Formulations that specifically bind cd137 and uses thereof

Abstract

The present application provides a preparation comprising an antibody or antigen-binding fragment thereof that specifically binds CD137, wherein the CD137 antibody comprises a light chain variable region VL and a heavy chain variable region VH, wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-11; the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12-22. The present application also provides a kit comprising the preparation, and use of the preparation or the kit in the preparation of a medicament for treating cancer.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a formulation that specifically binds to CD137 and its uses. Background Technology

[0002] Cancer is a disease that seriously threatens human health. In recent years, immunotherapy has shown great potential as a new treatment for cancer.

[0003] CD137 protein (also known as 4-1BB, TNFRSF9, etc.) is a member of the tumor necrosis factor receptor superfamily and belongs to type I transmembrane proteins. CD137L is a member of the tumor necrosis factor receptor superfamily and belongs to type II transmembrane proteins. Current research results indicate that CD137L is mainly expressed on activated APCs, such as dendritic cells (DCs), macrophages, and B cells (Pollok, KE, et al., 1994, Eur. J. Immunol. 24: 367-374); and that T cells can induce CD137 expression after receiving antigen-specific signals (Kwon, BS, et al., 1989, PNAS 86: 1963-67). The function of CD137 on T cells has been well demonstrated. In the presence of a certain amount of CD3 antibody, activation of CD137 signaling can induce T cell proliferation and cytokine (mainly IFN-γ) synthesis, and inhibit apoptosis of activated T cells, thereby prolonging the lifespan of T cells.

[0004] However, the anti-CD137 antibodies currently developed have low stability and limited ability to inhibit tumors. Therefore, there is an urgent need to develop new anti-CD137 drugs for new drug development. Summary of the Invention

[0005] On one hand, this application provides a formulation comprising an antibody or antigen-binding fragment thereof that specifically binds to CD137, wherein the CD137 antibody comprises a light chain variable region VL and a heavy chain variable region VH, wherein the VL comprises an amino acid sequence shown in any one of SEQ ID NO: 1-11; and the VH comprises an amino acid sequence shown in any one of SEQ ID NO: 12-22.

[0006] In some embodiments, in the formulation,

[0007] 1) The VL contains the amino acid sequence shown in SEQ ID NO: 1, and the VH contains the amino acid sequence shown in SEQ ID NO: 12;

[0008] 2) The VL contains the amino acid sequence shown in SEQ ID NO: 3, and the VH contains the amino acid sequence shown in SEQ ID NO: 14;

[0009] 3) The VL contains the amino acid sequence shown in SEQ ID NO: 5, and the VH contains the amino acid sequence shown in SEQ ID NO: 16;

[0010] 4) The VL contains the amino acid sequence shown in SEQ ID NO: 7, and the VH contains the amino acid sequence shown in SEQ ID NO: 18;

[0011] 5) The VL contains the amino acid sequence shown in SEQ ID NO: 8, and the VH contains the amino acid sequence shown in SEQ ID NO: 19;

[0012] 6) The VL contains the amino acid sequence shown in SEQ ID NO: 9, and the VH contains the amino acid sequence shown in SEQ ID NO: 20;

[0013] 7) The VL contains the amino acid sequence shown in SEQ ID NO: 10, and the VH contains the amino acid sequence shown in SEQ ID NO: 21;

[0014] 8) The VL contains the amino acid sequence shown in SEQ ID NO: 11, and the VH contains the amino acid sequence shown in SEQ ID NO: 22.

[0015] In some embodiments, the formulation comprises a buffer salt selected from one or more of the group consisting of phosphates, citrates, and acetates.

[0016] In some embodiments, the formulation comprises a buffer salt comprising disodium hydrogen phosphate, sodium dihydrogen phosphate, and / or sodium citrate.

[0017] In some embodiments, the formulation comprises a buffer salt at a concentration of 5-50 mM.

[0018] In some embodiments, the formulation comprises a stabilizer selected from one or more of the group consisting of sucrose, trehalose, mannitol, glycine, arginine hydrochloride, sodium citrate, histidine, sodium acetate, and sodium chloride.

[0019] In some embodiments, the formulation comprises a stabilizer, which comprises sucrose.

[0020] In some embodiments, the formulation comprises a stabilizer comprising sucrose and sodium citrate.

[0021] In some embodiments, the formulation comprises a stabilizer at a concentration of 1%-10% (wt).

[0022] In some embodiments, the formulation comprises a surfactant selected from one or more of the group consisting of polysorbate 20 and polysorbate 80.

[0023] In some embodiments, the formulation comprises a surfactant at a concentration of 0.01%-0.2% (wt).

[0024] In some embodiments, the pH of the formulation is 6.5-7.5.

[0025] In some embodiments, the formulation comprises:

[0026] Sucrose, polysorbate 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, and water.

[0027] On the other hand, this application provides a kit comprising the aforementioned formulation and a container holding the formulation.

[0028] On the other hand, this application provides the use of the said formulation or the said kit in the preparation of a medicament for the treatment of cancer.

[0029] In some embodiments, the cancer is selected from the group consisting of melanoma, prostate cancer, colorectal cancer, Merkel cell skin cancer, pancreatic cancer, non-Hodgkin lymphoma, squamous cell carcinoma, and breast cancer.

[0030] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0031] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0032] Figure 1 A-1B demonstrates that the formulation described in this application has a significant killing effect on tumors in vivo.

[0033] Figure 2 A-2C demonstrates that the formulation described in this application has a significant killing effect on tumors in vivo.

[0034] Figure 3 The results show that the formulation described in this application significantly promotes the secretion of cytokines by T cells. Detailed Implementation

[0035] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0036] The following further describes this application: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0037] In this application, the term "formulation" generally refers to a pharmaceutical composition that typically comprises an active ingredient and a pharmaceutically acceptable carrier or excipient. For example, in this application, the formulation may comprise an antibody that specifically binds to CD137 or an antigen-binding fragment thereof, wherein the CD137 antibody comprises a light chain variable region VL and a heavy chain variable region VH, wherein the VL comprises the amino acid sequence shown in any one of SEQ ID NO: 1-11; and the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 12-22. Furthermore, in some embodiments, the formulation of this application may also comprise at least one of a buffer salt, a stabilizer, and a surfactant.

[0038] In this application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule containing its antigen-binding portion. The term "antibody" includes monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies (e.g., dAb), single-chain antibodies (e.g., scFv), and antigen-binding antibody fragments (e.g., Fab, Fab', and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, and any antigen-binding antibody fragments and their derivatives described herein. Each heavy chain may consist of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may consist of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0039] In this application, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by the following fragments: (1) Fab fragment, i.e., a monovalent fragment consisting of VL, VH, CL and CH domains; (2) F(ab')2 fragment, a divalent fragment containing two Fab fragments connected by disulfide bonds at the hinge region; (3) Fd fragment consisting of VH and CH domains; (4) Fv fragment consisting of VL and VH domains of a single arm of the antibody; (5) dAb fragment consisting of VH domain (Ward et al., (1989) Nature 341: 544-546); (6) a separate complementarity-determining region (CDR) and (7) a combination of two or more separate CDRs optionally connected by a linker. In addition, it may include a monovalent single-chain molecule Fv (scFv) formed by pairing VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883). The “antigen-binding portion” may also include an immunoglobulin fusion protein containing a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The antibody or its antigen-binding fragment described in this application is selected from the group consisting of: Fab, scFv, Fab', F(ab)2, F(ab')2, and dAb.

[0040] In this application, the term "CD137 protein," also known as 4-1BB or TNFRS9, generally refers to a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), an activation-induced costimulatory molecule and an important regulator of the immune response. Studies have shown that CD137 agonist monoclonal antibodies increase the expression of costimulatory molecules in many models and significantly enhance cytolytic T lymphocyte responses, thus exerting antitumor effects. The antitumor effect of CD137-targeted therapy can be confirmed by in vivo antitumor efficacy studies in mice using agonist anti-mouse CD137 monoclonal antibodies. CD137 has become a powerful activator of immune cells and an important candidate antigen for the treatment of various diseases (see Vinay, Dass S., and Byoung S. Kwon. "4-1BB (CD137), an inducible costimulatory receptor, as a specific target for cancer therapy." BMB reports 47.3 (2014): 122.).

[0041] In this application, the term "antibody Fc domain" generally refers to the base region of the Y-shaped structure of an antibody, also known as the fragment crystallizable region (Fc region). In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody; the Fc regions of IgM and IgE contain three heavy chain constant domains in each polypeptide chain. The Fc region of IgG has a highly conserved N-glycosylation site. In this application, the Fc domain may contain the amino acid sequence shown in SEQ ID: 34.

[0042] In this application, the term "specific binding" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, in which the presence of the target is determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) binds to that target with greater affinity, strength, ease, and / or duration than it binds to other targets. In one embodiment, the degree to which the antibody binds to an irrelevant target is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA).

[0043] In this application, the term "kit" generally refers to a collection of various components, which may or may not be packaged together. For example, in this application, the kit may contain the stated formulation and a container holding the formulation. The components of the kit may be contained in separate vials (i.e., a kit with separate portions) or provided in a single vial. Additionally, the kit may include instructions, which may be provided in paper or electronic form as a user manual. For example, the manual may contain instructions for interpreting results obtained when using the kit of this application.

[0044] In this application, the term "cancer" generally refers to or describes a physiological condition in mammals characterized by disordered cell proliferation or survival. In this application, the term cancer refers to hyperproliferative diseases including, but not limited to, solid tumors, lymphoma, and leukemia.

[0045] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0046] In this application, the term "administration" generally refers to a method of giving a dose of a formulation or drug to a subject (e.g., a patient). Administerment can be performed in any suitable manner. This document covers various dosing schedules, including but not limited to single administration or multiple administrations at various time points.

[0047] In this application, the homology of amino acid or nucleotide sequences may be used interchangeably with the term "identity." The term "identity" generally refers to the percentage of identical residues in a pair. "Sequence identity percentage" is calculated as follows: two optimally aligned sequences are compared within a specific region; the number of positions in both sequences containing the same base or amino acid is determined to obtain the number of matching positions; this number of positions is divided by the total number of positions in the compared region, and the quotient is multiplied by 100. As described above, the procedure for determining homology (or identity) compares the aligned sequences on an amino acid-to-amino acid basis, and the procedure may set different levels of stringency for this comparison (e.g., identical amino acids, conserved amino acid substitutions, etc.). As used herein, two amino acids in discussion are considered to be "conserved substitutions" if they belong to the same chemical class (i.e., acidic, nonpolar / hydrophobic, uncharged polar, and basic). Non-limiting examples include two distinct amino acids belonging to the nonpolar amino acid family being considered a “conservative substitution” for each other, even if the two amino acids are not identical, whereas a nonpolar amino acid on one side and a basic amino acid on the other side are not considered a “conservative substitution” for each other. Section 3.1 of the 4th edition of *Molecular Biology of the Cell* (2002) by Alberts, Johnson, Lewis, Raff, Roberts, and Walter classifies amino acids into four main groups: acidic, nonpolar, uncharged polar, and basic. This grouping can be used in the context of this invention to determine whether a particular amino acid constitutes a conservative substitution for another amino acid under discussion. These main groups can be further subdivided, for example, into small and large nonpolar amino acids, large aromatic amino acids, etc. The term “conservative amino acid substitution” also refers to any substitution of a given amino acid residue where the substituted residue is so chemically similar to the given residue that there is no substantial reduction in polypeptide function (e.g., binding).

[0048] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0049] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0050] Formulations and kits

[0051] Antibody or its antigen-binding fragment

[0052] On one hand, this application provides a formulation comprising an antibody or antigen-binding fragment thereof that specifically binds to CD137, wherein the CD137 antibody comprises a light chain variable region VL and a heavy chain variable region VH, wherein the VL comprises an amino acid sequence shown in any one of SEQ ID NO: 1-11; and the VH comprises an amino acid sequence shown in any one of SEQ ID NO: 12-22.

[0053] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 1, and the VH may contain the amino acid sequence shown in SEQ ID NO: 12.

[0054] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 2, and the VH may contain the amino acid sequence shown in SEQ ID NO: 13.

[0055] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 3, and the VH may contain the amino acid sequence shown in SEQ ID NO: 14;

[0056] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 4, and the VH may contain the amino acid sequence shown in SEQ ID NO: 15.

[0057] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 5, and the VH may contain the amino acid sequence shown in SEQ ID NO: 16;

[0058] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 6, and the VH may contain the amino acid sequence shown in SEQ ID NO: 17.

[0059] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 7, and the VH may contain the amino acid sequence shown in SEQ ID NO: 18;

[0060] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 8, and the VH may contain the amino acid sequence shown in SEQ ID NO: 19;

[0061] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 9, and the VH may contain the amino acid sequence shown in SEQ ID NO: 20;

[0062] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 10, and the VH may contain the amino acid sequence shown in SEQ ID NO: 21;

[0063] In this application, in the antibody or antigen-binding fragment that specifically binds to CD137, the VL may contain the amino acid sequence shown in SEQ ID NO: 11, and the VH may contain the amino acid sequence shown in SEQ ID NO: 22.

[0064] In this application, the antibody that specifically binds to CD137 or its antigen-binding fragment may further include an Fc domain. For example, the antibody Fc region may comprise the Fc region of IgG. For example, the antibody Fc domain may comprise a constant region amino acid sequence selected from the following immunoglobulins: IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain may comprise an amino acid sequence as shown in SEQ ID NO: 34.

[0065] In this application, the Fc domain of the antibody that specifically binds to CD137 or its antigen-binding fragment may be located at the C-terminus of the antibody or its antigen-binding fragment.

[0066] In this application, the sequence of the Fc of the antibody that specifically binds to CD137 or its antigen-binding fragment may include the amino acid sequence shown in SEQ ID NO: 34 or a variant thereof.

[0067] In this application, the antibody that specifically binds to CD137 or its antigen-binding fragment may be a homodimeric protein composed of two polypeptide chains, wherein each polypeptide chain contains the light chain variable region VL, the heavy chain variable region VH, and the Fc domain, wherein the Fc domain is located at the C-terminus of the VL and the VH.

[0068] In this application, the antibody that specifically binds to CD137 or its antigen-binding fragment may further include a linker peptide 1 between the light chain variable region and the heavy chain variable region. The linker peptide 1 may include the amino acid sequence shown in SEQ ID NO: 35 or a variant thereof. For example, the linker peptide 1 may include (G4S)3.

[0069] In this application, the antibody that specifically binds to CD137 or its antigen-binding fragment may further include a linker peptide 2 between the light chain variable region and the Fc region. The linker peptide 2 may include the amino acid sequence shown in SEQ ID NO: 36 or a variant thereof. For example, the linker peptide 2 may include G4S.

[0070] In this application, the polypeptide chain in the antibody or antigen-binding fragment that specifically binds to CD137 may contain the amino acid sequence shown in any one of SEQ ID NO. 23-33.

[0071] For example, the antibody that specifically binds to CD137 or its antigen-binding fragment may include antibody C2-4A-Fc, C2-4B-Fc, C2-4C-Fc, C2-5A-Fc, C2-5B-Fc, C2-5C-Fc, C2-6A-Fc, C2-6B-Fc, C2-7A-Fc, C2-7B-Fc or C2-7BN78K-Fc.

[0072] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-4A-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0073] For example, the antibody described in this application can be C2-4A-Fc. The amino acid sequence of VL of antibody C2-4A-Fc is shown in SEQ ID NO: 1, the amino acid sequence of VH is shown in SEQ ID NO: 12, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 23.

[0074] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-4B-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0075] For example, the antibody described in this application can be C2-4B-Fc. The amino acid sequence of VL of antibody C2-4B-Fc is shown in SEQ ID NO: 2, the amino acid sequence of VH is shown in SEQ ID NO: 13, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 24.

[0076] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-4C-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0077] For example, the antibody described in this application can be C2-4C-Fc. The amino acid sequence of VL of antibody C2-4C-Fc is shown in SEQ ID NO: 3, the amino acid sequence of VH is shown in SEQ ID NO: 14, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 25.

[0078] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-5A-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0079] For example, the antibody described in this application can be C2-5A-Fc. The amino acid sequence of VL of antibody C2-5A-Fc is shown in SEQ ID NO: 4, the amino acid sequence of VH is shown in SEQ ID NO: 15, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 26.

[0080] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-5B-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0081] For example, the antibody described in this application can be C2-5B-Fc. The amino acid sequence of VL of antibody C2-5B-Fc is shown in SEQ ID NO: 5, the amino acid sequence of VH is shown in SEQ ID NO: 16, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 27.

[0082] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 17 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-5C-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0083] For example, the antibody described in this application can be C2-5C-Fc. The amino acid sequence of VL of antibody C2-5C-Fc is shown in SEQ ID NO: 6, the amino acid sequence of VH is shown in SEQ ID NO: 17, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 28.

[0084] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 18 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-6A-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0085] For example, the antibody described in this application can be C2-6A-Fc. The amino acid sequence of VL of antibody C2-6A-Fc is shown in SEQ ID NO: 7, the amino acid sequence of VH is shown in SEQ ID NO: 18, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 29.

[0086] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 19 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-6B-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0087] For example, the antibody described in this application can be C2-6B-Fc. The amino acid sequence of VL of antibody C2-6B-Fc is shown in SEQ ID NO: 8, the amino acid sequence of VH is shown in SEQ ID NO: 19, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 30.

[0088] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 20 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-7A-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0089] For example, the antibody described in this application can be C2-7A-Fc. The amino acid sequence of VL of antibody C2-7A-Fc is shown in SEQ ID NO: 9, the amino acid sequence of VH is shown in SEQ ID NO: 20, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 31.

[0090] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 10 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-7B-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0091] For example, the antibody described in this application can be C2-7B-Fc. The amino acid sequence of VL of antibody C2-7B-Fc is shown in SEQ ID NO: 10, the amino acid sequence of VH is shown in SEQ ID NO: 21, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 32.

[0092] In some embodiments, the light chain of the antibody or its antigen-binding fragment that specifically binds to CD137 described in this application may include a light chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof; and the heavy chain may include a heavy chain variable region, the amino acid sequence of which may include the amino acid sequence shown in SEQ ID NO: 22 or a variant thereof. For example, the antibody or its antigen-binding fragment may include antibody C2-7BN78K-Fc or the antibody described in this application having the same light chain variable region and heavy chain variable region.

[0093] For example, the antibody described in this application can be C2-7BN78K-Fc. The amino acid sequence of VL of antibody C2-7BN78K-Fc is shown in SEQ ID NO: 11, the amino acid sequence of VH is shown in SEQ ID NO: 22, and the Fc sequence is shown in SEQ ID NO: 34; the linker peptide between VL and VH is (G4S)3, the sequence of which is shown in SEQ ID NO: 35; the linker peptide between VL and Fc is G4S, the sequence of which is shown in SEQ ID NO: 36; and the complete sequence of each polypeptide chain is shown in SEQ ID NO: 33.

[0094] In this application, the variant of the amino acid sequence may be an amino acid sequence having substantially the same function (e.g., the ability to specifically bind to the CD137 protein) and having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence identity. In some embodiments, the variant of the amino acid sequence is an amino acid sequence having substantially the same function (e.g., the ability to specifically bind to the CD137 protein) and comprising one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid additions, deletions, or substitutions.

[0095] In some embodiments, the concentration of the antibody can be 5-50 mg / mL. For example, in some cases, the concentration of the antibody can be 5-50 mg / mL, 5-45 mg / mL, 5-40 mg / mL, 5-35 mg / mL, 5-30 mg / mL, 5-25 mg / mL, 5-20 mg / mL, 6-50 mg / mL, 6-45 mg / mL, 6-40 mg / mL, 6-35 mg / mL, 6-30 mg / mL, 6-25 mg / mL, 6-20 mg / mL, 7-50 mg / mL, 7-45 mg / mL, 7-40 mg / mL, 7-35 mg / mL, 7-30 mg / mL, 7-25 mg / mL, 7-20 mg / mL, 8-50 mg / mL, 8-45 mg / mL, 8-40 mg / mL, 8- 35mg / mL,8-30mg / mL,8-25mg / mL,8-20mg / mL,9-50mg / mL,9-45mg / mL,9-40mg / mL,9-35mg / mL,9-30mg / mL,9-25mg / mL,9-20mg / mL,10-50mg / mL,10-45mg / mL,1 0-40mg / mL, 10-35mg / mL, 10-30mg / mL, 10-25mg / mL, 10-20mg / mL, 10-50mg / mL, 15-45mg / mL, 15-40mg / mL, 15-35mg / mL, 15-30mg / mL, 15-25mg / mL, or 15-20mg / mL.

[0096] Buffer salts, stabilizers, surfactants, and reagent kits

[0097] In this application, the formulation may contain, in addition to an antibody or antigen-binding fragment thereof that specifically binds to CD137, a buffer salt selected from one or more of the group consisting of phosphates, citrates, and acetates.

[0098] In some embodiments, the buffer salt may comprise disodium hydrogen phosphate, sodium dihydrogen phosphate, and / or sodium citrate. For example, in some cases, the buffer salt may comprise disodium hydrogen phosphate and sodium dihydrogen phosphate. Yet another example, in some cases, the buffer salt may comprise disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium citrate.

[0099] In some embodiments, the concentration of the buffer salt can be 5-50 mM. For example, in some cases, the concentration of the buffer salt can be 5-10 mM, 5-20 mM, 5-30 mM, 5-40 mM, 5-50 mM, 10-20 mM, 10-30 mM, 10-40 mM, 10-50 mM, 15-20 mM, 15-30 mM, 15-40 mM, 15-50 mM, 20-30 mM, 20-40 mM, 20-50 mM, 25-30 mM, 25-40 mM, 25-50 mM, 30-40 mM, 30-50 mM, or 40-50 mM. For example, in some cases, the concentration of the buffer salt may be 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, or 35 mM.

[0100] In this application, the buffer salt may include disodium hydrogen phosphate and sodium dihydrogen phosphate. For example, in the formulation described in this application, the sum of the concentrations of the disodium hydrogen phosphate and sodium dihydrogen phosphate may be 10-20 mM, 10-30 mM, 10-40 mM, 10-50 mM, 15-20 mM, 15-30 mM, 15-40 mM, 15-50 mM, 20-30 mM, 20-40 mM, 20-50 mM, 25-30 mM, 25-40 mM, 25-50 mM, 30-40 mM, 30-50 mM, or 40-50 mM. For example, in the formulation described in this application, the sum of the concentrations of disodium hydrogen phosphate and sodium dihydrogen phosphate can be 1%-2% (wt), 1%-3% (wt), 1%-4% (wt), 1%-5% (wt), 1%-6% (wt), 1%-7% (wt), 1%-8% (wt), 1%-9% (wt), 1%-10% (wt), 2%-3% (wt), 2%-4% (wt), 2%-5% (wt), 2%-6% (wt), 3%-4% (wt), 3%-5% (wt), 3%-6% (wt), 3.5%-4% (wt), 3.5%-5% (wt), or 3%-6% (wt).

[0101] In this application, the buffer salt may include sodium citrate. For example, in the formulation described in this application, the concentration of sucrose may be 0.1%-6% (wt), 0.1%-6.5% (wt), 0.1%-7% (wt), 0.1%-7.5% (wt), 0.1%-8% (wt), 0.1%-8.5% (wt), 0.1%-9% (wt), 0.1%-9.5% (wt), 0.1%-10% (wt), 0.2%-6% (wt), 0.2%-6.5% (wt), 0.2%-7% (wt), 0.2%-7.5% (wt). % (wt), 0.2%-8% (wt), 0.2%-8.5% (wt), 0.2%-9% (wt), 0.2%-9.5% (wt), 0.2%-10% (wt), 0.3%-6% (wt), 0.3%-6.5% (wt) t), 0.3%-7% (wt), 0.3%-7.5% (wt), 0.3%-8% (wt), 0.3%-8.5% (wt), 0.3%-9% (wt), 0.3%-9.5% (wt) or 0.3%-10% (wt).

[0102] In this application, the formulation may contain, in addition to an antibody or antigen-binding fragment specifically binding to CD137, a stabilizer selected from one or more of the following: sucrose, trehalose, mannitol, glycine, arginine hydrochloride, sodium citrate, histidine, sodium acetate, and sodium chloride. For example, in some embodiments, the stabilizer may contain sucrose. In other embodiments, the stabilizer may contain both sucrose and sodium citrate.

[0103] In some embodiments, the concentration of the stabilizer can be 1%-10% (wt). For example, in some cases, the concentration of the stabilizer can be 1%-2% (wt), 1%-3% (wt), 1%-4% (wt), 1%-5% (wt), 1%-6% (wt), 1%-7% (wt), 1%-8% (wt), 1%-9% (wt), 1%-10% (wt), 2%-3% (wt), 2%-4% (wt), 2%-5% (wt), 2%-6% (wt), 2%-7% (wt), 2%-8% (wt), 2%-9% (wt), 2%-10% (wt), 3%-4% (wt), 3%-5% (wt), 3%-6% (wt), 3%-7% (wt), 3%-8% (wt), 3%-9% (wt), 3%-1 0% (wt), 4%-5% (wt), 4%-6% (wt), 4%-7% (wt), 4%-8% (wt), 4%-9% (wt), 4%-10% ( wt), 5%-6% (wt), 5%-7% (wt), 5%-8% (wt), 5%-9% (wt), 5%-10% (wt), 6%-7% (wt), 6%-8% (wt), 6%-9% (wt), 6%-10% (wt), 6.5%-7% (wt), 6.5%-8% (wt), 6.5%-9% (w t), 6.5%-10% (wt), 6.8%-7% (wt), 6.8%-8% (wt), 6.8%-9% (wt) or 6.8%-10% (wt).

[0104] In some embodiments, the concentration of the stabilizer can be 1%-10% (wt). For example, in some cases, the concentration of the stabilizer can be 5 mM-250 mM. For example, in some cases, the concentration of the buffer salt can be 5-250 mM, 5-240 mM, 5-230 mM, 5-220 mM, 5-210 mM, 5-200 mM, 5-190 mM, 5-180 mM, 5-170 mM, 5-160 mM, 5-150 mM, 10-250 mM, 10-240 mM, 10-230 mM, 10-220 mM, 10-210 mM, 10-200 mM, 10-190 mM, 10-180 mM, 10-170 mM, 10-160 mM, or 10-150 mM.

[0105] In this application, the stabilizer may include sucrose. For example, in some cases, the concentration of the sucrose may be 5-250mM, 5-240mM, 5-230mM, 5-220mM, 5-210mM, 5-200mM, 5-190mM, 10-250mM, 10-240mM, 10-230mM, 10-220mM, 10-210mM, 10-200mM, 10-190mM, 50-250mM, 50-240mM, 50-230mM, 50-220mM, 50-210mM, 50-200mM, or 50-190mM.

[0106] In this application, the stabilizer may include sodium citrate, for example, sodium citrate dihydrate. For example, in some cases, the concentration of the sodium citrate may be 5-250mM, 5-240mM, 5-230mM, 5-220mM, 5-210mM, 5-200mM, 5-190mM, 5-180mM, 5-170mM, 5-160mM, 5-150mM, 10-250mM, 10-240mM, 10-230mM, 10-220mM, 10-210mM, 10-200mM, 10-190mM, 10-180mM, 10-170mM, 10-160mM, or 10-150mM.

[0107] In this application, the stabilizer may include sucrose. For example, in the formulation described in this application, the concentration of sucrose may be 1%-6% (wt), 1%-6.5% (wt), 1%-7% (wt), 1%-7.5% (wt), 1%-8% (wt), 1%-8.5% (wt), 1%-9% (wt), 1%-9.5% (wt), 1%-10% (wt), 2%-6% (wt), 2%-6.5% (wt), 2%-7% (wt), or 2%-7%. 5% (wt), 2%-8% (wt), 2%-8.5% (wt), 2%-9% (wt), 2%-9.5% (wt), 2%-10% (wt), 3%-6% (wt), 3%-6.5% ( wt), 3%-7% (wt), 3%-7.5% (wt), 3%-8% (wt), 3%-8.5% (wt), 3%-9% (wt), 3%-9.5% (wt) or 3%-10% (wt).

[0108] In this application, the stabilizer may include sodium citrate. For example, in the formulation described in this application, the concentration of sucrose may be 0.1%-6% (wt), 0.1%-6.5% (wt), 0.1%-7% (wt), 0.1%-7.5% (wt), 0.1%-8% (wt), 0.1%-8.5% (wt), 0.1%-9% (wt), 0.1%-9.5% (wt), 0.1%-10% (wt), 0.2%-6% (wt), 0.2%-6.5% (wt), 0.2%-7% (wt), 0.2%-7.5% (wt). % (wt), 0.2%-8% (wt), 0.2%-8.5% (wt), 0.2%-9% (wt), 0.2%-9.5% (wt), 0.2%-10% (wt), 0.3%-6% (wt), 0.3%-6.5% (wt) t), 0.3%-7% (wt), 0.3%-7.5% (wt), 0.3%-8% (wt), 0.3%-8.5% (wt), 0.3%-9% (wt), 0.3%-9.5% (wt) or 0.3%-10% (wt).

[0109] In this application, the formulation may contain, in addition to an antibody or antigen-binding fragment thereof that specifically binds to CD137, a surfactant selected from one or more of the group consisting of polysorbate 20 and polysorbate 80. For example, the surfactant may contain polysorbate 20 (i.e., PS20). Another example is that the surfactant may contain polysorbate 80 (i.e., PS80). Yet another example is that the surfactant may contain both polysorbate 20 and polysorbate 80.

[0110] In some embodiments, the concentration of the surfactant can be 0.01%-0.2% (wt). For example, in some cases, the concentration of the surfactant can be 0.01%-0.2% (wt), 0.02%-0.2% (wt), 0.03%-0.2% (wt), 0.04%-0.2% (wt), 0.05%-0.2% (wt), 0.06%-0.2% (wt), 0.07%-0.2% (wt), 0.08%-0.2% (wt), 0.09%-0.2% (wt), 0.1%-0.2% (wt), or 0.15%-0.2% (wt).

[0111] In this application, the surfactant may include polysorbate 80. For example, in the formulation described in this application, the concentration of polysorbate 80 may be 0.01%-0.2% (wt), 0.02%-0.2% (wt), 0.03%-0.2% (wt), 0.04%-0.2% (wt), 0.05%-0.2% (wt), 0.06%-0.2% (wt), 0.07%-0.2% (wt), 0.08%-0.2% (wt), 0.09%-0.2% (wt), 0.1%-0.2% (wt), or 0.15%-0.2% (wt).

[0112] In this application, the pH of the formulation can be 6.5-7.5. For example, in some cases, the pH of the formulation can be 6.5-6.6, 6.5-6.7, 6.5-6.8, 6.5-6.9, 6.5-7.0, 6.5-7.1, 6.5-7.2, 6.5-7.3, 6.5-7.4, 6.5-7.5, 6.6-6.7, 6.6-6.8, 6.6-6.9, 6.6-7.0, 6.6-7.1, or 6.6-7. 2. pH values ​​of 6.6-7.3, 6.6-7.4, 6.6-7.5, 6.7-6.8, 6.7-6.9, 6.7-7.0, 6.7-7.1, 6.7-7.2, 6.7-7.3, 6.7-7.4, 6.7-7.5, 6.8-6.9, 6.8-7.0, 6.8-7.1, 6.8-7.2, 6.8-7.3, 6.8-7.4, or 6.8-7.5. For example, in some cases, the pH of the formulation may be 6.6-7.2.

[0113] In this application, the formulation may contain, in addition to an antibody that specifically binds to CD137 or its antigen-binding fragment, sucrose, polysorbate 80 (i.e., PS80), disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, and water.

[0114] In this application, the formulation may consist of an antibody that specifically binds to CD137 or its antigen-binding fragment, sodium dihydrogen phosphate, disodium hydrogen phosphate, sucrose, sodium citrate, histidine, polysorbate 80, and water.

[0115] In this application, the formulation may consist of an antibody that specifically binds to CD137 or its antigen-binding fragment, about 20 mM of stabilizers (sodium dihydrogen phosphate and disodium hydrogen phosphate), about 4.8%-6.2% (wt) of sucrose, about 10 mM of sodium citrate, about 50 mM of histidine, about 0.05% (wt) of polysorbate 80 and water.

[0116] On the other hand, this application provides a kit comprising the stated formulation and a container for holding the formulation. In this application, the kit generally refers to a collection of various components, which may or may not be packaged together. For example, in this application, the kit may comprise the stated formulation and a container for holding the formulation. The components of the kit may be contained in separate vials (i.e., a kit with separate portions) or provided in a single vial. Additionally, the kit may include instructions, which may be provided as a user manual in paper or electronic form. For example, the manual may contain instructions for interpreting results obtained when using the kit of this application.

[0117] use

[0118] On the other hand, this application provides the use of the said formulation or the said kit in the preparation of a medicament for the treatment of cancer.

[0119] On the other hand, this application provides a method for treating cancer, the method comprising administering the preparation or the kit described in this application to a subject in need.

[0120] On the other hand, the formulations or kits described in this application can be used to treat cancer.

[0121] In this application, the formulation or kit described herein can promote the secretion of cytokines by T cells (e.g., the secretion of IFN-γ).

[0122] In this application, the formulation can be formulated as an injectable preparation so that it can be used for intravenous or subcutaneous injection. In some embodiments, the formulation can also be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir.

[0123] In this application, the administration method may be intratumoral injection, such as injecting the formulation described in this application into the tumor. In some embodiments, the administration method may also be oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous storage bank.

[0124] In some embodiments, the dose level of the formulation described herein administered to the subject may vary depending on the delivered molecules, route of administration, and the patient’s size (weight, body surface or organ size) and / or physical condition (age and general health status).

[0125] In this application, the cancer may be selected from the group consisting of: melanoma, prostate cancer, colorectal cancer, Merkel cell skin cancer, pancreatic cancer, non-Hodgkin lymphoma, squamous cell carcinoma, and breast cancer.

[0126] Example

[0127] The following examples are provided merely to illustrate the formulations and uses of this application, and are not intended to limit the scope of the invention, nor are they intended to represent all or only the experiments conducted. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise stated, parts are by weight, molecular weights are weight-average molecular weights, temperatures are in Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, such as bp (base pair); kb (kilobase pair); pl (picolitrogen); s or sec (second); min (minute); h or hr (hour); aa (amino acid); nt (nucleotide); iv (intravenous); im (intramuscular); ip (intraperitoneal); sc (subcutaneous), etc.

[0128] Example 1. Preparation of anti-CD137 antibody

[0129] 1.1. Structure of anti-CD137 antibody

[0130] The amino acid sequence of the anti-CD137 antibody of this application is shown in Table 1.

[0131] Table 1. Amino acid sequence of anti-CD137 antibody

[0132]

[0133] 1.2. Expression and purification of anti-CD137 antibody

[0134] The amino acid sequences in Table 1 were used to design corresponding coding DNA sequences using the DNAworks online tool (http: / / helixweb.nih.gov / dnaworks / ). The obtained DNA sequences were cloned into the commercial vector pcDNA4 by double digestion with Hind III and EcoRI from Fermentas. Sequencing was used to verify the accuracy of the constructed plasmids, and the recombinant plasmid DNAs obtained were: pcDNA4-C2-4A-Fc, pcDNA4-C2-4B-Fc, pcDNA4-C2-4C-Fc, pcDNA4-C2-5A-Fc, pcDNA4-C2-5B-Fc, pcDNA4-C2-5C-Fc, pcDNA4-C2-6A-Fc, pcDNA4-C2-6B-Fc, pcDNA4-C2-7A-Fc, pcDNA4-C2-7B-Fc, and pcDNA4-C2-7BN78K-Fc. Plasmids were extracted according to standard molecular cloning procedures. The extracted plasmids were transiently expressed in HEK 293 cells and purified using a Protein A column to obtain C2-4A-Fc, C2-4B-Fc, C2-4C-Fc, C2-5A-Fc, C2-5B-Fc, C2-5C-Fc, C2-6A-Fc, C2-6B-Fc, C2-7A-Fc, C2-7B-Fc, and C2-7BN78K-Fc, which were used in the following examples. The obtained protein samples were initially analyzed using SDS-PAGE, and the target bands were clearly visible.

[0135] Example 2. Preparation and performance testing of the formulation

[0136] The anti-CD137 antibody prepared in Example 1 was mixed with one or more of buffer salts, stabilizers and surfactants, and the pH was adjusted to obtain formulation 1-14 (i.e. the formulation described in this application). The types and contents of each component are shown in Tables 2-1, 2-2 and 2-3, where “ / ” indicates that it is not contained.

[0137] Table 2-1. Components and their content of the formulation

[0138]

[0139] Table 2-2. Components and their content of the formulation

[0140]

[0141] Table 2-3. Components and their content of the formulation

[0142]

[0143] The performance of formulations 1-14 was tested, and the main testing contents and methods are briefly described below:

[0144] (1) Appearance

[0145] The appearance of the samples was inspected visually. The sample bottles were wiped clean and placed in front of a clarity meter (purchased from Tianda Tianfa Company) under a light intensity of 1000 lux to observe the color, clarity and visible foreign matter of the samples.

[0146] (2) pH value

[0147] The pH value of the samples was measured using a pH meter (purchased from Mettler). The pH meter was calibrated using pH correction solutions of pH 4.01, 7.00, and 9.21 to ensure the slope was above 95%.

[0148] (3) Protein concentration

[0149] Protein concentration was determined using a UV spectrophotometer (purchased from Agilent Technologies). Samples were diluted with ultrapure water to achieve absorbance values ​​within the range of 0.3-0.7 Abs. The absorbance of the sample at 280 nm was measured, and the sample concentration was calculated. The extinction coefficient was 1.749. Protein concentration = absorbance value / extinction coefficient * dilution factor.

[0150] (4) Size exclusion chromatography purity (SEC-HPLC)

[0151] SEC purity was determined using HPLC (Waters e2695 SEC) with an SEC column (TSKgel G3000SWXL, 7.8 mm ID × 30 cm, 5 µm). The mobile phase consisted of 1 × PBS, pH 6.8. The relative percentages of the main peak, aggregates, and degradation products were calculated using the area normalization method.

[0152] (5) Capillary isoelectric focusing electrophoresis (cIEF)

[0153] cIEF analysis was performed using a capillary electrophoresis apparatus (Beckman, PA800 plus) equipped with coated capillaries (µSIL-FC Capillaries 50µm ID×30.2cm).

[0154] (6) Capillary gel electrophoresis (CE-SDS-R / NR)

[0155] The purity analysis of CE-SDS-R / NR was performed using a capillary electrophoresis apparatus (Beckman, PA800 plus) equipped with uncoated quartz capillaries (30.2 cm total length, 50 µm ID).

[0156] (7) DSC (Differential Scanning Calorimetry)

[0157] The thermal transition temperature of proteins was determined using a differential calorimeter (TA), and the thermal stability of the samples was investigated. The scanning temperature ranged from 10 to 90 °C, and the scanning rate was 1 °C / min. The obtained spectra were integrated using software to obtain the midpoint value (Tm) of the thermal transition temperature.

[0158] (8) Insoluble microparticles

[0159] The optical obscuration method was used for detection. The instrument system of the insoluble particulate matter detector (purchased from HIAC) was checked with filtered purified water. Each sample was measured four times. The data from the first measurement was discarded, and the average value of the last three measurements was taken. The particle size ranges of 2~10µm, 10~25µm and ≥25µm were detected.

[0160] (9) Binding activity

[0161] Indirect enzyme-linked immunosorbent assay (ELISA) was used for detection in conjunction with the activity assay. The samples were first serially diluted using 1% BSA-PBST diluent, with an initial concentration of 10 µg / ml. Then, the samples were added to an ELISA plate pre-coated with 5 µg / ml of antigen CD137-muFc overnight and incubated. Horseradish peroxidase-labeled detection antibody (1:10000 dilution) was then added and incubated again. After TMB color development, the OD value at 450 nm was measured using an ELISA reader. Results were plotted using a four-parameter regression model, and the relative binding activity of the test samples was calculated by comparing the EC50 values ​​of the 0-day samples with those of the test samples. Detailed experimental parameters are shown in Table 3.

[0162] Table 3. Experimental parameters for binding activity

[0163]

[0164] The performance test results of formulation 1-14 are shown in Tables 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9 and 4-10. In the table, “√” indicates that the solution is colorless and clear and free of visible foreign matter, “-” indicates that no foreign matter was detected, “D” indicates day, “W” indicates week, and “T0” indicates the start time of the test.

[0165] Table 4-1. Performance test results (appearance) of the formulation

[0166]

[0167] It can be seen that formulations 1, 2, and 3, after being stored at high temperature (40℃) for 30 days, developed a small amount of white fibers and became cloudy. Formulations 4 and 5, after being observed at 40℃ for 2 weeks, became slightly cloudy; after being observed at 4℃ for 4 weeks, the solution became clear; and after 6 freeze-thaw cycles, formulation 4 developed a small amount of white particles. Formulations 6 and 7 remained clear regardless of whether they were stored at room temperature, shaken, or subjected to freeze-thaw cycles. Formulations 8, 9, and 10, after being observed at 37℃ for 3 weeks, became slightly cloudy. Formulations 11 and 12, after being observed at 37℃ for 3 weeks, became slightly cloudy; and after 6 freeze-thaw cycles, the solution of formulation 12 became clear. Formulations 13 and 14, after being observed at 40℃ for 2 weeks, became slightly cloudy; and after being observed at 40℃ for 4 weeks, the solution became cloudy.

[0168] Table 4-2. Performance test results (pH) of the formulation

[0169]

[0170] It can be seen that the pH of formulations 1-14 did not change significantly.

[0171] Table 4-3. Performance test results of the formulation (protein concentration)

[0172]

[0173] It can be seen that the protein concentrations of formulations 1-14 did not change significantly.

[0174] Table 4-4. Performance test results of the formulation (SEC-HPLC)

[0175]

[0176]

[0177] It can be seen that for formulations 1, 2, and 3, after storage at high temperature (40℃) for 30 days, the rate of increase in aggregates increased with increasing pH. For formulations 4 and 5, after 2 weeks at 40℃, the main peak purity was lower and the rate of aggregate increase was slower. After 4 weeks at 4℃, the main peak purity was not significantly different from T0. After 6 freeze-thaw cycles, the main peak content showed no significant difference. The SEC results for formulations 6 and 7 showed little difference between groups. For formulations 8, 9, and 10, after 4 weeks at 37℃, the main peak decreased more slowly and the increase in aggregates was less. For formulations 11 and 12, after 3 weeks at 37℃, the change in SEC main peak purity was not significantly different. After 6 freeze-thaw cycles, the change in SEC main peak purity for formulation 12 was not significantly different. For formulations 13 and 14, after 4 weeks at 40℃, the data showed that the lower the protein concentration, the lower the pH, and the higher the sucrose concentration, the higher the SEC main peak purity.

[0178] Table 4-5. Performance test results of the formulation (cIEF)

[0179]

[0180]

[0181] It can be seen that formulations 1, 2, and 3, after being stored at high temperature (40℃) for 30 days, all showed a significant decrease in the main peak and alkaline peak, and an increase in the acidic peak. Formulations 4 and 5, after being observed at 40℃ for 2 weeks, both showed a significant decrease in the main peak and alkaline peak, and an increase in the acidic peak. After being observed at 4℃ for 4 weeks, the purity of the main peak was not significantly different from that of T0. After 6 freeze-thaw cycles, the purity of the main peak was not significantly different from that of T0. The cIEF results for formulations 6 and 7 showed little difference between the groups. For formulations 13 and 14, after being observed at 40℃ for 4 weeks, the main peak decreased significantly, while the acidic and alkaline peaks increased significantly.

[0182] Table 4-6. Performance test results of the formulation (CE-SDS-R)

[0183]

[0184] It can be seen that after 30 days of storage at high temperature (40℃), the CE-SDS-R peak purity of formulations 1, 2, and 3 decreased by 5.5%, 8.9%, and 14.1%, respectively. Formulations 4 and 5 showed a significant decrease in peak purity after 2 weeks at 40℃; after 4 weeks at 4℃, there was no significant difference in peak purity compared to T0; and after 6 freeze-thaw cycles, there was no significant difference in peak purity compared to T0. The CE-SDS-R results for formulations 6 and 7 showed little difference between groups. After 4 weeks at 37℃, the CE-SDS-R peak purity of formulations 8, 9, and 10 decreased only slightly. After 3 weeks at 37℃, the peak purity of formulations 11 and 12 decreased by 6.2% and 6.3%, respectively; after 6 freeze-thaw cycles, the change in CE-SDS-R peak purity of formulation 12 was not significantly different. After 4 weeks at 40℃, the change in CE-SDS-R peak purity of formulations 13 and 14 was not significantly different.

[0185] Table 4-7. Performance test results of the formulation (CE-SDS-NR)

[0186]

[0187] It can be seen that after 30 days of storage at high temperature (40℃), the CE-SDS-NR peak purity of formulations 1, 2, and 3 decreased by 10.1%, 12.1%, and 18.1%, respectively. Formulations 4 and 5 showed a significant decrease in peak purity after 2 weeks at 40℃; after 4 weeks at 4℃, the peak purity was not significantly different from T0; and after 6 freeze-thaw cycles, the peak purity was not significantly different from T0. The CE-SDS-NR results for formulations 6 and 7 showed little difference between groups. After 4 weeks at 37℃, the CE-SDS-NR peak purity of formulations 8, 9, and 10 decreased only slightly. After 3 weeks at 37℃, the peak purity of formulations 11 and 12 decreased by 8.1% and 8.3%, respectively; after 6 freeze-thaw cycles, the change in CE-SDS-NR peak purity of formulation 12 was not significantly different. After 4 weeks at 40℃, the change in CE-SDS-NR peak purity of formulations 13 and 14 was not significantly different.

[0188] Table 4-8. Performance testing results (DSC) of the formulation

[0189]

[0190] It can be seen that formulations 4 and 5 have higher Tm values ​​and relatively better thermal stability.

[0191] Table 4-9. Performance test results of the formulation (insoluble microparticles)

[0192]

[0193] It can be seen that formulations 6 and 7 have fewer insoluble particles; formulation 12 also has fewer insoluble particles.

[0194] Table 4-10. Performance test results of the formulation (binding activity)

[0195]

[0196] It can be seen that formulations 13 and 14 have relatively better binding activity.

[0197] Example 3. Therapeutic effect of the formulation on tumors in vivo.

[0198] Constructing a CT26 tumor model mouse:

[0199] (1) Mouse colon cancer cells CT26 (purchased from ATCC) were resuscitated. Five days after resuscitation, the cells were digested and collected. The digested cells were washed with PBS, filtered through a sieve, counted, resuspended, and a concentration of 5 × 10⁻⁶ cells was obtained. 6 The cell suspension contained cells per ml, with a cell viability of approximately 94% for CT26 cells.

[0200] (2) Take 40 Balbc-KI mice (purchased from Jicui Yaokang) and subcutaneously inoculate each mouse with 100 μL of the cell suspension obtained in step (1). After inoculation, observe the mice until they bear tumors, thus obtaining CT26 tumor model mice.

[0201] Treatment of tumors with the formulation described in this application:

[0202] (1) The formulation 13 prepared in Example 2 and the solution containing human IgG4 of the control group (wherein all other components and contents of the solution are the same as those of formulation 13 except that the solute is human IgG4 and not CD137 antibody) were applied to the CT26 tumor model mice according to Table 5.

[0203] Table 5

[0204]

[0205] The situation after application is as follows Figure 1 A- Figure 1 As shown in B, cases 1-3 represent group 1-3 respectively. Figure 1 A and Figure 1 B shows the results of the two repeated trials. Figure 1 The results show that, compared with the control group, the formulation 13 prepared in Example 2 can significantly improve the killing effect on mouse colon cancer cells, and the higher the dosage, the more significant the killing effect.

[0206] Figure 2 Similarly, A-2C reflects the killing effect of groups 1-3 on mouse colon cancer cells, and reflects the tumor volume in each mouse in each group. Figure 2 The results showed that, compared with the control group, the formulation 13 prepared in Example 2 significantly improved the killing effect on mouse colon cancer cells, and the higher the dosage, the more significant the killing effect.

[0207] Example 4. Formulation to enhance cytokine secretion

[0208] (1) Pack 0.1 μg / mL of CD3 antibody (purchased from Biolegend 317304) onto a plate and incubate overnight at 4°C.

[0209] (2) Wash twice with PBS solution, and pack 13 of the formulations prepared in Example 2 at different concentrations into plates and place them at 37°C for 2 hours.

[0210] (3) Wash twice with PBS solution, isolate human T cells from fresh blood of healthy people at a concentration of 200,000 cells / well, add them to the plate treated in step (2) above, and react at 37°C for 3 hours.

[0211] (4) Collect the supernatant and detect the content of human IFN-g in it.

[0212] The results are as follows Figure 3 As shown, the control is a solution containing human IgG4 from Example 3, and the formulation is formulation 13 prepared in Example 2. Figure 3 The results show that, compared with the control group, formulation 13 prepared in Example 2 can significantly enhance the ability of T cells to secrete IFN-γ, which is beneficial for improving the tumor-killing ability.

[0213] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents. sequence list <110> Suzhou Dingfu Target Biotechnology Co., Ltd. <120> A formulation that specifically binds to CD137 and its uses <130> 0027-PA-036CN <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-4A VL <400> 1 Gln Ser Ala Leu Ile Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 2 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-4B VL <400> 2 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 3 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-4C VL <400> 3 Gln Ser Val Leu Ile Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 4 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-5A VL <400> 4 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 5 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-5B VL <400> 5 Gln Ser Val Leu Ile Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 6 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-5C VL <400> 6 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 7 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-6A VL <400> 7 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-6B VL <400> 8 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 111 <212> PRT <213]] Artificial Sequence <220> <223> C2-7A VL <400> 9 Gln Ser Ala Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Thr Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 10 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-7B VL <400> 10 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 11 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> C2-7BN78K VL <400> 11 Gln Ser Val Leu Ile Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser 85 90 95 Asn Asn Phe Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 12 <211> 124 <212> PRT <213]] Artificial Sequence <220> <223> C2-4A VH <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> C2-4B VH <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 124 <212> PRT <213]] Artificial Sequence <220> <223> C2-4C VH <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> C2-5A VH <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 124 <212> PRT <213]] Artificial Sequence <220> <223> C2-5B VH <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> C2-5C VH <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> C2-6A VH <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> C2-6B VH <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 124 <212> PRT <213]] Artificial Sequence <220> <223> C2-7A VH <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 21 <211> 124 <212> PRT <213]] Artificial Sequence <220> <223> C2-7B VH <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> C2-7BN78K VH <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-4A-Fc <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Ile 130 135 140 Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 24 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-4B-Fc <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 25 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-4C-Fc <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 26 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-5A-Fc <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 27 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-5B-Fc <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 28 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-5C-Fc <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 29 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-6A-Fc <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 30 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-6B-Fc <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 31 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-7A-Fc <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu His Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Phe Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Thr Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 32 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2-7B-Fc <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 33 <211> 484 <212> PRT <213]] Artificial Sequence <220> <223> C2-7BN78K-Fc <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ala Ser Gly Ser Thr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Val Val Pro Ala Gly Ser Gly Trp Arg Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Ile 130 135 140 Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser 145 150 155 160 Cys Thr Gly Ile Ser Ser Asp Val Gly Ala Tyr Asp Tyr Val Ser Trp 165 170 175 Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu Met Ile Tyr Glu Val 180 185 190 Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 195 200 205 Gly Asp Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ser Ser His Ala Gly Ser Asn Asn Phe Tyr Val 225 230 235 240 Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys <210> 34 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Fc <400> 34 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 1-(G4S)3 <400> 35 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide 2-G4S <400> 36 Gly Gly Gly Gly Ser 1 5

Claims

1. A formulation comprising an antibody or antigen-binding fragment thereof that specifically binds to CD137, wherein the CD137 antibody comprises a light chain variable region VL and a heavy chain variable region VH, wherein The VL contains the amino acid sequence shown in SEQ ID NO: 11, and the VH contains the amino acid sequence shown in SEQ ID NO:

22. The concentration of the antibody is 10 mg / mL. The formulation further comprises 20 mM buffer salt, 3%-7% wt sucrose, 10 mM sodium citrate, 50 mM histidine, 0.05% wt polysorbate 80 and water, wherein the buffer salt comprises sodium dihydrogen phosphate and disodium hydrogen phosphate.

2. The formulation according to claim 1, wherein the pH of the formulation is 6.5-7.

5.

3. A kit comprising the formulation of any one of claims 1-2 and a container for containing the formulation.

4. Use of the formulation of any one of claims 1-2 or the kit of claim 3 in the preparation of a medicament, wherein the medicament is used to treat colorectal cancer.

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