Immunoglobulin constructs having multiple binding domains

Abstract

Novel formats of multispecific molecules are described, as well as methods for their production. Further, uses in therapeutic indications are described.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 121,166, filed on 3 December 2020. The said application is incorporated herein by reference for any purpose herein.

[0002] This invention relates to the field of protein engineering. [Background technology]

[0003] Bispecific conjugating molecules have recently shown promising therapeutic potential. For example, bispecific molecules targeting both CD3 and CD19 in the form of a bispecific T cell engager (BiTE®) have shown remarkable efficacy at low doses. Bargou et al. (2008), Science 321:974~978. This BiTE® form contains two scFv' molecules, one targeting CD3 and the other targeting the tumor antigen CD19, linked by a mobile linker. This unique design allows the bispecific molecule to bring activated T cells closer to target cells, resulting in cytolytic death of the target cells. See, for example, International Publication No. 99 / 54440A1 (U.S. Patent No. 7,112,324B1) and International Publication No. 2005 / 040220 (U.S. Patent Application Publication No. 2013 / 0224205A1). Later, a bispecific construct was developed that binds to a context-independent epitope at the N-terminus of the CD3ε chain (see International Publication No. 2008 / 119567; U.S. Patent Application Publication No. 2016 / 0152707A1).

[0004] In certain therapeutic metrics, targeting more than two targets may be desirable. For example, in cancer immunotherapy, tumor escape is a known mechanism by which tumors lose the expression of targeted antigens through mutation and selective pressure of treatment. When this occurs, cancer immunotherapy loses its effectiveness against tumor cells. Adding additional antigenic targets related to the tumor is one way to address this type of tumor escape.

[0005] In addition to addressing tumor evasion, molecules containing multiple target binding sites may also be useful for targets expressed at relatively low levels by cells. In this type of scenario, perhaps due to their binding activity, multiple binding sites on a single molecule for the same target may help overcome this low expression and improve target binding. Several examples of molecules utilizing multiple binding sites are provided in U.S. Provisional Patent Application No. 63 / 110,957.

[0006] In the biopharmaceutical industry, molecules are generally fabricated on a large scale to meet the commercial demand for supplying many patients, and may be evaluated for numerous attributes to mitigate the fear that the molecules are unsuitable for mass production and purification. Efficiently expressing these complex recombinant polypeptides can be an ongoing challenge. Furthermore, even after expression, polypeptides are often not as stable as desired for pharmaceutical compositions. Various attempts have been made to address these difficulties by successfully altering the molecular form. See, for example, international patent applications PCT / US20 / 36464, "Bispecific Binding Constructs" and PCT / US20 / 36474, "Bispecific Binding Constructs with Selectively Cleavable Linkers". However, challenges remain for molecules with multiple binding sites. Therefore, there is a need in the art for therapeutic molecules with multiple binding sites that have a form that provides desirable pharmacokinetic properties, therapeutic efficacy, and improved production and stability. [Prior art documents]

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0009] A novel form of a binding molecule comprising a plurality of binding domains is described herein. In one embodiment, the present invention has the following structure: VH1-L1-VH2-L2-VL1-L3-VL2-L4-VH3-L1-VH4-L2-VL3-L3-VL4 or VH1-L1-VH2-L2-VL1-L3-VL2-L4-Half-life extension portion-L5-VH3-L1-VH4-L2-VL3-L3-VL4 The present invention provides a molecule comprising a polypeptide chain having (wherein VH1, VH2, VH3, and VH4 are immunoglobulin heavy chain variable regions, VL1, VL2, VH3, and VL4 are immunoglobulin light chain variable regions, and L1, L2, L3, L4, and L5 are linkers, where L1 is at least 10 amino acids, L2 is at least 15 amino acids, and L3 is at least 10 amino acids), the molecule being able to bind to immune effector cells and target cells.

[0010] In another embodiment, the present invention has the following structure: VH1-L1-VH2-L2-VL1-L3-VL2-L4-VH3-L1-VH4-L2-VL3-L3-VL4, or VH1-L1-VH2-L2-VL1-L3-VL2-L4-Half-life extension portion-L5-VH3-L1-VH4-L2-VL3-L3-VL4 The present invention provides a molecule comprising a polypeptide chain having (wherein VH1, VH2, VH3, and VH4 are immunoglobulin heavy chain variable regions, VL1, VL2, VL3, and VL4 are immunoglobulin light chain variable regions, and L1, L2, and L3 are linkers, where L1 has at least 10 amino acids, L2 has at least 10 amino acids, L3 has at least 10 amino acids, and the total amino acids of L1, L2, and L3 are at least 35 amino acids), the molecule being able to bind to immune effector cells and target cells.

[0011] The present invention further provides nucleic acids encoding molecules described herein, vectors containing these nucleic acids, and host cells containing these vectors.

[0012] In other embodiments, the present invention provides a method for producing the molecules described herein, comprising (1) culturing host cells under conditions for expressing the molecules, and (2) recovering the molecules from a cell aggregate or cell culture supernatant, wherein the host cells contain one or more nucleic acids encoding any of the molecules provided herein.

[0013] In other embodiments, the present invention provides a method for treating a cancer patient, comprising administering to the patient a therapeutically effective amount of the molecules provided herein.

[0014] In other embodiments, the present invention provides a method for treating a patient with an infectious disease, comprising administering to the patient a therapeutically effective dose of the molecule provided herein.

[0015] In further embodiments, the present invention provides a method for treating a patient having an autoimmune condition, an inflammatory condition, or a fibrotic condition, comprising administering to the patient a therapeutically effective dose of the molecules provided herein.

[0016] In another embodiment, the present invention further provides pharmaceutical compositions comprising molecules provided herein.

[0017] In another embodiment, the present invention provides the use of the molecules provided herein in the manufacture of agents for the prevention, treatment, or remission of disease. [Brief explanation of the drawing]

[0018] [Figure 1] This diagram shows a comparison between two different representative binding molecules—the structure of a representative (HLHL)2 molecule and the structure of a representative (HHLL)2 molecule. [Figure 2]This diagram shows two representative (HHLL) molecules with two different therapeutic targets and CD3-binding VH / VL domains, where both the VH2 / VL2 and VH4 / VL4 domains bind to CD3. Different representative linkers are represented by L# (e.g., L1, L2, L3, etc.), structure A shows a molecule with a linker as a spacer portion between the two (HHLL) components, and structure B shows a molecule with an optional scFc as a spacer portion. [Figure 3] This figure is a chromatographic reading showing the appropriate expression of two T6M(HHLL) molecules compared to two G7Q(HLHL) molecules. [Figure 4] This figure is an image of an SDS-PAGE analysis performed to assay purity and whether the molecule has a reasonable molecular weight, showing that the T6M molecule is expressed with a reasonable molecular weight. [Figure 5] This figure provides a graphical representation of the results of an in vitro TDCC assay, demonstrating the functionality and superior target cell death capabilities of T6M(HHLL)2 molecules compared to G7Q(HLHL)2 molecules at 48 hours. [Figure 6] This figure provides a graphical representation of the results of an in vitro TDCC assay, demonstrating the functionality and superior target cell death capabilities of T6M(HHLL)2 molecules compared to G7Q(HLHL)2 molecules at 72 hours. [Modes for carrying out the invention]

[0019] Novel forms of molecules having four different binding domains are described herein. These molecules comprise a single-chain polypeptide containing four immunoglobulin variable heavy (VH) domains, four immunoglobulin variable light (VL) domains, and optionally an Fc region (e.g., scFC), arranged in the following order: VH-linker-VH-linker-VL-linker-VL-linker-VH-linker-VH-linker-VL-linker-VL or VH-linker-VH-linker-VL-linker-VL-linker-scFc-VH-linker-VH-linker-VL-linker-VL (hereafter referred to herein, along with the representative form shown in Figure 2 herein, as "(HHLL)"). 2 (Also known as "squared format").

[0020] This (HHLL) 2 Depending on the format, for example (HLHL) 2 Compared to the original form, this method offers both improved stability and increased in vitro expression while maintaining the intended function of binding to desired targets on immune effector cells and target cells. Therefore, this (HHLL) 2 Depending on the format, molecules that can be produced more efficiently and have better stability and characteristics required in pharmaceutical compositions are provided.

[0021] The present invention provides a molecule having four unique binding domains, the molecule comprising at least one polypeptide, (HHLL) 2The molecule is characterized by comprising at least five characteristic structures that form together, namely (i) a first domain binding containing VH and VL, (ii) a second binding domain containing VH and VL, (iii) a spacer that links but also leaves a sufficient gap between the first (HHLL) domain and the second (HHLL) domain, (iv) a third binding domain, and (v) a fourth binding domain. Preferably, each of these domains is composed of a mobile peptide linker, as shown in Figure 2 herein, in which VH is linked to VH, then to VL, and then to the VL domain in the amino-to-carboxyl direction. In certain embodiments of the present invention, the first binding domain binds to an extracellular target other than CD3 (e.g., tumor-associated antigen, "TAA"), the second binding domain binds to extracellular epitopes of human and non-human (e.g., macaque) CD3ε chains, the third binding domain binds to the same or different extracellular target other than CD3 to which the first binding domain binds, and the fourth binding domain binds to extracellular epitopes of human and non-human (e.g., macaque) CD3ε chains.

[0022] Please understand that the above summary and the following detailed description are merely illustrative and descriptive, and do not limit the claimed invention. In this application, the use of the singular form includes the plural form unless otherwise specified. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "contains" is not limiting, as is the use of other forms such as "contains" and "includes". Also, terms such as "element" or "component" include both elements and components containing one unit and elements and components containing two or more subunits unless otherwise specified. Also, the use of the term "part" may include a part of a part or the whole of a part.

[0023] In this specification, unless otherwise defined, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless the context requires a different interpretation, singular terms shall include plural forms, and plural terms shall include singular forms. In general, nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids described herein are well known and commonly used in the art. The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art and described in various general and more specific references.

[0024] Polynucleotide and polypeptide sequences are indicated using standard one- or three-letter abbreviations. Unless otherwise specified, polypeptide sequences have an amino terminus on their left and a carboxyl terminus on their right, and the upper strand of single-stranded and double-stranded nucleic acid sequences has a 5' terminus on its left and a 3' terminus on its right. Specific sites in a polypeptide may be designated by amino acid residue numbers, such as amino acids 1-50, or by actual residues at sites such as asparagine to proline. A particular polypeptide or polynucleotide sequence may also be described by explaining how it differs from a reference sequence.

[0025] definition The term “isolated” with respect to a molecule (in this case, a molecule is, for example, a polypeptide, polynucleotide, polyspecific molecule, bispecific molecule, or antibody) is a molecule that, by its origin or source of origin, (1) does not associate with its naturally associated components in its natural state, (2) substantially does not contain other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cells in which it occurs naturally is “isolated” from its naturally associated components. A molecule can also be made substantially free of its naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assessed by many means well known in the art. For example, the purity of a polypeptide sample can be assayed by using polyacrylamide gel electrophoresis, staining the gel using techniques well known in the art, and visualizing the polypeptide. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art.

[0026] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and nucleotide analogs not found in nature), and hybrids thereof. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule of the present invention includes a continuous open reading frame encoding the binding molecule or fragment, derivative, mutain, or variant thereof of the present invention.

[0027] A "vector" is a nucleic acid that can be used to introduce another nucleic acid, to which it is ligated, into a cell. A "plasmid," a type of vector, refers to a linear or circular double-stranded DNA molecule to which further nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses) that can introduce further DNA segments into the viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0028] When a regulatory element affects the expression of a nucleotide sequence (e.g., the level, timing, or position of expression), the nucleotide sequence is "operably ligated" to the regulatory element. A "regulatory element" is a nucleic acid that affects the expression of the nucleic acid to which it is operably ligated (e.g., the level, timing, or position of expression). The regulatory element may exert its effect on the nucleic acid being regulated, for example, directly or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory element and / or nucleic acid). Examples of regulatory elements include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals).

[0029] A “host cell” is a cell that can be used to express nucleic acids, for example, the nucleic acids of the present invention. A host cell may be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells), or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transmigrated with a nucleic acid encoding a polypeptide, and subsequently express the nucleic acid in that host cell. The term “recombinant host cell” may be used to describe a host cell that has been transformed or transmigrated with the nucleic acid to be expressed. A host cell may also be a cell that contains nucleic acid but does not express the nucleic acid at the desired level unless a regulatory sequence is introduced into the host cell so that it becomes operablely linked to the nucleic acid. It is understood that the term host cell refers not only to the cell of a particular target, but also to the progeny or potential progeny of such a cell. For example, certain modifications may occur in later generations due to mutation or environmental influences, and such progeny may not be virtually identical to the parent cells, but they are still included within the scope of the terminology used herein.

[0030] A "single-chain variable fragment" ("scFv") is a fusion protein in which a VL region and a VH region are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, the linker being long enough to allow the protein chain to refold on its own to form a monovalent antigen-binding site or binding domain (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). In relation to other further parts (e.g., an Fc region), the scFv may be arranged, for example, VH-linker-VL or VL-linker-VH.

[0031] The term "CDR" refers to the complementarity-determining region (also called the "minimum recognition unit" or "hypervariable region") within an antibody variable sequence, and the molecule of the present invention includes heavy chain and / or light chain CDRs. These CDRs enable the binding molecule to specifically bind to a particular antigen of interest. Three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3) exist. Each CDR of the two chains is typically aligned by a framework region to form a structure that specifically binds to a particular epitope or domain on the target protein. From the N-terminus to the C-terminus, both the native light chain and heavy chain variable regions typically follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying each position in these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or in Chothia & Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883. This system can be used to identify the complementarity-determining region (CDR) and framework region (FR) of a given antibody. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (international ImMunoGeneTics information system; Lefranc et al., Dev.Comp.Immunol.29:185-203; 2005) and AHo (Honegger and Pluckthun, J.Mol.Biol.309(3):657-670; 2001). One or more CDRs can be incorporated into a molecule either covalently or noncovalently to form a binding molecule.

[0032] The "binding domain" of the binding molecule according to the present invention may include, for example, the CDRs of the group mentioned above. Preferably, these CDRs are included in the framework of the antibody light chain variable region (VL) and antibody heavy chain variable region (VH) contained by the molecule of the present invention, or, in the terminology used herein, the "L" and "H" variable regions (e.g., "HHLL").

[0033] The term "human antibody" includes antibodies having antibody regions such as variable and constant regions or domains that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) (cited above). Human antibodies as referred to herein may include amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, for example, by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo) in, for example, CDR, particularly CDR3. Human antibodies may have at least one, two, three, four, five or more positions replaced by amino acid residues not encoded by human germline immunoglobulin sequences. As used herein, the definition of a human antibody also includes fully human antibodies that consist only of human sequences of non-artificial and / or genetically modified antibodies, such as those that can be induced by using, for example, phage display technology or transgenic mouse technology, including, but not limited to, Xenomouse®. In connection with the present invention, variable regions derived from human antibodies can be used in the intended molecular form.

[0034] Humanized antibodies have a sequence that differs from that of non-human antibodies due to one or more amino acid substitutions, deletions, and / or additions. Therefore, when administered to human subjects, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response compared to non-human antibodies. In one embodiment, humanized antibodies are generated by mutating certain amino acids within the framework and the constant domains of the heavy and / or light chains of a non-human antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain from a non-human antibody. In yet another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are modified to increase the likelihood of reducing the immunogenicity of the non-human antibody when administered to a human subject, but none of these modified amino acid residues are important for the immunospecific binding of the antibody or binding molecule to the antigen, or the changes made to the amino acid sequence are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly worse than that of the non-human antibody to the antigen. Examples of methods for producing humanized antibodies can be found in U.S. Patent No. 6,054,297, No. 5,886,152, and No. 5,877,293. In connection with the present invention, the variable region derived from the humanized antibody may be used in the intended molecular form.

[0035] The term "chimeric antibody" refers to an antibody that contains one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. In one embodiment, one or more CDRs are derived from human antibodies. In another embodiment, all CDRs are derived from human antibodies. In yet another embodiment, CDRs derived from multiple human antibodies are mixed and fitted into the chimeric antibody. For example, a chimeric antibody may include CDR1 derived from the light chain of a first human antibody, CDR2 and CDR3 derived from the light chain of a second human antibody, and CDR derived from the heavy chain of a third antibody. Furthermore, the framework regions may come from one of the same antibodies, from one or more different antibodies such as human antibodies, or from humanized antibodies. In one example of a chimeric antibody, the heavy chain and / or part of the light chain are identical, homologous, or derived from antibodies from a particular species or belonging to a particular antibody class or antibody subclass, while the remainder of the chain is identical, homologous, or derived from antibodies from a different species or belonging to a different antibody class or antibody subclass. Furthermore, fragments of such antibodies exhibiting the desired biological activity are also included. In connection with the present invention, the variable region derived from the chimeric antibody may be used in the intended molecular form.

[0036] This invention (HHLL) 2 In relation to molecules, the "spacer" domain is (HHLL) 2 It is located between each of the two (HHLL) subunits that contain the molecule together. In some embodiments, the spacer is a half-life extension portion. In other embodiments, this spacer is a polypeptide linker.

[0037] For further examples of spacer domains, see U.S. Provisional Patent Application No. 63 / 110,957. In certain embodiments of the present invention, the molecular weight of the spacer domain is about 3.2 kDa, preferably greater than 10 kDa, more preferably at least 15 kDa, 20 kDa, or even more preferably 50 kDa, and / or the spacer domain contains an amino acid sequence comprising at least 50 amino acids, preferably 75 amino acids, more preferably at least 150 amino acids, and even more preferably at least 500 amino acids.

[0038] In other embodiments, the spacer domain that sufficiently separates the first and second (HHLL) domains is a programmed cell death protein 1 (PD1) domain, human serum albumin (HSA) or derivatives thereof, a multimer of a rigid linker, for example, (EAAAK) 10 and selected from the group consisting of a hinge, CH2 and CH3 domains, and an Fc domain containing two polypeptide monomers, each containing a hinge and further CH2 and CH3 domains, wherein the two polypeptide monomers are fused to each other via a peptide linker, or the two polypeptide monomers are linked together by non-covalent CH3-CDH3 interactions and / or covalent disulfide bonds to form a heterodimer.

[0039] In certain embodiments, the spacer entity is at least one domain, preferably one or two covalently bonded domains, which are arranged in the order of amino to carboxyl: Includes hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0040] In certain embodiments, the CH2 domain in the spacer may also include intradomain cysteine ​​disulfide bridges.

[0041] According to the present invention, in certain embodiments, the two bispecific entities must be spaced apart by a certain distance, preferably more than 35 Å, more preferably at least 40, 50, 60, 70, 80, 90 or at least 100 Å. This distance can be readily determined by crystallography, cryo-electron microscopy or nuclear magnetic resonance analysis techniques. This distance is driven by a spacer entity between the two (HHLL) domains that spaces apart the two domains, maintains them in the desired higher-order structure, and prevents unwanted interactions between the two separated (HHLL) domains. Generally, the more rigid the linker, the smaller the required minimum distance between the two (HHLL) domains.

[0042] The present compositions and the arrangement of these amino acids preferably confer a certain degree of rigidity and are not characterized by high mobility. In this regard, an amino acid spacer rich in proline and less in serine and glycine is preferred. Particularly envisioned are spacers that are, for example, two-dimensional (e.g., helical structures) or three-dimensional, folded polypeptides that form, for example, three-dimensional domains and subsequently ensure a certain degree of rigidity by their construction, preferably conferring further advantageous effects such as an extended in vivo half-life of a multi-target bispecific molecule as a therapeutic agent. In the context of the present invention, a spacer comprising an Fc domain or a part thereof is envisioned.

[0043] (HHLL) 2 In certain embodiments where the spacer is a half-life extension moiety, the half-life extension moiety is an Fc polypeptide chain. In other embodiments, the half-life extension moiety is a single-chain Fc (see, for example, SEQ ID NOs: 45 - 53). In yet other embodiments, the half-life extension moiety is a hetero-Fc (see, for example, SEQ ID NOs: 55 and 56). In yet other embodiments, the half-life extension moiety is human albumin or human serum albumin (see, for example, SEQ ID NO: 57). In other embodiments, the half-life extension moiety is an albumin-binding domain. Further specific examples and sequences of half-life extension moieties and spacers are provided in U.S. Provisional Patent Application No. 63 / 110,957 (see, for example, Table 17).

[0044] Linker A peptide linker exists between the immunoglobulin variable regions, and this peptide linker may be the same linker or different linkers of different lengths. In further embodiments, (HHLL) 2 The molecule further includes spacer regions between (HHLL) domains, which in certain embodiments are linkers. Linkers can play a crucial role in the structure of the binding molecule, and the present invention as described herein provides not only appropriate linker sequences but also appropriate linker lengths for each position in the binding molecule of the present invention. If the linker is too short, appropriate variable regions on the single-chain polypeptide cannot obtain sufficient mobility to interact to form an antigen-binding site (or "binding domain"). If the linker is of appropriate length, the variable regions can interact with other variable regions on the same polypeptide chain to form an antigen-binding site. In certain embodiments, the HHLL form includes disulfide bonds both within the domain (within H1, L1) and between the domains (between H1 and L1). In certain embodiments, specific linkers are used between various immunoglobulin regions to achieve appropriate expression and conformation of the molecule of the present invention (see, for example, Figure 1 herein). Exemplary linkers are provided in Table 1 herein. In certain embodiments, extending the linker length may result in the undesirable property of increased protein clipping. Therefore, it is desirable to strike an appropriate balance between linker lengths so as not to lead to increased clipping, while enabling appropriate polypeptide structure and activity.

[0045] As used herein, "linker" refers to a peptide that links two polypeptides. In certain embodiments, a linker may link multiple immunoglobulin variable regions in relation to the molecule. A linker may be 2 to 30 amino acid long. In some embodiments, a linker may be 2 to 25, 2 to 20, or 3 to 18 amino acid long. In some embodiments, a linker may be a peptide with 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 or fewer amino acid long. In other embodiments, a linker may be 5 to 25, 5 to 15, 4 to 11, 10 to 20, or 20 to 30 amino acid long. In other embodiments, the linker may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid lengths. Typical linkers include, for example, the amino acid sequences GGGGS (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2), GGGGSGGGGSGGGGS (SEQ ID NO: 3), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4), GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5), GGGGQ (SEQ ID NO: 6), GGGGQGGGGQ (SEQ ID NO: 7), GGGGQGGGGQGGGGQ (SEQ ID NO: 8), GGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 9), GGGGQGGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 10), GGGGSAAA (SEQ ID NO: 11), TVAAP (SEQ ID NO: 12), ASTKGP (SEQ ID NO: 13), AAA (SEQ ID NO: 14), SGGGGS (SEQ ID NO: 17), and SGGGGQ (SEQ ID NO: 18), and in particular include repeats of the above-mentioned amino acid sequences or subunits of amino acid sequences (for example, repeats of GGGGS (SEQ ID NO: 1) or GGGGQ (SEQ ID NO: 6)).

[0046] (HHLL) of the present invention 2In certain embodiments relating to the molecule, the linker sequence of linker 1 consists of at least 10 amino acids. In other embodiments, linker 1 consists of at least 15 amino acids. In other embodiments, linker 1 consists of at least 20 amino acids. In other embodiments, linker 1 consists of at least 25 amino acids. In other embodiments, linker 1 consists of at least 30 amino acids. In other embodiments, linker 1 consists of 10 to 30 amino acids. In other embodiments, linker 1 consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet another embodiment, linker 1 consists of more than 30 amino acids.

[0047] (HHLL) of the present invention 2 In certain embodiments in relation to the molecule, the linker sequence of linker 2 consists of at least 15 amino acids. In other embodiments, linker 2 consists of at least 20 amino acids. In other embodiments, linker 2 consists of at least 25 amino acids. In other embodiments, linker 2 consists of at least 30 amino acids. In other embodiments, linker 2 consists of 15 to 30 amino acids. In other embodiments, linker 2 consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet another embodiment, linker 2 consists of more than 30 amino acids.

[0048] (HHLL) of the present invention 2In certain embodiments in relation to the molecule, the linker sequence of linker 3 consists of at least 15 amino acids. In other embodiments, linker 3 consists of at least 20 amino acids. In other embodiments, linker 3 consists of at least 25 amino acids. In other embodiments, linker 3 consists of at least 30 amino acids. In other embodiments, linker 3 consists of 15 to 30 amino acids. In other embodiments, linker 3 consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet another embodiment, linker 3 consists of more than 30 amino acids.

[0049] The present invention (HHLL) is characterized by a molecule that does not have a spacer portion such as scFc, but instead contains only a linker at L4. 2 In certain embodiments in relation to the molecule, the L4 linker sequence consists of at least 5 amino acids. In preferred embodiments, the L4 linker sequence in this context is SGGGGS. In other embodiments, the L4 linker sequence in this context consists of at least 10 amino acids. In other embodiments in this context, linker 4 consists of at least 15 amino acids. In other embodiments in this context, linker 4 consists of at least 20 amino acids. In other embodiments in this context, linker 4 consists of at least 25 amino acids. In other embodiments in this context, linker 4 consists of at least 30 amino acids. In other embodiments in this context, linker 4 consists of 5 to 30 amino acids. In other embodiments in this context, linker 4 consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet another embodiment in this context, linker 4 consists of more than 30 amino acids.

[0050] The molecule contains a spacer portion such as scFc, according to the present invention (HHLL) 2In certain embodiments in relation to the molecule, the linker sequence of linker 4 is at least 5 amino acids. In other embodiments in this context, linker 4 is at least 10 amino acids. In other embodiments in this context, linker 4 is at least 15 amino acids. In certain embodiments, linker 4 in this context is (GGGGS)3. In other embodiments in this context, linker 4 is at least 20 amino acids. In other embodiments in this context, linker 4 is at least 25 amino acids. In other embodiments in this context, linker 4 is at least 30 amino acids. In other embodiments in this context, linker 4 is 5 to 30 amino acids. In other embodiments in this context, linker 4 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet another embodiment in this context, linker 4 consists of more than 30 amino acids.

[0051] The present invention (HHLL) includes a spacer portion such as scFc in the molecule. 2 In certain embodiments in relation to the molecule, the L5 linker sequence consists of at least 5 amino acids. In other embodiments in this context, linker 5 consists of at least 10 amino acids. In other embodiments in this context, linker 5 consists of at least 15 amino acids. In certain embodiments, linker 5 in this context is (GGGGS)3. In other embodiments in this context, linker 5 consists of at least 20 amino acids. In other embodiments in this context, linker 5 consists of at least 25 amino acids. In other embodiments in this context, linker 5 consists of at least 30 amino acids. In other embodiments in this context, linker 5 consists of 5 to 30 amino acids. In other embodiments in this context, linker 5 consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet another embodiment in this context, linker 5 is more than 30 amino acids.

[0052] (HHLL) of the present invention 2 For further guidance on linker length in relation to each HHLL subunit within the molecule, see Figures 4A–4D of the brochure for international patent application PCT / US20 / 36464, titled "Bispecific Binding Constructs," which shows molecular models of the HHLL molecule in various orientations and how linker lengths of specific lengths are required to enable the HHLL molecule to adopt the correct higher-order structure and for both HL-binding domains to function. A, B, and C represent the distance between the C-alpha atom of the terminal residue of one domain and the C-alpha atom of the initiation residue of the other domain. An experienced person can use this information to model the intended HHLL molecule and thus the HHLL molecule and therefore (HHLL) 2 The linker length can be adjusted to suit the specific HL-binding domain required for the molecule to express and function as desired.

[0053] The present invention includes a spacer such as scFc between two (HHLL) subunits. 2 In certain embodiments in relation to molecules, a sample of representative linker arrangements and positions is shown in Table 1, where the linker positions correspond to those shown in Figures 1B and 2B. In these particular embodiments, linkers 1, 2, and 3 are (GGGGS)4, and linkers 4 and 5 are (GGGGS)3. In other embodiments, the linker between two scFc subunits is preferably (GGGGS)6, although no numbered linker designation is given.

[0054] [Table 1]

[0055] amino acid sequence of the binding region In the exemplary embodiments described herein, the molecule maintains desired binding affinity to various desired targets, which is assumed to be enabled by an appropriate three-dimensional structure. The immunoglobulin variable region comprises VH and VL domains, which associate to form a variable domain that binds to the desired target.

[0056] Variable domains can be obtained from any immunoglobulin having desired properties, and methods for achieving this are further described herein. In one embodiment, VH1 and VL1 associate and bind to CD3ε, VH3 and VL3 associate and bind to CD3ε, VH2 and VL2 associate and bind to different targets, e.g., TAA, and VH4 and VL4 associate and bind to different targets, e.g., the same or different TAA.

[0057] In another embodiment, VH2 and VL2 associate and bind to CD3ε, VH4 and VL4 associate and bind to CD3ε, VH1 and VL1 associate and bind to different targets, and VH3 and VL3 associate and bind to different targets.

[0058] In certain embodiments, VH1 and VL1 associate with and bind to mesothelin, VH2 and VL2 associate with and bind to CD3ε, VH3 and VL3 associate with and bind to CDH3, and VH4 and VL4 associate with and bind to CD3ε.

[0059] In further specific embodiments, VH1 and VL1 associate and bind to CDH3, VH2 and VL2 associate and bind to CD3ε, VH3 and VL3 associate and bind to mesothelin, and VH4 and VL4 associate and bind to CD3ε.

[0060] In another specific embodiment, VH1 and VL1 associate and bind to CD3ε, VH2 and VL2 associate and bind to mesothelin, VH3 and VL3 associate and bind to CD3ε, and VH4 and VL4 associate and bind to CDH3.

[0061] In another specific embodiment, VH1 and VL1 associate and bind to CD3ε, VH2 and VL2 associate and bind to CDH3, VH3 and VL3 associate and bind to CD3ε, and VH4 and VL4 associate and bind to mesotheline.

[0062] In certain embodiments, VH1 (SEQ ID NO: 39) and VL1 (SEQ ID NO: 40) associate with and bind to mesothelin, VH2 (SEQ ID NO: 41) and VL2 (SEQ ID NO: 42) associate with and bind to CD3ε, VH3 (SEQ ID NO: 43) and VL3 (SEQ ID NO: 44) associate with and bind to CDH3, and VH4 (SEQ ID NO: 41) and VL4 (SEQ ID NO: 42) associate with and bind to CD3ε.

[0063] In further specific embodiments, VH1 (SEQ ID NO: 43) and VL1 (SEQ ID NO: 44) associate with and bind to CDH3, VH2 (SEQ ID NO: 41) and VL2 (SEQ ID NO: 42) associate with and bind to CD3ε, VH3 (SEQ ID NO: 39) and VL3 (SEQ ID NO: 40) associate with and bind to mesotheline, and VH4 (SEQ ID NO: 41) and VL4 (SEQ ID NO: 42) associate with and bind to CD3ε.

[0064] In another specific embodiment, VH1 (SEQ ID NO: 41) and VL1 (SEQ ID NO: 42) associate with and bind to CD3ε, VH2 (SEQ ID NO: 39) and VL2 (SEQ ID NO: 40) associate with and bind to mesotheline, VH3 (SEQ ID NO: 41) and VL3 (SEQ ID NO: 42) associate with and bind to CD3ε, and VH4 (SEQ ID NO: 43) and VL4 (SEQ ID NO: 44) associate with and bind to CDH3.

[0065] In another specific embodiment, VH1 (SEQ ID NO: 41) and VL1 (SEQ ID NO: 42) associate with and bind to CD3ε, VH2 (SEQ ID NO: 43) and VL2 (SEQ ID NO: 44) associate with and bind to CDH3, VH3 (SEQ ID NO: 41) and VL3 (SEQ ID NO: 42) associate with and bind to CD3ε, and VH4 (SEQ ID NO: 39) and VL4 (SEQ ID NO: 40) associate with and bind to mesotheline.

[0066] In further specific embodiments, representative (HHLL) 2 The molecular amino acid sequence is shown in SEQ ID NO: 37.

[0067] Further specific examples of sequences that may be incorporated in the molecular binding domain that binds to CD3ε are provided herein in Sequence IDs 58-97, including the designated VH, VL, and CDR.

[0068] In another embodiment, the light chain variable domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain variable domain sequence described herein.

[0069] In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence described herein. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes to a complement of the polynucleotide encoding the light chain variable domain selected from the sequences described herein under moderately stringent conditions. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes to a complement of the polynucleotide encoding the light chain variable domain selected from the group consisting of the sequences described herein under stringent conditions.

[0070] In another embodiment, the heavy chain variable domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the heavy chain variable domain selected from the sequences described herein. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence encoding the heavy chain variable domain selected from the sequences described herein. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes to the complement of the polynucleotide encoding the heavy chain variable domain selected from the sequences described herein under moderately stringent conditions. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes to the complement of the polynucleotide encoding the heavy chain variable domain selected from the sequences described herein under stringent conditions.

[0071] replacement It will be recognized that the molecules of the present invention may have at least one amino acid substitution, provided that the molecule retains the same or better desired binding specificity (e.g., binding to CD3). Therefore, modifications to the structure of the binding molecule are included within the scope of the present invention. In one embodiment, the binding molecule comprises sequences independently different by the addition, substitution, and / or deletion of 5, 4, 3, 2, 1, or 0 single amino acids from the CDR sequence described herein. As used herein, a CDR sequence different by the addition, substitution, and / or deletion of, for example, 4 or fewer amino acids in total from the CDR sequence described herein refers to a sequence having the addition, substitution, and / or deletion of 4, 3, 2, 1, or 0 single amino acids compared to the sequence described herein. These may include amino acid substitutions that are conserved or non-conservative, as long as they do not impair the desired binding ability of the binding molecule. Conservative amino acid substitutions may include non-natural amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimes and other forms in which the amino acid moieties are reversed or inverted. Conservative amino acid substitutions may also include substitutions of standard native amino acid residues that have little or no effect on the polarity or charge of the amino acid residue at that position.

[0072] Non-conservative substitutions may also include the exchange of a member of one class of amino acids or amino acid mimeographs with a member from another class that has different physical properties (e.g., size, polarity, hydrophobicity, charge). In certain embodiments, such substituted residues may be introduced into a region of a human antibody homologous to a non-human antibody, or into a non-homologous region of the molecule, which can then be used to generate the conjugation molecule of the present invention.

[0073] Furthermore, those skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art. Using such variants, information about suitable variants can be gathered. For example, if a change is found at a particular amino acid residue that is disrupted, undergoes undesirable reduction, or results in inappropriate activity, variants with such changes can be avoided. In other words, those skilled in the art can easily determine, based on information gathered from such routine experiments, which amino acids should be avoided for further substitution, either alone or in combination with other mutations.

[0074] A skilled technician will be able to determine preferred variants of the binding molecule described herein using well-known techniques. In certain embodiments, a person skilled in the art can identify preferred regions of the molecule that can be modified without impairing activity by targeting regions that are not considered important to activity. In certain embodiments, as described above, it is also possible to identify residues and portions of molecules that are conserved within similar polypeptides. In certain embodiments, even regions that may be important to biological activity or structure can be conserved amino acid substitutions without impairing biological activity or having a detrimental effect on the polypeptide structure.

[0075] Furthermore, those skilled in the art may consider structural-functional studies to identify residues in similar polypeptides that are important for activity or structure. By considering such comparisons, it may be possible to predict the importance of amino acid residues in a protein corresponding to amino acid residues that are important for the activity or structure of similar proteins. Those skilled in the art may select chemically similar amino acid substitutions for amino acid residues that are predicted to be important.

[0076] In some embodiments, those skilled in the art can identify modifiable residues that, if necessary, result in improved properties. For example, amino acid substitutions (conservative or non-conservative) may enhance binding affinity to a desired target.

[0077] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence of similar polypeptide structures. By considering such information, they can predict the alignment of amino acid residues of an antibody relative to its three-dimensional structure. In certain embodiments, those skilled in the art can make selections that avoid fundamental changes to amino acid residues predicted to be present on the protein surface, as these residues may be involved in important interactions with other molecules. Numerous scientific publications have contributed to the prediction of secondary structures. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). Furthermore, computer programs can now be used to assist in the prediction of secondary structures. One method of predicting secondary structures is based on homology modeling. For example, two polypeptides or proteins with more than 30% sequence homology or more than 40% similarity often have similar structural topologies. The development of protein databases (PDBs) has improved the accuracy of predicting secondary structures, including the potential number of folds within polypeptide or protein structures. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999).Further methods for predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87(1997); Sippl et al., Structure, 4(1):15-19(1996)), "profile analysis" (Bowie et al., Science, 253:164-170(1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and "evolutionary linkage" (see Holm (1999) and Brenner (1997) cited above).

[0078] In certain embodiments, variants of the binding molecule include glycosylated variants, in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the variant contains more or fewer N-linked glycosylation sites than the native protein. Alternatively, existing N-linked glycans are removed by substitution that deletes this sequence. Furthermore, N-linked glycan rearrangement occurs, resulting in the deletion of one or more N-linked glycosylation sites (typically naturally occurring) and the creation of one or more new N-linked sites. Further variants include cysteine ​​variants, in which one or more cysteine ​​residues are deleted or substituted by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when an antibody or other polypeptide molecule needs to be refolded into a biologically active conformation, such as after isolation of an insoluble inclusion body. Cysteine ​​variants generally have fewer cysteine ​​residues than the native protein and typically have an even number of cysteine ​​residues to minimize interactions arising from unpaired cysteine.

[0079] The desired amino acid substitutions (whether conserved or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify key residues of the binding molecule to the target of interest, or to increase or decrease the affinity of the binding molecule to the target of interest described herein.

[0080] According to certain embodiments, the desired amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity, and / or (4) confer or modify other physiological or functional properties to such polypeptides. According to certain embodiments, one or more amino acid substitutions (conservative amino acid substitutions in certain embodiments) may be made in the natural sequence (in certain embodiments, the portion of the polypeptide outside the domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically do not substantially alter the structural features of the parent sequence (for example, the substituted amino acid should not tend to disrupt helices present in the parent sequence or other types of secondary structures that characterize the parent sequence). Examples of secondary and tertiary structures of polypeptides that are recognizable to those skilled in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991), which are incorporated herein by reference, respectively.

[0081] Half-life extension and Fc region In certain embodiments, it is desirable to extend the in vivo half-life of the molecule of the present invention. This can be achieved by including a half-life extension portion as part of the molecule. Non-limiting examples of half-life extension portions include Fc polypeptides, albumin, albumin fragments, portions that bind to albumin or neonatal Fc receptors (FcRn), derivatives of fibronectin modified to bind to albumin or its fragments, peptides, single-domain protein fragments, or other polypeptides that can extend the serum half-life. In alternative embodiments, the half-life extension portion may be a molecule other than a polypeptide, such as polyethylene glycol (PEG).

[0082] When used herein, the term "Fc polypeptide" includes the native and mutaine forms of polypeptides derived from the Fc region of an antibody. Truncate forms of such polypeptides containing a hinge region that promotes dimerization are also included. In addition to other properties described herein, polypeptides containing the Fc moiety offer the advantage of being purifiable, for example, by affinity chromatography using a protein A or protein G column.

[0083] In a particular embodiment, the half-life extension portion is the Fc region of the antibody. The Fc region is (HHLL) 2 It may be located at the N-terminus of the molecule, or (HHLL) 2 It may be located at the C-terminus of the molecule. (HHLL) 2A linker may, but is not required, be present between the molecule and the Fc region. As described above, an Fc polypeptide chain may contain all or part of a hinge region, followed by CH2 and CH3 regions. Fc polypeptide chains may originate from mammals (e.g., humans, mice, rats, rabbits, dromedaries, or New World or Old World monkeys), birds, or sharks. In addition, as described above, an Fc polypeptide chain may contain a limited number of modifications. For example, an Fc polypeptide chain may contain one or more heterodimerization modifications, one or more modifications that inhibit or enhance binding to FcγR, or one or more modifications that increase binding to FcRn.

[0084] In certain embodiments, the Fc used for half-life extension is single-chain Fc ("scFc").

[0085] In some embodiments, the amino acid sequence of the Fc polypeptide may be that of a mammal, such as a human. The isotype of the Fc polypeptide may be IgG, IgA, IgD, IgE, or IgM, such as IgG1, IgG2, IgG3, or IgG4. The amino acid sequence alignment of the Fc polypeptide chains of human IgG1, IgG2, IgG3, and IgG4 is shown in Table 2 below.

[0086] Sequences of human IgG1, IgG2, IgG3, and IgG4 Fc polypeptides that can be used are provided in SEQ ID NOs.45-48. Other closely related variants are also conceivable, including those containing one or more heterodimerization changes, one or more Fc changes that prolong the half-life, one or more changes that enhance ADCC, and / or one or more changes that inhibit Fcγ receptor (FcγR) binding, as well as deletions, insertions, or substitutions of 10 or fewer single amino acids per 100 amino acids of the sequence.

[0087] [Table 2]

[0088] The numbering shown in Table 2 follows the EU numbering system based on the sequential numbering of the constant region of the IgG1 antibody. Edelman et al. (1969), Proc. Natl. Acad. Sci. 63:78~85. Therefore, this numbering does not adequately correspond to the extra length of the hinge of IgG3. Nevertheless, this numbering is used herein to represent the position within the Fc region, as this numbering is still commonly used in the art to indicate a position within the Fc region. The hinge regions of the Fc polypeptides of IgG1, IgG2, and IgG4 extend from approximately position 216 to approximately position 230. It is evident from the alignment that the hinge regions of IgG2 and IgG4 are three amino acids shorter than the hinge of IgG1, respectively. The hinge of IgG3 is considerably longer, extending an additional 47 amino acids upstream. The CH2 region extends from approximately position 231 to 340, and the CH3 region extends from approximately position 341 to 447.

[0089] The naturally occurring amino acids of Fc polypeptides can be slightly altered. Such alterations may include insertions, deletions, or substitutions of no more than 10 single amino acids per 100 amino acids in the sequence of the natural Fc polypeptide chain. Where substitutions exist, these may be conservative amino acid substitutions as defined above. The Fc polypeptides on the first and second polypeptide chains may differ in their amino acid sequences. In some embodiments, these may include "heterodimerization alterations," such as charge pairing substitutions that promote heterodimerization as defined above. Furthermore, the Fc polypeptide moiety of PABP may also include alterations that inhibit or enhance FcγR binding. Such mutations are described above and in Xu et al. (2000), Cell Immunol. 200(1):16-26, the relevant parts of which are incorporated herein by reference. Furthermore, the Fc polypeptide portion may include “Fc mutations that extend the half-life,” as described above, including, for example, those described in U.S. Patent No. 7,037,784, U.S. Patent No. 7,670,600, and U.S. Patent No. 7,371,827, U.S. Patent Publication No. 2010 / 0234575, and International Patent Application PCT / US2012 / 070146, all relevant portions thereof are incorporated herein by reference. In addition, the Fc polypeptide may include “changes that enhance ADCC,” as defined above.

[0090] Another suitable Fc polypeptide described in the PCT application publication, International Publication No. 93 / 10151 (incorporated herein by reference), is a single-chain polypeptide extending from the N-terminal hinge region of the Fc region of a human IgG1 antibody to the native C-terminus. Another useful Fc polypeptide is Fc mutein, described in U.S. Patent No. 5,457,035 and Baum et al., 1994, EMBO J.13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in International Publication No. 93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. This mutein exhibits reduced affinity for the Fc receptor.

[0091] By introducing one or more mutations into Fc, the effector function of an antibody can be increased or decreased. Embodiments of the present invention include IL-2 mutein Fc fusion proteins having Fc modified to increase effector function (U.S. Patent No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both are incorporated herein by reference in their entirety). For certain therapeutic indicators, it may be desirable to increase effector function. For other therapeutic indicators, it may be desirable to decrease effector function.

[0092] Examples of IgG1 Fc molecules with enhanced effector function include those having the following substituents: S239D / I332E S239D / A330S / I332E S239D / A330L / I332E S298A / D333A / K334A P247I / A339D P247I / A339Q D280H / K290S D280H / K290S / S298D D280H / K290S / S298V F243L / R292P / Y300L F243L / R292P / Y300L / P396L F243L / R292P / Y300L / V305I / P396L G236A / S239D / I332E K326A / E333A K326W / E333S K290E / S298G / T299A K290N / S298G / T299A K290E / S298G / T299A / K326E K290N / S298G / T299A / K326E

[0093] Another method to enhance the effector function of IgG Fc-containing proteins involves reducing Fc fucosylation. By removing core fucose from the branched complex oligosaccharide bound to Fc, ADCC effector function is significantly enhanced without altering antigen-binding or CDC effector function. Several methods are known for reducing or eliminating fucosylation of Fc-containing molecules, such as antibodies. These methods include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, variant CHO cell line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNAs specifically targeting the FUT8 gene, and cell lines co-expressing α-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules can be expressed in plant cells, yeast, or prokaryotic cells (e.g., non-mammalian cells such as E. coli).

[0094] In certain embodiments of the present invention, the molecule comprises Fc modified to reduce its effector function. Examples of Fc molecules having reduced effector function include those having the following substituents: N297A or N297Q (IgG1) L234A / L235A (IgG1) V234A / G237A(IgG2) L235A / G237A / E318A(IgG4) H268Q / V309L / A330S / A331S(IgG2) C220S / C226S / C229S / P238S(IgG1) C226S / C229S / E233P / L234V / L235A(IgG1) L234F / L235E / P331S(IgG1) S267E / L328F(IgG1)

[0095] Human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation is known to contribute to the effector function of IgG1 antibodies. An exemplary IgG1 sequence is provided in SEQ ID NO: 45. N297 can be mutated to produce aglycosylated antibodies. For example, the mutation can replace N297 with an amino acid whose physiological and chemical properties are similar to asparagine, such as glutamine (N297Q), or with alanine (N297A), which mimics asparagine without polar groups.

[0096] In certain embodiments, mutating the amino acid N297 of human IgG1 to glycine, i.e., N297G, results in far superior purification efficiency and biophysical properties compared to other amino acid substitutions at that residue. See, for example, U.S. Patent No. 9,546,203 and U.S. Patent No. 10,093,711. In certain embodiments, the molecule of the present invention comprises the Fc of human IgG1 having the N297G substitution.

[0097] The molecules of the present invention, comprising human IgG1 Fc having the N297G mutation, may also include further insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fc comprises the N297G substitution and is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in SEQ ID NO: 45. In particularly preferred embodiments, the C-terminal lysine residue is substituted or deleted.

[0098] In certain cases, the Fc-containing molecule of aglycosylated IgG1 may be less stable than the Fc-containing molecule of glycosylated IgG1. Therefore, the stability of the aglycosylated molecule may be further modified. In some embodiments, one or more amino acids are substituted with cysteine ​​to form a disulfide bond in the dimeric state. In certain embodiments, residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence described in SEQ ID NO: 45 may be substituted with cysteine. In other embodiments, specific pairs of residues are substituted so as to preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond scrambling. In certain embodiments, examples of pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[0099] As discussed above in the linker section, the molecules of the present invention have various domains and (HHLL) 2 The molecule includes a linker between the constituent parts and, for example, as shown in Figure 2 herein. In certain embodiments, the linker, when expressed in suitable cells, is glycosylated, and such glycosylation can promote protein stabilization when administered in solution and / or in vivo. Thus, in certain embodiments, the molecule of the present invention is (HHLL) 2The polypeptide contains at least one glycosylated linker between its domains.

[0100] Nucleic acid encoding this molecule In another embodiment, the present invention provides isolated nucleic acid molecules encoding the molecules of the present invention. In addition, vectors containing nucleic acids, cells containing nucleic acids, and methods for producing the binding molecules of the present invention are provided. The nucleic acids include, for example, polynucleotides encoding all or part of a molecule, e.g., fragments, derivatives, mutaines, or variants thereof; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers for identifying, analyzing, mutagenicating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting the expression of polynucleotides; and complementary sequences. The nucleic acids may be of any length suitable for the desired use or function and may include one or more further sequences, e.g., regulatory sequences, and / or larger nucleic acids, e.g., parts of a vector. The nucleic acids may be single-stranded or double-stranded and may include RNA and / or DNA nucleotides and their artificial variants (e.g., peptide nucleic acids).

[0101] Nucleic acids encoding polypeptides (e.g., heavy or light chains, variable domains only or full length) can be isolated from B cells of mice immunized with an antigen. These nucleic acids can be isolated by conventional procedures such as polymerase chain reaction (PCR).

[0102] Nucleic acid sequences encoding variable regions of the heavy chain and light chain variable regions are included herein. A skilled technician will recognize that, due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by a number of other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each binding molecule of the present invention.

[0103] The present invention further provides nucleic acids that hybridize to other nucleic acids under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, NY 1989; 6.3.1-6.3.6. As defined herein, for example, moderately stringent hybridization conditions include a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide, a hybridization buffer of 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions at a hybridization temperature of 42°C, such as those containing about 50% formamide), as well as washing conditions at 60°C in 0.5x SSC and 0.1% SDS. Under stringent hybridization conditions, hybridization occurs in 6x SSC at 45°C, followed by one or more washes in 0.1x SSC and 0.2% SDS at 68°C. Furthermore, those skilled in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization so that nucleic acids containing nucleotide sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to each other typically remain hybridized.Basic parameters influencing the selection of hybridization conditions and guidance for devising preferred conditions are shown, for example, by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those skilled in the art, for example, based on the length and / or base composition of the DNA. Changes can be introduced by mutations in nucleic acids, thereby altering the amino acid sequence of the polypeptide (e.g., binding molecule) encoded by the nucleic acid. Mutations can be introduced using any technique known in the art. In one embodiment, for example, a site-directed mutagenesis protocol is used to change one or more specific amino acid residues. In another embodiment, for example, a random mutagenesis protocol is used to change one or more randomly selected residues. Regardless of the method used, mutant polypeptides can be expressed and their desired properties can be screened.

[0104] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues can be performed. In one embodiment, a nucleotide sequence of the binding molecule of the present invention provided herein, or a desired fragment, variant, or derivative thereof, is mutated to encode an amino acid sequence containing the deletion or substitution of one or more amino acid residues shown herein for the light chain or heavy chain of the binding molecule of the present invention, such that two or more sequences are different residues. In another embodiment, this mutagenesis results in the insertion of two or more amino acids adjacent to one or more amino acid residues shown herein for the light chain or heavy chain of the binding molecule of the present invention, such that two or more sequences are different residues. Alternatively, one or more mutations can be introduced into nucleic acids to selectively alter the biological activity of the polypeptide encoded by the nucleic acid.

[0105] In another embodiment, the present invention provides a vector comprising a nucleic acid encoding the polypeptide of the present invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0106] The recombinant expression vector of the present invention may contain the nucleic acid of the present invention in a form suitable for expressing the nucleic acid in a host cell. The recombinant expression vector includes one or more regulatory sequences selected based on the host cell used for expression, and such regulatory sequences are operably ligated to the nucleic acid sequence to be expressed. Regulatory sequences may include those that induce constitutive expression of a nucleotide sequence in many types of host cells (e.g., SV40 early gene enhancer, Rouss sarcoma virus promoter, and cytomegalovirus promoter), or those that induce expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences; the entirety of which is incorporated herein by reference: Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science). (See 236:1237) and those that induce inducible expression of nucleotide sequences in response to specific treatments or conditions (e.g., metallothionin promoters in mammalian cells and tet-responsive and / or streptomycin-responsive promoters in both prokaryotes and eukaryotes (see same literature)). Those skilled in the art will recognize that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. The expression vectors of the present invention can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.

[0107] In another embodiment, the present invention provides host cells into which the recombinant expression vector of the present invention has been introduced. The host cells may be any prokaryotic or eukaryotic cell. Examples of prokaryotic host cells include Gram-negative or Gram-positive microorganisms, such as E. coli or bacilli. Examples of higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or derivatives thereof such as Veggie CHO and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or DXB-11 of the CHO line lacking DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20). Further CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862). Further host cells include the monkey kidney cell culture COS-7 (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), AM-1 / D cells (described in U.S. Patent No. 6,210,924), HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J.10:2821), 293, 293 EBNA, or MSR. Examples include human embryonic kidney cells such as 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissue, primary grafts, HL-60 cells, U937 cells, HaK cells, or Jurkat cells.Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0108] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or gene transfer techniques. In the case of stable translocation of mammalian cells, it is known that, depending on the expression vector and gene transfer technique used, foreign DNA can be incorporated into the genome of only a small number of cells. To identify and select these integrations, genes encoding selectable markers (e.g., for antibiotic resistance) are generally introduced into host cells along with the target gene. Further selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells stably translocated with the introduced nucleic acid can be identified, among other methods, particularly by drug selection (e.g., cells incorporating the selectable marker gene survive while other cells die).

[0109] Transformed cells can be cultured under conditions that promote polypeptide expression, and the polypeptides can be recovered by conventional protein purification procedures. Polypeptides intended for use herein include substantially homogeneous recombinant mammalian polypeptides that are substantially free from the contamination of endogenous material.

[0110] Cells containing nucleic acids encoding the molecules of the present invention also include hybridomas. The generation and culture of hybridomas are described herein.

[0111] In some embodiments, vectors comprising nucleic acid molecules as described herein are provided. In some embodiments, the present invention comprises a host cell comprising nucleic acid molecules as described herein.

[0112] In some embodiments, nucleic acid molecules encoding molecules such as those described herein are provided.

[0113] In some embodiments, a pharmaceutical composition comprising at least one molecule described herein is provided.

[0114] Method of production The molecules of the present invention can be produced by any method known in the art for the synthesis of proteins (e.g., antibodies), particularly by chemical synthesis techniques, or preferably by recombinant expression techniques.

[0115] Recombinant expression of this molecule requires the construction of an expression vector containing the polynucleotide encoding this molecule. Once the polynucleotide encoding this molecule is obtained, a vector for producing this molecule can be constructed using recombinant DNA technology. An expression vector containing the coding sequence of this molecule and appropriate transcription and translation regulatory signals is then constructed. Examples of these methods include in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination.

[0116] An expression vector is introduced into host cells using conventional techniques, and then these gene-transferred cells are cultured using conventional techniques to generate the molecule of the present invention.

[0117] Various host expression vector systems can be used to express the molecules of the present invention. Such host expression systems mean not only vehicles that can generate and subsequently purify the coding sequence of interest, but also cells that can express the molecules of the present invention in situ when transformed or transfused with the appropriate nucleotide coding sequence. Bacterial cells such as E. coli, and eukaryotic cells, are commonly used for the expression of recombinant binding molecules, particularly the entire recombinant binding molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) combined with vectors such as the promoter element of a major intermediate early gene derived from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio / Technology 8:2 (1990)).

[0118] In addition, host cell lines that regulate the expression of the inserted sequence or modify and process the gene product in a desired specific manner may be selected. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the protein to function. Different host cells have characteristic and specific mechanisms regarding post-translational processing and modification of proteins and gene products. To ensure the precise modification and processing of expressed foreign proteins, appropriate cell lines or host systems may be selected. To achieve this objective, eukaryotic host cells with cellular mechanisms for appropriate processing, glycosylation, and phosphorylation of the primary transcript of the gene product may be used. Examples of such mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3, or myeloma cells.

[0119] Stable expression is desirable for producing recombinant proteins in high yield over long periods. For example, cell lines that stably express the binding molecule can be modified. Instead of using expression vectors containing viral replication origins, host cells can be transformed with DNA and selectable markers controlled by appropriate expression regulatory elements (e.g., promoters, enhancer sequences, transcriptional terminators, polyadenylation sites, etc.). After introducing the foreign DNA, the manipulated cells can be grown in concentrated medium for 1-2 days, followed by a switch to selective medium. Selectable markers in the recombinant plasmid provide resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes, grow, and form accumulations, which can then be cloned and expanded into cell lines. This method can be advantageously used to modify cell lines that express the binding molecule. Such modified cell lines can be particularly useful in screening and evaluating compounds that directly or indirectly interact with the binding molecule.

[0120] Examples of selective enzymes that can be used include, but are not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)), and the genes can be utilized in tk, hgprt, or aprt- cells, respectively. Furthermore, antimetabolite resistance can be used as a selection criterion for the following genes: dhfr (Wigler et al., Proc.Natl.Acad.Sci.USA 77:357(1980); O'Hare et al., Proc.Natl.Acad.Sci.USA 78:1527(1981)), which gives resistance to methotrexate; gpt (Mulligan & Berg, Proc.Natl.Acad.Sci.USA 78:2072(1981)), which gives resistance to mycophenolate; neo (Wu and Wu, Biotherapy 3:87-95(1991)), which gives resistance to aminoglycoside G-418; and hygro (Santerre et al., Gene 30:147(1984)), which gives resistance to hygromycin.Methods commonly known in the field of recombinant DNA technology may be conventionally applied to select a desired recombinant clone, such as those described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); and Colberre-Garapin et al., J.Mol.Biol.150:1 (1981), which are incorporated herein by reference in their entirety.

[0121] The expression level of the binding molecule can be increased by vector amplification (see Bebbington and Hentschel, “The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells” (DNA Cloning, Vol.3, Academic Press, New York, 1987) for a review). If the marker in the vector system expressing binding is amplified, the copy number of the marker gene increases due to an increase in the level of the inhibitor present in the host cell culture. Since the amplified region associates with the gene, the protein product also increases (Crouse et al., Mol.Cell.Biol.3:257(1983)).

[0122] Host cells can be simultaneously gene-transferred using multiple expression vectors of the present invention. The vectors may contain identical selectable markers that enable similar expression of the polypeptides to be expressed. Alternatively, a single vector capable of encoding and expressing the polypeptide of the present invention may be used. The coding sequence may include cDNA or genomic DNA.

[0123] The conjugated molecules of the present invention may be produced in animals by chemical synthesis or expressed by recombinant DNA, and then purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography to specific antigens after protein A, and size exclusion chromatography), centrifugation, solubility differential chromatography, or any other standard technique for protein purification. In addition, the conjugated molecules of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. The purification technique may vary depending on whether or not an Fc region (e.g., scFC) is conjugated to the molecules of the present invention.

[0124] In some embodiments, the present invention encompasses conjugated molecules (including both covalent and noncovalent complexing) that are fused to polypeptides by recombinant technology or chemically complexed. For ease of purification, the conjugated constructs of the present invention that are fused or complexed may be used. See, for example, Harbor et al., cited above and International Publication No. 93 / 21232 of the PCT application; European Patent No. 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent No. 5,474,981; Gillies et al., Proc. Natl. Acad. Sci. 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991).

[0125] Furthermore, the binding molecules or fragments thereof of the present invention may be fused to marker sequences such as peptides to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 58), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. For example, hexahistidine (SEQ ID NO: 58), as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag (Wilson et al., Cell 37:767 (1984)) and the "flag" tag, which correspond to epitopes derived from influenza hemagglutinin protein.

[0126] Molecular fabrication In a general sense, the molecules of the present invention are formed by selecting the VH and VL regions from a desired antibody, linking them using a polypeptide linker as described herein, and optionally attaching an Fc region (HHLL). 2 It is constructed by forming molecules. More specifically, the molecules of the present invention are encoded by combining nucleic acids that encode VH, VL, and a linker, and optionally Fc. 2 Generate nucleic acid constructs.

[0127] Antibody production In certain embodiments, a monospecific antibody having binding specificity to a desired target is first prepared before the molecule of the present invention is constructed.

[0128] Antibodies useful for producing the molecules of the present invention can be prepared by techniques well known to those skilled in the art. For example, by immunizing an animal (e.g., mouse, rat, or rabbit), followed by immortalizing spleen cells taken from this animal after the completion of the immunization schedule. Spleen cells can be immortalized using any technique known in the art, for example, by fusing spleen cells with myeloma cells to produce hybridomas. See, for example, Antibodies; Harlow and Lane, Cold Spring Harbor Laboratory Press, 1st Edition (e.g., from 1988) or 2nd Edition (e.g., from 2014).

[0129] In one embodiment, the humanized monoclonal antibody comprises the variable domain (or all or part of its antigen-binding site) of a mouse antibody and a constant domain derived from a human antibody. Alternatively, the humanized antibody fragment may comprise the antigen-binding site of a mouse monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for producing manipulated monoclonal antibodies are described in Riechmann et al., 1988, Nature 332:323, Liu et al., 1987, Proc.Nat.Acad.Sci.USA 84:3439, Larrick et al., 1989, Bio / Technology 7:934, and Winter et al., 1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDR-transplanted antibody. Techniques for humanizing antibodies include, for example, U.S. Patent Nos. 5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557; Padlan et al., 1995, FASEB J.9:133-39; Tamura et al., 2000, J.Immunol.164:1432-41; Zhang, W., et al., Molecular Immunology.42(12):1445-1451, 2005; Hwang W. et al., Methods.36(1):35-42, 2005; Dall'Acqua WF, et al., Methods 36(1):43-60, 2005; and Clark, M., Immunology. This is discussed in Today.21(8):397-402, 2000.

[0130] The molecules of the present invention may also include regions of fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by many techniques that will be familiar to those skilled in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice having inserted human immunoglobulin genes, isolation of human immunoglobulin V regions from phage libraries, or other procedures based on those known in the art and disclosed herein.

[0131] Procedures have been developed to produce human monoclonal antibodies in non-human animals. For example, mice have been created in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes are introduced into the mice to replace the inactivated mouse genes. This technique involves introducing elements of human heavy and light chain loci into a mouse lineage derived from embryonic stem cells, which includes targeting and disrupting endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes may be minigene constructs or transgene loci on yeast artificial chromosomes that undergo B cell-specific DNA rearrangement and high-frequency mutations in mouse lymphoid tissue.

[0132] Antibodies produced in animals incorporate human immunoglobulin polypeptide chains encoded by human genetic material introduced into those animals. In one embodiment, non-human animals such as transgenic mice are immunized with a suitable immunogen.

[0133] Examples of techniques for producing and using transgenic animals to produce human antibodies or partial human antibodies include U.S. Patents No. 5,814,318, 5,569,825, and 5,545,806; Davis et al., Production of human antibodies from transgenic mice in Lo, ed. Antibody Engineering: Methods and Protocols, Humana Press, NJ:191-200 (2003); Kellermann et al., 2002, Curr Opin Biotechnol. 13:593-97; Russel et al., 2000, Infect Immun. 68:1820-26; Gallo et al., 2000, Eur J Immun. 30:534-40; Davis et al., 1999, Cancer Metastasis Rev.18:421-25, Green,1999, J Immunol Methods.231:11-23, Jakobovits,1998, Advanced Drug Delivery Reviews 31:33-42, Green et al.,1998, J Exp Med.188:483-95, Jakobovits A,1998,Exp.Opin.Invest.Drugs.7:607-14, Tsuda et al.,1997,Genomics.42:413-21, Mendez et al.,1997, Nat Genet.15:146-56, Jakobovits,1994,Curr Biol.4:761-63, Arbones et al., 1994, Immunity.1:247-60, Green et al., 1994, Nat. Genet.7:13-21, Jakobovits et al.,1993,Nature.362:255-58,Jakobovits et al.,1993,Proc Natl Acad Sci USA.90:2551-55.Chen, J., M. Trounstine, FWAlt, F. Young, C. Kurahara, J. Loring, D. Huszar."Immunoglobulin gene rearrangement in B-cell deficient mice generated by targeted deletion of the JH locus." International Immunology 5(1993):647-656, Choi et al., 1993, Nature Genetics 4:117-23, Fishwild et al., 1996, Nature Biotechnology 14:845-51, Harding et al., 1995, Annals of the New York Academy of Sciences, Lonberg et al., 1994, Nature 368:856-59, Lonberg, 1994, Transgenic Approaches to Human Monoclonal Antibodies in Handbook of Experimental Pharmacology 113:49-101, Lonberg et al., 1995, Internal Review of Immunology 13:65-93, Neuberger, 1996, Nature Biotechnology 14:826, Taylor et al., 1992, Nucleic Acids Research 20:6287-95, Taylor et al., 1994, International Immunology 6:579-91, Tomizuka et al., 1997, Nature Genetics 16:133-43, Tomizuka et al., 2000, Proceedings of the National Academy of Sciences USA 97:722-27, Tuaillon et al., 1993, Proceedings of the National Academy of Sciences USA 90:3720-24 and Tuaillon et al., 1994, Journal of Immunology 152:2912-20.; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun.This is described in 6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. NYAcad. Sci. 764:525-35. In addition, protocols including XenoMouse® (Abgenix, now Amgen, Inc.) are described, for example, in U.S. Patent Application No. 05 / 0118643 and International Publication No. 05 / 694879, International Publication No. 98 / 24838, International Publication No. 00 / 76310, and U.S. Patent No. 7,064,244.

[0134] For example, to produce hybridomas, lymphoid cells derived from immunized transgenic mice are fused with myeloma cells. Myeloma cells for use in the fusion procedure to produce hybridomas are preferably non-antibody-producing cells, have high fusion efficiency, and possess enzyme deficiencies that prevent growth in certain selective media, thereby promoting the proliferation of only the desired fusion cells (hybridoms). Examples of cell lines suitable for use in such fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XX0 Bul. Examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion include U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0135] Lymphoid (e.g., spleen) cells and myeloma cells can be combined with a membrane fusion promoter such as polyethylene glycol or a nonionic surfactant for several minutes, and then seeded at low density in a selective medium that helps hybridoma cells to proliferate but assists unfused myeloma cells. One such selective medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient amount of time, usually about 1-2 weeks, colonies of cells are observed. A single colony can be isolated, and antibodies produced by the cells can be tested for binding activity to a desired target using any of the various immunoassays known in the art and described herein. Hybridomas can be cloned (e.g., by limiting dilution cloning or by soft agar plaque isolation), and positive clones that produce molecules specific to the desired target can be selected and cultured. Binding molecules derived from the hybridoma culture can be isolated from the supernatant of the hybridoma culture. Thus, the present invention provides hybridomas containing polynucleotides encoding the binding molecules of the present invention in the chromosomes of the cells. These hybridomas can be cultured according to methods described herein and known in the art.

[0136] Another method for producing human antibodies useful for creating the binding molecules of the present invention involves immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. Such immortalized B cell lines (or lymphoblastoid cell lines) that produce monoclonal antibodies that specifically bind to a desired target can be identified by immunodetection methods such as those provided herein, e.g., ELISA, and subsequently isolated by standard cloning techniques. The stability of antibody-producing lymphoblastoid cell lines can be improved by fusing the transformed cell line with mouse myeloma to create a mouse-human hybrid cell line, according to methods known in the art (see, for example, Glasky et al., Hybridoma 8:377-89 (1989)). A further alternative method for producing human monoclonal antibodies is in vitro immunization, which involves priming human spleen B cells with an antigen and then fusing the primed B cells with a heterohybrid fusion partner. For example, see Boerner et al., 1991 J.Immunol. 147:86-95.

[0137] In certain embodiments, B cells producing a desired antibody are selected, and the light chain and heavy chain variable regions are cloned from these B cells according to molecular biological techniques known in the art (International Publication No. 92 / 02551; U.S. Patent No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. By selecting cells producing a desired antibody, B cells derived from immunized animals can be isolated from spleen, lymph node, or peripheral blood samples. B cells can also be isolated from humans, for example, from peripheral blood samples. Methods for detecting a single B cell producing an antibody with desired specificity are well known in the art, and include, for example, plaque formation, fluorescence-activated cell sorting, and detection of specific antibodies after in vitro stimulation. Methods for selecting B cells producing specific antibodies include, for example, preparing a single cell suspension of B cells in soft agar containing the antigen. When specific antibodies produced by B cells bind to an antigen, complex formation occurs, which may be visible as immunoprecipitates. After selecting B cells that produce the desired antibody, the specific antibody gene can be cloned by isolating and amplifying its DNA or mRNA, and this can be used to construct the molecule of the present invention according to methods known in the art and described herein.

[0138] A further method for obtaining antibodies useful for constructing the molecules of the present invention is by phage display. See, for example, Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Combinatorial libraries of human or mouse immunoglobulin variable region genes can be constructed in phage vectors, which can then be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to TGF-β binding proteins or their variants or fragments. For example, see U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc.Natl.Acad.Sci.USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc.Natl.Acad.Sci.USA 88:4363-66; Hoogenboom et al., 1992 J.Molec.Biol.227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and the references cited therein. For example, a library containing multiple polynucleotide sequences encoding Ig variable region fragments can be inserted in frame with a sequence encoding a phage coat protein into the genome of a filamentous bacteriophage such as M13 or a variant thereof. The fusion protein may be a fusion of the coat protein with the light chain variable region domain and / or the heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments can also be displayed on phage particles (see, for example, U.S. Patent No. 5,698,426).

[0139] Heavy and light chain immunoglobulin cDNA expression libraries can also be prepared using lambda phages, such as λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B cell population and used to construct heavy and light chain immunoglobulin cDNA expression libraries in λImmunoZap(H) and λImmunoZap(L) vectors. These vectors can be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., op. cit.; also see Sastry et al., op. cit.). Subsequently, positive plaques can be converted into insoluble plasmids, which allows for high-level expression of monoclonal antibody fragments derived from E. coli.

[0140] In one embodiment, in a hybridoma, the variable region of the gene expressing the monoclonal antibody of interest is amplified using nucleotide primers. These primers can be synthesized by those skilled in the art or purchased from commercial sources. (See, for example, Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including primers for the VHa, VHb, VHc, VHd, CH1, VL, and CL regions.) Using these primers, the heavy-chain or light-chain variable region can be amplified and subsequently inserted into vectors such as ImmunoZAPTMH or ImmunoZAPTML (Stratagene), respectively. These vectors can then be introduced into E. coli, yeast, or mammal-based systems for expression. Using these methods, large quantities of single-chain proteins containing fusions of the VH and VL domains can be produced (see Bird et al., Science 242:423-426, 1988).

[0141] In certain embodiments, the conjugated molecules of the present invention are obtained from transgenic animals (e.g., mice) that produce “heavy chain only” antibodies or “HCAb”. HCAb are similar to the single-chain VHH antibodies of natural camels and llamas. See, for example, U.S. Patent No. 8,507,748 and 8,502,014 and U.S. Patent Publication No. 2009 / 0285805A1, U.S. Patent Publication No. 2009 / 0169548A1, U.S. Patent Publication No. 2009 / 0307787A1, U.S. Patent Publication No. 2011 / 0314563A1, U.S. Patent Publication No. 2012 / 0151610A1, International Publication No. 2008 / 122886A2 and International Publication No. 2009 / 013620A2.

[0142] Once cells producing antibodies according to the present invention are obtained using either the immunization or other techniques described above, specific antibody genes can be cloned therefrom by isolating and amplifying DNA or mRNA according to standard procedures as described herein, and subsequently used to construct molecules of the present invention. The antibodies produced therefrom can be sequenced, the CDRs identified, and the DNA encoding the CDRs can be manipulated as described above to construct other molecules according to the present invention.

[0143] Molecular evolution of the complementarity-determining region (CDR) at the center of the antibody binding site has also been used to isolate antibodies with increased affinity, such as those described in Schier et al., 1996, J.Mol.Biol.263:551. Therefore, such techniques are useful in preparing the conjugation constructs of the present invention.

[0144] Human antibodies, partially human antibodies, or humanized antibodies are suitable for many applications, particularly for the applications of the present invention, but other types of binding molecules are preferred for certain specific applications. These non-human antibodies may be derived, for example, from any antibody-producing animal, such as a mouse, rat, rabbit, goat, donkey, or a non-human primate (e.g., a monkey such as a crab-eating macaque or rhesus macaque) or ape (e.g., a chimpanzee). Antibodies of a specific species can be produced by converting an antibody from one species to one of another, for example, by immunizing the animal of that species with a desired immunogen, or by using an artificial system for producing antibodies of that species (e.g., a bacterial or phage display-based system for producing antibodies of a specific species), or by, for example, replacing the constant region of the antibody with a constant region from another species, or by substituting one or more amino acid residues of the antibody to more closely resemble the sequence of an antibody from another species. In one embodiment, the antibody is a chimeric antibody containing amino acid sequences derived from two or more antibodies from different species. Subsequently, the molecule of the present invention can be produced using a desired binding region sequence.

[0145] If it is desired to improve the affinity of the binding molecule according to the present invention, which contains one or more of the above-mentioned CDRs, the following methods may be used: maintenance of CDRs (Yang et al., J.Mol.Biol., 254, 392-403, 1995), chain shuffling method (Marks et al., Bio / Technology, 10, 779-783, 1992), use of E. coli mutant strains (Low et al., J.Mol.Biol., 250, 350-368, 1996), DNA shuffling method (Patten et al., Curr.Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J.Mol.Biol., 256, 7-88, 1996), and further PCR techniques (Crameri, et al.) This can be achieved by several affinity maturation protocols, including those described in Vaughan et al., Nature, 391, 288-291, 1998). All of these methods for affinity maturation are discussed in Vaughan et al., Nature biotechnology 16;535:539-1998.

[0146] In certain embodiments, the present invention (HHLL) 2To construct molecules, it may be desirable to first produce a more typical single-chain antibody, which can be formed by linking heavy-chain and light-chain variable domain (Fv region) fragments via amino acid crosslinks (short peptide linkers), thereby generating a single-chain polypeptide. Such a single-chain Fv (scFv) is prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). The resulting polypeptides can be refolded on their own, and depending on the length of the mobile linker between the two variable domains, they may form antigen-binding monomers or multimers (e.g., dimers, trimers, or tetramers) (Kortt et al., 1997, Prot.Eng.10:423; Kortt et al., 2001, Biomol.Eng.18:95-108). Techniques developed for producing single-chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544; and de Graaf et al., 2002, Methods Mol Biol. 178:379-87. These single-chain antibodies are distinct from and separate from the molecules of the present invention.

[0147] Antigen-binding fragments derived from antibodies can also be obtained according to conventional methods, for example, by proteolytic hydrolysis of the antibody, or by pepsin or papain digestion of the whole antibody. For example, an antibody fragment may be generated by enzymatic cleavage of the antibody with pepsin, providing a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce a 3,5S Fab' monovalent fragment. Optionally, the cleavage reaction may be carried out using a blocking group for the sulfhydryl group resulting from the cleavage of the disulfide bond. Alternatively, two monovalent Fab and Fc fragments can be directly synthesized by enzymatic cleavage using papain. These methods are described, for example, in Goldenberg, U.S. Patent No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Methods in Enzymology 1:422 (Academic Press 1967) by Edelman et al.; and Current Protocols in Immunology (Coligan JE, et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5 by Andrews, SMand Titus, JA. To form a monovalent light-heavy chain fragment (Fd), the heavy chain may be separated, or the fragment may be further cleaved. Other methods for cleaving the antibody, such as other enzymatic, chemical, or genetic techniques, may also be used, as long as the fragment binds to the antigen recognized by the intact antibody.

[0148] In certain embodiments, the molecule comprises one or more complementarity-determining regions (CDRs) of an antibody. CDRs can be obtained by constructing a polynucleotide encoding the CDR of interest. Such polynucleotides are prepared, for example, by using a polymerase chain reaction to synthesize the variable region using mRNA from an antibody-producing cell as a template (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)). The antibody fragment may further comprise at least one variable region domain of the antibody described herein. For example, the V region domain may be a monomer and a VH or VL domain, which can independently bind to a desired target (e.g., human CD3) with an affinity of at least 10⁻⁷ M or less, as described herein.

[0149] The variable region may be any natural variable domain or a modified version thereof. A modified version means a variable region produced using recombinant DNA modification techniques. Such modified versions include, for example, those produced from the variable region of a specific antibody by insertion, deletion, or modification within or to the amino acid sequence of the specific antibody. Those skilled in the art may use any known method to identify amino acid residues suitable for modification. Further examples include modified variable regions containing at least one CDR and optionally one or more framework amino acids from the first antibody and the remainder of the variable region domain from the second antibody. Modified versions of antibody variable domains can be produced by many techniques familiar to those skilled in the art.

[0150] The variable region can be covalently bound to at least one other antibody domain or fragment thereof at its C-terminal amino acid. For example, the VH domain present in the variable region can be linked to the CH1 domain of immunoglobulin. Similarly, the VL domain can be linked to the CK domain. Thus, for example, an antibody may be a Fab fragment containing associated VH and VL domains, where the antigen-binding domain is covalently bound to the CH1 and CK domains at the C-terminuses of the VH and VL domains, respectively. The CH1 domain can be extended with further amino acids to provide, for example, a hinge region or a portion of a hinge region domain as seen in the Fab' fragment, or to provide further domains such as the CH2 and CH3 domains of the antibody.

[0151] Binding specificity Antibody or (HHLL) 2 A molecule "specifically binds" to an antigen when it has a tight binding affinity, as determined by an equilibrium dissociation constant (KD) of 10⁻⁷ M or less, or the corresponding KD as defined below.

[0152] Affinity can be measured using various techniques known in the art, for example, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol.373:52-60, 2008; or radioimmunoassay (RIA)), or by surface plasmon resonance assay or other kinetics-based assay mechanisms (e.g., BIACORE® analysis or Octet® analysis (forteBIO)), as well as other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling on one or more of the components being examined, and / or may use various detection methods, including but not limited to chromogenic labeling, fluorescent labeling, luminescence labeling, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. An example of a competitive binding assay is a radioimmunoassay that involves incubating a labeled antigen with an increasing amount of unlabeled antigen alongside an antibody of interest and detecting the antibody bound to the labeled antigen. The affinity and binding-dissociation rate of the antibody of interest to a specific antigen can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using a radioimmunoassay. In this case, the antigen is incubated with an antibody of interest conjugated to a labeled compound while increasing the amount of the unlabeled second antibody. This type of assay can be readily adapted for use with the molecules of the present invention.

[0153] Further embodiments of the present invention provide molecules that bind to a desired target with an equilibrium dissociation constant or KD (koff / kon) of less than 10⁻⁷ M, or less than 10⁻⁸ M, or less than 10⁻⁹ M, or less than 10⁻¹¹ M, or less than 10⁻¹³ M, or less than 5 x 10⁻¹³ M (lower values ​​indicate higher binding affinity). Further embodiments of the present invention provide molecules that bind to a desired target (with an with an) with an equilibrium dissociation constant or KD (koff / kon) of less than about 10⁻⁷ M, or less than about 10⁻⁸ M, or less than about 10⁻⁹ M, or less than about 10⁻¹¹ M, or less than about 10⁻¹² M, or less than about 10⁻¹³ M, or less than 5 x 10⁻¹³ M.

[0154] In yet another embodiment, the molecule that binds to the desired target has equilibrium dissociation constants or KD (koff / kon) of approximately 10⁷M to approximately 10⁻⁸M, approximately 10⁸M to approximately 10⁻⁹M, approximately 10⁻¹⁰M to approximately 10⁻¹¹M, approximately 10⁻¹¹M to approximately 10⁻¹²M, and approximately 10⁻¹²M to approximately 10⁻¹³M. In yet another embodiment, the molecule of the present invention has equilibrium dissociation constants or KD (koff / kon) of 10⁷M to 10⁸M, 10⁸M to 10⁹M, 10⁻¹⁰M to 10⁻¹¹M, 10⁻¹¹M to 10⁻¹²M, and 10⁻¹²M to 10⁻¹³M.

[0155] molecular stability Particularly in relation to therapeutic molecules of biopharmaceuticals, various aspects of molecular stability may be desired. For example, stability at various temperatures ("thermal stability") may be desired. In some embodiments, this may include stability within the physiological temperature range, e.g., 37°C or approximately 37°C, or 32°C to 42°C. In other embodiments, this may include stability within a higher temperature range, e.g., 42°C to 60°C. In yet another embodiment, this may include stability within a lower temperature range, e.g., 20°C to 32°C. In yet another embodiment, this may include stability during freezing, e.g., below 0°C.

[0156] Assays for measuring the thermal stability of protein molecules are known in the art. For example, the fully automated UNcle platform (Unchained Labs), which allows simultaneous acquisition of endogenous protein fluorescence and static light scattering (SLS) data during a thermal gradient, is used and further described in the examples. Furthermore, both thermal melting (Tm) and thermal aggregation (Tagg) can also be measured, respectively, using the thermal stability and aggregation assays described herein, such as differential scanning fluorescence (DSF) and static light scattering (SLS), as described in the examples.

[0157] Alternatively, accelerated stress studies can be performed on this molecule. In summary, this involves warming the protein molecule at a specific temperature (e.g., 40°C) and then measuring aggregation at various time points by size exclusion chromatography (SEC), in which case a lower level of aggregation indicates better protein stability.

[0158] Alternatively, the thermal stability parameter can be determined from the perspective of molecular aggregation temperature as follows: A molecular solution with a concentration of 250 μg / ml is transferred to a single-use cuvette and placed in a dynamic light scattering (DLS) apparatus. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min while maintaining a constant radius. An increase in radius indicates the melting and aggregation of the protein, which is used to calculate the molecular aggregation temperature.

[0159] Alternatively, the melting temperature curve can be determined by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of the binding molecule. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC device. Energy uptake of the sample containing the binding molecule is recorded from 20°C to 90°C and compared with a sample containing only the formulation buffer. The binding molecule is adjusted to a final concentration of, for example, 250 μg / ml in SEC running buffer. The temperature of the entire sample is increased stepwise to record each melting curve. Energy uptake of the sample and the formulation buffer standard is recorded at each temperature T. The difference in energy uptake Cp (kcal / mole / °C) obtained by subtracting the standard from the sample is plotted against each temperature. The melting temperature is defined as the temperature at which energy uptake is first maximized.

[0160] In further embodiments, the molecule according to the present invention is stable at physiological pH or approximately physiological pH, i.e., about pH 7.4. In other embodiments, the molecule is stable at lower pH, for example, down to pH 6.0. In other embodiments, the molecule is stable at higher pH, for example, up to pH 9.0. In one embodiment, the molecule is stable at pH 6.0 to 9.0. In another embodiment, the molecule is stable at pH 6.0 to 8.0. In yet another embodiment, the molecule is stable at pH 7.0 to 9.0.

[0161] In certain embodiments, the more tolerant the molecule is to non-physiological pH (e.g., pH 6.0), the higher the recovery rate of the molecule eluted from the ion-exchange column relative to the total amount of protein loaded. In one embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥30%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥40%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥50%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥60%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥70%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥80%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥90%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥95%. In another embodiment, the recovery rate of the molecule from the ion (e.g., cation) exchange column is ≥99%.

[0162] In certain embodiments, it may be desirable to measure the chemical stability of a molecule. The chemical stability of a molecule can be measured by isothermal denaturation ("ICD") by monitoring endogenous protein fluorescence, as further described herein in the examples. ICD yields C1 / 2 and ΔG, which can be excellent metrics for protein stability. C1 / 2 is the amount of denaturant required to denature 50% of the protein, and this is used to derive ΔG (i.e., unfolding energy).

[0163] Protein chain clipping is another critical product quality characteristic that is carefully observed or reported with respect to biological drugs. Typically, long linkers and / or less structured linkers are expected to exhibit increased clipping as a function of incubation time and temperature. Clipping to linkers connecting either the target domain or the T cell binding domain is a significant problem for molecules because it ultimately has detrimental effects on drug potency and efficacy. Clipping to further sites, including scFc, can affect pharmacodynamic / kinetic properties. Increased clipping is a characteristic to be avoided in pharmaceuticals. Therefore, in certain embodiments, protein clipping can be assayed as described herein in the examples.

[0164] Immune effector cells and effector cell proteins The molecule may bind to a molecule expressed on the surface of an immune effector cell (referred to herein as "effector cell protein") and to another molecule expressed on the surface of a target cell (referred to herein as "target cell protein"). Immune effector cells may be T cells, NK cells, macrophages, or neutrophils. In some embodiments, the effector cell protein is a protein included in the T cell receptor (TCR)-CD3 complex. The TCR-CD3 complex is a heteromultimer containing heterodimers that include TCRα and TCRβ or TCRγ and TCRδ, in addition to various CD3 chains from among the CD3 zeta (CD3ζ), CD3 epsilon (CD3ε), CD3 gamma (CD3γ), and CD3 delta (CD3δ) chains.

[0165] The CD3 receptor complex is a protein complex composed of four chains. In mammals, this complex contains a CD3γ (gamma) chain, a CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor CD3 complex, which generates an activation signal in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are cell surface proteins of a very closely related immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor activation tyrosine motif, or ITAM for short, which is essential for TCR signaling. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on chromosome 11 in humans. The most preferred CD3 epsilon epitopes are those within the amino acid residue range of 1-27 of the human CD3 epsilon extracellular domain. The molecules according to the present invention are expected to exhibit typically and advantageously minimal nonspecific T cell activation, which is undesirable in specific immunotherapy. In other words, this reduces the risk of side effects.

[0166] In some embodiments, the effector cell protein may be a human CD3 epsilon (CD3ε) chain (whose mature amino acid sequence is disclosed in SEQ ID NO: 40), which may be part of a multimeric protein. Alternatively, the effector cell protein may be a human and / or cynomolgus monkey TCRα, TCRβ, TCRδ, TCRγ, CD3 beta (CD3β) chain, CD3 gamma (CD3γ) chain, CD3 delta (CD3δ) chain, or CD3 zeta (CD3ζ) chain.

[0167] Furthermore, in some embodiments, the molecule may also bind to CD3ε chains derived from non-human species such as mice, rats, rabbits, New World monkeys, and / or Old World monkey species. Such species include, but are not limited to, the following mammalian species: Mus musculus; Rattus rattus; Rattus norvegicus; Crab-eating macaque, Macaca fascicularis; Hamadryas baboon, Papio hamadryas; Guinea baboon, Papio papio; Anubis baboon, Papio anubis; Yellow baboon, Papio cynocephalus; Chacma baboon, Papio ursinus; Callisrix jacchus; Saguinus Oedipus; and Saimiri ssiureus. (Sciureus). The mature amino acid sequence of the CD3ε chain in cynomolgus monkeys is provided in SEQ ID NO: 34. Therapeutic molecules with equivalent activity in humans and species commonly used in preclinical trials, such as mice and monkeys, can simplify, accelerate, and ultimately lead to improved outcomes in drug development. Such advantages can be significant in the long and costly process of bringing a drug to market.

[0168] In a particular embodiment, (HHLL) 2The molecule may bind to an epitope within the first 27 amino acids (SEQ ID NO: 36) of a CD3ε chain, which may be a human CD3ε chain or a CD3ε chain from a different species, particularly one of the mammalian species listed above. The epitope may contain the amino acid sequence Gln-Asp-Gly-Asn-Glu. The advantages of a molecule that binds to such an epitope are described in detail in U.S. Patent Application Publication No. 2010 / 0183615A1, the relevant portion of which is incorporated herein by reference. The epitope to which the antibody or molecule binds may be determined, for example, by alanine scanning as described in U.S. Patent Application Publication No. 2010 / 0183615A1, the relevant portion of which is incorporated herein by reference. In other embodiments, the molecule may bind to an epitope within the extracellular domain of CD3ε (SEQ ID NO: 35).

[0169] In embodiments where T cells are immune effector cells, the effector cell proteins to which the molecule can bind include, but are not limited to, CD3ε chain, CD3γ, CD3δ chain, CD3ζ chain, TCRα, TCRβ, TCRγ, and TCRδ. In embodiments where NK cells or cytotoxic T cells are immune effector cells, for example, NKG2D, CD352, NKp46, or CD16a may be effector cell proteins. In embodiments where CD8+ T cells are immune effector cells, for example, 4-1BB or NKG2D may be effector cell proteins. Alternatively, in other embodiments, the molecule may bind to other effector cell proteins expressed on T cells, NK cells, macrophages, or neutrophils.

[0170] Target cells and target cell proteins expressed on target cells As described above, the molecule can bind to effector cell proteins and target cell proteins. Target cell proteins may be expressed on the surface of cells involved in diseases, such as cancer cells, pathogen-infected cells, or disease-mediated conditions such as inflammatory states, autoimmune states, and / or fibrotic states. In some embodiments, target cell proteins may be highly expressed on target cells, but high levels of expression are not necessarily required.

[0171] If the target cells are cancer cells, molecules such as those described herein may bind to cancer cell antigens as described above. Cancer cell antigens or tumor-associated antigens ("TAAs") may be human proteins or proteins of another species. For example, molecules may bind to target cell proteins of various species, including, in particular, mouse, rat, rabbit, New World monkey, and / or Old World monkey species. Such species include, but are not limited to, the following: Mus musculus; Rattus rattus; Rattus norvegicus; Crab-eating macaque, Macaca fascicularis; Hamadryas baboon, Papio hamadryas; Guinea baboon, Papio papio; Anubis baboon, Papio anubis; Yellow baboon, Papio cynocephalus; Chacma baboon, Papio ursinus; Callisrix jacchus, Saguinus Oedipus, and Saimiri ssiureus. (sciureus). Preferred target cell surface antigens in connection with the present invention are MSLN, CDH3, FLT3, CLL1, EpCAM, CD20, and CD22. Typically, target cell surface antigens in connection with the present invention are tumor-associated antigens (TAAs). B lymphocyte antigen CD20 or CD20 is expressed on the surface of all B cells, starting in pre-B phase (CD45R+, CD117+) and gradually increasing in concentration until maturity. CD22 or surface antigen classification 22 is a molecule belonging to the SIGLEC family of lectins. This molecule is found on the surface of mature B cells and is present to a relatively low degree on some immature B cells. Fms-like tyrosine kinase 3 (FLT3) is also known as surface antigen classification 135 (CD135), receptor tyrosine-protein kinase FLT3, or fetal liver kinase-2 (Flk2). FLT3 is a cytokine receptor belonging to receptor tyrosine kinase class III. CD135 is a receptor for the cytokine Flt3 ligand (FLT3L).The FLT3 gene is frequently mutated in acute myeloid leukemia (AML). C-type lectin-like receptor (CLL1), also known as CLEC12A or MICL, contains an ITIM motif in its cytoplasmic tail that may be associated with the signaling phosphatases SHP-1 and SHP-2. Human MICL is expressed primarily as a monomer on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells, and is associated with AML. Mesoserine (MSLN) is a 40 kDa protein expressed in mesothelial cells and overexpressed in several human tumors. Cadherin-3 (CDH3), also known as P-cadherin, is a calcium-dependent cell-to-cell adhesion glycoprotein consisting of five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail. It is associated with certain types of tumors. Epithelial cell adhesion molecules (EpCAMs) are transmembrane glycoproteins that mediate Ca2+-independent isoplastic cell-cell adhesion in epithelium. EpCAMs are oncogenic and are thought to contribute to tumorigenesis and metastasis in carcinomas.

[0172] In some examples, the target cell protein may be a protein selectively expressed on infected cells. For example, in the case of HBV or HCV infection, the target cell protein may be the HBV or HCV envelope protein expressed on the surface of the infected cell. In other embodiments, the target cell protein may be gp120 encoded by HIV on human immunodeficiency virus (HIV) infected cells.

[0173] In other embodiments, target cells may be cells associated with autoimmune or inflammatory diseases. For example, human eosinophils in asthma may be target cells, in which case, for example, EGF-like module-containing mucin-like hormone receptor (EMR1) may be the target cell protein. Alternatively, excess human B cells in patients with systemic lupus erythematosus may be target cells, in which case, for example, CD19 or CD20 may be the target cell proteins. In other autoimmune conditions, excess human Th2 T cells may be target cells, in which case, for example, CCR4 may be the target cell protein. Similarly, target cells may be fibrous cells associated with diseases such as atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, renal transplant fibrosis, allogeneic kidney transplant nephropathy, or idiopathic pulmonary fibrosis and / or idiopathic pulmonary hypertension. In such fibrous conditions, for example, fibroblast-activating protein alpha (FAP alpha) may be the target cell protein.

[0174] Treatment method and composition Molecules can be used to treat a variety of conditions, including, for example, various forms of cancer, infections, autoimmune or inflammatory conditions, and / or fibrotic conditions.

[0175] Accordingly, in one embodiment, molecules for use in the prevention, treatment, or remission of a disease are provided herein.

[0176] Another embodiment provides the use of the binding molecule of the present invention (or a binding molecule produced according to the process of the present invention) in the manufacture of a medicine for the prevention, treatment, or remission of a disease.

[0177] Pharmaceutical compositions comprising molecules are provided herein. These pharmaceutical compositions comprise a therapeutically effective amount of the molecule and one or more further components, such as physiologically acceptable carriers, excipients, or diluents. In some embodiments, these additional components may include, among many possibilities, buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and / or preservatives.

[0178] In some embodiments, molecules may be used to treat cell proliferation disorders, including cancer, that involve unregulated and / or inappropriate cell proliferation, sometimes accompanied by the destruction of adjacent tissue and the growth of neovascularization, which can allow cancer cells to invade new areas, i.e., metastasize. Conditions treatable by molecules include non-malignant conditions involving inappropriate cell proliferation, such as colorectal polyps, cerebral ischemia, gross cystic disease, polycystic kidney disease, benign prostatic hyperplasia, and endometriosis. A preferred method of targeting cancer is to target the molecule to cancer cell surface antigens, i.e., tumor-associated antigens (TAAs). These can be proteins, preferably the extracellular components of proteins, or carbohydrate structures, preferably carbohydrate structures of proteins such as glycoproteins.

[0179] The molecules of the present invention can be used to treat hematological malignancies or solid tumor malignancies. More specifically, cell proliferative diseases that can be treated using the molecules include, for example, mesothelioma, squamous cell carcinoma, myeloma, osteosarcoma, glioblastoma, glioma, carcinoma, adenocarcinoma, melanoma, sarcoma, acute and chronic leukemia, lymphoma and meningioma, Hodgkin's disease, Sézary syndrome, multiple myeloma and lung cancer, non-small cell lung cancer, small cell lung cancer, laryngeal cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, skin cancer, Cancers include prostate cancer, cervical cancer, vaginal cancer, gastric cancer, renal cell carcinoma, kidney cancer, pancreatic cancer, colorectal cancer, endometrial cancer, esophageal cancer, hepatobiliary cancer, bone cancer, skin cancer, and hematological cancers, as well as cancers of the nasal cavity and sinuses, nasopharynx, oral cavity, oropharynx, larynx, inferior larynx, salivary glands, mediastinum, stomach, small intestine, colon, rectum and anus, ureters, urethra, penis, testes, vulva, endocrine system, central nervous system, and plasma cells.

[0180] A text that provides a guideline for cancer therapy is *Cancer, Principles and Practice of Oncology, 4th Edition*, DeVita et al., Eds. JBLippincott Co., Philadelphia, PA (1993). The appropriate treatment approach is selected according to the specific type of cancer and other factors such as the patient's overall condition, as recognized in the relevant field. When treating cancer patients, molecules may be added to treatment regimens that use other anti-cancer agents.

[0181] In some embodiments, molecules may be administered simultaneously with, before, or after, various drugs and treatments widely used in cancer treatment, such as chemotherapeutic agents, non-chemotherapeutic anti-cancer agents, and / or radiation. For example, chemotherapy and / or radiation therapy may be performed before, during, and / or after any treatment described herein. Examples of chemotherapeutic agents are listed above and include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone (Novantrone®), actinomycin D, cycloheximide, camptothecin (or their water-soluble derivatives), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine (DTIC), anti-cancer antibiotics, such as doxorubicin and daunomycin, and all of the aforementioned chemotherapeutic agents.

[0182] Molecules can also be used to treat infectious diseases, including, among many others, chronic hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection, Epstein-Barr virus (EBV) infection, or cytomegalovirus (CMV) infection.

[0183] If a molecule is useful for depleting a particular cell type, it may find further use in other types of conditions. For example, it may be useful for depleting human eosinophils in asthma, excess human B cells in systemic lupus erythematosus, excess human Th2 T cells in autoimmune conditions, or pathogen-infected cells in infectious diseases. In fibrous conditions, it may be useful for depleting cells that form fibrous tissue.

[0184] A therapeutically effective dose of the molecule may be administered. The amount of molecule constituting the therapeutic dose may vary depending on the symptoms being treated, the patient's weight, and the calculated patient's skin surface area. The dosage of the molecule may be adjusted to achieve the desired effect. Repeated administration may often be necessary.

[0185] Molecules or pharmaceutical compositions containing such molecules may be administered by any feasible method. Unless there is a specific prescription or special circumstances, protein-based therapeutics are usually administered via parenteral routes, such as injection, because oral administration would result in hydrolysis of the protein in the acidic environment of the stomach. Possible routes of administration include subcutaneous, intramuscular, intravenous, intra-arterial, intrafocal, or peritoneal bolus injections. Molecules may also be administered by infusion, such as intravenous or subcutaneous infusion. Topical administration is also possible, particularly for diseases involving the skin. Alternatively, molecules may be administered through contact with mucous membranes, such as intranasal, sublingual, vaginal, or rectal administration, or as an inhalant. Alternatively, certain suitable pharmaceutical compositions containing molecules may be administered orally.

[0186] The term “treatment” encompasses the alleviation of at least one symptom or embodiment of another disorder, or a reduction in disease severity. Molecules according to the present invention do not need to result in a complete cure or the elimination of any symptoms or manifestations of any disease to constitute a viable therapeutic agent. As recognized in the relevant art, a drug used as a therapeutic agent may reduce the severity of a given condition, but does not need to eliminate all symptoms of a disease to be considered a useful therapeutic agent. It is sufficient to simply mitigate the effects of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reduce the likelihood of the disease developing or worsening in a subject. One embodiment of the present invention relates to a method comprising administering a patient a sufficient amount and time to induce a sustained improvement above a baseline in an index reflecting the severity of a particular disorder.

[0187] The term "prevention" encompasses the prevention of at least one symptom or embodiment of other disorders. A preventive measure incorporating the molecules according to the present invention does not need to be completely effective in preventing the onset of a condition in order to constitute a viable preventive agent. It is sufficient to simply reduce the likelihood that the disease will develop or worsen in the subject.

[0188] As understood in the relevant fields, pharmaceutical compositions containing this molecule are administered to the target in a manner appropriate to the indication and composition. Pharmaceutical compositions may be administered by any preferred technique, including but not limited to parenteral, topical, or inhalation. When administered by injection, the pharmaceutical composition may be administered by bolus injection or continuous infusion, for example, via intra-articular, intravenous, intramuscular, intrafocal, intraperitoneal, or subcutaneous routes. Inhalation delivery includes, for example, nasal or oral inhalation, the use of a nebulizer, or inhalation of the conjugated molecule in aerosol form. Other alternatives include oral formulations, including pills, syrups, or lozenges.

[0189] The molecule may be administered in the form of a composition comprising one or more further components, such as a physiologically acceptable carrier, excipient, or diluent. Optionally, the composition further comprises one or more physiological activators. In various specific embodiments, the composition comprises one or more molecules in addition to one, two, three, four, five, or six physiological activators.

[0190] A kit for physician use is provided, comprising one or more molecules and a label or other instruction manual, for use in the treatment of any of the conditions discussed herein. In one embodiment, the kit may be in the form of a composition as disclosed herein and may comprise a sterile formulation of one or more molecules in one or more vials.

[0191] Dosage and frequency of administration may vary depending on factors such as the route of administration, the specific molecule used, the nature and severity of the disease being treated, whether the condition is acute or chronic, and the patient's physique and overall health.

[0192] The above has described the present invention using general terminology and is not intended to limit it; rather, the following examples are provided as practical illustrations. [Examples]

[0193] Example 1: (HHLL) 2 Formation and expression of binding molecules Figure 1 is (HLHL) 2 and (HHLL) 2 It possesses representative structures for both forms. We have constructed both versions of these molecules.

[0194] (HHLL) 2Version (T6M) includes the following domains from N- to C-terminus: anti-MSLN VH-(GGGS)4 linker-anti-CD3 VH-(GGGS)4 linker-anti-MSLN VL-(GGGS)4 linker-anti-CD3 VL-(GGGS)3 linker-scFc-(GGGS)3 linker-anti-CDH3 VH-(GGGS)4 linker-anti-CD3 VH-(GGGS)4 linker-anti-CDH3 VL-(GGGS)4 linker-anti-CD3 VL.

[0195] Produced (HLHL) 2 Version (G7Q) contains the following domains from N- to C-terminus: anti-MSLN VH-(GGGS)3 linker-anti-MSLN VL-(SGGGS)1 linker-anti-CD3 VH-(GGGS)3 linker-anti-CD3 VL-(GGGS)3 linker-scFc-(GGGS)3 linker-anti-CDH3 VH-(GGGS)3 linker-anti-CDH3 VL-(SGGGS)1 linker-anti-CD3 VH-(GGGS)3 linker-anti-CD3 VL.

[0196] Two different forms of open reading frames, as shown in Figure 1, were ordered for gene synthesis and subcloned into mammalian expression vectors containing IgG-derived signal peptides for secretory expression in cell culture supernatant. The sequenced plasmid clones were stably transferred into CHO cells, and the cell culture supernatant was collected after 6 days and stored at -80°C until protein purification. Tables 3 and 4 provide summaries of production runs for both T6M and G7Q, respectively, and demonstrate comparable protein yields for both molecules.

[0197] [Table 3]

[0198] [Table 4]

[0199] Example 2 Chromatography analysis Protein purification was performed by protein A (GE Healthcare, mAb SelectSuRE) affinity chromatography of filtered cell culture supernatant followed by size exclusion chromatography in pH 7 buffer solution (Error! Reference source not found. e3). Peaks were pooled using OD280nm signaling, and MW was analyzed by SDS-PAGE. Protein monomer peaks (G7Q peaked at 159.9 ml or T6M peaked at 166.08 ml, respectively) were formulated in buffer solution and aliquoted for storage at -80°C.

[0200] SDS-PAGE analysis Purified monomer samples were subjected to SDS-PAGE analysis to determine purity and accurate molecular weight (MW) (Figure 4). 60 μl of sample was mixed with 20 μl (4X) of LDS sample buffer and 10 μl of 1M DTT, and incubated at 70°C for 10 minutes. 15 μl of sample was placed in each lane using a Bolt 4-12% Bis-Tris Plus 12-well gel (NW04122BOX, Invitrogen). For the marker, 7.5 μl (Sharp Pre-Stained Protein Standard (LC5800, Invitrogen)) was placed in a separate lane. The running buffer was 1x MES (20x MES SDS Running Buffer, Invitrogen, NP0002-02), and the gel was electrophoresed at 200V-120mA max-60 minutes. The final results revealed the migration of T6M at the expected molecular weight.

[0201] Example 3 Cytotoxicity assay using unstimulated human PBMCs (TDCC) Isolation of effector cells Human peripheral blood mononuclear cells (PBMCs) were prepared from concentrated lymphocyte preparations (buffycoat), a byproduct of blood banks that collect blood for transfusion, by Ficol density gradient centrifugation. Buffycoat was supplied by local blood banks, and PBMCs were prepared the day after blood collection. After Ficol density centrifugation and thorough washing with Dulbecco's PBS (Gibco), residual red blood cells were removed from the PBMCs by warming with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 100 μM EDTA). Residual lymphocytes mainly consisted of B lymphocytes, T lymphocytes, NK cells, and monocytes. PBMCs were maintained under culture conditions of 37°C / 5% CO2 in RPMI medium (Gibco) containing 10% FCS (Gibco).

[0202] Depletion of CD14+ and CD56+ cells For CD14+ cell depletion, human CD14 microbeads (Milteny Biotec, MACS, #130-050-201) were used to deplete NK cell human CD56 microbeads (MACS, #130-050-401). PBMC counts were taken, and the cells were centrifuged at 300xg for 10 minutes at room temperature. The supernatant was discarded, and MACS isolation buffer (60 μL / 10) was used. 7 The cell pellet was resuspended in (10⁷ cells). CD14 microbeads and CD56 microbeads (20 μL / 10⁷ cells) were added and incubated at 4-8°C for 15 minutes. AutoMACS rinse buffer (Milteny #130-091-222) (1-2 ml / 10⁷) 7 The cells were washed with (100 μL / 100 μL). After centrifugation (see above), the supernatant was discarded and MACS isolation buffer (500 μL / 1 8Cells were resuspended in (cells). Next, CD14 / CD56-negative cells were isolated using an LS column (Milteny Biotec, #130-042-401). PBMC w / o CD14+ / CD56+ cells were adjusted to 1.2 x 10⁶ cells / mL and cultured in an incubator at 37°C until required in RPMI 1640 (Biochrom AG, #FG1215) supplemented with 10% FBS (Bio West, #S1810), 1x non-essential amino acids (Biochrom AG, #K0293), 10 mM Hepes buffer (Biochrom AG, #L1613), 1 mM sodium pyruvate (Biochrom AG, #L0473), and 100 U / mL penicillin / streptomycin (Biochrom AG, #A2213).

[0203] Preparation of target cells The cells were harvested, precipitated by centrifugation, and then incubated in complete RPMI medium at a rate of 1.2 x 10⁴ 5 The cells were adjusted to 1 / mL. Cell activity was measured using Nucleocounter NC-250 (Chemometec) and Solution18 Dye (Chemometec) containing acridine orange and DAPI.

[0204] Luciferase-based analysis This assay was designed to quantify the lysis of target cells in the presence of serial dilutions of a multispecific antibody construct. Equivolutes of luciferase-positive target cells and effector cells (i.e., PBMCs without CD14+ cells; CD56+ cells) were mixed to a 10:1 E:T cell ratio. 42 μL of this suspension was transferred to each well of a 384-well plate. 8 μL of serial dilutions of the corresponding molecule and a negative control molecule (a CD3-based molecule that also recognizes unrelated target antigens) or RPMI complete medium as a further negative control were added. Molecularly mediated cytotoxicity was allowed to proceed for 48 hours in a humidified incubator at 5% CO2. Next, 25 μL of substrate (Steady-Glo® reagent, Promega) was transferred to a 384-well plate. Only living luciferase-positive cells were allowed to react with the substrate, thus generating a luminescence signal. The samples were measured using a SPARK microplate reader (TECAN) and analyzed using Spark Control Magellan software (TECAN).

[0205] The percentage of cytotoxicity was calculated as follows:

number

[0206] Using GraphPad Prism 7.04 software (Graph Pad Software, San Diego), the percentage of cytotoxicity was plotted against the corresponding multispecific antibody construct concentration. Dose-response curves were analyzed using four parametric logistic regression models for evaluating sigmoid dose-response curves with fixed Hill slopes, and EC50 values ​​were calculated. The results of this experiment are shown in Figures 5 and 6, which reveal the in vitro functionality of the tested molecule (HHLL). 2 The molecule exhibits excellent activity at both 48 hours (Figure 5) and 72 hours (Figure 6).

[0207] The following target cell lines were used for the luciferase-based cytotoxic assay: GSU-LUC wt(CDH3+ and MSLN+) GSU-LUC KO CDH3 (CDH3- and MSLN+) GSU-LUC KO MSLN (CDH3+ and MSLN-)

[0208] Any references cited herein are incorporated herein by reference in their entirety for any purpose.

[0209] The present invention is not limited in scope by a single description of each individual embodiment of the invention, nor by any specific embodiment described herein intended to be functionally equivalent as a method and component of the invention. In fact, various modifications of the invention beyond those shown and described herein will become apparent to those skilled in the art from the above description and accompanying drawings. Such modifications are intended to be included within the claims.

[0210] array Typical linker sequences GGGGS (Sequence No. 1) GGGGSGGGGS (Sequence 2) GGGGSGGGGSGGGGS(Sequence 3) GGGGSGGGGSGGGGSGGGGS(Sequence 4) GGGGSGGGGSGGGGSGGGGSGGGGS(CR5) GGGGQ (Sequence No. 6) GGGGQGGGGQ (Sequence 7) GGGGQGGGGQGGGGQ(Sequence 8) GGGGQGGGGQGGGGQGGGGQ(Sequence 9) GGGGQGGGGQGGGGQGGGGQGGGGQ(Sequence 10) GGGGSAAA (Sequence ID 11) TVAAP (Sequence ID 12) ASTKGP (Sequence ID 13) AAA (Sequence ID 14) GGNGT (Sequence ID 15) YGNGT (Sequence ID 16) SGGGGS (Sequence No. 17) SGGGGQ (Sequence No. 18) GGGG (Sequence No. 19) (GGGG)2 (Sequence No. 20) (GGGG)3 (Sequence No. 21) (GGGG)4 (Sequence No. 22) (GGGG)5 (Sequence No. 23) (GGGG)1-10 (Sequence No. 24) (GGGG)2-10 (Sequence No. 25) (GGGG)3-10 (Sequence No. 26) (GGGGS)1-10 (Sequence No. 27) (GGGGS)2-10 (Sequence No. 28) (GGGGS)3-10 (Sequence No. 29) (GGGGQ)1-10 (Sequence ID 30) (GGGGQ)2-10 (Sequence ID 31) (GGGGQ)3-10 (Sequence ID 32)

[0211] Amino acid sequence of mature human CD3ε (SEQ ID NO: 33) [ka]

[0212] Amino acid sequence of mature CD3ε in cynomolgus monkeys (SEQ ID NO: 34) [ka]

[0213] Amino acid sequence of the extracellular domain of human CD3ε (SEQ ID NO: 35) QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMS

[0214] Amino acids 1-27 of human CD3ε (SEQ ID NO: 36) QDGNEEMGGITQTPYKVSISGTTVILT

[0215] T6M maturation (SEQ ID NO: 37)

Chem.

[0216] G7Q maturation (SEQ ID NO: 38)

Chem.

[0217] Anti-methotrexate 15-B12 CC VH (SEQ ID NO: 39)

Chem.

[0218] Anti-methotrexate 15-B12 CC VL (SEQ ID NO: 40) DIVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGCGTKVEIK

[0219] Anti-CD3 6H10.09 VH (SEQ ID NO: 41)

Chem.

[0220] Anti-CD3 6H10.09 VL (SEQ ID NO: 42) QTVVTQEPSLTVSPGGTVTITCGSSTGAVTSGNYPNWIQKKPGQAPRGLIGGTKFLAPGTPARFSGSLEGGKAALTLSGVQPEDEAEYYCVLYYSNRWVFGSGTKLTVL

[0221] Anti-CDH3 15-E11 CC VH (SEQ ID NO: 43)

Chem.

[0222] Anti-CDH3 15-E11 CC VL (SEQ ID NO: 44) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCVQYAQFPLTFGCGTKVEIK

[0223] scFv (SEQ ID NO: 45)

Chem.

[0224] scFv-2 (SEQ ID NO: 46)

Chem.

[0225] scFv-3 (SEQ ID NO: 47)

Chem.

[0226] scFv-4 (SEQ ID NO: 48)

Chem.

[0227] scFv-5 (SEQ ID NO: 49)

Chem.

[0228] scFv-6 (SEQ ID NO: 50)

Chem.

[0229] scFv-7 (SEQ ID NO: 51)

Chemical Structure

[0230] scFv-8 (SEQ ID NO: 52)

Chemical Structure

[0231] scFv Variant (SEQ ID NO: 53)

Chemical Structure

[0232] 2X scFc (SEQ ID NO: 54)

Chemical Structure

[0233] ' heteroFc(A) (SEQ ID NO: 55)

Chemical Structure

[0234] heteroFc(B) (SEQ ID NO: 56)<(

Chemical Structure

[0235] Human Serum Albumin (HSA) (SEQ ID NO: 57)

Chemical Structure

Table 5

[0237] Table 6

Claims

1. Next structure: a. VH1-L1-VH2-L2-VL1-L3-VL2-L4-VH3-L1-VH4-L2-VL3-L3-VL4 or b. VH1-L1-VH2-L2-VL1-L3-VL2-L4-Half-life extension portion-L5-VH3-L1-VH4-L2-VL3-L3-VL4 A molecule comprising a polypeptide chain having, VH1, VH2, VH3, and VH4 are immunoglobulin heavy chain variable regions, VL1, VL2, VL3, and VL4 are immunoglobulin light chain variable regions, and L1, L2, L3, L4, and L5 are linkers, where L1 is 10 to 30 amino acids, L2 is 15 to 30 amino acids, and L3 is 15 to 30 amino acids. The molecule can bind to immune effector cells and target cells, and the half-life extension portion is a single-chain immunoglobulin Fc region ("scFc"). (i) VH1 and VL1 associate and bind to mesothelin, VH2 and VL2 associate and bind to CD3ε, VH3 and VL3 associate and bind to CDH3, and VH4 and VL4 associate and bind to CD3ε, or (ii) A molecule in which VH1 and VL1 associate and bind to CDH3, VH2 and VL2 associate and bind to CD3ε, VH3 and VL3 associate and bind to mesothelin, and VH4 and VL4 associate and bind to CD3ε.

2. The molecule according to claim 1, wherein the half-life extension portion is scFc derived from human IgG1, IgG2, or IgG4 antibody.

3. The molecule according to claim 2, wherein the scFc polypeptide chain comprises one or more modifications that inhibit Fc gamma receptor (FcγR) binding and / or one or more modifications that extend the half-life.

4. The molecule according to claim 1, wherein all of the VH1, VH2, VH3, VH4, VL1, VL2, VL3, and VL4 have different sequences.

5. The molecule according to claim 1, wherein the VH2 and VH4 sequences include sequence number 41, and the VL2 and VL4 sequences include sequence number 42.

6. VH1 (SEQ ID NO: 39) and VL1 (SEQ ID NO: 40) associate and bind to mesothelin, VH2 (SEQ ID NO: 41) and VL2 (SEQ ID NO: 42) associate and bind to CD3ε, VH3 (SEQ ID NO: 43) and VL3 (SEQ ID NO: 44) associate and bind to CDH3, Furthermore The molecule according to claim 1, wherein VH4 (SEQ ID NO: 41) and VL4 (SEQ ID NO: 42) associate and bind to CD3ε.

7. The molecule according to claim 1, wherein L1, L2, and L3 are of different lengths or the same length.

8. The molecule according to claim 1, wherein L1 and L2 are of the same length, or L1 and L3 are of the same length, or L2 and L3 are of the same length.

9. The molecule according to claim 1, which exhibits improved stability when compared to a molecule having the structure VH1-linker-VL1-linker-VH2-linker-VL2-linker-VH3-linker-VL3-linker-VH4-linker-VL4 or VH1-linker-VL1-linker-VH2-linker-VL2-linker-half-life extension portion-linker-VH3-linker-VL3-linker-VH4-linker-VL4.

10. The molecule according to claim 1, which shows improved in vitro expression when compared to a molecule having the structure VH1-linker-VL1-linker-VH2-linker-VL2-linker-VH3-linker-VL3-linker-VH4-linker-VL4 or VH1-linker-VL1-linker-VH2-linker-VL2-linker-half-life extension portion-linker-VH3-linker-VL3-linker-VH4-linker-VL4.

11. The molecule according to claim 1, wherein the effector cell expresses an effector cell protein which is part of the human T cell receptor (TCR)-CD3 complex, and the effector cell protein is a CD3ε chain.

12. A method for producing the molecule according to claim 1, comprising (1) culturing host cells under conditions for expressing the molecule, and (2) recovering the molecule from a cell aggregate or cell culture supernatant, wherein the host cells contain one or more nucleic acids encoding the molecule according to any one of claims 1 to 11.

13. A composition comprising the molecule described in any one of Claims 1 to 11, for the purpose of preventing, treating, or improving cancer.

14. The composition according to claim 13, wherein, in the prevention, treatment, or improvement of symptoms of cancer, a chemotherapeutic agent, a non-chemotherapeutic anti-cancer agent, and / or radiation are administered to the patient simultaneously with, before or after, the administration of the molecule.

15. A pharmaceutical composition comprising the molecule described in any one of claims 1 to 11.

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