Transthyretin-Immunoglobulin Fusion

Abstract

The present invention relates to transthyretin (TTR) fusions that can be used for dimerization and tetramerization of antibodies and antibody fragments such as Fab. The TTR fusion proteins described herein are particularly useful in increasing antibody avidity and enhancing antigen clustering. Methods of treating diseases using the fusion proteins of the present invention are described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 568,217, filed on October 4, 2017, under 35 U.S.C. 119(e), which is incorporated herein by reference. Technical Field

[0003] The present invention relates to transthyretin (TTR) fusions that can be used for dimerization and tetramerization of antibodies and antibody fragments such as Fab. The TTR fusion proteins described herein are particularly useful in increasing antibody avidity and enhancing antigen clustering. Methods of treating diseases using the fusion proteins of the present invention are described herein.

[0004] Reference sequence list

[0005] This application is submitted in electronic format via ePCT together with the sequence listing. The sequence listing is provided as a text file entitled A-2196-WO-PCT_Sequence_Listing_ST25.txt, which was created on October 2, 2018 and is 69,791 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Background Art

[0006] Transthyretin (TTR) is a noncovalent tetrameric human serum and cerebrospinal fluid protein responsible for extending the serum half-life of retinol-binding protein and carrying a portion of circulating thyroxine. The molecular weight of the native human monomer is approximately 14 kDa, although TTR is normally present as a 56 kDa tetrameric serum protein.

[0007] TTR and TTR variants have previously been fused to biologically active agents to increase the serum half-life of such active agents. For example, substantially homogeneous formulations of TTR- (or TTR variant-) biologically active agent fusions and PEG-TTR- (or PEG-TTR variant-) biologically active agent fusions have been developed that exhibit increased serum half-life compared to the biologically active agent alone. See, e.g., US20030191056, which is incorporated herein by reference in its entirety.

[0008] In addition, previous efforts to polymerize proteins include the use of streptavidin (Kipriyanov et al., Protein Engineering, 9(2):203-211 (1996)), helix-turn-helix constructs (Kriangkum et al., Biomolecular Engineering, 18:31-40 (2001)), leucine zippers (Kruif et al., The Journal of Biological Chemistry, 271(13):7630-7634, 1996 (1996)), barnase / barnase inhibitor complexes (Deyev et al., Nature Biotechnology, 21(12):1486-1492 (2003)), and the Dock NLock technique (protein kinase and A kinase anchor protein anchor domain interaction) (Goldenberg et al., Journal of Nuclear Medicine, 2003). Medicine, 49(1):158-163 (2008).

[0009] However, there is still a need for multimeric proteins, such as multimeric whole antibodies and antibody fragments (e.g., Fab), that exhibit enhanced biological and therapeutic properties. For example, there is still a need for multimeric proteins that have increased antibody avidity and enhanced antigen clustering compared to their non-multimeric counterparts. Summary of the invention

[0010] In one aspect, the present invention relates to a homodimeric fusion protein comprising two antigen binding proteins, wherein the antigen binding proteins are linked to a protein complex. In a specific aspect, the protein complex is a TTR protein complex. In a specific aspect, the antigen binding protein is an antibody. In another specific aspect, the antigen binding protein is directly fused to the protein complex without a linker. In another specific aspect, the C-terminus of the antigen binding protein is directly fused to the N-terminus present in the TTR protein complex. In some embodiments, the antigen binding protein is fused to the protein complex via a linker. In another specific aspect, the C-terminus of the antigen binding protein is connected to the N-terminus present in the TTR protein complex. The linker can be an amino acid linker, such as an amino acid linker with a length of 1-20 amino acids. In a specific embodiment, the amino acid linker is GGGGS, (GGGGS) 2 、(GGGGS) 3 、(GGGGS) 4 、(GGGGS) 5 or (GGGGS) 6 .

[0011] In another aspect, the present invention relates to a homotetrameric fusion protein comprising four antigen binding proteins, wherein the antigen binding proteins are linked to a protein complex. In one aspect, the protein complex is a TTR protein complex. In another aspect, the antigen binding protein is an antibody. The antigen binding protein may be a Fab. In some embodiments, the antigen binding protein is directly fused to the protein complex without a linker. In specific embodiments, the C-terminus of the antigen binding protein is directly fused to the N-terminus present in the TTR protein complex. In other embodiments, the antigen binding protein is fused to the protein complex via a linker. The C-terminus of the antigen binding protein may be linked to the N-terminus present in the TTR protein complex. In some embodiments, the linker is an amino acid linker, such as an amino acid linker of 1-20 amino acids in length. In specific embodiments, the amino acid linker is GGGGS, (GGGGS) 2 、(GGGGS) 3 、(GGGGS) 4 、(GGGGS) 5 or (GGGGS) 6 .

[0012] The present invention also relates to pharmaceutical compositions comprising any of the homodimeric or homotetrameric fusion proteins discussed above.

[0013] In addition, the present invention relates to a method for treating cancer using any of the above-mentioned homodimers or homotetramer fusion proteins. Moreover, the present invention relates to the use of any of the above-discussed homodimers or homotetramer fusion proteins in cancer treatment. On the other hand, the present invention relates to any of the above-discussed homodimers or homotetramer fusion proteins for treating cancer.

[0014] In some respects, the present invention relates to one or more separated nucleic acids encoding any homodimer or homotetramer fusion proteins discussed above. Expression vectors comprising such nucleic acids, and recombinant host cells comprising nucleic acids and / or vectors discussed herein are also contemplated. In a specific embodiment, the recombinant host cell is a Chinese hamster ovary (CHO) cell, an E5 cell, a baby hamster kidney (BHK) cell, a monkey kidney (COS) cell, a human hepatocellular carcinoma cell, or a human embryonic kidney 293 (HEK 293) cell.

[0015] Methods for preparing any of the homodimer or homotetramer fusion proteins discussed above are also part of the present invention. Such methods may include: a) culturing the recombinant host cell of claim 32 or 33; and b) isolating the homodimer or homotetramer fusion protein from the culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . Figure 1is a schematic diagram of a homomultimeric construct of the present invention. Figure 1 a is an exemplary TTR antibody homodimeric fusion protein, in which the C-termini of two antibody heavy chains are linked to the N-termini of each TTR subunit. Figure 1 b is an exemplary TTR antibody homotetrameric fusion protein, in which one of the two heavy chains at the C-terminus of each antibody is linked to the N-terminus of each TTR subunit. The "+" and "-" symbols represent Fc charge pairs, which allow each intact antibody to always be linked to one TTR subunit. Figure 1 c is an exemplary TTRFab homotetrameric fusion protein, in which the C-terminus of each Fab fragment is linked to the N-terminus of each TTR subunit. Figure 1 Each of a-1c shows an optional linker between the heavy chain and TTR.

[0017] Figure 2 . Figure 2 is a series of SDS-PAGE gels showing the following: anti-CB1 TTR antibody homodimers without linkers or with linkers of different lengths expressed in large quantities in HEK 293 cells ( Figure 2 a), anti-CB1 TTR antibody homotetramer without linker ( Figure 2 b), and anti-CB1 TTR Fab homotetramer without linker ( Figure 2 c). Figure 2 This is discussed further in Example 2.

[0018] Figure 3 . Figure 3 Without connector, with (G 4 S) connector, belt (G 4 S) 2 Connector, belt (G 4 S) 3 Connector, or (G 4 S) 4 A series of HPLC size exclusion chromatography (SEC) analyses of the anti-CB1 TTR antibody homodimer fusion proteins with linkers. Figure 3 This is discussed further in Example 2.

[0019] Figure 4 . Figure 4 showed that TTR anti-CB1 antibody homotetramer and TTR anti-CB1Fab homotetramer fusion protein improved EC compared with the parental CB1 Ab 50 . Figure 4 This is discussed further in Example 3.

[0020] Figure 5 . Figure 5is an SDS-PAGE gel showing the following expressed in HEK 293 cells: anti-GITR TTR antibody homodimer (lane 1), anti-GITR TTR antibody homotetramer (lane 2), and anti-GITR TTR Fab homotetramer protein (lane 3), respectively. Lanes 4-7 are anti-dinitrophenyl (anti-DNP) antibodies. Figure 5 Further discussed in Example 4.

[0021] Figure 6 . Figure 6 Is an SDS-PAGE gel showing that anti-GITR TTR antibody homodimers (lanes 1 and 4), anti-GITR TTR antibody homotetramers (lanes 2 and 5), and anti-GITR TTR Fab homotetramer proteins (lanes 3 and 6) are correctly assembled based on the unheated non-reducing lanes. After heating and reduction, these three protein fusion constructs are resolved into their expected component chains (one or more upper bands are heavy chains, and the lowest bands are light chains). Figure 6 Further discussed in Example 4.

[0022] Figure 7 . Figure 7 is an HPLC SEC analysis of each of the anti-GITR TTR antibody homodimer (middle peak), TTR antibody homotetramer (left peak), and TTRFab homotetramer (right peak) fusion proteins. Figure 7 Further discussed in Example 4.

[0023] Figure 8 . Figure 8 Differential scanning calorimetry (DSC) analysis of anti-GITR TTR parent mAb ("1"), anti-GITR TTR antibody homotetramer ("2"), anti-GITR TTR Fab homotetramer ("3"), and anti-GITR TTR antibody homodimer ("4") fusion proteins. Figure 8 The melting temperature of the TTR fusion protein was shown to be comparable or better than that of the parental Ab, indicating that the TTR fusion protein formed is robust.

[0024] Fig. 9 . Fig. 9 a) is the result of in vivo (mouse) pharmacokinetic (PK) analysis of total anti-GITR antibody species. Fig. 9 b) is the result of in vivo (mouse) PK analysis of the intact anti-GITR TTR fusion protein.

[0025] Fig.10 . Fig.10a) shows that the binding affinity of anti-GITR TTR Fab homotetramer ("2"), anti-GITR TTR antibody homodimer ("3"), and anti-GITR TTR antibody homotetramer ("4") fusion proteins is better than that of the parental anti-GITR mAb ("1"). Fig.10 b) Demonstration that higher affinity does not translate into higher potency in cell-based assays.

[0026] Fig.11 . Fig.11 is a series of SDS-PAGE gels showing that the anti-TRAILR2 TTR antibody homodimer (1) and anti-TRAILR2 TTR Fab homotetramer (2) proteins are well expressed and correctly assembled in mammalian cells, respectively.

[0027] Fig.12 . Fig.12 is a series of SDS-PAGE gels demonstrating that anti-TRAILR2 TTR antibody homotetramers are well expressed and assembled correctly in CHO-K1 cells.

[0028] Fig.13 . Fig.13 Is a series of SDS-PAGE gels showing that anti-TRAILR2 TTR Fab homotetramers, anti-TRAILR2 TTR antibody homodimers, and anti-TRAILR2 TTR antibody homotetramers are correctly assembled based on the unheated non-reducing lanes. After heating and reduction, the molecules resolve into their expected component chains (one or more upper bands are heavy chains, and the lowest bands are light chains).

[0029] Fig.14 . Fig.14 is an HPLC SEC analysis of each of the anti-TRAILR2 TTR antibody homodimer (middle chromatogram), TTR antibody homotetramer (right chromatogram), and TTR Fab homotetramer (left chromatogram) fusion proteins.

[0030] Fig.15 . Fig.15 a) is the result of in vivo (mouse) PK analysis of total anti-TRAILR2 antibody species. Fig.15 b) is the result of in vivo (mouse) PK analysis of the intact anti-TRAILR2 TTR fusion protein.

[0031] Fig.16 . Fig.16 Shown is the potency of anti-TRAILR2 TTR fusion protein compared to the parental mAb (conatumumab) in a WM35 cell killing assay.

[0032] Fig.17 . Fig.17 Shown are the potency of anti-TRAILR2 TTR Fab homotetramer ("2") and TTR antibody homotetramer ("3") compared to the parental mAb (conatumumab; ("1") in a primary human keratinocyte killing assay.

[0033] Fig.18 . Fig.18 The ability of anti-TRAILR2TTR fusion protein to inhibit tumor growth in the murine colo205 model compared to the parental mAb (conatumumab) is shown.

[0034] Fig.19 . Fig.19 The ability of anti-TRAILR2TTR fusion protein to inhibit tumor growth in the murine SW403 model compared to the parental mAb (conatumumab) is shown.

[0035] Fig. 20 . Fig. 20 It was shown that body weights of murine colo205 model mice and murine SW403 model mice were similar for all tested compounds. DETAILED DESCRIPTION

[0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0037] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms will include pluralities and plural terms will include the singular.

[0038] Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present application can be carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992); and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein and the laboratory procedures and techniques thereof are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0039] It should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein and thus may vary. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the claims.

[0040] Except in the operating examples, or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” The term “about” when used in conjunction with a percentage may mean ±1%.

[0041] All embodiments that are narrower in any way than the scope of variation defined by a particular paragraph herein should be considered to be included in the present disclosure. For example, certain aspects are described as a class, and it should be understood that each member belonging to a class can be an embodiment. Similarly, aspects described as a class or members selected to belong to a class should be understood to include combinations of two or more members in the class. It should also be understood that although multiple embodiments in the specification are presented using "comprising" language, in many cases, "consisting of" or "consisting essentially of" language can also be used to describe related embodiments.

[0042] In this application, the use of "or" means "and / or" unless otherwise specified. In addition, the use of the term "including", as well as other forms such as "includes" and "included", is not limiting. Likewise, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising more than one subunit, unless otherwise expressly specified.

[0043] definition

[0044] "Amino acid" includes its standard meaning in the art. Twenty natural amino acids and their abbreviations follow conventional usage. See, Immunology-A Synthesis [immunology-A synthesis], 2nd edition, (ES Golub and DR Green, ed.), Sinauer Associates: Sunderland, Massachusetts. (1991), which is incorporated herein by reference for any purpose. Twenty conventional amino acids, non-natural amino acids (e.g., [α]-, [α]-disubstituted amino acids), N-alkyl amino acids, and other unconventional amino acid stereoisomers (e.g., D-amino acids) can also be suitable components of polypeptides and are included in the term "amino acid". Examples of unconventional amino acids include: 4-hydroxyproline, [γ]-carboxyglutamate, [ε]-N,N,N-trimethyllysine, [ε]-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, [σ]-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction in accordance with standard usage and convention.

[0045] As used herein, "antagonists" generally refer to molecules that can bind to an antigen and inhibit, reduce or eliminate biological signaling associated with the antigen, such as the antigen binding proteins provided herein.

[0046] The term "antibody" refers to an immunoglobulin of any isotype, or a fragment thereof that can compete with a complete antibody to bind to a target antigen. "Antibody" is a type of antigen binding protein. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies. The antibody can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the antibody comprises at least two full-length heavy chains and two full-length light chains. In other embodiments, the antibody comprises fewer chains, such as antibodies naturally present in camelids, which may comprise only heavy chains. The antibody may be derived only from a single source, or may be a "chimeric" antibody, in which different parts of the antibody are derived from two different antibodies, as further described below. Antigen binding proteins, antibodies, or binding fragments may be produced, for example, in hybridomas, by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody.

[0047] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a binding agent such as an antigen binding protein (including, for example, an antibody) and can additionally be used in an animal to produce antibodies that can bind to the antigen. An antigen may have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0048] As used herein, "antigen binding protein" means any protein that specifically binds to a particular target antigen. The term includes polypeptides that include at least one antigen binding region. The term also encompasses intact antibodies that include at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutations thereof. Antigen binding proteins also include Fab, Fab', F(ab') 2 , Fv fragments, domain antibodies (e.g. ) and single-chain antibodies, as described in more detail below.

[0049] "Antigen binding region" or "antigen binding domain" means the portion of a protein (e.g., an antibody or fragment, derivative, or variant thereof) that specifically binds to, interacts with, or recognizes a given epitope or site on a molecule (e.g., an antigen). For example, the portion of an antigen binding protein that contains amino acid residues that interact with an antigen and impart its specificity and affinity to the antigen is called an "antigen binding region." An antigen binding region may include one or more "complementarity determining regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions. The "framework" region may directly contribute to the specific binding of the antigen binding protein, but generally helps to maintain the proper conformation of the CDRs to promote binding between the antigen binding region and the antigen.

[0050] The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, epithelial cancers, including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More specific examples of such cancers include melanoma, lung cancer, head and neck cancer, renal cell carcinoma, colon cancer, colorectal cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (e.g., hepatocellular carcinoma and hepatoma), bladder cancer, breast cancer, endometrial cancer, myeloma (e.g., multiple myeloma), salivary gland cancer, kidney cancer (e.g., renal cell carcinoma and Wilms' tumor), basal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, and esophageal cancer.

[0051] The term "CDR" and its plural "multiple CDRs" (also called "hypervariable regions") refer to the complementary determining regions of a protein, such as an antibody, or a fragment, derivative, or variant thereof. The light chain variable region and the heavy chain variable region each contain three CDRs. For example, the light chain variable region contains the following CDRs: CDR-L1, CDR-L2, and CDR-L3; and the heavy chain variable region contains the following CDRs: CDR-H1, CDR-H2, and CDR-H3. CDRs contain most of the residues that are responsible for the specific interaction of the antibody with the antigen, and therefore contribute to the functional activity of the antibody molecule. CDRs are the main determinants of antigen specificity.

[0052] Accurately defined CDR boundaries and lengths are subject to different classifications and numbering systems. Therefore, CDRs can be referenced by Kabat, Chothia, contact or any other boundary definition (including numbering systems described herein). The Kabat numbering scheme (system) is a widely used standard for numbering the amino acid residues of antibody variable domains in a consistent manner, and is a preferred embodiment of the present invention, as also mentioned elsewhere herein. Other structural considerations can also be used to determine the canonical structure of antibodies. For example, those differences that are not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al., and / or revealed by other techniques (such as crystallography and two-dimensional or three-dimensional computational modeling). Despite different boundaries, each of these systems has a certain degree of overlap in terms of constituting the "CDR" in the variable sequence. Therefore, the CDR definitions according to these systems can be different in length and boundary regions relative to adjacent framework regions. See, e.g., Kabat (a method based on cross-species sequence variability), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., loc. cit.; Chothia et al., J. Mol. Biol., 1987, 196:901-917; and MacCallum et al., J. Mol. Biol., 1996, 262:732). Yet another standard for characterizing antigen binding sites is the AbM definition used by the AbM antibody modeling software of Oxford University Molecular. See, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains In: Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow et al., eds., 1988.

[0053] Typically, CDR forms a loop structure that can be classified as a canonical structure. The term "canonical structure" refers to the main chain conformation used by the antigen binding (CDR) loop. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited set of available conformational repertoires. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Therefore, the corresponding loops between antibodies may have very similar three-dimensional structures, but most parts of the loops have high amino acid sequence variability (Chothia and Lesk, J.MoI.Biol. [Molecular Biology Journal], 1987, 196: 901; Chothia et al., Nature [Nature], 1989, 342: 877; Martin and Thornton, J.MoI.Biol [Molecular Biology Journal], 1996, 263: 800). In addition, there is a relationship between the loop structure used and the amino acid sequence around it. The conformation of a specific canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop and within the conservative framework (i.e., outside the loop). Therefore, assignment to a specific canonical class can be made based on the presence of these key amino acid residues.

[0054] When used in the context of antigen binding proteins (e.g., antibodies or fragments thereof) that compete for the same epitope, the term "competition" means competition between the antigen binding proteins and is determined by an assay in which the antigen binding protein (e.g., antibody or fragment thereof) being tested prevents or inhibits specific binding of a reference antigen binding protein to a common antigen. Many types of competitive binding assays can be used, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct labeled assays, solid phase direct labeled sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1996). Press [Antibodies, a laboratory manual, Cold Spring Harbor Press]); solid phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. [Molecular Immunology] 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology [Virology] 176:546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. [Scandinavian Journal of Immunology] 32:77-82). Typically, such assays involve the use of purified antigen or cells expressing the antigen bound to a solid surface, an unlabeled test antigen binding protein, and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein and antigen binding proteins that bind to the epitope that is close enough to the reference antigen binding protein to bind to the adjacent epitope (to cause steric hindrance). Other details about the method for determining competitive binding are provided herein. For example, in one embodiment, competition is determined according to BiaCore determination. Typically, when competitive antigen binding proteins are present in excess, they will inhibit the specific binding of the reference antigen binding protein to the common antigen by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or more than 97%.

[0055] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of the coding sequence linked thereto. The nature of such control sequences may depend on the host organism. In a particular embodiment, the control sequence of a prokaryotic organism may include a promoter, a ribosome binding site, and a transcription termination sequence. For example, the control sequence of a eukaryotic organism may include a promoter comprising one or more transcription factor recognition sites, a transcription enhancer sequence, and a transcription termination sequence. A "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0056] A "derivative" of a polypeptide is a polypeptide that has been chemically modified in some manner other than insertion, deletion or substitution variants, such as by conjugation to another chemical moiety.

[0057] "Domain antibodies" are immunologically functional immunoglobulin fragments that contain only the variable region of a heavy chain or the variable region of a light chain. Examples of domain antibodies include In some cases, two or more V H The two V regions of the bivalent domain antibody are H The regions may target the same or different antigens.

[0058] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or potential causes, prevent the occurrence of symptoms and / or their potential causes, and / or improve or cure the damage caused by or associated with cancer. In some embodiments, an effective amount is a therapeutically effective amount or a preventive effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state (e.g., cancer) or symptom, a state or symptom particularly associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "preventive effective amount" is an amount of a pharmaceutical composition that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of cancer, or reducing the likelihood of the onset (or recurrence) of cancer or cancer symptoms. A single dose may not result in a complete therapeutic or preventive effect, and may only occur after a series of doses are administered. Therefore, a therapeutic or preventive effective amount may be administered once or multiple times.

[0059] The term "epitope" refers to an antigenic part that can be recognized and specifically bound by an antigen binding protein (e.g., an antibody). In the case of a polypeptide, an epitope can be formed from continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by continuous amino acids are usually retained when the protein is denatured, while epitopes formed by tertiary folding are usually lost after protein denaturation. An epitope usually contains at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. A "linear epitope" or "sequential epitope" is an epitope that an antigen binding protein (e.g., an antibody) recognizes by its linear sequence or primary structure of amino acids. A "conformational epitope" or "non-sequential epitope" is an epitope that an antigen binding protein (e.g., an antibody) recognizes by its tertiary structure. The residues that make up these epitopes may not be continuous in the primary amino acid sequence, but are close to each other in the tertiary structure of the molecule. When a protein is denatured, broken, or reduced, linear and conformational epitopes usually behave differently.

[0060] The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or controls (together with the host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of a coding region operably linked thereto.

[0061] The "Fab fragment" or "Fab" consists of a light chain and a heavy chain C H 1 and variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0062] A "Fab' fragment" or "Fab" contains one light chain and one heavy chain. H Domain and C H 1 domain and between C H 1 and C H The region between the two Fab' domains allows for the formation of an interchain disulfide bond between the two heavy chains of the two Fab' fragments, thereby forming a F(ab') 2 molecular.

[0063] “F(ab') 2 Fragment" or "F(ab') 2 " contains two light chains and two heavy chains, the heavy chain containing C H 1 and C H A portion of the constant region between the two heavy chains that allows the formation of interchain disulfide bonds. 2 The fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0064] The "Fc" region contains two C H 2 and C H The two heavy chain fragments are linked by two or more disulfide bonds and C H The 3 domains are held together by hydrophobic interactions.

[0065] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0066] The term "heavy chain" when used with respect to an antigen binding protein, antibody, or fragment thereof includes a full-length heavy chain and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length heavy chain includes a variable region domain (V H ) and three constant region domains (C H 1. C H 2. and C H 3). V H The domain is located at the amino terminus of the polypeptide, and the C H The domain is located at the carboxyl terminus, where C H 3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can be of any isotype, such as IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

[0067] "Hematological cancers" are cancers that begin in blood-forming tissues (such as the bone marrow) or in cells of the immune system. Examples of blood cancers are leukemias, lymphomas, and multiple myeloma.

[0068] The term "homodimeric fusion protein" refers to a fusion protein comprising two identical antigen binding proteins. For example, an antibody homodimeric fusion protein refers to a fusion protein comprising two identical antibodies. In a specific example, the homodimer can be a TTR homodimeric fusion protein comprising two identical antibodies linked via a TTR protein as described herein.

[0069] The term "homotoquemer fusion protein" refers to a fusion protein comprising four identical antigen binding proteins. For example, an antibody homotetraquemer fusion protein refers to a fusion protein comprising four identical antibodies. In another example, a Fab homotetraquemer fusion protein refers to a fusion protein comprising four identical Fab fragments. In a specific example, the homotetramer can be a TTR homotetraquer fusion protein, which comprises two identical antigen binding proteins (e.g., two identical antibodies, or two identical Fab fragments) connected via a TTR protein as described herein.

[0070] The term "host cell" means a cell that has been transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes progeny of a parent cell, whether or not the progeny is identical to the original parent cell in morphology or genetic composition, as long as the gene of interest is present.

[0071] The term "identity" means a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of sequences. "Identity percentage" means the percentage of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the compared molecules. For these calculations, gaps in the alignment, if any, must be resolved using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer [Introduction to Sequence Analysis], (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0072] When calculating the percent consistency, the compared sequences are aligned in a manner that achieves the maximum match between each sequence. The computer program used to determine the percent identity is the GCG software package, which includes GAP (Devereux et al., 1984 Nucl. Acid Res. [Nucleic Acids Research] 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wisconsin). The computer algorithm GAP is used to compare two polypeptides or polynucleotides to be determined for the percent identity of the sequence. These sequences are aligned for the best match of their respective amino acids or nucleotides (such as the "matching range" determined by the algorithm). Gap open penalty (calculated as 3x diagonal average, where "diagonal average" is the average of the diagonal of the comparison matrix used; "diagonal" is the score or number given to each perfect amino acid match by a specific comparison matrix) and gap extension penalty (usually 1 / 10 times the gap open penalty) and comparison matrix, such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm. In certain embodiments, the algorithm also uses a standard comparison matrix (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0073] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program are as follows:

[0074] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

[0075] Comparison matrix: BLOSUM 62, from Henikoff et al., 1992, supra;

[0076] Gap Penalty: 12 (but no penalty for gap at the end)

[0077] Gap length penalty: 4

[0078] Similarity threshold: 0

[0079] Certain alignment schemes used to align two amino acid sequences can result in only a shorter region of the two sequences being matched, and this smaller alignment region can have a very high sequence identity, even though there is no obvious relationship between the two full-length sequences. Therefore, if necessary, the selected alignment method (GAP program) can be adjusted to align over at least 50 consecutive amino acids of the target polypeptide.

[0080] The phrase "immunomodulator" refers to a molecule that induces, enhances, or suppresses an immune response. An immunoactivator is a molecule that induces or amplifies an immune response. An immunosuppressant is a molecule that reduces or suppresses an immune response. Thus, activation immunotherapy is a therapy that involves administering one or more molecules to induce or enhance a subject's immune system. Suppression immunotherapy is a therapy that treats a subject with one or more molecules to reduce or suppress the subject's immune system.

[0081] As used herein, the term "fragment" of an antibody or immunoglobulin chain (heavy chain or light chain) is an antigen binding protein that comprises a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but is capable of specific binding to an antigen (regardless of how the portion is obtained or synthesized). Such fragments have biological activity because they specifically bind to the target antigen and can compete with other antigen binding proteins (including intact antibodies) for binding to a given epitope. In one aspect, such fragments will retain at least one CDR present in a full-length light or heavy chain, and in some embodiments will comprise a single heavy and / or light chain or a portion thereof. These biologically active fragments can be produced by recombinant DNA technology, or can be produced by enzymatic or chemical cleavage of antigen binding proteins (including intact antibodies). Immunoglobulin fragments with immune function include, but are not limited to, Fab, Fab', F(ab') and Fab'. 2 , Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to humans, mice, rats, camelids or rabbits. It is further contemplated that the functional portion of the antigen binding protein disclosed herein, such as one or more CDRs, can be covalently bound to a second protein or small molecule to produce a therapeutic agent against a specific target in vivo or with an extended serum half-life.

[0082] "Isolated nucleic acid molecule" means a DNA or RNA of genomic, mRNA, cDNA or synthetic origin or some combination thereof, which is not associated with all or part of a polynucleotide (where the isolated polynucleotide is found in nature) or is linked to a polynucleotide to which it is not linked in nature. For the purposes of this disclosure, it is understood that a "nucleic acid molecule" that "comprising" a specific nucleotide sequence does not encompass a complete chromosome. An isolated nucleic acid molecule that "comprising" a specified nucleic acid sequence may include, in addition to these specified sequences, coding sequences for up to ten or even up to twenty other proteins or portions thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences.

[0083] As used herein, the terms "isolated polypeptide," "purified polypeptide," "isolated protein," or "purified protein" are intended to refer to a composition separated from other components, wherein the polypeptide is purified to any degree relative to its naturally accessible state. Thus, a purified polypeptide also refers to a polypeptide that is not in the environment in which it is naturally present. Generally, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components and that substantially retains the biological activity expressed by it. When the term "substantially purified" is used, the name will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, e.g., constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0084] The term "light chain" when used in reference to an antigen binding protein, antibody, or fragment thereof includes a full-length light chain and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length light chain includes a variable region domain (V L ) and constant region domain (C L ). The variable region domain of the light chain is located at the amino terminus of the polypeptide. The light chain includes a kappa chain and a lambda chain.

[0085] The term "naturally occurring" as used throughout this specification in conjunction with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials found in nature.

[0086] The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotides. In certain embodiments, the length of the oligonucleotide is 10 to 60 bases. In other embodiments, the length of the oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 nucleotides. The oligonucleotide can be single-stranded or double-stranded, for example, for constructing a mutant gene. The oligonucleotide can be a sense or antisense oligonucleotide. The oligonucleotide can include a label, including a radioactive label, a fluorescent label, a hapten or an antigen label, for detection and determination. The oligonucleotide can be used as, for example, a PCR primer, a cloning primer or a hybridization probe.

[0087] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under suitable conditions. For example, a control sequence "operably linked" to a protein coding sequence in a vector is linked to the coding sequence so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0088] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. Modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide bond modifications, such as phosphorothioates, phosphorodithioates, selenophosphates, diselenphosphates, phenylamidophosphorothioates, phenylamidophosphorothioates, and phosphoramidates.

[0089] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as 5'. The 5' to 3' direction of addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region that is the 5' end of the 5' end of the RNA transcript on the same DNA strand as the RNA transcript is referred to as the "upstream sequence"; the sequence region that is the 3' end of the 3' end of the RNA transcript on the same DNA strand as the RNA transcript is referred to as the "downstream sequence".

[0090] The terms "polypeptide" or "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms are also applicable to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. These terms may also encompass amino acid polymers that have been modified, for example, by adding carbohydrate residues (to form glycoproteins) or by phosphorylation. Polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein or molecules having one or more amino acids of a native sequence that are deleted, added and / or substituted. The term "polypeptide fragment" refers to a polypeptide having an amino terminal deletion, a carboxyl terminal deletion and / or an internal deletion compared to the full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein. In certain embodiments, the length of the fragment is about 5 to 500 amino acids. For example, a fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0091] A "recombinant protein," including a recombinant TTR protein, is a protein produced using recombinant technology, ie, by expressing a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

[0092] "Single chain antibody" refers to an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0093] "Solid tumor" refers to an abnormal growth or mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign (noncancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, epithelial cancers, and lymphomas. Leukemias (cancers of the blood) do not usually form solid tumors.

[0094] An antigen binding protein "specifically binds" an antigen when it exhibits little to no binding to molecules other than the antigen. However, an antigen binding protein that specifically binds to an antigen may cross-react with antigens from different species. Typically, the dissociation constant (K) is measured as measured by surface plasmon resonance technology (e.g., BIACore, GE Healthcare, Uppsala, Sweden). D )≤10 -7When the antigen binding protein is at K, the antigen binding protein specifically binds to the antigen. D ≤5x10 -8 When the antigen binding protein binds to the antigen with K, the antigen binding protein specifically binds to the antigen with "high affinity", and when the antigen binding protein binds to the antigen with K, the antigen binding protein specifically binds to the antigen with "high affinity". D Yes ≤5x 10 -9 When M binds to an antigen, the antigen binding protein specifically binds to the antigen with "very high affinity" (as measured using methods such as BIACore).

[0095] As used herein, a "subject" or "patient" can be any mammal. In a typical embodiment, the subject or patient is a human.

[0096] As used herein, "substantially pure" means that the molecule of the described species is the predominant species present, i.e., on a molar basis, it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the target species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will constitute at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the target species is purified to substantial homogeneity, wherein contaminating species cannot be detected in the composition by conventional detection methods and thus the composition consists of a single detectable macromolecular species.

[0097] The term "treatment" refers to any sign of successful treatment or improvement of an injury, lesion or condition, including any objective or subjective parameter, such as alleviation; relief; weakening symptoms or making an injury, lesion or condition more tolerable to the patient; slowing the rate of degeneration or weakness; making the degeneration endpoint less debilitating; improving the patient's physical or mental health. The basis for the treatment or relief of symptoms can be objective or subjective parameters; including the results of physical examinations, neuropsychiatric examinations and / or psychiatric assessments. For example, certain methods proposed herein successfully treat cancer and tumors by, for example, reducing the progression or spread of cancer, inhibiting tumor growth, causing remission of tumors and / or alleviating symptoms associated with cancer or tumors. Similarly, other methods provided herein treat infectious diseases by reducing the progression or spread of infection, reducing the extent of infection and / or alleviating symptoms associated with infection.

[0098] As used herein, the term "TTR" refers to "transthyretin". Human TTR is described in Mita et al., Biochem. Biophys. Res. Commun., 124(2):558-564 (1984) (which is incorporated herein by reference). The amino acid sequence of human TTR is also described in the UniProt knowledgebase (www.uniprot.org / uniprot / P02766#sequences) and is cited herein as SEQ ID NO:43. The nucleic acid sequence of human TTR is also described in NCBI (www.ncbi.nlm.nih.gov / gene / 7276). See also GenBank Accession No. K02091.1. The amino acid and nucleic acid sequences of mouse TTR are listed in SEQ ID NOs:3 and 4, respectively.

[0099] The term "TTR variant" refers to a protein having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to TTR having SEQ ID NO: 1. The invention also includes nucleic acids encoding such TTR variants. Specific variants include, for example, TTR proteins that are truncated at the C-terminus or N-terminus.

[0100] A "tumor" is a mass of tissue that forms as cancer cells grow and multiply, and can invade and destroy normal adjacent tissue. Cancer cells can break away from a malignant tumor and enter the blood or lymphatic system, allowing the cancer cells to spread from the primary tumor to form new tumors in other organs.

[0101] A "variant" of a polypeptide comprises an amino acid sequence in which one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0102] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include expression vectors of other forms with equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0103] Homodimeric fusion protein

[0104] As described herein, the present invention relates in part to the use of TTR in the multimerization of antigen binding proteins, such as antibodies. Because TTR is a human extracellular protein found in human serum, its content in the human body is relatively high. Therefore, when compared to, for example, non-human, intracellular and rare proteins, TTR is less likely to elicit an immune response when present in the multimeric constructs of the present invention. Therefore, its use in the multimerization technology of the present invention is advantageous.

[0105] For example, TTR can be used for dimerization of antibodies. In such a homodimeric fusion protein, TTR (SEQ ID NO: 1) or a variant thereof exists in a tetrameric form, wherein the TTR subunit is linked to the C-terminus of the antibody heavy chain to form a TTR antibody homodimer. For example, the C-terminus of each antibody heavy chain (each antibody contains two such C-termini) can be linked to the N-terminus of each TTR subunit (see Figure 1 a) Thus, each antibody is linked to two TTR subunits in the TTR tetramer, generating TTR antibody homodimers.

[0106] Thus, the present invention relates to homodimeric fusion proteins comprising two antigen binding proteins. In some embodiments, the homodimeric fusion protein comprises an antigen binding protein linked to a protein complex. In some embodiments, the protein complex is a TTR protein complex, wherein the TTR protein complex is a TTR tetramer. In some embodiments, the antigen binding protein is an antibody.

[0107] In a specific embodiment, the invention relates to a homodimeric fusion protein comprising two antibodies linked to a TTR tetramer. The antibody can be linked to the TTR tetramer without a linker (ie, the antibody is directly linked to TTR).

[0108] In other embodiments, the antibody is connected to the TTR tetramer via a linker. For example, an amino acid linker can be used to connect the C-terminus of the antibody heavy chain to the N-terminus of the TTR subunit. In some embodiments, the length of the linker is 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35 or 1-40 amino acids. In some embodiments, the length of the linker is 0, 1, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. In other embodiments, the length of the linker is 0, 1, 5, 10, 15, 20, 25, 30, 35 or 40 amino acids. In other embodiments, the length of the linker is up to 5, 10, 15, 20, 25, 30, 35 or 40 amino acids. In some embodiments, the length of the linker is up to 5, 10, 15 or 20 amino acids. In specific embodiments, the length of the linker is 0, 5, 10, 15 or 20 amino acids.

[0109] In some embodiments, the linker is GGGGS, GGGGSGGGGS (i.e., (GGGGS) 2 ), GGGGSGGGGSGGGGS (ie (GGGGS)3), GGGGSGGGSGGGGSGGGGS (ie (GGGGS) 4 ), GGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 5 ), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 6 In other embodiments, it is GGGGS, GGGGSGGGGS (i.e. (GGGGS) 2 ), GGGGSGGGGSGGGGS (i.e. (GGGGS) 3 ), or GGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 4 ).

[0110] Other suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n=1-10), (EAAAK) n (n=1-5), A(EAAAK) 4ALEA(EAAAK) 4 A. PAPAP, AEAAAKEAAAKA, (Ala-Pro) n (n=1-20), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG and LE. Suitable non-amino acid linkers include polyethylene glycol (PEG).

[0111] In some embodiments, the antibody is linked to a truncated TTR subunit with or without a linker. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be removed from the N-terminus of one or more TTR subunits, and the antibody can be attached to the N-terminus of the truncated TTR subunit.

[0112] The present invention also relates to nucleic acid molecules encoding the homodimeric fusion proteins described herein.Details on exemplary methods of producing homodimeric fusion proteins can be found in the Examples.

[0113] Tetramerization of Antibodies and Fabs

[0114] As described herein, the present invention relates in part to the use of TTR in the multimerization of antigen binding proteins, such as antibodies. As described above, the use of TTR in the multimerization techniques of the present invention is advantageous because TTR is less likely to cause an immune response when present in the multimerization constructs of the present invention when compared to, for example, non-human intracellular and rare proteins.

[0115] The present invention also relates in part to the use of TTR in the tetramerization of antigen binding proteins such as antibodies. In such homotetrameric fusion proteins, TTR (SEQ ID NO: 1) or its variants are again present in tetrameric form. However, in the case of TTR antibody homotetramers, a single antibody heavy chain (i.e., only one of the two heavy chains present in a single antibody) is attached to each TTR subunit, thereby allowing four antibodies to be attached to a TTR tetramer (see Figure 1 b). One of the two heavy chains at the C-terminus of the antibody can be linked to the N-terminus of each TTR subunit (see Figure 1 b) Thus, each antibody is linked to one TTR subunit in the TTR tetramer, generating a TTR antibody homotetramer.

[0116] In this homotetrameric fusion protein, the formation of Fc homodimers (as described above) is unfavorable by mutations in Fc. Such modifications include Fc mutations such as knob-hole, DuoBodies, Azymetric, charge pairs, HA-TF, SEEDbody, and modifications with differential protein A affinity. See, e.g., Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. Knob-hole mutations include T366W in the first heavy chain, and T366S, L368A, and / or Y407V in the second heavy chain. See, e.g., Ridgway et al., Protein Eng., 9(1996), pp. 617-621; and Atwell et al., J. Mol. Biol., 270(1997), pp. 26-35. DuoBody mutations include F405L in the first heavy chain and K409R in the second heavy chain. See, e.g., Labrijn et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 110 (2013), pp. 5145-5150. Azymetric mutations include T350V, L351Y, F405A and / or Y407V in the first heavy chain and T350V, T366L, K392L and / or T394W in the second heavy chain. See, e.g., Von Kreudenstein et al., mAbs, 5 (2013), pp. 646-654. HA-TF mutations include S364H and / or F405A in the first heavy chain, and Y349T and / or T394F in the second heavy chain. See, e.g., Moore et al., mAbs, 3 (2011), pp. 546-557. SEEDbody mutations include IgG / A chimeric mutations in the first heavy chain and IgG / A chimeric mutations in the second heavy chain. See, e.g., Davis et al., Protein Eng. Des. Sel. [Protein Engineering Design and Selection], 23 (2010), pp. 195-202. Differential protein A affinity mutations include H435R in one heavy chain and no mutation in the other heavy chain. See, e.g., U.S. Patent No. 8,586,713. Each of these documents is incorporated by reference in its entirety.

[0117] In certain embodiments, it is possible to drive homotetramerization of antibodies by using Fc charge pairs that disfavor homodimerization of antibody heavy chains and thus favor heavy chain heterodimerization between one antibody heavy chain attached to a TTR subunit and one antibody heavy chain not attached to TTR (see Figure 1 b) For example, a set of charged mutations can be incorporated into the C HIn the 3 domains, one heavy chain carries a negative charge and the corresponding heavy chain carries a positive charge, or a mixture of negative and positive charges on one heavy chain (which is paired with the corresponding positive and negative charges on the corresponding heavy chain). Exemplary negative charges include K392D and K409D, and exemplary positive charges include E356K and D399K. H 3. Similar charges on the interface repel each other while heterogeneous charges attract, so homodimerization is unfavorable, while heterodimerization is favorable. TTR is fused to heavy chains of only one charge type (positive or negative, but not both), so this results in a complete antibody consisting of four chains (two light chains, one unfused heavy chain, and one TTR fused heavy chain) for one TTR subunit. Additional charge pair mutations are discussed, for example, in USP 9,546,203. Charge pair mutations (including D221E, P228E, and / or L368E in the first heavy chain and D221R, P228R, and / or K409R in the second heavy chain) are also described, for example, in Strop et al., J. Mol. Biol [Journal of Molecular Biology]., 420 (2012), pp. 204-219. Each of these documents is incorporated by reference in its entirety.

[0118] The present invention also relates in part to the use of TTR in tetramerization of Fab fragments. In such homotetrameric fusion proteins, TTR (SEQ ID NO: 1), or a variant thereof, is again present in tetrameric form, wherein each TTR subunit is linked to the C-terminus of each Fab fragment to form a TTR Fab homotetramer (see Figure 1 c) Thus, each Fab fragment antibody is linked to a single TTR subunit in the TTR tetramer, thereby generating a TTR Fab homotetramer.

[0119] Thus, the present invention relates to homotetrameric fusion proteins comprising four antigen binding proteins (e.g., Fab tetramers) or eight antigen binding proteins (e.g., Ab tetramers). In some embodiments, the homotetrameric fusion protein comprises an antigen binding protein linked to a protein complex. In some embodiments, the protein complex is a TTR protein complex, wherein the TTR protein complex is a TTR tetramer. In some embodiments, the antigen binding protein is an antibody. In other embodiments, the antigen binding protein is a Fab fragment.

[0120] In specific embodiments, the invention relates to homotetrameric fusion proteins comprising four antibodies linked to a TTR tetramer. In other embodiments, the invention relates to homotetrameric fusion proteins comprising four Fab fragments linked to a TTR tetramer. In some embodiments, the antibody or Fab is linked to the TTR tetramer without a linker (i.e., the antibody or Fab is directly linked to TTR).

[0121] In other embodiments, the antibody or Fab is connected to the TTR tetramer via a linker. For example, an amino acid linker can be used to connect the C-terminus of the antibody heavy chain to the N-terminus of the TTR subunit. In some embodiments, the length of the linker is 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35 or 1-40 amino acids. In some embodiments, the length of the linker is 0, 1, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. In other embodiments, the length of the linker is 0, 1, 5, 10, 15, 20, 25, 30, 35 or 40 amino acids. In other embodiments, the length of the linker is up to 5, 10, 15, 20, 25, 30, 35 or 40 amino acids. In some embodiments, the length of the linker is up to 5, 10, 15 or 20 amino acids. In specific embodiments, the length of the linker is 0, 5, 10, 15 or 20 amino acids.

[0122] In some embodiments, the linker is GGGGS, GGGGSGGGGS (i.e., (GGGGS) 2 ), GGGGSGGGGSGGGGS (ie (GGGGS)3), GGGGSGGGSGGGGSGGGGS (ie (GGGGS) 4 ), GGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 5 ), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 6 In other embodiments, it is GGGGS, GGGGSGGGGS (i.e. (GGGGS) 2 ), GGGGSGGGGSGGGGS (i.e. (GGGGS) 3 ), or GGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 4 ).

[0123] Other suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n=1-10), (EAAAK) n (n=1-5), A(EAAAK) 4 ALEA(EAAAK) 4 A. PAPAP, AEAAAKEAAAKA, (Ala-Pro) n(n=1-20), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG and LE. Suitable non-amino acid linkers include polyethylene glycol (PEG) and triazine-containing moieties (contained in constructs having terminal groups capable of reacting with proteins; see, e.g., PCT Publication No. WO / 2017 / 083604, which is incorporated herein by reference in its entirety).

[0124] In some embodiments, the antibody or Fab is linked to a truncated TTR subunit with or without a linker. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be removed from the N-terminus of one or more TTR subunits, and the antibody or Fab can be attached to the truncated TTR subunit N-terminus.

[0125] The present invention also relates to nucleic acid molecules encoding the homodimeric fusion proteins described herein.Details on exemplary methods of producing homotetrameric (TTR and Ab) fusion proteins can be found in the Examples.

[0126] Antigen Binding Protein

[0127] Any antigen binding protein (e.g., Fab or antibody) can be used for the TTR fusion protein of the present invention. Because the fusion protein of the present invention allows antigen binding protein multimerization, antigen binding proteins that target / bind to antigens (wherein antigen binding protein clustering or affinity is required to be active) can particularly benefit from the fusion protein of the present invention. Therefore, in some embodiments, the antigen binding protein (e.g., antibody or Fab) targets / binds to: 4-1BB (CD137), CD20, GITR, DR5, OX40 (CD134), ICOS (CD278) or CD27. Such proteins / targets have been shown to play a role in cancer pathways. In other embodiments, the antigen binding protein (e.g., antibody or Fab) targets / binds to: ErbB-1 (epidermal growth factor receptor (EGFR)), ErbB-2 (HER2 in humans and neu in rodents), ErbB-3 (HER3), ErbB-4 (HER4), FGFR (fibroblast growth factor receptor), VEGFR (vascular endothelial growth factor), RET protein product, EGFR, KIT protein product, Abl (Abelson murine leukemia viral oncogene homolog 1), Raf (rapidly accelerated fibrosarcoma) kinase, or PDGFR (platelet-derived growth factor receptor).

[0128] In one embodiment, the antigen binding protein (e.g., antibody or Fab) specifically binds to CB1R (anti-cannabinoid receptor-1; gene name Cnrl). The CB1 receptor is a Gi-coupled G protein receptor widely expressed in the CNS and peripheral nervous system. Agonist stimulation of the CB1 receptor results in inhibition of adenylate cyclase activity and activation of mitogen-activated protein (MAP) kinase. Endogenous agonists of the CB1 receptor may include anandamide and anandamide glycerol. Exogenous agonists may include A9-tetrahydrocannabinol. Antagonists or inverse agonists have been shown to reduce body weight and improve metabolic parameters, such as lowering plasma glucose and insulin levels. Therefore, in a specific embodiment, the antigen binding portion of the TTR fusion protein is an anti-CB1R antibody (e.g., anti-CB1R antibody 10D10 having a heavy chain of SEQ ID NO: 5 and a light chain of SEQ ID NO: 11; or an anti-CB1R antibody having a heavy chain of SEQ ID NO: 6 or 7 and a light chain of SEQ ID NO: 11). In other specific embodiments, the antigen binding protein of the TTR fusion protein is an anti-CB1R Fab (e.g., a Fab derived from 10D10, such as a Fab heavy chain of SEQ ID NO: 44 and a Fab light chain of SEQ ID NO: 11). See, e.g., U.S. Patent Publication No. 20160145333, which is incorporated herein by reference.

[0129] In one embodiment, the antigen binding protein (e.g., antibody or Fab) specifically binds to GITR (glucocorticoid-induced TNFR-related protein; TNFRSF18). GITR is sometimes also referred to as activation-induced TNFR family member (AITR), which is a receptor belonging to the TNF receptor superfamily (TNFRSF). It is activated by its cognate ligand GITR ligand (GITRL, TNFSF18). GITR is a type I transmembrane protein containing a cysteine-rich extracellular domain, which is a feature of TNFR family members. For example, the cytoplasmic domain of GITR has close homology with some other TNFR family members (e.g., 4-1BB and CD27) (Nocentini, et al. (1997) Proc. Natl. Acad. Sci [Proceedings of the National Academy of Sciences of the United States]. 94: 6216-6221, which is incorporated herein by reference). GITR activation leads to an enhanced immune response, and this activation has the potential to restore immune responses against infection and tumors. Therefore, molecules capable of activating GITR can be used as immunostimulants in environments where it is desired to trigger an enhanced immune response. Therefore, in specific embodiments, the antigen binding portion of the TTR fusion protein is an anti-GITR antibody (e.g., an anti-GITR antibody 9H6 having a heavy chain SEQ ID NO: 18 and a light chain SEQ ID NO: 25; or an anti-GITR antibody having a heavy chain SEQ ID NO: 19 or 20 and a light chain SEQ ID NO: 25). In other specific embodiments, the antigen binding protein of the TTR fusion protein is an anti-GITR Fab (e.g., a Fab derived from 9H6, such as a Fab heavy chain SEQ ID NO: 21 and a Fab light chain SEQ ID NO: 26). See, e.g., U.S. Patent Publication 20150064204, which is incorporated herein by reference.

[0130] In one embodiment, the antigen binding protein (e.g., antibody or Fab) specifically binds to TRAILR2 (TRAIL receptor 2; also known as DR5 (death receptor 5)). The interaction between TR-2 (tumor necrosis factor (TNF)-related apoptosis-inducing ligand ("TRAIL") receptor-2) and its ligand TRAIL plays a role in inducing apoptosis (see, e.g., Almasan et al., Cytokine & Growth Factor Reviews 14:337-348 (2003)). TRAIL (also known as Apo2 ligand) is a homopolymeric ligand that interacts with four members of the TNF receptor superfamily (TRAIL receptor ("TR") 1 to 4), as well as with the related, soluble, osteoprotegerin ("OPG") receptor. TRAIL binds to TR-1 or TR-2 on the cell surface to trigger apoptosis of the cell. After the initial binding of TRAIL to TR-1 or TR-2, intracellular proteins are recruited to the intracellular death domain of the receptor to form a signaling complex. Certain intracellular caspases are recruited to the complex; here they are automatically activated, in turn activating other caspases and the intracellular apoptotic cascade. TR-3 and TR-4 and OPG lack the intracellular domain responsible for transmitting apoptotic signals. Therefore, the binding of TRAIL to TR-3, TR-4 or OPG does not trigger apoptosis. TR-3 and TR-4 are also known as "decoy" receptors, and their overexpression has been shown to protect cells from apoptosis induction by TRAIL. TR-2 is expressed in a variety of cells, including liver, brain, breast, kidney, colon, lung, spleen, thymus, peripheral blood lymphocytes, prostate, testis, ovary, uterus, and various tissues of the gastrointestinal tract. (See, e.g., Walczak et al., EMBO J. [Journal of the European Molecular Biology Association] 16:5386-5397 (1997); Spierings et al., J. Histochem. Cytochem. [Journal of Histochemistry and Cytochemistry] 52:821-831 (2004), each of which is incorporated herein by reference). Although TRAIL and TRAIL receptors are widely expressed, they are most active in inducing apoptosis in transformed cells. (See, e.g., Daigle et al., Swiss Med. Wkly. [Swiss Medical Weekly] 131:231-237 (2001), which is incorporated herein by reference). Conatumumab, an anti-TRAILR2 monoclonal antibody, is already in development for the treatment of cancer.Thus, in specific embodiments, the antigen binding portion of the TTR fusion protein is an anti-TRAILR2 antibody (e.g., conatumumab having a heavy chain of SEQ ID NO: 31 and a light chain of SEQ ID NO: 38; or an anti-TRAILR2 antibody having a heavy chain of SEQ ID NO: 32 or 33 and a light chain of SEQ ID NO: 38). In other specific embodiments, the antigen binding protein of the TTR fusion protein is an anti-TRAILR2 Fab (e.g., a Fab derived from conatumumab, such as a Fab heavy chain of SEQ ID NO: 34 and a Fab light chain of SEQ ID NO: 39).

[0131] TTR variants

[0132] As described above, TTR variants can also be used in the present invention. Any of the TTR variants discussed herein can be used in combination with each other. TTR variants include proteins having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 86%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a TTR protein having, for example, SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 43.

[0133] In some embodiments, the TTR of the invention comprises the amino acid sequence of human TTR SEQ ID NO: 43. In specific embodiments, the TTR of the invention comprises the amino acid sequence of SEQ ID NO: 43, wherein there is a mutation at K15, C10, or both K15 / C10 (e.g., K15A, C10A, or both K15A / C10A). In specific embodiments, the TTR of the invention comprises both K15A and C10A mutations, and thus has the amino acid sequence of SEQ ID NO: 1.

[0134] Cysteines present in human TTR (e.g., SEQ ID NO: 1 or SEQ ID NO: 43) can be used as conjugation sites with antigen binding proteins (e.g., antibodies and Fabs). In addition, TTR variants capable of site-specific conjugation, such as TTR variants with engineered cysteines, can be used in the present invention. See, e.g., USP 8,633,153, which is incorporated herein by reference. For example, a TTR variant can comprise one or more of the following cysteine ​​mutations: A37C, D38C, A81C, or G83C.

[0135] Other variants useful in the present invention include, for example, TTR proteins with truncations at the C-terminus or N-terminus. Such TTR proteins include those in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids are removed from the C-terminus or N-terminus of the TTR protein. In some embodiments, the fusion protein of the present invention comprises a TTR protein in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acids are removed from the C-terminus or N-terminus of the TTR protein. In other embodiments, the fusion protein of the present invention comprises a TTR protein in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acids are removed from the N-terminus of the TTR protein.

[0136] Other TTR variants useful in the present invention include those that reduce or block TTR binding to thyroxine. Each TTR tetramer contains two thyroxine binding sites located in the central channel of the TTR tetramer. For example, such variants can avoid interfering with the patient's thyroxine biology and can avoid thyroxine metabolic pathways from contributing to TTR fusion. Other TTR variants useful in the present invention include those that reduce or eliminate TTR proteolytic activity.

[0137] Additionally, TTR-His tag fusions can be used in the present invention. For example, TTR-His tag fusions can be used to purify TTR Fab constructs (where the Fab lacks Fc), or to purify TTR Ab constructs (where it is advantageous to avoid the low pH purification environment of a protein A affinity column). In some embodiments, the His tag is removed after purification. The His tag can also be present in the final therapeutic molecule (i.e., the tag can remain after purification). In some embodiments, the His tag is His, (His) 2 、(His) 3 、(His) 4 、(His) 5 、(His) 6 、(His) 7 、(His) 8 、(His) 9 or (His) 10 In certain embodiments, the His tag is (His) 6 or (His) 7 Tag. In a specific embodiment, the His tag is (His) 6 Tags. In some embodiments, the His tag comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycine amino acids as a linker. In specific embodiments, the His tag comprises two glycines (e.g., GGHHHHHH).

[0138] In some embodiments, two glycine amino acid linkers can be inserted between the TTR variant and the heavy or light chain.

[0139] In addition, the TTR variants of the invention may include variants that incorporate glycosylation sites, which may help modulate the PK or solubility of the TTR fusion. Additionally, the TTR variants or TTR fusion proteins of the invention may be modified to include moieties that confer beneficial PK properties, such as triazine-containing moieties (contained in a construct having a terminal group capable of reacting with a protein; see, e.g., PCT Publication No. WO / 2017 / 083604, which is incorporated herein by reference in its entirety).

[0140] Methods for preparing homodimeric and homotetrameric fusion proteins

[0141] Methods for making homodimer and homotetramer fusions of the invention are discussed in the Examples.

[0142] Generally, homodimers and homotetramer fusions of the present invention can be produced using recombinant methods. Therefore, the present invention includes polynucleotides encoding homodimers and homotetramer fusions. On the other hand, the present invention includes expression vectors, which include polynucleotides encoding homodimers and homotetramer fusions. In certain embodiments, the expression vector includes control sequences (e.g., promoters, enhancers) that are operably connected to polynucleotides encoding homodimers and homotetramer fusions to support expression in suitable host cells. In certain embodiments, the expression vector also includes polynucleotide sequences that allow chromosome-independent replication in host cells. Exemplary vectors include but are not limited to plasmids, cosmids, and YACS. In a specific embodiment, the carrier is pTT5.

[0143] Typically, mammalian host cells are utilized when producing Ab TTR homodimers or Ab TTR homotetramer fusion constructs. Mammalian host cells are also suitable for producing Fab TTR homotetramer fusion constructs, although non-mammalian cells, such as prokaryotic (bacterial) and non-mammalian (e.g., yeast) host cells, may also be used.

[0144] On the other hand, the present invention includes host cells comprising expression vectors of the present invention. Methods of transfecting host cells with expression vectors and culturing transfected host cells under conditions suitable for expressing homodimers and homotetramers are known in the art. The transfection procedure used may depend on the host to be transformed. Certain methods for introducing heterologous polynucleotides into mammalian cells are known in the art, and include but are not limited to: dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, one or more polynucleotides are encapsulated in liposomes, and direct microinjection of DNA into the nucleus. Certain mammalian cell lines that can be used as hosts for expression are well-known in the art and include but are not limited to many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO, such as CHO-K1) cells, E5 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (such as Hep G2), human embryonic kidney cells 293 (HEK293) and a variety of other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and produce homodimer and homotetramer fusions.

[0145] Therefore, the present invention also relates to the method for preparing homodimer and homotetramer fusion protein as described herein.For example, homodimer and homotetramer fusion protein can be prepared by the following method:

[0146] a) culturing a recombinant host cell comprising a polynucleotide encoding the homodimer and homotetramer fusions; and

[0147] b) isolating the homodimeric or homotetrameric fusion protein from the culture.

[0148] Pharmaceutical composition

[0149] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the homomultimeric fusion proteins of the present invention (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetrameric fusion proteins), and a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0150] Preferably, acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, a pharmaceutical composition is provided that comprises a therapeutically effective amount of a homomultimeric fusion protein (e.g., a TTR antibody homodimer, a TTR antibody homotetramer, or a TTR Fab homotetramer fusion protein).

[0151] In certain embodiments, pharmaceutical compositions may contain formulation materials to adjust, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers; Compounds (e.g., polyvinyl pyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic acid, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, trimethylamine, lecithin, cholesterol, tiloxapor); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (AR Genrmo ed.), 1990, Mack Publishing Company.

[0152] In certain embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art according to, for example, a predetermined route of administration, a delivery form, and a desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the present invention. In certain embodiments, the main vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, a physiological saline solution, or an artificial cerebrospinal fluid, which may be supplemented with other substances common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is another exemplary vehicle. In a particular embodiment, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute thereof. In certain embodiments of the invention, homomultimeric compositions (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) can be prepared for storage by mixing selected components having a desired degree of purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of lyophilized cakes or aqueous solutions. Additionally, in certain embodiments, homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) can be formulated into lyophilizates using suitable excipients (e.g., sucrose).

[0153] The pharmaceutical composition of the present invention can be selected for parenteral delivery. Alternatively, the composition can be selected for suction or for delivery via the digestive tract (e.g., oral). The preparation of such pharmaceutically acceptable compositions is within the technical scope of those skilled in the art. The formulation components are preferably present in concentrations acceptable to the site of administration. In certain embodiments, a buffer is used so that the composition is maintained at a physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0154] When parenteral administration is contemplated, the therapeutic compositions for use in the present invention can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired homopolymer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the homopolymer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) is formulated into a sterile isotonic solution that is appropriately preserved. In certain embodiments, the preparation may involve formulating the desired molecule with an agent that can provide a controlled or sustained release of the product (which can be delivered via reservoir injection), such as an injectable microsphere, a bioerodible particle, a polymer compound (e.g., polylactic acid or polyglycolic acid), a bead, or a liposome. In certain embodiments, hyaluronic acid may also be used, which has the effect of promoting duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired antigen binding protein.

[0155] The pharmaceutical compositions of the invention can be formulated for inhalation. In these embodiments, the homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) are advantageously formulated as dry, inhalable powders. In specific embodiments, homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) inhalation solutions can also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be atomized. Therefore, International Patent Application No. PCT / US 94 / 001875 further describes pulmonary administration and formulation methods, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins.

[0156] It is also contemplated that the formulations can be administered orally. Homopolymers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) administered in this manner can be formulated in the presence or absence of carriers that are commonly used to compound solid dosage forms (e.g., tablets and capsules). In certain embodiments, the capsules can be designed to release the active portion of the formulation in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to promote absorption of the homopolymers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins). Diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders can also be used.

[0157] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means (e.g., liposomal carriers, bioerodible microparticles or porous beads, and depot injections) are also known to those skilled in the art. See, e.g., International Patent Application No. PCT / US93 / 00829, which is incorporated herein by reference and describes controlled release of porous polymer microparticles for delivery of pharmaceutical compositions. Sustained release formulations can include a semipermeable polymer matrix in the form of a shaped article (e.g., a film or microcapsule). Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated herein by reference), copolymers of L-glutamic acid and ethyl gamma-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949, which are incorporated by reference.

[0158] Pharmaceutical compositions for in vivo administration are typically provided in the form of sterile preparations. Sterilization can be achieved by filtering through a sterile filtration membrane. When the composition is freeze-dried, the method can be used for sterilization before or after freeze-drying and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution form. Parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle.

[0159] Aspects of the invention include self-buffered homomultimeric (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetrameric fusion protein) formulations that can be used as pharmaceutical compositions as described in International Patent Application WO06138181A2 (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0160] As described above, certain embodiments provide homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) compositions, in particular pharmaceutical homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) compositions, which, in addition to the homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein), further comprise one or more excipients (e.g., excipients exemplarily described in this section and elsewhere herein). In this regard, excipients can be used in the present invention for a variety of purposes, such as adjusting the physical, chemical or biological properties of the formulation, such as adjusting the viscosity and / or the methods of the present invention to improve effectiveness and / or stabilize such formulations and methods to resist degradation and spoilage due to, for example, stresses occurring during manufacture, transportation, storage, preparation before use, administration and thereafter.

[0161] There are various discussions on protein stabilization and formulation materials and methods useful in this regard, for example, Arakawad et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3):285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution," in RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002), and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13: 159-75 (2002), each of which is incorporated herein by reference in its entirety, particularly with respect to excipients for self-buffering protein formulations according to the present invention and methods for their preparation, especially protein pharmaceutical products and methods for veterinary and / or human medical use.

[0162] According to certain embodiments of the invention, salts may be used, for example, to adjust the ionic strength and / or isotonicity of the formulation and / or to improve the solubility and / or physical stability of proteins or other ingredients of the compositions according to the invention.

[0163] As is well known, ions can stabilize the native state of proteins by binding to charged residues on the protein surface and by shielding the charged groups and polar groups in the protein and reducing the intensity of their electrostatic interactions, attraction and repulsion interactions. Ions can also stabilize the denatured state of proteins by binding to the denatured peptide bonds (--CONH) of proteins in particular. In addition, the ionic interactions with the charged groups and polar groups in the protein can also reduce the intermolecular electrostatic interactions, and thereby prevent or reduce protein aggregation and insolubility.

[0164] Ionic species have significantly different effects on proteins. A variety of ions that can be used to prepare pharmaceutical compositions according to the present invention and their classification ratings for their effects on proteins have been developed. An example is the Hofmeister series, which rates ions and polar non-ionic solutes by their effects on the conformational stability of proteins in solution. Stable solutes are called "lyophilic." Unstable solutes are called "chaotropic." High concentrations of lyophiles (e.g., >1 molar ammonium sulfate) are typically used to precipitate proteins from solution ("salting out"). Chaotropic agents are typically used to denature and / or dissolve proteins ("salting out"). The relative effectiveness of ions for "salting out" and "salting out" defines their position in the Hofmeister series.

[0165] According to various embodiments of the invention, free amino acids can be used in homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) formulations as fillers, stabilizers, and antioxidants, as well as other standard uses. Lysine, proline, serine, and alanine can be used to stabilize the protein in the formulation. Glycine can be used for lyophilization to ensure the correct cake structure and properties. Arginine can be used to inhibit protein aggregation in both liquid formulations and lyophilized formulations. Methionine can be used as an antioxidant.

[0166] Polyols include sugars such as mannitol, sucrose and sorbitol and polyols such as, for example, glycerol and propylene glycol, and for the purposes of this article, polyethylene glycol (PEG) and related substances. Polyols are lyophilic. They are useful stabilizers in both liquid formulations and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols can also be used to adjust the tonicity of the formulation.

[0167] A useful polyol in the selected embodiment of the present invention is mannitol, which is generally used to ensure the structural stability of the cake in the freeze-dried formulation. It ensures the structural stability of the cake. It is generally used together with a lyophilization protective agent (e.g., sucrose). Sorbitol and sucrose are preferred agents for adjusting tension and as stabilizers to prevent freeze-thaw stress during transportation or to prevent the preparation of agglomerates during manufacturing. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycosylate surface lysine and arginine residues. Therefore, they are generally not preferred polyols for use according to the present invention. In addition, in this regard, sugars that form such reactive substances are also not preferred polyols of the present invention, such as sucrose, which is hydrolyzed to fructose and glucose under acidic conditions and thus produces glycosylation. PEG can be used to stabilize proteins and act as a cryoprotectant, and can be used in this regard for the present invention.

[0168] Embodiments of homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer or TTR Fab homotetramer fusion protein) formulations further comprise a surfactant. Protein molecules can be easily adsorbed on surfaces and denatured and subsequently aggregated at air-liquid, solid-liquid and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These deleterious interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical oscillations such as those generated during product transportation and handling.

[0169] Surfactants are routinely used to prevent, minimize or reduce surface adsorption. In this regard, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188.

[0170] Surfactants are also commonly used to control protein conformational stability. The use of surfactants in this regard is protein specific, in that any given surfactant will typically stabilize some proteins and destabilize others.

[0171] Polysorbates are susceptible to oxidative degradation and are usually supplied with sufficient amounts of peroxide to cause oxidation of protein residue side chains, especially methionine. Therefore, polysorbates should be used with caution and, when used, should be used at their lowest effective concentration. In this regard, polysorbates exemplify the general rule that excipients should be used at their lowest effective concentration.

[0172] Embodiments of homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer or TTR Fab homotetramer fusion protein) formulations also include one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. In this regard, useful antioxidants are reducing agents, oxygen / free radical scavengers and chelating agents. Antioxidants used in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity throughout the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0173] Antioxidants can damage proteins. For example, reducing agents, such as glutathione in particular, can damage intramolecular disulfide bonds. Therefore, the antioxidants selected for use in the present invention are particularly useful for eliminating or sufficiently reducing the possibility that they themselves will damage proteins in the formulation.

[0174] The formulations according to the invention may contain metal ions which are protein cofactors and are necessary for the formation of protein coordination complexes, such as zinc which is necessary for the formation of certain insulin suspensions. Metal ions may also inhibit some processes that degrade proteins. However, metal ions also catalyze physical and chemical processes that degrade proteins.

[0175] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartate to isoaspartate. +2 ions (up to 100 mM) can increase the stability of human DNAse. +2 , Mn +2 and Zn +2 can destabilize rhDNase. Similarly, Ca +2 and Sr +2 It can stabilize factor VIII, which can be affected by Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 destabilization, and its aggregation can be achieved by Al +3 Ions increase.

[0176] Embodiments of homopolymer (e.g., TTR antibody homodimer, TTR antibody homotetramer or TTR Fab homotetramer fusion protein) formulations also include one or more preservatives. When developing a multi-dose parenteral formulation involving extraction from the same container more than once, a preservative is necessary. Its main function is to inhibit microbial growth and ensure product sterility throughout the shelf life or service life of the drug product. Commonly used preservatives include benzyl alcohol, phenol and meta-cresol. Although preservatives have a long history of use in small molecule parenterals, the development of protein formulations containing preservatives can be challenging. Preservatives almost always have an unstable effect (aggregation) on proteins, and this has become a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs are formulated only for one-time use. However, when multi-dose formulations are possible, they have the added advantage of making patient convenience and increased marketability. A good example is human growth hormone (hGH), where the development of preserved formulations has led to the commercialization of a more convenient, multiple-use injection pen presentation. At least four such pen devices containing preserved formulations of hGH are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized--dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with meta-cresol.

[0177] Several aspects need to be considered during the formulation and development of a preservative dosage form. Effective preservative concentrations in the drug product must be optimized. This requires testing a given preservative in the dosage form at a concentration range that imparts antimicrobial effectiveness without compromising protein stability.

[0178] As might be expected, the development of liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with a diluent containing a preservative at the time of use. This shortens the time that the preservative is in contact with the protein, thereby significantly minimizing the associated stability risks. In the case of liquid formulations, preservative effectiveness and stability should be maintained throughout the product shelf life (approximately 18 to 24 months). An important point to note is that preservative effectiveness should be demonstrated in the final formulation containing the active drug and all excipient components.

[0179] Homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) formulations will typically be designed for a specific route and method of administration, for a specific administration dose and frequency of administration, for a specific treatment of a specific disease, depending on, among other things, the range of bioavailability and persistence. Thus, formulations can be designed in accordance with the present invention for delivery by any suitable route, including, but not limited to, oral, intraaural, ophthalmic, rectal, and vaginal, as well as by parenteral routes, including intravenous and intraarterial injections, intramuscular injections, and subcutaneous injections.

[0180] Once the pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or lyophilized powder. Such preparations can be stored in a ready-to-use form or in a form (e.g., lyophilized form) reconstituted before administration. The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous preparation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0181] The therapeutically effective amount of the pharmaceutical composition containing a homomultimer (e.g., containing a TTR antibody homodimer, containing a TTR antibody homotetramer, or containing a TTR Fab homotetramer fusion protein) to be used will depend, for example, on the therapeutic setting and goal. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will vary depending in part on the delivered molecule, the indication for the homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) used, the route of administration, and the patient's size (weight, body surface area, or organ size) and / or status (age and general health). In certain embodiments, the clinician can titrate the dose and modify the route of administration to obtain the optimal therapeutic effect. Typical dosage ranges can be from about 0.1 μg / kg to up to about 30 mg / kg or more, depending on the above factors. In specific embodiments, dosages may range from 1.0 μg / kg to about 20 mg / kg, optionally from 10 μg / kg up to about 10 mg / kg or from 100 μg / kg up to about 5 mg / kg.

[0182] A therapeutically effective amount of a homomultimer (e.g., a TTR antibody homodimer, a TTR antibody homotetramer, or a TTR Fab homotetramer fusion protein) preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or the prevention of impairment or disability resulting from the disease.

[0183] The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163, all of which are incorporated herein by reference.

[0184] Therapeutic uses of homodimeric and homotetrameric fusion proteins

[0185] As shown in the Examples, it has been found that dimerization of antibodies with TTR (to produce TTR homodimeric fusion proteins) and tetramerization of antibodies and Fab fragments with TTR (to produce TTR homotetrameric fusion proteins) result in TTR-containing fusion proteins with increased avidity compared to the individual antibody(s) and / or Fab fragment(s).

[0186] In addition, compared to one or more antibodies and / or one or more Fab fragments alone, TTR homodimers and TTR homotetramer fusion proteins exhibit improved antigen clustering. When an antibody (e.g., an IgG antibody) binds to an antigen on a target cell (e.g., a tumor cell), the clustered Fc domain formed will engage with FcγRs on immune effector cells (e.g., NK cells and macrophages). This clustering helps signal transduction through FcγRs, thereby initiating cell-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, TTR homodimers and TTR homotetramer fusion proteins are particularly useful in targeting ligands, where high antibody or Fab affinity / avidity leads to enhanced biological effects. The TTR homodimer and TTR homotetramer constructs of the present invention enhance cell-mediated effector functions resulting in enhanced ability to kill cells, which can be used, for example, in the treatment of cancer.

[0187] The TTR homodimer and TTR homotetramer fusion proteins of the present invention can be used to bind to a variety of targets / antigens. For example, TTR homodimers and TTR homotetramer fusion proteins can be used as agonists in targeting the following: TRAIL, TRAIL2R, GITR, OX40, GLP1, TREM2, and 4-1BB. In addition, TTR homodimers and TTR homotetramer fusion proteins can be used as antagonists in targeting the following: GIPR, TNFR, integrin receptors, PD-1, PD-L1, TIGIT, LAG-3, and TIM-3.

[0188] Therefore, the present invention also relates to methods of treating cancer using the homodimeric fusion proteins and homotetrameric fusion proteins described herein.

[0189] In other embodiments, the present invention relates to the use of the homodimeric fusion proteins and homotetrameric fusion proteins described herein in cancer treatment.

[0190] In other embodiments, the present invention relates to homodimeric fusion proteins and homotetrameric fusion proteins described herein for use in treating cancer.

[0191] Examples

[0192] The following examples are provided to illustrate specific embodiments or features of the present invention and are not intended to limit the scope thereof.

[0193] Example 1: Overview of generated TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins

[0194] Cloning of TTR antibody homodimer, TTR antibody homotetramer and TTR Fab homotetramer fusion protein

[0195] The following hybridoma-derived antibodies and Fabs were used to generate fusion proteins:

[0196] Anti-CB1R antibodies and Fab from hybridoma CB10 (also known as hybridoma 10D10)

[0197] Anti-GITR antibody and Fab from hybridoma 9H6

[0198] Anti-TRAILR2 antibody conatumumab and the corresponding conatumumab Fab

[0199] The following TTR fusion proteins were generated. [TTR] = TTR tetramer. For simplicity, the His tag is not shown in the summary of Example 1 (for the Fab constructs).

[0200] Specifically, the following TTR antibody homodimer fusion proteins were constructed. In these constructs, the C-termini of the two antibody heavy chains were linked to the N-termini of each TTR subunit.

[0201] a.[Anti-CB1R antibody] 2 -[TTR]

[0202] "CB1R TTR antibody homodimer fusion protein"

[0203] The generated sequence pairs are:

[0204] TTR-heavy chain fusion Light chain <![CDATA[[SEQ ID NO:5] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:11 <![CDATA[[[SEQ ID NO:5]–[GGGGS]] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:11 <![CDATA[[[SEQ ID NO:5]–[(GGGGS) 2 ]] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:11 <![CDATA[[[SEQ ID NO:5]–[(GGGGS) 3 ]] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:11 <![CDATA[[[SEQ ID NO:5]–[(GGGGS) 4 ]] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:11

[0205] b.[Anti-GITR antibodies]2 -[TTR]

[0206] "GITR TTR antibody homodimer fusion protein"

[0207] The resulting sequence pairs are:

[0208] TTR-heavy chain fusion Light chain <![CDATA[[SEQ ID NO:18] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:25

[0209] c.[Anti-TRAILR2 antibody] 2 -[TTR]

[0210] "Conatumumab TTR antibody homodimer fusion protein"

[0211] The resulting sequence pairs are:

[0212] TTR-heavy chain fusion Light chain <![CDATA[[SEQ ID NO:31] 2 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:38

[0213] In addition, the following TTR antibody homotetrameric fusion proteins were constructed. In these constructs, one of the two heavy chains at the C-terminus of the antibody (comprising the charge pair mutations D399K and E356K (SEQ ID NO: 6, 19, or 32)) was linked (with or without a linker) to the N-terminus of the TTR subunit. The remaining heavy chain contained a complementary set of K392D and K409D charge pair mutations (SEQ ID NO: 7, 20, or 33) associated with the linked heavy chain. Note that in the discussion of the constructs in the Examples, the discussion of E416K and D459K refers to EU E356K and EU D399K, respectively. Similarly, in the discussion of the constructs in the Examples, the discussion of K420D and K437D refers to EU K392D and EU K409D, respectively.

[0214] a.[Anti-CB1R antibody] 4 -[TTR]

[0215] "CB1R TTR antibody homotetrameric fusion protein"

[0216] The resulting sequence pairs are:

[0217]

[0218] b.[Anti-GITR antibodies] 4 -[TTR]

[0219] "GITR TTR antibody homotetrameric fusion protein"

[0220] The resulting sequence pairs are:

[0221]

[0222] c.[Anti-TRAILR2 antibody] 4 -[TTR]

[0223] "Conatumumab TTR antibody homotetrameric fusion protein"

[0224] The generated sequence pairs are:

[0225]

[0226] In addition, the following TTR Fab antibody homotetrameric fusion proteins were constructed. In these constructs, the C-terminus of each Fab fragment was linked to the N-terminus of each TTR subunit.

[0227] a.[Anti-CB1R Fab] 4 –[TTR]

[0228] · "CB1R TTR Fab homotetrameric fusion protein"

[0229] Note that in the discussion of constructs in the Examples, discussion of S215E refers to EU S183E. Similarly, in the discussion of constructs in the Examples, discussion of S203K refers to EU S176K.

[0230] · In [SEQ ID NO:44] 4 –[SEQ ID NO:1] 4 + In the fusion of SEQ ID NO:11, no charge pair mutation was used. In [SEQ ID NO:45] 4 –[SEQ ID NO:1] 4 &In the fusion of SEQ ID NO:46, SEQ ID NO:45 comprises a S215E charge pair mutation and SEQ ID NO:46 comprises a charge pair S203K mutation, both numbered according to the EU system.

[0231] The generated sequence pairs are:

[0232] TTR-heavy chain fusion Light chain <![CDATA[[SEQ ID NO:44] 4 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:11 SEQ ID NO:44 <![CDATA[[SEQ ID NO:11] 4 –[SEQ ID NO:1] 4 ]]> <![CDATA[[SEQ ID NO:45] 4 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:46 SEQ ID NO:45 <![CDATA[[SEQ ID NO:46] 4 –[SEQ ID NO:1] 4 ]]>

[0233] b.[Anti-GITR Fab] 4 –[TTR]

[0234] · "GITR TTR Fab homotetrameric fusion protein"

[0235] The generated sequence pairs are:

[0236] TTR-heavy chain fusion Light chain <![CDATA[[SEQ ID NO:21] 4 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:26

[0237] c.[Anti-TRAILR2 Fab]4 –[TTR]

[0238] · "Conatumumab TTR Fab homotetrameric fusion protein"

[0239] The resulting sequence pairs are:

[0240] TTR-heavy chain fusion Light chain <![CDATA[[SEQ ID NO:34] 4 –[SEQ ID NO:1] 4 ]]> SEQ ID NO:39

[0241] For a TTR antibody homodimer fusion protein, the C-terminus of each antibody heavy chain is linked to the N-terminus of each TTR subunit. For a TTR antibody homotetramer fusion protein, one of the two heavy chains at the antibody C-terminus is linked to the N-terminus of each TTR subunit. For a TTR Fab homotetramer fusion protein, the C-terminus of each Fab fragment is linked to the N-terminus of each TTR subunit.

[0242] The fusion protein was generated using standard molecular biology techniques including polymerase chain reaction (PCR), site-directed PCR mutagenesis, restriction endonuclease digestion, and enzymatic ligation into a mammalian expression plasmid. Polyhistidine-tagged Fab-TTR molecules were also generated, where (His) 6 The tag was added to the Fab C-terminus.

[0243] The cloned TTR fusion variant heavy chain and Fab DNAs are combined with their respective cloned anti-CB1, anti-GITR and anti-TR2 antibody light chain (LC) DNAs and used to transfect mammalian cells to express TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins. The technique is generally performed according to the methods referenced in Molecular Cloning: A Laboratory Manual, 3rd ed., Sambrook et al., 2001, Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY.

[0244] Example 2: Cloning, expression and purification of anti-CB1 TTR antibody homodimers, TTR antibody homotetramers and TTR Fab homotetramer fusion proteins

[0245] Cloning of anti-CB1 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins Anti-CB1 TTR antibody homodimers were typically cloned as follows. pTT5-del-Bsm-BI:VK1O2O12::[hu <hucb1>10D10.1 VH]::huIgG2TO (construct C59477) was used as a template for the following constructs. The pTT5-del-Bsm-BI vector is a derivative of the pTT5 vector in which the BsmBI restriction site has been removed.

[0246] [SEQ ID NO:5] 2 –[SEQ ID NO:1] 4 The assembly is as follows. PCR1: Using C59477 as a template, a 5' PCR primer (5'AGT TTA AACGAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CC 3') encoding the amino terminus of the signal sequence, the SalI restriction site, and the optimized Kozak sequence was combined with a 3' primer (5'GTG CTG GCG AAT CCA GCT CCA ATA GTC ACC 3') designed to eliminate the existing BamHI restriction site in C59477. PCR1 produces a product of approximately 220 base pairs. PCR2a: Using C59477 as a template, a 5' PCR primer designed to eliminate the existing BamHI restriction site (5'GAG CTG GAT TCG CCA GCA CCC AGG 3') was combined with a 3' primer (5'GGT GCC CGT AGG GCCACC CGG AGA CAG GGA G 3') encoding the carboxyl-terminal human heavy chain constant region (CH3) and the amino terminus of TTR (SEQ ID NO: 1). PCR2a produced a product of approximately 1250 base pairs. PCR3a: Using TTR (SEQ ID NO: 1) as a template, a 5' PCR primer (5'TCCCTG TCT CCG GGT GGC CCT ACG GGC ACC G 3') encoding the carboxyl terminal human heavy chain constant region (CH3) and the amino terminus of TTR was combined with a 3' primer (5'AAC GAT ATC GCT AGC GCG GCC GCT CAT TCCTTG GGA TTG GTG 3') encoding the carboxyl terminal region of TTR (SEQ ID NO: 1), the stop codon and the NotI restriction site. PCR3a produces a product of approximately 400 base pairs. PCR reactions 1, 2a and 3a were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT15d digested with SalI and NotI using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was called C73494:pTT15d:VK1O2O12::[hu <hucb1>10D10.1(huIgG2-TO desK)VH]::TTR3.

[0247] [[SEQ ID NO:5]–[GGGGS]] was assembled using PCR1 as described above 2 –[SEQ ID NO:1] 4 PCR2b: Using C59477 as a template, a 5' PCR primer (5'GAG CTG GATTCG CCA GCA CCC AGG 3') designed to eliminate the existing BamHI restriction site was combined with a 3' primer (5'GGA TCC GCC ACC ACC ACC CGG AGA CAG GGA G 3') encoding the carboxyl-terminal human heavy chain constant region (CH3), the G4S linker, and the amino terminus of TTR (SEQ ID NO: 1). PCR2b produced a product of approximately 1250 base pairs. PCR3b: Using TTR (SEQ ID NO: 1) as a template, a 5' PCR primer (5'GGT GGT GGC GGA TCC GGCCCT ACG GGC ACC G 3') encoding the carboxyl-terminal human heavy chain constant region (CH3), the G4S linker, and the amino terminus of TTR was combined with a 3' primer (5'AAC GAT ATC GCT AGC GCG GCC GCT CAT TCC TTG GGA TTG GTG3') encoding the carboxyl-terminal region of TTR, the stop codon, and the NotI restriction site. PCR3b produces a product of approximately 400 base pairs. PCR reactions 1, 2b, and 3b were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT15d digested with SalI and NotI using the GeneArt seamless cloning and assembly kit. The resulting construct was called C73499: pTT15d: VK1O2O12:: [huantibody <hucb1>10D10.1(huIgG2-TO desK)VH]::G4S::TTR3.

[0248] [[SEQ ID NO:5]–[(GGGGS)] were assembled using PCR1 as described above 2 ]] 2 –[SEQ ID NO:1] 4 PCR2c: Using C59477 as template, a 5' PCR primer (5'GAGCTG GAT TCG CCA GCA CCC AGG 3') was designed to eliminate the existing BamHI restriction site and a primer encoding the carboxyl-terminal human heavy chain constant region (CH3), (GGGGS) 2 The 3' primer (5'GCC GGA CCC TCC CCC ACC GGA TCC GCC ACCTCC ACC CGG AGA CAG GGA G 3') of the linker and the amino terminus of TTR (SEQ ID NO: 1) was combined. PCR2c produced a product of approximately 1250 base pairs. PCR3c: Using TTR as a template, encoding the carboxyl-terminal human heavy chain constant region (CH3), (GGGGS) 2 The 5' PCR primer (5'CCG GTG GGG GAG GGT CCG GCC CTA CGG GCA CCG GTG AAT CCA AGG CTC CT 3') for the linker and the amino terminus of TTR was combined with a 3' primer (5'AAC GAT ATC GCT AGCGCG GCC GCT CAT TCC TTG GGA TTG GTG 3') encoding the carboxyl terminal region of TTR, the stop codon and the NotI restriction site. PCR3c produced a product of approximately 400 base pairs. PCR reactions 1, 2c and 3c were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT15d digested with SalI and NotI using the GeneArt seamless cloning and assembly kit. The resulting construct was called C73500: pTT15d: VK1O2O12:: [huantibody <hucb1>10D10.1(huIgG2-TO desK)VH]::(G4S)2::TTR3.

[0249] [[SEQ ID NO:5]–[(GGGGS) 3 ]] 2 –[SEQ ID NO:1] 4 The assembly of PCR4 was as follows: Using C73500 as template, 5' PCR primers (5'-AGT TTA AAC GAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGGGTG CC-3') and encoding the carboxyl-terminal human heavy chain constant region (CH3), (GGGGS) 3 The 3' primer (5'CGT AGG GCC GGA CCC TCC CCC ACC GGA GCC CCC GCC CCC GGA TCCGCC ACC TCC 3') of the linker and the amino terminus of TTR (SEQ ID NO: 1) was combined. PCR4 produced a product of approximately 1500 base pairs. PCR5: Using C73500 as a template, encoding (GGGGS) 3 The 5' PCR primer for the linker (5'TCC GGG GGC GGG GGC TCC GGT GGG GGA GGG T 3') was combined with a 3' primer (5'AAC GAT ATC GCT AGC GCGGCC GCT CAT TCC TTG GGA TTG GTG 3') encoding the carboxyl terminal region of TTR, the stop codon and the NotI restriction site. PCR5 produced a product of approximately 450 base pairs. PCR reactions 4 and 5 were gel separated and purified on a Qiagen column. The fragments were then mixed and ligated into the linearized mammalian expression vector pTT15d digested with SalI and NotI using the GeneArt seamless cloning and assembly kit. The resulting construct was called C73690: pTT15d: VK1O2O12:: [huantibody <hucb1>10D10.1(huIgG2-TO desK)VH]::(G4S)3::TTR3.

[0250] [[SEQ ID NO:5]–[(GGGGS) 4 ]] 2 –[SEQ ID NO:1] 4 PCR6: Using C73690 as template, 5' PCR primers (5'-AGT TTA AAC GAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGGGTG CC-3') and (GGGGS) encoding the carboxyl-terminal human heavy chain constant region (CH3) 4 3' primers (5'GGA ACC ACCTCC GCC GGA TCC GCC ACC TCC A 3') of the adapter. PCR6 produces a product of about 1500 base pairs. PCR7: Use C73690 as template, encoding (GGGGS) 4 A 5' PCR primer (5'GGC GGA GGT GGT TCC GGGGGC GGG GGC TCC G3') encoding a portion of the linker was combined with a 3' primer (5'AAC GAT ATC GCT AGC GCG GCC GCT CAT TCC TTG GGA TTG GTG 3') encoding the carboxyl terminal region of TTR (SEQ ID NO: 1), the stop codon and the NotI restriction site. PCR7 produced a product of approximately 450 base pairs. PCR reactions 6 and 7 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT15d digested with SalI and NotI using the GeneArt seamless cloning and assembly kit. The resulting construct was called C73729: pTT15d: VK1O2O12:: [hu anti <hucb1>10D10.1(huIgG2-TO desK)VH]::(G4S)4::TTR3.

[0251] [SEQ ID NO:6] 4 –[SEQ ID NO:1] 4 The assembly is as follows. PCR8: Using C73494 as a template, the 5'PCR primer (5'-AGT TTA AAC GAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CC-3') was combined with a 3' primer designed to convert glutamic acid residue 356 (EU) to lysine in huIgG2 CH3 (5'CTT GGT CATCTC CTT CCG GGA TGG GGG CAG G 3'). PCR8 produced a product of approximately 1200 base pairs. PCR9: Using C73494 as template, a 5' PCR primer designed to convert glutamic acid residue 356 (EU) to lysine in huIgG2CH3 (5'-CTG CCC CCA TCC CGG AAG GAG ATG ACC AAG AAC CA-3') was combined with a 3' primer designed to convert aspartic acid residue 399 (EU) to lysine in huIgG2CH3 (5'GAA GGA GCC GTC GGA CTT CAGCAT GGG AGG TGT 3'). PCR9 generated a product of approximately 160 base pairs. PCR10: Using C73494 as a template, a 5' PCR primer (5'CCT CCCATG CTG AAG TCC GAC GGC TCC TTC T 3') designed to convert aspartic acid residue 399 (EU) to lysine in huIgG2 CH3 was combined with a 3' primer encoding the carboxyl-terminal region of TTR, a stop codon, and a NotI restriction site. PCR10 produced a product of approximately 600 base pairs. PCR reactions 8, 9, and 10 were gel separated and purified on a Qiagen column. The fragments were then mixed and ligated into the SalI and NotI digested linearized mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was called C73730:pTT15d:VK1O2O12::[huanti <hucb1>10D10.1(E416K,D459K)(huIgG2-TO desK)VH]::TTR3.

[0252] [[SEQ ID NO:6]–[GGGGS]] 4 –[SEQ ID NO:1] 4 The assembly is as follows. PCR11: Using C73499 as a template, the 5'PCR primer (5'-AGT TTA AAC GAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGG GTGCC-3') was combined with a 3' primer designed to convert glutamic acid residue 356 (EU) to lysine in huIgG2 CH3 (5'CTTGGT CAT CTC CTT CCG GGA TGG GGG CAG G 3'). PCR11 produced a product of approximately 1200 base pairs. PCR12: Using C73499 as template, a 5' PCR primer designed to convert glutamic acid residue 356 (EU) to lysine in huIgG2 CH3 (5'CTG CCC CCA TCC CGG AAG GAG ATG ACC AAG AAC CA 3') was combined with a 3' primer designed to convert aspartic acid residue 399 (EU) to lysine in huIgG2 CH3 (5'GAA GGA GCC GTC GGACTT CAG CAT GGG AGG TGT 3'). PCR12 generated a product of approximately 160 base pairs. PCR13: Using C73499 as a template, a 5' PCR primer designed to convert aspartic acid residue 399 to lysine in huIgG2 CH3 (5'CCTCCC ATG CTG AAG TCC GAC GGC TCC TTC T3') was combined with a 3' primer encoding the carboxyl-terminal region of TTR, a stop codon, and a NotI restriction site (5'AAC GAT ATC GCT AGC GCG GCC GCT CAT TCC TTG GGA TTGGTG 3'). PCR13 produced a product of approximately 600 base pairs. PCR reactions 11, 12, and 13 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the SalI and NotI digested linearized mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The amino acid numbering and naming conventions are the same as in the previous construct description. The resulting construct is called C73731:pTT15d:VK1O2O12::[huanti <hucb1>10D10.1(E416K,D459K)(huIgG2-TO desK)VH]::G4S::TTR3.

[0253] SEQ ID NO:7 was constructed as follows. PCR14: Using C73494 as a template, a 5' PCR primer (5'-AGT TTA AACGAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CC-3') was combined with a 3' primer designed to convert lysine residue 392 (EU) to aspartic acid in huIgG2 CH3 (5'GGG AGG TGT GGT ATC GTA GTT GTTCTC CGG CTG C ​​3'). PCR14 produced a product of approximately 1300 base pairs. PCR15: Using pTT5-del-Bsm-BI:VK1O2O12::huFc_(IgG2)(K392D K409D)("C59541") as template, a 5' PCR primer designed to convert residue lysine 392 (EU) to aspartic acid in huIgG2 CH3 (5'CCG GAG AAC AAC TAC GAT ACCACA CCT CCC ATG C 3') was combined with a 3' primer encoding the carboxy-terminal region of huIgG2 minus the carboxy-terminal lysine, a stop codon, and a NotI restriction site (5'AAC GAT ATC GCT AGC GCG GCC GCT CAA CCC GGA GAC AGGGAG 3'). PCR15 produced a product of approximately 200 base pairs. PCR reactions 14 and 15 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the SalI and NotI digested linearized mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73513:pTT15d:VK1O2O12::[huantibody <hucb1>10D10.1(K420D,K437D)(huIgG2-TO desK)VH].

[0254] SEQ ID NO:11 was constructed as follows. PCR16: The 5' PCR primer (5'-TTT TTT TTG CGC GCT GTG ATA TTG TGA TGA CTC AGT C) encoding the amino terminus of the variable region and the BssHII restriction site was combined with the 3' primer (5'-AAA AAA CGT ACG TTT GAT TTC CAC CTT GGTCC) encoding the carboxyl terminus of the variable region and the BsiWI restriction site. PCR16 was purified by a Qiagen column. The fragment was then digested with BssHII and BsiWI and purified on a Qiagen column. The fragments were then mixed and ligated into the linearized mammalian expression vector pTT5-del-Bsm-BI digested with BssHII and BsiWI containing the signal peptide and kappa constant region. The resulting construct was called C59474: pTT5-del-Bsm-BI: VK1O2O12:: [hu anti <hucb1>10D10.1VL]::huKLC.

[0255] Expression of anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and anti-CB1 TTR Fab homotetramer fusion proteins

[0256] Anti-CB1 TTR antibody homodimers, TTR antibody homotetramers, and anti-CB1 TTR Fab homotetramer proteins are typically expressed as follows.

[0257] HEK 293 6E cells were cultured in suspension on a platform shaker at 37 °C in a humidified atmosphere of 5% CO. 2 Grow in an incubator at 120 RPM. Cell culture medium is FreeStyle F-17 + 0.1% (10% of 10mL / L) Kolliphor P188 + 500μL / L G418 + 6mM (30mL / L) L-glutamine. Subculture cells 1-2 days before transfection so that the cell density at transfection is about 1.5e 6 VC / mL. The transfection complex was mixed in Freestyle F17 medium without supplements at a volume of 10% of the final culture volume. 0.5 μg DNA was added to Freestyle F17 medium per mL of culture, and then 1.5 μl PEImax reagent was added per mL of culture. The culture medium was then mixed and incubated at room temperature for 10 minutes before being added to the cell culture and returned to the flask on a shaker platform in the incubator. 1-4 hours after transfection, 25 μL Yeastolate solution was added per mL of culture. Conditioned culture medium was harvested on day 6 after transfection.

[0258] Freestyle F-17 (Catalog No. 13835), L-glutamine (Catalog No. 25030), Geneticin G418 (Catalog No. 10131027, liquid) were obtained from Life Technologies. Kolliphor P 188 (Catalog No. K4894) was obtained from Sigma-Aldrich. Difco TC Yeastolate UF (Catalog No. 292805) was obtained from BD Biosciences. PEI Max (Catalog No. 24765-2) was obtained from Polysciences.

[0259] exist Figure 2 In gels of 2a, 2b and 2c, 10 μl of sample (unheated) was loaded into each lane of 4-20% SDS-PAGE and developed with a Coomassie blue-based dye system. Figure 2 a and 2b show that anti-CB1 TTR antibody homodimer and anti-CB1 TTR antibody homotetramer proteins were robustly expressed in HEK 293 cells, respectively. Figure 2 a shows the anti-CB1 TTR antibody homodimer ([SEQ ID NO:5] 2 –[SEQ ID NO:1] 4 and [[SEQ ID NO:5]–[(GGGGS) 1-4 ]] 2 –[SEQ ID NO:1] 4 ) resists SDS denaturation and thus can form strong non-covalent complexes. Various linker lengths (from the shortest in lane 2 ([GGGGS] 1 ) to the longest in lane 5 ([GGGGS] 4 )); linker length does not appear to substantially affect expression. Figure 2 b shows that the formation of anti-CB1 TTR antibody homotetramers using V1 Fc charge pair mutations (K409D and K392D in one heavy chain, and D399K and E356K) (SEQ ID NOs: 6 and 7) also appears to be SDS resistant. Various heavy and light chain DNA transfection ratios are shown (from 1:9 LC:HC in lane 2 to 9:1 LC:HC in lane 10). LC:HC ratios of 1:1 (lane 6) and 9:1 (lane 10) resulted in the most robust expression.

[0260] Figure 2 c shows that the anti-CB1 TTR Fab homotetramer construct was robustly expressed in HEK 293 cells and was resistant to SDS. TTR fusion was better tolerated at the heavy chain C-terminus (lanes 2, 4, 6, and 8 - fewer bands) compared to the light chain C-terminus (lanes 3, 5, 7, and 9 - more bands). The position of the His tag did not appear to affect fusion. The heavy chain-TTR used in lanes 2 and 4 was [SEQ ID NO: 44] 4 –[SEQ ID NO:1] 4 , and the light chain is SEQ ID NO: 11. The heavy chain used in lanes 3 and 5 is SEQ ID NO: 44, and the light chain-TTR is [SEQ ID NO: 11] 4 –[SEQ ID NO:1] 4 The heavy chain-TTR used in lanes 6 and 8 is [SEQ ID NO: 45] 4 –[SEQ ID NO:1] 4 , and the light chain is SEQ ID NO: 46. The heavy chain used in lanes 7 and 9 is SEQ ID NO: 45, and the light chain-TTR is [SEQ ID NO: 46] 4 –[SEQ ID NO:1] 4 Charge pair mutations were included in the anti-CB1 TTR Fab homotetramer constructs tested in lanes 6-9 (heavy chain having SEQ ID NO:45 and light chain having SEQ ID NO:46). SEQ ID NO:45 contained the S215E charge pair mutation and SEQ ID NO:46 contained the S203K charge pair mutation. A His tag was also used (see Figure 2 c).

[0261] Purification of anti-CB1 TTR Fab homotetrameric fusion protein

[0262] Anti-CB1 TTR Fab homotetrameric protein was generally purified as follows.

[0263] The cell culture medium was diluted with 2 L of 50 mM NaHCO using a 10 kDa MWCO slide analyzer (Thermo Fisher Scientific). 2 PO 4 , 300mM NaCl, 10mM imidazoles, pH 8.0 were dialyzed for at least 2 hours, twice.Use AKTA purifier (GM Medical Group) tandem liquid chromatography system (with 1mL Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany) as the first column, and 5mL desalting HiTrap (GM Medical Group) as the second column) to purify molecules from the cell culture medium of buffer exchange.Culture medium is directly loaded on Ni-NTA post, washed with 50mM sodium phosphate, 300mM NaCl, 10mM imidazoles, pH 8.0 of 8CV, and eluted with 50mM sodium phosphate, 300mM NaCl, 250mM imidazoles, pH 8.0 of 2CV.Ni-NTA post eluent is automatically introduced into desalting column, wherein by 10mM sodium acetate, 150mM NaCl (pH 5.2) of 4CV, protein is carried out isocratic elution. The samples were then sterilized by filtration through a 3.0 μm glass fiber / 0.2 μm Supor membrane (Pall Corporation, Port Washington, NY, USA).

[0264] The protein concentration of each purified molecule was determined by UV absorbance at 280 nM (A280) using a NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). 3 μg of each final purified molecule was subjected to SDS-PAGE analysis on a denaturing, non-reducing 4-12% Bis-Tris NuPAGE gel using MES running buffer (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. 20 μg of each final purified molecule was subjected to HPLC size exclusion chromatography, which was run as follows: on a Phenomenon SEC 3000 column (7.8 x 300 mm) (Phenomenon, Torrance, CA, USA) at 1 mL / min in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9, observing the absorbance at 280 nm.

[0265] Purification of anti-CB1 TTR antibody homodimer and TTR antibody homotetramer fusion proteins

[0266] Anti-CB1 TTR antibody homodimers and TTR antibody homotetramer fusion proteins are generally purified as follows.

[0267] First, the fusion protein was purified from the cell culture medium using an AKTA purifier (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) tandem liquid chromatography system with 1 mL MabSelect SuRe (MSS) HiTrap (GE Healthcare Life Sciences) as the first column and 5 mL Desalting HiTrap (GE Healthcare Life Sciences) as the second column. The culture medium was directly loaded onto the MSS column, washed with 8 column volumes (CV) of 25 mM Tris-HCl, 100 mM NaCl (pH 7.4), and eluted with 2 CV of 100 mM acetic acid. The MSS column eluate was automatically introduced into the desalting column, where the protein was isocratically eluted by 4 CV of 10 mM sodium acetate, 150 mM NaCl (pH 5.2). The samples were then sterilized by filtration through a 3.0 μm glass fiber / 0.2 μm Supor membrane (Pall Corporation, Port Washington, New York, USA). The protein concentration of each purified molecule was determined by UV absorbance at 280 nM (A280) using a NanoDrop 2000 (Thermo Fisher Scientific, Rockford, Illinois, USA). 3 μg of each final purified molecule was subjected to SDS-PAGE analysis using MES running buffer (Life Technologies, Carlsbad, California, USA) and run on a denaturing non-reducing 4%-12% Bis-Tris NuPAGE gel according to the manufacturer's instructions. 30 μg of each final purified molecule was subjected to HPLC size exclusion chromatography, which was run as follows: on a Pheromone SEC 3000 column (7.8 x 300 mm) (Pheromone Corporation, Torrance, California, USA) in 50 mM NaH 2 PO 4 , 250 mM NaCl, pH 6.9 at 1 mL / min, and the absorbance was observed at 280 nm. Figure 3 Shown without connector, with (G 4 S) connector, belt (G 4 S) 2 Connector, belt (G 4 S) 3 Connector, or belt (G 4 S) 4 Representative HPLC SEC analysis of anti-CB1 TTR antibody homodimer fusion proteins with linkers.

[0268] Example 3: Activity of anti-CB1 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins

[0269] The activity of anti-CB1 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins can be obtained using the CB1 cAMP assay.

[0270] CHO cells stably expressing hCB1 (Euroscreen) were grown in DMEM containing 10% FBS, 1% Pen / Strep / L-glutamine, 25 mM Hepes, 0.1 mM NEAA, 1 mM sodium pyruvate, and 400 μg / ml G418. To determine antibody activity, cells were seeded at a density of 10,000 cells / well in 80 μl DMEM containing 0.5% FBS, 1% Pen / Strep / L-glutamine, 25 mM Hepes, 0.1 mM NEAA, 1 mM sodium pyruvate, and 400 μg / ml G418 in 96-well plates. After overnight incubation, the culture medium was removed and replaced with 5 μl of fresh culture medium, followed by 5 μl of culture medium plus 15 μM forskolin (EMD Chemicals Cat#344273) and 250 pM CP55,940 (TOCRIS Cat#0949), followed by 40 μl of antibody (in 10 mM acetic acid, 150 mM NaCl, pH 5.0). The cells were then incubated at 37°C for 30 minutes. The culture medium was then aspirated and cAMP levels were measured using the DiscoverX XS+cAMP assay kit (90-0075-03) according to the manufacturer's protocol. The plate was read for 30 seconds on a PerkinElmer ViewLux microplate imager.

[0271] The results of the test are as follows Figure 4 EC of TTR antibody homotetramer 50 The advantage was 3.9-fold higher than that of the CB1 parent antibody. Some Fabs used charge pair mutations, while others did not (see Example 2 Figure 2 These results demonstrate that TTR antibody homotetramers and TTR Fab homotetramer fusion proteins have improved EC compared to the parental CB1 Ab. 50 Interestingly, the EC of anti-CB1TTR antibody homodimers 50 appears to be less favorable than the CB1 parent antibody, where EC 50 Get worse.

[0272] Example 4: Cloning, expression and purification of anti-GITR TTR antibody homodimers, TTR antibody homotetramers and TTR Fab homotetramer fusion proteins

[0273] Cloning of anti-GITR TTR antibody homodimer, TTR antibody homotetramer and TTR Fab homotetramer fusion proteins

[0274] Anti-GITR TTR antibody homodimer and TTR antibody homotetramer fusion proteins were generally cloned as follows. Construct C74201 (pSLX240p: native:: [hu <hugitr>9H6(D72(62)E)VH]::huIgG1z-N297G); C143046(pTT5d:VK1O2O12::[hu antibody <hugitr>9H6(S183E,N297G,E356K,D399K)VH]::TTR(C10A,K15A); C143048(pTT5d:VK1O2O12::[hu anti <hugitr>9H6(S183K,N297G,K392D,K409D)VH]::TTR(C10A,K15A); C137324(VK1O2O12::[hu anti <hugitr>9H6 VL]) and C73877 (pTT5: natural:: [hu <hugitr>9H6 VL]::huKLC) was used as template for all constructs described below.

[0275] [SEQ ID NO: 18] 2 –[SEQ ID NO:1] 4 The assembly is as follows. PCR17: Using C74201 as a template, the signal peptide of anti-GITR MAb 9H6 (with N297G) was replaced with the VK1 signal peptide (MDMRVPAQLLGLLLLWLRGARC) by three-step overlapping PCR extension, and then the product of the final PCR was amplified using a 5' signal peptide primer (5'-GTC GAC TAG GCC ACC ATG GACATG AGG GTG CCC GCT CAG CTC CTG GGG CT-3') and a 3' C-terminal heavy chain / N-terminal TTR primer (GGT GCCCGT AGG GCC ACC CGG AGA CAG GGA GAG G) to generate a 1450 base pair product. PCR18: Using TTR (SEQ ID NO: 1) as a template, a 5' PCR primer encoding the amino terminus of TTR (5'-GGC CCTACG GGC ACC G-3') was combined with a 3' primer encoding the carboxyl terminal region of TTR, a stop codon, and a NotI restriction site (5'-TCG CTA GCG CGG CCG CTC ATT CCT TGG GAT TGG TGA CG-3'). PCR18 produced a product of approximately 400 base pairs. PCR reactions 17 and 18 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT5d digested with SalI and NotI using the GeneArt seamless cloning and assembly kit. The resulting construct was called C143043:pTT5d:VK1O2O12::[huantibody <hugitr>9H6(N297G)VH]::TTR(C10A,K15A).

[0276] [SEQ ID NO:21] 4 –[SEQ ID NO:1] 4 The assembly is as follows. PCR19: Using C74201 as a template, the signal peptide of anti-GITR MAb 9H6 (C74201) was replaced with the VK1 signal peptide (MDMRVPAQLLGLLLLWLRGARC) by three-step overlapping PCR extension, and then the product of the final PCR was amplified using a 5' signal peptide primer (5'-GTC GAC TAG GCC ACC ATG GACATG AGG GTG CCC GCT CAG CTC CTG GGG CT-3') and a 3' C-terminal heavy chain / N-terminal TTR primer (5'-GCTCTC GAG GGA GTA GAG TCC TGA GGA CTG TAG G-3') to generate a 650 base pair product. PCR20: Using C74201 as a template, a 5'PCR primer encoding the carboxyl-terminal human heavy chain constant region (5'-CTCTAC TCC CTC GAG AGC GTG GTG ACC GTG CC-3') was combined with a 3' primer encoding the human heavy chain constant region (5'-CCTCCT CCA CAA GAT TTG GGC TCA ACT TTC TTG TC-3'). PCR20 produced a product of approximately 130 base pairs. PCR21: Using TTR3 as template, a 5' PCR primer encoding the amino terminus of TTR (5'-CAA ATC TTG TGG AGGAGG CCC TAC GGG CAC CG-3') was combined with two 3' primers encoding the carboxyl terminal region of TTR, the stop codon and the NotI restriction site (5'-ATG GTG ATG GTG ACC GCC TTC CTT GGG ATT GGT GAC GAC A-3') and (5'-ATC GCT AGC GCG GCC GCC TAG TGG TGA TGG TGA TGG TGA CC-3'). PCR21 produced a product of approximately 450 base pairs. PCR reactions 19, 20 and 21 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT5d digested with SalI and NotI using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was called C144132:pTT5d:VK1O2O12::[hu <hugitr>9H6(S183E)scFab]::TTR(C10A,K15A)::G::G::6xHis (note, (His) is included for purification purposes) 6 Label).

[0277] [SEQ ID NO: 19] 4 –[SEQ ID NO:1] 4 The assembly is as follows. PCR22: Using C74201 as a template, the signal peptide of anti-GITR MAb 9H6 (C74201) was replaced with the VK1 signal peptide (MDMRVPAQLLGLLLLWLRGARC) by three-step overlapping PCR extension, and then the product of the final PCR was amplified using a 5' signal peptide primer (5'-GTC GAC TAG GCC ACC ATG GACATG AGG GTG CCC GCT CAG CTC CTG GGG CT-3') and a 3' C-terminal heavy chain / N-terminal TTR primer (5'-TAGGTG CTT CCG TAC TGT TCC TCC CGG GGC TT-3') to generate a 990 base pair product. PCR23: Using C143046 as a template, a 5' PCR primer (5'-CCT GAG CAG CGT CGT CAC CGT CCC-3') encoding the carboxyl-terminal human heavy chain constant region (CH3) was combined with a 3' primer (5'-TAG GTG CTT CCG TAC TGT TCC TCC CGG GGC TT-3') encoding the carboxyl-terminal human heavy chain constant region (CH3). PCR23 produced a product of approximately 370 base pairs. PCR24: Using TTR3 as a template, a 5' PCR primer (5'-CAG TAC GGAAGC ACC TAC CGG GTG GTG TC-3') encoding the carboxyl-terminal region of TTR was combined with a 3' primer (5'-TCG CTA GCG CGG CCG CTC ATT CCT TGG GAT TGG TGA CG-3') encoding the carboxyl-terminal region of TTR. PCR24 produced a product of approximately 900 base pairs. PCR reactions 22, 23, and 24 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the linearized mammalian expression vector pTT5d digested with SalI and NotI using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C144127:pTT5d:VK1O2O12::[huantibody <hugitr>9H6(N297G,E356K,D399K)VH]::TTR(C10A,K15A).

[0278] SEQ ID NO: 20 was assembled as follows. PCR25: Using C143048 as a template, a 5' PCR primer encoding the amino terminus of the signal peptide (5'-GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTG GGGCT) was combined with a 3' primer encoding the carboxyl-terminal human heavy chain constant region (CH3) (5'-ACG GTG ACG ACG CTG CTC AGG CTGTAC AGG CCG CTG-3'). PCR25 produced a product of approximately 650 base pairs. PCR26: Using C143048 as a template, a 5' PCR primer (5'-CCT GAG CAG CGT CGT CAC CGTCCC-3') encoding the carboxyl-terminal human heavy chain constant region (CH3) was combined with a 3' primer (5'-TAG GTG CTT CCG TAC TGT TCCTCC CGG GGC TT-3') encoding the carboxyl-terminal human heavy chain constant region (CH3). PCR26 produced a product of approximately 370 base pairs. PCR27: Using TTR3 as a template, a 5' PCR primer (5'-CAG TAC GGA AGC ACC TAC CGG GTG GTG TC-3') encoding the carboxyl-terminal region of TTR was combined with a 3' primer (5'-TCG CTA GCG CGG CCGCTC ATT CCT TGG GAT TGG TGA CG-3') encoding the carboxyl-terminal region of TTR. PCR27 produced a product of approximately 900 base pairs. PCR reactions 25, 26, and 27 were gel separated and purified on a Qiagen column. These fragments were then mixed and ligated into the SalI and NotI digested linearized mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was called C144130:pTT5d:VK1O2O12::[huantibody <hugitr>9H6(N297G,K392D,K409D)VH].

[0279] SEQ ID NO:25 was assembled as follows. PCR28: Using C137324 as a template, the 5' PCR primer encoding the amino terminus of the signal sequence (5'-GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTGGGG CT) was combined with the 3' primer encoding the carboxyl-terminal human light chain constant region (5'-TAT CGC TAG CGC GGC CGC-3'). PCR28 produced a product of approximately 800 base pairs. PCR 28 was gel separated and purified on a Qiagen column. The fragments were then mixed and ligated into the linearized mammalian expression vector pTT5d digested with SalI and NotI using the GeneArt seamless cloning and assembly kit. The resulting construct was called C143044:pTT5d:VK1O2O12::[huantibody <hugitr>9H6 VL].

[0280] SEQ ID NO:26 was assembled as follows. PCR29: Using C137324 as a template, a 5' PCR primer encoding the amino terminus of the signal sequence (5'-GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTGGGG CT) was combined with a 3' primer encoding the carboxyl-terminal human light chain constant region (5'-TGG TGC AGC CAC CGT ACG TTT GATTTC CAC CTT GGT CC-3'). PCR29 produced a product of approximately 400 base pairs. PCR30: Using C73877 as a template, a 5' PCR primer encoding the carboxyl-terminal human light chain constant region (5'-ACG GTG GCT GCA CCA TCT G-3') was combined with a 3' primer encoding the carboxyl-terminal human light chain constant region (5'-TAT CGC TAG CGC GGC CGC-3'). PCR reactions 29 and 30 were gel separated and purified on a Qiagen column. The fragments were then mixed and ligated into the SalI and NotI digested linearized mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C143049:pTT5d:VK1O2O12::[huantibody <hugitr>9H6(S176K)VL].

[0281] Expression of anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins

[0282] Anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins are typically expressed as follows.

[0283] Freestyle F-17 (Catalog No. 13835), L-glutamine (Catalog No. 25030), Geneticin G418 (Catalog No. 10131027, liquid) were obtained from Life Technologies. Kolliphor P 188 (Catalog No. K4894) was obtained from Sigma-Aldrich. Difco TC Yeastolate UF (Catalog No. 292805) was obtained from BD Biosciences. PEI Max (Catalog No. 24765-2) was obtained from Polysciences. HEK 293 6E cells were cultured in suspension on a platform shaker at 37°C in a humidified atmosphere of 5% CO. 2 Grow in an incubator at 120 RPM. Cell culture medium is FreeStyle F-17 + 0.1% (10% of 10mL / L) Kolliphor P188 + 500uL / L G418 + 6mM (30mL / L) L-glutamine. Subculture cells 1-2 days before transfection so that the cell density at transfection is about 1.5e 6 VC / mL. The transfection complex was mixed in Freestyle F17 medium without supplements at a volume of 10% of the final culture volume. 0.5 μg DNA was added to Freestyle F17 medium per mL of culture; then 1.5 μl PEImax reagent was added per mL of culture. The culture was mixed and incubated at room temperature for 10 minutes before adding to the cell culture. The culture flask was then returned to the shaker platform in the incubator. 1-4 hours after transfection, 25 μL Yeastolate solution was added per mL of culture. The conditioned medium was then harvested on day 6 after transfection.

[0284] Anti-GITR TTR antibody homodimers can alternatively be expressed as follows. HEK293 cells were transiently transfected with the corresponding cDNA. 1.0x10 6 HEK 293-6E cells were suspended at 10 cells / mL and 0.5 mg / L DNA (0.25 mg / L hu antibody in pTT5d vector) was used. <hugitr>9H6(N297G)VH]::TTR(C10A,K15A) and 0.25mg / L hu antibody in pTT5d vector <hugitr>9H6VL) (Durocher et al., NRCC, Nucleic Acids. Res. [Nucleic Acids Research] (2002) 30, e9) (with 4mg PEI (Polyscience)) to 1mg DNA for transfection and incubated at 36°C at 150RPM in a shake flask. 4 hours after transfection, Yeasolate (BD Biosciences) for the final 0.5% of the transfection was added to the culture. The cells were grown in suspension in FreeStyle F17 medium (supplemented with 0.1% Pluronic F68 and 50μg / ml Geneticin) for 6 days and harvested for purification.

[0285] Conditioned media were analyzed using 4%-20% SDS-PAGE and Quick Blue Stain gels. Figure 5 It is shown that anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins can be expressed in HEK 293 cells. Anti-GITR TTR antibody homotetramers and TTR Fab homotetramers are expressed most robustly. Lane 1 is 10.3 mg anti-GITR TTR antibody homodimer (356 KDa); lane 2 is 29.3 mg anti-GITR TTR antibody homotetramer (656 KDa); lane 3 is 143.6 mg anti-GITR TTR Fab homotetramer (256 KDa); lane 4 is 100 ng anti-DNP antibody; lane 5 is 250 ng anti-DNP antibody; lane 6 is 500 ng anti-DNP antibody; and lane 7 is 1000 ng anti-DNP antibody. The anti-DNP antibody provides information on how the unconjugated antibody performs in this assay. 10 μl of protein in non-reducing loading buffer was added to each lane. The gel was run without heating.

[0286] Purification and characterization of anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins

[0287] Anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins are generally purified and characterized as follows.

[0288] Anti-GITR TTR antibody homodimer and TTR antibody homotetramer fusion proteins were purified from cell culture media using an AKTA purifier (GE Healthcare) liquid chromatography system with two sequentially connected 1 mL rProtein A Sepharose FastFlow (ProA FF) HiTrap (GE Healthcare) columns. The culture medium was directly loaded onto the ProA FF column, then washed with 5 CV of Dulbecco's phosphate buffered saline (DPBS) (Life Technologies) and eluted with 8 CV of 100 mM acetic acid. The purified sample was dialyzed twice with 2 L of 10 mM sodium acetate, 9% sucrose, pH 5.2 using a 10 kDa MWCO slide analyzer (Thermo Fisher Scientific).

[0289] TTR Fab homotetrameric fusion protein was purified from cell culture medium using an AKTA purifier liquid chromatography system with a 5 mL HisTrap excel HiTrap (GE Healthcare) column. The culture medium was loaded directly onto the HisTrap column, then washed with 20 CV of 20 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole, pH 7.4, and eluted with a 20 CV gradient of imidazole from 10 mM to 500 mM. The flow-through fraction was re-purified under the same above conditions, except that an 8 CV 20 mM NaH2PO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4 step elution was used instead of a gradient elution (due to the lack of chromatographic separation in the previous method). The purified sample was dialyzed twice against 2 L of 10 mM sodium acetate, 9% sucrose, pH 5.2 using a 10 kDa MWCO slide analyzer.

[0290] The protein concentrations of anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins were determined by UV absorbance at 280 nM (A280) using a NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA).

[0291] SDS-PAGE analysis (3 μg each of anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion protein) was run on denaturing non-reducing 4%-12% Bis-Tris NuPAGE gels using MES running buffer (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Figure 6 Lanes 1 and 4 are anti-GITR TTR antibody homodimers; lanes 2 and 5 are anti-GITR TTR antibody homotetramers; lanes 3 and 6 are anti-GITR TTR Fab homotetramers. Figure 6 The partially purified products of all three protein fusion constructs were shown to be correctly assembled based on the non-heated, non-reducing lanes. After heating and reduction, the three protein fusion constructs resolved into their expected component chains (the upper band is the heavy chain and the lowest band is the light chain).

[0292] 30 μg of each of the anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion protein were subjected to HPLC size exclusion chromatography. Chromatography was performed using a Phenomenon SEC 3000 column (7.8 x 300 mm) (Phenomenon, Torrance, CA, USA) (in 50 mM NaH 2 PO 4 , 250 mM NaCl, pH 6.9, at 1 mL / min), and the absorbance was observed at 280 nm.

[0293] Anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins were analyzed by reducing LCMS analysis. 20 μg of material was denatured in 8M guanidine HCl / TRIS (pH 8.0, Teknova, Hollister, CA) and reduced at 50°C for 20 minutes using 10 mM DTT (EMD Millipore, Darmstadt, Germany). The samples were acidified with trifluoroacetic acid and 10 μg was injected on a Zorbax reverse phase C8 column using an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, Canada). The column effluent was introduced into the electrospray source of an Agilent 6230ESI-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA) and mass spectra were collected. The relevant mass spectra were deconvoluted using the MaxEnt algorithm in the Agilent MassHunter software package. The resulting mass spectra of LC and HC were then compared with the theoretically calculated masses for each chain.

[0294] Purified anti-GITR TTR antibody homodimers were further purified isocratically (to remove aggregates accumulated over time) on a 320 mL Superdex 200 (GE Healthcare) with 1.4 CV of 10 mM sodium acetate, 150 mM NaCl, pH 5.0. Fractions were selected by HPLC-SEC purity for pooling. The Superdex pool was concentrated using a VivaSpin 10 kDa MWCO centrifugal filter device (Sartorius) and then sterile filtered through a 0.2 μm Supor syringe filter (Pall). HPLC size exclusion chromatography was performed as follows: on a Sepax Zenix-C, 7.8x300 mm column (Sepax, Newark, DE, USA), absorbance was observed at 280 nm at 1 mL / min in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9.

[0295] Figure 7 Results of HPLC size exclusion chromatography (SEC) analysis of each of the anti-GITR TTR antibody homodimer (middle peak), TTR antibody homotetramer (left peak), and TTR Fab homotetramer (right peak) fusion proteins are shown. The SEC chromatograms show that the major peak elutes at the expected position consistent with a correctly assembled molecule. Furthermore, native SEC purification (unlike SDS-PAGE) supports the view that the fusion protein does not aggregate.

[0296] The repurified anti-GITR TTR antibody homodimer was reanalyzed by reducing LCMS. About 10 μg of anti-GITR TTR antibody homodimer was dried in a Speed-Vac and then resuspended in 20 μL of 8M Gu-HCl, pH 8.0, with 20 mM DTT. The samples were incubated at 37°C for 1 hour to reduce each sample to individual protein chain components. Each reduced sample was then acidified by adding 0.1% TFA. For LC-MS, about 5 μg of the reduced sample was injected onto an Agilent Technologies 1100 capillary HPLC and sprayed into an Agilent Technologies 6224ESI-TOF mass spectrometer. Capillary HPLC used a 1.0 mmX 50 mm Agilent Zorbax 300SBC8 column with a flow rate of 50 μL / min and a column temperature of 75°C. The following buffers were used for HPLC: Buffer A-0.1% TFA / H2O; Buffer B-0.1% TFA / H2O / 90% n-propanol. The gradient consisted of the following: initial conditions were 2% B for 5 minutes; ramp up to 45% B over 20 minutes; reach 95% B over 3 minutes; isocratic at 95% B for 4 minutes; then fall back to 2% B over 1 minute. The MS method for the ESI-TOF instrument scanned m / z [750-6000] at a rate of 1 spectrum / second. Other MS instrument parameters included capillary voltage (VCap) = 3200 V, fragmentor voltage = 225 V, skimmer = 60 V and OCT 1RF Vpp = 800 V. For data analysis of the LC-MS data, the appropriate LC-MS spectra for each protein chain were merged and deconvoluted using Agilent MassHunter software. The output mass range of the deconvolution was [15,000-75,000], with a mass step size of 1.0 Da and a signal / noise (S / N) threshold of 30.0. Correct reduced chain masses were observed for each sample. Minor Asp-Pro cleavage was observed in the anti-GITR TTR antibody homodimer heavy chain, a result of using an analysis temperature of 75°C.

[0297] Differential scanning calorimetry (DSC) of anti-GITR TTR parent mAb 9H6 ("1"), anti-GITR TTR antibody homodimer ("4"), TTR antibody homotetramer ("2"), and TTR Fab homotetramer ("3") fusion proteins were performed on a Malvern Microcalorimeter VP-capillary DSC. Figure 8 The following parameters were used: Scan range: 10-100°C; Scan rate: 1°C / min; Pre-scan thermostat for 15 minutes. Each analysis typically consumed 400 μL of 1 mg / mL sample. Data were processed in Origin 7 software. The analyzed proteins were expressed in HEK 293-6E cells. Although the parental Ab ("1") was glycosylated, the TTR fusion was a N297Ga-glycosyl variant. Figure 8 The results in show that the melting temperature of the TTR fusion protein is comparable to or better than that of the parent Ab, indicating that the TTR fusion protein formed is robust. The expected order of heat-induced transitions is CH2, Fab, CH3.

[0298] Example 5: Activity and PK profiles of anti-GITR TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins

[0299] The activities and PK profiles of the parental anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, and anti-GITR TTR Fab homotetramer fusion protein were evaluated.

[0300] Activity evaluation

[0301] The binding and potency activities of the parental anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, and anti-GITR TTR Fab homotetramer fusion proteins were evaluated via the following assays. The results of these assays are shown in Table 1. Fig.10 shown.

[0302] Assay under cross-linking. 96-well high binding plates (Corning 3369) were coated overnight at 4°C with anti-CD3 Ab (OKT3) and cross-linker Ab (goat anti-human IgG Fc cross-adsorbed Ab (Thermo Fisher 31125) or goat anti-human IgG (H+L) (Pierce 31119)) at 1 μg / ml and 0.3 μg / ml, respectively. The next day, the Ab was washed out, and anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, anti-GITR TTR Fab homotetramer fusion protein TTR or isotype control mAb were added to the plate for capture at 37°C for 1 hour. After incubation, plates were washed and naive T cells were added to the wells (50K / well in RPMI1640 supplemented with 10% FBS, 2mM L-Glut, 10mM HEPES, 1mM NaPyr, 0.1mM NEAA and 50μM 2ME) and cultured for 4 days. Proliferation was measured using CellTiterGlo (Promega).

[0303] Determination without cross-linking. A 96-well high binding plate (Corning 3369) was coated with 1 μg / ml of anti-CD3 Ab at 4°C overnight. The next day, the anti-CD3 Ab was washed out, and anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, anti-GITR TTR Fab homotetramer fusion protein TTR, or isotype control mAb was added to the plate, and then the original T cells were added (50K / well in RPMI1640 supplemented with 10% FBS, 2mM L-Glut, 10mM HEPES, 1mM NaPyr, 0.1mM NEAA, and 50μM2ME). After 4 days of culture, proliferation was measured using CellTiterGlo (Promega).

[0304] PK profile assessment

[0305] PK profiles were determined by intravenous injection of anti-GITR TTR antibody homotetramer at 8.75 mg / kg, anti-GITR TTR antibody homodimer at 4.75 mg / kg, anti-GITR TTR Fab homotetramer at 3.4 mg / kg, and anti-GITR antibody at 2 mg / kg in male CD-1 mice (n=3 / group). Serum samples were collected from 75 μl blood samples at 0.5, 2, 8, 24, 48, 72, 96, 168, 336, 504, 672, and 840 hours after dosing. Each blood sample was kept at room temperature after collection and, after a clotting period of 30-40 minutes, centrifuged at 11,500 RPM for approximately 10 minutes at 2-8°C using a calibrated Eppendorf 5417R centrifugal system (Brinkmann Instruments, Inc., Westbury, NY). The collected serum was then transferred to pre-labeled (for each rat) cryogenic storage tubes and stored at -60°C to -80°C for future bioanalysis.

[0306] The following PK assay was used to measure the total anti-GITR species (i.e., any anti-GITR binding species present) of the parental anti-GITR mAb, anti-GITR TTR antibody homodimers, anti-GITR TTR antibody homotetramers, and anti-GITR TTR Fab homotetramer fusion proteins in mouse serum as determined by Meso Scale Discovery (MSD). Conventional binding 96-well MSD plates (Meso Scale Discovery, Gaithersburg, MD) were coated with 2 μg / ml of mouse anti-idiotypic anti-GITR antibody, Mab 1.2.1 (Amgen Inc., Thousand Oaks, CA) in PBS and then incubated overnight at 4°C. The plates were then washed and incubated with I-Block TM (Life Technologies, Carlsbad, CA) and blocked overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted in virgin CD-1 mouse serum if dilution was required. Standards, QCs, and samples were then diluted 1:20 in a buffer containing PBS, 1 M NaCl, 0.5% Tween 20, and 1% bovine serum albumin. The plates were washed three times with approximately 200 μl of 1X KPL buffer (KPL, Gaithersburg, MD), and 50 μl aliquots of the diluted standards, QCs, and samples were transferred to the Mab 1.2.1 antibody-coated plates and incubated at room temperature (approximately 25°C) for 1.5 hours. The plates were washed three times with approximately 200 μl of 1X KPL wash buffer, and then 50 μl of 250 ng / ml mouse anti-idiotypic anti-GITR antibody, Mab 1.1.1, conjugated to biotin was added and incubated for 1.5 hours. After washing the plate three times with 1X KPL wash buffer, add 50 μl of 100 ng / ml streptavidin conjugated with MSDSULFO-tag (Amgen, USA) and incubate for 15 minutes. Wash the plate six times with about 200 μl of 1X KPL wash buffer, then add 150 μl of 1x read buffer T (Mesoscale Discovery), and measure the electrochemiluminescent signal using an MSD 6000 plate reader (Mesoscale Discovery). Use Serum concentration data were analyzed by a non-compartmental method using ANALYZER(R) (Phoenix64, version 6.4.0.768, Pharsight Corp., Mountain View, CA).

[0307] The following PK assay was used to measure the presence of anti-GITR TTR binding species and TTR species in anti-GITR TTR antibody homodimers, anti-GITR TTR antibody homotetramers, and anti-GITR TTR Fab homotetramer fusion proteins in mouse serum by MSD assay of mouse serum samples. Conventional binding 96-well MSD plates (Mesoscale Discovery, Gaithersburg, MD) were coated with 2 μg / ml rabbit anti-TTR polyclonal antibody (Amgen Inc., Thousand Oaks, CA) in PBS and then incubated overnight at 4°C. The plates were then washed and blocked with I-Block™ (Life Technologies, Carlsbad, CA) at 4°C overnight. Standards and quality controls (QC) were prepared in mouse serum, and PK samples were diluted in virgin CD-1 mouse serum if dilution was required. The standards, QCs, and samples were then diluted 1:20 in a buffer containing PBS, 1M NaCl, 0.5% Tween 20, and 1% bovine serum albumin buffer. The plate was washed 3 times with approximately 200 μl of 1X KPL buffer (KPL, Gaithersburg, Maryland), and then 50 μl of the diluted standards, QCs, and samples were transferred to the anti-TTR antibody-coated plate and incubated at room temperature for 1.5 hours. The plate was washed 3 times with approximately 200 μl of 1X KPL wash buffer, and then 50 μl of 250 ng / ml of mouse anti-idiotypic anti-GITR antibody, Mab1.1.1, conjugated to biotin was added, and incubated for 1.5 hours. After washing the plate 3 times with 1X KPL wash buffer, 50 μl of 100 ng / ml of streptavidin conjugated to MSD SULFO-tag (Amgen, USA) was added and incubated for or 15 minutes. The plate was washed six times with approximately 200 μl of 1X KPL Wash Buffer, then 150 μl of 1X Read Buffer T (Mesoscale Discovery) was added and the electrochemiluminescent signal was measured using an MSD 6000 plate reader (Mesoscale Discovery). Serum concentration data were analyzed by a non-compartmental method using ELISA® (Phoenix 64, version 6.4.0.768, Pharsight Corp., Mountain View, CA).

[0308] The results of the PK analysis can be found in Fig. 9 Found in Fig. 9 a) shows (measuring the presence of any anti-GITR binding species): [1] the PK of the anti-GITR TTR antibody homodimer ("1") is more optimal than that of the parental Ab ("2"); [2] the PK of the anti-GITR TTRFab homotetramer ("4") is less optimal and this may be attributed to the Fab's lack of an Fc region and therefore lack of the ability to mediate / extend its half-life; [3] the PK of the anti-GITR TTR antibody homotetramer ("3"), while not as robust as that of the parental mAb, shows significantly enhanced PK compared to the Fab. Fig. 9 b) (measuring the presence of anti-GITR-bound species and TTR species) shows that the PK of the intact anti-GITR TTR fusion protein is similar to Fig. 9 This is consistent with what was observed in a), indicating that in vivo proteolysis is unable to release the anti-GITR binding moiety from each TTR fusion protein.

[0309] like Fig.10 As shown, multimerization of the parental anti-GITR mAb 9H6 (via TTR fusion) improved binding, but not potency. Activation of GITR is known to prevent suppression of regulatory T cells. It is also known that anti-GITR antibody-mediated cell proliferation, cytokine production, and induction of CD4+Th9 cells require clustering and FcγR binding. Fig.10 a) shows that the binding affinity of the anti-GITR TTR antibody homodimer ("3"), TTR antibody homotetramer ("4"), and TTR Fab homotetramer ("2") fusion proteins is more optimal than that of the parental anti-GITR mAb 9H6 ("1"). It is worth noting that the higher affinity did not translate into higher potency in the cell-based assay, as shown in Figure 2. Fig.10 b). Fig.10 The control Ab in b) is a non-binding control version of the anti-GITR mAb.

[0310] Fig. 9 Comparison of the data in a and 9b shows that there is almost no partial molecular degradation of the TTR construct.

[0311] Example 6: Cloning, expression and purification of anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers and TTR Fab homotetramer fusion proteins

[0312] Cloning of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer and TTR Fab homotetramer fusion protein

[0313] Anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers and TTR Fab homotetramer fusion proteins were generally cloned as follows: In these experiments, the anti-TRAILR2 antibody was conatumumab (AMG 655) and the tag anti-TRAILR2Fab was the Fab portion of conatumumab.

[0314] The GITR TTR expression plasmid (see Example 4) was used as a template for the construction of these anti-TRAILR2 TTR fusions. Construct C36606 (pTT5: native:: [hu <hutrailr2>XG1048(W)VH]::huIgG1(f)) and C9448(VK3_A27L::[huanti <hutrailr2>XG1048 VL]::huKLC) were templates for the anti-TRAILR2 heavy and light chain variable region sequences, respectively. In these experiments, the anti-TRAILR2 antibody was conatumumab (AMG 655). Since the anti-TRAILR2 sequence template did not have a VK1 (VK1O2O12) signal peptide, the pSLX240puro, pSLX240hygro, and pSLX240neo constructs were found to contain a VK1 with a BssHII site at the carboxyl terminus of the signal peptide sequence.

[0315] [SEQ ID NO:31] 2 –[SEQ ID NO:1] 4 The assembly of is as follows. PCR31: Using C36606 as a template, a 5'PCR primer (5'-CTG CTG TGG CTG AGAGGT GCG CGC TGT CAG GTG CAG CTG CAG GAG-3') encoding the amino terminus of the heavy chain sequence and the VK1 (VK1O1O12) signal sequence was combined with a 3' primer (5'-GCT GAG GAG ACG GTG ACC GT-3') designed to amplify the anti-TRAILR2 variable heavy chain region. PCR31 generates a product of 395 base pairs. PCR32: Using C-143043 as template, a 5' primer (5'-GGT CAC CGT CTC CTC AGC TAG CAC CAA GGG CCC A-3') encoding the amino terminus of the heavy chain constant region and the last 18 bases of the anti-TRAILR2 variable heavy chain was combined with a 3' primer (5'-TTA AAC GAT ATC GCT AGC GCG GCC GCT CAT TCC TTG GGA TTG GTG ACG-3') encoding the carboxyl terminus of TTR, the stop codon, the NotI site, and an 18 base overhang encoding the amino terminus of the linearized pSLX240p plasmid. PCR 32 produced a product of 1390 bases. PCR reactions 31 and 32 were purified on a Qiagen column. These fragments were then combined with the pSLX240p:VK1 plasmid linearized at the BssHII and NotI sites in a ligation-independent cloning reaction (Geneart seamless cloning and assembly kit). The resulting construct was called C-150225:pSLX240p:VK1O2O12::[hu <hutrailr2>AMG 655VH]::IgG1z_SEFL(desK)::TTR(C10A,K15A).

[0316] SEQ ID NO:38 was assembled as follows. PCR33: Using C9448 as a template, the 5' PCR primer (5'-CTG CTG TGG CTG AGA GGT GCG CGC TGT GAAATT GTG TTG ACG CAG-3') encoding the amino terminus and signal sequence of the anti-TRAILR2 light chain variable region was combined with a 3' primer (5'-AGC CAC CGT TCG TTT GAT TTC CAC CTT-3') to amplify the anti-TRAILR2 light chain variable region to generate a 363 base pair product. PCR34, using C-143044 (pTT5d:VK1O2O12::[huanti <hugitr>9H6 VL]) as a template, the amino terminus of the κ light chain constant region and the last 9 bases of the anti-TRAILR2 variable region are encoded by a 5' primer (5'-ATC AAA CGA ACG GTG GCT GCA CCA TCT-3') and a 3' primer (5'-TGT TTA AAC GAT ATCGCT AGC GCG GCC GCC TAA CAC TCT CCC CTG TTG AAG-3') encoding the carboxyl terminus of the human κ light chain constant region, the stop codon and the NotI restriction enzyme. PCR34 produces a product of about 330 base pairs. PCR reactions 33 and 34 are purified on a Qiagen column. These fragments are then combined with the pSLX240h:VK1 plasmid linearized at the BssHII and NotI sites in a ligation-independent cloning reaction (Geneart seamless cloning and assembly kit). The resulting construct was called C-150226:pSLX240h:VK1O2O12::[hu <hutrailr2>AMG655 VL]::huKLC.

[0317] [SEQ ID NO:32] 4 –[SEQ ID NO:1] 4 The assembly is as follows. PCR 31 was used as described above. PCR35: Using C-144127 as a template, the 5' primer (5'-GGT CAC CGT CTC CTC AGC CTC CAC CAA GGG CCC C-3') encoding the amino terminus of the heavy chain constant region and the last 18 bases of the anti-TRAILR2 heavy chain variable region was combined with the 3' primer (5'-TTAAACGATATCGCTAGCGCGGCCGCTCATTCCTTGGGATTGGTGACG-3') encoding the carboxyl terminus of TTR to produce a product of about 1390 base pairs. PCR reactions 31 and 35 were purified on a Qiagen column. These fragments were then combined with the pSLX240p:VK1 plasmid linearized at the BssHII and NotI sites in a cloning reaction (Geneart seamless cloning and assembly kit) that was unrelated to the connection. The resulting construct was called C-150227:pSLX240p:VK1O2O12::[hu <hutrailr2>AMG 655VH]::IgG1z(N297G,KK)::TTR(C10A,K15A).

[0318] SEQ ID NO:33 was assembled as follows. PCR31 was used as described above. PCR36: Using C-144130 as a template, the 5' primer (5'-GGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCC-3') was combined with the 3' primer (5'-TTA AAC GAT ATC GCT AGC GCG GCC GCT CAA CCC GGGGAG AGG CTC A-3') encoding the carboxyl terminus, stop codon and NotI site of the heavy chain constant region to produce a product of approximately 1 kb in size. PCR reactions 31 and 36 were purified on a Qiagen column. These fragments were then combined with the pSLX240n:VK1 plasmid linearized at the BssHII and NotI sites in a ligation-independent cloning reaction (GeneArt Seamless Cloning and Assembly Kit). The resulting construct was called C-150228:pSLX240n:VK1O2O12::[huantibody <hutrailr2>AMG 655VH]::IgG1z(N297G,DD).

[0319] [SEQ ID NO:34] 4 –[SEQ ID NO:1] 4 The assembly is as follows. PCR31 was used as described above. PCR37: Using C144132 as a template, the 5' primer (5'-GGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCA-3') was combined with the 3' primer (5'-TTA AAC GAT ATC GCT AGC GCG GCC GCC TAG TGGTGA TGG TGA TGG TGA CC-3') encoding 6xHis, a stop codon and a NotI site to produce a product of about 740 base pairs. PCR reactions 31 and 37 were purified on a Qiagen column. These fragments were then combined with the pSLX240p:VK1 plasmid linearized at the BssHII and NotI sites in a ligation-independent cloning reaction (Geneart seamless cloning and assembly kit). The resulting construct is called C-150237:pSLX240p:VK1O2O12::[hu-anti <hutrailr2>AMG 655(S183E) scFab]::G2::TTR(C10A,K15A)::G2::6xHis (note, (His) is included for purification purposes) 6 Label).

[0320] SEQ ID NO:39 was assembled as follows. PCR33 was used as described above. PCR38: Using C-143049 as a template, the 5' primer (6186-65) was combined with a 3' primer (5'-TGTTTA AAC GAT ATC GCT AGC GCG GCC GCT CAA CAC TCT CCC CTG TTG AA-3') encoding the carboxyl terminus, stop codon and NotI site of the kappa light chain constant region to produce a product of approximately 330 base pairs. PCR reactions 33 and 38 were purified on a Qiagen column. These fragments were then combined with the pSLX240h:VK1 plasmid linearized at the BssHII and NotI sites in a ligation-independent cloning reaction (Geneart seamless cloning and assembly kit). The resulting construct was called C-150238:pSLX240h:VK1O2O12::[huantibody <hutrailr2>AMG655VL]::huKLC-S176K.

[0321] Expression of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins

[0322] Anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins are typically expressed as follows.

[0323] TTR fusion protein was stably expressed in suspension-adapted CHO-K1 cells. Lipofectamine LTX (Invitrogen) was used according to the manufacturer's protocol. TM (Invitrogen TM )) was transfected. A total of 30 μg-36 μg of mammalian expression plasmid DNA was used, with a 1:1 ratio (1 HC or Fab-HC to 1 Lc) for anti-TRAILR2 TTR antibody homodimers and TTR Fab homotetramers, or a 1:1:1 ratio (1 HC+ to 1 HC- to 1 LC) for TTR antibody homotetramers. In each case, plasmid DNA was added to 3-4 ml OPTI-MEM (Gibco) and mixed. In a separate test tube, 72-75 μl Lipofectamine LTX was added to 3-4 ml OPTI-MEM. The solution was incubated at room temperature for 5 minutes. To form a transfection complex, the DNA and Lipofectamine LTX mixtures of each test tube were combined and incubated for another 20 minutes at room temperature. Logarithmic phase CHO-K1 cells were pelleted by centrifugation (1200-1500 RPM for 5 minutes), washed once with 1X PBS (Gibco), and then resuspended in OPTI-MEM to 1.5-2e 6 For each transfection, add 5-6 mL of washed cells to a 125 mL shake flask. For each transfection, add DNA transfection complex to the cells. Incubate at 36°C, 5% CO 2 Incubate the shake flasks at 150 RPM for 6 hours. To stop the transfection, add 9-12 ml of growth medium to each flask and incubate for 48-72 hours.

[0324] To start selection, cells were pelleted by centrifugation (1200-1500 RPM for 5 minutes) 72 hours after transfection and the medium was then replaced with 23-25 ​​mL of growth medium supplemented with antibiotics. Selection medium was changed 2-3 times per week, and cultures were diluted when necessary to ensure that the cultures did not overgrow (<5-6e 6 vc / mL) until cell viability and density are restored.

[0325] Large-scale production (2.3 L-2.5 L) was performed in shake flasks at 36°C. 6 The production was inoculated at a concentration of 1.5 vc / mL. The conditioned medium was harvested on day 5 by centrifugation followed by filtration (0.45 μm).

[0326] like Fig.11 As shown, anti-TRAILR2 TTR antibody homodimer and TTR Fab homotetramer fusion proteins were well expressed and assembled correctly (see Fig.11 a). Fig.11 b) shows the presence of the expected fusion protein components after heating and reduction. 5 μl of conditioned medium was loaded into each lane and run on a 4%-20% TG gel. Fig.11 As shown in a), anti-TRAILR2 TTR antibody homodimers and TTR Fab tetramers migrate on SDS-PAGE as larger complexes than the standard antibody, as expected for fully assembled complexes. Fig.11 As shown in b), anti-TRAILR2 TTR antibody homodimers and TTR Fab homotetramers migrate on reducing SDS-PAGE as expected for fusion molecules with heavy chain (upper band) and free light chain (lower band).

[0327] like Fig.12 As shown, anti-TRAILR2 TTR antibody homotetramers were well expressed (about 150 mg / L) and assembled correctly in CHO-K1 cells (see Fig.12 a), upper strip). Fig.12 b) shows that upon heating the homotetrameric complex dissociates into their expected components, whereas Fig.12 c) shows that the homotetrameric complexes resolved into their expected components upon heating and reduction. 2-5 μl of conditioned media was loaded per lane and run on a 4%-20% TG gel.

[0328] Fig.13 It is shown that anti-TRAILR2 TTR Fab homotetramers, anti-TRAILR2 TTR antibody homodimers, and anti-TRAILR2 TTR antibody homotetramers assemble correctly and that upon heating and reduction the molecules resolve into their expected component chains (the upper band or bands are heavy chains, and the lowest bands are light chains). Fig.13 It was shown that TTR constructs can be generated with a variety of antibodies.

[0329] Purification and characterization of anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins

[0330] Anti-TRAILR2 TTR antibody homodimers and TTR antibody homotetramer fusion proteins are usually purified as follows. The molecules are purified from the cell culture medium using an AKTA purifier (Universal Medical Group) liquid chromatography system with two 5mL ProA FF HiTrap (Universal Medical Group) columns connected in sequence. The culture medium is directly loaded onto the ProA FF column, then washed with 5CV of DPBS (Life Technologies), and eluted with 8CV of 50mM acetic acid, pH 3.2. The ProA FF elution pool is titrated to pH 5.0 with 2M Tris-HCl, pH 9.2, and then diluted with 9 volumes of sterile water. The conditioned ProA FF pool is dialyzed twice with 2L of 20mM HEPES, 150mM NaCl, pH 7.0 using a 10kDa MWCO sliding analyzer (Thermo Fisher Scientific). Dialysis pool is purified on 18mL SP agarose high performance (SP HP) (General Medical Group) column, wherein uses 20mMNaH2PO4, pH 7.0 5CV washing, and uses the 20CV NaCl gradient elution from 0mM to 500mM.By SDS-PAGE and HPLC-SEC purity selection fraction for pooling.Use VivaSpin 10kDa MWCO centrifugal filter assembly (Sartorius, Gottingen, Germany) to concentrate SP HP pool, and then on 320mL Superdex200 (General Medical Group) with 1.4CV 20mM HEPES, 300mM NaCl, pH 7.0, carry out isocratic purification.By SDS-PAGE and HPLC-SEC purity selection fraction for pooling.Use 10kDa MWCO sliding analyzer (Thermo Fisher Scientific) by SP HP pool 2L 10mMMES, 150mM NaCl, pH 7.0 dialyzed twice. Dialyzed samples were concentrated using VivaSpin 10 kDa MWCO centrifugal filter devices (Sartorius) and then sterile filtered through 0.2 μm Supor syringe filters (Pall).

[0331] Anti-TRAILR2 Fab homotetrameric fusion proteins were typically purified as follows. The molecules were purified from cell culture media using an AKTA purifier (GE Healthcare) liquid chromatography system with a 50 mL Ni Sepharoseexcel (Ni excel) (GE Healthcare) column. The culture media was directly loaded onto the HisTrap column and then eluted with 10 CV of 20 mM NaH 2 PO 4 , 0.5MNaCl, 10mM imidazole, pH 7.4 washing, and 8CV imidazole gradient elution from 10mM to 500mM. Using 10kDa MWCO sliding analyzer (Thermo Fisher Scientific) Ni excel pool is dialyzed twice with 2L of 20mM HEPES, 150mM NaCl, pH 7.0. Dialysis pool is purified on 18mL SP agarose high performance (SP HP) (General Medical Group) column, wherein using 20mMNaH2PO4, 5CV washing of pH 7.0, and using 20CV NaCl gradient elution from 0mM to 500mM. Purity selection fractions are used for pooling by SDS-PAGE and HPLC-SEC. Using 10kDa MWCO sliding analyzer (Thermo Fisher Scientific) SP HP pool is dialyzed twice with 2L of 10mM MES, 150mM NaCl, pH 7.0. The dialyzed samples were concentrated using a VivaSpin 10 kDa MWCO centrifugal filter device (Sartorius) and then sterile filtered through a 0.2 μm Supor syringe filter (Pall Corporation). Protein concentration was determined by UV absorbance at 280 nM (A280) using a NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Samples were analyzed by denaturing non-reducing 4%–12% Bis-Tris NuPAGE gels using MES running buffer (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The samples were analyzed as follows: on a Pheromones SEC 3000 column (7.8 x 300 mm) (Peromones, Torrance, CA, USA), absorbance was observed at 280 nm at 1 mL / min in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9.

[0332] Fig.14 HPLC size exclusion chromatography (SEC) analysis results for each of the anti-TRAILR2 TTR antibody homodimer (middle chromatogram), TTR antibody homotetramer (right chromatogram), and TTR Fab homotetramer (left chromatogram) fusion proteins are shown. The SEC chromatograms show that the major peak elutes at the expected position consistent with a correctly assembled molecule.

[0333] Reducing LC / MS analysis was performed on TRAILR2 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins. Samples were buffer exchanged into 200 mM ammonium acetate and made into 5 μM as the final MS working solution for native MS studies. Samples were also denatured in 8 M guanidine hydrochloride and reduced in 20 mM DDT for LC-MS analysis. 2 μg of material was then injected into the LC-MS system.

[0334] Native MS and ion mobility analysis of anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins were performed on a modified Synapt G1 HDMS instrument operated in positive nanoflow ESI mode on an RF limited drift tube. Critical instrument voltages and pressures were as follows: capillary voltage 0.8 kV to 1.0 kV; sample cone 40 V, extraction cone 1 V; source block temperature 30 °C; trap collision energy 4.0 V; transmission collision energy 60 V to 200 V, equivalent to 3.3 V to 11.1 V / cm applied to the RF-confined drift chamber; trap inlet 3.0 V; trap bias 16 V (N2; when operating with N2, an increased potential is required to inject ions into the drift chamber); IMS DC inlet 5.0 V; IMS DC outlet 0.0 V; transmission DC inlet 0.0 V; transmission DC outlet 2.0 V; transmission wave velocity 70 m / sec; transmission wave amplitude 4.0 V; mobility capture release time 250 μsec; trap height 20.0 V; extraction height 0.0 V; source RF amplitude (peak to peak) 450 V; three-wave RF amplitude (peak to peak), trap 380 V, IMS 150V, transmission 380V; source back pressure 6.0mbar; sink / transmission pressure SF6, 3.3e -2 mbar (indicating Pirani gauge; flow rate 3.0mL / min); IMS pressure N2 2.05mbar (1.54 Torr; flow rate 38mL / min); IMS pressure He 2.70mbar (2.03 Torr; flow rate 70mL / min). The pressure in the RF confined drift chamber can be accurately measured using an MKS Baratron 626 capacitance manometer (range 10 Torr; accurate to 0.25%) and an MKS PDR2000 power supply. The ambient temperature is measured using an Oakton Temp10T thermocouple. The temperature is measured at the location where the capacitance manometer is connected to the instrument ion optical mirror cover. Instrument control and data acquisition are performed by MassLynx 4.1SCN 639, SCN744. By accurately measuring the pressure inside the drift chamber assembly and the ambient temperature of the drift chamber, and performing up to 10 mobility measurements at different drift chamber voltages (60V to 200V), the mobility and Ω values ​​are obtained. Individual temperature and pressure measurements were made for each acquisition, and for the final Ω value calculation, the average temperature and pressure values ​​were used. Typical temperature and pressure changes were ≤ 0.2°C and ≤ 0.002 Torr over 10 minutes.

[0335] Denaturing LC-MS analysis of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins was performed by injecting 2 μg of denatured and reduced protein directly onto a C4BEH 2.1 x 50 mm analytical column operated at 45 °C and a flow rate of 400 μL / min. MS detection was performed on a Waters XevoQ-ToF mass spectrometer. The sample cone and extraction cone were set to 35 V and 1 V, respectively.

[0336] Mass spectrometry and ion mobility results: anti-TRAILR2 TTR antibody homotetramer: the MW measured by native-MS is 645.9 kDa, and the N2-CCS value measured by ion mobility is 190.1 nm2, and the denatured and reduced subunit MWs are 23,388 Da, 49,180 Da, and 62,872 Da. Anti-TRAILR2 TTR Fab homotetramer fusion protein: the MW measured by native-MS is 250.5 kDa, and the N2-CCS value measured by ion mobility is 130.3 nm2, and the denatured and reduced subunit MWs are 23,429 Da and 38,582 Da. The MW measured by native-MS of the anti-TRAILR2 TTR antibody homodimer fusion protein is 347.2 kDa, and the N2-CCS value measured by ion mobility is 124.0 nm2, and the denatured and reduced subunit MWs are 23,391 Da and 62,867 Da.

[0337] SEC multi-angle light scattering (MALS) of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer and TTR Fab homotetramer fusion protein was performed on an Agilent 1100HPLC equipped with Wyatt Heleos II and OptiLab-TrEX detector. The column used was Superdex 200 (10 / 300GL). The mobile phase was 2X PBS and the flow rate was 0.4ml / min. The running time of each sample was 70 minutes. The injection amounts of the four SP fractions (A3, A8, A12 and C1) of anti-TRAILR2 TTR antibody homodimer and TTR antibody homotetramer fusion protein were 81μg, 63μg, 75μg and 75μg respectively, and the injection amount of the final pool of anti-TRAILR2-TTR was 77ug. Run setup, data collection and analysis procedures were performed on Agilent's Chemstation (v B.04.02 96) and Wyatt's ASTRA (v 6.1.1.17) software.

[0338] Example 7: Activity and PK profiles of anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins

[0339] The activities and PK profiles of the parental anti-TRAILR2 mAb (conatumumab), anti-TRAILR2 TTR antibody homodimer, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer fusion protein were evaluated.

[0340] PK spectrum

[0341] The PK profiles of anti-TRAILR2 TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins were determined by intravenous injection in male CD-1 mice (n=3 / group) at 6.5 mg / kg of TTR antibody homotetramers, 3.5 mg / kg of TTR antibody homodimers, 2.5 mg / kg of TTR Fab homotetramers, and 1.5 mg / kg of conatumumab normalized to the molar equivalent of conatumumab 341-G1 (conatumumab 341-G1 is an Ab engineered to no longer bind TRAILR2) antibody. Serum samples were collected from 75 μl blood samples at 0.5, 2, 8, 24, 48, 72, 96, 192, 336, 504, 672, and 840 hours after dosing. Each blood sample was kept at room temperature after collection and, after a 30-40 minute clotting period, centrifuged at 11,500 rpm for approximately 10 minutes at 2-8° C. using a calibrated Eppendorf 5417R centrifugal system (Brinkman Instruments, Westbury, NY). The collected serum was then transferred to pre-labeled (for each rat) cryogenic storage tubes and stored at −60° C. to −80° C. for future bioanalysis.

[0342] The following PK assay was used to measure the total anti-TRAILR2 species (i.e., any anti-TRAILR2 binding species present) of conatumumab, anti-TRAILR2 TTR antibody homodimers, anti-TRAILR2 TTR antibody homotetramers, and anti-TRAILR2 TTR Fab homotetramer fusion proteins in mouse serum as determined by Mesoscale Discovery (MSD). To measure the total amount of conatumumab in mouse serum samples, conventional binding 96-well MSD plates (Mesoscale Discovery, Gaithersburg, Maryland) were coated with 2 μg / ml of rabbit anti-conatumumab polyclonal antibody (Amgen Inc., Thousand Oaks, California) in PBS and then incubated overnight at 4°C. The plates were then washed and incubated with I-Block TM (Life Technologies, Carlsbad, CA) and blocked overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted in virgin CD-1 mouse serum if dilution was required. Standards, QCs, and samples were then diluted 1:20 in a buffer containing PBS, 1 M NaCl, 0.5% Tween 20, and 1% bovine serum albumin. The plates were washed three times with approximately 200 μl of 1X KPL buffer (KPL, Gaithersburg, MD), and 50 μl aliquots of the diluted standards, QCs, and samples were transferred to the anti-conatumumab antibody-coated plates and incubated at room temperature (approximately 25°C) for 1.5 hours. The plates were washed three times with approximately 200 μl of 1X KPL wash buffer and then added to the I-Block 5000 antibody containing 5% BSA. TM 50 μl of 100 ng / ml mouse anti-huFc antibody (clone 1.35.1) conjugated with MSD SULFO-tag (Amgen, Thousand Oaks, CA) was added and incubated at room temperature for 1.5 h. For the anti-TRAILR2 TTR Fab homotetramer construct, 50 μl of 250 ng / ml mouse anti-κLC, clone KCF-9 conjugated with biotin was added and incubated for 1.5 hours; then, after washing the plate with 1X KPL wash buffer, 50 μl of 100 ng / ml streptavidin conjugated with MSD SULFO-tag (Amgen) was added and incubated for or 15 minutes. The plate was then washed six times with approximately 200 μl of 1X KPL wash buffer, followed by the addition of 150 μl of 1X read buffer T (Mid-Scale Discovery), and the electrochemiluminescent signal was measured using an MSD 6000 plate reader (Mid-Scale Discovery). Use Serum concentration data were analyzed by a non-compartmental method using ELISA® (Phoenix 64, version 6.4.0.768, Pharsight Corp., Mountain View, CA).

[0343] The following PK assay was used to measure the presence of anti-TRAILR2 TTR binding species and TTR species in anti-TRAILR2 TTR antibody homodimers, anti-TRAILR2 TTR antibody homotetramers, and anti-TRAILR2 TTR Fab homotetramer fusion proteins in mouse serum by MSD assay of mouse serum samples. Conventional binding 96-well MSD plates (Mesoscale Discovery, Gaithersburg, Maryland) were coated with 2 μg / ml rabbit anti-TTR polyclonal antibody (Amgen Inc., Thousand Oaks, California) in PBS and then incubated overnight at 4°C. The plates were then washed and incubated with I-Block TM (Life Technologies, Carlsbad, CA) and blocked overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted in virgin CD-1 mouse serum if dilution was required. Standards, QCs, and samples were then diluted 1:20 in a buffer containing PBS, 1 M NaCl, 0.5% Tween 20, and 1% bovine serum albumin buffer. The plates were washed three times with approximately 200 μl of 1X KPL buffer (KPL, Gaithersburg, MD), and 50 μl aliquots of the diluted standards, QCs, and samples were transferred to the anti-TTR antibody-coated plates and incubated at room temperature for 1.5 hours. The plates were washed three times with approximately 200 μl of 1X KPL wash buffer, and then 50 μl of 250 ng / ml of rabbit anti-conatumumab polyclonal antibody conjugated to biotin was added and incubated for 1.5 hours. After washing the plate three times with 1X KPL wash buffer, add 50 μl of 100 ng / ml streptavidin conjugated with MSD SULFO-tag (Amgen, USA) and incubate for 15 minutes. Wash the plate six times with about 200 μl of 1X KPL wash buffer, then add 150 μl of 1x read buffer T (Middle Scale Discovery), and measure the electrochemiluminescent signal using an MSD 6000 plate reader (Middle Scale Discovery). Use Serum concentration data were analyzed by a non-compartmental method using ELISA® (Phoenix 64, version 6.4.0.768, Pharsight Corp., Mountain View, CA).

[0344] The results of the PK analysis can be found in Fig.15 Found in Fig.15 a) shows (measured for any anti-TRAILR2 binding species): [1] the PK of the anti-TRAILR2 TTR antibody homodimer ("1") is better than that of the parental Ab (conatumumab ("3"); and conatumumab 341-G1 ("2")); [2] the PK of the anti-TRAILR2 TTR Fab homotetramer ("5") is poor, which is likely attributed to the lack of the Fc region of the Fab and therefore the lack of the ability to mediate / extend its half-life; and [3] the PK of the anti-TRAILR2 TTR antibody homotetramer ("4") is similar to that of the parental mAb (conatumumab). Notably, the anti-TRAILR2 TTR antibody homotetramer showed improved PK compared to its parent (conatumumab), which is different from what was observed for the anti-GITR TTR antibody homotetramer (which was not as robust as the parental mAb, although it still showed significantly enhanced PK compared to the Fab). This observation suggests that the PK of TTR antibody homotetrameric fusion proteins may be antibody and / or target dependent. Fig.15 b) (measuring anti-TRAILR2 TTR-bound species and TTR species) shows that the PK of the intact anti-TRAILR2 TTR fusion protein is similar to Fig.15 This is consistent with what was observed in a), indicating that in vivo proteolysis cannot release the anti-TRAILR2 binding moiety from each TTR fusion protein. Fig.15 In b), anti-TRAILR2 TTR antibody homodimer ("1"); anti-TRAILR2 TTR antibody homotetramer ("2"); and TRAILR2 TTR Fab homotetramer ("3") were evaluated.

[0345] Cell-based activity assays

[0346] The activity of anti-TRAILR2 TTR fusion protein was evaluated compared to conatumumab + protein G on the melanoma cell line WM35 (ATCC). WM35 was evaluated for expression of DR5 but not DR4. TRAIL activates both DR5 and DR4. WM35 cells were cultured at 10 4 Cells / well were plated in microtiter plates and then incubated with conatumumab, anti-TRAILR2 TTR antibody homodimers, anti-TRAILR2 TTR antibody homotetramers, or anti-TRAILR2 TTR Fab homotetramer fusion proteins in the absence or presence of 1 μg / ml protein G (Pierce) (to promote cross-linking). Triplicate samples were analyzed. The cells were incubated at 37°C with 5% CO 2 After 24 h of incubation in , cell viability was assessed using the CellTiter-Glo assay (Promega). CellTiter-Glo reactions were performed according to the manufacturer's instructions, and luminescence was measured using PerkinElmer Envision. The y-axis displays the luminescence signal as relative light units (RLU); a decrease in RLU reflects a decrease in ATP production and a decrease in the number of viable cells. The x-axis displays protein concentration.

[0347] Fig.16 Results of the WM35 assay are shown. Conatumumab ("1") is ineffective at killing cells unless it is clustered with protein G (which has two Ig binding sites; "5"). Anti-TRAILR2 TTR antibody homodimers ("2") are slightly better than parent conatumumab at killing WM35 cells. Anti-TRAILR2 TTR Fab homotetramers ("4") have slightly better WM35 cell killing ability than conatumumab. However, anti-TRAILR2 TTR antibody homotetramers ("3") are significantly more effective than conatumumab at killing WM35 cells, and even more effective than conatumumab / protein G clustered complexes. In addition, clustering of anti-TRAILR2 TTR antibody homodimers with protein G ("6") improves the potency of the fusion protein, while clustering of anti-TRAILR2 TTR Fab homotetramers with protein G ("8") has little effect. Interestingly, clustering of anti-TRAILR2 TTR antibody homotetramers with protein G ("7") slightly impaired the potency of the protein fusion. These results suggest that clustering mediated by TTR antibody homotetramers can enhance antitumor activity.

[0348] The activity of anti-TRAILR2 TTR fusions was also evaluated compared to conatumumab + protein G on primary human keratinocytes (Lonza). Primary keratinocytes were cultured at 10 4 Cells / well were plated in microtiter plates and then incubated with: TRAIL alone, conatumumab (with 1 μg / ml protein G (Pierce) to facilitate cross-linking), or TRAIL plus 1 ug / ml conatumumab. Samples were prepared in triplicate for each condition. After incubation for 24 hours at 37°C, 5% CO2, cell viability was assessed using the Cell Titer-glo assay (Promega). Primary keratinocytes were also cultured at 10 4 Cells / well were plated in microtiter plates and then incubated with anti-TRAILR2 TTR antibody homotetramer, anti-TRAILR2 TTR Fab homotetramer, conatumumab, or TRAIL at the indicated concentrations. The combination of TRAIL and 1 μg / ml conatumumab was also tested. Samples were prepared in triplicate. Incubate at 37°C with 5% CO 2 After 24 h of incubation, cell viability was measured using the CellTiter-glo assay (Promega).

[0349] Fig.17 Results of keratinocyte assays are shown. Again, conatumumab ("1") was ineffective in killing primary human keratinocytes. Anti-TRAILR2 TTR Fab homotetramers ("2") showed slightly better potency than conatumumab. Again, anti-TRAILR2 TTR antibody homotetramers ("3") were significantly more effective than conatumumab in killing primary human keratinocytes. These results again show that clustering mediated by TTR antibody homotetramers can enhance activity in in vitro cell killing assays.

[0350] Activity assay based on the murine colo 205 adenocarcinoma model

[0351] The activity of conatumumab, anti-TRAILR2 TTR antibody homodimers, anti-TRAILR2 TTR antibody homotetramers, and anti-TRAILR2 TTR Fab homotetramer fusion proteins were evaluated in the murine Colo205 human colon adenocarcinoma model. Colo205 human colon adenocarcinoma cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Sigma, 2442-500 mL), 4 mM L-glutamine (Hyclone, SH30034.01), 1 mM HEPES (Hyclone, SH30237.01), 1 mM sodium pyruvate (Sigma, S8636-100 mL), and 2.5 g / L glucose (Sigma, G8769) at 5% CO. 2 At passage 3, cells were harvested and resuspended in serum-free medium to generate 10x10 6 The final concentration was 10 cells / mL. The cell viability was 98% as determined by trypan blue exclusion.

[0352] Sixty female NU / NU nude mice were injected subcutaneously on the right flank with 1x10 6 cells (100 μL volume) while the mice were anesthetized with isoflurane. On day 7 after tumor implantation, the tumor volume was 34 mm 3 and 99mm 3 50 mice were divided into 5 treatment groups with 10 mice in each group, so that each group had a 3 Up to 48mm 3 Similar mean tumor volumes. Ten mice with smaller or larger tumors were excluded from the study.

[0353] Five groups of animals received intraperitoneal administration of conatumumab (0.69 μM / animal), conatumumab-341-G1 (0.69 μM / animal), anti-TRAILR2 TTR antibody homodimer (0.34 μM / animal), anti-TRAILR2 TTR antibody homotetramer (0.17 μM / animal) and anti-TRAILR2 TTR Fab homotetramer (0.17 μM / animal) twice a week (biw) for 3 weeks and a total of 6 treatments. Treatments were standardized so that all had the same number of binding sites as conatumumab, except for anti-TRAILR2 TTR Fab homotetramer (which had only half the binding sites compared to the rest of the treatments). All treatments started on day 8 after tumor implantation and ended on day 25 (treatments were performed on days 8, 11, 15, 19, 22 and 25, respectively). All treatments were freshly prepared in diluent (DPBS) on the treatment date before injection.

[0354] Tumor volume measurement: The length and width of the tumor were measured using an ABS solar-powered digital caliper model #Cd-S6"C (Mitutoyo Corporation, Japan). Tumor volume was calculated as 0.5×L×W2, where W is the smaller of the two measurements and is expressed in mm. 3 express.

[0355] Body weight measurement: To measure body weight, animals were placed in a weighing pan on a balance (Mettler Toledo model PB602-S, Switzerland). The average body weight over 3 seconds was determined using the dynamic weight function of the balance. Body weight was reported as body weight change, which was calculated as 100x (Wc / Wi), where Wc is the current body weight and Wi is the body weight at the start of treatment. For this study, the body weight on day 4 was used as Wi for all animals.

[0356] Euthanasia: Five animals were euthanized before the end of the study because tumor size reached 1500 mm 3 -2000mm 3 Conatumumab-341-G1 group: On day 25, two mice were euthanized due to excessive tumor volume; on day 35, another two mice were euthanized due to excessive tumor volume. On day 42, due to average tumor volume >1200mm 3 Ten mice in the anti-TRAILR2 TTR antibody homodimer group and the remaining six mice in the conatumumab-341-G1 group were euthanized when more than half of the mice in the cohort were lost. 3 , so one mouse from the anti-TRAILR2 TTR Fab homotetramer group was euthanized. One mouse from the anti-TRAILR2 TTR Fab homotetramer group was euthanized due to excessive tumor volume on day 49. All remaining animals were euthanized at the end of the study on day 63. Animals were euthanized by an overdose of isoflurane, and blood was collected by cardiac puncture.

[0357] Tumor Measurements: Data from days 4 to 63 are expressed as the mean plus or minus the standard error and plotted as a function of time. The statistical significance of the observed differences between growth curves was assessed by repeated measures analysis of variance on transformed tumor volume data with Dunnett's adjustment for multiple comparisons. Analyses were performed using the SAS PROC MIXED procedure (with model effects of transformed baseline tumor volume, day, treatment, and day by treatment interaction; REPEATED formulation with day as the replicate, animal subject, and Toeplitz covariance structure; and LSMEANS formulation for Dunnett's analysis comparing control to other treatment groups). Data were log- or square-root transformed according to the Horwitz method, and transformed baseline tumor volume was included in the model as a covariate to account for possible pre-treatment differences in tumor volume. If log- or square-root transformation failed to obtain an appropriate residual distribution, a nonparametric repeated measures analysis of variance model with the same model effect for rank of tumor volume was used. P values ​​< 0.05 were considered statistically significant. Conatumumab (wild type) and conatumumab-341-G1 groups were used as control groups for analysis.

[0358] Fig.18 Results from the mouse colo205 model test are shown. Conatumumab ("Group 1"; 0.69 μM / animal), anti-TRAILR2 TTR antibody homotetramer ("Group 4"; 0.17 μM / animal), and anti-TRAILR2 TTR Fab homotetramer ("Group 5"; 0.17 μM / animal) all inhibited tumor growth in vivo during the dosing period, with the anti-TRAILR2 TTR antibody homotetramer showing the best level of inhibition during the dosing period. The anti-TRAILR2 TTR antibody homodimer ("Group 3"; 0.34 μM / animal) was only slightly better than the vehicle control (conatumumab-341-G1, "Group 2"; 0.69 μM / animal) in inhibiting tumor growth. Notably, the anti-TRAILR2 TTR antibody homotetramer continued to inhibit tumor growth long after dosing was stopped. In addition, surprisingly, although the anti-TRAILR2 TTR Fab homotetramer had a poor PK profile ( Fig.15 ), but the construct was as effective as conatumumab in inhibiting tumor growth. Without being bound by a particular theory, it is possible that initial exposure to the anti-TRAILR2 TTR Fab homotetramer triggers a series of events in the target cell that leads to death and does not require long-term binding. In addition, the relatively small size of the Fab construct may allow it to better enter the tumor environment, thereby compensating for poor PK. Finally, the physical configuration of the Fab construct may be more effective in vivo.

[0359] Activity assay based on the murine SW403 adenocarcinoma model

[0360] SW403 human colon adenocarcinoma cells were cultured at 37°C in 5% CO 2 Maintained in RPMI-1640 medium (Sigma R0883) supplemented with 10% FBS (Sigma, 2442-500 mL), 4 mM L-glutamine (Hyclone, SH30034.01). At passage 5, cells were harvested and resuspended in serum-free medium to generate 50×10 6 The final concentration was 10 cells / mL. The cell viability was 95% as determined by trypan blue exclusion.

[0361] Seventy-five female NU / NU nude mice were injected subcutaneously on the right flank with 5x10 6 cells (100 μL volume) while the mice were anesthetized with isoflurane. On day 7 after tumor implantation, the tumor volume was 21 mm 3 and 160mm 3 The 60 mice were divided into 6 treatment groups with 10 mice in each group, so that each group had a mean of 58 mm 3 Up to 66mm 3 Similar mean tumor volumes. Fifteen mice with smaller or larger tumors were excluded from the study.

[0362] Six groups of animals received intraperitoneal administration of conatumumab wild-type (1.38 μM / animal), conatumumab-341-G1 (1.38 μM / animal), anti-TRAILR2 TTR antibody homodimer (0.69 uM / animal), anti-TRAILR2 TTR antibody homotetramer (0.34 μM / animal), anti-TRAILR2 TTR Fab homotetramer (0.34 μM / animal) and DPBS (vehicle control) twice a week (biw) for 3 weeks and a total of 6 treatments. Treatments were standardized so that each had an equal number of binding sites as conatumumab wild-type, except for anti-TRAILR2 TTR Fab homotetramer (which had only half the binding sites compared to the rest of the treatments). All treatments started on day 15 after tumor implantation and ended on day 32 (treatments were performed on days 15, 19, 22, 26, 29 and 32, respectively). All treatments were prepared fresh in diluent (DPBS) on the treatment date prior to injection.

[0363] Tumor volume measurement: The length and width of the tumor were measured using an ABS solar-powered digital caliper model #Cd-S6"C (Mitutoyo, Japan). Tumor volume was calculated as 0.5×L×W2, where W is the smaller of the two measurements and is expressed in mm. 3 express.

[0364] Body weight measurement: Animals were placed in a weighing pan on a balance (Mettler Toledo model PB602-S, Switzerland). The average body weight over 3 seconds was determined using the dynamic weight function of the balance. Body weight was reported as body weight change, which was calculated as 100x (Wc / Wi), where Wc is the current body weight and Wi is the body weight at the start of treatment. For this study, the day 4 body weight was used as Wi for all animals.

[0365] Euthanasia and tissue collection: Eight animals were euthanized before the end of the study because tumor size reached 1500 mm 3 -2000 mm 3 . One mouse from the Conatumumab-341-G1 group was euthanized due to excessive tumor volume on day 33. On day 36, another mouse from the Conatumumab-341-G1 group, two mice from the DPBS vehicle control group, and one mouse from the anti-TRAILR2 TTR antibody homodimer group were euthanized. The remaining animals in the Conatumumab-341-G1, DPBS vehicle control, and anti-TRAILR2 TTR antibody homodimer groups were all euthanized on day 39. One mouse from the Conatumumab WT group was euthanized on day 39. Another mouse from the Conatumumab WT group and one mouse from the anti-TRAILR2 TTR Fab homotetramer group were euthanized on day 46. At the end of the study, all remaining animals in the conatumumab WT, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer groups were euthanized on day 49. All euthanasias were performed by an overdose of isoflurane followed by blood collection by cardiac puncture.

[0366] Tumor Measurements: Data between days 7 and 49 are expressed as the mean plus or minus the standard error and plotted as a function of time. The statistical significance of the observed differences between growth curves was assessed by repeated measures analysis of variance on transformed tumor volume data with Dunnett's adjustment for multiple comparisons. Analyses were performed using the SAS PROC MIXED procedure (with model effects of transformed baseline tumor volume, day, treatment, and day by treatment interaction; REPEATED formulation, where day is the replicate value, animal subject, and Toeplitz covariance structure; and LSMEANS formulation for Dunnett's analysis comparing control and other treatment groups). Data were log- or square-root transformed according to the Horwitz method, and transformed baseline tumor volume was included in the model as a covariate to account for possible pre-treatment differences in tumor volume. If log- or square-root transformation failed to obtain an appropriate residual distribution, a nonparametric repeated measures analysis of variance model with the same model effect for rank of tumor volume was used. P values ​​< 0.05 were considered statistically significant. All statistical calculations were performed using the AMG Biostatistics analysis tool (cld-pweb-taivd.amgen.com / biostats / Statistics). The Conatumumab WT group was used as the control group in the analysis.

[0367] Fig.19 The results of the mouse SW403 model test are shown. Anti-TRAILR2 TTR antibody homotetramers ("Group 5") and anti-TRAILR2 TTR Fab homotetramers ("Group 6") inhibited tumor growth in vivo better than conatumumab ("Group 1") during the dosing period. Anti-TRAILR2 TTR antibody homodimers ("Group 4") inhibited growth at about the same level as conatumumab during the dosing period, although neither was significantly better than the vehicle control ("Group 3") in terms of growth inhibition. It is worth noting that even after stopping dosing, anti-TRAILR2 TTR antibody homotetramers and anti-TRAILR2 TTR Fab homotetramers slowed tumor growth. Again, it was surprisingly observed that although anti-TRAILR2 TTR Fab homotetramers had a poor PK profile ( Fig.15 ), but even after dosing was stopped, the construct was as effective as the anti-TRAILR2 TTR antibody homotetramer in slowing tumor growth.

[0368] Fig. 20 It was shown that the body weights of the murine colo205 model mice and the murine SW403 model mice were similar for all tested compounds and appeared to be on a normal upward trajectory. This result suggests that the tested compounds were not overtly toxic (as the mice were healthy enough to eat and gain weight normally).

[0369] Summary of SEQ ID NO:

[0370]

[0371] Sequence Listing <110> Amgen K.W.Walker <120> Transthyretin-Immunoglobulin Fusion <130> A-2196-WO-PCT <150> 62 / 568,217 <151> 2017-10-04 <160> 47 <170> PatentIn Version 3.5 <210> 1 <211> 127 <212> PRT <213> Homo sapiens <400> 1 Gly Pro Thr Gly Thr Gly Glu Ser Lys Ala Pro Leu Met Val Ala Val 1 5 10 15 Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val 20 25 30 Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys 35 40 45 Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe 50 55 60 Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys 65 70 75 80 Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr 85 90 95 Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser 100 105 110 Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu 115 120 125 <210> 2 <211> 381 <212> DNA <213> Homo sapiens <400> 2 ggccctacgg gcaccggtga atccaaggct cctctgatgg tcgccgttct agatgctgtc 60 cgaggcagtc ctgccatcaa tgtggccgtg catgtgttca gaaaggctgc tgatgacacc 120 tgggagccat ttgcctctgg gaaaaccagt gagtctggag agctgcatgg gctcacaact 180 gaggaggaat ttgtagaagg gatatacaaa gtggaaatag acaccaaatc ttactggaag 240 gcacttggca tctccccatt ccatgagcat gcagaggtgg tattcacagc caacgactcc 300 ggcccccgcc gctacaccat tgccgccctg ctgagcccct actcctattc caccacggct 360 gtcgtcacca atcccaagga a 381 <210> 3 <211> 127 <212> PRT <213> Mus musculus <400> 3 Gly Pro Ala Gly Ala Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val 1 5 10 15 Leu Asp Ala Val Arg Gly Ser Pro Ala Val Asp Val Ala Val Lys Val 20 25 30 Phe Lys Lys Thr Ser Glu Gly Ser Trp Glu Pro Phe Ala Ser Gly Lys 35 40 45 Thr Ala Glu Ser Gly Glu Leu His Gly Leu Thr Thr Asp Glu Lys Phe 50 55 60 Val Glu Gly Val Tyr Arg Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys 65 70 75 80 Thr Leu Gly Ile Ser Pro Phe His Glu Phe Ala Asp Val Val Phe Thr 85 90 95 Ala Asn Asp Ser Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser 100 105 110 Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn 115 120 125 <210> 4 <211> 615 <212> DNA <213> Mus musculus <400> 4 acagaagtcc actcattctt ggcaggatgg cttctcatcg tctgctcctc ctctgccttg 60 ctggactggt atttgtgtct gaggctggcc ctacgggcac cggtgaatcc aagtgtcctc 120 tgatggtcaa agttctagat gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg 180 tgttcagaaa ggctgctgat gacacctggg agccatttgc ctctgggaaa accagtgagt 240 ctggagagct gcatgggctc acaactgagg aggaatttgt agaagggata tacaaagtgg 300 aaatagacac caaatcttac tggaaggcac ttggcatctc cccattccat gagcatgcag 360 aggtggtatt cacagccaac gactccggcc cccgccgcta caccattgcc gccctgctga 420 gcccctactc ctattccacc acggctgtcg tcaccaatcc caaggaatga gggacttctc 480 ctccagtgga cctgaaggac gagggatggg atttcatgta accaagagta ttccattttt 540 actaaagcac tgttttcacc tcatatgcta tgttagaagt ccaggcagag acaataaaac 600 attcctgtga aaggc 615 <210> 5 <211> 452 <212> PRT <213> Homo sapiens <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Arg Gly 20 25 30 Gly Asp Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Tyr Ser Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135 140 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 195 200 205 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr 210 215 220 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 225 230 235 240 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 290 295 300 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 6 <211> 452 <212> PRT <213> Homo sapiens <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Arg Gly 20 25 30 Gly Asp Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Tyr Ser Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135 140 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 195 200 205 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr 210 215 220 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 225 230 235 240 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 290 295 300 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Met Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 7 <211> 452 <212> PRT <213> Homo sapiens <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Arg Gly 20 25 30 Gly Asp Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Tyr Ser Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135 140 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 195 200 205 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr 210 215 220 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 225 230 235 240 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 290 295 300 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr 385 390 395 400 Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 8 <211> 7 <212> PRT <213> Homo sapiens <400> 8 Arg Gly Gly Asp Tyr Trp Ser 1 5 <210> 9 <211> 16 <212> PRT <213> Homo sapiens <400> 9 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 10 <211> 17 <212> PRT <213> Homo sapiens <400> 10 Asp Tyr Asp Ile Leu Thr Gly Tyr Ser Tyr Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val <210> 11 <211> 219 <212> PRT <213> Homo sapiens <400> 11 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Arg Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 12 <211> 16 <212> PRT <213> Homo sapiens <400> 12 Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Homo sapiens <400> 13 Leu Gly Ser Asn Arg Ala Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Met Gln Ala Leu Gln Thr Pro Arg Thr 1 5 <210> 15 <211> 1956 <212> DNA <213> Homo sapiens <400> 15 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcaga agaggtggtg actattggag ctggattcgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtactaac 180 tacaacccgt ccctcaagag tcgagctacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tctattactg tgcgagagat 300 tacgatattt tgactggtta ttcctactac tactacggta tggacgtctg gggccaaggg 360 accacggtca ccgtctctag tgcctccacc aagggcccat cggtcttccc cctggcgccc 420 tgctccagga gcacctccga gagcacagcg gccctgggct gcctggtcaa ggactacttc 480 cccgaaccgg tgacggtgtc gtggaactca ggcgctctga ccagcggcgt gcacaccttc 540 ccagctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600 agcaacttcg gcacccagac ctacacctgc aacgtagatc acaagcccag caacaccag acctgcactg tctctggtgg ctccatcaga agaggtggtg actattggag ctggattcgc 720 cagcacccag ggaagggcct ggagtggatt gggtacatct attackcagtgg gagtactac 780 840. ccctcaagag tcgagctacc atatcagtag acacgtctaa gaaccagttc tccctgaagc tgagctctgt gactgccgcg gacacggccg tctattactg tgcgagagat 900 tacgatattt tgactggtta ttcctactac tactacggta tggacgtctg gggccaaggg accacggtca ccgtctctag tgcctccacc aagggcccat cggtcttccc cctggcgccc 1020 tgctccagga gcacctccga gagcacagcg gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca ggcgctctga ccagcggcgt gcacaccttc 1140 ccagctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc aacgtagatc acaagcccag caacaccag gtggacaaga cagttgagcg caaatgttgt gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 1380 cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc acgaagaccc cgaggtccag 1440 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc acgggaggag 1500 cagttcaaca gcacgttccg tgtggtcagc gtcctcaccg ttgtgcacca ggactggctg 1560 aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccagcccc catcgagaaa 1620 accatctcca aaaccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1680 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1740 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccaca 1800 cctcccatgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1860 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1920 cactacacgc agaagagcct ctccctgtct ccgggt 1956 <210> 16 <211> 1356 <212> DNA <213> Homo sapiens <400> 16 60. caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc acctgcactg tctctggtgg ctccatcaga agaggtggtg actattggag ctggattcgc cagcacccag ggaagggcct ggagtggatt gggtacatct attackcagtgg gagtactac 240. ccctcaagag tcgagctacc atatcagtag acacgtctaa gaaccagttc tccctgaagc tgagctctgt gactgccgcg gacacggccg tctattactg tgcgagagat 300 tacgatattt tgactggtta ttcctactac tactacggta tggacgtctg gggccaaggg 360 accacggtca ccgtctctag tgcctccacc aagggcccat cggtcttccc cctggcgccc 420 tgctccagga gcacctccga gagcacagcg gccctgggct gcctggtcaa ggactacttc 480 cccgaaccgg tgacggtgtc gtggaactca ggcgctctga ccagcggcgt gcacaccttc 540 ccagctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600 agcaacttcg gcacccagac ctacacctgc aacgtagatc acaagcccag caacaccag gtggacaaga cagttgagcg caaatgttgt gtcgagtgcc caccgtgccc agcaccacct 720 gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc acgaagaccc cgaggtccag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc acgggaggag 900 cagttcaaca gcacgttccg tgtggtcagc gtcctcaccg ttgtgcacca ggactggctg 960 aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccagcccc catcgagaaa 1020 accatctcca aaaccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cggaaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccaca 1200 cctcccatgc tgaagtccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacgc agaagagcct ctccctgtct ccgggt 1356 <210> 17 <211> 1356 <212> DNA <213> Homo sapiens <400> 17 60. caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc acctgcactg tctctggtgg ctccatcaga agaggtggtg actattggag ctggattcgc cagcacccag ggaagggcct ggagtggatt gggtacatct attackcagtgg gagtactac 240. ccctcaagag tcgagctacc atatcagtag acacgtctaa gaaccagttc tccctgaagc tgagctctgt gactgccgcg gacacggccg tctattactg tgcgagagat 300 tacgatattt tgactggtta ttcctactac tactacggta tggacgtctg gggccaaggg 360 accacggtca ccgtctctag tgcctccacc aagggcccat cggtcttccc cctggcgccc 420 tgctccagga gcacctccga gagcacagcg gccctgggct gcctggtcaa ggactacttc 480 cccgaaccgg tgacggtgtc gtggaactca ggcgctctga ccagcggcgt gcacaccttc 540 ccagctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600 agcaacttcg gcacccagac ctacacctgc aacgtagatc acaagcccag caacaccag gtggacaaga cagttgagcg caaatgttgt gtcgagtgcc caccgtgccc agcaccacct 720 gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc acgaagaccc cgaggtccag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc acgggaggag 900 cagttcaaca gcacgttccg tgtggtcagc gtcctcaccg ttgtgcacca ggactggctg 960 aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccagcccc catcgagaaa 1020 accatctcca aaaccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta cgataccaca 1200 cctcccatgc tggactccga cggctccttc ttcctctaca gcgatctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacgc agaagagcct ctccctgtct ccgggt 1356 <210> 18 <211> 452 <212> PRT <213> Homo sapiens <400> 18 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Leu Gly Lys Asp Tyr Tyr Ser Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 19 <211> 452 <212> PRT <213> Homo sapiens <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Leu Gly Lys Asp Tyr Tyr Ser Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 20 <211> 452 <212> PRT <213> Homo sapiens <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Leu Gly Lys Asp Tyr Tyr Ser Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 21 <211> 228 <212> PRT <213> Homo sapiens <400> 21 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Leu Gly Lys Asp Tyr Tyr Ser Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Glu Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Gly Gly 225 <210> 22 <211> 5 <212> PRT <213> Homo sapiens <400> 22 Ser Tyr Gly Met His 1 5 <210> 23 <211> 17 <212> PRT <213> Homo sapiens <400> 23 Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 24 <211> 14 <212> PRT <213> Homo sapiens <400> 24 Gly Gly Arg Leu Gly Lys Asp Tyr Tyr Ser Gly Met Asp Val 1 5 10 <210> 25 <211> 214 <212> PRT <213> Homo sapiens <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 26 <211> 214 <212> PRT <213> Homo sapiens <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Lys 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 27 <211> 11 <212> PRT <213> Homo sapiens <400> 27 Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10 <210> 28 <211> 7 <212> PRT <213> Homo sapiens <400> 28 Ala Thr Ser Ser Leu Gln Ser 1 5 <210> 29 <211> 9 <212> PRT <213> Homo sapiens <400> 29 Leu Gln His Asn Thr Tyr Pro Trp Thr 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 30 Gly Gly His His His His His 1 5 <210> 31 <211> 451 <212> PRT <213> Homo sapiens <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe 65 70 75 80 Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 32 <211> 451 <212> PRT <213> Homo sapiens <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe 65 70 75 80 Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 33 <211> 451 <212> PRT <213> Homo sapiens <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe 65 70 75 80 Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 34 <211> 227 <212> PRT <213> Homo sapiens <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe 65 70 75 80 Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Glu Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Gly Gly 225 <210> 35 <211> 7 <212> PRT <213> Homo sapiens <400> 35 Ser Gly Asp Tyr Phe Trp Ser 1 5 <210> 36 <211> 16 <212> PRT <213> Homo sapiens <400> 36 His Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 37 <211> 12 <212> PRT <213> Homo sapiens <400> 37 Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 38 <211> 215 <212> PRT <213> Homo sapiens <400> 38 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Arg Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ser Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 39 <211> 215 <212> PRT <213> Homo sapiens <400> 39 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Arg Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ser Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Lys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 40 <211> 12 <212> PRT <213> Homo sapiens <400> 40 Arg Ala Ser Gln Gly Ile Ser Arg Ser Tyr Leu Ala 1 5 10 <210> 41 <211> 7 <212> PRT <213> Homo sapiens <400> 41 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 42 <211> 9 <212> PRT <213> Homo sapiens <400> 42 Gln Gln Phe Gly Ser Ser Pro Trp Thr 1 5 <210> 43 <211> 127 <212> PRT <213> Homo sapiens <400> 43 Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val 1 5 10 15 Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val 20 25 30 Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys 35 40 45 Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe 50 55 60 Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys 65 70 75 80 Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr 85 90 95 Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser 100 105 110 Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu 115 120 125 <210> 44 <211> 231 <212> PRT <213> Homo sapiens <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Arg Gly 20 25 30 Gly Asp Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Tyr Ser Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135 140 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 195 200 205 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr 210 215 220 Val Glu Arg Lys Cys Gly Gly 225 230 <210> 45 <211> 231 <212> PRT <213> Homo sapiens <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Arg Gly 20 25 30 Gly Asp Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Tyr Ser Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135 140 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Glu Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 195 200 205 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr 210 215 220 Val Glu Arg Lys Cys Gly Gly 225 230 <210> 46 <211> 221 <212> PRT <213> Homo sapiens <400> 46 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Arg Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Lys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly 210 215 220 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 47 Gly Gly Gly Gly Ser 1 5 < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr>

Claims

1. A homotetrameric fusion protein comprising four antigen binding proteins, wherein the antigen binding proteins are linked to a transthyretin protein (TTR) protein complex, wherein the C-terminus of the antigen binding protein is linked to the N-terminus present in the TTR protein complex, and wherein the antigen binding protein is conatumumab or a Fab fragment thereof, and when the antigen binding protein is conatumumab, the antigen binding protein comprises an Fc mutation that is not conducive to the formation of Fc homodimers, and wherein the amino acid sequence of TTR is as shown in SEQ ID NO.

1.

2. The homotetrameric fusion protein of claim 1, wherein the antigen binding protein is directly fused to the TTR protein complex without a linker.

3. The homotetrameric fusion protein of claim 2, wherein the C-terminus of the antigen binding protein is directly fused to the N-terminus present in the TTR protein complex.

4. The homotetrameric fusion protein of claim 1, wherein the antigen binding protein is fused to the TTR protein complex via a linker.

5. The homotetrameric fusion protein of claim 4, wherein the C-terminus of the antigen binding protein is linked to the N-terminus present in the TTR protein complex. The homotetrameric fusion protein according to claim 5 , wherein the linker is an amino acid linker.

7. The homotetrameric fusion protein of claim 6, wherein the amino acid linker has a length of 1-40 amino acids.

8. The homotetrameric fusion protein of claim 7, wherein the amino acid linker is GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5 or (GGGGS)6.

9. The homotetrameric fusion protein according to any one of claims 1 to 8, for use in treating cancer.

10. A pharmaceutical composition comprising the homotetrameric fusion protein according to any one of claims 1-8.

11. Use of the homotetrameric fusion protein according to any one of claims 1 to 8 in the preparation of a medicament for treating melanoma or colon adenocarcinoma.

12. One or more isolated nucleic acids encoding the homotetrameric fusion protein according to any one of claims 1-8.

13. An expression vector comprising the nucleic acid according to claim 12.

14. A recombinant host cell comprising the nucleic acid according to claim 12 or the vector according to claim 13.

15. The recombinant host cell of claim 14, wherein the host cell is a Chinese hamster ovary (CHO) cell, an E5 cell, a baby hamster kidney (BHK) cell, a monkey kidney (COS) cell, a human hepatocellular carcinoma cell, or a human embryonic kidney 293 (HEK 293) cell.

16. A method for preparing a homotetrameric fusion protein according to any one of claims 1 to 8, the method comprising: a) culturing the recombinant host cell according to claim 14 or 15; and b) isolating the homotetrameric fusion protein from the culture.

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