CN113166264B

CN113166264B - Isolated antigen binding proteins and uses thereof

Info

Publication number: CN113166264B
Application number: CN201980082803.7A
Authority: CN
Inventors: 张骅; 华坚
Original assignee: Shanghai Pharmaceutical Group Biotherapy Technology Co ltd; Shangyao Biotherapy Hong Kong Ltd
Current assignee: Shanghai Pharmaceutical Group Biotherapy Technology Co ltd; Shangyao Biotherapy Hong Kong Ltd
Priority date: 2019-09-19
Filing date: 2019-09-19
Publication date: 2024-02-27
Anticipated expiration: 2039-09-19
Also published as: WO2021051351A1; CN113166264A

Abstract

An isolated antigen binding protein is provided comprising at least one CDR in a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 16 and at least one CDR in a light chain variable region VL comprising the amino acid sequence shown in SEQ ID No. 15. Also provided are nucleic acid molecules encoding the isolated antigen binding proteins, vectors comprising the isolated antigen binding proteins, cells comprising the nucleic acid molecules or the vectors, methods of preparing the isolated antigen binding proteins, pharmaceutical compositions, and uses of the isolated antigen binding proteins.

Description

Isolated antigen binding proteins and uses thereof

Technical Field

The present application relates to the field of biological medicine, and in particular to an isolated antigen binding protein and uses thereof.

Background

Tumors are a disease that severely threatens human health, and immunotherapy has shown great potential in tumor therapy as a new therapy in recent years.

T cell immunoglobulin and mucin molecule 3 (T cell immunoglobulin and mucin-domain containing molecule 3, tim-3) is an important immune checkpoint molecule discovered in recent years, which, unlike other "immune checkpoint" molecules, is primarily inducible expressed on the T cell surface in inflammatory and tumor microenvironments. It is a type I membrane surface molecule, highly expressed in Th1 cells, and produces an inhibitory signal that leads to apoptosis of Th1 cells. In the course of cancer and chronic viral infection, tim-3 binds to its ligand galectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group protein 1 (high mobility group box protein, HMGB1), carcinoembryonic antigen-related cell adhesion molecule 1 (cecino-embryonic antigen related cellular adhesion molecule 1, CEACAM1), which in turn causes T-cell depletion and apoptosis, is one of the important causes of immune escape of tumor cells. Meanwhile, selective activation of Tim-3, resulting in immune escape, is the primary mechanism for drug resistance during PD-1/PD-L1 antibody immunotherapy.

Disclosure of Invention

In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH and at least one CDR in a light chain variable region VL, wherein the VH comprises the amino acid sequence of SEQ ID NO:16, and the VL comprises the amino acid sequence set forth in SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

In certain embodiments, the isolated antigen binding proteins described herein have a Tim-3 binding capacity.

In certain embodiments, the isolated antigen binding proteins described herein have one or more of the following properties:

1) Capable of binding human-derived Tim-3 with a KD value of 10nM or less, wherein said KD value is determined by surface plasmon resonance;

2) In FACS assays, it is capable of specifically binding human Tim-3 but not mouse Tim-3;

3) In ELISA assay, tim-3 binding to PtdSer can be inhibited; and

4) Promote secretion of IFN-gamma and/or TNF-alpha.

The isolated antigen binding protein of any one of claims 1-3, comprising an antibody or antigen binding fragment thereof.

In certain embodiments, the antibodies include humanized and murine antibodies.

In certain embodiments, the antigen binding fragment comprises a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, and/or dAb.

In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

In certain embodiments, the HCDR1 comprises SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

In certain embodiments, the HCDR2 comprises SEQ ID NO: 5.

In certain embodiments, the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

In certain embodiments, the LCDR2 comprises SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

In certain embodiments, the LCDR3 comprises SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

In certain embodiments, the isolated antigen binding protein described herein competes for binding to the Tim-3 protein with a reference antibody comprising a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises SEQ ID NO:1, and a polypeptide sequence shown in the specification; the LCDR2 comprises SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2); the LCDR3 comprises SEQ ID NO:3, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence of SEQ ID NO:4, and a polypeptide sequence shown in the figure; the HCDR2 comprises SEQ ID NO:5, and a polypeptide sequence shown in the figure; the HCDR3 comprises SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

In certain embodiments, the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

In certain embodiments, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence of SEQ ID NO: 7.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of SEQ ID NO: 9.

In certain embodiments, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

In certain embodiments, the VL comprises SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

In certain embodiments, the isolated antigen binding proteins described herein comprise an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region.

In certain embodiments, the antibody light chain constant region comprises SEQ ID NO:17, and a sequence of amino acids shown in seq id no.

In certain embodiments, the isolated antigen binding proteins described herein comprise an antibody light chain LC, and the LC comprises the amino acid sequence of SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no.

In certain embodiments, the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence of SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

In certain embodiments, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In certain embodiments, the VH comprises SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

In certain embodiments, the isolated antigen binding proteins described herein comprise an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.

In certain embodiments, the antibody heavy chain constant region comprises SEQ ID NO:18, and a sequence of amino acids shown in seq id no.

In certain embodiments, the isolated antigen binding proteins described herein comprise an antibody heavy chain HC, and the HC comprises the amino acid sequence of SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no.

In another aspect, the present application also provides an isolated nucleic acid molecule or molecules encoding an isolated antigen binding protein described herein.

In another aspect, the present application also provides vectors comprising the nucleic acid molecules described herein, or expressing the antigen binding proteins described herein.

In another aspect, the present application also provides a cell comprising a nucleic acid molecule as described herein or a vector as described herein.

In another aspect, the present application also provides a method of making an isolated antigen binding protein described herein, comprising culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In another aspect, the present application also provides a pharmaceutical composition comprising an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein and/or a cell described herein, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application also provides the use of said isolated antigen binding protein, said nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a tumor.

In certain embodiments, the tumor comprises a solid tumor and/or a hematological tumor.

In another aspect, the present application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a Tim-3 related disease.

In another aspect, the present application also provides a method of inhibiting binding of Tim-3 to PtdSer comprising administering an isolated antigen binding protein described herein.

In another aspect, the present application also provides a method of preventing, alleviating and/or treating a tumor, the method comprising administering to a subject in need thereof an isolated antigen binding protein as described herein or a pharmaceutical composition as described herein.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The brief description of the drawings is as follows:

FIG. 1 shows the binding of an isolated antigen binding protein Z1 described herein to a cell line expressing human Tim-3;

FIG. 2 shows the binding of the isolated antigen binding protein Z1 described herein to a strain of cells expressing mouse Tim-3;

FIG. 3 shows the binding of the comparative antibodies to a mouse Tim-3 expressing cell line;

FIGS. 4A-4C show that the isolated antigen binding protein Z1 described herein blocks binding of human Tim-3 to the ligand PtdSer.

Detailed Description

The present invention provides for the first time an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH and at least one CDR in a light chain variable region VL, wherein said VH comprises the amino acid sequence of SEQ ID NO:16, and the VL comprises the amino acid sequence set forth in SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no. The isolated antigen binding proteins described herein are capable of binding human-derived Tim-3 with KD values of 10nM or less, and furthermore, are capable of specifically binding human Tim-3 but not mouse Tim-3. In addition, the isolated antigen binding proteins described herein are capable of inhibiting the binding of Tim-3 to PtdSer and also promote secretion of IFN-gamma and/or TNF-alpha.

Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.

The present application is further described below: in the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In this application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability obtained from a natural state by artificial means. The "isolated antigen binding protein" may comprise an antigen-binding moiety and optionally, a scaffold or framework moiety that allows the antigen-binding moiety to adopt a conformation that promotes binding of the antigen by the antigen-binding moiety. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, scaffolds comprising antibody sources that are introduced, for example, to stabilize mutations in the three-dimensional structure of the antigen binding protein, as well as fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., korndorfer et al, 2003, proteins: structure, function, andbrio information, 53 (1): 121-129 (2003); roque et al, biotechnol. Prog.20:639-654 (2004). In addition, peptide antibody mimetics ("PAMs") and scaffolds based on antibody mimetics using fibronectin components may be used as scaffolds.

In this application, the term "KD" (likewise, "K _D "or" K _D ") generally refers to an" affinity constant "or" equilibrium dissociation constant "and refers to the dissociation rate constant (K) at equilibrium, or by passing the dissociation rate constant (K) in a titration measurement _dissoc ) Divided by the binding rate constant (K _assoc ) The obtained value. Using a binding rate constant (K _assoc ) Dissociation rate constant (K) _dissoc ) And equilibrium dissociation constant (KD) represents a junctionBinding affinity of a synthetic protein (e.g., an isolated antigen binding protein described herein) to an antigen (e.g., tim-3). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be determined by surface plasmon resonance _D Value, the K can also be determined by Octet _D Values may also be determined using other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) (e.g., instruments available from BIAcoreIntemationalAB, aGEHealthcarecompany, uppsala, sweden). In addition, the K can also be determined using KinExA (kinetic exclusion assay) available from Sapidyneinstruments (Boise, idaho) _D Values.

In this application, 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 paired. "percent sequence identity" is calculated as: comparing the two optimally aligned sequences within a specific region; determining the number of positions in the two sequences at which the same base or amino acid occurs to obtain the number of matched positions; the number of such positions is divided by the total number of positions in the compared section, and the resulting quotient is multiplied by 100. As described above, the procedure for determining homology (or identity) compares aligned sequences on an amino acid-to-amino acid basis, and the procedure can set different levels of stringency (e.g., identical amino acids, conservative amino acid substitutions, etc.) for the comparison. As the term is used herein, two amino acids in question are considered to be "conservative substitutions" of each other if they each belong to the same chemical class (i.e. acidic, non-polar/hydrophobic, uncharged polar and basic). Non-limiting examples are that two different amino acids belonging to a non-polar amino acid will be considered as "conservative substitutions" of each other, even if the two amino acids are not identical, but on the one hand a non-polar amino acid and on the other hand a basic amino acid will not be considered as "conservative substitutions" of each other. Alberts, johnson, lewis, raff, roberts and Walter, molecularBi ology of the Cell, 4 th edition (2002), column 3.1, divide amino acids into four major groups: acidic, non-polar, uncharged polar and basic. This grouping can be used in the context of the present invention for the purpose of determining whether a particular amino acid is a conservative substitution for another amino acid in question. The main groups mentioned above can be further sub-classified into, for example, small nonpolar and large nonpolar amino acids, large aromatic amino acids, etc. The term "conservative amino acid substitution" also refers to any amino acid substitution of a given amino acid residue, wherein the substitution residue is chemically similar to the given residue without a substantial decrease in the functional (e.g., binding) result of the polypeptide.

In the present application, the term "Tim-3" is an abbreviation for "T cell immunoglobulin and mucin molecule 3 (T cell immunoglobulin and mucin-domain containing molecule 3)", also known as TIM-3, HAVCR2, KIM-3, TIMD3, and FLJ14428, which generally refers to a T helper cell type I specific cell surface protein that regulates macrophage activation and the severity of inflammatory conditions. Tim-3 is also associated with cancer, particularly cancer stem cells, which are primarily inducibly expressed on the surface of T cells in the inflammatory and oncological microenvironment, are highly expressed on Th1 cells, and produce inhibitory signals leading to apoptosis of Th1 cells. In the course of cancer and chronic viral infection, tim-3 binds to its ligand galectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group protein 1 (high mobility group box protein, HMGB1), carcinoembryonic antigen-related cell adhesion molecule 1 (cecino-embryonic antigen related cellular adhesion molecule 1, CEACAM1), which in turn causes T-cell depletion and apoptosis, is one of the important causes of immune escape of tumor cells. Meanwhile, selective activation of Tim-3, resulting in immune escape, is the primary mechanism for drug resistance during PD-1/PD-L1 antibody immunotherapy. Tim-3 may include variants, isoforms, species homologs, and analogs of human Tim-3 that have at least one epitope in common with Tim-3. For example, the amino acid sequence of human-derived Tim-3 is set forth in SEQ ID NO:25, the amino acid sequence of Tim-3 from the mouse is shown as SEQ ID NO: shown at 26.

In this application, the term "PtdSer" generally refers to phosphatidylserine, a ligand of Tim-3. Tim-3 binds to PtdSer, which can cause apoptosis or cause autoimmune, allergic and viral infection-related diseases.

In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds a target (which may be an epitope) is one that binds the target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent of antibody binding to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). For example, in the present application, the isolated antigen binding protein is capable of binding to human-derived Tim-3 with a KD value of 10nM or less. For another example, the isolated antigen binding protein is capable of specifically binding human Tim-3 and not mouse Tim-3. In certain embodiments, the antibodies specifically bind to epitopes on proteins that are conserved among proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusively binding.

In this application, the term "inhibit" generally refers to reducing the growth rate of a cell or the number of cells. For example, the isolated antigen binding proteins described herein are capable of inhibiting tumor growth and/or tumor cell proliferation. For another example, the isolated antigen binding proteins described herein are capable of inhibiting the binding of Tim-3 to PtdSer.

In the present application, the term "IFN-gamma" is a type of Interferon (IFN), IFN-gamma is a type II interferon and binds to a type II interferon antibody. IFN-gamma regulates a variety of biological functions such as antiviral response, cell growth, immune response and tumor suppression.

In this application, the term "TNF- α" generally refers to tumor necrosis factor α (also known as cachectin), a naturally occurring mammalian cytokine produced by a number of cell types, including monocytes and macrophages in response to endotoxin or other stimuli. TNF- α is the primary mediator of inflammatory, immunological and pathophysiological responses (Grell, M.et al, (1995) Cell [ Cell ], 83:793-802). In this application, TNF- α can include wild-type TNF- α from various species (e.g., human, mouse, and monkey), polymorphic variants of TNF- α, and functional equivalents of TNF- α.

In this application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, the tumor may be a solid tumor or a hematological tumor.

In this application, the term "variable domain" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable portions of an antibody (relative to other antibodies of the same type) and comprise antigen binding sites.

In the present application, the term "variable" generally refers to the fact that there is a large difference in sequence in certain segments of the variable domain between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. Instead, it concentrates in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al, sequences of Immunological Interest, fifth Edition, national Institute of Health, bethesda, md. (1991)). The constant domains are not directly involved in binding of antibodies to antigens, but exhibit various effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity.

In this application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present invention the term "antibody" also includes antibody fragments, such as Fab, F (ab') ₂ Fv, scFv, fd, dAb and other antibody fragments that retain antigen binding function (e.g., specifically bind Tim-3). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, whereas IgA antibodies comprise 2-5 basic 4-chain units that can polymerize in conjunction with the J chain to form multivalent combinations. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, followed by four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The VH and VL pairs together form a single antigen binding site. For the structure and properties of antibodies of different classes, see e.g. Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw (eds), appleton & Lange，Norwalk，Conn.，1994, pages 71 and chapter 6. L chains from any vertebrate species can be divided into one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of their heavy Chain (CH) constant domain. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM, have heavy chains named α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA1 and IgK1.

In this application, the term "CDR" generally refers to a region of an antibody variable domain whose sequence is highly variable and/or forms a structurally defined loop. Typically, an antibody comprises six CDRs; three in VH (HCDR 1, HCDR2, HCDR 3), and three in VL (LCDR 1, LCDR2, LCDR 3). In natural antibodies, HCDR3 and LCDR3 show most of the diversity of the six CDRs, and in particular HCDR3 is thought to play a unique role in conferring fine specificity to antibodies. See, e.g., xu et al, immunity 13:37-45 (2000); johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., human Press, totowa, n.j., 2003). In fact, naturally occurring camelid antibodies consisting of heavy chains only function normally and stably in the absence of light chains. See, for example, hamers-masterman et al, nature 363:446-448 (1993); shereoff et al, nature Structure. Biol.3:733-736 (1996).

In this application, the term "FR" generally refers to the more highly conserved portion of the antibody variable domain, which is referred to as the framework region. Typically, the variable domains of the natural heavy and light chains each comprise four FR regions, namely four in VH (H-FR 1, H-FR2, H-FR3, and H-FR 4), and four in VL (L-FR 1, L-FR2, L-FR3, and L-FR 4). For example, VL of an isolated antigen binding protein described herein may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen binding proteins described herein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In the present application,the term "antigen-binding fragment" generally refers to a fragment having antigen-binding activity. In the present application, the antigen binding fragment may include Fab, fab', F (ab) ₂ Fv fragment, F (ab') ₂ scFv, di-scFv and/or dAb.

In this application, the term "competitive binding" generally refers to the ability of an antibody or fragment thereof to interfere with another antibody's ability to bind directly or indirectly to a target/antigen (e.g., tim-3) by allosteric modulation of the other antibody (e.g., a reference antibody). For example, in the present application, the isolated antigen binding protein may compete with the reference antibody for binding to Tim-3. Furthermore, the extent to which an antibody or fragment thereof is able to interfere with the binding of another antibody or fragment thereof to a target, and thus whether or not it can be considered blocking or competing according to the invention, can be determined using a competitive binding assay. A particularly suitable quantitative competition assay uses FACS-based or AlphaScreen-based methods to measure competition between a labeled (e.g., his-tagged, biotinylated, or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof in terms of binding to a target. Typically, the competing antibody or fragment thereof is, for example, one of the following: the target is bound in a competition assay such that the recorded substitution of the isolated antigen binding proteins of the invention during the assay and in the presence of a second antibody or fragment thereof reaches at most 100% of the theoretical substitution (e.g., substitution by a cold (e.g., unlabeled) antibody or fragment thereof that needs to be blocked) resulting from the presence of a given amount of the detected potential blocking antibody or fragment thereof (e.g., in a FACS-based competition assay). Preferably, the competing antibody or fragment thereof has a recorded substitution of between 10% and 100%, such as between 50% and 100%.

In this application, the term "directly coupled" is used to refer to a direct connection as opposed to the term "indirectly coupled". For example, the direct linkage may be where there is no spacer between the substances. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirect linkage" generally refers to the situation where the materials are not directly linked. For example, the indirect connection may be the case where the connection is through a spacer. For example, in the isolated antigen binding proteins described herein, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly linked.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or an analogue isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. For example, the carrier comprises: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may or may not be the recipient of a subject plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors of the invention. The cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as COS cells, chinese Hamster Ovary (CHO) cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.

In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, preferably a human patient. For example, a pharmaceutical composition described herein may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In this application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration, are generally safe, nontoxic, and neither biologically nor otherwise undesirable.

In the present application, the term "Tim-3 related disease" generally refers to a disease associated with Tim-3 expressing cells. Such as cancer, autoimmune diseases and allergic diseases. In the present application, the Tim-3 related disease may be a tumor and/or leukemia.

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.

In this application, the term "comprising" is generally intended to include the features specifically recited, but does not exclude other elements.

In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 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 the specified value.

Isolated antigen binding proteins

For example, in the present application, the isolated antigen binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: HCDR1 in VH shown in 16. For example, in the present application, the isolated antigen binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: HCDR2 in VH shown in 16. For example, in the present application, the isolated antigen binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: HCDR3 in VH shown in 16. For another example, in the present application, the isolated antigen binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: LCDR1 in VL shown at 15. For example, in the present application, the isolated antigen binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: LCDR2 in VL shown at 15. For example, in the present application, the isolated antigen binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: LCDR3 in VL shown at 15.

In the present application, the isolated antigen binding protein further comprises a polypeptide sequence that hybridizes to SEQ ID NO:16, and at least one CDR in a heavy chain variable region VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:15, at least one CDR in the light chain variable region VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity. In the present application, the isolated antigen binding protein has Tim-3 binding capacity.

In the present application, the isolated antigen binding protein may bind to the amino acid sequence of SEQ ID NO:25, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to an epitope within a Tim-3 fragment.

Properties of the isolated antigen binding proteins

In the present application, the isolated antigen binding protein may have one or more of the following properties:

3) In ELISA assay, tim-3 binding to PtdSer can be inhibited; and

4) Promote secretion of IFN-gamma and/or TNF-alpha.

In the present application, the isolated antigen binding protein is capable of binding human-derived Tim-3 with a KD of 10nM or less, wherein the KD can be determined by table-and-plasma resonance methods. For example, the number of the cells to be processed, the isolated antigen binding proteins described herein bind human-derived Tim-3 with KD values of 10nM or less, 9.5nM or less, 9nM or less, 8.9nM or less, 8.8nM or less, 8.7nM or less, 8.6nM or less, 8.5nM or less, 8.4nM or less, 8.3nM or less, 8.2nM or less, 8.1nM or less, 8nM or less, 7.9nM or less, 7.8nM or less, 7.7nM or less, 7.6nM or less, 7.5nM or less, and with a high binding potential. 7.4nM or less, 7.3nM or less, 7.2nM or less, 7.1nM or less, 7nM or less, 6.9nM or less, 6.5nM or less, 5.5nM or less, 5nM or less, 4.5nM or less, 3.5nM or less, 3nM or less, 2.9nM or less, 2.8nM or less, 2.7nM or less, 2.6nM or less, 2.5nM or less, 2.4nM or less, 2.3nM or less, 2.1nM or less, 2nM or less, 1.5nM or less, 1nM or less, or less.

In this application, the KD values can also be determined by FACS, ELISA, competition ELISA or BIACORE or KINEXA.

In the present application, the isolated antigen binding protein is capable of specifically binding human Tim-3 but not mouse Tim-3, which specific binding can be determined by FACS. For example, the case where an isolated antigen binding protein described herein specifically binds human Tim-3 can be reflected in the half maximal effect concentration (EC 50) in a FACS assay, e.g., a lower half maximal effect concentration (EC 50) indicates better specific binding.

In the present application, said human Tim-3 may comprise a sequence as set forth in SEQ ID NO:25, said mouse Tim-3 may comprise the amino acid sequence set forth in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no.

In this application, the isolated antigen binding protein is capable of inhibiting the binding of Tim-3 to PtdSer, as determined by flow cytometry.

In the present application, the isolated antigen binding protein is capable of promoting secretion of IFN-gamma and/or TNF-alpha. For example, the isolated antigen binding proteins described herein increase the amount of IFN-gamma and/or TNF-alpha.

The species of the isolated antigen binding protein

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof. For example, isolated antigen binding proteins described herein may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, fv fragments, scFv fragments, fab 'fragments, F (ab') 2 fragments, and camelized single domain antibodies.

In this application, the antibodies may include murine antibodies. For example, the murine antibody can be prepared by injecting a test subject (e.g., a mouse) with Tim-3 and then isolating a hybridoma expressing an antibody having the desired sequence or functional property. In certain embodiments, the murine antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa, lambda chains or variants thereof, or a heavy chain constant region comprising murine IgGl, igG2, igG3 or IgG4 or variants thereof.

In the present application, the antibody may be a humanized antibody. In other words, the isolated antigen binding proteins described herein may be antibodies or variants, derivatives, analogs or fragments thereof that immunospecifically bind to a related antigen (e.g., human Tim-3) and that comprise a Framework (FR) region having substantially the amino acid sequence of a human antibody and a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. "substantially" herein in the context of a CDR means that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a CDR of a non-human antibody. The humanized antibody may comprise substantially all of at least one and typically two variable domains (Fab, fab ', F (ab') 2, fabC, fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., an antibody) and all or substantially all of the framework regions are those having a human immunoglobulin consensus sequence. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., an Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises at least a variable domain of a light chain and a heavy chain. Antibodies may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only humanized light chains. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In particular embodiments, the humanized antibody comprises only a humanized variable domain of a light chain and/or a humanized heavy chain.

In the present application, the antigen binding fragment may include Fab, fab', F (ab) ₂ Fv fragment, F (ab') ₂ scFv, di-scFv and/or dAb.

Reference antibody

In the present application, the isolated antigen binding protein may compete for binding to the Tim-3 protein with a reference antibody, wherein the reference antibody may comprise a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:1, and a polypeptide sequence shown in the specification; the LCDR2 can comprise SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2); the LCDR3 can comprise SEQ ID NO:3, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:4, and a polypeptide sequence shown in the figure; the HCDR2 may comprise SEQ ID NO:5, and a polypeptide sequence shown in the figure; the HCDR3 may comprise SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

CDR

In this application, for the variable region sequences of the isolated antigen binding proteins (i.e., the sequences of the VH or VL) the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition (see, e.g., kabat, "Sequences of Proteins of ImmunologicalInterest", national Institutes of Health, bethesda, md. (1991); A1-Lazikani et al, J. Mol. Biol.273:927-948 (1997); and Martin et al, proc. Natl. Acad. Sci. USA86:9268-9272 (1989)).

In this application, for the variable region sequences of the isolated antigen binding proteins (i.e., the sequences of the VH or VL), the CDR region sequences in the VH and VL sequences may also be determined according to the Combined definition rules, which include Kabat definition and Chothia definition.

In the present application, the VH may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 may comprise SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

In the present application, the HCDR3 may comprise a sequence identical to SEQ ID NO:6 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

In the present application, the HCDR1 may comprise SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

In the present application, the HCDR1 may comprise a sequence identical to SEQ ID NO:4 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

In the present application, the HCDR2 may comprise SEQ ID NO: 5.

In the present application, the HCDR2 may comprise a sequence identical to SEQ ID NO:5 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

For example, the HCDR1 of an isolated antigen binding protein described herein can comprise SEQ ID NO:4, the HCDR2 may comprise the amino acid sequence of SEQ ID NO:5, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

In the present application, the VL may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

In the present application, the LCDR1 may comprise a sequence identical to SEQ ID NO:1 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

In the present application, the LCDR2 can comprise SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

In the present application, the LCDR2 can comprise a sequence identical to SEQ ID NO:2 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

In the present application, the LCDR3 can comprise SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

In the present application, the LCDR3 can comprise a sequence identical to SEQ ID NO:3 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

For example, LCDR1 of an isolated antigen binding protein described herein can comprise SEQ ID NO:1, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

For another example, an isolated antigen binding protein described herein may comprise HCDR1 of SEQ ID NO:4, the HCDR2 may comprise the amino acid sequence of SEQ ID NO:5, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6, and LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:1, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

FR

In the present application, the VL of the isolated antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

In the present application, the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 7.

In the present application, the L-FR1 may comprise a sequence identical to SEQ ID NO:7 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

For example, the C-terminus of the L-FR1 can be directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 can comprise the amino acid sequence of SEQ ID NO: 7.

In the present application, the L-FR2 may comprise the amino acid sequence of SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.

In the present application, the L-FR2 may comprise a sequence identical to SEQ ID NO:8 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

For example, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 can comprise the amino acid sequence of SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.

In the present application, the L-FR3 may comprise the amino acid sequence of SEQ ID NO: 9.

In the present application, the L-FR3 may comprise a nucleotide sequence identical to SEQ ID NO:9 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

For example, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 can comprise the amino acid sequence of SEQ ID NO: 9.

In the present application, the L-FR4 may comprise the amino acid sequence of SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

In the present application, the L-FR4 may comprise a nucleotide sequence identical to SEQ ID NO:10 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

For example, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 can comprise the amino acid sequence of SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

For another example, the L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence of SEQ ID NO:7, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:9, and L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

In the present application, the VH of the isolated antigen binding protein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In the present application, the H-FR1 may comprise the sequence of SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.

In the present application, the H-FR1 may comprise a sequence identical to SEQ ID NO:11 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

For example, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 can comprise the amino acid sequence of SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.

In the present application, the H-FR2 may comprise the sequence of SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

In the present application, the H-FR2 may comprise a sequence identical to SEQ ID NO:12 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

For example, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence of SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

In the present application, the H-FR3 may comprise the sequence of SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

In the present application, the H-FR3 may comprise a sequence identical to SEQ ID NO:13 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

For example, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence of SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

In the present application, the H-FR4 may comprise the amino acid sequence of SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In the present application, the H-FR4 may comprise a sequence identical to SEQ ID NO:14 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

For example, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 can comprise the amino acid sequence of SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

For example, the H-FR1 of the isolated antigen binding proteins described herein may comprise the amino acid sequence of SEQ ID NO:11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:12, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:13, the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

For another example, the L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence of SEQ ID NO:7, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:9, and L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:10, and H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:12, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:13, the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

VL and VH

The isolated antigen binding proteins described herein may comprise an antibody light chain variable region VL and an antibody heavy chain variable region VH. For example, the VL may comprise SEQ ID NO:15, said VH may comprise the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

For example, the VL may comprise SEQ ID NO:15, and the VH may comprise the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

Light and heavy chains

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human igkappa constant region.

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human igλ constant region.

In the present application, the antibody light chain constant region may comprise SEQ ID NO:17, and a sequence of amino acids shown in seq id no.

In the present application, the antibody light chain constant region may comprise a sequence identical to SEQ ID NO:17, has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

In the present application, the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG2 heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG3 heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG4 heavy chain constant region.

In the present application, the antibody heavy chain constant region may comprise SEQ ID NO:18, and a polypeptide having the amino acid sequence shown in seq id no.

In the present application, the antibody heavy chain constant region may comprise a sequence identical to SEQ ID NO:18 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

In the present application, the isolated antigen binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence of SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no.

In the present application, the LC may comprise a sequence identical to SEQ ID NO:19 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence of SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no.

In the present application, the HC may comprise a nucleotide sequence identical to SEQ ID NO:20 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

The isolated antigen binding proteins described herein may comprise an antibody light chain and an antibody heavy chain.

For example, the light chain may comprise SEQ ID NO:19, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence of SEQ ID NO:19, and the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no. Wherein the HCDR1 of the isolated antigen binding protein may comprise SEQ ID NO:4, the HCDR2 may comprise the amino acid sequence of SEQ ID NO:5, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6, and LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:1, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3. Wherein the L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence of SEQ ID NO:7, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:9, and L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:10, and H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:12, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:13, the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no. The VL may comprise SEQ ID NO:15, and the VH may comprise the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no. For example, the isolated antigen binding protein may be Z1.

Nucleic acid molecules, vectors, cells, methods of preparation and pharmaceutical compositions

In another aspect, the present application also provides an isolated nucleic acid molecule or molecules, which may encode an isolated antigen binding protein as described herein. The isolated nucleic acid molecule or molecules described herein may be any length of isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide, or an analogue thereof either isolated from the natural environment or synthesized, but may encode an isolated antigen binding protein described herein. Furthermore, given that the same amino acid has two or more codons, i.e., the degeneracy of the codons, nucleic acid molecules encoding the same isolated antigen binding protein described herein are not unique.

In another aspect, the present application also provides vectors that may comprise a nucleic acid molecule as described herein, or, alternatively, express an antigen binding protein as described herein. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. For example, the carrier may comprise: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40). For another example, the vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may further contain a replication origin. In addition, the vector may include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not exclusively.

In another aspect, the present application also provides a cell, which may comprise a nucleic acid molecule as described herein or a vector as described herein. The cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. In certain embodiments, the cells may also include cells transfected in vitro with the vectors of the invention. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell. In certain embodiments, the cell may be a mammalian cell. In certain embodiments, the mammalian cell may be a HEK293 cell. The purification and separation method of the protein can be salting out method, isoelectric point precipitation method, low temperature organic solvent precipitation method, dialysis and ultrafiltration method, gel filtration method, electrophoresis method, ion exchange chromatography method or affinity chromatography method.

In another aspect, the present application also provides methods of making an isolated antigen binding protein described herein, which methods can comprise culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In certain embodiments, the isolated antigen binding proteins described herein can be expressed by induction of a foreign gene (e.g., IPTG induction).

In another aspect, the present application also provides a pharmaceutical composition, which may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein and/or a cell described herein, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (safe and effective amount of) pharmaceutically acceptable carriers, such as suitable formulations of stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, nontoxic, and neither biologically nor otherwise undesirable.

In certain embodiments, the pharmaceutical composition may comprise a parenteral and/or a parenteral route of administration, such as subcutaneous, transdermal, intracavity, intravenous, intra-arterial, intrathecal, intratumoral, intraperitoneal, and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient, as described in WO 2015/036583.

Use and application

In another aspect, the present application also provides the use of an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein in the manufacture of a medicament for the prevention, alleviation and/or treatment of a tumor.

In another aspect, the present application also provides the use of an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein and/or a pharmaceutical composition described herein in the manufacture of a medicament for the prevention, alleviation and/or treatment of a Tim-3 related disease.

In another aspect, the present application also provides methods of preventing, alleviating and/or treating a tumor, which methods may comprise administering to a subject in need thereof an isolated antigen binding protein described herein. In the present application, the administration may be performed in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the present application also provides methods of preventing, alleviating and/or treating a Tim-3 related disorder, which methods may comprise administering to a subject in need thereof an isolated antigen binding protein described herein. In the present application, the administration may be performed in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the isolated antigen binding proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the cells described herein, and/or the pharmaceutical compositions described herein can be used to prevent, ameliorate or treat a tumor.

In another aspect, the isolated antigen binding proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the cells described herein, and/or the pharmaceutical compositions described herein can be used to prevent, ameliorate or treat a Tim-3 related disease.

In this application, the tumor may include a solid tumor and/or a hematological tumor. For example, the tumor may be leukemia.

In the present application, the Tim-3 related diseases may include diseases related to Tim-3 expressing cells. Such as cancer, autoimmune diseases and allergic diseases. In the present application, the Tim-3 related disease may be leukemia and/or tumor.

In this application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In another aspect, the present application also provides a method of inhibiting binding of Tim-3 to PtdSer comprising administering an isolated antigen binding protein described herein. In the present application, the administration may be performed in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In certain embodiments, the isolated antigen binding proteins described herein may also be administered to a subject in need thereof with one or more effective amounts of a therapeutic agent, which may include a chemotherapeutic agent, a cytotoxic agent, an immunosuppressant, a steroid, an antiemetic, a cancer vaccine, an analgesic, or another antibody.

In certain embodiments, the isolated antigen binding proteins described herein can also be fused to toxins to create immunoconjugates to exert cytotoxic activity on cells expressing the Tim-3 protein upon specific binding to the cells, specifically killing cancer cells.

In certain embodiments, the isolated antigen binding proteins described herein can also be formulated as oncolytic viruses, whereby the virus is invaded into tumor cells by cell surface molecules, and then targeted to specific receptors that are overexpressed in tumor cells, and performs subsequent functions.

In certain embodiments, the isolated antigen binding proteins described herein may also be fused to antibodies capable of specifically binding other antigens (i.e., other than Tim-3) to produce bispecific antibodies, thereby specifically binding two different antigens simultaneously for better tumor therapeutic effects.

The isolated antigen binding proteins described herein have at least one of the following beneficial effects:

1) The isolated antigen binding proteins described herein are capable of binding human-derived Tim-3 with a KD value of 10nM or less, wherein the KD value is determined by surface plasmon resonance;

2) In FACS assays, the isolated antigen binding proteins described herein are capable of specifically binding human Tim-3 but not mouse Tim-3, and thus have species specificity;

3) In ELISA assays, the isolated antigen binding proteins described herein are capable of inhibiting the binding of Tim-3 to PtdSer, blocking the Tim-3 signaling pathway, and thereby inhibiting tumor cell growth;

4) The isolated antigen binding proteins described herein are capable of promoting secretion of IFN-gamma and/or TNF-alpha, which may result in immunostimulation, thereby inhibiting or eliminating a tumor.

Without intending to be limited by any theory, the following examples are meant to illustrate the protein molecules, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the invention of the present application. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells.

Examples

Example 1 Structure of isolated antigen binding protein Z1 described herein

The light chain of the isolated antigen binding protein Z1 described herein comprises the amino acid sequence of SEQ ID NO:19, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no. Wherein the HCDR1 of the isolated antigen binding protein Z1 comprises SEQ ID NO:4, HCDR2 comprises the amino acid sequence of SEQ ID NO:5, and HCDR3 comprises the amino acid sequence of SEQ ID NO:6, and LCDR1 comprises the amino acid sequence of SEQ ID NO:1, LCDR2 comprises the amino acid sequence of SEQ ID NO:2, LCDR3 comprises the amino acid sequence of SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3. Furthermore, the L-FR1 of the isolated antigen binding protein Z1 comprises the amino acid sequence of SEQ ID NO:7, and L-FR2 comprises the amino acid sequence shown in SEQ ID NO:8, and L-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9, and L-FR4 comprises the amino acid sequence set forth in SEQ ID NO:10, and H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:11, and H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:12, and H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:13, and H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no. Furthermore, the VL of the isolated antigen binding protein Z1 comprises the amino acid sequence of SEQ ID NO:15, and VH comprises the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

The nucleotide sequence of VL encoding the isolated antigen binding protein Z1 described herein is set forth in SEQ ID NO:21, the nucleotide sequence encoding the VH of the isolated antigen binding protein Z1 described herein is set forth in SEQ ID NO:22, and the nucleotide sequence encoding the light chain constant region of the isolated antigen binding protein Z1 described herein is set forth in SEQ ID NO:23, the nucleotide sequence encoding the heavy chain constant region of the isolated antigen binding protein Z1 described herein is set forth in SEQ ID NO: shown at 24.

Example 2 detection of binding affinity of isolated antigen binding protein Z1 described herein to human Tim-3

2.1 Coupling of Protein A

Protein A (Thermo Fisher # 21181) was coupled to 4 channels of CM5 using 1 XHBS-EP+ (GE Healthcare #BR-1006-69) as running buffer at a flow rate of 10. Mu.l/min: 1) Setting the injection time to 800s, mixing 50mM NHS and 200mM EDC in a volume ratio of 1:1, and injecting into 4 channels; 2) Protein A was diluted to 20. Mu.g/ml with sodium acetate pH4.5 and injected for 800s; 3) 1M ethanolamine is injected for 800s to block the residual active carboxyl groups on the chip surface. The instrument was equilibrated with 1 XHBS-EP+ buffer for two hours after blocking and the final coupling of Protein A was about 2000RU.

2.2 kinetic testing

A multi-cycle kinetic mode is set, each cycle comprising capture of antibodies, binding of analytes and regeneration of the chip. The antibodies were diluted to 1. Mu.g/ml and injected into the 2, 3, 4 channels at a flow rate of 10. Mu.l/min for 40s, each antibody was captured by pre-conjugated Protein A in an amount of about 200RU. Human Tim-3-hIg Fc fusion protein (i.e., human Tim-3 protein, product No. 2365-TM-050 of R & D systems, U.S.A.) was injected into the four channels in a concentration gradient of 0nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, 40nM, at a flow rate of 30ul/min, setting an injection time of 180s, and a dissociation time of 900s, respectively. Wherein the amino acid sequence of the human Tim-3 protein is shown as SEQ ID NO: 25. Finally glycine (10 mM, pH 1.5) was injected at the same flow rate for 30s to regenerate the chip.

2.3 data analysis

The experimental result is analyzed by Biacore T200 analysis software 2.0, the 1 channel is used as a reference channel for deduction, and a 1:1 dynamics fitting model is selected as an analysis model.

The results are shown in Table 1. Experimental results show that the isolated antigen binding protein Z1 disclosed in the application has better binding with human Tim-3.

TABLE 1 binding affinity of antigen binding protein Z1 to human Tim-3

Antigen binding proteins	K _assoc (1/Ms)	K _dissoc (1/s)	KD(nM)	Same type (Isotype)
					Z1	2.3×10 ⁴	1.9×10 ^-4	8.1	IgG2b，κ

In Table 1, K _assoc : a binding rate constant; k (K) _dissoc : dissociation rate constant; KD: affinity constant equal to K _dissoc /K _assoc 。

Example 3 detection of binding of isolated antigen binding protein Z1 to cell surface Tim-3 as described herein

Cell surface target antigen (Tim-3, including human derived Tim-3 and mouse derived Tim-3) was tested for binding affinity to antibodies (i.e., isolated antigen binding protein Z1 described herein or a comparative antibody) using an iQue Screen flow machine (available from IntelliCyt Corp.) using PBS containing 0.1% BSA as buffer, wherein the amino acid sequence of human Tim-3 is set forth in SEQ ID NO:25, the amino acid sequence of Tim-3 of the mouse is shown as SEQ ID NO: shown at 26. The comparative antibody is represented by Anti-mTim-3Ab, which is a commercial antibody (Rat IgG2a Clone #215008, available from R & D). The specific detection process is as follows:

1. concentration was 1 x 10 using buffer ⁶ Target cells (i.e., transgenic K562 cells expressing human Tim-3, or transgenic K562 cells expressing mouse Tim-3) at cells/ml were added to a 96 Kong Jian bottom plate (corn 3894) at 30 μl per well;

2. the concentration of the detection antibody is 3 mug/ml by using buffer solution, and the antibody is diluted according to a 3-fold ratio to form 8 concentration gradients;

3. Adding the prepared antibodies with different concentrations into paved target cells according to 30 μl/hole, and uniformly mixing;

incubating for 1 hour at 4.4 ℃ in a refrigerator;

5. 150 μl of buffer is added to each well, 300g is centrifuged for 5 minutes, and the cells are loosened after the supernatant is discarded;

6. repeating the step 5;

7. preparing a fluorescent secondary antibody (ab 98593) by using a buffer solution according to the ratio of 1:200, adding 30 mu l of each hole into cells, uniformly mixing, and incubating for 30 minutes by a 4-DEG refrigerator;

8. 150 μl of buffer is added to each well, 300g is centrifuged for 5 minutes, and the cells are loosened after the supernatant is discarded;

9. repeating the step 8;

10. adding 35 mu l of buffer solution into each hole, uniformly mixing, and detecting by using a flow instrument;

11. the data were analyzed using Graphpad software.

The results of the flow affinity binding assay are shown in fig. 1-3, wherein fig. 1 shows the binding of isolated antigen binding protein Z1 described herein to a strain of cells expressing human Tim-3, fig. 2 shows the binding of isolated antigen binding protein Z1 described herein to a strain of cells expressing mouse Tim-3, and fig. 3 shows the binding of the antibody of the comparative example to a strain of cells expressing mouse Tim-3. Furthermore, igG in fig. 1-3 represents the isotype control antibody Mouse IgG2b in the experiments, which is a commercial antibody (Mouse IgG2b Clone #133303, purchased from R & D).

As can be seen from fig. 1-3, antigen binding protein Z1 has biological activity of specifically binding to human Tim-3.

Example 4 isolated antigen binding protein Z1 described herein blocks the detection of binding of human Tim-3 to PtdSer

Phosphatidylserine (PtdSer) is another ligand of Tim-3, commonly exposed on the surface of apoptotic cell membranes, and binds to the Tim-3IgV domain, mediating apoptotic cell phagocytosis, promoting apoptotic body clearance and Dendritic Cell (DCs) antigen cross presentation. The present application dilutes Jurkat cells to 5X 10 ⁵ Cells/ml and 5. Mu.M Camptothecin were added and incubated at 37℃for 5 hours to induce apoptosis, expressing phosphatidylserine. Thereafter, the cells were washed 2 times with PBS plus 2% FSA. Finally apoptotic Jurkat cells were plated at 1X 10 cells per well ⁵ Each was added to a 96-well plate. The isolated antigen binding protein Z1 described herein and isotype control antibody Mouse IgG2b (Mouse IgG2b Clone #133303, purchased from R&D) Diluted to 20. Mu.g/ml with PBS, or T im-3-hIg Fc fusion protein (i.e., human Tim-3 protein, U.S. R&D system Co., ltd., cat. No. 2365-TM-050) was diluted to 4. Mu.g/ml with PBS, and Tim-3-hIg Fc fusion protein alone or Z1 and Tim-3-hIg Fc fusion protein were mixed together, respectively, and co-cultured with the above cells at room temperature for 30 minutes. After incubation, centrifugation and washing 2 times with PBS plus 2% FSA. Adding secondary antibody APC-Conjugated anti-hIgG Fc Ab (R) &D system, # FAB 110A) 100 μl, washed 3 times and subjected to flow cytometry on-machine test. The results of the assays are shown in FIGS. 4A-4C, and it can be seen from FIGS. 4A-4C that the corresponding receptor phosphatidylserine (PtdSer) expressed by apoptotic cells was not detected by the Tim-3-hIg Fc fusion protein (i.e., human Tim-3 protein) only in the presence of the isolated antigen binding protein Z1 described herein, whereas isotype control mouse antibodies were not effective. The isolated antigen binding protein Z1 described herein thus blocks binding of human Tim-3 to PtdSer.

The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently exemplified embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and equivalents thereof.

Sequence listing

<110> Shanghai pharmaceutical group biological treatment Co., ltd

<120> an isolated antigen binding protein and uses thereof

<130> 0130-PA-002CN

<160> 26

<170> PatentIn version 3.5

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Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

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Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp

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Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe

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Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser

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Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser

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Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr

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Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu

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Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys

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Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val

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Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr

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Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

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Glu Ile Lys Arg Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro

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Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys

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Glu Trp Ile Gly Ser Ile Asp Pro Ala His Gly Glu Thr Arg Tyr Asp

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Ser Lys Phe Gln Asp Lys Ala Thr Met Thr Ala Asp Pro Ser Ser Asn

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Thr Ala Phe Leu Gln Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala Val

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Tyr Tyr Cys Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

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Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp

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Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe

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Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser

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Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser

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Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr

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Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu

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Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys

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Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val

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Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr

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Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp

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Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

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Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser

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Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

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Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile

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Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro

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Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu

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Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn

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Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser

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Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys

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Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu

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Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu Asp

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Leu Asp Asp Ile Cys Ala Glu Ala Lys Asp Gly Glu Leu Asp Gly Leu

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Trp Thr Thr Ile Thr Ile Phe Ile Ser Leu Phe Leu Leu Ser Val Cys

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Tyr Ser Ala Ser Val Thr Leu Phe Lys Val Lys Trp Ile Phe Ser Ser

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Val Val Glu Leu Lys Gln Lys Ile Ser Pro Asp Tyr Arg Asn Met Ile

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Gly Gln Gly Ala

530

<210> 21

<211> 396

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> code Z1 VL

<400> 21

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagtagtgaa 60

gtttttatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagct cattgtacat agtaatggaa acacctattt acaatggtac 180

ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa tcgatttttt 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat tactgctttc aaggatcaca tgttccgtac 360

acgttcggag gggggaccaa gctggaaata aaacgg 396

<210> 22

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> code for Z1 VH

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atgaaatgga gctgggttat tctcttcctg atggcagtgg ttacaggggt caattcagag 60

gttcagctgc agcagtctgg gtcagagctt gtgaagccag gggcctcagt cattttgtcc 120

tgcacagctt ctggcttcaa cattaaagtc gcctatattc actgggtgaa gcagaggcct 180

gaacagggcc tggagtggat tggaagtatt gatcctgcgc atggtgaaac tagatatgac 240

tcgaagttcc aggacaaggc cactatgaca gcggacccat cctccaacac agccttcctg 300

cagctcatta gcctgacatc tgaggacact gccgtctatt actgtactac ggctatggac 360

tactggggtc aaggaacctc agtc 384

<210> 23

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> coding for the Z1 light chain constant region

<400> 23

cgcgcggatg cggcgccgac cgtgagcatt tttccgccga gcagcgaaca gctgaccagc 60

ggcggcgcga gcgtggtgtg ctttctgaac aacttttatc cgaaagatat taacgtgaaa 120

tggaaaattg atggcagcga acgccagaac ggcgtgctga acagctggac cgatcaggat 180

agcaaagata gcacctatag catgagcagc accctgaccc tgaccaaaga tgaatatgaa 240

cgccataaca gctatacctg cgaagcgacc cataaaacca gcaccagccc gattgtgaaa 300

agctttaacc gcaacgaatg c 321

<210> 24

<211> 1212

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> coding for the Z1 heavy chain constant region

<400> 24

aaaaccaccc cgccgagcgt gtatccgctg gcgccgggct gcggcgatac caccggcagc 60

agcgtgaccc tgggctgcct ggtgaaaggc tattttccgg aaagcgtgac cgtgacctgg 120

aacagcggca gcctgagcag cagcgtgcat acctttccgg cgctgctgca gagcggcctg 180

tataccatga gcagcagcgt gaccgtgccg agcagcacct ggccgagcca gaccgtgacc 240

tgcagcgtgg cgcatccggc gagcagcacc accgtggata aaaaactgga accgagcggc 300

ccgattagca ccattaaccc gtgcccgccg tgcaaagaat gccataaatg cccggcgccg 360

aacctggaag gcggcccgag cgtgtttatt tttccgccga acattaaaga tgtgctgatg 420

attagcctga ccccgaaagt gacctgcgtg gtggtggatg tgagcgaaga tgatccggat 480

gtgcagatta gctggtttgt gaacaacgtg gaagtgcata ccgcgcagac ccagacccat 540

cgcgaagatt ataacagcac cattcgcgtg gtgagcaccc tgccgattca gcatcaggat 600

tggatgagcg gcaaagaatt taaatgcaaa gtgaacaaca aagatctgcc gagcccgatt 660

gaacgcacca ttagcaaaat taaaggcctg gtgcgcgcgc cgcaggtgta tattctgccg 720

ccgccggcgg aacagctgag ccgcaaagat gtgagcctga cctgcctggt ggtgggcttt 780

aacccgggcg atattagcgt ggaatggacc agcaacggcc ataccgaaga aaactataaa 840

gataccgcgc cggtgctgga tagcgatggc agctatttta tttatagcaa actgaacatg 900

aaaaccagca aatgggaaaa aaccgatagc tttagctgca acgtgcgcca tgaaggcctg 960

aaaaactatt atctgaaaaa aaccattagc cgcagcccgg gcctggatct ggatgatatt 1020

tgcgcggaag cgaaagatgg cgaactggat ggcctgtgga ccaccattac catttttatt 1080

agcctgtttc tgctgagcgt gtgctatagc gcgagcgtga ccctgtttaa agtgaaatgg 1140

atttttagca gcgtggtgga actgaaacag aaaattagcc cggattatcg caacatgatt 1200

ggccagggcg cg 1212

<210> 25

<211> 179

<212> PRT

<213> Homo sapiens

<400> 25

Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu Pro

1 5 10 15

Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys Trp

20 25 30

Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu Arg

35 40 45

Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu Asn

50 55 60

Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val Thr

65 70 75 80

Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly Ile

85 90 95

Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala Lys

100 105 110

Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe Pro

115 120 125

Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr Leu

130 135 140

Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala Asn

145 150 155 160

Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly Ala

165 170 175

Thr Ile Arg

<210> 26

<211> 281

<212> PRT

<213> Mus musculus

<400> 26

Met Phe Ser Gly Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu

1 5 10 15

Leu Leu Ala Arg Ser Leu Glu Asp Gly Tyr Lys Val Glu Val Gly Lys

20 25 30

Asn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pro Thr Ser Gly Thr Leu

35 40 45

Val Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr

50 55 60

Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser

65 70 75 80

Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Leu

85 90 95

Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys Arg

100 105 110

Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu Leu Lys Leu

115 120 125

Asp Ile Lys Ala Ala Lys Val Thr Pro Ala Gln Thr Ala His Gly Asp

130 135 140

Ser Thr Thr Ala Ser Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Ser

145 150 155 160

Glu Thr Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys Ile

165 170 175

Ser Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Thr

180 185 190

Ala Ile His Ile Gly Val Gly Val Ser Ala Gly Leu Thr Leu Ala Leu

195 200 205

Ile Ile Gly Val Leu Ile Leu Lys Trp Tyr Ser Cys Lys Lys Lys Lys

210 215 220

Leu Ser Ser Leu Ser Leu Ile Thr Leu Ala Asn Leu Pro Pro Gly Gly

225 230 235 240

Leu Ala Asn Ala Gly Ala Val Arg Ile Arg Ser Glu Glu Asn Ile Tyr

245 250 255

Thr Ile Glu Glu Asn Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr Tyr

260 265 270

Cys Tyr Val Asn Ser Gln Gln Pro Ser

275 280

Claims

1. An isolated antigen binding protein comprising HCDR1, HCDR2, HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2 and LCDR3 of the light chain variable region VL; the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 5, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 6, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 3; the isolated antigen binding protein is an antibody or antigen binding fragment thereof, and the isolated antigen binding protein has a Tim-3 protein binding capacity.

2. The isolated antigen binding protein of claim 1, wherein the antibody comprises humanized and murine antibodies.

3. The isolated antigen binding protein of claim 1, wherein the antigen binding fragment comprises Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, and/or di-scFv.

4. The isolated antigen binding protein of claim 1, wherein the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

5. The isolated antigen binding protein of claim 4, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1 and the L-FR1 has the amino acid sequence of SEQ ID No. 7.

6. The isolated antigen binding protein of claim 4, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 has the amino acid sequence of SEQ ID No. 8.

7. The isolated antigen binding protein of claim 4, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 has the amino acid sequence of SEQ ID No. 9.

8. The isolated antigen binding protein of claim 4, wherein the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3 and the L-FR4 has the amino acid sequence of SEQ ID No. 10.

9. The isolated antigen binding protein of claim 1, wherein the amino acid sequence of VL is set forth in SEQ ID No. 15.

10. The isolated antigen binding protein of claim 1, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region.

11. The isolated antigen binding protein of claim 10, wherein the antibody light chain constant region has the amino acid sequence set forth in SEQ ID No. 17.

12. The isolated antigen binding protein of claim 1, comprising an antibody light chain LC, and said LC has the amino acid sequence shown in SEQ ID No. 19.

13. The isolated antigen binding protein of claim 1, wherein the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

14. The isolated antigen binding protein of claim 13, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1 and the H-FR1 has the amino acid sequence of SEQ ID No. 11.

15. The isolated antigen binding protein of claim 13, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 has the amino acid sequence of SEQ ID No. 12.

16. The isolated antigen binding protein of claim 13, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 has the amino acid sequence of SEQ ID No. 13.

17. The isolated antigen binding protein of claim 13, wherein the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3 and the H-FR4 has the amino acid sequence of SEQ ID No. 14.

18. The isolated antigen binding protein of claim 1, wherein the amino acid sequence of VH is set forth in SEQ ID No. 16.

19. The isolated antigen binding protein of claim 1, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.

20. The isolated antigen binding protein of claim 19, wherein the antibody heavy chain constant region has the amino acid sequence set forth in SEQ ID No. 18.

21. The isolated antigen binding protein of claim 1, comprising an antibody heavy chain HC, and said HC has the amino acid sequence set forth in SEQ ID No. 20.

22. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-21.

23. A vector comprising the nucleic acid molecule of claim 22, or expressing the antigen binding protein of any one of claims 1-21.

24. A cell comprising the nucleic acid molecule of claim 22 or the vector of claim 23.

25. A method of making the isolated antigen binding protein of any one of claims 1-21, the method comprising culturing the cell of claim 24 under conditions such that the isolated antigen binding protein of any one of claims 1-21 is expressed.

26. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23 and/or the cell of claim 24, and optionally a pharmaceutically acceptable carrier.

27. Use of the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or the pharmaceutical composition of claim 26 in the manufacture of a medicament for the prevention, alleviation and/or treatment of a tumor.

28. The use of claim 27, wherein the tumor comprises a solid tumor and/or a hematological tumor.

29. Use of the isolated antigen binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or the pharmaceutical composition of claim 26 in the manufacture of a medicament for preventing, alleviating and/or treating a Tim-3 related disease.