / β-actin promoter or an immunoglobulin promoter or an active fragment thereof. Examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV_{40} (COS-7, ATCC CRL1651); human fetal-derived kidney cell lines (subcloned 293 cells or 293 cells for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL10); or Chinese hamster ovary cells (CHO).

[0240] Methods for introducing isolated nucleic acids or expression constructs containing them into host cells for expression are known to those skilled in the art. The technique used for a given cell depends on the technique known to be successful. Methods for introducing recombinant DNA into cells include, among others, microinjection, DEAE-dextran-induced transfection, liposome-induced transfection using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-induced DNA uptake, retroviral gene transfer, electroporation, and injection of microparticles such as DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0241] After introduction, expression from nucleic acids can be induced or enabled, for example, by culturing host cells under expression conditions.

[0242] The disclosure also provides a method that includes using the above-mentioned constructs in an expression system to express antigen-binding fragment chains of antibodies or immunoglobulins and BDM.

[0243] This disclosure also provides recombinant host cells containing one or more nucleic acid sequences described herein. Host cells used for protein production can be cultured in a wide variety of media depending on the cell type used. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Culture media for other cell types discussed herein are known in the art.

[0244] A wide variety of host / expression vector combinations can be utilized when expressing the nucleic acid sequences of this disclosure. Useful expression vectors may consist of sequences of chromosomal DNA, non-chromosomal DNA, and synthetic DNA, for example. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids such as col El, Perl, Pbr322, Pmb9 and their derivatives, and RP4; phage DNA, e.g., phage λ, e.g., NM989, and many other phage DNAs, e.g., M13 and numerous derivatives of filamentous single-stranded phage DNA; yeast plasmids such as 2u plasmids or their derivatives; vectors useful in eukaryotic cells, e.g., vectors useful in insect or mammalian cells; vectors derived from combinations of plasmid and phage DNA, such as plasmids modified to utilize phage DNA or other expression regulatory sequences; and the like.

[0245] Recombinant host cells containing one or more polynucleotides described herein are also provided herein.

[0246] A wide variety of expression regulatory sequences, that is, sequences that control the expression of nucleic acid sequences functionally linked to them, can be used in these vectors to express those nucleic acid sequences. Such useful expression regulatory sequences include, for example, early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, lac, tip, TAC, TRC, LTR systems, major operator and promoter regions of phage λ, regulatory regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycoseptic enzymes, promoters of acid phosphatases (e.g., Pho5), promoters of yeast junction factors, and other sequences, as well as various combinations thereof, whose gene expression regulation in prokaryotic or eukaryotic cells or their viruses is known.

[0247] Those skilled in the art will be able to select appropriate vectors, regulatory sequences, and hosts without departing from the scope of this disclosure and without excessive experimentation to achieve the desired expression. For example, when selecting a vector, the host must be considered because the vector needs to function in a host. The copy number of the vector, the ability to control its copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, may also be considered. Those skilled in the art will be able to select appropriate vectors, regulatory sequences, and hosts without departing from the scope of this disclosure to achieve the desired expression. For example, when selecting a vector, the host must be considered because the vector needs to function in a host. The copy number of the vector, the ability to control its copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, may also be considered.

[0248] This disclosure provides isolated polynucleotides (nucleic acids) encoding polypeptides as described herein, vectors containing such polynucleotides, and host cells and expression systems for transcribing such polynucleotides and translating them into polypeptides.

[0249] This disclosure also provides constructs comprising at least one of the above-described polynucleotides in the form of plasmids, vectors, transcriptions, or expression cassettes, as described elsewhere in this specification.

[0250] This disclosure also provides host cells containing one or more polynucleotides disclosed herein.

[0251] After being produced by expression, the molecules can be isolated and / or purified using any suitable technique. Since nucleic acids are encoded, the molecules and vectors described herein may be provided, for example, from their natural environment in a substantially pure or homogeneous form, or, in the case of nucleic acids, isolated and / or purified without containing or substantially without the original nucleic acid or genes other than the sequence encoding the polypeptide having the required function. Nucleic acids may include DNA or RNA and may be entirely or partially synthetic.

[0252] When expressing nucleic acid sequences in Saccharomyces cerevisiae, a 2μ ring origin of replication derived from an endogenous yeast plasmid can be used (Broach, (1983) Meth. Enz. 101:307). Alternatively, sequences derived from a yeast genome that can promote autonomous replication can be used (see, for example, Stinchcomb et al., (1979) Nature 282:39; Tschemper et al., (1980) Gene 10:157; and Clarke et al., (1983) Meth. Enz. 101:300).

[0253] The transcriptional regulatory sequences of yeast vectors include promoters for glycosphagase synthesis (Hess et al., (1968) J.Adv.Enzyme Reg.7:149; Holland et al., (1978) Biochemistry 17:4900). Further promoters known in the art include the CMV promoter provided in the CDM8 vector (Toyama and Okayama, (1990) FEBS 268:217-221); the promoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J.Biol.Chem.255:2073); and promoters for other glycosphagases.

[0254] Polypeptide expression can be detected by methods known in the art. For example, molecules can be detected by Coomassie-stained SDS-PAGE gel and immunoblotting using an antibody, an antigen-binding fragment of an immunoglobulin, or an antibody that binds to BDM. Protein recovery can be performed using standard protein purification methods, such as affinity chromatography or ion-exchange chromatography, to obtain a substantially pure product (R. Scopes in: “Protein Purification, Principles and Practice”, Third edition, Springer-Verlag (1994)).

[0255] This disclosure also provides a method for producing the multispecific molecules described herein, and this method is: (i) Provide a vector (or more) containing a protein (e.g., an antibody) and a polynucleotide encoding BDM, (ii) Using such vectors, transform mammalian host cells (e.g., CHO), (iii) The host cells from step (ii) are cultured under conditions that promote protein secretion from the host cells into the culture medium, and (iv) Collect the secreted protein from step (iii). This includes the following step.

[0256] In one example, the method is: (i) To provide a first vector encoding the heavy chain of a multispecific molecule, (ii) To provide a second vector encoding the light chain of a multispecific molecule, and (iii) Transforming mammalian host cells (e.g., CHO) using the first and second vectors. Includes.

[0257] Synthesis Nucleic acids encoding the polypeptides of this disclosure can be prepared by cloning, or by synthesis rather than by cloning. Nucleic acids can be designed with appropriate codons for the polypeptide portion (e.g., immunoglobulins and BDMs). Generally, if the sequence is to be used for expression, codons preferred for a given intended host will be selected. Generally, if the sequence is to be used for expression, codons preferred for that intended host will be selected. Complete polynucleotides can be assembled from duplicate oligonucleotides prepared by standard methods and constructed into complete coding sequences. See, for example, Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

[0258] Protein isolation Methods for isolating polypeptides are known in the art and / or described herein.

[0259] If polypeptides are secreted into the culture medium, the supernatant obtained from such an expression system can be initially concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit proteolysis, a protease inhibitor such as PMSF may be included in one of the above steps, and an antibiotic may be included to prevent the growth of exogenous contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the polypeptide-expressing cells, for example, by serial centrifugation.

[0260] Polypeptides prepared from host cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination thereof. These methods are known in the art and are described, for example, in WO99 / 57134 or in Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0261] In some examples, the polypeptides of this disclosure are fused to heterologous amino acid sequences, such as signal sequences or affinity tags, without affecting their biological activity (i.e., their ability to bind to their targets).

[0262] Those skilled in the art will also recognize that polypeptides can be modified to include affinity tags that facilitate purification or detection, such as polyhistidine tags, such as hexahistidine tags, or influenza virus hemagglutinin (HA) tags, or Simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting polypeptides are then purified using methods known in the art, such as affinity purification. For example, polypeptides containing hexa-his tags are purified by contacting a sample containing such polypeptides with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to hexa-his tags immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins.

[0263] Binding of pharmacologically active proteins to BDM The binding of a pharmacologically active protein or peptide to at least the BDM according to this disclosure can be prepared using chemical binding (Brennan et al (1985) Science 229:81), chemical coupling (Shalaby et al (1992) J Exp Med 175:217-225), or gene fusion.

[0264] Furthermore, fusion or binding between a protein (e.g., an antibody) and a BDM can be achieved by conventional covalent or ionic bonds, protein fusion, or heterobifunctional crosslinkers, such as carbodiimide, glutaraldehyde, etc. Alternatively, a conventional inactive linker sequence (e.g., a peptide linker) that simply provides the desired amount of space between the protein and the BDM may be used. Designs of such linkers are well known to those skilled in the art and are described, for example, in US8,580,922; US5,525,491; and US6,165,476.

[0265] Various coupling or crosslinking agents can be used for the covalent complexation of proteins. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (sulfo-SMCC) (see, for example, Karpovsky el al. (1984) J. Exp. Med 160 1686, Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82 8648). Other methods include those described in Paulus (1985) Behring Ins Mitt No 78, 1 18-132, Brennan et al. (1985) Science 229 81-83, and Glennie et al. (1987). J Immunol 39 2367-2375).

[0266] Linkers enable increased flexibility and / or reduced steric hindrance between any two proteins. Linkers can be naturally occurring, for example, sequences determined to be present in random coils between two domains of a protein. An exemplary linker sequence is the one found between the C-terminal and N-terminal domains of the RNA polymerase alpha subunit. Other examples of naturally occurring linkers include those found in the 1CI and LexA proteins.

[0267] Within the linker, the amino acid sequence may vary based on preferred linker properties determined empirically or revealed by modeling. Considerations during linker selection include linker flexibility, linker charge, and the presence of some amino acids in naturally occurring subunits. Linkers can also be designed so that residues within the linker come into contact with DNA, thereby influencing binding affinity or specificity, or interacting with other proteins. In some cases, particularly when long distances between subunits are required, or when domains must be maintained in a specific configuration, linkers may optionally contain further folded domains.

[0268] In some examples, the linker design is preferably such that the domain arrangement requires the linker to extend over a relatively short distance, preferably less than about 10 angstroms (Å). However, in some embodiments, the linker extends over a distance of up to about 50 Å or more.

[0269] The term "peptide linker" refers to a short peptide fragment that connects or links a protein to the BDM (biodynamic dimethyl) portion of a polypeptide molecule. The linker is preferably composed of amino acids linked to each other by peptide bonds. For example, a peptide linker may include low molecular weight amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). For example, a peptide linker is a peptide having an amino acid sequence of at least 5 amino acids, or about 5 to about 100 amino acids, or about 10 to 50 amino acids, or about 10 to 15 amino acids.

[0270] In one example, the linker is composed mostly of sterically unhinged amino acids, such as glycine and alanine. Therefore, in further examples, the linker is polyglycine, polyalanine, or polyserine.

[0271] Those skilled in the art will understand that many commonly used peptide linkers may be used in embodiments of this disclosure. In some embodiments, a short peptide linker may include repeating units that extend the linker length, for example, a linker that is repeated two, three, or four times. In one example, the linker includes the formula (Gly-Gly-Gly-Gly-Ser)n or the formula (Ser-Gly-Gly-Gly-Gly)n Ser, where n is a number from 3 to 6.

[0272] For example, the linker may contain or be a component of the sequence SGGGGSGGGGSGGGGS (sequence number 16) or SGGGGSGGGGSGGGGSGGGGS (sequence number 17).

[0273] Non-peptide linkers are also possible. For example, -NH-(CH2) with s=2~20 sAlkyl linkers such as -C(O)- may be used. These alkyl linkers may be further substituted with any sterically unhindered group, such as lower alkyls (e.g., C1-C6), lower acyls, halogens (e.g., Cl, Br), CN, NH2, phenyl, etc. An example of a non-peptide linker is a PEG linker with a molecular weight of 100-5000 kD, preferably 100-500 kD.

[0274] Other examples of linkers suitable for use include GSTVAAPS, TVAAPSGS, or GSTVAAPSGS or polyploids of such linkers. Another example is (TVSDVP)n(GS)m, where n=1 and m=1, or n=2 and m=1, or n=2 and m=0.

[0275] In another example, the linker is GS.

[0276] Evaluation of polypeptide activity The proteins (or peptides) and BDM portions of this disclosure can be evaluated by various means according to known methods. Such evaluation methods may include functional analyses, e.g., cell killing assays, cAMP or calcium flux assays, or binding assays, e.g., ELISA or competitive assays.

[0277] The type of functional analysis used will depend on the target to which the polypeptide protein or peptide and BDM portion bind.

[0278] Half-life assays may be used. Such methods are known in the art. Two commonly used methods for measuring the half-life of proteins are radioactive pulse chase analysis and cycloheximide chase (Zhou P (2004) Methods Mol. Biol. 284:67-77).

[0279] Measurement of binding affinity Epitope binding can be measured by conventional antigen-binding assays such as ELISA, fluorescence-based techniques such as FRET, or techniques such as surface plasmon resonance that measure molecular mass. Specific binding of antigen-binding proteins (e.g., BDM) to antigens or epitopes can be determined by suitable assays, including competitive binding assays such as scatchard analysis and / or radioimmunoassays (RIA), ELISA, and sandwich competitive assays.

[0280] Competitive assays, such as surface plasmon resonance, can be used to determine whether BDM, which has been engineered to bind to a specific target, can bind in that manner. For example, BDM can be engineered to bind to a stem cell factor receptor (CSFR or c-kit receptor) and its ability to compete for binding with the native ligand (c-kit) can be tested. This allows for the determination of the ability of modified BDM to compete for binding to the target, as well as the dissociation constant (K). D In vitro competitive assays for determining ) are known in the art.

[0281] The binding affinity or dissociation constant (K) of the interaction between the BDM portion of a protein or polypeptide and its respective target. D ) can be measured by a number of methods known in the art. Such methods include, but are not limited to, fluorescence titration, competitive ELISA, calorimetry, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (BIAcore) or biolayer interferometry (e.g., the Blitz system (ForteBio)).

[0282] A preferred surface plasmon resonance method is BIAcore, which is known in the art.

[0283] Most of the bonding parts are K D The value is low micromolar (10 -6 ) ~ nanomoles (10 -7 ~10 -9The high affinity binding portion is generally within the range of picomoles. -12 ) Low nanomolar range (10) with a binding region having very high affinity in that range -9 It is thought that this is the case.

[0284] Complex formation between each part and its target depends on many different factors, such as the concentration of each binding partner, the presence or absence of competitors, the pH and ionic strength of the buffer system used, and K D It is influenced by the experimental method used to determine (e.g., fluorescence titration, competitive ELISA, or surface plasmon resonance) or the mathematical algorithm used to evaluate the experimental data.

[0285] Therefore, depending on the method and experimental setup used to determine the affinity of a particular immunoglobulin or BDM to a given target, K D It is obvious to those skilled in the art that values ​​can vary within a specific experimental range. This means that there may be slight deviations in the measured KD value or its acceptable range depending on whether the KD value was determined by surface plasmon resonance (Biacore), competitive ELISA, or "direct ELISA."

[0286] In a preferred example, surface plasmon resonance (SRP) is used to determine the KD value, for example, by using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) on an immobilized target.

[0287] The affinity may be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold or more than the affinity of the protein or BDM to an unrelated amino acid sequence. The affinity of the protein or BDM to a target (e.g., a protein antigen) may be, for example, about 100 nanomoles (nM) to about 0.1 nM, about 100 nM to about 1 picomole (pM), or about 100 nM to about 1 femtomol (fM) or more.

[0288] For example, proteins with affinity levels measured by KD are approximately 200 nM or less, approximately 100 nM or less, approximately 50 nM or less, approximately 25 nM or less, approximately 10 nM or less, approximately 5 nM or less, approximately 1 nM or less, or approximately 0.5 nM or less.

[0289] For example, BDM has affinity levels of approximately 200 nM or less, approximately 100 nM or less, approximately 50 nM or less, approximately 25 nM or less, approximately 10 nM or less, approximately 5 nM or less, approximately 1 nM or less, or approximately 0.5 nM or less, as measured by KD.

[0290] Biolayer interferometry is a label-free technique for measuring biomolecular interactions within an interactome. It is an optical analysis method that analyzes the interference patterns of white light reflected from two surfaces: a layer of immobilized proteins at the tip of a biosensor and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern, which can be measured in real time.

[0291] When a ligand immobilized on the biosensor tip surface binds to the analyte in solution, the optical thickness of the biosensor tip increases, resulting in a wavelength shift Δλ, which directly measures the change in the thickness of the biological layer. This allows for real-time measurement of the interaction and precise and accurate monitoring of binding specificity, association and dissociation rates, or concentrations.

[0292] Only molecules that bind to or dissociate from the biosensor shift the interference pattern, enabling the creation of a reaction profile. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate do not affect the interference pattern. This is a characteristic unique to biolayer interferometry and can be widely implemented with crude samples used in applications related to protein-protein interactions, quantification, affinity, and kinetics.

[0293] target The targets of this disclosure are preferably antigens. Antigens may be selected from proteins, glycans, lipids, lipoproteins, or nucleic acids. Proteins may be human proteins, non-human proteins (e.g., primate, canid, feline, etc.), viral proteins, yeast proteins, bacterial proteins, algal proteins, plant proteins, or protozoan proteins. Proteins may be soluble proteins or membrane-bound proteins. Examples of soluble proteins include, but are not limited to, transcription factors, antibodies, growth factors, blood proteins (e.g., albumin), or drugs (e.g., steroids, pharmaceuticals, etc.). Types of membrane-bound proteins include growth factor receptors, tumor markers, or markers that mediate intracellular transport (e.g., transferrin), or Fc receptors.

[0294] The nucleic acid target may be DNA, RNA, or a combination of DNA and RNA.

[0295] More specifically, the target is an epitope present on the antigen. Those skilled in the art will understand that the antigen may contain numerous specific epitopes, each of which will be recognized by a protein (e.g., an antibody) or BDM.

[0296] The polypeptides of this disclosure can bind to different targets. In one example, the different targets are two or three different antigens, each of which may be present in a different cell. In another example, the polypeptide may bind to two or three different targets (e.g., epitopes) on the same antigen. Preferably, the target to which the protein binds is different from the target to which the BDM binds.

[0297] The “target antigen” as defined herein may be a protein, peptide, glycoprotein, polysaccharide, glycan, lipid, lipoprotein, or nucleic acid. The target antigen as defined herein may be secreted or membrane-bound. Such antigens may be derived from bacterial, mammalian (human and non-human), fungal, algal, or protozoan material. Proteins may be human proteins, non-human proteins (e.g., primate, canid, feline, etc.), viral proteins, yeast proteins, bacterial proteins, algal proteins, plant proteins, or protozoan proteins.

[0298] The references to identical "first target antigen," "second target antigen," etc., should be understood to mean that while the pharmacologically active protein and at least one BDM moiety bind to the same antigen, they can bind to different epitopes present on that antigen.

[0299] The references to different "first target antigens," "second target antigens," etc., should be understood as meaning that the pharmacologically active protein portion and at least one BDM portion bind to different antigens, and therefore different epitopes, that are present on different cells.

[0300] (i) Bacterial antigens Antigens may be derived from bacteria, including Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholera, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium Examples include, but are not limited to, difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, hemophilus influenzae, Escherichia coli, Shigella spp., Erlicia spp., and Rickettsia spp.

[0301] (ii) Viral antigens Antigens may originate from viruses, including, but are not limited to, influenza viruses, parainfluenza viruses, mumps viruses, adenoviruses, polynuclear respiratory viruses, Epstein-Barr virus, rhinoviruses, polioviruses, coxsackieviruses, echoviruses, measles viruses, rubella viruses, varicella-zoster viruses, herpesviruses (human and animal), herpes simplex viruses, parvoviruses (human and animal), cytomegaloviruses, hepatitis viruses, human papillomaviruses, alphaviruses, flaviviruses, bunyaviruses, rabies viruses, arenaviruses, filoviruses, HIV1, HIV2, HTLV-1, HTLV-II, FeLV, bovine LV, FeIV, canine distemper virus, canine infectious hepatitis virus, feline calicivirus, feline rhinotracheitis virus, TGE virus (swine), and foot-and-mouth disease.

[0302] (iii) Tumor antigen The target antigen may be a tumor-associated antigen. Such tumor-associated antigens include MUC-1 and its peptide fragment, protein MZ2-E, pleomorphic epithelial mucin, folate-binding protein LK26, MAGE-1 or MAGE-3 and its peptide fragment, human chorionic gonadotropin (HCG) and its peptide fragment, carcinoembryonic antigen (CEA) and its peptide fragment, alpha-fetoprotein (AFP) and its peptide fragment, pancreatic carcinoembryonic antigen and its peptide fragment, CA125, CA15-3, CA19-9, CA549, CA195 and their peptide fragments, prostate-specific antigen (PSA) and its peptide fragment, prostate-specific membrane antigen (PSMA) and its peptide fragment, squamous cell carcinoma antigen (SC Examples include, but are not limited to, the following: CA) and its peptide fragments, ovarian cancer antigen (OCA) and its peptide fragments, pancreatic cancer-associated antigen (PaA) and its peptide fragments, Her1 / neu and its peptide fragments, gp-100 and its peptide fragments, mutant K-ras protein and its peptide fragments, mutant p53 and its peptide fragments, non-mutant p53 and its peptide fragments, cleaved epidermal growth factor receptor (EGFR), chimeric protein p210BCR-ABL, telomerase and its peptide fragments, Survivin and its peptide fragments, Melan-A / MART-1 protein and its peptide fragments, WT1 protein and peptide fragments, LMP2 protein and peptide fragments, HPV E6 E7 protein and peptide fragments, idiotype protein and peptide fragments, NY-ESO-1 protein and peptide fragments, PAP protein and peptide fragments, cancer / testicular protein and peptide fragments, and 5T4 protein and peptide fragments.

[0303] (iv) Other mammalian antigens The target antigen may be an antigen or epitope present on cells located in the heart, blood system, lungs, intestines, stomach, rectum, prostate, thyroid, liver, or esophagus. The target antigen may also be an antigen or epitope present on a secreted protein. Examples of secreted proteins include, but are not limited to, hormones, enzymes, toxins, antimicrobial agents, and peptides. Alternatively, the antigen or epitope may be present on a non-membrane-bound protein.

[0304] Selective binding For example, a molecule can selectively bind to cells that express two or more of the target antigens of a multispecific molecule, rather than to cells that express only one of the target antigens of that molecule.

[0305] Such cell selectivity can be achieved by regulating the binding affinity of each part of a bispecific or triplicate molecule (e.g., a protein or BDM), where each individual part is not sufficiently bound to its target to enable fluorescence-activated cell sorting (FACS), immunofluorescence labeling, or cell killing, or where a cell expresses its target in the absence of other targets bound to the bispecific or triplicate molecule. However, the combination of these weakly bound parts promotes sufficient avidity to the bispecific or triplicate molecule, allowing it to selectively bind to cells co-expressing the relevant target molecule rather than cells expressing only one such target, thus enabling selective FACS sorting, immunofluorescence labeling, or cell killing.

[0306] composition The multispecific molecules of this disclosure can be used as compositions when combined with pharmacologically acceptable carriers or additives. Such pharmaceutical compositions are useful for in vivo administration of the subject.

[0307] Pharmacologically acceptable carriers are physiologically acceptable to the patient receiving the administration and retain the therapeutic properties of the molecules used in combination with the administration. Pharmacologically acceptable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences 18. thThis is outlined in edn. Ed. A. Gennaro, Mack Publishing Co., Easton PA 1990. One exemplary carrier is physiological saline. As used herein, the term “pharmacologically acceptable carrier” means a pharmacologically acceptable substance, composition, or vehicle, such as a liquid or solid excipient, diluent, additive, solvent, or containment agent, that carries a polypeptide from one organ or body part to another organ or body part. Each carrier must be acceptable in the sense that it is compatible with the other components of the formulation and must not be harmful to the patient.

[0308] Pharmacologically acceptable additives may include preservatives or cryopreservatives.

[0309] Pharmaceutical compositions can be formulated to suit a specific route of administration, whether for systemic or topical administration.

[0310] For example, the compositions described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, or via implantable reservoir, in a conventionally pharmacologically acceptable non-toxic carrier-based dosage form or any other convenient dosage form. As used herein, the term "parenteral" includes injection or infusion methods into the subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial regions.

[0311] Methods for preparing molecules into forms suitable for administration to a target (e.g., pharmaceutical compositions) are known in the art and include, for example, the methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the United States Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0312] The pharmaceutical compositions of this disclosure are particularly useful for parenteral administration, such as intravenous administration or administration into body cavities or lumens of organs or joints. The administration compositions will generally comprise a pharmacologically acceptable carrier, such as a polypeptide solution dissolved in an aqueous carrier. A wide variety of aqueous carriers, such as buffered saline, may be used. The compositions may contain pharmacologically acceptable adjuvants, such as pH adjusters and buffers, toxicity modifiers, etc., to approximate physiological conditions as needed, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The protein concentrations in these formulations may vary considerably and will be selected primarily based on volume, viscosity, body weight, etc., according to the specific administration method chosen and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain small amounts of additives, such as buffers, preservatives, or other additives, to enhance its isotonic and chemical stability.

[0313] During formulation, the polypeptides of this disclosure will be administered in a manner compatible with the administered formulation and in a therapeutically / prophylactically effective amount. The formulations are readily administered in a wide variety of dosage forms, such as the injectable type described above, but other pharmacologically acceptable forms, such as tablets, pills, capsules or other solid preparations for oral administration, suppositories, pessaries, nasal drops or sprays, aerosols, inhalants, liposomes, etc., are also intended. Pharmaceutical "sustained-release" capsules or compositions may be used. Sustained-release formulations are generally designed to provide a certain level of drug over an extended period and may be used to deliver the compounds of this disclosure.

[0314] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size during dispersion, and using a surfactant. Antimicrobial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), and sodium chloride, may be included in the composition. The resulting solution can be packaged for immediate use or lyophilized; the lyophilized preparation can be combined with a sterile solution before administration.

[0315] Pharmacologically acceptable carriers may contain compounds that stabilize, enhance, or delay absorption or clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or additives or other stabilizers and / or buffers. Drugs that delay absorption include, for example, aluminum monostearate and gelatin. Surfactants may also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions, such as liposome carriers. To protect against digestion, compounds may be complexed with the composition to make it resistant to acid and enzymatic hydrolysis, or compounds may be complexed with a suitable resistant carrier, such as liposomes. Means for protecting polypeptides from digestion are known in the art (see, for example, Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and U.S. Patent No. 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).

[0316] Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations are known to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes directed to cells or tissues using antibody or viral coating proteins) can also be used as pharmacologically acceptable carriers. These can be prepared according to methods known in the art, for example, U.S. Patent Nos. 4,235,871; 4,501,728; 4,522,811; 4,837,028; 6,110,490; 6,096,716; 5,283,185; 5,279,833; Akimaru (1995) Cytokines Mol. Ther. 1:197 210; Alving (1995) Immunol. Rev. 145:5 31; and Szoka (1980) Ann. Rev. Biophys. Bioeng. 9:467). Other biodegradable polymer configurations capable of sustainably delivering biodegradable microparticles or capsules or low molecules such as peptides are known in the art (see, for example, Putney (1998) Nat. Biotechnol. 16:153 157).

[0317] The molecules of this disclosure can be incorporated into micelles (see, e.g., Suntres (1994) J. Pharm. Pharmacol. 46:23 28; Woodle (1992) Pharm. Res. 9:260 265). The molecules can be bound to the surface of a lipid monolayer or bilayer. For example, the molecules can be bound to hydrazide-PEG-(distearoylphosphatidyl)ethanolamine-containing liposomes (see, e.g., Zalipsky (1995) Bioconjug. Chem. 6:705 708). Alternatively, any form of lipid membrane, such as a lipid planar membrane or the cell membrane of an intact cell, such as an erythrocyte, can be used. Liposome formulations and lipid-containing formulations can be delivered by any means, such as intravenous, transdermal absorption (see, e.g., Vutla (1996) J. Pharm. Sci. 85:5 8), transmucosal, or oral administration.

[0318] The compositions of this disclosure can be combined with other therapeutic or contrast / diagnostic components as provided herein. The therapeutic and / or contrast components may be provided as separate compositions or as a composite component. Linkers may be included in the composite component as needed, as described elsewhere herein.

[0319] The molecules disclosed herein may be formulated as immunoliposomes. Liposomes containing polypeptides can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with long circulation times are disclosed in U.S. Patent No. 5,013,556.

[0320] The formulation for in vivo administration is sterile. Sterilization can be easily achieved through filtration using a sterile filtration membrane.

[0321] The compositions of this disclosure can be administered together with other therapeutic agents, such as chemotherapeutic agents. Chemotherapeutic agents are known in the art and include cytotoxic drugs and cell proliferation inhibitors. Non-limiting examples include paclitaxel, cisplatin, methotrexate, doxorubicin, and fludarabine. Other therapeutic agents are intended depending on the condition to be treated.

[0322] In one embodiment of the present disclosure, any pharmaceutical composition of the present disclosure is intended to be used to manufacture a pharmaceutical for the treatment of a disorder. The pharmaceutical may be packaged in a suitable pharmaceutical package with appropriate labeling relating to instructions for the treatment of a disorder in a subject.

[0323] Labeling and detection This disclosure provides the molecules described herein that are labeled with a drug. In one example, the drug is a contrast / detection moiety. In another example, the drug is a therapeutic moiety. Methods for labeling polypeptides will be familiar to those skilled in the art.

[0324] The terms “labeling” or “labeled” encompass both direct labeling of a protein (e.g., an antibody) or BDM by binding (i.e., physical linking) a detectable substance to said protein or BDM, and indirect labeling by reactivity with another directly labeled reagent. Such terms include both covalent and non-covalent bonding.

[0325] For example, molecules can be labeled using toxins, radionuclides, iron-related compounds, dyes, contrast agents, or fluorescently labeled or chemotherapeutic drugs.

[0326] Alternatively, molecules can be labeled with detectable labels, such as radionuclides, iron-related compounds, dyes, contrast agents, or fluorescent substances for immunological detection of target antigens.

[0327] Non-exclusive examples of radioactively labeled objects include, for example, 32 P, 33 P, 43 K, 52 Fe,57 Co, 64 Cu, 67 Ga, 67 Cu, 68 Ga, 71 Enjoy, 75 Br, 76 Br, 77 Br, 77 As, 81 Rb / 81 MKr, 87 MSr, 90 Y, 97 Ru, 99 Tc, 100 Pd, 101 Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb, 121 Sn, 123 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 I, 131 Cs, 143 Pr, 153 Sm, 161 Tb, 166 Ho, 169 EU, 177 Lu, 186 Re, 188 Re, 189Re, 191Os, 193Pt, 194 Ir, 197 Hg, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi and 213 It contains Bi.

[0328] Various radionuclides can be used to create radioactive complex proteins. For example, 13 C, 15 N, 2 H, 125 I, 123 I, 99 Tc, 43 K, 52Fe, 67 Ga, 68 Ga, 111 Examples include, but are not limited to, low-energy radioactive nuclei such as In, etc. (for example, suitable for diagnosis). For example, the radionuclide is a radionuclide that emits gamma rays, photons, or positrons having a suitable half-life that enables action or detection after the passage of time from administration to localization at the imaging site. The present disclosure also includes 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 high-energy radioactive nuclei such as Re, etc. (for example, for treatment). These isotopes typically generate short-range, high-energy alpha or beta particles. Such radionuclides kill cells in their proximity, such as tumor cells bound or invaded by the complex. They have little or no effect on non-localized cells and are essentially non-immunogenic. Alternatively, high-energy isotopes can be produced by thermally irradiating an otherwise stable isotope, such as in the case of boron neutron capture therapy (Guan et al., 1998). Other isotopes that may be suitable are described in Carter. (2001) Nature Reviews Cancer 1, 118 - 129, Goldmacher et al. (2011) Therapeutic Delivery 2; 397 - 416, Payne (2003) Cancer Cell 3, 207 - 212, Schrama et al, (2006) Nature Rev. Drug Discov. 5, 147 - 159, Reichert et al. (2007) Nature Reviews Drug Discovery 6; 349 - 356.

[0329] Toxins include any drug that is harmful to cells (e.g., kills them). Further techniques related to the preparation of antibody-immunotoxin complexes are provided, for example, in US5,194,594, which may be utilized in this disclosure. Non-exclusive examples of toxins include, for example, diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin and trichothecenes, Clostridium perfringens phospholipase C (PLC), bovine pancreatic ribonuclease (BPR), antiviral protein (PAP), abrin, cobra venom factor (CVF), geronin (GEL), saporin (SAP), and viscumin.

[0330] Non-limiting examples of iron-related compounds include, for example, magnetic iron oxide particles, ferric or ferrous particles, Fe2O3, and Fe3O4. Iron-related compounds and methods for labeling polypeptides, proteins, and peptides can be found, for example, in U.S. Patents 4,101,435 and 4,452,773, and U.S. Publication Applications 20020064502 and 20020136693, all of which are incorporated herein by reference in their entirety.

[0331] In certain cases, molecules can be labeled with cytotoxins or other cell proliferation inhibitors to pinpoint the delivery site of the drug to tumor cells. For example, the drug may be selected from the group consisting of drugs, enzyme inhibitors, proliferation inhibitors, solubilants, DNA or RNA synthesis inhibitors, membrane permeability modifiers, DNA metabolites, dichloroethyl sulfide derivatives, protein synthesis inhibitors, ribosome inhibitors, apoptosis inducers, and neurotoxins.

[0332] For example, the molecules described herein include one or more detectable markers that facilitate detection and / or isolation. For example, polypeptides include fluorescent labels such as fluorescein (FITC), 5,6-carboxymethylfluorescein, Texas red, nitrobenzo-2-oxa-1,3-diazole-4-yl (NBD), coumarin, dancylcloride, rhodamine, 4'-6-diamidino-2-phenylindole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), and rhodamine (5,6-tetramethylrhodamine). For these fluorescent dyes, the absorption and emission maxima are FITC (490nm; 520nm), Cy3 (554nm; 568nm), Cy3.5 (581nm; 588nm), Cy5 (652nm; 672nm), Cy5.5 (682nm; 703nm), and Cy7 (755nm; 778nm), respectively.

[0333] In certain cases, molecules can be combined with agents useful in tumor imaging. Such agents include metals, metal chelators, lanthanides, lanthanide chelators, radiometals, radiometal chelators, positron-emitting nuclei, microbubbles (for ultrasound), liposomes, molecules microencapsulated in liposomes or nanoparticles, single-crystal iron oxide nanocompounds, contrast agents for magnetic resonance imaging, absorbers, reflectors and / or scatterers, colloidal particles, and fluorophores such as near-infrared fluorophores. In many cases, such secondary functionalities / parts are relatively large, for example, at least 25 amu in size, and often at least 50 amu, 100 amu, or 250 amu in size. In certain cases, the secondary functionality is a chelating part for chelating metals, e.g., a chelator for radiometallic ions or paramagnetic ions. In further cases, the secondary functionality is a chelator for radionuclides useful in radiotherapy or imaging.

[0334] Alternatively or additionally, the molecules may be labeled with magnetic or paramagnetic compounds, such as iron, steel, nickel, cobalt, rare earth elements, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt-platinum, or strontium ferrite.

[0335] In another example, a molecule is complexed with a "receptor" (such as streptavidin) and used in cell pretargeting. In this case, the complex is administered to the patient, a clearing agent is used to remove the unbound complex from circulation, and then a "ligand" (e.g., avidin) complexed with a therapeutic agent (e.g., a radionucleotide) is administered.

[0336] Examples of therapeutic agents include, but are not limited to, anti-angiogenic agents, anti-angiogenic and / or other angiogenic agents, antiproliferative agents, apoptosis promoters, chemotherapeutic agents, antimitotic agents (e.g., the antimitotic agent auristatin, (MMAF / MMAE according to Angew. Chem. Int. Ed. 2014, 53, 1-6) or therapeutic nucleic acids).

[0337] Chemotherapy agents useful as agents in this specification include cytotoxic and cell proliferation inhibitory drugs. Chemotherapy agents may also have other effects on cells, such as reversing a transformed state to a differentiated state, or inhibiting cell replication. Examples of known cytotoxic agents useful in the present invention are, for example, published in Goodman et al., “The Pharmacological Basis of Therapeutics,” Sixth Edition, AB Gilman et al., eds. / Macmillan Publishing Co., New York, 1980. These include taxanes such as paclitaxel and docetaxel; nitrogen compounds such as mechloretamine, melphalan, uracil mustard, and chlorambucil; ethyleneimine derivatives such as thiotepa; alkyl sulfonic acids such as busulfan; nitrosourea compounds such as lomustine, semustine, and streptozocin; triazenes such as dacarbazine; folic acid analogs such as methotrexate; pyrimidine analogs such as fluorouracil, cytarabine, and azalibine; purine analogs such as mercaptopurine and thioguanine; vinca alkaloids such as vinblastine and vincristine; and dactyl This includes antibiotics such as nomycin, daunorubicin, doxorubicin, and mitomycin; enzymes, platinum-coordinated complexes such as cisplatin; urea derivatives such as hydroxyurea; methylhydrazine derivatives such as procarbazine; adrenal cortical depressants such as mitotane; and hormones and antagonists such as corticosteroids (prednisone), progestins (hydroxyprogesterone caproate, acetate, and megestrol acetate), estrogens (diethylstilbestrol and ethinylestradiol), and androgens (testosterone propionate and fluoxymesterone).

[0338] In one example, the multispecific molecules described herein are further complexed or linked with another protein (e.g., human serum albumin or HSA). In another example, the non-antibody protein is HSA.

[0339] The molecule may be complexed or linked with a therapeutic agent or liposome (e.g., a drug-containing liposome).

[0340] Drugs that interfere with protein synthesis may be used, and such drugs are known to those skilled in the art, including puromycin, cycloheximide, and ribonucleases.

[0341] Furthermore, other labels, such as biotin, followed by streptavidin-alkaline phosphatase (AP), horseradish peroxidase, etc., are intended by this disclosure.

[0342] The molecules of this disclosure can be modified to include further non-proteinoid moieties that are known and readily available in the art. For example, moieties suitable for protein derivatization are physiologically acceptable polymers, such as water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polypropylene glycol (PPG).

[0343] use For example, the molecules of this disclosure can be used for affinity purification or for the detection of a desired antigen carrying an epitope to which a protein (or peptide) and / or BDM binds.

[0344] This disclosure also provides a method for detecting targets to which either or both the protein and / or BDM portions of a polypeptide bind. Such a method may, for example, utilize the use of detectable labels as described above. In one example, different labels could be used for the protein and / or BDM to determine whether the bound target is the protein or the BDM.

[0345] Various formats can be used to determine whether a sample contains a target (e.g., a protein) that binds to a protein or peptide and / or BDM. Examples of such formats include, but are not limited to, enzyme-linked immunoassay (EIA), radioimmunoassay (RIA), Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0346] In one format, a polypeptide can be used to detect a target using methods such as Western blotting or immunofluorescence antibody testing.

[0347] Proteins (or peptides) or polypeptides or target antigens to which BDMs are bound may be immobilized on a solid support. Alternatively, multispecific molecules may be bound to a solid support. Suitable solid supports or carriers include any support capable of binding to antigens (i.e., targets), immunoglobulins, or BDMs. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. Those skilled in the art will know many other suitable carriers for binding to proteins, BDMs, or target antigens, and such supports can be adapted to use with the present disclosure. For example, a target protein can be run on a solid support such as nitrocellulose by polyacrylamide gel electrophoresis. The support can then be washed with a suitable buffer and treated with a detectably labeled polypeptide. The solid support can then be washed a second time with buffer to remove unbound polypeptides. The amount of label bound to the solid support can then be detected by conventional means.

[0348] For example, the molecules of this disclosure can be used as pharmaceuticals.

[0349] Advantages of the multispecific molecules of this disclosure With regard to non-antibody proteins, the addition of BDM to such proteins can promote increased protein stability, circulation time, and / or biological activity. For example, factor VIII protein can be bound to human serum albumin (VLD) to extend the half-life of such protein. This is clearly advantageous for individuals with hemophilia in terms of reducing the frequency of administration.

[0350] With respect to antibodies, the molecules described herein offer advantages over therapeutic agents using bispecific antibodies. A simple and efficient method for improving one or more characteristics of an antibody, for example, an antibody with insufficient therapeutic effect, is to conjugate one or more single-binding domain molecules (BDMs) that bind to a specific therapeutic target to an antibody or immunoglobulin antigen-binding fragment that is specific to the target.

[0351] The advantages of this bispecific method are that it allows for the simple and highly efficient production of bispecific antibodies; the method is applicable to any antibody sequence; the parent antibody retains its original binding site and specificity; the parent antibody retains its avidity; the parent antibody retains its original structure and function, and retains its effector function for selected applications; the pharmacokinetics of the antibody, such as its serum half-life, are preserved; and the bispecific product can be seamlessly integrated into existing IgG production processes, such as manufacturing, purification, formulation, and any other processes based on standard IgG production procedures.

[0352] For selective applications, bispecific molecules using antibody Fab fragments would also be desirable due to their small molecular weight, short half-life in the bloodstream, and improved molecular weight-to-binding site ratio compared to all antibodies.

[0353] For example, the neutralization of toxic substances, which has traditionally been treated with all antibodies, can be more effectively treated with Fab. Since the molecular weight of antibodies (150 kDa) is relatively large compared to toxic substances (typically less than 1 kDa), a large amount of antibody is required to bind to the toxin stoichiometrically. The bispecific or triplicate Fabs described herein can be used at low doses because the bispecific or triplicate molecules provide multiple binding sites. With Fabs that are small in size relative to antibodies, rapid excretion of the toxic substance-Fab complex via the kidney is observed. Indeed, an efficient and simple method for creating bispecific molecules using Fab is to conjugate a BDM that binds to a specific target to an existing Fab that is specific to the target.

[0354] kit This disclosure also provides kits containing the multispecific molecules described herein, along with instructions for use. If the compositions are lyophilized, the kits may contain further solution preparations for reconstituting the preparations. Instructions may be in the form of “printed material,” for example, on paper or cardboard included in or attached to the kit, on labels affixed to the kit or packaging materials, or affixed to vials or tubes containing the components of the kit.

[0355] Furthermore, the kit may further include any other components provided herein, such as chemotherapy drugs.

[0356] The kit may also include components for assays provided herein, such as those for ELISA assays.

[0357] The kit may further include a label that clearly states, for example, the product overview, administration method, and therapeutic indications. The label or accompanying leaflet may also include appropriate written instructions.

[0358] Those skilled in the art will understand that numerous variations and / or modifications may be made to the above embodiments without departing from the broad general scope of this disclosure. Accordingly, the embodiments of this application should be considered in all respects as illustrative and not restrictive. [Examples]

[0359] Example 1: Preparation of bispecific and tripspecific antibody molecules i) Vector creation Gene construction and cloning of CTLA-4 BDM followed standard, well-documented techniques, as well as the previously described US7,166,697. CTLA-4 BDM was obtained from a library (Geneart). Gene library 1696327 was constructed using degenerate oligonucleotide synthesis to incorporate different substitutions in the DNA sequence corresponding to the binding loop region.

[0360] The CTLA-4 sequences used in these examples include a C-terminal modification of the natural sequence, where the natural sequence PEPCPDSDGSTG is replaced with PEPSPDSN. Because these sequences do not contain a C-terminal Cys residue, BDM can remain in monomeric form.

[0361] Nucleic acid constructs encoding an anti-lysozyme IgG1 heavy chain sequence fused with a B7-1-binding CTLA-4 V-like domain sequence (represented as D1.3 IgG-VLD×2(HC)) and a nucleic acid construct encoding an anti-lysozyme IgG kappa light chain sequence fused with a B7-1-binding V-like domain sequence (represented as D1.3 IgG-VLD×2(LC)) were constructed. The VLDs were conjugated to either the heavy chain (H) or light chain (L) of the antibody via a Gly-Ser linker sequence. All DNA constructs were validated by restriction enzyme analysis and DNA sequencing, and recombinant protein expression was tested using standard, well-understood techniques. The sequences of these components are shown below. The antigen-binding loop is underlined in the relevant section: Sequence of B7-1 binding V-like domain (VLD) (SEQ ID NO: 13) [Table 7] The VLD coupling loop is underlined. Linker sequence (sequence number 16) [Table 8] Linker array (sequence number 17) [Table 9] Anti-lysozyme IgG single-chain sequence (SEQ ID NO: 18) used in antibody-VLD fusions [Table 10] Sequence of anti-lysozyme IgG single-chain linked to B7-1 bound VLD by a linker (D1.3 IgG-VLDx2(HC)) (SEQ ID NO: 19) [Table 11] The VLD coupling loop is underlined. Anti-lysozyme IgG kappa light chain (SEQ ID NO: 20) [Table 12] Anti-lysozyme IgG kappa light chain (represented as D1.3 IgG-VLD×2(LC)) conjugated to a B7-1 linked VLD having a 21-amino acid Gly-Ser linker (SEQ ID NO: 21) [Table 13] The VLD coupling loop is underlined. Anti-lysozyme Fab-Kappa light chain (represented as D1.3 Fab-VLD×1(LC)) is linked to a B7.1-linked VLD containing a 21-amino acid Gly-Ser linker (SEQ ID NO: 22). [Table 14] The VLD coupling loop is underlined. Anti-lysozyme Fab heavy chain (represented as D1.3 Fab-VLD×1(HC) (SEQ ID NO: 23) is linked to a B7.1-linked VLD having a 21-amino acid Gly-Ser linker. [Table 15] The VLD coupling loop is underlined. Anti-lysozyme Fab heavy chain (with His6 and myc tags at the C-terminus) (SEQ ID NO: 24) [Table 16]

[0362] ii) Preparation of bispecific and tripspecific molecules Individual heavy and light chains encoding antibody-VLD sequences or Fab-VLD sequences were cloned into the expression vector pcDNA3.4 (Thermo Fisher). Vector preparation and amplification were performed in E. coli cells (Electro Ten Blue, Agilent Technologies).

[0363] Schematic diagrams of bispecific and triplicate molecules according to this disclosure are shown as examples in Figures 2-4 for antibody-VLD molecules and in Figures 5-7 for Fab-VLD molecules.

[0364] Figure 2 shows a schematic diagram of an antibody-VLD bispecific molecule [D1.3 IgG-VLD×2(HC)] created by linking a target-specific VLD (e.g., bound to target B) with the ends of the constant region (CH3) sequences of each heavy chain of the anti-lysozyme IgG1 antibody heavy chain. The antibody molecule binds to target A via the variable regions of the antibody's heavy and light chains, and to target B via the VLD of the heavy chain.

[0365] Figure 3 shows a schematic diagram of an antibody-VLD bispecific molecule [D1.3 IgG-VLD×2(LC)] created by linking a target-specific VLD (e.g., bound to target B) with the terminals of each light chain constant region (CL) sequence of the anti-lysozyme IgG1 antibody light chain. The molecule binds to target A via the variable regions of the antibody's heavy and light chains, and to target B via the VLD of the light chain.

[0366] Figure 4 shows a schematic diagram of an antibody-VLD tripspecific molecule D1.3 IgG-VLD×4(LC+HC) created by binding a target-specific VLD (e.g., bound to target B) to both the heavy and light chains, in which case the VLD is bound to the terminal CL sequence and also to the terminal heavy chain CH3 sequence. The molecule binds to target A via the variable regions of the antibody's heavy and light chains, to target B via the VLD on the light chain, and to target C via the same or a different VLD as the one to which target B binds. The tripspecific molecule can bind to targets A, B, and C individually, simultaneously, or in combination of the three targets (e.g., target A and B, or target A and C, or target B and C).

[0367] Figure 5 shows a schematic diagram of a bispecific Fab-VLD molecule (D1.3 Fab-VLD×1(HC)) according to an example of this disclosure. In this schematic diagram, bispecificity is created by fusing a target-specific VLD (for example, in this example, the VLD is specific to target B) with the terminal of the constant region (CH1) sequence of a Fab that binds to target A (for example, in this example, the Fab is specific to target A). The bispecific molecule can bind to both target A and target B individually or simultaneously.

[0368] Figure 6 shows a schematic diagram of a bispecific Fab-VLD molecule (D1.3 Fab-VLD×1(LC)) according to an example of this disclosure. In this schematic diagram, bispecificity is created by fusing a target-specific VLD (for example, in this example, the VLD is specific to target B) with the terminal CL sequence of a Fab that binds to target A (for example, in this example, the Fab is specific to target A). The bispecific molecule can bind to both target A and target B individually or simultaneously.

[0369] Figure 7 shows a schematic diagram of a triple-specific Fab-VLD molecule (D1.3 Fab-VLD×2(LC+HC)) according to this disclosure. In this schematic diagram, triple specificity is created by fusing a target-specific VLD (for example, in this example, the VLD is specific to target B) with the end of a Fab CL sequence, and by fusing a VLD specific to a second target (for example, in this example, the VLD is specific to target C) with the end of a Fab CH3 sequence that binds to target A (for example, in this example, Fab is specific to target A). The triple-specific molecule can bind to targets A, B, and C individually, simultaneously, or in combination of the three targets (for example, target A and B, or target A and C, or target B and C).

[0370] Bacterial transformation with a vector was performed using a standard technique employing 2 ng of DNA in an electroporation-competent cell line (e.g., ElectroTen-Blue cells, Stratagene catalog number 200159).

[0371] After vector amplification, DNA was extracted and prepared using either the Qiagen HiSpeed ​​Plasmid Maxi Kit (catalog number 12663) or the Qiafilter Plasmid Mega Kit (catalog number 12281). The DNA was then eluted in purified water that did not contain nucleases.

[0372] iii) DNA transfection into mammalian cells for protein expression Using the Expi293 Expression System (ThermoFisher Scientific), approximately 2 × 10 6 Transfection of Expi293 cells / mL (mammalian cell viability over 95%) was performed. Bispecific or tripspecific DNA vectors were prepared with an HC DNA:LC DNA ratio of 1:3. The DNA was heated to 65°C for 5 minutes, cooled to room temperature, and then Opti-MEM I (Life Technologies catalog number 31985070) and ExpiFectamine293 reagent (ThermoFisher catalog number A14525) were added. The mixture was incubated at room temperature for 30 minutes. The transfection complex was added to the cells and incubated at 37°C, 5% CO2, and 120 rpm. After approximately 4-5 days, the supernatant containing protein was collected.

[0373] Tables 1 and 2 show the protein expression levels of bispecific and tripspecific mutants obtained from transfection of antibody-VLD and Fab-VLD using the parental anti-lysozyme D1.3 antibody and Expi293 expression system, respectively. The antibody or Fab binds to lysozyme, while VLD binds to B7.1. Table 1 Protein expression levels in bispecific and tripspecific antibody-VLD mutants [Table 17] Table 2 Protein expression levels in bispecific and tripspecific Fab-VLD mutants [Table 18]

[0374] iv) Purification ProsepA purification was used to purify bispecific and tripspecific molecules. The ProsepA column was equilibrated with PBS / Tris (2 mM Tris, pH 8) at 5 times the column volume. The supernatant of transfected cells containing the bispecific molecules at pH 8 was loaded onto the column, and the column was washed with PBS, 2 mM Tris, pH 8 at 10 times the column volume. The proteins were eluted with 0.1 M glycine (pH 3). The eluted protein fraction was neutralized to approximately pH 7 with 1 M Tris, pH 8, and dialyzed according to standard procedures.

[0375] The expression levels of the mutant protein were determined by SDS-PAGE. Each value was obtained from 30 ml of expression culture purified by affinity chromatography (not shown).

[0376] Example 2 Expression of Fab molecule The expression of parental D1.3 Fab and its bispecific and triplicate mutants, expressed in an unoptimized standard Expi293 expression system, was analyzed by SDS-PAGE under non-reducing and reducible conditions. Values ​​were obtained from 30 ml of expression culture purified by affinity chromatography. The parental Fab is D1.3 Fab. The bispecific mutants are D1.3 Fab-VLD×1(HC), which is a combination of D1.3 Fab and a VLD fused to the CH sequence of D1.3 Fab, and D1.3 Fab-VLD×1(CL), which is a combination of D1.3 Fab and a VLD fused to the CL sequence of D1.3 Fab. The triplicate molecule is D1.3 Fab-VLD×2(HC+LC), which is a combination of D1.3 Fab and each VLD fused to both the CH and CL sequences of D1.3 Fab. The results under non-reducing conditions are shown in Figure 8. The results under reducing conditions are shown in Figure 9. The analysis indicated that the appropriate heavy-chain and light-chain fusion was within the predicted size range.

[0377] Example 3: Binding evaluation of bispecific and tripspecific molecules Using standard chemistry and reagents, the binding properties of purified bispecific and tripspecific molecules were characterized using a ForteBio Blitz biosensor.

[0378] Antibodies, antibody-VLD bispecific molecules, and antibody-VLD triplicate molecules purified by affinity chromatography were used with commercially available lysozyme (Sigma Aldrich catalog no. L4919) and B7-1-Fc (R&D Systems catalog no. 140-B1). Biotin-labeled lysozyme was captured using a streptavidin capture surface (SA Sensor ForteBio catalog no. 18-5019, or Sensor Chip SA, GE catalog no. BR-1000-32). Antibodies, antibody-VLD bispecific molecules, and antibody-VLD triplicate molecules were passed over targets captured on the biosensor surface to create a binding sensorgram (i.e., first specificity). Against this same target, B7-1-Fc was passed over already bound antibodies, antibody-VLD bispecific molecules, and antibody-VLD triplicate molecules (i.e., second specificity) while they were in the dissociation phase to demonstrate a second binding interaction.

[0379] Figure 10 shows a preliminary analysis using a ForteBio Blitz biosensor, demonstrating that the bispecific molecule [IgG VLD×2(HC)] binds to biotin-labeled lysozyme captured with streptavidin, and subsequently to B7.1-Fc. The bispecific molecule has a B7-1 conjugated VLD attached to the heavy chain of the D1.3 antibody [D1.3 IgG]. Trace 1 is a sensorgram of the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific molecule. It shows the antibody binding to lysozyme immobilized on the biosensor surface, followed by the addition of buffer at point 1. Trace 2 is a sensorgram of the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific molecule. It shows the antibody binding to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc at point 1. B7-1-Fc was replaced with buffer at point 2. Trace 3 is a sensorgram of a bispecific D1.3 anti-lysozyme antibody [bispecific molecule-D1.3 IgG-VLD×2(HC)] with a VLD conjugated to the constant heavy chain (CH) of the antibody. It shows the case where the bispecific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of buffer at point 1. Trace 4 is a sensorgram of a bispecific D1.3 anti-lysozyme antibody [bispecific molecule-D1.3 IgG-VLD×2(HC)] with a VLD conjugated to the CH chain of the antibody. It shows the case where the bispecific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc at point 1. At point 2, B7-1-Fc was replaced with buffer.

[0380] Figure 11 shows a preliminary analysis using a ForteBio Blitz biosensor, demonstrating that a bispecific molecule [IgG VLD×2(LC)] binds to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to B7-1-Fc. The bispecific molecule has a B7-1-conjugated VLD conjugated to the light chain of a D1.3 antibody [D1.3 IgG]. Trace 1 is a sensorgram of the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific molecule. It shows the antibody binding to lysozyme immobilized on the biosensor surface, followed by the addition of buffer at point 1. Trace 2 is a sensorgram of the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific molecule. It shows the antibody binding to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc at point 1. B7-1-Fc was replaced with buffer at point 2. Trace 3 is a sensorgram of a bispecific D1.3 anti-lysozyme antibody [bispecific molecule-D1.3 IgG-VLD×2(LC)] with a VLD conjugated to the constant region (CL) of the antibody light chain. It shows the case where the bispecific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of buffer at point 1. Trace 4 is a sensorgram of a bispecific D1.3 anti-lysozyme antibody [bispecific molecule-D1.3 IgG-VLD×2(LC)] with a VLD conjugated to the antibody CL chain. It shows the case where the bispecific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc at point 1. At point 2, B7-1-Fc was replaced with buffer.

[0381] Figure 12 shows a preliminary analysis using a ForteBio Blitz biosensor, demonstrating that the triple-specific [IgG VLD×4(HC+LC)] molecule binds to biotin-labeled lysozyme captured with streptavidin, followed by secondary binding to B7-1-Fc. The triple-specific molecule has each B7-1-binding VLD bound to both the heavy and light chains of the D1.3 antibody [D1.3 IgG]. Trace 1 is a sensorgram of the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific molecule. It shows the antibody binding to lysozyme immobilized on the biosensor surface, followed by the addition of buffer at point 1. Trace 2 is a sensorgram of the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific molecule. It shows the antibody binding to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc at point 1. B7-1-Fc was replaced with buffer at point 2. Trace 3 is a sensorgram of a triply specific D1.3 anti-lysozyme antibody [triply specific molecule-D1.3 IgG-VLD×4(HCLC)] having VLDs bound to the CH and CL chains of the antibody. It shows the case where the triply specific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of buffer at point 1. Trace 4 is a sensorgram of a triply specific D1.3 anti-lysozyme antibody [triply specific molecule-D1.3 IgG-Im×4(HC+LC)] having VLDs bound to the CH and CL chains of the antibody. It shows the case where the triply specific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc at point 1. At point 2, B7-1-Fc was replaced with buffer.

[0382] Figure 13 shows a preliminary analysis using the ForteBio Blitz biosensor demonstrating that the triplicate molecule has higher binding affinity than the bispecific molecule. The figure demonstrates that the triplicate molecule has a higher binding level to B7-1-Fc than the bispecific molecule. When bispecific and triplicate molecules with equivalent antibody counts were captured on biotin-labeled lysozyme conjugated to the biosensor surface, and then buffer or B7-1-Fc was added (point 1), the triplicate molecule showed higher binding affinity than the bispecific molecule. Trace 1 shows the D1.3 anti-lysozyme antibody [D1.3 IgG] used to construct the bispecific and triplicate molecules. Point 1 shows the antibody bound after injecting only buffer. Trace 2 shows the D1.3 anti-lysozyme antibody [D1.3 IgG], after injecting B7-1-Fc at point 1. At point 2, B7-1-Fc was replaced with buffer. Trace 3 is a bispecific D1.3 anti-lysozyme antibody [bispecific molecule-D1.3 IgG-VLD×2(LC)] with each VLD fused to the antibody CL chain. It shows that the captured bispecific molecule bound to B7-1-Fc injected at point 1. At point 2, B7-1-Fc was replaced with buffer. Trace 4 is a triplicate D1.3 anti-lysozyme antibody [triplicate molecule-D1.3 IgG-VLD×4(HCLC)] with each VLD fused to the antibody's CH and CL chains. It shows that the captured bispecific molecule bound to B7-1-Fc injected at point 1. At point 2, B7-1-Fc was replaced with buffer.

[0383] Example 4 Molecular binding affinity to B7.1-Fc determined by surface plasmon resonance (SPR) The binding analysis in this section was performed using a Biacore X100SPR instrument (GE) with standard chemistry and reagents. Concentration series traces are displayed superimposed, and all data are presented as response units (RUs) after subtracting the response from a reference surface that did not contain lysozyme.

[0384] Figure 14 shows superimposed binding sensorgrams for each concentration series, determined by SPR analysis that the bispecific [IgG VLD×2(HC)] binds to biotin-labeled lysozyme captured with streptavidin, and then secondarily binds to a certain concentration series of B7-1-Fc (50, 25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). The bispecific molecule has each B7-1 binding VLD fused to both the heavy and light chains of the D1.3 antibody [D1.3 IgG], as illustrated in Figure 2. Injection of a bispecific molecule: This is the point where IgG VLD×2(HC) is added to the sensor surface. The trace shows the binding of IgG VLD×2(HC) to the lysozyme immobilized on the biosensor surface. Buffer injection 1: This is the point where the injection of IgG VLD×2(HC) is stopped and replaced with buffer injection. The trace shows the dissociation of IgG VLD×2(HC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: Points where the second analyte, B7-1-Fc, is added at specific concentrations (50, 25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). Traces show the binding of B7-1-Fc to IgG VLD×2(HC) that remains bound to lysozyme immobilized on the biosensor surface. Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with buffer injection. The trace shows the dissociation of B7-1-Fc from IgG VLD×2(HC) still bound to the lysozyme immobilized on the biosensor surface.

[0385] Figure 15 shows superimposed binding sensorgrams for each concentration series, determined by SPR analysis that the bispecific molecule [IgG VLD×2(LC)] binds to biotin-labeled lysozyme captured with streptavidin, and then secondarily binds to a certain concentration series of B7-1-Fc (50, 25, 12.5, 6.25, 3.125, 1.56, and 0 μug / ml). The bispecific molecule has each B7-1 binding VLD fused to the light chain of the D1.3 antibody [D1.3 IgG], as illustrated in Figure 3. Injection of bispecific molecules: This is the point where IgG VLD×2(LC) is added to the sensor surface. The trace shows the binding of IgG VLD×2(LC) to lysozyme immobilized on the biosensor surface. Buffer injection 1: The point at which the injection of IgG VLD×2(LC) is stopped and replaced with buffer injection. The trace shows the dissociation of IgG VLD×2(LC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: Points where the second analyte, B7-1-Fc, is added at specific concentrations (50, 25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). Traces show the binding of B7-1-Fc to IgG VLD×2(LC) still bound to lysozyme immobilized on the biosensor surface. Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with buffer injection. The trace shows the dissociation of B7-1-Fc from IgG VLD×2(LC) still bound to lysozyme immobilized on the biosensor surface.

[0386] Figure 16 shows the superimposed binding sensorgrams for each concentration series, determined by SPR analysis that the triple-specific [IgG VLD×4(HC+LC)] binds to biotin-labeled lysozyme captured with streptavidin, and then secondarily binds to B7-1-Fc at a certain concentration series (25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). The triple-specific molecule has each B7-1 binding VLD fused to both the heavy and light chains of the D1.3 antibody [D1.3 IgG], as illustrated in Figure 4. Injection of a triple-specific molecule: The point where IgG VLD×4(HC+LC) is added to the sensor surface. The trace shows the binding of IgG Im×4(HCLC) to lysozyme immobilized on the biosensor surface. Buffer injection 1: This is the point where the injection of IgG VLD×4(HC+LC) is stopped and replaced with buffer injection. The trace shows the dissociation of IgG VLD×4(HC+LC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: Points where the second analyte, B7-1-Fc, is added at specific concentrations (25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). Traces show the binding of B7-1-Fc to IgG VLD×4(HC+LC) still bound to lysozyme immobilized on the biosensor surface. Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with buffer injection. The trace shows the dissociation of B7-1-Fc from IgG VLD×4(HC+LC) still bound to the lysozyme immobilized on the biosensor surface.

[0387] Surface plasmon resonance (SPR) was used to characterize the binding properties of purified Fab, Fab-VLD bispecific and tripspecific molecules.

[0388] Figure 17 shows a superimposed sensorgram of a series of SPR bindings, demonstrating that the bispecific [Fab-VLD×1(HC)] first binds to biotin-labeled lysozyme captured with streptavidin, and then secondarily binds to a certain concentration series of B7-1-Fc (25, 12.5, 6.25, 3.125, 1.56, and 0 ug / ml). The bispecific molecule has a B7-1 bound VLD fused to a D1.3 Fab [D1.3 Fab] heavy chain, as illustrated in Figure 5. Injection of bispecific molecules: Points where Fab-VLD×1(HC) is added to the sensor surface. Traces show the binding of Fab-VLD×1(HC) to lysozyme immobilized on the biosensor surface. Buffer injection 1: The point at which the injection of Fab-VLD×1(HC) is stopped and replaced with buffer injection. The trace shows the dissociation of Fab-VLD×1(HC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: Points where the second analyte, B7-1-Fc, is added at specific concentrations (25, 12.5, 6.25, 3.125, 1.56, and 0 ug / ml). Traces show the binding of B7-1-Fc to Fab-VLD×1(HC) which remains bound to the lysozyme immobilized on the biosensor surface. Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with buffer injection. The trace shows the dissociation of B7-1-Fc from Fab-VLD×1(HC) which is still bound to the lysozyme immobilized on the biosensor surface.

[0389] Figure 18 shows a superimposed sensorgram of a series of SPR bindings, demonstrating that the bispecific [Fab-VLD×1(LC)] first binds to streptavidin-captured biotin-labeled lysozyme, followed by secondary binding to a certain concentration series of B7-1-Fc (25, 12.5, 6.25, 3.125, 1.56, and 0 ug / ml). The bispecific molecule has a B7-1 bound VLD fused to a D1.3 Fab[D1.3 Fab] light chain, as illustrated in Figure 6. Injection of bispecific molecules: Points where Fab-VLD×1(LC) is added to the sensor surface. Traces show the binding of Fab-VLD×1(LC) to lysozyme immobilized on the biosensor surface. Buffer injection 1: The point at which the injection of Fab-VLD×1(LC) is stopped and replaced with buffer injection. The trace shows the dissociation of Fab-VLD×1(LC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: Points where the second analyte, B7-1-Fc, is added at specific concentrations (25, 12.5, 6.25, 3.125, 1.56, and 0 ug / ml). Traces show the binding of B7-1-Fc to Fab-VLD×1(LC), which is still bound to the lysozyme immobilized on the biosensor surface. Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with buffer injection. The trace shows the dissociation of B7-1-Fc from Fab-VLD×1(LC) which is still bound to the lysozyme immobilized on the biosensor surface.

[0390] Figure 19 shows a superimposed sensorgram of a series of SPR bindings, demonstrating that the triple-specific [Fab-VLD×2(HC+LC)] first binds to biotin-labeled lysozyme captured with streptavidin, and then secondarily binds to a certain concentration series of B7-1-Fc (25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). The triple-specific molecule has each B7-1 bound VLD fused to both the heavy and light chains of D1.3 Fab [D1.3 Fab], as illustrated in Figure 7. Injection of a triple-specific molecule: Points where Fab-VLD×2(HC+LC) is added to the sensor surface. Traces show the binding of Fab-VLD×2(HC+LC) to lysozyme immobilized on the biosensor surface. Buffer injection 1: This is the point where the injection of Fab-VLD×2(HC+LC) is stopped and replaced with buffer injection. The trace shows the dissociation of Fab-VLD×2(HC+LC) from lysozyme immobilized on the biosensor surface. Injection of B7-1-Fc: Points where the second analyte, B7-1-Fc, is added at specific concentrations (25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml). Traces show the binding of B7-1-Fc to Fab-VLD×2(HC+LC) that remains bound to the lysozyme immobilized on the biosensor surface. Buffer injection 2: Point where the injection of B7-1-Fc is stopped and replaced with buffer injection. The trace shows the dissociation of B7-1-Fc from Fab-VLD×2(HC+LC) which is still bound to the lysozyme immobilized on the biosensor surface.

[0391] Example 5: Molecular bonding stoichiometry The binding stoichiometry of the bispecific molecule [IgG VLD×2(HC)] and the tripspecific molecule [IgG Im×4(HC+LC)] for B7-1-Fc was also determined by SPR (Figure 20). Kinetic analysis of IgG-VLD×2(HC), IgG-VLD×2(LC), and IgG-VLD×4(HCLC) was performed using a series of concentrations of B7-1-Fc (50, 25, 12.5, 6.25, 3.125, 1.56, and 0 μg / ml) as the analyte after capture on a biotin-lysozyme surface. Considering the molecular weight of the analyte and ligand, the theoretical maximum binding signal (Rmax) of the analyte was calculated based on the amount of captured IgG-VLD protein.

[0392] Assuming a 1:1 stoichiometry for all samples, the binding level of the analyte to the ligand was plotted as a percentage of the Rmax value for each concentration. As the analyte concentration increased to 25 μg / ml, the binding level approached the saturation or equilibrium level. The equilibrium binding level (Rmax: 103.9%) of the tetravalent protein IgG-VLD×4(HC+LC) was approximately twice that of the bivalent proteins IgG-VLD×2(HC) (56.7%) and IgG-VLD×2(LC) (51.4%). This suggests that IgG-VLD×4(HC+LC) can bind to B7.1-Fc simultaneously via the VLD domain fused to both the heavy and light chains.

[0393] Figure 21 shows the binding stoichiometry of the bispecific [Fab-VLD×1(HC)], [Fab-VLD×1(LC)], and [Fab-VLD×2(HC+LC)] to B7-1-Fc. The results suggest that Fab-VLD×2(HC+LC) can bind to B7.1 simultaneously via the VLD domain fused to both the heavy and light chains.

[0394] Example 6 Molecular binding affinity to B7.1-Fc The binding kinetics of the bispecificity and tripspecificity molecules of antibody-VLD and the bispecificity and tripspecificity molecules of Fab-VLD were determined by surface plasmon resonance. The association constant (Ka), dissociation constant (Kd), and equilibrium dissociation constant / binding constant K were determined. DThese are shown in Tables 3 and 4, respectively. Table 3 Affinity of antibody-VLD molecule against B7.1-Fc [Table 19]

[0395] The results demonstrate that the binding affinity of the triplicate molecule IgG VLD×4(HC+LC) to B7-1-Fc is comparable to that of the bispecific molecule IgG VLD×2(HC). Table 4 Affinity of Fab-VLD molecule for B7.1-Fc [Table 20]

[0396] The results demonstrate that the binding affinity of the triplicate molecule Fab VLD×4(HC+LC) to B7-1-Fc is comparable to that of the bispecific molecule Fab VLD×1(HC).

[0397] Example 7: Analysis of Triple Specific Binding The IgG VLD×4(HCLC) construct was modified by attaching a sclerostin (Scl)-conjugated VLD to the C-terminus of the heavy chain of the anti-lysozyme antibody D1.3, and a B7-1-conjugated VLD to the constant region (CL) of the antibody light chain of the anti-lysozyme antibody. This triple-specific molecule was named [IgG VLD×4(Scl-HC)(B7-LC)]. The sequence of the anti-lysozyme IgG single heavy chain fused with the anti-sclerostin VLD "1E1" is shown below (SEQ ID NO: 25):

[0398] [Table 21] The sequence of anti-sclerostin VLD is shown below: [Table 22] (Sequence ID 14) The VLD join loops in sequence numbers 25 and 14 are underlined.

[0399] To analyze the simultaneous triple binding of lysozyme, B7-1, and sclerostin, analysis was performed using a Biacore X100SPR instrument (GE). Antibody-Imunexin protein was injected at 35 μg / ml onto the surface of a streptavidin sensor chip (approximately 200 RU) that had previously captured biotinylated lysozyme in flow cell 2. After injection of the antibody-Imunexin protein, a stabilization period was allowed. The first analyte, B7.1-Fc, was injected at a concentration of 25 μg / ml. A control cycle was performed in which only buffer was injected instead of B7.1-Fc. Next, the second analyte, sclerostin (R&D Systems catalog number 1406-ST / CF), was rapidly injected at 15 μg / ml for 60 seconds, followed by a 120-second dissociation period. A control cycle was performed in which only buffer was injected instead of sclerostin.

[0400] Surface regeneration was performed by injecting 10 mM glycine buffer at pH 2.1 for 30 seconds.

[0401] All data are presented as response units (RUs) after subtracting the response from the reference surface (flow cell 1) that did not contain lysozyme.

[0402] (a) Analysis of triple-specific binding of full-length antibody constructs Figure 22 shows a superimposed sensorgram of a series of SPR bindings, demonstrating that the triple-specific [IgG VLD×4(Scl-HC)(B7-LC)] first binds to biotin-labeled lysozyme captured with streptavidin, and then co-binds to B7-1-Fc and sclerostin. The triple-specific molecule has a sclerostin-binding VLD fused to the heavy chain of the D1.3 antibody [D1.3 IgG] and a B7-1-binding VLD fused to the light chain.

[0403] The trace of B7-1-Fc alone is a sensorgram showing the case where the triple-specific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc. The sensorgram demonstrates dual-target simultaneous binding to both lysozyme and B7-1-Fc.

[0404] The trace of sclerostin alone is a sensorgram showing the case where the tripspecific molecule binds to lysozyme immobilized on the biosensor surface, followed by the addition of sclerostin. The sensorgram demonstrates dual target simultaneous binding to both lysozyme and sclerostin.

[0405] The traces of B7-1-Fc and sclerostin are sensorgrams showing the case where the triple-specific molecules bind to lysozyme immobilized on the biosensor surface, followed by the addition of B7-1-Fc, and then sclerostin. The sensorgrams demonstrate the simultaneous triple-target binding of lysozyme, B7-1-Fc, and sclerostin.

[0406] As shown in Figure 22, the binding of the molecule to B7-1 and sclerostin was investigated by SPR. The results show that the triple-specific molecule can simultaneously bind to both B7-1 and sclerostin.

[0407] (b) Analysis of triple-specific binding of Fab components The Fab VLD×2(HC+LC) construct was modified by attaching a B7-1-bound VLD to the C-terminus of the heavy chain of anti-lysozyme Fab D3.1, and a sclerostin (Scl)-bound VLD to the constant region (CL) of the Fab light chain of anti-lysozyme Fab. This triple-specific molecule was named [Fab VLD×2(B7-1-HC)(Scl-LC)]. A second construct was prepared by attaching a sclerostin-bound VLD to the C-terminus of the heavy chain of anti-lysozyme Fab D3.1, and a B7-1-bound VLD to the constant region (CL) of the Fab light chain of anti-lysozyme Fab. This triple-specific molecule was named [Fab VLD×2(Scl-HC)(B7-1-LC)].

[0408] Figure 23 shows the binding analysis of the triple-specific Fab VLD×2(B7-1-HC)(Scl-LC), and Figure 24 shows the binding analysis of the triple-specific Fab VLD×2(Scl-HC)(B7-1-LC). Binding analysis was performed using ForteBio Blitz. The binding traces show that the triple-specific molecules (Fab VLD×2(B7-HC)(Scl-LC)(Figure 23) and Fab VLD×2(Scl-HC)(B7-1-LC)(Figure 24)) first bind to biotin-labeled lysozyme captured with streptavidin, and then simultaneously bind to B7-1-Fc and sclerostin in sequence. The biosensor traces show that the triple-specific molecules first bind to lysozyme immobilized on the biosensor surface, and then bind to B7-1-Fc (B7-1-Fc was added at the specified points). The binding trace shows dual-target simultaneous binding to lysozyme and B7-1-Fc. Subsequently, sclerostin is added at the specified point, and the biosensor trace shows that both Fab VLD×2(B7-1-HC)(Scl-LC) and Fab VLD×2(Scl-HC)(B7-1-LC) molecules exhibit triple-target simultaneous binding to lysozyme, B7-1-Fc, and sclerostin. Finally, the sclerostin is replaced with a buffer, and the dissociation rate is shown.

[0409] Example 8: Preparation of human serum albumin-VLD fusion protein method Figure 25 shows a schematic diagram of a protein conjugated with a VLD according to an example of this disclosure. The bispecific molecule can conjugate to both target A and target B individually or simultaneously.

[0410] Vector creation A sequence encoding human serum albumin (HSA) fused with one or two VLDs was constructed, containing a 16-amino acid linker sequence (SGGGGSGGGGSGGGGS) (highlighted) and a C-terminal histidine tag. The sequence was cloned into a mammalian expression vector. The vector used was pcDNA3.4 vector (Thermo Fisher). The sequence was cloned using a signal peptide to induce protein secretion. The peptide sequence is MAWMMLLLGLAYGSG (SEQ ID NO: 8).

[0411] Bacterial transformation with vectors was performed using electroporation-competent cells (e.g., ElectroTen-Blue cells, Stratagene catalog number 200159) according to standard techniques.

[0412] After vector amplification, plasmid DNA was extracted and prepared using either the Qiagen HiSpeed ​​Plasmid Maxi Kit (catalog number 12663) or the Promega PureYield® Plasmid Midiprep System (catalog number A2492). The DNA was eluted in purified water that did not contain nucleases.

[0413] Transfection Using the Expi293 Expression System (ThermoFisher Scientific catalog number A14635), density 3 × 10 6 ~5×10 6Transfection of Expi293 mammalian cells with a cell / mL concentration and viability >95% was performed. DNA vectors were added to Opti-MEM I Reduced Serum Medium (Life Technologies catalog number 31985070) and ExpiFectamine 293 reagent (ThermoFisher catalog number A14525) and incubated at room temperature for 20 minutes. The transfection complex was added to the cells and incubated at 37°C, 5% CO2, and 120 rpm. After 20 hours, Expifectamine 293 Transfection Enhancer 1 and 2 (ThermoFisher catalog number A14525) were added to the cells. After approximately 4-5 days, the supernatant containing protein was collected.

[0414] purification Proteins were purified by affinity chromatography using Nickel Sepharose Excel (GE, catalog number 17-3712-01). The Nickel Sepharose column was equilibrated with 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4 at 5 times the column volume. Transfected cell culture supernatant containing His-tagged HSA-VLD protein was loaded onto the column, and the column was washed with 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4 at 10 times the column volume. The proteins were eluted using 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4. The eluted fractions were pooled and dialyzed with PBS according to standard procedures.

[0415] Figure 26 shows the Western blot analysis and detection of purified HSA fusion proteins using anti-His HRP (Sigma Aldrich, catalog number 11965085001), which contain VLDs fused to the C-terminus, N-terminus, or both the C-terminus and N-terminus of the HSA. The estimated sizes of the proteins are 67 kDa (predicted size: 81.9 kDa); 69.3 kDa (predicted size: 81.8 kDa); and 94.4 kDa (predicted size: 96.2 kDa).

[0416] Evaluation of the combination Using standard chemistry and reagents, the binding properties of the purified molecules were characterized using a ForteBio Blitz biosensor.

[0417] Molecules purified using affinity-purified commercially available B7.2-Fc protein (R&D Systems, catalog number 141-B2), CD3 (Acrobiosystems, catalog number CDD-H52W3), and biotinylated anti-HSA affibody (Abcam, catalog number 31898) were tested.

[0418] CD3 and B7.2Fc proteins were biotinylated in a 1:1 molar ratio using EZ-Link Sulfo-NHS-LC-Biotin (ThermoFisher, catalog number 21327) according to standard procedures.

[0419] A streptavidin-capturing surface (SA Sensor, ForteBio catalog number 18-5019) was used to capture biotin-labeled proteins. The HSA-VLD molecule was passed through the target captured on the biosensor surface, and a binding sensorgram was created.

[0420] Figure 27 shows analysis using the ForteBio Blitz biosensor, demonstrating the binding of the HSA-VLD molecule to biotin-labeled B7.2-Fc captured with streptavidin. Trace 1 is a sensorgram of the HSA-VLD fusion protein with B7-binding VLD bound to the C-terminus. Trace 2 is a sensorgram of the VLD-HSA-VLD fusion protein with B7-binding VLD bound to both the N-terminus and C-terminus. This shows the molecule binding to B7.2-Fc immobilized on the biosensor surface, followed by the addition of buffer at point 1.

[0421] Figure 28 shows an analysis using the ForteBio Blitz biosensor, demonstrating the binding of the HSA-VLD molecule to a biotin-labeled CD3 delta / epsilon(de) heterodimer captured with streptavidin. The illustrated trace is a sensorgram of the HSA-VLD fusion protein with a CD3-binding VLD attached to its C-terminus. The image shows the molecule binding to CD3de immobilized on the biosensor surface, followed by the addition of buffer at point 1.

[0422] Figure 29 shows an analysis using the ForteBio Blitz biosensor, demonstrating the binding of the HSA-VLD molecule to a biotin-labeled anti-HSA affibodi captured with streptavidin. The illustrated trace is a sensorgram of an HSA-VLD fusion protein with a B7-binding VLD attached to its C-terminus. Point 1 shows the molecule binding to an anti-HSA affibodi immobilized on the biosensor surface, followed by the addition of buffer. Point 2 shows the attachment of the B7.2Fc protein to the molecule captured on the sensor surface. Point 3 shows the replacement of the B7.2Fc with buffer. result (i) CTLA4 VLDs in which HSA is fused with human serum albumin at either the C-terminus, N-terminus, or both the C-terminus and N-terminus. The array is shown below: B7-2 linked VLD fused to the C-terminus of HSA (SEQ ID NO: 26): [Table 23] B7-1 bonded VLD fused to the N-terminus of HSA (SEQ ID NO: 27): [Table 24] B7-2 linked VLD (SEQ ID NO: 28) fused to both the N-terminus and C-terminus of HSA: [Table 25] (ii) A CD3-bound VLD (called AF3) fused to the C-terminus of human serum albumin. The array is shown below: CD3-bound VLD "AF3" (SEQ ID NO: 29) fused to the C-terminus of HSA: [Table 26]

Claims

1. A multispecific molecule capable of binding to two or more different target antigens or target epitopes, (i) A first target antigen or target epitope-binding domain molecule (BDM) comprising a V-like domain (VLD) scaffold having three exposed binding loops (BLs) contained therein, wherein at least two of the three BLs are modified or substituted with respect to their corresponding native sequences within the scaffold so as to selectively bind to a heterogeneous target antigen or target epitope, (ii) A pharmacologically active protein or peptide which is an antibody or its antigen-binding fragment or a non-antibody protein or peptide that binds to a second target antigen or target epitope. Includes, Herein, the molecule wherein at least one BDM is bound via its N-terminus to the C-terminus of a polypeptide present in the pharmacologically active protein or peptide.

2. (i) antibody heavy chain polypeptides, and / or antibody light chain polypeptides, and / or all antibody heavy chain polypeptides and antibody light chain polypeptides, (ii) Each polypeptide chain of a non-antibody protein or polypeptide The molecule according to claim 1, wherein at least one BDM is bound to the C-terminus.

3. The molecule according to claim 1 or 2, wherein the pharmacologically active protein is a full-length antibody.

4. The ratio of antibody chain to BDM is 4:2 ｎ The numerator according to claim 2, wherein n is a number from 1 to 5.

5. The aforementioned pharmacologically active proteins are Fab, Fab', and F(ab'). ２ or chemically bonded F(ab') ２ A molecule according to any one of claims 1 to 4, which is an antigen-binding fragment selected from the group consisting of the following.

6. The molecule according to any one of claims 1 to 5, wherein at least one BDM is bound to the C-terminus of the CH1 domain, CH2 domain, or CH3 domain of the heavy chain polypeptide.

7. (i) The first target antigen and the second target antigen are different, or (ii) The first target epitope and the second target epitope are located on the same antigen or different antigens. The molecule according to any one of claims 1 to 6.

8. The molecule according to any one of claims 1 to 7, wherein the molecule comprises one or two pairs of BDM, wherein the BDM in the pair are identical.

9. The aforementioned VLD scaffolding is Table 1 The molecule according to claim 1, comprising a framework sequence corresponding to residues 1-25, 34-54, 60-97, and 106-126 of Sequence ID No. 1 described above.

10. The aforementioned BDM is Table 2 (Sequence No. 5) Includes or consists of the sequence described above, Here, X is any amino acid residue, n1, n2, and n3 are numbers, and 1, 2, and 3 refer to BL1, BL2, and BL3, respectively. Xn １ It consists of 5 to 8 amino acids, Xn ２ It consists of 5 to 8 amino acids, Xn ３ The molecule according to any one of claims 1 to 9, wherein is 10 to 15 amino acids.

11. (i) Xn １ It has 8 amino acids. (ii) Xn ２ It is a 5-amino acid, and (iii) Xn ３ is 10 to 15 amino acids, The molecule according to claim 10.

12. The molecule according to claim 1, wherein the binding of the at least one BDM to the pharmacologically active protein or peptide is by linker means, direct fusion, or covalent bond.

13. The molecule according to claim 12, wherein the linker is a Gly-Ser peptide linker.

14. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 13, together with a pharmacologically acceptable carrier and / or additive.

15. The composition according to claim 14 for use in the manufacture of pharmaceuticals.

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