Transthyretin immunoglobulin fusions

Abstract

Multimerized proteins are provided that have increased antibody binding activity and enhanced antigen clustering compared to their non-multimerized counterparts. A homodimeric fusion protein is provided, comprising two antigen-binding proteins, wherein the antigen-binding proteins are linked into a protein complex.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 568,217, filed October 4, 2017, which is incorporated herein by reference.

[0002] The present invention relates to transthyretin (TTR) fusions useful in the dimerization and tetramerization of antibodies and antibody fragments, such as Fabs. The TTR fusion proteins described herein are particularly useful in increasing antibody avidity and enhancing antigen clustering. Methods for treating disease using the fusion proteins of the invention are described herein.

[0003] Sequence Listing Reference This application has been filed with an electronic Sequence Listing via ePCT. The Sequence Listing is provided as a text file entitled A-2196-WO-PCT_Sequence_Listing_ST25.txt, created on October 2, 2018, and is 69,791 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Transthyretin (TTR) is a noncovalent tetrameric human serum and cerebrospinal fluid protein responsible for extending the serum half-life of retinol-binding protein and transporting a portion of circulating thyroxine. The native human monomer has a molecular weight of approximately 14 kDa, but TTR typically exists as a 56 kDa tetrameric serum protein.

[0005] TTR and TTR variants have previously been fused to biologically active agents to extend the serum half-life of such 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 extended serum half-lives compared to the biologically active agent alone. See, for example, U.S. Patent Application Publication No. 20030191056, the entire contents of which are incorporated herein by reference.

[0006] Additionally, previous attempts to multimerize proteins have included 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 / barstar complexes (Deyev et al., Nature Biotechnology, 21(12):1486-1492 (2003)), and Dock N Lock technology (protein kinase A-kinase anchor domain interactions) (Goldenberg et al., Journal of Nuclear Medicine, 49(1):158-163(2008)).

[0007] However, there remains a need for multimerized proteins, such as multimerized whole antibodies and antibody fragments (e.g., Fabs), that exhibit enhanced biological and therapeutic properties, e.g., increased antibody binding activity and enhanced antigen clustering compared to their non-multimerized counterparts. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 20030191056 [Non-patent literature]

[0009] [Non-Patent Document 1] Kipriyanov et al., Protein Engineering, 9(2):203-211(1996) [Non-patent document 2] Kriangkum et al., Biomolecular Engineering, 18:31-40(2001) [Non-patent document 3] Kruif et al., The Journal of Biological Chemistry, 271(13):7630-7634, 1996(1996) [Non-patent document 4] Deyev et al.,Nature Biotechnology,21(12):1486-1492(2003) [Non-patent document 5] Goldenberg et al., Journal of Nuclear Medicine, 49(1):158-163(2008) Summary of the Invention [Means for solving the problem]

[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 fused directly to the protein complex without a linker. In another specific aspect, the C-terminus of the antigen-binding protein is fused directly 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 other embodiments, the C-terminus of the antigen-binding protein is linked to the N-terminus present in the TTR protein complex. The linker may be an amino acid linker, such as an amino acid linker of 1 to 20 amino acids in length. 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 fused directly to the protein complex without a linker. In certain embodiments, the C-terminus of the antigen-binding protein is fused directly 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 between 1 and 20 amino acids in length. In certain 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 described above.

[0013] Additionally, the present invention relates to a method for treating cancer using any of the above-mentioned homodimeric or homotetrameric fusion proteins. Furthermore, the present invention relates to the use of any of the above-mentioned homodimeric or homotetrameric fusion proteins in the treatment of cancer. In another aspect, the present invention relates to any of the above-mentioned homodimeric or homotetrameric fusion proteins for use in the treatment of cancer.

[0014] In some aspects, the present invention relates to one or more isolated nucleic acids encoding any of the above-described homodimeric or homotetrameric fusion proteins. Expression vectors containing such nucleic acids are also contemplated, as are recombinant host cells containing the above-described nucleic acids and / or vectors. In certain embodiments, the recombinant host cells are Chinese hamster ovary (CHO) cells, E5 cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells, or human embryonic kidney 293 (HEK293) cells.

[0015] A method for producing any of the above-mentioned homodimeric or homotetrameric fusion proteins is also part of the present invention. Such a method may comprise: a) culturing a recombinant host cell according to claim 32 or 33; and b) isolating the homodimeric or homotetrameric fusion protein from the culture. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1 is a schematic diagram of a homomultimeric construct of the invention. Figure 1a is an exemplary TTR antibody homodimeric fusion protein in which the C-terminus of both antibody heavy chains is linked to the N-terminus of each TTR subunit. Figure 1b is an exemplary TTR antibody homotetrameric fusion protein in which one of the two heavy chains of each antibody C-terminus is linked to the N-terminus of each TTR subunit. The "+" and "-" symbols indicate Fc charge pairs that allow consistent attachment of one TTR subunit across the antibody. Figure 1c is an exemplary TTR Fab homotetrameric fusion protein in which the C-terminus of each Fab fragment is linked to the N-terminus of each TTR subunit. Each of Figures 1a-c shows an optional linker between the heavy chain and TTR. [Figure 2] Figure 2 is a series of SDS-PAGE gels showing that linker-free and linker-length-varying anti-CB1 TTR antibody homodimers (Figure 2a), linker-free anti-CB1 TTR antibody homotetramers (Figure 2b), and linker-free anti-CB1 TTR Fab homotetramer proteins (Figure 2c) are robustly expressed in HEK 293 cells. Figure 2 is further discussed in Example 2. [Figure 3] Figure 3 is a series of HPLC size exclusion chromatography (SEC) analyses of anti-CB1 TTR antibody homodimeric fusion proteins with no linker, with a (G4S) linker, with a (G4S)2 linker, with a (G4S)3 linker, or with a (G4S)4 linker. Figure 3 is further discussed in Example 2. [Figure 4] Figure 4 shows that the TTR anti-CB1 antibody homotetramer and TTR anti-CB1 Fab homotetramer fusion proteins have improved EC50 compared to the parental CB1 Ab. Figure 4 is further discussed in Example 3. [Figure 5] Figure 5 is an SDS-PAGE gel showing the expression of anti-GITR TTR antibody homodimer (lane 1), anti-GITR TTR antibody homotetramer (lane 2), and anti-GITR TTR Fab homotetramer protein (lane 3) in HEK293 cells. Lanes 4-7 are anti-dinitrophenyl (anti-DNP) antibodies. Figure 5 is further discussed in Example 4. [Figure 6] Figure 6 is an SDS-PAGE gel showing that the anti-GITR TTR antibody homodimer (lanes 1 and 4), anti-GITR TTR antibody homotetramer (lanes 2 and 5), and anti-GITR TTR Fab homotetramer proteins (lanes 3 and 6) are correctly assembled based on the unheated, non-reduced lanes. Upon heating and reduction, the three protein fusion constructs are resolved into the expected component chains (the upper band is the heavy chain, and the lowest band is the light chain). Figure 6 is further discussed in Example 4. [Figure 7] Figure 7 shows HPLC SEC analyses of the anti-GITR TTR antibody homodimer (center peak), anti-GITR TTR antibody homotetramer (left peak), and anti-GITR TTR Fab homotetramer (right peak) fusion proteins, respectively. Figure 7 is further discussed in Example 4. [Figure 8] Figure 8 shows differential scanning calorimetry (DSC) analyses of anti-GITR TTR parental 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 shows that the melting temperatures of the TTR fusion proteins are comparable to or better than those of the parental Ab, indicating that the TTR fusion proteins formed are robust. [Figure 9] Figure 9a) shows the results of in vivo (mouse) pharmacokinetic (PK) analysis of the entire anti-GITR antibody species. Figure 9b) shows the results of in vivo (mouse) PK analysis of the intact anti-GITR TTR fusion protein. [Figure 10] Figure 10a) shows that the binding affinity of the anti-GITR TTR Fab homotetramer ("2"), anti-GITR TTR antibody homodimer ("3"), and anti-GITR TTR antibody homotetramer ("4") fusion proteins is superior to that of the parent anti-GITR mAb ("1"). Figure 10b) shows that higher affinity does not translate into higher potency in cell-based assays. [Figure 11]FIG. 11 is a series of SDS-PAGE gels showing that anti-TRAILR2 TTR antibody homodimer (1) and anti-TRAILR2 TTR Fab homotetramer (2) proteins, respectively, are well expressed and correctly assembled in mammalian cells. [Figure 12] FIG. 12 is a series of SDS-PAGE gels showing that the anti-TRAILR2 TTR antibody homotetramer is well expressed and assembled correctly in CHO-K1 cells. [Figure 13] Figure 13 is an SDS-PAGE gel showing that the anti-TRAILR2 TTR Fab homotetramer, anti-TRAILR2 TTR antibody homodimer, and anti-TRAILR2 TTR antibody homotetramer are correctly assembled based on the unheated, unreduced lane. Heating and reduction resolves the molecules into the expected component chains (the upper band is the heavy chain, and the lowest band is the light chain). [Figure 14] FIG. 14 shows HPLC SEC analyses of anti-TRAILR2 TTR antibody homodimer (center chromatogram), anti-TRAILR2 TTR antibody homotetramer (right chromatogram), and anti-TRAILR2 TTR Fab homotetramer (left chromatogram) fusion proteins, respectively. [Figure 15] Figure 15a) shows the results of in vivo (mouse) PK analysis of all anti-TRAILR2 antibody species. Figure 15b) shows the results of in vivo (mouse) PK analysis of intact anti-TRAILR2 TTR fusion protein. [Figure 16] FIG. 16 shows the efficacy of anti-TRAILR2 TTR fusion proteins compared to the parental mAb (conatumumab) in a WM35 cell killing assay. [Figure 17] Figure 17 shows 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 cell killing assay. [Figure 18]FIG. 18 shows the tumor growth suppression ability of anti-TRAILR2 TTR fusion proteins compared to the parental mAb (conatumumab) in the murine colo205 model. [Figure 19] FIG. 19 shows the tumor growth suppression ability of anti-TRAILR2 TTR fusion proteins compared to the parental mAb (conatumumab) in the murine SW403 model. [Figure 20] FIG. 20 shows that the body weights of the murine colo205 and SW403 model mice were similar for all compounds tested. DETAILED DESCRIPTION OF THE INVENTION

[0017] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0018] As used herein, unless otherwise defined, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0019] Generally, the nomenclatures and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present application are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the 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 specifications, as commonly accomplished in the art, or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein 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.

[0020] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0021] Except as otherwise noted in the examples, all numbers indicating quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0022] All embodiments narrower in scope, specifically narrower than the variations defined by certain paragraphs herein, should be considered within the present disclosure. For example, certain aspects are described as genus, and it should be understood that all members of the genus may individually be embodiments. Also, aspects described as genus, or aspects selecting members of the genus, should be understood to encompass combinations of two or more members of the genus. Also, while various embodiments are presented using the language "comprising" in various contexts, it should be understood that related embodiments may also be described using the language "consisting of" or "consisting essentially of."

[0023] In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "comprising" and other forms such as "comprises" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components that contain a single unit and elements and components that contain two or more subunits, unless otherwise specified.

[0024] definition "Amino acid" has its standard meaning in the art. The 20 naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis, 2nd Edition, (ES Golub and DR Green, eds.), Sinauer Associates: Sunderland, Mass. (1991), which is incorporated herein by reference for any purpose. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as [α]-,[α]-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable building blocks for polypeptides and are encompassed by the term "amino acid." Examples of unconventional amino acids include: 4-hydroxyproline, [γ]-carboxyglutamic acid, [ε]-N,N,N-trimethyllysine, [ε]-N-acetyllysine, 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.

[0025] As used herein, "antagonist" generally refers to a molecule, e.g., an antigen-binding protein as provided herein, that is capable of binding to an antigen and inhibiting, reducing, or eliminating the biological signal associated with the antigen.

[0026] The term "antibody" refers to an immunoglobulin of any isotype, or a fragment thereof that can compete with an intact antibody for binding to a target antigen. An "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-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, an antibody comprises at least two full-length heavy chains and two full-length light chains. In other embodiments, an antibody comprises fewer chains, such as antibodies naturally occurring in camelids, which may comprise only heavy chains. An antibody can be derived from only a single source or can be "chimeric," where different portions of the antibody are derived from two different antibodies, as further described herein below. Antigen binding proteins, antibodies, or binding fragments may be produced, for example, in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.

[0027] The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a binding agent, such as an antigen-binding protein (including, for example, an antibody), and that can be used in an animal to generate antibodies capable of binding to that antigen. An antigen can have one or more epitopes that are capable of interacting with different antigen-binding proteins (e.g., antibodies).

[0028] As used herein, "antigen-binding protein" refers to any protein that specifically binds to a particular target antigen. The term includes polypeptides comprising at least one antigen-binding region. The term also encompasses antibodies comprising at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutants thereof. Antigen-binding proteins also include Fab, Fab', F(ab')2, Fv fragments, domain antibodies such as Nanobodies®, and single-chain antibodies, as described in more detail below.

[0029] "Antigen-binding region" or "antigen-binding domain" refers to a portion of a protein, such as 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, that portion of an antigen-binding protein that comprises amino acid residues that interact with an antigen and confer its specificity and affinity for the antigen to the antigen-binding protein is referred to as the "antigen-binding region." An antigen-binding region may comprise one or more "complementarity-determining regions" ("CDRs"). A particular antigen-binding region also comprises one or more "framework" regions. "Framework" regions may directly contribute to the specific binding of the antigen-binding protein, but typically also help maintain the proper conformation of the CDRs, thereby facilitating binding between the antigen-binding region and the antigen.

[0030] 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, carcinomas, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. Further 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 and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, kidney cancer such as renal cell carcinoma and Wilms' tumor, basal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, and esophageal cancer.

[0031] The terms "CDR" and its plural "CDRs" (also referred to as "hypervariable region") refer to the complementarity-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; the heavy chain variable region contains the following CDRs: CDR-H1, CDR-H2, and CDR-H3. CDRs contain most of the residues responsible for specific interactions between the antibody and the antigen and thus contribute to the functional activity of the antibody molecule. CDRs are the primary determinants of antigen specificity.

[0032] The precise definition of the boundaries and lengths of CDRs follows various classifications and numbering systems. Thus, CDRs may be represented by Kabat, Chothia, contact, or any other boundary definition, including the numbering systems described herein. The Kabat numbering scheme is a widely adopted standard for consistently numbering amino acid residues in antibody variable domains and, as noted elsewhere herein, is the preferred scheme for application in the present invention. Additional structural considerations may be used to determine the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering system can be described by the Chothia et al. numbering system and / or revealed by other techniques, such as crystallography and two-dimensional or three-dimensional computer modeling. Although the boundaries differ, each of these systems has some overlap in the portions that constitute CDRs within the variable sequences. Thus, CDR definitions according to these systems may differ in length and in the boundary regions relative to the adjacent framework regions. See, e.g., Kabat (an approach based on interspecies sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; 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 Oxford Molecular's AbM antibody modeling software. See, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Eds.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0033] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main-chain conformation adopted by the antigen-binding (CDR) loop. Comparative structural studies have found that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Thus, corresponding loops between antibodies can have very similar three-dimensional structures, despite the high degree of amino acid sequence variability observed in the majority of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J. Mol. Biol., 1996, 263:800). Furthermore, there is a relationship between the adopted loop structure and the surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and by the amino acid residues present at key positions within the loop and within the conserved framework (i.e., outside the loop). Thus, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0034] When used in the context of antigen-binding proteins (e.g., antibodies or fragments thereof) that compete for the same epitope, the term "compete" refers to competition between the antigen-binding proteins, as determined by an assay in which the antigen-binding protein (e.g., antibody or fragment thereof) under test blocks or inhibits specific binding of a reference antigen-binding protein to a common antigen. Many types of competitive binding assays can be used, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (see, e.g., Stahl 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 label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1986, J. Immunol. 137:3614-3619); al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays use purified antigen bound to a solid surface or cells expressing such antigen, 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 adjacent epitopes that are sufficiently close to the epitope bound by the reference antigen-binding protein for steric hindrance to occur.Further details regarding methods for determining competitive binding are provided herein. For example, in one embodiment, competition is determined according to a BiaCore assay. Typically, when a competing antigen-binding protein is present in excess, the competing antigen-binding protein will inhibit specific binding of a reference antigen-binding protein to a common antigen by at least 20%, 25%, 30%, 35%, 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some cases, binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% or more.

[0035] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes may include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. "Control sequences" may include leader sequences and / or fusion partner sequences.

[0036] A "derivative" of a polypeptide is a polypeptide that has been chemically modified in some way that differs from an insertion, deletion, or substitution variant, for example, by conjugation to another chemical moiety.

[0037] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or only the variable region of a light chain. Examples of domain antibodies include Nanobodies®. In some cases, two or more V H The domains are covalently linked via a peptide linker to create a bivalent domain antibody. H The regions may target the same or different antigens.

[0038] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, prevent the occurrence of symptoms and / or their underlying causes, and / or ameliorate or repair damage caused by or associated with cancer. In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a condition (e.g., cancer) or symptom, specifically a condition or symptom associated with the condition, or to prevent, prevent, delay, or reverse the progression of any other undesirable symptoms associated with the condition or disease, however caused. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of cancer or reducing the likelihood of the onset (or recurrence) of cancer or cancer symptoms. A complete therapeutic or prophylactic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0039] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by an antigen-binding protein (e.g., an antibody). In the context of a polypeptide, an epitope can be formed from contiguous or noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon protein denaturation, while epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least three, more typically at least five, or 8-10 amino acids in a unique spatial conformation. A "linear epitope" or "continuous epitope" is an epitope recognized by an antigen-binding protein (e.g., an antibody) in its linear sequence of amino acids or primary structure. A "conformational epitope" or "discontinuous epitope" is an epitope recognized by an antigen-binding protein (e.g., an antibody) in its tertiary structure. The residues that make up these epitopes may not be adjacent in the primary amino acid sequence, but are close to each other in the tertiary structure of the molecule. Linear and conformational epitopes generally behave differently when a protein is denatured, fragmented, or reduced.

[0040] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in cooperation with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of the coding regions operably linked thereto.

[0041] A "Fab fragment" or "Fab" is a fragment of one light chain and one heavy chain. H Fab molecules consist of a heavy chain and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0042] A "Fab' fragment" or "Fab" is a fragment of a human Fab that contains one light chain and one V HDomain and C H 1 domain plus C H 1 Domain and C H The two Fab' fragments each contain a portion of one heavy chain, including the region between the two domains, so that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0043] "F(ab')2 fragment" or "F(ab')2" refers to a fragment of two light chains and two heavy chains separated by a C cleavage reaction so that interchain disulfide bonds are formed between them. H 1 Domain and C H The F(ab')2 fragment contains two heavy chains with part of the constant region between the two domains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0044] The "Fc region" is the C region of an antibody. H 2 domain and C H The two heavy chain fragments contain two or more disulfide bonds and a C H The three domains are held together by hydrophobic interactions.

[0045] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0046] The term "heavy chain" as used with respect to an antigen-binding protein, antibody, or fragment thereof, includes a full-length heavy chain and fragments thereof having sufficient variable region sequence 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 is C H The domain is located at the carboxyl terminus and is C H3 is closest to the carboxy 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.

[0047] A "blood cancer" is a cancer that originates in blood-forming tissues, such as the bone marrow, or in cells of the immune system. Examples of blood cancers are leukemia, lymphoma, and multiple myeloma.

[0048] The term "homodimeric fusion protein" refers to a fusion protein containing two identical antigen-binding proteins. For example, an antibody homodimeric fusion protein refers to a fusion protein containing two identical antibodies. In a specific example, the homodimer may be a TTR homodimeric fusion protein containing two identical antibodies linked via a TTR protein, as described herein.

[0049] The term "homotetrameric fusion protein" refers to a fusion protein comprising four identical antigen-binding proteins. For example, an antibody homotetrameric fusion protein refers to a fusion protein comprising four identical antibodies. In another example, a Fab homotetrameric fusion protein refers to a fusion protein comprising four identical Fab fragments. In a specific example, the homotetramer may be a TTR homotetrameric fusion protein comprising two identical antigen-binding proteins (e.g., two identical antibodies or two identical Fab fragments) linked via the TTR protein, as described herein.

[0050] The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0051] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of residues that are identical among the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignment, if any, must be addressed by a particular 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, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0052] When calculating percent identity, the sequences being compared are aligned in a manner that maximizes the match between the sequences. A computer program used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides are optimally matched (the "match span" determined by the algorithm). A gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used, and "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and gap extension penalty (usually 1 / 10 of the gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62, are used with the algorithm. In certain embodiments, standard comparison matrices (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) are also used by the algorithm.

[0053] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992 (supra); Gap penalty: 12 (but no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0

[0054] A particular alignment scheme for aligning two amino acid sequences may result in matching only a short region of the two sequences, and this small aligned region may have very high sequence identity despite the lack of significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) may be adjusted, if desired, to produce an alignment over at least 50 consecutive amino acids of the target polypeptide.

[0055] The phrase "immunomodulatory agent" refers to a molecule that induces, enhances, or suppresses an immune response. An immunoactivator is a molecule that induces or enhances an immune response. An immunosuppressant is a molecule that decreases or suppresses an immune response. Thus, activating immunotherapy is a therapy that involves administering a molecule to induce or enhance a subject's immune system. Suppressive immunotherapy is a therapy in which a subject is treated with a molecule to decrease or suppress the subject's immune system.

[0056] As used herein, the term "fragment" of an antibody or immunoglobulin chain (heavy or light chain) refers to an antigen-binding protein lacking at least some of the amino acids present in the full-length chain, but comprising a portion of an antibody (regardless of how the portion is obtained or synthesized) that is capable of specifically binding to an antigen. Such a fragment is biologically active in that it specifically binds to a target antigen and can compete with other antigen-binding proteins, including intact antibodies, for binding to a given epitope. In one aspect, such a fragment retains 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 portion thereof. Such biologically active fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and may be derived from any mammalian source, including, but not limited to, human, mouse, rat, camel, or rabbit. It is further contemplated that functional portions of the antigen binding proteins disclosed herein, such as, for example, one or more CDRs, can be covalently linked to a second protein or small molecule to create therapeutic agents that are directed to specific targets in the body or to extend serum half-life.

[0057] An "isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, where the isolated polynucleotide is unaccompanied by all or part of polynucleotides with which it is found in nature or is linked to polynucleotides with which it is not naturally linked. For purposes of this disclosure, a "nucleic acid molecule comprising" a particular nucleotide sequence should be understood to not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include sequences encoding up to 10 or even up to 20 other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0058] As used herein, the terms "isolated polypeptide," "purified polypeptide," "isolated protein," or "purified protein" are intended to refer to a composition isolatable from other components, where the polypeptide has been purified to any degree relative to the state in which it is available in nature. As such, a purified polypeptide also refers to a polypeptide that has been released from the environment in which it may naturally occur. Generally, "purified" refers to a polypeptide composition that has undergone fractionation to remove various other components, and the composition substantially maintains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms the majority of the composition, e.g., comprising about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0059] The term "light chain" as used in reference to an antigen-binding protein, antibody, or fragment thereof, includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain (V L ) and constant region domain (C LThe variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0060] The term "naturally occurring" as used throughout this specification in reference to biological material, such as a polypeptide, nucleic acid, host cell, etc., refers to material that is found in nature.

[0061] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. In some embodiments, oligonucleotides are 10-60 bases in length. In other embodiments, oligonucleotides are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can contain a label, including a radiolabel, fluorescent label, hapten, or antigenic label for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

[0062] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their specific functions under appropriate conditions. For example, a control sequence in a vector "operably linked" to a protein-coding sequence is ligated to the protein-coding sequence so that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0063] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising a 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 linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0064] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand with the same sequence as the RNA transcript that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand with the same sequence as the RNA transcript that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0065] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. These terms also encompass amino acid polymers that have been modified, for example, by the addition of carbohydrate residues (to form glycoproteins) or phosphorylation. Polypeptides and proteins can be naturally occurring and produced by non-recombinant cells, or by genetic engineering or recombinant cells, and include molecules with the amino acid sequence of a native protein or molecules with one or more amino acid deletions, additions, and / or substitutions from the native sequence. The term "polypeptide fragment" refers to a polypeptide that has 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, fragments are about 5-500 amino acids in length. For example, fragments 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.

[0066] A "recombinant protein," including recombinant TTR protein, is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

[0067] A "single-chain antibody" is an Fv molecule in which the heavy chain variable region and the light chain variable region are linked by a flexible linker to form a single polypeptide chain, and this single polypeptide chain forms the antigen-binding region. Single-chain antibodies are discussed in detail in WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

[0068] A "solid tumor" refers to an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign (not cancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.

[0069] An antigen-binding protein "specifically binds" to an antigen if it exhibits little or no binding to molecules other than the antigen. However, antigen-binding proteins that specifically bind to an antigen may cross-react with antigens from different species. Typically, the dissociation constant (K) is measured by surface plasma resonance technology (e.g., BIACore, GE-Healthcare Uppsala, Sweden). D ) is ≦10 -7 An antigen-binding protein specifically binds to an antigen when M is greater than K (as measured using a method such as BIACore). D ≦5×10 -8 M is "high affinity" and K is "high affinity." D ≦5×10 -9 When binding occurs at M, it specifically binds to the antigen with "very high affinity."

[0070] As used herein, a "subject" or "patient" can be any mammal. In typical embodiments, the subject or patient is a human.

[0071] As used herein, "substantially pure" means that the recited molecular species is the predominant species present, i.e., more abundant on a molar basis 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 comprises 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 such that contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

[0072] The term "treating" refers to any indication of success in treating or ameliorating an injury, condition, or pathology, including any objective or subjective parameter, such as relief, remission, or reduction in symptoms, or improved patient tolerance of the injury, condition, or pathology; a slowing of the rate of deterioration or decline; a less debilitating end point of deterioration; or an improvement in the patient's physical or mental well-being. Treating or ameliorating symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric testing, and / or psychiatric evaluation. For example, certain methods presented herein have been successful in treating cancer and tumors, e.g., by reducing the progression or spread of cancer, inhibiting tumor growth, causing tumor remission, and / or improving symptoms associated with cancer or tumor. Similarly, other methods presented herein treat infectious diseases by reducing the progression or spread of infection, reducing the extent of infection, and / or improving symptoms associated with infection.

[0073] 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), incorporated herein by reference. The amino acid sequence for human TTR is also described in the UniProt Knowledgebase (www.uniprot.org / uniprot / P02766#sequences) and is referred to herein as SEQ ID NO: 43. Nucleic acids for human TTR are also described in NCBI (www.ncbi.nlm.nih.gov / gene / 7276). See also GenBank deposit K02091.1. The amino acid and nucleic acid sequences for murine TTR are set forth in SEQ ID NOs: 3 and 4, respectively.

[0074] 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 present invention also includes nucleic acids encoding such TTR variants. Specific variants include, for example, TTR proteins with truncations at the C- or N-terminus.

[0075] "Tumor" refers to a mass of tissue that forms when cancerous cells grow and multiply, potentially invading and destroying normal adjacent tissue. Cancer cells can break away from a malignant tumor and enter the bloodstream or lymphatic system, or they can spread from the primary tumor to form new tumors in other organs.

[0076] A "variant" of a polypeptide includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another polypeptide sequence. Variants include fusion proteins.

[0077] The term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector, a "plasmid," refers to a circular double-stranded DNA loop to which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0078] Homodimeric fusion proteins As described herein, the present invention relates in part to the use of TTR in the multimerization of antigen-binding proteins such as antibodies.TTR is a human extracellular protein found in human serum, and therefore exists in relatively large amounts throughout the human body.Therefore, when TTR is present in the multimerization construct of the present invention, it is less likely to induce an immune response, for example, compared with non-human proteins, intracellular proteins, and rare proteins.Therefore, its use in the multimerization technology of the present invention is advantageous.

[0079] For example, TTR can be used in antibody dimerization. In such homodimeric fusion proteins, TTR (SEQ ID NO: 1), or a variant thereof, exists as a tetramer, in which a TTR subunit is linked to the C-terminus of an 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-terminus) may be linked to the N-terminus of each TTR subunit (see Figure 1a). Thus, each antibody is linked to two TTR subunits in the TTR tetramer, resulting in a TTR-antibody homodimer.

[0080] 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.

[0081] In a specific embodiment, the present invention relates to a homodimeric fusion protein comprising two antibodies that bind to a TTR tetramer. The antibodies may bind to the TTR tetramer without a linker (i.e., the antibodies bind directly to TTR).

[0082] In other embodiments, the antibody is linked 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 linker is 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, or 1 to 40 amino acids in length. In some embodiments, 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 length. In other embodiments, the linker is 0, 1, 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. In other embodiments, the linker is up to 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. In some embodiments, the linker is up to 5, 10, 15, or 20 amino acids in length. In particular embodiments, the linker is 0, 5, 10, 15, or 20 amino acids in length.

[0083] In some embodiments, the linker is GGGGS, GGGGSGGGGS (i.e., (GGGGS)2), GGGGSGGGGSGGGGGS (i.e., (GGGGS)3), GGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)4), GGGGSGGGGSGGGGSGGGGSGGGGGS (i.e., (GGGGS)5), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)6). In other embodiments, it is GGGGS, GGGGSGGGGS (i.e., (GGGGS)2), GGGGSGGGGSGGGGGS (i.e., (GGGGS)3), or GGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)4).

[0084] Other suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n=1-10), (EAAAK) n(n=1~5), A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) n (n=1 to 20), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG, and LE. Suitable non-amino acid linkers include polyethylene glycol (PEG).

[0085] In some embodiments, the antibody is attached 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 may be removed from the N-terminus of one or more TTR subunits, and the antibody may be attached to the truncated TTR subunit N-terminus.

[0086] The present invention also relates to nucleic acid molecules encoding the homodimeric fusion proteins described herein. Details regarding exemplary methods for generating homodimeric fusion proteins can be found in the Examples.

[0087] Tetramerization of antibodies and Fabs 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 noted above, the use of TTR in the multimerization techniques of the present invention is advantageous because, when present in the multimerization constructs of the present invention, TTR is less likely to elicit an immune response, as compared to, for example, non-human proteins, intracellular proteins, and rare proteins.

[0088] 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 still exist as a tetramer. However, in the context of a TTR antibody homotetramer, a single antibody heavy chain (i.e., only one of the two heavy chains present in a single antibody) is linked to each TTR subunit, allowing for the linking of four antibodies into a TTR tetramer (see Figure 1b). One of the two heavy chains at the C-terminus of the antibody may be linked to the N-terminus of each TTR subunit (see Figure 1b). Thus, each antibody is linked to one TTR subunit in the TTR tetramer, resulting in a TTR antibody homotetramer.

[0089] In such homotetrameric fusion proteins, the formation of Fc homodimers (described above) is inhibited by mutations in the Fc. Such modifications include Fc mutations such as knobs-into-holes, DuoBody, Azymetric, charge-pair, HA-TF, SEEDbody, and modifications with different Protein A affinities. See, for example, Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. Knobs-into-holes 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, 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., 23 (2010), pp. 195-202. Mutations with different Protein A affinities 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 references is incorporated by reference in its entirety.

[0090] In certain embodiments, antibody homotetramerization can be promoted by the use of Fc charge pairs that inhibit antibody heavy chain homodimerization, thus favoring heavy chain heterodimerization between one antibody heavy chain linked to a TTR subunit and one antibody heavy chain that is not linked to TTR (see Figure 1b). For example, one set of charge mutations can be a C heavy chain mutation with either a negative charge on one heavy chain and a positive charge on the corresponding heavy chain, or a mixture of negative and positive charges on one heavy chain that pair with corresponding positive and negative charges on the corresponding heavy chain. H 3 domain. Exemplary negative charges include K392D & K409D, and exemplary positive charges include E356K & D399K. While dissimilar charges are attracted to each other, C H Because the charges at the three interfaces are repulsive, homodimerization is inhibited, but heterodimerization is favored. TTR is fused to a heavy chain of only one charge type (either positive or negative, but not both); thus, resulting in a complete antibody TTR subunit consisting of four chains (two light chains, one unfused heavy chain, and one TTR-fused heavy chain). Further, charge pair mutations are described, for example, in U.S. Pat. No. 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., 420 (2012), pp. 204-219. Each of these references is incorporated by reference in its entirety.

[0091] 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, also exists as a tetramer, in which each TTR subunit is linked to the C-terminus of each Fab fragment to form a TTR Fab homotetramer (see Figure 1c). Thus, each Fab fragment antibody is linked to a single TTR subunit in the TTR tetramer, resulting in a TTR Fab homotetramer.

[0092] Thus, the present invention relates to homotetrameric fusion proteins comprising four antigen-binding proteins (e.g., a Fab tetramer) or eight antigen-binding proteins (e.g., an Ab tetramer). 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.

[0093] In certain embodiments, the present invention relates to homotetrameric fusion proteins comprising four antibodies linked to a TTR tetramer. In other embodiments, the present invention relates to homotetrameric fusion proteins comprising four Fab fragments linked to a TTR tetramer. In some embodiments, the antibodies or Fabs are bound to the TTR tetramer without a linker (i.e., the antibodies or Fabs are directly bound to TTR).

[0094] In other embodiments, the antibody or Fab is linked 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 linker is 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, or 1 to 40 amino acids in length. In some embodiments, 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 length. In other embodiments, the linker is 0, 1, 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. In other embodiments, the linker is up to 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. In some embodiments, the linker is up to 5, 10, 15, or 20 amino acids in length. In particular embodiments, the linker is 0, 5, 10, 15, or 20 amino acids in length.

[0095] In some embodiments, the linker is GGGGS, GGGGSGGGGS (i.e., (GGGGS)2), GGGGSGGGGSGGGGGS (i.e., (GGGGS)3), GGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)4), GGGGSGGGGSGGGGSGGGGSGGGGGS (i.e., (GGGGS)5), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)6). In other embodiments, it is GGGGS, GGGGSGGGGS (i.e., (GGGGS)2), GGGGSGGGGSGGGGGS (i.e., (GGGGS)3), or GGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)4).

[0096] Other suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n=1-10), (EAAAK) n(n=1~5), A(EAAAK)4ALEA(EAAAK)4A, 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 within a construct with a terminal group capable of reacting with a protein; see, e.g., WO 2017 / 083604, incorporated herein by reference in its entirety).

[0097] In some embodiments, the antibody or Fab is conjugated 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 may be removed from the N-terminus of one or more TTR subunits, and the antibody or Fab may be conjugated to the truncated TTR subunit N-terminus.

[0098] The present invention also relates to nucleic acid molecules encoding the homodimeric fusion proteins described herein. Details regarding exemplary methods for generating homotetrameric (TTR and Ab) fusion proteins can be found in the Examples.

[0099] antigen-binding proteins Any antigen binding protein (e.g., Fab or antibody) can be used in the TTR fusion proteins of the invention. Because the fusion proteins of the invention allow for multimerization of the antigen binding protein, antigen binding proteins that target / bind to antigens for which antigen binding protein clustering or avidity is required for activity may particularly benefit from the fusion proteins of the invention. Thus, 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 rodents), ErbB-3 (HER3), ErbB-4 (HER4), FGFR (fibroblast growth 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).

[0100] In one embodiment, the antigen-binding protein (e.g., antibody or Fab) specifically binds to CB1R (anti-cannabinoid receptor-1; gene name Cnrl). CB1 receptors are Gi-coupled G-protein receptors widely expressed in the CNS and peripheral nervous system. Agonist stimulation of CB1 receptors leads to inhibition of adenylyl cyclase activity and activation of mitogen-activated protein (MAP) kinase. Endogenous agonists of CB1 receptors may include anandamide and arachidonoylglycerol. Exogenous agonists may include A9-tetrahydrocannabinol. Antagonists or inverse agonists have been shown to reduce body weight and improve metabolic parameters (e.g., reduced plasma glucose and insulin levels). Thus, in certain embodiments, the antigen-binding portion of the TTR fusion protein is an anti-CB1R antibody (e.g., anti-CB1R antibody 10D10 having a heavy chain SEQ ID NO: 5 and a light chain SEQ ID NO: 11; or an anti-CB1R antibody having a heavy chain SEQ ID NO: 6 or 7 and a light chain 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 the Fab heavy chain SEQ ID NO: 44 and the Fab light chain SEQ ID NO: 11). See, e.g., U.S. Patent Application Publication No. 20160145333, incorporated herein by reference.

[0101] In one embodiment, the antigen binding protein (e.g., antibody or Fab) specifically binds to GITR (glucocorticoid-induced TNFR-related protein; TNFRSF18). GITR, sometimes called activation-induced TNFR family member (AITR), is a receptor belonging to the TNF receptor superfamily (TNFRSF). It is activated by its cognate ligand, the GITR ligand (GITRL, TNFSF18). GITR is a type I transmembrane protein containing a cysteine-rich extracellular domain, characteristic of TNFR family members. For example, the cytoplasmic domain of GITR shares close homology with certain other TNFR family members, such as 4-1BB and CD27 (Nocentini, et al. (1997) Proc. Natl. Acad. Sci. 94:6216-6221, incorporated herein by reference). Activation of GITR enhances immune responses, and such activation has the potential to restore immune responses against infections and tumors. Thus, molecules capable of activating GITR may be useful as immunostimulants in situations where it is desirable to induce an enhanced immune response. Thus, in certain embodiments, the antigen-binding portion of the TTR fusion protein is an anti-GITR antibody (e.g., 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 Application Publication No. 20150064204, incorporated herein by reference.

[0102] In one embodiment, an antigen binding protein (e.g., an 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 homomeric ligand that interacts with four members of the TNF receptor superfamily (TRAIL receptors ("TR") 1-4) as well as the related soluble opteoprotegerin ("OPG") receptor. Binding of TRAIL to TR-1 or TR-2 on the cell surface triggers apoptosis of that cell. Following initial binding of TRAIL to TR-1 or TR-2, intracellular proteins are recruited to the intracellular death domain of the receptor, forming a signaling complex. Specific intracellular caspases are recruited to the complex, where they autoactivate and sequentially activate additional caspases and the intracellular apoptotic cascade. TR-3, TR-4, and OPG lack the intracellular domains involved in transmitting the apoptotic signal. Therefore, binding of TRAIL to TR-3, TR-4, or OPG does not induce apoptosis. TR-3 and TR-4 are also called "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 the liver, brain, breast, kidney, colon, lung, spleen, thymus, peripheral blood lymphocytes, prostate, testis, ovary, uterus, and various tissues along the gastrointestinal tract (see, e.g., Walczak et al., EMBO J. 16:5386-5397 (1997); Spierings et al., J. Histochem. Cytochem. 52:821-831 (2004), each of which is incorporated by reference).TRAIL and TRAIL receptors are widely expressed but are most active in inducing apoptosis in transformed cells (see, e.g., Daigle et al., Swiss Med. Wkly. 131:231-237 (2001), each of which is incorporated by reference). Conatumumab, an anti-TRAILR2 monoclonal antibody, has been developed for the treatment of cancer. Thus, in certain embodiments, the antigen-binding portion of the TTR fusion protein is an anti-TRAILR2 antibody (e.g., conatumumab having a heavy chain SEQ ID NO: 31 and a light chain SEQ ID NO: 38; or an anti-TRAILR2 antibody having a heavy chain SEQ ID NO: 32 or 33 and a light chain SEQ ID NO: 38). In other specific embodiments, the antigen-binding portion of the TTR fusion protein is an anti-TRAILR2 Fab (e.g., a Fab derived from conatumumab, such as a Fab heavy chain SEQ ID NO: 34 and a Fab light chain SEQ ID NO: 39).

[0103] TTR variants As mentioned above, TTR variants can also be used in the present invention. Any of the TTR variants discussed herein may be used in combination with each other. TTR variants include proteins having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a TTR protein having SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 43.

[0104] In some embodiments, the TTR of the present invention comprises the amino acid sequence of human TTR SEQ ID NO: 43. In certain embodiments, the TTR of the present invention comprises the amino acid sequence of SEQ ID NO: 43 with mutations at K15, C10, or both K15 / C10 (e.g., K15A, C10A, or both K15A / C10A). In certain embodiments, the TTR of the present invention comprises both K15A and C10A mutations, and thus has the amino acid sequence of SEQ ID NO: 1.

[0105] Cysteines present in human TTR (e.g., SEQ ID NO: 1 or SEQ ID NO: 43) can be used as sites of attachment to antigen-binding proteins (e.g., antibodies and Fabs). In addition, the present invention may utilize TTR variants that allow for site-specific attachment, such as TTR variants with engineered cysteines. See, e.g., U.S. Patent No. 8,633,153, incorporated herein by reference. For example, TTR variants may include one or more of the following cysteine ​​mutations: A37C, D38C, A81C, or G83C.

[0106] Additional variants useful in the present invention include TTR proteins with truncations at the C- or N-terminus, such as 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 have been removed from the C- or N-terminus of the TTR protein. In some embodiments, fusion proteins of the present invention comprise TTR proteins in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acids have been removed from the C- or N-terminus of the TTR protein. In other embodiments, fusion proteins of the present invention comprise TTR proteins in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acids have been removed from the N-terminus of the TTR protein.

[0107] Additional TTR variants that can be used 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. Such variants can, for example, avoid interference with a patient's thyroxine biology and may prevent TTR fusions from acting in the thyroxine metabolic pathway. Still other TTR variants that can be used in the present invention include those that reduce or eliminate the proteolytic activity of TTR.

[0108] Additionally, TTR-His tag fusions may be used in the present invention. For example, TTR-His tag fusions may be used in the purification of TTR Fab constructs, where the Fab lacks Fc, or in the purification of TTR Ab constructs, where it is beneficial to avoid the low pH purification environment of a Protein A affinity column. In some embodiments, the His tag is removed after purification. A His tag may also be present in the final therapeutic molecule (i.e., the tag may 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 a (His)6 or (His)7 tag. In specific embodiments, the His tag is a (His)6 tag. 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 certain embodiments, the His tag comprises two glycines (e.g., GGHHHHHH).

[0109] In some embodiments, two glycine amino acid linkers may be inserted between the TTR variant and the heavy or light chain.

[0110] Furthermore, the TTR variants of the present invention may include variants incorporating glycosylation sites, which may be useful for modulating the PK or solubility properties of the TTR fusion. In addition, the TTR variants or TTR fusion proteins of the present invention may be modified to include moieties that confer beneficial PK properties, such as triazine-containing moieties (contained within constructs with terminal groups capable of reacting with proteins; see, e.g., WO 2017 / 083604, incorporated herein by reference in its entirety).

[0111] Methods for producing homodimeric and homotetrameric fusion proteins Methods for making the homodimer and homotetramer fusions of the invention are discussed in the Examples.

[0112] Generally, the homodimer and homotetramer fusions of the present invention can be produced using recombinant methods. Accordingly, the present invention includes polynucleotides encoding the homodimer and homotetramer fusions. In another aspect, the present invention includes expression vectors comprising polynucleotides encoding the homodimer and homotetramer fusions. In certain embodiments, the expression vector comprises control sequences (e.g., promoters, enhancers) operably linked to the polynucleotides encoding the homodimer and homotetramer fusions to facilitate expression in a suitable host cell. In certain embodiments, the expression vector also comprises a polynucleotide sequence enabling chromosome-independent replication in the host cell. Exemplary vectors include, but are not limited to, plasmids, cosmids, and YACS. In certain embodiments, the vector is pTT5.

[0113] Generally, mammalian host cells are utilized when producing Ab TTR homodimer or Ab TTR homotetramer fusion constructs. Mammalian host cells are also suitable for producing Fab TTR homotetramer fusion constructs, although non-mammalian host cells, such as prokaryotes (bacteria) and non-mammalian (e.g., yeast) host cells can also be used.

[0114] In yet another embodiment, the present invention includes a host cell comprising the expression vector of the present invention. Methods for transfecting host cells with expression vectors and culturing the transfected host cells under conditions suitable for expression of the homodimer and homotetramer fusions are known in the art. The transfection procedure used may depend on the host being transformed. Specific methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include, but are not limited to, dextran-mediated gene transfer, calcium phosphate precipitation, polybrene-mediated gene transfer, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Certain mammalian cell lines available as hosts for expression are 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; e.g., CHO-K1) cells, E5 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells (HEK293), and many other cell lines. In certain embodiments, cell lines may be selected by determining which cell lines express and produce high levels of homodimer and homotetramer fusions.

[0115] Thus, the present invention also relates to methods of making the homodimeric and homotetrameric fusion proteins described herein. For example, homodimeric and homotetrameric fusion proteins may comprise: a) culturing recombinant host cells containing polynucleotides encoding the homodimer and homotetramer fusions; b) isolating the homodimeric or homotetrameric fusion protein from the culture; and It can be made by

[0116] Pharmaceutical Composition In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or more of the homomultimeric fusion proteins of the present invention (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion proteins) in association with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0117] Preferably, formulation materials are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions are provided comprising 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).

[0118] In certain embodiments, pharmaceutical compositions may include formulation materials intended to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); injectables; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium phosphate). preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilization enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as 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.

[0119] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In specific embodiments, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or 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, homomultimer compositions (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) can be prepared for storage by combining selected compositions having a desired degree of purity, optionally with a formulating agent (REMINGTON'S PHARMACEUTICAL SCIENCES, supra), in the form of a lyophilized cake or aqueous solution. Furthermore, in certain embodiments, homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) can be formulated as lyophilizates using appropriate excipients, such as sucrose.

[0120] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Alternatively, the compositions may be selected for delivery via the digestive tract, such as by inhalation or orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are preferably present in concentrations acceptable to the site of administration. In certain embodiments, a buffering agent is used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8.

[0121] When parenteral administration is contemplated, therapeutic compositions for use in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired homomultimer (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, in which the homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, preparations may include formulation of the desired molecule with an agent, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which may provide sustained or sustained release of the product, which may be delivered via depot injection. In certain embodiments, hyaluronic acid, which has the effect of enhancing duration in the circulation, may also be used. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antigen binding protein.

[0122] The pharmaceutical compositions of the present invention can be formulated for inhalation. In these embodiments, the homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) is advantageously formulated as a dry, inhalable powder. In specific embodiments, the homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) inhalation solution may be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be nebulized. Pulmonary administration, and therefore formulation methods, are further described in International Patent Application No. PCT / US94 / 001875 (incorporated by reference), which describes pulmonary delivery of chemically modified proteins.

[0123] It is also contemplated that the formulation may be orally administrable. Homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) administered in this manner may be formulated with or without carriers customarily used in compounding solid dosage forms such as tablets and capsules. In certain embodiments, capsules may be designed to release the active portion of the formulation at a time when bioavailability is maximized in the gastrointestinal tract and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the homomultimer (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins). Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[0124] Additional pharmaceutical compositions, including formulations involving homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins) in sustained- or controlled-delivery formulations, will be apparent to those skilled in the art. Various other formulation techniques for sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829 (incorporated by reference), which describes the controlled release of porous polymeric microparticles for delivering pharmaceutical compositions. Sustained-release preparations may include, for example, semipermeable polymer matrices in the form of shaped articles such as films or microcapsules. Sustained-release matrices can include polyesters, hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919 and EP 058481, each of which is incorporated by reference), copolymers of L-glutamic acid and gamma-ethyl-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 (EP 133,988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Applications Nos. 036,676; 088,046 and 143,949, which are incorporated by reference.

[0125] Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are generally filled into a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0126] Embodiments of the present invention include self-buffering homomultimers (e.g., TTR antibody homodimers, TTR antibody homotetramers, or TTR Fab homotetramer fusion proteins), which can be used as pharmaceutical compositions as described in WO 06138181 A2 (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0127] As discussed above, certain embodiments provide homomultimeric (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) compositions, particularly pharmaceutical homomultimeric (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) compositions, which, in addition to a homomultimer (e.g., a TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein), comprise one or more excipients, such as those illustratively described in this section and elsewhere herein. Excipients can be used in the present invention for a wide range of purposes, including methods of the invention for adjusting the physical, chemical, or biological properties of the formulations, such as adjusting viscosity, and / or improving efficacy, and / or stabilizing such formulations, as well as methods for preventing degradation and damage due to stresses encountered during and after manufacturing, transportation, storage, preparation prior to use, and administration.

[0128] Various descriptions are available for protein stabilization and formulation materials and methods useful in this regard, see, e.g., Arakawa 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 protein pharmaceuticals and processes for veterinary and / or human medical use, and particularly the sections regarding excipients and processes similar to those for self-buffering protein formulations according to the present invention.

[0129] Salts may be used in accordance with certain embodiments of the invention, for example, to adjust the ionic strength and / or tonicity of the formulation and / or to improve the solubility and / or physical stability of proteins or other components of compositions according to the invention.

[0130] As is well known, ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and by shielding charged and polar groups in the protein, reducing the strength of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize proteins in their denatured state, particularly by binding to the denatured peptide bond (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0131] Different ionic species have significantly different effects on proteins. Several taxonomic rankings of ions and their effects on proteins have been developed and can be used in formulating pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropics." Destabilizing solutes are called "chaotropics." Kosmotropes are commonly used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate ("salting out") proteins from solution. Chaotropes are commonly used to denature and / or solubilize ("salting in") proteins. The relative effects of an ion on "salting in" and "salting out" define its position on the Hofmeister series.

[0132] Free amino acids can be used in homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) formulations according to various embodiments of the present invention as bulking agents, stabilizers, and antioxidants, as well as for other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful for ensuring proper cake structure and properties during lyophilization. Arginine can be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0133] Polyols include sugars such as mannitol, sucrose, and sorbitol, and polyhydric alcohols such as glycerol and propylene glycol, and for purposes of this discussion, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. They are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful for adjusting the isotonicity of the formulation.

[0134] Among polyols useful in selected embodiments of the present invention, mannitol is commonly used to ensure cake structural stability in lyophilized formulations. Mannitol ensures cake structural stability. It is generally used in conjunction with a lyoprotectant, such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting isotonicity and for protecting against freeze-thaw stress during transportation or bulk preparation in the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they are generally not among the preferred polyols for use in the present invention. Additionally, sugars that form such reactive species, such as sucrose, are also not among the preferred polyols of the present invention, as they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a lyoprotectant, and in this regard can be used in the present invention.

[0135] 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 prone to adsorption to surfaces and to denaturation and subsequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These adverse interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical agitation, such as that encountered during product transportation and handling.

[0136] Surfactants are traditionally used to prevent, minimize, or reduce surface adsorption. Surfactants useful in the present invention in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

[0137] Detergents are also commonly used to control the conformational stability of proteins: in this respect, detergent use is protein-specific, as any given detergent typically stabilizes some proteins and destabilizes others.

[0138] Polysorbates are prone to oxidative degradation and often contain sufficient peroxides as supplied to cause oxidation of protein residue side chains, particularly methionine. Therefore, polysorbates should be used with caution and, when used, at the lowest possible concentrations. In this respect, polysorbates exemplify the general rule that excipients should be used at the lowest possible concentrations.

[0139] Embodiments of homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) formulations further comprise one or more antioxidants. Detrimental oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and by avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Particularly useful antioxidants in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain activity over the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0140] Antioxidants can damage proteins. For example, reducing agents such as glutathione can, inter alia, disrupt intramolecular disulfide bonds. Therefore, antioxidants for use in the present invention are selected, inter alia, to eliminate or sufficiently reduce the possibility of damaging proteins in the formulation.

[0141] The formulations according to the present invention may contain metal ions, which are cofactors for proteins and are required to form protein coordination complexes, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some processes that degrade proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins.

[0142] 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 deoxyribonuclease. +2 , Mn +2 and Zn +2 can destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, which is due to the presence of Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 The aggregation can be destabilized by Al +3 It can be increased by ions.

[0143] Embodiments of homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) formulations further comprise one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations involving more than one withdrawal from the same container. Their primary function is to inhibit microbial growth and ensure product sterility over the shelf life or usage period of the formulation. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Preservatives have a long history of use with small molecule parenteral drugs, but developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single use only. However, the possibility of multi-dose formulations offers the added benefits of patient convenience and increased marketability. Human growth hormone (hGH) is a good example, where the development of a preservative formulation has led to the commercialization of a more convenient multi-use injection pen. 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 m-cresol.

[0144] Several aspects must be considered during the formulation and development of a preserved dosage form. The effective preservative concentration in the formulation must be optimized. This requires testing a given preservative in the dosage form over a range of concentrations that impart antimicrobial efficacy without compromising protein stability.

[0145] As might be expected, developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, significantly minimizing the associated stability risks. For liquid formulations, preservative effectiveness and stability should be maintained throughout the product's shelf life (approximately 18-24 months). It is important to note that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0146] Homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) formulations will generally be designed for a particular route and method of administration, a particular dosage and frequency of administration, and a particular treatment of a particular disease, particularly in the areas of bioavailability and duration. Thus, formulations may be designed in accordance with the present invention for delivery by any suitable route, including, but not limited to, oral, oral, ocular, rectal, and vaginal, as well as by parenteral routes, including intravenous and intraarterial injection, intramuscular injection, and subcutaneous injection.

[0147] Once the pharmaceutical composition is formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystalline, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. The present invention also provides kits for producing single-dose administration units. The kits of the present invention each include both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single- and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0148] The therapeutically effective amount of a homomultimer-containing (e.g., TTR antibody homodimer-containing, TTR antibody homotetramer-containing, or TTR Fab homotetramer-containing fusion protein) pharmaceutical composition used will depend, for example, on the context and purpose of the treatment. Those skilled in the art will understand that appropriate therapeutic dosage levels will vary, in part, depending on the molecule being delivered, the indication for which the homomultimer (e.g., TTR antibody homodimer, TTR antibody homotetramer, or TTR Fab homotetramer fusion protein) is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and / or condition (age and general health). In certain embodiments, a clinician may titrate the dosage and modify the route of administration to obtain optimal therapeutic effect. Typical dosages can range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors described above. In certain embodiments, dosages may range from 1.0 μg / kg up 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.

[0149] 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 decrease in the severity of disease symptoms, an increase in the frequency or duration of disease-free periods, or prevention of disability or incapacity due to disease affliction.

[0150] Pharmaceutical compositions can be administered using medical device.The example of medical device for administering pharmaceutical compositions is described in United States Patent (USP) 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 United States Patent (USP) 5,399,163 (all are incorporated herein by reference).

[0151] Therapeutic Uses of Homodimeric and Homotetrameric Fusion Proteins As shown in the Examples, it has been found that dimerization of antibodies with TTR to generate TTR homodimeric fusion proteins, and tetramerization of antibodies and Fab fragments with TTR to generate TTR homotetrameric fusion proteins, result in TTR-containing fusion proteins with increased binding activity compared to the individual antibodies and / or Fab fragments.

[0152] Additionally, TTR homodimer and TTR homotetramer fusion proteins exhibit improved antigen clustering compared to individual antibodies and / or Fab fragments. When an antibody (e.g., an IgG antibody) binds to an antigen on a target cell (e.g., a tumor cell), the resulting clustered Fc domains bind to FcγRs found on immune effector cells such as NK cells and macrophages. This clustering facilitates FcγR-mediated signaling, initiating cell-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, TTR homodimer and TTR homotetramer fusion proteins are particularly useful for targeting ligands where high antibody or Fab affinity / avidity leads to enhanced biological effects. Enhanced cell-mediated effector function by the TTR homodimer and TTR homotetramer constructs of the present invention results in increased cell-killing potential, useful, for example, in cancer treatment.

[0153] The TTR homodimer and TTR homotetramer fusion proteins of the present invention can be used to bind to various targets / antigens. For example, TTR homodimer and TTR homotetramer fusion proteins can be used as agonists to target TRAIL, TRAIL2R, GITR, OX40, GLP1, TREM2, and 4-1BB. In addition, TTR homodimer and TTR homotetramer fusion proteins can be used as antagonists to target GIPR, TNFR, integrin receptors, PD-1, PD-L1, TIGIT, LAG-3, and TIM-3.

[0154] Accordingly, the present invention also relates to methods of treating cancer using the homodimeric and homotetrameric fusion proteins described herein.

[0155] In other embodiments, the present invention relates to the use of the homodimeric and homotetrameric fusion proteins described herein in the treatment of cancer.

[0156] In yet other embodiments, the present invention relates to the homodimeric and homotetrameric fusion proteins described herein for use in the treatment of cancer. [Example]

[0157] The following examples are provided for the purpose of illustrating specific embodiments or features of the present invention, and are not intended to limit its scope.

[0158] Example 1: Summary of generated TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Cloning of TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins The following hybridoma-derived antibodies and Fabs were used to generate fusion proteins: Anti-CB1R antibody and Fab derived from hybridoma CB10 (also called hybridoma 10D10) Anti-GITR antibody and Fab derived from hybridoma 9H6 The anti-TRAILR2 antibody conatumumab and the corresponding conatumumab Fab

[0159] The following TTR fusion proteins were generated: [TTR] = TTR tetramer. For simplicity, the His tag (for the Fab construct) is not shown in the summary in Example 1.

[0160] Specifically, the following TTR antibody homodimeric fusion proteins were constructed: In these constructs, the C-terminus of both antibody heavy chains was linked to the N-terminus of each TTR subunit. a.[Anti-CB1R antibody]2-[TTR] "CB1R TTR antibody homodimer fusion protein" Generated sequence pair:

[0161] [Table 1]

[0162] b.[Anti-GITR antibody]2-[TTR] "GITR TTR antibody homodimer fusion protein" Generated sequence pair:

[0163] [Table 2]

[0164] c.[Anti-TRAILR2 antibody]2-[TTR] Conatumumab TTR antibody homodimer fusion protein Generated sequence pair:

[0165] [Table 3]

[0166] Additionally, the following TTR antibody homotetramer fusion proteins were constructed. In these constructs, one of the two heavy chains at the C-terminus of the antibody, containing 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, containing the complementary set of K392D and K409D charge-pair mutations (SEQ ID NO: 7, 20, or 33), was linked with the linked heavy chain. Note that in the discussion of the constructs in the Examples, discussion of E416K and D459K refers to EU E356K and EU D399K, respectively. Similarly, in the discussion of the constructs in the Examples, discussion of K420D and K437D refers to EU K392D and EU K409D, respectively. a.[Anti-CB1R antibody]4-[TTR] "CB1R TTR antibody homotetramer fusion protein" Generated sequence pair:

[0167] [Table 4]

[0168] b.[Anti-GITR antibody]4-[TTR] "GITR TTR antibody homotetramer fusion protein" Generated sequence pair:

[0169] [Table 5]

[0170] c.[Anti-TRAILR2 antibody]4-[TTR] Conatumumab TTR antibody homotetramer fusion protein Generated sequence pair:

[0171] [Table 6]

[0172] Additionally, the following TTR Fab homotetrameric fusion proteins were constructed, in which the C-terminus of each Fab fragment was linked to the N-terminus of each TTR subunit: a.[Anti-CB1R Fab]4-[TTR] CB1R TTR Fab homotetramer fusion protein Note that in the discussion of constructs in the examples, the discussion of S215E refers to EU S183E. Similarly, in the discussion of constructs in the examples, the discussion of S203K refers to EU S176K. In the [SEQ ID NO:44]4-[SEQ ID NO:1]4+SEQ ID NO:11 fusion, no charge pair mutation was used. In the [SEQ ID NO:45]4-[SEQ ID NO:1]4&SEQ ID NO:46 fusion, SEQ ID NO:45 contains the S215E charge pair mutation and SEQ ID NO:46 contains the charge pair S203K mutation, both numbered according to the EU system. Generated sequence pair:

[0173] [Table 7]

[0174] b.[Anti-GITR Fab]4-[TTR] GITR TTR Fab homotetramer fusion protein Generated sequence pair:

[0175] [Table 8]

[0176] c.[Anti-TRAILR2 Fab]4-[TTR] Conatumumab TTR Fab homotetramer fusion protein Generated sequence pair:

[0177] [Table 9]

[0178] For the TTR antibody homodimer fusion protein, the C-terminus of each antibody heavy chain was linked to the N-terminus of each TTR subunit. For the TTR antibody homotetramer fusion protein, one of the two antibody C-terminal heavy chains was linked to the N-terminus of each TTR subunit. For the TTR Fab homotetramer fusion protein, the C-terminus of each Fab fragment was linked to the N-terminus of each TTR subunit.

[0179] Fusion proteins were generated using standard molecular biology techniques, including polymerase chain reaction (PCR), site-directed PCR mutagenesis, restriction endonuclease digestion, and enzymatic ligation into mammalian expression plasmids. Polyhistidine-tagged Fab-TTR molecules, with a (His)6 tag added to the C-terminus of the Fab, were also generated.

[0180] The cloned TTR fusion variant heavy chain and Fab DNAs in combination with the respective cloned anti-CB1, anti-GITR, and anti-TR2 antibody light chain (LC) DNAs were used to transfect mammalian cells for the expression of TTR antibody homodimers, TTR antibody homotetramers, and TTR Fab homotetramer fusion proteins. These procedures were generally performed according to methods found in Molecular Cloning: A Laboratory Manual, 3rd ed., Sambrook et al., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0181] Example 2: Cloning, expression, and purification of anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Cloning of anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins The anti-CB1 TTR antibody homodimer was generally cloned as follows: pTT5-del-Bsm-BI:VK1O2O12::[hu anti <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.

[0182] [SEQ ID NO:5]2-[SEQ ID NO:1]4 were constructed as follows: PCR1: Using C59477 as a template, a 5' PCR primer (5'-AGT TTA AAC GAA TTC GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CC-3') encoding the amino terminus of the signal sequence, a SalI restriction enzyme site, and an optimized Kozak sequence was combined with a 3' primer (5'-GTG CTG GCG AAT CCA GCT CCA ATA GTC ACC-3') designed to eliminate a pre-existing BamHI restriction site within C59477. PCR1 yielded an approximately 220 base pair product. PCR2a: Using C59477 as a template, a 5' PCR primer (5'-GAG CTG GAT TCG CCA GCA CCC AGG-3') designed to eliminate an existing BamHI restriction site was combined with a 3' primer (5'-GGT GCC CGT AGG GCC ACC 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 yielded an approximately 1250 base pair product. PCR3a: Using TTR (SEQ ID NO: 1) as a template, a 5' PCR primer (5'-TCC CTG TCT CCG GGT GGC CCT ACG GGC ACC G-3') encoding the carboxyl-terminal human heavy chain constant region (CH3) and amino-terminus of TTR 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-terminus of TTR (SEQ ID NO: 1), a stop codon, and a NotI restriction site. PCR3a yielded an approximately 400 base pair product. PCR reactions 1, 2a, and 3a were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73494:pTT15d:VK1O2O12::[huantigen] <hucb1>10D10.1(huIgG2-TO desK)VH]::TTR3.

[0183] [[SEQ ID NO:5]-[GGGGS]]2-[SEQ ID NO:1]4 was constructed using PCR1, as described above. PCR2b: Using C59477 as a template, a 5' PCR primer (5'-GAG CTG GAT TCG CCA GCA CCC AGG-3') designed to eliminate an 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), a G4S linker, and the amino terminus of TTR (SEQ ID NO:1). PCR2b yielded an approximately 1250 base pair product. PCR3b: Using TTR (SEQ ID NO: 1) as a template, a 5' PCR primer (5'-GGT GGT GGC GGA TCC GGC CCT ACG GGC ACC G-3') encoding the carboxyl-terminal human heavy chain constant region (CH3), a 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 GTG-3') encoding the carboxyl-terminal region of TTR, a stop codon, and a NotI restriction site. PCR3b yielded an approximately 400 base pair product. PCR reactions 1, 2b, and 3b were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73499:pTT15d:VK1O2O12::[huantigen <hucb1>10D10.1(huIgG2-TO desK)VH]::G4S::TTR3.

[0184] [[SEQ ID NO:5]-[(GGGGS)2]]2-[SEQ ID NO:1]4 was constructed using PCR1, as described above. PCR2c: Using C59477 as a template, a 5' PCR primer (5'-GAG CTG GAT TCG CCA GCA CCC AGG-3') designed to eliminate an existing BamHI restriction site was combined with a 3' primer (5'-GCC GGA CCC TCC CCC ACC GGA TCC GCC ACC TCC ACC CGG AGA CAG GGA G-3') encoding the carboxyl-terminal human heavy chain constant region (CH3), a (GGGGS)2 linker, and the amino terminus of TTR (SEQ ID NO:1). PCR2c yielded an approximately 1250 base pair product. PCR3c: Using TTR as a template, a 5' PCR primer (5'-CCG GTG GGG GAG GGT CCG GCC CTA CGG GCA CCG GTG AAT CCA AGG CTC CT-3') encoding the carboxyl-terminal human heavy chain constant region (CH3), a (GGGGS)2 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 GTG-3') encoding the carboxyl-terminal region of TTR, a stop codon, and a NotI restriction site. PCR3c yielded an approximately 400 base pair product. PCR reactions 1, 2c, and 3c were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73500:pTT15d:VK1O2O12::[huantigen <hucb1>10D10.1(huIgG2-TO desK)VH]::(G4S)2::TTR3.

[0185] [[SEQ ID NO:5]-[(GGGGS)3]]2-[SEQ ID NO:1]4 was constructed as follows: PCR4: Using C73500 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 (5'-CGT AGG GCC GGA CCC TCC CCC ACC GGA GCC CCC GCC CCC GGA TCC GCC ACC TCC-3') encoding the carboxyl-terminal human heavy chain constant region (CH3), (GGGGS)3 linker, and the amino terminus of TTR (SEQ ID NO:1). PCR4 yielded an approximately 1500 base pair product. PCR5: Using C73500 as a template, a 5' PCR primer encoding a (GGGGS)3 linker (5'-TCC GGG GGC GGG GGC TCC GGT GGG GGA GGG T-3') 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 TTG GTG-3'). PCR5 yielded an approximately 450 base pair product. PCR reactions 4 and 5 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73690:pTT15d:VK1O2O12::[huantigen]. <hucb1>10D10.1(huIgG2-TO desK)VH]::(G4S)3::TTR3.

[0186] [[SEQ ID NO:5]-[(GGGGS)4]]2-[SEQ ID NO:1]4 was constructed as follows. PCR6: Using C73690 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 the 3' primer (5'-GGA ACC ACC TCC GCC GGA TCC GCC ACC TCC A-3') encoding the carboxyl-terminal human heavy chain constant region (CH3) and the (GGGGS)4 linker. PCR6 yielded an approximately 1500 base pair product. PCR7: Using C73690 as a template, a 5' PCR primer (5'-GGC GGA GGT GGT TCC GGG GGC GGG GGC TCC G-3') encoding part of the (GGGGS)4 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), a stop codon, and a NotI restriction site. PCR7 yielded an approximately 450 base pair product. PCR reactions 6 and 7 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73729:pTT15d:VK1O2O12::[huantigen]. <hucb1>10D10.1(huIgG2-TO desK)VH]::(G4S)4::TTR3.

[0187] [SEQ ID NO:6]4-[SEQ ID NO:1]4 were constructed 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 (5'-CTT GGT CAT CTC CTT CCG GGA TGG GGG CAG G-3') designed to convert residue glutamic acid 356 (EU) in huIgG2 CH3 to lysine. PCR8 yielded an approximately 1200 base pair product. PCR9: Using C73494 as a template, a 5' PCR primer (5'-CTG CCC CCA TCC CGG AAG GAG ATG ACC AAG AAC CA-3') designed to convert residue glutamic acid 356 (EU) to lysine in huIgG2 CH3 was combined with a 3' primer (5'-GAA GGA GCC GTC GGA CTT CAG CAT GGG AGG TGT-3') designed to convert residue aspartic acid 399 (EU) to lysine in huIgG2 CH3. PCR9 yielded an approximately 160 base pair product. PCR10: Using C73494 as a template, a 5' PCR primer (5'-CCT CCC ATG CTG AAG TCC GAC GGC TCC TTC T-3') designed to convert residue aspartic acid 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. PCR 10 yielded an approximately 600 base pair product. PCR reactions 8, 9, and 10 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73730:pTT15d:VK1O2O12::[huantigen]. <hucb1>10D10.1(E416K,D459K)(huIgG2-TO desK)VH]::TTR3.

[0188] [[SEQ ID NO:6]-[GGGGS]]4-[SEQ ID NO:1]4 was constructed as follows: PCR11: Using C73499 as a template, a 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 (5'-CTT GGT CAT CTC CTT CCG GGA TGG GGG CAG G-3') designed to convert residue glutamic acid 356 (EU) in huIgG2 CH3 to lysine. PCR11 yielded an approximately 1200 base pair product. PCR12: Using C73499 as a template, a 5' PCR primer (5'-CTG CCC CCA TCC CGG AAG GAG ATG ACC AAG AAC CA-3') designed to convert residue glutamic acid 356 (EU) to lysine in huIgG2 CH3 was combined with a 3' primer (5'-GAA GGA GCC GTC GGA CTT CAG CAT GGG AGG TGT-3') designed to convert residue aspartic acid 399 (EU) to lysine in huIgG2 CH3. PCR12 yielded an approximately 160 base pair product. PCR13: Using C73499 as a template, a 5' PCR primer (5'-CCT CCC ATG CTG AAG TCC GAC GGC TCC TTC T-3') designed to convert residue aspartic acid 399 in huIgG2 CH3 to lysine 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, a stop codon, and a NotI restriction site. PCR13 yielded an approximately 600 base pair product. PCR reactions 11, 12, and 13 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. Amino acid numbering and nomenclature are the same as described for the previous construct.The resulting construct was designated C73731:pTT15d:VK1O2O12::[huanti. <hucb1>10D10.1(E416K,D459K)(huIgG2-TO desK)VH]::G4S::TTR3.

[0189] SEQ ID NO:7 was constructed as follows: PCR14: Using C73494 as a template, a 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 (5'-GGG AGG TGT GGT ATC GTA GTT GTT CTC CGG CTG C-3') designed to convert residue lysine 392 (EU) in huIgG2 CH3 to an aspartic acid. PCR14 yielded an approximately 1300 base pair product. PCR15: Using pTT5-del-Bsm-BI:VK1O2O12::huFc_(IgG2)(K392D K409D) ("C59541") as template, a 5' PCR primer (5'-CCG GAG AAC AAC TAC GAT ACC ACA CCT CCC ATG C-3') designed to convert residue lysine 392 (EU) in huIgG2 CH3 to aspartic acid was combined with a 3' primer (5'-AAC GAT ATC GCT AGC GCG GCC GCT CAA CCC GGA GAC AGG GAG-3') encoding the carboxyl-terminal region of huIgG2 minus the carboxyl-terminal lysine, a stop codon, and a NotI restriction site. PCR15 yielded an approximately 200 base pair product. PCR reactions 14 and 15 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into the SalI- and NotI-digested linear mammalian expression vector pTT15d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C73513:pTT15d:VK1O2O12::[huantigen] <hucb1>10D10.1(K420D,K437D)(huIgG2-TO desK)VH].

[0190] 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 a BssHII restriction site was combined with the 3' primer (5'-AAA AAA CGT ACG TTT GAT TTC CAC CTT GGT CC) encoding the carboxyl terminus of the variable region and a BsiWI restriction site. PCR16 was purified on 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 BssHII and BsiWI digested linear mammalian expression vector pTT5-del-Bsm-BI, which contains the signal peptide and kappa constant region. The resulting construct was designated C59474:pTT5-del-Bsm-BI:VK1O2O12::[hu <hucb1>10D10.1 VL]::huKLC.

[0191] Expression of anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and anti-CB1 TTR Fab homotetramer fusion proteins Anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and anti-CB1 TTR Fab homotetramer proteins were generally expressed as follows.

[0192] HEK293 6E cells were grown in suspension on a platform shaker in a humidified 37°C, 5% CO2 incubator rotating at 120 RPM. Cell culture passaging medium was FreeStyle F-17 + 0.1% (10 mL / L) Kolliphor P188 + 500 μL / L G418 + 6 mM (30 mL / L) L-glutamine. Cells were passaged 1-2 days before transfection to ensure a cell density of approximately 1.5 e at the time of transfection. 6 The transfection complex was mixed in supplement-free Freestyle F17 medium at a volume of 10% of the final culture volume. 0.5 μg of DNA per mL of culture was added to Freestyle F17 medium, followed by 1.5 μL of PEImax reagent per mL of culture. The medium was then mixed and incubated at room temperature for 10 minutes, then added to the cell culture, and the flask was returned to the shaker platform in the incubator. 1–4 hours after transfection, 25 μL of yeastolate solution was added per mL of culture. Conditioned medium was harvested 6 days after transfection.

[0193] Freestyle F-17 (catalog no. 13835), L-glutamine (catalog no. 25030), and Geneticin G418 (catalog no. 10131027, liquid) were obtained from Life Technologies. Kolliphor P188 (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.

[0194] In the gels shown in Figures 2a, 2b, and 2c, 10 μL of sample (unheated) was loaded into each lane of a 4-20% SDS-PAGE gel and developed using a Coomassie Blue dye system. Figures 2a and 2b show that the anti-CB1 TTR antibody homodimer and anti-CB1 TTR antibody homotetramer proteins, respectively, are robustly expressed in HEK293 cells. Figure 2a shows the expression of the anti-CB1 TTR antibody homodimer ([SEQ ID NO: 5]-[SEQ ID NO: 1]) and [[SEQ ID NO: 5]-[(GGGGS) 1-4 Figure 2b shows that the V1 Fc homotetramer formed with the V1 Fc charge pair mutations K409D & K392D and D399K & E356K in one heavy chain (SEQ ID NOs: 6 and 7) also appears to be SDS-resistant. The transfection ratios of various heavy and light chain DNAs 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 strongest expression.

[0195] Figure 2c shows that the anti-CB1 TTR Fab homotetramer construct was robustly expressed in HEK293 cells and was SDS-resistant. The TTR fusion was more resistant to SDS 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 the fusion. The heavy chain-TTR used in lanes 2 and 4 was [SEQ ID NO:44]-[SEQ ID NO:1], and the light chain was SEQ ID NO:11. The heavy chain used in lanes 3 and 5 was SEQ ID NO:44, and the light chain-TTR was [SEQ ID NO:11]-[SEQ ID NO:1]. The heavy chain-TTR used in lanes 6 and 8 was [SEQ ID NO:45]-[SEQ ID NO:1], and the light chain was SEQ ID NO:46. The heavy chain used in lanes 7 and 9 was SEQ ID NO:45, and the light chain-TTR was [SEQ ID NO:46]-[SEQ ID NO:1]. Charge pair mutations were included in the anti-CB1 TTR Fab homotetramer constructs tested in lanes 6-9 (heavy chain with SEQ ID NO: 45 and light chain with SEQ ID NO: 46). SEQ ID NO: 45 contains the S215E charge pair mutation, and SEQ ID NO: 46 contains the S203K charge pair mutation. A His tag was also used (see Figure 2c).

[0196] Purification of anti-CB1 TTR Fab homotetramer fusion protein Anti-CB1 TTR Fab homotetramer protein was generally purified as follows.

[0197] Cell culture media was dialyzed twice for a minimum of 2 hours against 2 L of 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 using 10 kDa MWCO Slide-a-lyzers (Thermo Fisher Scientific). Molecules were purified from the buffer-exchanged cell culture media using an AKTA purification device (GE Healthcare Life Sciences) tandem liquid chromatography system equipped with a 1 mL Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany) as the first column and a 5 mL Desalting HiTrap (GE Healthcare Life Sciences) as the second column. The medium was loaded directly onto a Ni-NTA column, washed with 8 CV of 50 mM Na phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0, and eluted with 2 CV of 50 mM Na phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0. The Ni-NTA column eluate was automatically transferred to a desalting column, where the protein was eluted isocratically with 4 CV of 10 mM Na acetate, 150 mM NaCl, pH 5.2. The sample was sterile filtered through a 3.0 μm glass fiber / 0.2 μm Supor membrane (Pall Corporation, Port Washington, NY, USA).

[0198] The protein concentration of each purified molecule was measured by UV absorbance at 280 nM (A280) using a NanoDrop2000 (Thermo Fisher Scientific, Rockford, Illinois, USA). SDS-PAGE analysis was performed on 3 μg of each final purified molecule on a denaturing, non-reducing 4-12% Bis-Tris NuPAGE gel using MES running buffer (Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions. HPLC size-exclusion chromatography analysis was performed on 20 μg of each final purified molecule. This was run at 1 mL / min on a Phenomenex SEC3000 column (7.8 x 300 mm, Phenomenex, Torrance, California, USA) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9, and the absorbance at 280 nm was monitored.

[0199] Purification of anti-CB1 TTR antibody homodimer and TTR antibody homotetramer fusion proteins Anti-CB1 TTR antibody homodimer and TTR antibody homotetramer fusion proteins were generally purified as follows.

[0200] The fusion protein was initially purified from cell culture medium using an AKTA purification device (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) tandem liquid chromatography system equipped with a 1 mL MabSelect SuRe (MSS) HiTrap (GE Healthcare Life Sciences) as the first column and a 5 mL Desalting HiTrap (GE Healthcare Life Sciences) as the second column. The medium was loaded directly 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 transferred to a desalting column, where the protein was eluted isocratically with 4 CV of 10 mM Na acetate, 150 mM NaCl, pH 5.2. The sample was sterile filtered 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 measured by UV absorbance at 280 nM (A280) using a NanoDrop2000 (Thermo Fisher Scientific, Rockford, Illinois, USA). SDS-PAGE analysis was performed by running 3 μg of each final purified molecule on a denaturing, non-reducing 4-12% Bis-Tris NuPAGE gel using MES running buffer (Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions. HPLC size-exclusion chromatography analysis was performed on 30 μg of each final purified molecule. This was run at 1 mL / min on a Phenomenex SEC3000 column (7.8 x 300 mm, Phenomenex, Torrance, California, USA) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9, and the absorbance at 280 nm was monitored.Figure 3 shows a representative HPLC SEC analysis of anti-CB1 TTR antibody homodimer fusion proteins with no linker, with a (G4S) linker, with a (G4S)2 linker, with a (G4S)3 linker, or with a (G4S)4 linker.

[0201] Example 3: Activities of anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins The activity of anti-CB1 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins was assessed by the CB1 cAMP assay.

[0202] 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 measure antibody activity, cells were seeded into 96-well plates at a density of 10,000 cells per well in 80 μL of DMEM containing 0.5% FBS, 1% Pen / Strep / L-glutamine, 25 mM Hepes, 0.1 mM NEAA, 1 mM sodium pyruvate, and 400 μg / L G418. After overnight incubation, the medium was removed and replaced with 5 μL of fresh medium, followed by 5 μL of medium plus 15 μM forskolin (EMD Chemicals Catalog No. 344273) and 250 pM CP55,940 (TOCRIS Catalog No. 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 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.

[0203] The results of the assay are shown in Table 4. The TTR antibody homotetramer had a 3.9-fold better EC than the CB1 parent antibody. 50 Some Fabs used charge pair mutations, while others did not (see discussion of Figure 2 in Example 2). These results demonstrate that the TTR antibody homotetramer and TTR Fab homotetramer fusion proteins have EC 50 Interestingly, the anti-CB1 TTR antibody homodimer showed a significantly improved EC than the CB1 parent antibody. 50 is not preferable, and the longer the linker, the lower the EC 50 appears to be getting worse.

[0204] Example 4: Cloning, expression, and purification of anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Cloning of anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Anti-GITR TTR antibody homodimer and TTR antibody homotetramer fusion proteins were generally cloned as follows: Construct C74201 (pSLX240p:Native::[huAnti <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>9H6VL]) and C73877 (pTT5:Native::[hu antibody <hugitr>9H6VL]::huKLC) was used as the template for all constructs described below.

[0205] [SEQ ID NO:18]2-[SEQ ID NO:1]4 were constructed as follows: PCR17: Using C74201 as a template, the signal peptide of anti-GITR MAb 9H6 (w / N297G) was replaced with the VK1 signal peptide (MDMRVPAQLLGLLLLWLRGARC) by a three-step overlapping PCR extension, and the product of the final PCR was then amplified using a 5' signal peptide primer (5'-GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTG GGG CT-3') and a 3' C-terminal heavy chain / N-terminal TTR primer (GGT GCC CGT AGG GCC ACC CGG AGA CAG GGA GAG G) to yield 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 CCT ACG 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 yielded an approximately 400 base pair product. PCR reactions 17 and 18 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C143043:pTT5d:VK1O2O12::[huantigen]. <hugitr>9H6(N297G)VH]::TTR(C10A,K15A).

[0206] [SEQ ID NO:21]4-[SEQ ID NO:1]4 were constructed 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 a three-step overlapping PCR extension, and the product of the final PCR was then amplified using a 5' signal peptide primer (5'-GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTG GGG CT-3') and a 3' C-terminal heavy chain / N-terminal TTR primer (5'-GCT CTC GAG GGA GTA GAG TCC TGA GGA CTG TAG G-3') to yield 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'-CTC TAC TCC CTC GAG AGC GTG GTG ACC GTG CC-3') was combined with a 3' primer encoding the human heavy chain constant region (5'-CCT CCT CCA CAA GAT TTG GGC TCA ACT TTC TTG TC-3'). PCR20 yielded an approximately 130 base pair product. PCR21: Using TTR3 as a template, a 5' PCR primer encoding the amino terminus of TTR (5'-CAA ATC TTG TGG AGG AGG CCC TAC GGG CAC CG-3') was combined with two 3' primers encoding the carboxyl-terminal region of TTR, a stop codon, and a 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 yielded an approximately 450 base pair product. PCR reactions 19, 20, and 21 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit.The resulting construct was C144132:pTT5d:VK1O2O12::[hu anti. <hugitr>9H6(S183E)scFab]::TTR(C10A,K15A)::G::G::6xHis (note that the (His)6 tag was included for purification purposes).

[0207] [SEQ ID NO:19]4-[SEQ ID NO:1]4 were constructed 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 a three-step overlapping PCR extension, and the product of this final PCR was then amplified using a 5' signal peptide primer (5'-GTC GAC TAG GCC ACC ATG GAC ATG AGG GTG CCC GCT CAG CTC CTG GGG CT-3') and a 3' C-terminal heavy chain / N-terminal TTR primer (5'-TAG GTG CTT CCG TAC TGT TCC TCC CGG GGC TT-3') to yield 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 yielded an approximately 370 base pair product. PCR24: 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 CCG CTC ATT CCT TGG GAT TGG TGA CG-3') encoding the carboxyl-terminal region of TTR. PCR 24 yielded an approximately 900 base pair product. PCR reactions 22, 23, and 24 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C144127:pTT5d:VK1O2O12::[huantigen]. <hugitr>The clone was named 9H6(N297G,E356K,D399K)VH]::TTR(C10A, K15A).

[0208] SEQ ID NO:20 was constructed 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 GGG CT) was combined with a 3' primer encoding the carboxyl-terminal human heavy chain constant region (CH3) (5'-ACG GTG ACG ACG CTG CTC AGG CTG TAC AGG CCG CTG-3'). PCR25 yielded an approximately 650 base pair product. PCR26: Using C143048 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). PCR26 yielded an approximately 370 base pair product. 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 CCG CTC ATT CCT TGG GAT TGG TGA CG-3') encoding the carboxyl-terminal region of TTR. PCR 27 yielded an approximately 900 base pair product. PCR reactions 25, 26, and 27 were gel separated and purified on Qiagen columns. These fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C144130:pTT5d:VK1O2O12::[huantigen] <hugitr>The clone was named 9H6(N297G,K392D,K409D)VH.

[0209] SEQ ID NO:25 was constructed as follows: PCR28: 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 CTG GGG CT) was combined with a 3' primer encoding the carboxyl-terminal human light chain constant region (5'-TAT CGC TAG CGC GGC CGC-3'). PCR28 yielded an approximately 800 base pair product. PCR reaction 28 was gel separated and purified on a Qiagen column. The fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C143044:pTT5d:VK1O2O12::[huantigen]. <hugitr>9H6 VL].

[0210] SEQ ID NO:26 was constructed 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 CTG GGG CT) was combined with a 3' primer encoding the carboxyl-terminal human light chain constant region (5'-TGG TGC AGC CAC CGT ACG TTT GAT TTC CAC CTT GGT CC-3'). PCR29 yielded an approximately 400 base pair product. 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 Qiagen columns. The fragments were then mixed and ligated into SalI- and NotI-digested linear mammalian expression vector pTT5d using the GeneArt Seamless Cloning and Assembly Kit. The resulting construct was designated C143049:pTT5d:VK1O2O12::[huantigen]. <hugitr>It was named 9H6(S176K)VL.

[0211] Expression of anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were generally expressed as follows.

[0212] Freestyle F-17 (catalog no. 13835), L-glutamine (catalog no. 25030), and Geneticin G418 (catalog no. 10131027, liquid) were obtained from Life Technologies. Kolliphor P188 (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. HEK293 6E cells were grown in suspension on a platform shaker in a humidified 37°C, 5% CO2 incubator rotating at 120 RPM. Cell culture passaging medium was FreeStyle F-17 + 0.1% (10 mL / L) Kolliphor P188. The medium was P188 + 500 μL / L G418 + 6 mM (30 mL / L) L-glutamine. Cells were passaged 1–2 days before transfection to ensure a cell density of approximately 1.5 e at the time of transfection. 6 The transfection complexes were mixed in supplement-free Freestyle F17 medium at a volume of 10% of the final culture volume. 0.5 μg of DNA per mL of culture was added to Freestyle F17 medium, followed by 1.5 μL of PEImax reagent per mL of culture. The culture was mixed, incubated at room temperature for 10 minutes, and then added to the cell culture. The culture flask was then returned to the shaker platform in the incubator. 1–4 hours after transfection, 25 μL of Yeastolate solution was added per mL of culture. Conditioned medium was then harvested 6 days after transfection.

[0213] Alternatively, anti-GITR TTR antibody homodimers were expressed as follows: HEK293 cells were transiently transfected with the corresponding cDNA. 1.0 × 10 6 A suspension of HEK293-6E cells at 3700 cells / mL was incubated with 0.5 mg / L of DNA (0.25 mg / L hu-antibody in pTT5d vector) containing 4 mg of PEI (Polysciences). <hugitr>9H6(N297G)VH]::TTR(C10A,K15A) and 0.25 mg / L of hu antigen in pTT5d vector <hugitr>9H6VL) (Durocher et al. NRCC, Nucleic Acids. Res. (2002) 30, e9) were transfected with 1 mg of DNA in FreeStyle F17 medium (Life Technologies) and incubated at 36°C in a shake flask at 150 RPM. A final 0.5% transfection-grade yeastolate (BD Biosciences) was added to the culture 4 hours after transfection. Cells were grown in suspension in FreeStyle F17 medium supplemented with 0.1% Pluronic F68 and 50 μg / mL Geneticin for 6 days and harvested for purification.

[0214] Conditioned medium was analyzed using 4-20% SDS-PAGE and Quick Blue Stain gels. Figure 5 shows that anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins could be expressed in HEK293 cells. Expression was strongest for the anti-GITR TTR antibody homotetramer and TTR Fab homotetramer. Lane 1 contains 10.3 mg of anti-GITR TTR antibody homodimer (356 KDa); lane 2 contains 29.3 mg of anti-GITR TTR antibody homotetramer (656 KDa); lane 3 contains 143.6 mg of anti-GITR TTR Fab homotetramer (256 KDa); lane 4 contains 100 ng of anti-DNP antibody; lane 5 contains 250 ng of anti-DNP antibody; lane 6 contains 500 ng of anti-DNP antibody; and lane 7 contains 1000 ng of anti-DNP antibody. The anti-DNP antibody provides information about how unlabeled antibodies function in this assay. 10 μL of protein in non-reducing loading buffer was added to each lane. The gel was run without heating.

[0215] Purification and characterization of anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were generally purified and characterized as follows.

[0216] Anti-GITR TTR antibody homodimer and TTR antibody homotetramer fusion proteins were purified from cell culture medium using an AKTA purification device (GE Healthcare Life Sciences) liquid chromatography system equipped with two sequentially connected 1 mL Protein A Sepharose Fast Flow (ProA FF) HiTrap (GE Healthcare Life Sciences) columns. The medium was loaded directly onto the ProA FF column, 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 against 2 L of 10 mM sodium acetate, 9% sucrose, pH 5.2, using 10 kDa MWCO Slide-a-lyzers (Thermo Fisher Scientific).

[0217] TTR Fab homotetramer fusion protein was purified from cell culture medium using an AKTA Purifier liquid chromatography system equipped with a 5 mL HisTrap Excel HiTrap (GE Healthcare Life Sciences) column. The medium was loaded directly onto the HisTrap column, washed with 20 CV of 20 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole, pH 7.4, and eluted with a 20 CV imidazole gradient from 10 mM to 500 mM. Because the former method lacks chromatographic resolution, repurification of the flow-through fraction was performed under the same conditions as above, except that a step elution of 8 CV of 20 mM NaH2PO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4 was used instead of the gradient elution. The purified sample was dialyzed twice against 2 L of 10 mM sodium acetate, 9% sucrose, pH 5.2 using 10 kDa MWCO Slide-a-lyzers.

[0218] The protein concentrations of the anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were measured by UV absorbance at 280 nM (A280) using a NanoDrop 2000 (Thermo Fisher Scientific, Rockford, Illinois, USA).

[0219] SDS-PAGE analysis (performed with 3 μg each of the anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins) was performed on a denaturing, non-reducing 4-12% Bis-Tris NuPAGE gel using MES running buffer (Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions. See Figure 6. Lanes 1 and 4 are the anti-GITR TTR antibody homodimer; lanes 2 and 5 are the anti-GITR TTR antibody homotetramer; and lanes 3 and 6 are the anti-GITR TTR Fab homotetramer. Figure 6 shows that the partially purified products for all three protein fusion constructs are correctly assembled based on the unheated, non-reduced lanes. Upon heating and reduction, the three protein fusion constructs are resolved into the expected component chains (the upper bands are the heavy chains, and the lowest bands are the light chains).

[0220] 30 μg of each of the anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins was analyzed by HPLC size-exclusion chromatography using a Phenomenex SEC3000 column (7.8 × 300 mm, Torrance, California, USA) in 50 mM NaHPO, 250 mM NaCl, pH 6.9, at 1 mL / min, and absorbance was monitored at 280 nm.

[0221] Anti-GITR TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were analyzed by reduced LCMS analysis. 20 μg of material was denatured in 8 M guanidine HCl / TRIS pH 8.0 (Teknova, Hollister, CA) and reduced with 10 mM DTT (EMD Millipore, Darmstadt, Germany) at 50°C for 20 min. Samples were acidified with trifluoroacetic acid, and 10 μg was loaded onto a Zorbax reverse-phase C8 column using an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA). The column eluate was introduced into the electrospray source of an Agilent 6230 ESI-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA), and mass spectra were collected. The relevant spectra were deconvoluted using the MaxEnt algorithm within the Agilent MassHunter software package. The mass spectra obtained for LC and HC were compared with the theoretically calculated masses of each chain.

[0222] The purified anti-GITR TTR antibody homodimer was further purified at constant concentration with 320 mL of Superdex 200 (GE Healthcare Life Sciences) in 1.4 CV of 10 mM Na acetate, 150 mM NaCl, pH 5.0 (to remove aggregates that accumulated over time). Fractions were selected for pooling based on HPLC-SEC purity. The Superdex pool was concentrated using a VivaSpin 10 kDa MWCO centrifugal filtration unit (Sartorius) and then sterile filtered through a 0.2 μm Supor syringe filter (Pall). HPLC size-exclusion chromatography analysis was performed on a Sepax Zenix-C, 7.8 x 300 mm column (Sepax, Newark, Delaware, USA) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 mL / min, and absorbance was monitored at 280 nm.

[0223] Figure 7 shows the results of HPLC size-exclusion chromatography (SEC) analysis of the anti-GITR TTR antibody homodimer (center peak), TTR antibody homotetramer (left peak), and TTR Fab homotetramer (right peak) fusion proteins. The SEC chromatograms show that the main peak elutes at the expected position, consistent with a correctly assembled molecule. Furthermore, non-denaturing SEC purification (unlike SDS-PAGE) supports the notion that the fusion proteins do not aggregate.

[0224] Repurified anti-GITR TTR antibody homodimers were reanalyzed by reduced LCMS. Approximately 10 μg of anti-GITR TTR antibody homodimer was dried in a Speed-Vac and then resuspended in 20 μL of 8 M Gu-HCl, pH 8.0, containing 20 mM DTT. The samples were incubated at 37°C for 1 hour to reduce each sample to its individual protein chain components. Each reduced sample was then acidified by adding 0.1% TFA. For LC-MS, approximately 5 μg of reduced sample was injected into an Agilent Technologies 1100 capillary HPLC and sprayed into an Agilent Technologies 6224 ESI-TOF mass spectrometer. The capillary HPLC used a 1.0 mm x 50 mm Agilent Zorbax 300SBC8 column with a flow rate of 50 μL / min and a column temperature of 75°C. The HPLC was performed using the following buffers: Buffer A - 0.1% TFA / HO; Buffer B - 0.1% TFA / HO / 90% n-propanol. The gradient consisted of an initial condition of 2% B for 5 min, increasing to 45% B over 20 min, increasing to 95% B over 3 min, remaining at 95% B for 4 min, and then decreasing to 2% B over 1 min. The MS method for the ESI-TOF instrument scanned m / z [750–6000] at a rate of 1 spectrum / s. Other MS instrument parameters included a capillary voltage (Vcap) of 3200 V, a fragmentor voltage of 225 V, a skimmer voltage of 60 V, and an OCT 1RF voltage of 800 V. For data analysis of the LC-MS data, the appropriate LC-MS spectra for each protein chain were combined and deconvoluted using Agilent MassHunter software. The deconvoluted output mass range was [15,000-75,000] with a mass step of 1.0 Da and a signal-to-noise (S / N) threshold of 30.0. Correctly reduced chain masses were observed for each sample. Using an analysis temperature of 75°C, Asp-Pro cleavage was confirmed in the heavy chain of the anti-GITR TTR antibody homodimer.

[0225] Differential scanning calorimetry (DSC) of the anti-GITR TTR parent mAb 9H6 ("1"), anti-GITR TTR antibody homodimer ("4"), TTR antibody homotetramer ("2"), and TTR Fab homotetramer ("3") fusion proteins was performed on a Malvern MicroCal VP-capillary DSC (see Figure 8). The following parameters were used: scan range: 10-100°C; scan rate: 1°C / min; prescan thermostat: 15 min. Typically, 400 μL of 1 mg / mL sample was consumed per analysis. Data were processed with Origin7 software. Analyzed proteins were expressed in HEK293-6E cells. The parent Ab ("1") is glycosylated, while the TTR fusion is the N297G α-glycovariant. The results in Figure 8 show that the melting temperatures of the TTR fusion proteins were comparable to or better than those of the parent Ab, demonstrating that the TTR fusion proteins were robust. The predicted order of thermally induced transitions was CH2, Fab, CH3.

[0226] Example 5: Activity and PK Profiles of Anti-GITR TTR Antibody Homodimer, TTR Antibody Homodimer, and TTR Fab Homodimer Fusion Proteins The activity and PK profiles of the parent anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, and anti-GITR TTR Fab homotetramer fusion proteins were evaluated.

[0227] Activity evaluation The binding and potency activities of the parent anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, and anti-GITR TTR Fab homotetramer fusion proteins were assessed by the following assays, and the results of these assays are shown in Figure 10.

[0228] Crosslinking assay. A 96-well high-binding plate (Corning 3369) was coated overnight at 4°C with an anti-CD3 antibody (OKT3) and a cross-linker antibody (either goat anti-human IgG Fc Cross-Adsorbed Ab (Thermo Scientific 31125) or goat anti-human IgG (H+L) (Pierce 31119)) at 1 and 0.3 μg / mL, respectively. The next day, the Abs were washed away, and anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, anti-GITR TTR Fab homotetramer fusion protein TTR, or an isotype control mAb was added to the capture plate for 1 hour at 37°C. After incubation, plates were rinsed, and naive T cells (50K / well in RPMI 1640 supplemented with 10% FBS, 2 mM L-Glut, 10 mM HEPES, 1 mM NaPyr, 0.1 mM NEAA, and 50 μM 2ME) were added to the wells and cultured for 4 days. Proliferation was measured using CellTiterGlo (Promega).

[0229] Crosslinking-free assay. 96-well high-binding plates (Corning 3369) were coated with 1 μg / mL anti-CD3 Ab overnight at 4°C. The following day, the anti-CD3 Ab was washed away, 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, followed by naive T cells (50 kJ / well in RPMI 1640 supplemented with 10% FBS, 2 mM L-Glut, 10 mM HEPES, 1 mM NaPyr, 0.1 mM NEAA, and 50 μM 2ME). After 4 days of culture, proliferation was measured using CellTiterGlo (Promega).

[0230] PK profile evaluation PK profiles were measured in male CD-1 mice (n = 3 per group) by intravenous injection of 8.75 mg / kg anti-GITR TTR antibody homotetramer, 4.75 mg / kg anti-GITR TTR antibody homodimer, 3.4 mg / kg anti-GITR TTR Fab homotetramer, and 2 mg / kg anti-GITR antibody normalized to the GITR antibody. Serum samples were collected from 75 μL blood samples collected at 0.5, 2, 8, 24, 48, 72, 96, 168, 336, 504, 672, and 840 hours post-dose. After collection, each blood sample was maintained at room temperature. After a 30-40 minute clotting period, the samples were centrifuged at 11,500 RPM for approximately 10 minutes at 2-8°C using a calibrated Eppendorf 5417R centrifuge system (Brinkmann Instruments, Inc., Westbury, NY). The collected serum was then transferred to pre-labeled (for each rat) cryopreservation tubes and stored at −60°C to −80°C for later bioanalysis.

[0231] The following PK assay was used to measure total anti-GITR species (i.e., any anti-GITR-binding species present) in mouse serum using the Meso Scale Discovery (MSD) assay: parental anti-GITR mAb, anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, and anti-GITR TTR Fab homotetramer fusion protein. A standard binding 96-well MSD plate (Meso Scale Discovery, Gaithersburg, MD) was coated with 2 μg / mL of mouse anti-idiotypic anti-GITR antibody, Mab1.2.1 (Amgen Inc., Thousand Oaks, CA) in PBS and then incubated overnight at 4°C. The plate was then washed and blocked with I-Block™ (Life Technologies, Carlsbad, CA) overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted in naive CD-1 mouse serum when dilutions were required. The 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 plate was washed three times with approximately 200 μL of 1×KPL buffer (KPL, Gaithersburg, MD), and then 50 μL of the diluted standards, QCs, and samples were transferred to the Mab1.2.1 antibody-coated plate and incubated at room temperature (approximately 25°C) for 1.5 hours. The plate was washed three times with approximately 200 μL of 1×KPL wash buffer, and then 50 μL of 250 ng / mL mouse anti-idiotypic anti-GITR antibody, Mab1.1.1, conjugated to biotin was added and incubated for 1.5 hours. After washing the plate three times with 1X KPL wash buffer, 50 μL of 100 ng / mL streptavidin conjugated to MSD SULFO-TAG (Amgen, Inc.) was added and incubated for 15 minutes. The plate was washed six times with approximately 200 μL of 1X KPL wash buffer, followed by the addition of 150 μL of 1X Read Buffer T (Meso Scale Discovery), and the electrochemiluminescence signal was measured using an MSD6000 plate reader (Meso Scale Discovery).Serum concentration data were analyzed using non-compartmental methods in Phoenix® (Phoenix 64, Build 6.4.0.768, Pharsight® Corp., Mountain View, Calif.).

[0232] The following PK assay was used to measure the presence of both anti-GITR and TTR species of anti-GITR TTR antibody homodimer, anti-GITR TTR antibody homotetramer, and anti-GITR TTR Fab homotetramer fusion proteins in mouse serum by MSD assay of mouse serum samples. A regular binding 96-well MSD plate (Meso Scale Discovery, Gaithersburg, MD) was 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 plate was then washed and blocked with I-Block™ (Life Technologies, Carlsbad, CA) overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted with naive CD-1 mouse serum when dilutions were required. The 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 plate was washed three times with approximately 200 μL of 1×KPL buffer (KPL, Gaithersburg, MD), 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 three times with approximately 200 μL of 1×KPL wash buffer, and then 50 μL of 250 ng / mL mouse anti-idiotypic anti-GITR antibody, Mab1.1.1, conjugated to biotin was added and incubated for 1.5 hours. After washing the plate three times with 1X KPL wash buffer, 50 μL of 100 ng / mL streptavidin conjugated to MSD SULFO-TAG (Amgen, Inc.) was added and incubated for 15 minutes. The plate was washed six times with approximately 200 μL of 1X KPL wash buffer, followed by the addition of 150 μL of 1X Read Buffer T (Meso Scale Discovery), and the electrochemiluminescence signal was measured using an MSD6000 plate reader (Meso Scale Discovery).Serum concentration data were analyzed using non-compartmental methods in Phoenix® (Phoenix 64, Build 6.4.0.768, Pharsight® Corp., Mountain View, Calif.).

[0233] The results of the PK analysis can be seen in Figure 9. As shown by Figure 9a) (measurement of the presence of any anti-GITR binding species): [1] the PK of the anti-GITR TTR antibody homodimer ("1") is more favorable than that of the parental Ab ("2"); [2] the PK of the anti-GITR TTR Fab homotetramer ("4") is less favorable, likely due to the fact that the Fab lacks an Fc region and therefore lacks the ability to mediate / extend its half-life; and [3] the PK of the anti-GITR TTR antibody homotetramer ("3"), while not as potent as the parental mAb, shows significantly enhanced PK compared to the Fab. Figure 9b) (measurement of the presence of both anti-GITR binding species and TTR species) shows that the PK of the intact anti-GITR TTR fusion proteins is consistent with that observed in Figure 9a), indicating that the anti-GITR binding moiety is not liberated from each TTR fusion protein by in vivo proteolysis.

[0234] As shown in Figure 10, multimerization of the parent anti-GITR mAb 9H6 with TTR fusions improves binding but not potency. Activation of GITR is known to prevent suppression by regulatory T cells. Clustering and FcγR binding are also known to be required for anti-GITR antibody-mediated activation of cell proliferation, cytokine production, and induction of CD4+ Th9 cells. Figure 10a) shows that the binding affinities of the anti-GITR TTR antibody homodimer ("3"), TTR antibody homotetramer ("4"), and TTR Fab homotetramer ("2") fusion proteins are more favorable than those of the parent anti-GITR mAb 9H6 ("1"). Notably, as shown in Figure 10b), cell-based assays demonstrate that higher affinity does not translate into higher potency. The control Ab in Figure 10b) is a non-binding control version of the anti-GITR mAb.

[0235] Comparison of the data in Figures 9a and 9b shows that there is little partial molecular degradation of the TTR construct.

[0236] Example 6: Cloning, expression, and purification of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Cloning of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were generally cloned as follows: In these experiments, the anti-TRAILR2 antibody was conatumumab (AMG655) and the anti-TRAILR2 Fab was the Fab portion of conatumumab.

[0237] 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::[huanti <hutrailr2>XG1048(W)VH]::huIgG1(f)) and C9448 (VK3_A27L::[hu antibody <hutrailr2>XG1048VL]::huKLC) were the templates used for anti-TRAILR2 heavy and light chain variable region sequences, respectively. In these experiments, the anti-TRAILR2 antibody was conatumumab (AMG655). Because the anti-TRAILR2 sequence template lacks the VK1 (VK1O2O12) signal peptide, the pSLX240puro, pSLX240hygro, and pSLX240neo constructs were found to contain VK1 with a BssHII site at the carboxy-terminus of the signal peptide sequence.

[0238] [SEQ ID NO:31]2-[SEQ ID NO:1]4 were constructed as follows: PCR31: Using C36606 as a template, a 5' PCR primer (5'-CTG CTG TGG CTG AGA GGT 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 yielded a 395 base pair product. PCR32: Using C-143043 as a 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, a stop codon, a NotI site, and an 18-base overhang encoding the amino terminus of the linearized pSLX240p plasmid. PCR32 yielded a 1390-base product. PCR reactions 31 and 32 were purified using Qiagen columns. 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 C-150225:pSLX240p:VK1O2O12::[huanti <hutrailr2>AMG 655 VH]::IgG1z_SEFL(desK)::TTR(C10A,K15A).

[0239] SEQ ID NO:38 was constructed as follows: PCR33: Using C9448 as a template, the 5' PCR primer (5'-CTG CTG TGG CTG AGA GGT GCG CGC TGT GAA ATT GTG TTG ACG CAG -3') encoding the amino terminus and signal sequence of the anti-TRAILR2 light chain variable region was combined with the 3' primer (5'-AGC CAC CGT TCG TTT GAT TTC CAC CTT-3') to amplify the anti-TRAILR2 light chain variable region, generating a 363 base pair product. PCR34: Using C-143044 (pTT5d:VK1O2O12::[huanti <hugitr>PCR 34 was used (Aggregate PCR Reaction [9H6VL]) to combine a 5' primer (5'-ATC AAA CGA ACG GTG GCT GCA CCA TCT-3') encoding the amino terminus of the kappa light chain constant region and the last 9 bases of the anti-TRAILR2 variable region with a 3' primer (5'-TGT TTA AAC GAT ATC GCT AGC GCG GCC GCC TAA CAC TCT CCC CTG TTG AAG-3') encoding the carboxyl terminus of the human kappa light chain constant region, a stop codon, and the NotI restriction enzyme. PCR 34 generated an approximately 330 base pair product. PCR reactions 33 and 34 were purified on Qiagen columns. 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 C-150226:pSLX240h:VK1O2O12::[huanti <hutrailr2>AMG655 VL]::huKLC.

[0240] [SEQ ID NO:32]4-[SEQ ID NO:1]4 were constructed as follows. PCR 31 was utilized as described above. PCR 35: Using C-144127 as a template, a 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 a 3' primer (5'-TTAAACGATATCGCTAGCGCGGCCGCTCATTCCTTGGGATTGGTGACG-3') encoding the carboxyl terminus of TTR to generate an approximately 1390 base pair product. PCR reactions 31 and 35 were purified on Qiagen columns. 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 C-150227:pSLX240p:VK1O2O12::[huanti <hutrailr2>AMG 655 VH]::IgG1z(N297G,KK)::TTR(C10A,K15A).

[0241] SEQ ID NO:33 was constructed as follows. PCR31 was utilized as described above. PCR36: Using C-144130 as a template, a 5' primer (5'-GGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCC-3') was combined with a 3' primer (5'-TTA AAC GAT ATC GCT AGC GCG GCC GCT CAA CCC GGG GAG AGG CTC A-3') encoding the carboxyl terminus of the heavy chain constant region, a stop codon, and a NotI site, resulting in an approximately 1 kb product. PCR reactions 31 and 36 were purified using Qiagen columns. 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 C-150228:pSLX240n:VK1O2O12::[huanti <hutrailr2>AMG 655 VH]::IgG1z(N297G,DD).

[0242] [SEQ ID NO:34]4-[SEQ ID NO:1]4 were constructed as follows. PCR 31 was utilized as described above. PCR 37: Using C144132 as template, the 5' primer (5'-GGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCA-3') was combined with the 3' primer (5'-TTA AAC GAT ATC GCT AGC GCG GCC GCC TAG TGG TGA TGG TGA TGG TGA CC-3') encoding 6xHis, a stop codon, and a NotI site to generate an approximately 740 base pair product. PCR reactions 31 and 37 were purified on Qiagen columns. 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 C-150237:pSLX240p:VK1O2O12::[huanti <hutrailr2>AMG655(S183E)scFab]::G2::TTR(C10A,K15A)::G2::6xHis (note that the (His)6 tag was included for purification purposes).

[0243] SEQ ID NO:39 was constructed as follows. PCR33 was utilized as described above. PCR38: Using C-143049 as template, the 5' primer (6186-65) was combined with the 3' primer (5'-TGT TTA AAC GAT ATC GCT AGC GCG GCC GCT CAA CAC TCT CCC CTG TTG AA-3') encoding the carboxyl terminus of the kappa light chain constant region, a stop codon, and a NotI site to generate an approximately 330 base pair product. PCR reactions 33 and 38 were purified on Qiagen columns. 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 C-150238:pSLX240h:VK1O2O12::[huantigen] <hutrailr2>It was named AMG 655 VL]::huKLC-S176K.

[0244] Expression of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were generally expressed as follows.

[0245] TTR fusion proteins were stably expressed in suspension with CHO-K1 cells. Transfections were performed using Lipofectamine LTX (Invitrogen™) according to the manufacturer's protocol. A total of 30–36 μg of mammalian expression plasmid DNA was used at a 1:1 ratio (1HC or Fab-HC to 1LC) for the anti-TRAILR2 TTR antibody homodimer and TTR Fab homotetramer, or a 1:1:1 ratio (1HC+ to 1HC- to 1LC) for the TTR antibody homotetramer. For each, the plasmid DNA was added to 3–4 mL of OPTI-MEM (Gibco) and mixed. In a separate tube, 72–75 μL of Lipofectamine LTX was added to 3–4 mL of OPTI-MEM. The solution was incubated at room temperature for 5 minutes. To form the transfection complex, the DNA and Lipofectamine LTX mixtures were combined and incubated for an additional 20 minutes at room temperature. Log-phase CHO-K1 cells were pelleted by centrifugation (1200-1500 RPM for 5 minutes), washed once with 1X PBS (Gibco), and stored for 1.5-2 e in OPTI-MEM. 6 The cells were resuspended to 100% viable cells / mL. For each transfection, 5-6 mL of washed cells were added to a 125 mL shake flask. The DNA transfection complex was added to each cell. The flasks were incubated at 36°C, 5% CO2, and shaken at 150 RPM for 6 hours. To stop the transfection, 9-12 mL of growth medium was added to each flask and incubated for 48-72 hours.

[0246] To begin selection, 72 hours after transfection, cells were pelleted by centrifugation (1200-1500 RPM for 5 minutes) and the medium was replaced with 23-25 ​​mL of growth medium supplemented with antibiotics. The selection medium was changed 2-3 times per week, ensuring that the culture was not overgrown (<5-6 e) until cell viability and density had recovered. 6 vc / mL) and diluted the culture medium when necessary.

[0247] Large-scale production (2.3 L to 2.5 L) was carried out in shake flasks at 36°C. 6 On day 5, the conditioned medium was collected by centrifugation followed by filtration (0.45 μm).

[0248] As shown in Figure 11, the anti-TRAILR2 TTR antibody homodimer and TTR Fab homotetramer fusion proteins are well expressed and correctly assembled (see Figure 11a). Figure 11b shows that the expected fusion protein components are present upon heating and reduction. 5 μL of conditioned medium was loaded per lane and run on a 4-20% TG gel. As seen in Figure 11a, the anti-TRAILR2 TTR antibody homodimer and TTR Fab homotetramer migrate on SDS-PAGE as larger complexes than the standard antibody, as expected for a fully assembled complex. As seen in Figure 11b, the anti-TRAILR2 TTR antibody homodimer and TTR Fab homotetramer migrate on reducing SDS-PAGE as expected for a fusion molecule with a heavy chain (upper band) and a free light chain (lower band).

[0249] As shown in Figure 12, the anti-TRAILR2 TTR antibody homotetramer is well expressed (at approximately 150 mg / L) and assembles correctly in CHO-K1 cells (see Figure 12a, top band). Figure 12b) shows that the homotetramer complex disassembles into its expected components upon heating, and Figure 12c) shows that the homotetramer complex disassembles into its expected components upon heating and reduction. 2-5 μL of conditioned medium was loaded per lane and run on a 4-20% TG gel.

[0250] Figure 13 shows that the anti-TRAILR2 TTR Fab homotetramer, anti-TRAILR2 TTR antibody homodimer, and anti-TRAILR2 TTR antibody homotetramer are correctly assembled, and that upon heating and reduction, the molecules are resolved into the expected component chains (the upper band is the heavy chain, and the lowest band is the light chain). Figure 13 demonstrates that TTR constructs can be generated with a variety of antibodies.

[0251] Purification and characterization of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins Anti-TRAILR2 TTR antibody homodimer and TTR antibody homotetramer fusion proteins were generally purified as follows. The molecules were purified from cell culture medium using an AKTA purification device (GE Healthcare Life Sciences) liquid chromatography system equipped with two sequentially connected 5 mL ProA FF HiTrap (GE Healthcare Life Sciences) columns. The medium was loaded directly onto the ProA FF column, washed with 5 CV of DPBS (Life Technologies), and eluted with 8 CV of 50 mM acetic acid, pH 3.2. The ProA FF elution pool was titrated to pH 5.0 using 2 M Tris-HCl, pH 9.2, and then diluted with 9 volumes of sterile water. The adjusted ProA FF pool was dialyzed twice against 2 L of 20 mM HEPES, 150 mM NaCl, pH 7.0 using 10 kDa MWCO Slide-a-lyzers (Thermo Fisher Scientific). The dialyzed pool was purified on an 18 mL SP Sepharose High Performance (SP HP) (GE Healthcare Life Science) column, employing a 5 CV wash with 20 mM NaHPO, pH 7.0, and eluting with a 20 CV NaCl gradient from 0 mM to 500 mM. Fractions were selected for pooling based on SDS-PAGE and HPLC-SEC purity. The SP HP pool was concentrated using a VivaSpin 10 kDa MWCO centrifugal filtration unit (Sartorius, Göttingen, Germany) and then purified isocratically on a 320 mL Superdex 200 (GE Healthcare Life Science) with 1.4 CV of 20 mM HEPES, 300 mM NaCl, pH 7.0. Fractions were selected for pooling based on SDS-PAGE and HPLC-SEC purity. The SP HP pool was dialyzed twice against 2 L of 10 mM MES, 150 mM NaCl, pH 7.0 using 10 kDa MWCO Slide-a-lyzers (Thermo Fisher Scientific).The dialyzed sample was concentrated using a VivaSpin 10 kDa MWCO centrifugal filtration unit (Sartorius) and then sterile filtered through a 0.2 μm Supor syringe filter (Pall).

[0252] The anti-TRAILR2 Fab homotetramer fusion protein was generally purified as follows. The molecule was purified from cell culture medium using an AKTA purification apparatus (GE Healthcare Life Sciences) liquid chromatography system equipped with a 50 mL Ni Sepharose excel (Ni excel) (GE Healthcare Life Sciences) column. The medium was loaded directly onto the HisTrap column, then washed with 10 CV of 20 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole, pH 7.4, and eluted with an 8 CV imidazole gradient from 10 mM to 500 mM. The Ni excel pool was dialyzed twice against 2 L of 20 mM HEPES, 150 mM NaCl, pH 7.0 using 10 kDa MWCO Slide-a-lyzers (Thermo Fisher Scientific). The dialyzed pool was purified on an 18 mL SP Sepharose High Performance (SP HP) (GE Healthcare Life Science) column, employing a 5 CV wash with 20 mM NaH2PO4, pH 7.0, and eluting with a 20 CV NaCl gradient from 0 mM to 500 mM. Fractions were selected for pooling by SDS-PAGE and HPLC-SEC purity. The SP HP pool was dialyzed twice against 2 L of 10 mM MES, 150 mM NaCl, pH 7.0 using a 10 kDa MWCO Slide-a-lyzer (Thermo Fisher Scientific). The dialyzed sample was concentrated using a VivaSpin 10 kDa MWCO centrifugal filtration unit (Sartorius) and then sterile filtered through a 0.2 μm Supor syringe filter (Pall). Protein concentration was measured by UV absorbance at 280 nM (A280) using a NanoDrop2000 (Thermo Fisher Scientific, Rockford, Illinois, USA). Samples were analyzed on denaturing, non-reducing 4–12% Bis-Tris NuPAGE gels using MES running buffer (Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions.Samples were analyzed on a Phenomenex SEC3000 column, 7.8 x 300 mm (Phenomenex, Torrance, California, USA) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 mL / min, and absorbance at 280 nm was monitored.

[0253] Figure 14 shows the results of HPLC size-exclusion chromatography (SEC) analysis of each of the anti-TRAILR2 TTR antibody homodimer (center chromatogram), anti-TRAILR2 TTR antibody homotetramer (right chromatogram), and anti-TRAILR2 TTR Fab homotetramer (left chromatogram) fusion proteins. The SEC chromatograms show that the main peak elutes at the expected position, consistent with a correctly assembled molecule.

[0254] Reduced LC / MS analysis of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins was performed. Samples were buffer exchanged into 200 mM ammonium acetate and made to 5 μM as the final MS working solution for native MS studies. Samples were also denatured with 8 M guanidine HCl and reduced with 20 mM DDT for LC-MS analysis. 2 μg of material was then injected into the LC-MS system.

[0255] Native MS and ion mobility analyses of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were performed in a radio frequency confinement drift tube using a modified Synapt G1 HDMS instrument operated in positive nanoflow ESI mode. Key instrument voltages and pressures were as follows: capillary voltage 0.8-1.0 kV; sample cone 40 V, extraction cone 1 V; source block temperature 30 °C; trap collision energy 4.0 V; transfer collision energy 60-200 V, corresponding to 3.3 V-11.1 V / cm applied across the RF confinement drift cell; trap inlet 3.0 V; trap bias 16 V (N2; increased potential required to inject ions into the drift cell when operated with N2); IMS DC inlet 5.0 V; IMS DC outlet 0.0 V; transfer DC inlet 0.0 V; transfer DC outlet 2.0 V; travel wave velocity 70 m / sec; travel wave amplitude 4.0 V; mobility trapping release time 250 μsec; trap height 20.0 V; extraction height 0.0 V; source RF amplitude (maximum amplitude) 450 V; Triwave RF amplitude (maximum amplitude), trap 380 V, IMS 150V, transfer 380V; source backing pressure 6.0mbar; trap / transfer pressure SF6, 3.3e -2 mbar (Pirani gauge reading: flow rate 3.0 mL / min); IMS pressure N2 2.05 mbar (1.54 Torr; flow rate 38 mL / min); IMS pressure He 2.70 mbar (2.03 Torr; flow rate 70 mL / min). An MKS Baratron capacitance manometer, type 626 (range 10 Torr; accurate to 0.25%) and an MKS PDR2000 power supply were used to accurately measure the pressure in the RF confinement drift cell. Ambient temperature was measured using an Oakton Temp10T thermocouple. Temperature was measured at the point where the capacitance manometer was connected to the ion optics lid. Instrument control and data acquisition were performed with MassLynx 4.1 SCN 639, SCN 744. Therefore, mobility and Ω values ​​were generated by precisely measuring the pressure within the drift cell device and the ambient temperature of the drift cell, and performing up to 10 mobility measurements at different drift cell voltages (60 V to 200 V). Individual temperature and pressure measurements were performed for each collection and for the final Ω value calculation, and the average temperature and pressure values ​​were used. Typical temperature and pressure changes over a 10-minute period were ≤0.2 °C and ≤0.002 Torr.

[0256] Denaturing LC-MS analysis of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins was performed by directly injecting 2 μg of denatured and reduced protein onto a C4 BEH 2.1 × 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 and extraction cones were set at 35 V and 1 V, respectively.

[0257] Mass spectrometry and ion mobility results: Anti-TRAILR2 TTR antibody homotetramer: native-MS measured MW was 645.9 kDa, ion mobility measured N2-CCS value was 190.1 nm2, and the denatured and reduced MW of the subunits were 23,388 Da, 49,180 Da, and 62,872 Da. Anti-TRAILR2 TTR Fab homotetramer fusion protein: native-MS measured MW was 250.5 kDa, ion mobility measured N2-CCS value was 130.3 nm2, and the denatured and reduced MW of the subunits were 23,429 Da and 38,582 Da. The MW of the anti-TRAILR2 TTR antibody homodimer fusion protein measured by native-MS was 347.2 kDa, the N2-CCS value measured by ion mobility measurement was 124.0 nm2, and the denatured and reduced MW of the subunits were 23,391 Da and 62,867 Da.

[0258] SEC-multiangle light scattering (MALS) analysis of the anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins was performed on an Agilent 1100 HPLC equipped with a Wyatt Heleos II and OptiLab-TrEX detector. The column used was a Superdex200 (10 / 300GL). The mobile phase was 2x PBS with a flow rate of 0.4 mL / min. The analysis time was 70 min per sample. The injection amounts for the four SP fractions (A3, A8, A12, and C1) of the anti-TRAILR2 TTR antibody homodimer and TTR antibody homotetramer fusion proteins were 81, 63, 75, and 75 μg, respectively, and the injection amount for the final anti-TRAILR2-TTR pool was 77 μg. Run setup, data collection, and analysis procedures were performed with Agilent's Chemstation (v B.04.02 96) and Wyatt's ASTRA (v6.1.1.17) software.

[0259] Example 7: Activity and PK profiles of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins The activity and PK profiles of the parent anti-TRAILR2 mAb (conatumumab), anti-TRAILR2 TTR antibody homodimer, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer fusion proteins were evaluated.

[0260] PK Profile The PK profiles of anti-TRAILR2 TTR antibody homodimer, TTR antibody homotetramer, and TTR Fab homotetramer fusion proteins were measured by intravenous injection of 6.5 mg / kg TTR antibody homotetramer, 3.5 mg / kg TTR antibody homodimer, 2.5 mg / kg TTR Fab homotetramer, and 1.5 mg / kg conatumumab and conatumumab-341-G1 (conatumumab-341-G1 is an Ab designed not to bind TRAILR2) antibodies into male CD-1 mice (n=3 per group) at molar equivalent doses. Serum samples were collected from 75 μL blood samples collected 0.5, 2, 8, 24, 48, 72, 96, 192, 336, 504, 672, and 840 hours after administration. After collection, each blood sample was maintained at room temperature, and after a 30-40 minute clotting time, the sample was centrifuged at 11,500 rpm for approximately 10 minutes at 2-8°C using a calibrated Eppendorf 5417R centrifuge system (Brinkmann Instruments, Inc., Westbury, NY). The collected serum was then transferred to pre-labeled cryogenic tubes (for each rat) and stored at -60°C to -80°C for later bioanalysis.

[0261] The following PK assay was used to measure total anti-TRAILR2 species (i.e., any anti-TRAILR2-binding species present) of conatumumab, anti-TRAILR2 TTR antibody homodimer, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer fusion proteins in mouse serum using a Meso Scale Discovery (MSD) assay. To measure the total amount of conatumumab in mouse serum samples, a standard binding 96-well MSD plate (Meso Scale Discovery, Gaithersburg, MD) was coated with 2 μg / mL rabbit anti-conatumumab polyclonal antibody (Amgen Inc., Thousand Oaks, CA) in PBS and then incubated overnight at 4°C. The plate was then washed and blocked with I-Block™ (Life Technologies, Carlsbad, CA) overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted with naive CD-1 mouse serum when necessary. The standards, QCs, and samples were then diluted 1:20 with a buffer containing PBS, 1 M NaCl, 0.5% Tween 20, and 1% bovine serum albumin. The plate was washed three times with approximately 200 μL of 1X KPL buffer (KPL, Gaithersburg, MD). Subsequently, 50 μL of diluted standards, QCs, and samples were transferred to the anti-conatumumab antibody-coated plate and incubated at room temperature (approximately 25°C) for 1.5 hours. The plate was washed three times with approximately 200 μL of 1×KPL wash buffer, and then 50 μL of 100 ng / mL mouse anti-hu Fc antibody, clone 1.35.1, conjugated to MSD SULFO-TAG (Amgen Inc., Thousand Oaks, CA) in 5% BSA with I-Block™ was added and incubated at room temperature for 1.5 hours.For the anti-TRAILR2 TTR Fab homotetramer construct, 50 μL of 250 ng / mL mouse anti-κLC, clone KCF-9, conjugated to biotin was added and incubated for 1.5 hours; the plate was then washed with 1×KPL wash buffer, followed by the addition of 50 μL of 100 ng / mL streptavidin conjugated to MSD SULFO-TAG (Amgen, Inc.) and incubation for 15 minutes. The plate was washed six times with approximately 200 μL of 1×KPL wash buffer, followed by the addition of 150 μL 1×Read Buffer T (Meso Scale Discovery), and the electrochemiluminescence signal was measured using an MSD6000 plate reader (Meso Scale Discovery). Serum concentration data were analyzed using non-compartmental methods in Phoenix® (Phoenix 64, Build 6.4.0.768, Pharsight® Corp., Mountain View, Calif.).

[0262] The following PK assay was used to measure the presence of both anti-TRAILR2 TTR-binding and TTR species of anti-TRAILR2 TTR antibody homodimer, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer fusion proteins in mouse serum by MSD assay of mouse serum samples. A regular binding 96-well MSD plate (Meso Scale Discovery, Gaithersburg, MD) was 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 plate was then washed and blocked with I-Block™ (Life Technologies, Carlsbad, CA) overnight at 4°C. Standards and quality controls (QCs) were prepared in mouse serum, and PK samples were diluted in naive CD-1 mouse serum when dilutions were required. The standards, QCs, and samples were then diluted 1:20 with a buffer containing PBS, 1 M NaCl, 0.5% Tween 20, and 1% bovine serum albumin buffer. The plate was washed three times with approximately 200 μL of 1× KPL buffer (KPL, Gaithersburg, MD), 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 three times with approximately 200 μL of 1× KPL wash buffer, and then 50 μL of 250 ng / mL 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, 50 μL of 100 ng / mL streptavidin conjugated to MSD SULFO-TAG (Amgen, Inc.) was added and incubated for 15 minutes. The plate was washed six times with approximately 200 μL of 1X KPL wash buffer, followed by the addition of 150 μL of 1X Read Buffer T (Meso Scale Discovery), and the electrochemiluminescence signal was measured using an MSD6000 plate reader (Meso Scale Discovery).Serum concentration data were analyzed using non-compartmental methods in Phoenix® (Phoenix 64, Build 6.4.0.768, Pharsight® Corp., Mountain View, Calif.).

[0263] The results of the PK analysis can be found in Figure 15. As shown by Figure 15a) (measurement of any anti-TRAILR2 binding species): [1] the PK of the anti-TRAILR2 TTR antibody homodimer ("1") is better than that of the parental Abs (conatumumab ("3"); and conatumumab-341-G1 ("2")); [2] the PK of the anti-TRAILR2 TTR Fab homotetramer ("5") is poor, likely due to the fact that the Fab lacks an Fc region and therefore the ability to mediate / extend its half-life; and [3] the PK of the anti-TRAILR2 TTR antibody homotetramer ("4") shows similar PK to the parental mAb. Notably, the anti-TRAILR2 TTR antibody homotetramer exhibited better PK compared to its parent (conatumumab), which differs from that observed for the anti-GITR TTR antibody homotetramer (which, while not as potent as the parent mAb, showed significantly enhanced PK compared to the Fab). This observation suggests that the PK of TTR antibody homotetramer fusion proteins may be antibody- and / or target-dependent. Figure 15b) (measurement of the presence of both anti-TRAILR2 TTR-binding and TTR species) shows that the PK of intact anti-TRAILR2 TTR fusion proteins is consistent with that observed in Figure 15a), indicating that the anti-TRAILR2-binding moiety is not liberated from each TTR fusion protein by in vivo proteolysis. The anti-TRAILR2 TTR antibody homodimer ("1"), anti-TRAILR2 TTR antibody homotetramer ("2"), and anti-TRAILR2 TTR Fab homotetramer ("3") are evaluated in Figure 15b).

[0264] Cell-based activity assays The activity of anti-TRAILR2 TTR fusion protein versus conatumumab + Protein G on the melanoma cell line WM35 (ATCC) was evaluated. WM35 was assessed to express DR5 but not DR4. TRAIL activates both DR5 and DR4. 10 cells per well of a microtiter plate were used. 4 WM35 cells were plated at 1000 x g of TRAILR2 TTR antibody homodimer, anti-TRAILR2 TTR antibody homotetramer, or anti-TRAILR2 TTR Fab homotetramer fusion protein in the absence or presence of 1 μg / mL Protein G (Pierce) to facilitate cross-linking. Triplicate samples were analyzed. Incubation was continued at 37°C, 5% CO. 2 After 24 hours of incubation with HCl, cell viability was assessed using the Cell Titer-glo assay (Promega). Cell Titer-glo reactions were performed according to the manufacturer's instructions, and luminescence was measured using a Perkin Elmer Envision. The y-axis shows the luminescence signal as relative luminescence units (RLU); a decrease in RLU reflects a decrease in ATP production and a decrease in viable cell number. The x-axis shows protein concentration.

[0265] Figure 16 shows the results of the WM35 assay. Conatumumab ("1") is ineffective at killing cells unless clustered with Protein G (which has two Ig binding sites; "5"). The anti-TRAILR2 TTR antibody homodimer ("2") is slightly better at killing WM35 cells than parent conatumumab. The anti-TRAILR2 TTR Fab homotetramer ("4") has slightly better WM35 cell killing ability than conatumumab. However, the anti-TRAILR2 TTR antibody homotetramer ("3") is much more potent than conatumumab and even more potent than the conatumumab / Protein G clustered complex in killing WM35 cells. In addition, clustering of anti-TRAILR2 TTR antibody homodimers with Protein G ("6") improved the potency of the fusion protein, whereas clustering of anti-TRAILR2 TTR Fab homotetramers with Protein G ("8") had little effect. Interestingly, clustering of anti-TRAILR2 TTR antibody homotetramers with Protein G ("7") slightly impaired the potency of the protein fusion. These results indicate that TTR antibody homotetramer-mediated clustering can improve anti-tumor activity.

[0266] The activity of anti-TRAILR2 TTR fusions versus conatumumab + Protein G on primary human keratinocytes (Lonza) was also assessed. 4 Primary keratinocytes were plated at 10 cells per well of a microtiter plate and then incubated with TRAIL alone, conatumumab containing 1 μg / mL Protein G (Pierce) to promote cross-linking, or TRAIL plus 1 μg / mL conatumumab. Samples were prepared in triplicate for each condition. After 24 hours of incubation at 37°C and 5% CO2, cell viability was assessed using the Cell Titer-glo assay (Promega). Additionally, 10 cells per well of a microtiter plate were plated. 4 Primary keratinocytes were plated at 0.05% cells and then incubated with anti-TRAILR2 TTR antibody homotetramer, anti-TRAILR2 TTR Fab homotetramer, conatumumab, or TRAIL at the indicated concentrations. TRAIL in combination with 1 μg / mL conatumumab was also tested. Samples were prepared in triplicate. After 24 hours of incubation at 37°C and 5% CO2, cell viability was measured using the Cell Titer-glo assay (Promega).

[0267] Figure 17 shows the results of a keratinocyte assay. Conatumumab ("1") was ineffective at killing primary human keratinocyte cells. The anti-TRAILR2 TTR Fab homotetramer ("2") showed slightly greater efficacy than conatumumab. The anti-TRAILR2 TTR antibody homotetramer ("3") was significantly more potent than conatumumab at killing primary human keratinocyte cells. These results also indicate that TTR antibody homotetramer-mediated clustering can improve activity in in vitro cell killing assays.

[0268] Activity assay in murine colo205 adenocarcinoma model system The activity of conatumumab, anti-TRAILR2 TTR antibody homodimer, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer fusion proteins was evaluated in a murine Colo205 human colon adenocarcinoma cancer model. Colo205 human colon adenocarcinoma cells were maintained at 37°C and 5% CO in RPMI-1640 culture 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 passage 3, cells were harvested and resuspended in serum-free medium to a concentration of 10 × 10 6 A final concentration of 100 cells / mL was obtained. Cell viability was determined to be 98% by trypan blue exclusion.

[0269] 1 × 10 into the right flank of 60 female NU / NU nude mice. 6 Cells (volume 100 μL) were injected subcutaneously and the mice were anesthetized with isofluorane. On day 7 after tumor implantation, the tumor volume was 34 mm 3 ~99mm 3 50 mice were placed in groups of 40 mm 3 ~48mm 3 The mice were divided into five treatment groups of 10 each to achieve similar mean tumor volumes in the range of 10. Ten mice with small or large tumors were excluded from the study.

[0270] Five groups of animals were intraperitoneally administered twice weekly (biw) with 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) for a total of six treatments over three weeks. Treatments were normalized so that all had the same number of binding sites as conatumumab, except for the anti-TRAILR2 TTR Fab homotetramer, which had only half the binding sites compared to the remaining treatments. All treatments began on day 8 and ended on day 25 after tumor implantation (treatments were on days 8, 11, 15, 19, 22, and 25). All treatments were freshly prepared in diluent (DPBS) with treatment immediately prior to injection.

[0271] Tumor volume measurement: The length and width of the tumor were measured with an ABS Digimatic solar 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 in mm 3 It was expressed as:

[0272] Weight Measurement: For weight measurement, animals were placed on the pan of a weighing scale (Mettler Toledo model PB602-S, Switzerland). The average weight over a 3-second period was determined using the dynamic weight function of the weighing scale. Weights were reported as weight change, calculated as 100 × (Wc / Wc), where Wc is the current weight and Wc is the weight at the start of treatment. In this study, the weight on day 4 was used as Wc for all animals.

[0273] Euthanasia: Tumor size 1500–2000 mm 3 Five animals were euthanized before the end of the study due to tumor volume reaching 1200 mm. Conatumumab-341-G1 group: Two mice were euthanized due to excessive tumor volume on day 25. Two additional mice were euthanized due to excessive tumor volume on day 35. Ten mice in the anti-TRAILR2 TTR antibody homodimer group and the remaining six mice in the conatumumab-341-G1 group had a mean tumor volume >1200 mm. 3 or loss of more than half of the mice in the cohort was euthanized on day 42. One mouse from the anti-TRAILR2 TTR Fab homotetramer group was euthanized on day 42 due to tumor size >2000 mm. 3 One mouse from the anti-TRAILR2 TTR Fab homotetramer group was euthanized on day 49 due to excessive tumor volume. All remaining animals were euthanized on day 63 at the end of the study. Animals were euthanized with an overdose of isofluorane, followed by blood collection by cardiac puncture.

[0274] Tumor measurements: Data from days 4 to 63 are expressed as mean ± standard error and plotted as a function of time. The statistical significance of observed differences between growth curves was assessed by repeated measures analysis of variance for transformed tumor volume data using Dunnett's adjustment for multiple comparisons. Analyses were performed using the SAS PROC MIXED procedure with model effects for transformed baseline tumor volume, day, treatment, and treatment-by-day interaction: a REPEATED statement where day was a repeated value, animal-subject, and a Toeplitz covariance structure; and an LSMEANS statement to perform Dunnett analyses comparing control groups with other treatment groups. Data were log- or square-root transformed according to the Horwitz method, and transformed baseline tumor volumes were included as model covariates to account for potential differences in pretreatment tumor volume. When log or square-root transformations failed to achieve an appropriate residual distribution, nonparametric repeated measures analysis of variance was used with the same model effect on tumor volume rank. A P value of less than 0.05 was considered statistically significant. The conatumumab (wild-type) and conatumumab-341-G1 groups were used as control groups in the analysis.

[0275] Figure 18 shows the results of a murine colo205 model study. 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 in vivo tumor growth during treatment, with the anti-TRAILR2 TTR antibody homotetramer demonstrating the highest level of inhibition during treatment. The anti-TRAILR2 TTR antibody homodimer ("Group 3"; 0.34 μM / animal) was slightly superior to 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 treatment was discontinued. Additionally, surprisingly, the anti-TRAILR2 TTR Fab homotetramer has a poor PK profile (Figure 15), yet this construct is as effective as conatumumab in inhibiting tumor growth. Without being bound by any particular theory, it is possible that initial exposure to the anti-TRAILR2 TTR Fab homotetramer triggers a cascade of events in target cells that leads to death and does not require long-term binding. Additionally, the relatively small size of the Fab construct may improve access to the tumor environment, compensating for poor PK. Finally, the physical configuration of the Fab construct may be more effective in vivo.

[0276] Murine SW403 adenocarcinoma model system activity assay SW403 human colon adenocarcinoma cells were maintained in RPMI-1640 culture medium (Sigma R0883) supplemented with 10% FBS (Sigma, 2442-500 mL), 4 mM L-glutamine (Hyclone, SH30034.01) at 37°C and 5% CO. At passage 5, cells were harvested and resuspended in serum-free medium to give 50 × 10 6 A final concentration of 100 cells / mL was obtained. Cell viability was determined to be 95% by trypan blue exclusion.

[0277] 5 × 10 into the right flank of 75 female NU / NU nude mice. 6 Cells (100 μL volume) were injected subcutaneously and the mice were anesthetized with isofluorane. On day 7 after tumor implantation, the tumor volume was 21 mm 3 ~160mm 3 Sixty mice were placed in groups of 58 mm 3 ~66mm 3 The mice were divided into six treatment groups of 10 each to achieve similar mean tumor volumes in the range of 1. Fifteen mice with small or large tumors were excluded from the study.

[0278] Six groups of animals were administered twice weekly (biw) intraperitoneally with conatumumab wild-type (1.38 μM / animal), conatumumab-341-G1 (1.38 μM / animal), anti-TRAILR2 TTR antibody homodimer (0.69 μM / animal), anti-TRAILR2 TTR antibody homotetramer (0.34 μM / animal), anti-TRAILR2 TTR Fab homotetramer (0.34 μM / animal), and DPBS (vehicle control) for a total of six treatments over three weeks. Treatments were normalized so that each had an equal number of binding sites as conatumumab wild-type, except for the anti-TRAILR2 TTR Fab homotetramer, which had only half the binding sites compared to the remaining treatments. All treatments began on day 15 after tumor implantation and ended on day 32 (treatments were on days 15, 19, 22, 26, 29, and 32). All treatments were freshly prepared in diluent (DPBS) with treatments immediately prior to injection.

[0279] Tumor volume measurement: The length and width of the tumor were measured with an ABS digimatic solar caliper, model #Cd-S6"C (Mitutoyo Corporation, Japan). Tumor volume was calculated as 0.5 × L × W, where W is the smaller of the two measurements in mm. 3 It was expressed as:

[0280] Weight measurement: Animals were placed on the pan of a weighing scale (Mettler Toledo model PB602-S, Switzerland). The average weight over a 3-second period was determined using the dynamic weight function of the weighing scale. Weights were reported as weight change, calculated as 100 × (Wc / Wi), where Wc is the current weight and Wi is the weight at the start of treatment. In this study, the weight on day 4 was used as Wi for all animals.

[0281] Euthanasia and tissue collection: Tumor size 1500-2000 mm 3 Eight animals were euthanized before the end of the study because tumor volume reached 100%. One mouse from the conatumumab-341-G1 group was euthanized on day 33 due to excessive tumor volume. 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 on day 36. 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. All remaining animals in the conatumumab WT, anti-TRAILR2 TTR antibody homotetramer, and anti-TRAILR2 TTR Fab homotetramer groups were euthanized at the end of the study on day 49. All were euthanized with an overdose of isofluorane, followed by blood collection by cardiac puncture.

[0282] Tumor measurements: Data from days 7 to 49 are expressed as mean ± standard error and plotted as a function of time. The statistical significance of observed differences between growth curves was assessed by repeated measures analysis of variance for transformed tumor volume data using Dunnett's adjustment for multiple comparisons. Analyses were performed using the SAS PROC MIXED procedure with model effects for transformed baseline tumor volume, day, treatment, and treatment-by-day interaction: a REPEATED statement where day was a repeated value, animal-subject, and a Toeplitz covariance structure; and an LSMEANS statement to perform Dunnett analyses comparing control groups with other treatment groups. Data were log- or square-root transformed according to the Horwitz method, and transformed baseline tumor volumes were included as model covariates to account for potential differences in pretreatment tumor volume. When log or square-root transformations failed to achieve an appropriate residual distribution, nonparametric repeated measures analysis of variance was used with the same model effect on tumor volume rank. A P value of less than 0.05 was considered statistically significant. All statistical calculations were performed using the AMG biostatistical analysis tool (cld-pweb-taivd.amgen.com / biostats / Statistics). The conatumumab WT group was used as the control group in the analysis.

[0283] Figure 19 shows the results of a murine SW403 model study. The anti-TRAILR2 TTR antibody homotetramer ("Group 5") and anti-TRAILR2 TTR Fab homotetramer ("Group 6") suppress in vivo tumor growth during administration more effectively than conatumumab ("Group 1"). The anti-TRAILR2 TTR antibody homodimer ("Group 4") suppressed growth during administration at approximately the same level as conatumumab, but was not significantly superior to the vehicle control ("Group 3") in growth suppression. Notably, both the anti-TRAILR2 TTR antibody homotetramer and the anti-TRAILR2 TTR Fab homotetramer slowed tumor growth even after administration was stopped. Furthermore, although the anti-TRAILR2 TTR Fab homotetramer has a poorer PK profile (Figure 15), it was surprising to observe that the construct was as effective as the anti-TRAILR2 TTR antibody homotetramer in slowing tumor growth, even after administration was stopped.

[0284] Figure 20 shows that the weight gain of the murine colo205 and SW403 model mice was similar for all compounds tested and appeared to be on a normal upward trajectory, indicating that the compounds tested were not overtly toxic (as the mice were healthy enough to eat normally and gain weight).

[0285] [Table 10]

[0286] [Table 11] < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr> < / hugitr>

Claims

1. A homodimeric fusion protein comprising two antigen-binding proteins, wherein said antigen-binding proteins are linked into a protein complex.

2. 2. The homodimeric fusion protein of claim 1, wherein the protein complex is a TTR protein complex.

3. The homodimeric fusion protein of claim 1 or 2, wherein the antigen-binding protein is an antibody.

4. The homodimeric fusion protein of any one of claims 1 to 3, wherein the antigen-binding protein is fused directly to the protein complex without a linker.

5. 5. The homodimeric fusion protein of claim 4, wherein the C-terminus of the antigen binding protein is fused directly to the N-terminus present in the FAP protein complex.

6. The homodimeric fusion protein of any one of claims 1 to 3, wherein the antigen-binding protein is fused to the protein complex via a linker.

7. 7. The homodimeric fusion protein of claim 6, wherein the C-terminus of the antigen binding protein is linked to the N-terminus present in the FAP protein complex.

8. The homodimeric fusion protein of claim 7 , wherein the linker is an amino acid linker.

9. 9. The amino acid linker of claim 8, which is 1 to 40 amino acids in length.

10. GGGGS, (GGGGS) ２ , (GGGGS) ３ , (GGGGS) ４ , (GGGGS) ５ , or (GGGGS) ６ 10. The amino acid linker of claim 9, wherein:

11. A homotetrameric fusion protein comprising four antigen-binding proteins, wherein said antigen-binding proteins are linked into a protein complex.

12. 12. The homotetrameric fusion protein of claim 11, wherein the protein complex is a TTR protein complex.

13. 13. The homotetrameric fusion protein of claim 11 or 12, wherein the antigen-binding protein is an antibody.

14. 13. The homotetrameric fusion protein of claim 11 or 12, wherein the antigen-binding protein is a Fab.

15. The homotetrameric fusion protein of any one of claims 11 to 14, wherein the antigen-binding protein is fused directly to the protein complex without a linker.

16. 16. The homotetrameric fusion protein of claim 15, wherein the C-terminus of the antigen binding protein is fused directly to the N-terminus present in the FAP protein complex.

17. The homotetrameric fusion protein of any one of claims 11 to 14, wherein the antigen-binding protein is fused to the protein complex via a linker.

18. 18. The homotetrameric fusion protein of claim 17, wherein the C-terminus of the antigen binding protein is linked to the N-terminus present in the FAP protein complex.

19. 19. The homotetrameric fusion protein of claim 18, wherein the linker is an amino acid linker.

20. 20. The amino acid linker of claim 19, which is 1 to 40 amino acids in length.

21. The amino acid linker is GGGGS, (GGGGS) ２ , (GGGGS) ３ , (GGGGS) ４ , (GGGGS) ５ , or (GGGGS) ６ 21. The amino acid linker of claim 20, wherein:

22. A pharmaceutical composition comprising the homodimeric fusion protein of any one of claims 1 to 10.

23. A pharmaceutical composition comprising the homotetrameric fusion protein of any one of claims 11 to 22.

24. A method for treating cancer using the homodimeric fusion protein according to any one of claims 1 to 10.

25. A method for treating cancer using the homotetrameric fusion protein according to any one of claims 11 to 22.

26. 11. Use of the homodimeric fusion protein of any one of claims 1 to 10 in the treatment of cancer.

27. Use of the homotetrameric fusion protein of any one of claims 11 to 22 in the treatment of cancer.

28. 11. The homodimeric fusion protein of any one of claims 1 to 10 for use in the treatment of cancer.

29. The homotetrameric fusion protein of any one of claims 11 to 22 for use in the treatment of cancer.

30. One or more isolated nucleic acids encoding the homodimeric fusion protein of any one of claims 1 to 10.

31. An expression vector comprising the nucleic acid of claim 30.

32. 32. A recombinant host cell comprising the nucleic acid of claim 30 or the vector of claim 31.

33. 33. The recombinant host cell of claim 32, 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 (HEK293) cell.

34. A method for producing a homodimeric fusion protein according to any one of claims 1 to 10 or a homotetrameric fusion protein according to any one of claims 11 to 22, said method comprising: a) culturing a recombinant host cell according to claim 32 or 33; b) isolating the homodimeric or homotetrameric fusion protein from said culture; A method comprising:

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