CN116063528A - Isolated antigen binding proteins and uses thereof - Google Patents

Isolated antigen binding proteins and uses thereof Download PDF

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Publication number
CN116063528A
CN116063528A CN202210907154.3A CN202210907154A CN116063528A CN 116063528 A CN116063528 A CN 116063528A CN 202210907154 A CN202210907154 A CN 202210907154A CN 116063528 A CN116063528 A CN 116063528A
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China
Prior art keywords
seq
acid sequence
amino acid
antigen binding
isolated antigen
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Chinese (zh)
Inventor
何晓文
郭昊
王华菁
杨月
杨棋
李慧姣
李霄培
李博华
杨焕凤
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Oricell Therapeutics Co Ltd
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Oricell Therapeutics Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present application relates to an isolated antigen binding protein comprising at least one CDR in VH with the amino acid sequence shown in SEQ ID No. 53; and which comprises at least one CDR in the VL having the amino acid sequence shown in SEQ ID NO. 49. The present application also relates to nucleic acids encoding the isolated antigen binding proteins, vectors comprising the isolated nucleic acids, cells comprising the nucleic acids or the vectors, methods of preparing the isolated antigen binding proteins, and uses of the isolated antigen binding proteins.

Description

Isolated antigen binding proteins and uses thereof
The present application is a divisional application of chinese patent application with application number 2020108200514, entitled "an isolated antigen binding protein and use thereof", having application number 2020, month 08 and 14.
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
A tumor is a disease that severely threatens human health, wherein liver cancer is a widely-compromised malignancy. Liver cancer caused by hepatitis B virus has the characteristic of long latency period, and once the liver cancer is found to be late, the liver cancer is fast in progress after the liver cancer is ill, and the treatment prognosis is poor.
Glypican3 (GPC 3) is a heparan sulfate proteoglycan on the surface of cell membranes, which is found in many tumors and is particularly common in liver cancer. In recent years, with the progress of molecular biology, genomics and proteomics, a series of molecular targeted drugs for the treatment of liver cancer have been continuously developed. The molecular targeting treatment uses some marker molecules over-expressed by tumor cells as targets, and selects targeted blocking agents to perform effective intervention, so as to achieve the effect of inhibiting tumor growth, progress and metastasis. Compared with three traditional treatment means of operation, radiotherapy and chemotherapy, the molecular targeting treatment can efficiently and selectively kill tumor cells, and reduce the damage to normal tissues. However, the effect of tumor treatment by using the molecular targeting technology is still not satisfactory, and there are many improvements.
Disclosure of Invention
The present application provides an isolated antigen binding protein comprising at least one CDR in a VH having the amino acid sequence shown in SEQ ID No. 53; and which comprises at least one CDR in the VL having the amino acid sequence shown in SEQ ID NO. 49.
In certain embodiments, the isolated antigen binding protein has one or more of the following properties:
1) Can be 6×10 -9 M or lower K D Binding to GPC3 protein, wherein said K D The values were determined by Octet;
2) GPC3 proteins capable of specifically binding to the surface of HepG2 cells and/or Huh7 cells in FACS assays; and, a step of, in the first embodiment,
3) Can inhibit tumor growth and/or tumor cell proliferation.
In certain embodiments, the GPC3 protein comprises human GPC3 protein.
In certain embodiments, the human GPC3 protein comprises the amino acid sequence shown as SEQ ID NO. 74.
In certain embodiments, the tumor comprises a GPC3 positive tumor.
In certain embodiments, the GPC 3-positive tumor comprises liver cancer.
In certain embodiments, the isolated antigen binding protein comprises HCDR1 in a VH having an amino acid sequence set forth in SEQ ID NO. 53.
In certain embodiments, the isolated antigen binding protein comprises HCDR2 in a VH having an amino acid sequence as set forth in SEQ ID NO. 53.
In certain embodiments, the isolated antigen binding protein comprises HCDR3 in a VH having an amino acid sequence set forth in SEQ ID NO. 53.
In certain embodiments, the HCDR1 comprises X 1 YX 2 MH, wherein X 1 Can be D or A, X 2 May be a or E.
In certain embodiments, the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 4 and 12.
In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 20.
In certain embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 5, 10 and 13.
In certain embodiments, the HCDR3 comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 The amino acid sequence shown, wherein X 1 Can be D or T, X 2 Can be H or R, X 3 Can be T or F, X 4 Can be I or Y, X 5 Can be G or S, X 6 Can be V or Y, X 7 Can be G or A, X 8 Can be A, Y or H, X 9 Can be F or blank, X 10 Can be D or blank, X 11 May be I or blank.
In certain embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 6, 11 and 14.
In certain embodiments, the isolated antigen binding protein comprises LCDR1 in VL having the amino acid sequence set forth in SEQ ID NO. 49.
In certain embodiments, the isolated antigen binding protein comprises LCDR2 in VL having the amino acid sequence set forth in SEQ ID NO. 49.
In certain embodiments, the isolated antigen binding protein comprises LCDR3 in VL having the amino acid sequence set forth in SEQ ID NO. 49.
In certain embodiments, the LCDR1 comprises X 1 X 2 X 3 X 4 SX 5 VX 6 X 7 X 8 X 9 YX 10 X 11 X 12 X 13 The amino acid sequence shown, wherein X 1 Can be T or R, X 2 Can be G or S, X 3 Can be T or S, X 4 Can be S or Q, X 5 Can be D or L, X 6 Can be G or H, X 7 Can be G or S, X 8 Can be Y or N, X 9 Can be N or G, X 10 Can be V or T, X 11 Can be S or Y, X 12 Can be blank or L, X 13 May be blank or H.
In certain embodiments, the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 1 and 7.
In certain embodiments, the LCDR2 comprises X 1 X 2 SX 3 RX 4 S, wherein X 1 Can be D or K, X 2 Can be V or G, X 3 Can be N, Y or Q, X 4 May be P or G.
In certain embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 2, 8 and 15.
In certain embodiments, the LCDR3 comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 The amino acid sequence shown, wherein X 1 Can be S or G, X 2 Can be S or Q, X 3 Can be Y or S, X 4 Can be A or G, X 5 Can be S or L, X 6 Can be G or T, X 7 Can be S or P, X 8 Can be T or P, X 9 Can be L or T, X 10 May be V or blank.
In certain embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 3 and 9.
In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
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 VL of the isolated antigen-binding protein 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 set forth in any one of SEQ ID NOs 22 and 30.
In certain embodiments, the L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 22 and 30.
In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 23 and 31.
In certain embodiments, the L-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 23 and 31.
In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 24 and 32.
In certain embodiments, the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 24 and 32.
In certain embodiments, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3 and the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 25 and 33.
In certain embodiments, the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 25 and 33.
In certain embodiments, the VL of the isolated antigen-binding protein comprises an amino acid sequence set forth in any one of SEQ ID NOs 46-48.
In certain embodiments, the isolated antigen binding protein comprises 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 the amino acid sequence set forth in SEQ ID NO. 54.
In certain embodiments, the isolated antigen binding protein comprises an antibody light chain LC, and the LC comprises the amino acid sequence set forth in any one of SEQ ID NOs 56-58.
In certain embodiments, said VH of said isolated antigen binding protein 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 set forth in any one of SEQ ID NOs 26 and 34.
In certain embodiments, the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs 26 and 34.
In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 27 and 35.
In certain embodiments, the H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 27 and 35.
In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 28 and 36.
In certain embodiments, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs 28 and 36.
In certain embodiments, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3 and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 29 and 37.
In certain embodiments, the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs 29 and 37.
In certain embodiments, said VH of said isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 50-52.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.
In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 55.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in any one of SEQ ID NOs 59-61.
The present application also provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins described herein.
The present application also provides a vector comprising a nucleic acid molecule as described herein.
The present application also provides a cell comprising a nucleic acid molecule as described herein or a vector as described herein.
The present application also provides methods of making the isolated antigen binding proteins described herein, comprising culturing the cells described herein under conditions such that the isolated antigen binding proteins are expressed.
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 adjuvant.
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.
The present application also provides 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 for use in preventing, alleviating and/or treating a tumor.
The present application also provides methods of preventing, alleviating or treating a tumor comprising administering to a subject in need thereof an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein and/or a pharmaceutical composition as described herein.
The present application also provides methods of detecting GPC3 in a sample, the methods comprising administering an isolated antigen binding protein 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 C-terminal sequence of wild-type human GPC3 protein and its mutated mutants 1-11, wherein the underlined sites indicate that the sites were mutated to the amino acids corresponding to the murine GPC3 protein.
FIG. 2A shows epitope analysis results for detection of L1H2, L2H6 and L1H6 intact antibodies and GC33 control antibodies using wild-type human GPC3 protein or mutants 1-5, respectively; FIG. 2B shows the results of epitope analysis using mutants 6-11 for detection of L1H2, L2H6 and L1H6 intact antibodies, and GC33 control antibodies, respectively.
FIG. 3 shows the results of the ADCC activity assay for L1H2, L2H6 and L1H6 whole antibodies.
Figure 4 shows the tumor growth inhibitory effect of L1H2 whole antibodies.
Detailed Description
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, andBioiInformatics, 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 the present application, the term "K D "(likewise," K) D "or" KD ") generally refers to an" affinity constant "or" equilibrium dissociation constant "and refers to the dissociation rate constant (k) at equilibrium in a titration measurement, or by d ) Divided by the binding rate constant (k a ) The obtained value. Using a binding rate constant (k a ) Dissociation rate constant (k) d ) And equilibrium dissociation constant (K) D ) Represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) to an antigen (e.g., GPC3 protein). 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 Octet D Values may also be determined using other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) (e.g., instruments available from BIAcoreInternationalAB, aGEHealthcarecompany, uppsala, sweden). In addition, the K can also be determined using KinExA (kinetic exclusion assay) available from Sapidyneinstruments (Boise, idaho) D Values.
In the present application, the term "GPC3 protein" generally refers to glypican 3 (GPC 3), a heparan sulfate proteoglycan on the surface of cell membranes, which is present in various tumors, and is particularly common in liver cancer. GPC3 protein is highly expressed in the liver during fetal period. The abnormal expression of GPC3 protein after birth has a close relationship with the development of tumorigenesis, and GPC3 protein is highly expressed in primary liver cancer (PHC), but is lowly expressed or expressed in other tumors or benign liver diseases. For example, a GPC3 protein described herein can comprise a human GPC3 protein.
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 can be as follows<6x10 -9 And a dissociation constant (KD) of M or less is bound to the GPC3 protein. 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.
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. For example, in the present application, the tumor may be a GPC 3-positive tumor, wherein the GPC 3-positive tumor may include liver cancer.
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 GPC 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 include 2-5 chains that can be polymerized in conjunction with the J chainBasic 4-chain units in multivalent combination. 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, e.g., hamers-Casterman et al, nature 363:446-448 (1993); sheiff 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 the binding of another antibody to a target/antigen (e.g., GPC 3), either directly or indirectly, 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 GPC3 protein. 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 adjuvant. 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 adjuvant" 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 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
In one aspect the present application provides an isolated antigen binding protein comprising at least one CDR in a VH having an amino acid sequence as shown in SEQ ID NO 53; and which comprises at least one CDR in the VL having the amino acid sequence shown in SEQ ID NO. 49.
X 1 VQLVQSGX 2 X 3 X 4 X 5 X 6 PGX 7 SX 8 X 9 X 10 SCX 11 ASGX 12 X 13 FX 14 X 15 YX 16 MHWVRQAPGX 17 GLEWX 18 X 1 9 X 20 LX 21 X 22 X 23 X 24 GX 25 X 26 X 27 YX 28 X 29 X 30 X 31 X 32 GRX 33 TX 34 X 35 X 36 DX 37 X 38 X 39 X 40 X 41 X 42 YX 43 X 44 X 45 X 46 X 47 LRX 48 X 49 DTX 50 X 51 YYCX 52 X 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 X 61 X 62 X 63 X 64 WGQGTX 65 VTVSS (SEQ ID NO: 53), wherein X 1 May be E or Q, X 2 Can be G or A, X 3 Can be G or E, X 4 Can be L or V, X 5 Can be V or K, X 6 Can be Q or K, X 7 Can be R or A, X 8 Can be L or V, X 9 Can be R or K, X 10 Can be L or V, X 11 Can be A or K, X 12 Can be F or Y, X 13 Can be T or D, X 14 Can be D or T, X 15 Can be D or A, X 16 Can be A or E, X 17 Can be K or Q, X 18 Can be V or M, X 19 Can be S or G, X 20 Can be G or A, X 21 Can be S or D, X 22 Can be W or P, X 23 Can be N or K, X 24 Can be S or T, X 25 Can be S or Q, X 26 Can be I or T, X 27 Can be G or A, X 28 Can be A or S, X 29 Can be D or Q, X 30 Can be S or K, X 31 Can be V or F, X 32 Can be K or Q, X 33 Can be F or V, X 34 Can be I or L, X 35 Can be S or T, X 36 Can be R or A, X 37 Can be N or K, X 38 Can be A or S, X 39 Can be K or I, X 40 Can be N or S, X 41 Can be S or T, X 42 Can be L or A, X 43 Can be L or M, X 44 Can be Q or E, X 45 Can be M or L, X 46 Can be N or S, X 47 Can be S or R, X 48 Can be A or S, X 49 Can be E or D, X 50 Can be A or V, X 51 Can be L or V, X 52 Can be A or blank, X 53 Can be K or blank, X 54 Can be D or T, X 55 Can be H or R, X 56 Can be T or F, X 57 Can be I or Y, X 58 Can be G or S, X 59 Can be V or Y, X 60 Can be G or A, X 61 Can be A, Y or H, X 62 Can be F or blank, X 63 Can be D or blank, X 64 Can be I or blank, X 65 May be M or L.
For example, the VH of an isolated antigen binding protein described herein may comprise the amino acid sequence set out in any one of SEQ ID NOs 50 to 52.
X 1 X 2 VX 3 TQX 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 SX 25 VX 26 X 27 X 28 X 29 YX 30 X 31 X 32 X 33 WYQQX 34 PGX 35 X 36 PX 37 LX 38 IYX 39 X 40 SX 41 RX 42 SGVX 43 X 44 RFSGSX 45 SGX 46 X 47 X 48 X 49 LX 50 ISX 5 1 X 52 X 53 AEDX 54 X 55 X 56 YYCX 57 X 58 X 59 X 60 X 61 X 62 X 63 X 64 X 65 X 66 FGX 67 GTKLX 68 X 69 X 70 (SEQ ID NO: 49), wherein X 1 Can be Q or D, X 2 Can be S or V, X 3 Can be L or M, X 4 Can be P or S, X 5 Can be A or P, X 6 Can be S or L, X 7 Can be V or S, X 8 Can be S or L, X 9 Can be G or P, X 10 Can be S or V, X 11 Can be P or T, X 12 Can be G or L, X 13 Can be Q or G, X 14 Can be S or Q, X 15 Can be I or P, X 16 Can be T or A, X 17 Can be I or S, X 18 Can be S or I, X 19 May be C orS，X 20 Can be blank or C, X 21 Can be T or R, X 22 Can be G or S, X 23 Can be T or S, X 24 Can be S or Q, X 25 Can be D or L, X 26 Can be G or H, X 27 Can be G or S, X 28 Can be Y or N, X 29 Can be N or G, X 30 Can be V or T, X 31 Can be S or Y, X 32 Can be blank or L, X 33 Can be blank or H, X 34 Can be H or R, X 35 Can be K or Q, X 36 Can be A or S, X 37 Can be K or R, X 38 Can be M or L, X 39 Can be D or K, X 40 Can be V or G, X 41 Can be N, Y or Q, X 42 Can be P or G, X 43 Can be S or P, X 44 Can be N or D, X 45 Can be K or G, X 46 Can be N or T, X 47 Can be T or D, X 48 Can be A or F, X 49 Can be S or T, X 50 Can be T or K, X 51 Can be G or R, X 52 Can be L or V, X 53 Can be Q or E, X 54 May be E or V, X 55 Can be A or G, X 56 Can be D or V, X 57 Can be S or G, X 58 Can be S or Q, X 59 Can be Y or S, X 60 Can be A or G, X 61 Can be S or L, X 62 Can be G or T, X 63 Can be S or P, X 64 Can be T or P, X 65 Can be L or T, X 66 Can be V or blank, X 67 Can be G or S, X 68 Can be T or E, X 69 Can be V or I, X 70 May be L or K.
For example, the VL of the isolated antigen-binding proteins described herein may comprise the amino acid sequence set forth in any one of SEQ ID NOS.46-48.
For example, in the present application, the isolated antigen binding protein may comprise HCDR1 in a VH having the amino acid sequence shown in SEQ ID NO 53. For example, in the present application, the isolated antigen binding protein may comprise HCDR2 in a VH having the amino acid sequence shown in SEQ ID NO 53. For example, in the present application, the isolated antigen binding protein may comprise HCDR3 in a VH having the amino acid sequence shown in SEQ ID NO 53. For another example, in the present application, the isolated antigen binding protein may comprise LCDR1 in VL having the amino acid sequence shown in SEQ ID NO. 49. For example, in the present application, the isolated antigen binding protein may comprise LCDR2 in VL having the amino acid sequence shown in SEQ ID NO. 49. For example, in the present application, the isolated antigen binding protein may comprise LCDR3 in VL having the amino acid sequence shown in SEQ ID NO. 49.
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:
1) Can be 6×10 -9 M or lower K D Binding to GPC3 protein, wherein said K D The values were determined by Octet;
2) GPC3 proteins capable of specifically binding to the surface of HepG2 cells and/or Huh7 cells in FACS assays; and, a step of, in the first embodiment,
3) Can inhibit tumor growth and/or tumor cell proliferation.
In the present application, the isolated antigen binding protein can be present in a 6X10 ratio -9 M or lower K D Binding to GPC3 protein, wherein said K D The value can be determined by Octet. For example, the isolated antigen binding proteins described herein bind to K derived from human GPC3 protein D The value may be 6x10 or less -9 M、≤5x10 -9 M、≤4x10 -9 M、≤3x10 -9 M、≤2x10 -9 M、≤1x10 -9 M、≤1x10 -10 M、≤2x10 -10 M、≤3x10 -10 M、≤4x10 -10 M、≤5x10 -10 M、≤6x10 -10 M、≤7x10 -10 M、≤8x10 -10 M、≤9x10 -10 M. For another example, an isolated antigen binding protein described herein binds to K of a murine-derived GPC3 protein D The value may be 6x10 or less -9 M、≤5x10 -9 M、≤4x10 -9 M、≤3x10 -9 M、≤2x10 -9 M、≤1x10 -9 M、≤1x10 - 10 M、≤2x10 -10 M、≤3x10 -10 M、≤4x10 -10 M、≤5x10 -10 M、≤6x10 -10 M、≤7x10 -10 M、≤8x10 -10 M、≤9x10 -10 M. For another example, an isolated antigen binding protein described herein binds to K of a monkey-derived GPC3 protein D The value may be 6x10 or less -9 M、≤5x10 -9 M、≤4x10 -9 M、≤3x10 -9 M、≤2x10 -9 M、≤1x10 -9 M、≤1x10 -10 M、≤2x10 -10 M、≤3x10 -10 M、≤4x10 -10 M、≤5x10 -10 M、≤6x10 -10 M、≤7x10 -10 M、≤8x10 -10 M、≤9x10 -10 M。
In the present application, the K D The values may also be determined by ELISA, competition ELISA or BIACORE or KINEXA.
In the present application, the isolated antigen binding protein is capable of specifically binding to GPC3 protein on the surface of HepG2 cells and/or Huh7 cells, which specific binding can be determined by FACS. For example, the case where an isolated antigen binding protein described herein specifically binds to GPC3 protein on the surface of HepG2 cells and/or Huh7 cells can be reflected in the half maximal effector concentration (EC 50) in a FACS assay, e.g., lower half maximal effector concentration (EC 50) indicates better specific binding. For example, the number of the cells to be processed, the EC50 value of the isolated antigen binding protein for binding GPC3 proteins on the surface of HepG2 cells and/or Huh7 cells in a FACS assay may be from 0.01. Mu.g/ml to 0.20. Mu.g/ml, from 0.01. Mu.g/ml to 0.02. Mu.g/ml, from 0.01. Mu.g/ml to 0.03. Mu.g/ml, from 0.01. Mu.g/ml to 0.04. Mu.g/ml, from 0.01. Mu.g/ml to 0.05. Mu.g/ml, from 0.01. Mu.g/ml to 0.06. Mu.g/ml, from 0.01. Mu.g/ml to 0.07. Mu.g/ml, from 0.01. Mu.g/ml to 0.08. Mu.g/ml 0.01 to 0.09. Mu.g/ml, 0.01 to 0.10. Mu.g/ml, 0.01 to 0.11. Mu.g/ml, 0.01 to 0.12. Mu.g/ml, 0.01 to 0.13. Mu.g/ml, 0.01 to 0.14. Mu.g/ml, 0.01 to 0.15. Mu.g/ml, 0.01 to 0.16. Mu.g/ml, 0.01 to 0.17. Mu.g/ml, 0.01 to 0.18. Mu.g/ml, 0.01 to 0.19. Mu.g/ml or 0.01 to 0.20. Mu.g/ml.
In the present application, the isolated antigen binding proteins described herein are capable of inhibiting tumor growth and/or tumor cell proliferation, e.g., capable of reducing tumor volume by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%. For another example, the number of tumor cells can be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.
In the present application, the GPC3 protein may comprise a human GPC3 protein, and the human GPC3 protein may comprise an amino acid sequence as shown in SEQ ID NO: 74.
In the present application, the tumor may include a GPC 3-positive tumor, and the GPC 3-positive tumor may include liver cancer.
In the present application, the isolated antigen binding protein is capable of inhibiting the growth of tumor cells by inducing antibody dependent cell-mediated cytotoxicity (ADCC). For example, the isolated antigen binding proteins described herein are capable of bringing cytotoxic infiltrating T lymphocytes into tumor tissue, thereby inhibiting the growth of tumor cells. For another example, the isolated antigen binding proteins described herein may also indirectly inhibit proliferation of GPC 3-positive cells through macrophage-related mechanisms.
In this application, the isolated antigen binding protein has a certain endocytic activity. The endocytic activity can be detected by the Confocal method.
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 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 GPC 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%, preferably 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.
CDR
In the present application, the LCDR1 may comprise X 1 X 2 X 3 X 4 SX 5 VX 6 X 7 X 8 X 9 YX 10 X 11 X 12 X 13 The amino acid sequence shownWherein X is 1 Can be T or R, X 2 Can be G or S, X 3 Can be T or S, X 4 Can be S or Q, X 5 Can be D or L, X 6 Can be G or H, X 7 Can be G or S, X 8 Can be Y or N, X 9 Can be N or G, X 10 Can be V or T, X 11 Can be S or Y, X 12 Can be blank or L, X 13 May be blank or H.
In the present application, the LCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 1 and 7.
In the present application, the LCDR2 may comprise X 1 X 2 SX 3 RX 4 S, wherein X 1 Can be D or K, X 2 Can be V or G, X 3 Can be N, Y or Q, X 4 May be P or G.
In the present application, the LCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 2, 8 and 15.
In the present application, the LCDR3 may comprise X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 The amino acid sequence shown, wherein X 1 Can be S or G, X 2 Can be S or Q, X 3 Can be Y or S, X 4 Can be A or G, X 5 Can be S or L, X 6 Can be G or T, X 7 Can be S or P, X 8 Can be T or P, X 9 Can be L or T, X 10 May be V or blank.
In the present application, the LCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 3 and 9.
For example, LCDR1 of the isolated antigen binding proteins described herein may comprise the amino acid sequence shown in SEQ ID NO. 1, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 2, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 3.
For example, LCDR1 of an isolated antigen binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 8, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 9.
For example, LCDR1 of an isolated antigen binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 15, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 9.
In the present application, the HCDR1 may comprise X 1 YX 2 MH, wherein X 1 Can be D or A, X 2 May be a or E.
In the present application, the HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 4 and 12.
In this application, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 20.
X 1 LX 2 X 3 X 4 X 5 GX 6 X 7 X 8 YX 9 X 10 X 11 X 12 X 13 G (SEQ ID NO: 20), wherein X 1 Can be G or A, X 2 Can be S or D, X 3 Can be W or P, X 4 Can be N or K, X 5 Can be S or T, X 6 Can be S or Q, X 7 Can be I or T, X 8 Can be G or A, X 9 Can be A or S, X 10 Can be D or Q, X 11 Can be S or K, X 12 Can be V or F, X 13 May be K or Q.
In the present application, the HCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 5, 10 and 13.
In the present application, the HCDR3 may comprise X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 The amino acid sequence shown, wherein X 1 Can be D or T, X 2 Can be H or R, X 3 Can be T or F, X 4 Can be I or Y, X 5 Can be G or S, X 6 Can be V or Y, X 7 Can be G or A, X 8 May be AY or H, X 9 Can be F or blank, X 10 Can be D or blank, X 11 May be I or blank.
In the present application, the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs 6, 11 and 14.
For example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 5, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 6.
For example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 10, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 11.
For example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 13, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 14.
For another example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 1, LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 2, LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 5, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 6.
For another example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 8, LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 9, and HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 10, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 11.
For another example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 8, LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 9, and HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 13, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 14.
For another example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 15, LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 9, and HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 13, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 14.
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 X 1 X 2 VX 3 TQX 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 1 9 X 20 (SEQ ID NO: 38) wherein X is the amino acid sequence shown 1 Can be Q or D, X 2 Can be S or V, X 3 Can be L or M, X 4 Can be P or S, X 5 Can be A or P, X 6 Can be S or L, X 7 Can be V or S, X 8 Can be S or L, X 9 Can be G or P, X 10 Can be S or V, X 11 Can be P or T, X 12 Can be G or L, X 13 Can be Q or G, X 14 Can be S or Q, X 15 Can be I or P, X 16 Can be T or A, X 17 Can be I or S, X 18 Can be S or I, X 19 Can be C or S, X 20 May be blank or C.
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 set forth in any one of SEQ ID NOs 22 and 30.
For example, the L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs 22 and 30.
In the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 39.
WYQQX 1 PGX 2 X 3 PX 4 LX 5 IY (SEQ ID NO: 39), wherein X 1 Can be H or R, X 2 Can be K or Q, X 3 Can be A or S, X 4 Can be K or R, X 5 May be M or L.
For example, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 can comprise the amino acid sequence shown in any one of SEQ ID NOs 23 and 31.
For example, the L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs 23 and 31.
In the present application, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 40.
GVX 1 X 2 RFSGSX 3 SGX 4 X 5 X 6 X 7 LX 8 ISX 9 X 10 X 11 AEDX 12 X 13 X 14 YYC (SEQ ID NO: 40), wherein X 1 Can be S or P, X 2 Can be N or D, X 3 Can be K or G, X 4 Can be N or T, X 5 Can be T or D, X 6 Can be A or F, X 7 Can be S or T, X 8 Can be T or K, X 9 Can be G or R, X 10 Can be L or V, X 11 Can be Q or E, X 12 May be E or V, X 13 Can be A or G, X 14 May be D or V.
For example, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs 24 and 32.
For example, the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs 24 and 32.
In the present application, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 41.
FGX 1 GTKLX 2 X 3 X 4 (SEQ ID NO: 41) wherein X 1 Can be G or S, X 2 Can be T or E, X 3 Can be V or I, X 4 May be L or K.
For example, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs 25 and 33.
For example, the L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs 25 and 33.
For another example, an isolated antigen binding protein described herein may comprise the amino acid sequence set forth in SEQ ID NO. 22, L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO. 23, L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO. 24, and L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO. 25.
For another example, an isolated antigen binding protein described herein may have an L-FR1 that comprises the amino acid sequence shown in SEQ ID NO. 30, an L-FR2 that comprises the amino acid sequence shown in SEQ ID NO. 31, an L-FR3 that comprises the amino acid sequence shown in SEQ ID NO. 32, and an L-FR4 that comprises the amino acid sequence shown in SEQ ID NO. 33.
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 amino acid sequence shown in SEQ ID NO. 42.
X 1 VQLVQSGX 2 X 3 X 4 X 5 X 6 PGX 7 SX 8 X 9 X 10 SCX 11 ASGX 12 X 13 FX 14 (SEQ ID NO: 42), wherein X 1 May be E or Q, X 2 Can be G or A, X 3 Can be G or E, X 4 Can be L or V, X 5 Can be V or K, X 6 Can be Q or K, X 7 Can be R or A, X 8 Can be L or V, X 9 Can be R or K, X 10 Can be L or V, X 11 Can be used forIs A or K, X 12 Can be F or Y, X 13 Can be T or D, X 14 May be D or T.
In the present application, the C-terminal end of the H-FR1 is directly or indirectly linked to the N-terminal end of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs 26 and 34.
In the present application, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs 26 and 34.
In the present application, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 43.
WVRQAPGX 1 GLEWX 2 X 3 (SEQ ID NO: 43), wherein X 1 Can be K or Q, X 2 Can be V or M, X 3 May be S or G.
In the present application, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs 27 and 35.
In the present application, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs 27 and 35.
In the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 44.
RX 1 TX 2 X 3 X 4 DX 5 X 6 X 7 X 8 X 9 X 10 YX 11 X 12 X 13 X 14 X 15 LRX 16 X 17 DTX 18 X 19 YYCX 20 X 21 (SEQ ID NO: 44), wherein X 1 Can be F or V, X 2 Can be I or L, X 3 Can be S or T, X 4 Can be R or A, X 5 Can be N or K, X 6 Can be A or S, X 7 Can be K or I, X 8 Can be N or S, X 9 Can be S or T, X 10 Can be L or A, X 11 Can be L or M, X 12 Can be Q or E, X 13 Can be M or L, X 14 Can be N or S, X 15 Can be S or R, X 16 Can be A or S, X 17 Can be E or D, X 18 Can be A or V, X 19 Can be L or V, X 20 Can be A or blank, X 21 May be K or blank.
In the present application, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs 28 and 36.
In the present application, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs 28 and 36.
In the present application, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45.
WGQGTX 1 VTVSS (SEQ ID NO: 45), wherein X 1 May be M or L.
In the present application, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs 29 and 37.
In the present application, the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs 29 and 37.
For example, the H-FR1 of the isolated antigen binding proteins described herein may comprise the amino acid sequence shown in SEQ ID NO. 26, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 28, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 29.
For example, the H-FR1 of the isolated antigen binding proteins described herein may comprise the amino acid sequence shown in SEQ ID NO. 34, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 35, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 36, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 37.
For another example, the isolated antigen binding protein described herein may have L-FR1 comprising the amino acid sequence shown in SEQ ID NO. 22, L-FR2 comprising the amino acid sequence shown in SEQ ID NO. 23, L-FR3 comprising the amino acid sequence shown in SEQ ID NO. 24, L-FR4 comprising the amino acid sequence shown in SEQ ID NO. 25, and H-FR1 comprising the amino acid sequence shown in SEQ ID NO. 26, H-FR2 comprising the amino acid sequence shown in SEQ ID NO. 27, H-FR3 comprising the amino acid sequence shown in SEQ ID NO. 28, and H-FR4 comprising the amino acid sequence shown in SEQ ID NO. 29.
For another example, the isolated antigen binding protein described herein may have L-FR1 comprising the amino acid sequence shown in SEQ ID NO. 30, L-FR2 comprising the amino acid sequence shown in SEQ ID NO. 31, L-FR3 comprising the amino acid sequence shown in SEQ ID NO. 32, L-FR4 comprising the amino acid sequence shown in SEQ ID NO. 33, and H-FR1 comprising the amino acid sequence shown in SEQ ID NO. 34, H-FR2 comprising the amino acid sequence shown in SEQ ID NO. 35, H-FR3 comprising the amino acid sequence shown in SEQ ID NO. 36, and H-FR4 comprising the amino acid sequence shown in SEQ ID NO. 37.
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 the amino acid sequence shown in SEQ ID NO. 49 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 53. For another example, the VL of the isolated antigen-binding protein may comprise an amino acid sequence set forth in any one of SEQ ID NOs 46-48. The VH of the isolated antigen binding protein may comprise an amino acid sequence set out in any one of SEQ ID NOs 50 to 52.
For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 46 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 50.
For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 47 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 51.
For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 47 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 52.
For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 48 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 52.
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. For example, the antibody light chain constant region may comprise the amino acid sequence shown in SEQ ID NO. 54.
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. For example, the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO. 55.
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 shown in any one of SEQ ID NOs 56 to 58.
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 set forth in any one of SEQ ID NOs 59-61.
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 the amino acid sequence shown in SEQ ID NO. 56 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 59.
For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 57 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 60.
For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 57 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 61.
For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 58 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 61.
In this application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 56, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 59. Wherein, LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 1, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 2, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 5, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 6. Wherein the isolated antigen binding protein L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 22, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 23, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 24, L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 25, and H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 26, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 28, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 29. The VL may comprise the amino acid sequence shown in SEQ ID NO. 46, and the VH may comprise the amino acid sequence shown in SEQ ID NO. 50. For example, the isolated antigen binding protein may be 204A.
In this application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 57, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 60. Wherein, LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 8, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 9, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 10, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 11. Wherein the isolated antigen binding protein L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 30, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 31, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32, L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 33, and H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 34, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 35, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 36, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 37. The VL may comprise the amino acid sequence shown in SEQ ID NO. 47, and the VH may comprise the amino acid sequence shown in SEQ ID NO. 51. For example, the isolated antigen binding protein may be L1H2.
In this application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 57, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 61. Wherein, LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 8, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 9, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 13, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 14. Wherein the isolated antigen binding protein L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 30, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 31, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32, L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 33, and H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 34, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 35, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 36, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 37. The VL may comprise the amino acid sequence shown in SEQ ID NO. 47, and the VH may comprise the amino acid sequence shown in SEQ ID NO. 52. For example, the isolated antigen binding protein may be L1H6.
In this application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 58 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 61. Wherein, LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 15, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 9, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 12, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 13, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 14. Wherein the isolated antigen binding protein L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 30, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 31, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32, L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 33, and H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 34, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 35, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 36, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 37. The VL may comprise the amino acid sequence shown in SEQ ID NO. 48 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 52. For example, the isolated antigen binding protein may be L2H6.
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.
In another aspect, the present application also provides vectors, which may comprise the nucleic acid molecules 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.
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 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 adjuvant.
In certain embodiments, the pharmaceutical compositions 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 certain embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, 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 parenteral, transdermal, endoluminal, intra-arterial, intrathecal 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 a method of preventing, alleviating or treating a tumor, which method may comprise administering to a subject in need thereof an isolated antigen binding protein as 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 this application, the tumor may be a solid tumor or a hematological tumor. For example, the tumor may comprise a GPC 3-positive tumor, and the GPC 3-positive tumor may comprise liver cancer.
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 detecting GPC3 in a sample, the method 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.
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. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: a Laboratory Manual,2nd edition,Cold spring Harbor Laboratory Press.
Description of the embodiments
11 comprising at least one CDR in a VH having the amino acid sequence shown in SEQ ID No. 53; and which comprises at least one CDR in the VL having the amino acid sequence shown in SEQ ID NO. 49.
The isolated antigen binding protein of embodiment 1 having one or more of the following properties:
1) Can be 6×10 -9 M or lower K D Binding to GPC3 protein, wherein said K D The values were determined by Octet;
2) GPC3 proteins capable of specifically binding to the surface of HepG2 cells and/or Huh7 cells in FACS assays;
3) Can inhibit tumor growth and/or tumor cell proliferation.
13 the isolated antigen binding protein of embodiment 2, wherein the GPC3 protein comprises human GPC3 protein.
14 the isolated antigen binding protein of any of embodiments 2-3, wherein said human GPC3 protein comprises the amino acid sequence set forth in SEQ ID No. 74.
The isolated antigen binding protein of any of embodiments 2-4, wherein the tumor comprises a GPC 3-positive tumor.
The isolated antigen binding protein of embodiment 5, wherein the GPC 3-positive tumor comprises liver cancer.
The isolated antigen binding protein of any of embodiments 1-6 comprising HCDR1 in VH having an amino acid sequence shown in SEQ ID No. 53.
The isolated antigen binding protein of any of embodiments 1-7 comprising HCDR2 in VH having an amino acid sequence shown as SEQ ID No. 53.
19 the isolated antigen binding protein of any of embodiments 1-8 comprising HCDR3 in VH having an amino acid sequence shown as SEQ ID No. 53.
20 the isolated antigen binding protein of embodiment 7, wherein said HCDR1 comprises X 1 YX 2 MH, wherein X 1 Can be D or A, X 2 May be a or E.
The isolated antigen binding protein of embodiment 10, wherein said HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 4 and 12.
22 the isolated antigen binding protein of embodiment 8, wherein said HCDR2 comprises the amino acid sequence of SEQ ID No. 20.
23 the isolated antigen binding protein of embodiment 12, wherein said HCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 5, 10 and 13.
24 the isolated antigen binding protein of embodiment 9, wherein the HCDR3 comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 Shown in the figureAmino acid sequence, wherein X 1 Can be D or T, X 2 Can be H or R, X 3 Can be T or F, X 4 Can be I or Y, X 5 Can be G or S, X 6 Can be V or Y, X 7 Can be G or A, X 8 Can be A, Y or H, X 9 Can be F or blank, X 10 Can be D or blank, X 11 May be I or blank.
25 the isolated antigen binding protein of embodiment 14, wherein said HCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 6, 11 and 14.
The isolated antigen binding protein of any of embodiments 1-15, comprising LCDR1 in VL having an amino acid sequence as shown in SEQ ID No. 49.
The isolated antigen binding protein of any of embodiments 1-16, comprising LCDR2 in VL having an amino acid sequence as shown in SEQ ID No. 49.
The isolated antigen binding protein of any of embodiments 1-17, comprising LCDR3 in VL having an amino acid sequence as shown in SEQ ID No. 49.
29 the isolated antigen binding protein of embodiment 16, wherein said LCDR1 comprises X 1 X 2 X 3 X 4 SX 5 VX 6 X 7 X 8 X 9 YX 10 X 11 X 12 X 13 The amino acid sequence shown, wherein X 1 Can be T or R, X 2 Can be G or S, X 3 Can be T or S, X 4 Can be S or Q, X 5 Can be D or L, X 6 Can be G or H, X 7 Can be G or S, X 8 Can be Y or N, X 9 Can be N or G, X 10 Can be V or T, X 11 Can be S or Y, X 12 Can be blank or L, X 13 May be blank or H.
The isolated antigen binding protein of embodiment 19, wherein said LCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 1 and 7.
31 according to embodiment 17The isolated antigen binding protein, wherein the LCDR2 comprises X 1 X 2 SX 3 RX 4 S, wherein X 1 Can be D or K, X 2 Can be V or G, X 3 Can be N, Y or Q, X 4 May be P or G.
32 the isolated antigen binding protein of embodiment 21, wherein said LCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 2, 8 and 15.
33 the isolated antigen binding protein of embodiment 18, wherein said LCDR3 comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 The amino acid sequence shown, wherein X 1 Can be S or G, X 2 Can be S or Q, X 3 Can be Y or S, X 4 Can be A or G, X 5 Can be S or L, X 6 Can be G or T, X 7 Can be S or P, X 8 Can be T or P, X 9 Can be L or T, X 10 May be V or blank.
34 the isolated antigen binding protein of embodiment 23, wherein said LCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 3 and 9.
The isolated antigen binding protein of any of embodiments 1-24, comprising an antibody or antigen binding fragment thereof.
The isolated antigen binding protein of embodiment 25, wherein said antigen binding fragment comprises Fab, fab', F (ab) 2 Fv fragment, F (ab') 2 scFv, di-scFv and/or dAb.
The isolated antigen binding protein of any one of embodiments 1-26, wherein the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
38, wherein 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 any one of SEQ ID NOs 22 and 30.
39 according to any one of embodiments 27 to 28, wherein said L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 22 and 30.
40 the isolated antigen binding protein of embodiment 27, wherein said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises the amino acid sequence of any one of SEQ ID NOs 23 and 31.
41 the isolated antigen binding protein of any one of embodiments 27 and 30, wherein said L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 23 and 31.
The isolated antigen binding protein of embodiment 27, wherein said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises the amino acid sequence of any one of SEQ ID NOs 24 and 32.
The isolated antigen binding protein of any one of embodiments 27 and 32, wherein said L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 24 and 32.
44 the isolated antigen binding protein of embodiment 27, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3 and the L-FR4 comprises the amino acid sequence of any one of SEQ ID NOs 25 and 33.
45 the isolated antigen binding protein of any one of embodiments 27 and 34, wherein the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 25 and 33.
The isolated antigen binding protein of any one of embodiments 1-35, wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs 46-48.
47, which comprises an antibody light chain constant region, and which comprises a human igκ constant region.
48 the isolated antigen binding protein of embodiment 37, wherein said antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 54.
49 comprising an antibody light chain LC and said LC comprising the amino acid sequence shown in any one of SEQ ID NOs 56-58.
The isolated antigen binding protein of any one of embodiments 1-39, wherein said VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
51 the isolated antigen binding protein of embodiment 40, wherein 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 set forth in any one of SEQ ID NOs 26 and 34.
52 the isolated antigen binding protein of any of embodiments 40-41, wherein said H-FR1 comprises the amino acid sequence set forth in any of SEQ ID NOs 26 and 34.
53 the isolated antigen binding protein of embodiment 40, wherein said H-FR2 is located between said HCDR1 and said HCDR2 and said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 27 and 35.
54 the isolated antigen binding protein of any one of embodiments 40 and 43, wherein said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 27 and 35.
55, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of any one of SEQ ID NOs 28 and 36.
56 the isolated antigen binding protein of any one of embodiments 40 and 45, wherein said H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 28 and 36.
57 the isolated antigen binding protein of embodiment 40, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3 and the H-FR4 comprises the amino acid sequence of any one of SEQ ID NOs 29 and 37.
58 the isolated antigen binding protein of any one of embodiments 40 and 47, wherein said H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 29 and 37.
59 the isolated antigen binding protein of any one of embodiments 1-48, wherein said VH comprises the amino acid sequence set forth in any one of SEQ ID NOs 50-52.
60 the isolated antigen binding protein of any of embodiments 1-49, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
The isolated antigen binding protein of any of embodiments 1-50, comprising an antibody heavy chain constant region, and wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.
62 the isolated antigen binding protein of any of embodiments 1-51, wherein the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID No. 55.
63 the isolated antigen binding protein of any of embodiments 1-52, comprising an antibody heavy chain HC, and said HC comprises an amino acid sequence as set forth in any of SEQ ID NOs 59-61.
64 encoding the isolated antigen binding protein of any one of embodiments 1-53.
65 vector comprising the nucleic acid molecule according to embodiment 54.
66 cells comprising the nucleic acid molecule of embodiment 54 or the vector of embodiment 55.
67. a method of making the isolated antigen binding protein of any one of embodiments 1-53, the method comprising culturing the cell of embodiment 56 under conditions such that the isolated antigen binding protein of any one of embodiments 1-53 is expressed.
A pharmaceutical composition comprising the isolated antigen binding protein of any one of embodiments 1-53, the nucleic acid molecule of embodiment 54, the vector of embodiment 55, and/or the cell of embodiment 56, and optionally a pharmaceutically acceptable adjuvant.
69 the use of the isolated antigen binding protein of any one of embodiments 1-53, the nucleic acid molecule of embodiment 54, the vector of embodiment 55, the cell of embodiment 56, and/or the pharmaceutical composition of embodiment 58 in the manufacture of a medicament for preventing, alleviating and/or treating a tumor.
70 a method of preventing, alleviating or treating a tumor, the method comprising administering to a subject in need thereof the isolated antigen binding protein of any one of embodiments 1-53.
71 a method of detecting GPC3 in a sample, the method comprising administering the isolated antigen binding protein of any of embodiments 1 to 53.
The present application also provides the following embodiments:
1. an isolated antigen binding protein capable of binding to a GPC3 protein, the antigen binding protein comprising an antibody light chain or fragment thereof comprising LCDR1, LCDR2 and LCDR3 and an antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from the group consisting of:
(1)HCDR1：SEQ ID NO:12、HCDR2：SEQ ID NO:13、HCDR3：SEQ ID NO:14、LCDR1：SEQ ID NO:7、LCDR2：SEQ ID NO:8、LCDR3：SEQ ID NO:9；
(2) HCDR1: SEQ ID NO. 12, HCDR2: SEQ ID NO. 13, HCDR3: SEQ ID NO. 14, LCDR1: SEQ ID NO. 7, LCDR2: SEQ ID NO. 15, LCDR3: SEQ ID NO. 9; and
(3)HCDR1：SEQ ID NO:4、HCDR2：SEQ ID NO:5、HCDR3：SEQ ID NO:6、LCDR1：SEQ ID NO:1、LCDR2：SEQ ID NO:2、LCDR3：SEQ ID NO:3。
2. The isolated antigen binding protein of embodiment 1, having one or more of the following properties:
1) Can be 6×10 -9 M or lower K D Binding to GPC3 protein, wherein said K D The values were determined by Octet;
2) GPC3 proteins capable of specifically binding to the surface of HepG2 cells and/or Huh7 cells in FACS assays;
3) Can inhibit tumor growth and/or tumor cell proliferation.
3. The isolated antigen binding protein of embodiment 2, wherein the GPC3 protein comprises human GPC3 protein.
4. The isolated antigen binding protein of any of embodiments 2-3, wherein the human GPC3 protein comprises an amino acid sequence set forth in SEQ ID No. 74.
5. The isolated antigen binding protein of any of embodiments 2-4, wherein the tumor comprises a GPC 3-positive tumor.
6. The isolated antigen binding protein of embodiment 5, wherein the GPC 3-positive tumor comprises liver cancer.
7. The isolated antigen binding protein of any one of embodiments 1-6, comprising an antibody or antigen binding fragment thereof.
8. The isolated antigen binding protein of embodiment 7, wherein said antigen binding fragment comprises Fab, fab', F (ab) 2 Fv fragment, F (ab') 2 scFv, di-scFv and/or dAb.
9. The isolated antigen binding protein of any of embodiments 1-8, wherein the antibody light chain or fragment thereof comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
10. The isolated antigen binding protein of embodiment 9, wherein 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 any one of SEQ ID NOs 22 and 30.
11. The isolated antigen binding protein of any one of embodiments 9-10, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of any one of SEQ ID NOs 23 and 31.
12. The isolated antigen binding protein of any one of embodiments 9-11, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of any one of SEQ ID NOs 24 and 32.
13. The isolated antigen binding protein of any one of embodiments 9-12, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3 and the L-FR4 comprises the amino acid sequence of any one of SEQ ID NOs 25 and 33.
14. The isolated antigen binding protein of any one of embodiments 1-13, wherein the antibody light chain or fragment thereof comprises a light chain variable region VL, and wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs 46-48.
15. The isolated antigen binding protein of any of embodiments 1-14, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region.
16. The isolated antigen binding protein of embodiment 15, wherein said antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 54.
17. The isolated antigen binding protein of any of embodiments 1-16, wherein the antibody light chain comprises the amino acid sequence set forth in any of SEQ ID NOs 56-58.
18. The isolated antigen binding protein of any one of embodiments 1-17, wherein the antibody heavy chain or fragment thereof comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
19. The isolated antigen binding protein of embodiment 18, wherein 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 any one of SEQ ID NOs 26 and 34.
20. The isolated antigen binding protein of any one of embodiments 18-19, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises an amino acid sequence of any one of SEQ ID NOs 27 and 35.
21. The isolated antigen binding protein of any one of embodiments 18-20, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises an amino acid sequence of any one of SEQ ID NOs 28 and 36.
22. The isolated antigen binding protein of any one of embodiments 18-21, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3 and the H-FR4 comprises the amino acid sequence of any one of SEQ ID NOs 29 and 37.
23. The isolated antigen binding protein of any one of embodiments 1-22, wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 50 or 52.
24. The isolated antigen binding protein of any of embodiments 1-23, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
25. The isolated antigen binding protein of any of embodiments 1-24, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.
26. The isolated antigen binding protein of any of embodiments 24-25, wherein the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID No. 55.
27. The isolated antigen binding protein of any of embodiments 1-26, wherein the antibody heavy chain comprises the amino acid sequence set forth in SEQ ID NO 59 or 61.
28. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of embodiments 1-27.
29. A vector comprising the nucleic acid molecule of embodiment 28.
30. A cell comprising the nucleic acid molecule of embodiment 28 or the vector of embodiment 29.
31. A method of making the isolated antigen binding protein of any one of embodiments 1-27, comprising culturing the cell of embodiment 30 under conditions such that the isolated antigen binding protein of any one of embodiments 1-27 is expressed.
32. A pharmaceutical composition comprising the isolated antigen binding protein of any one of embodiments 1-27, the nucleic acid molecule of embodiment 28, the vector of embodiment 29 and/or the cell of embodiment 30, and optionally a pharmaceutically acceptable adjuvant.
33. Use of the isolated antigen binding protein of any one of embodiments 1-27, the nucleic acid molecule of embodiment 28, the vector of embodiment 29, the cell of embodiment 30 and/or the pharmaceutical composition of embodiment 32 in the manufacture of a medicament for preventing, alleviating and/or treating a tumor.
34. A method of detecting GPC3 in a sample, the method comprising administering the isolated antigen binding protein of any of embodiments 1 to 27.
Examples
EXAMPLE 1 screening of anti-GPC 3 antibodies Using phage antibody library
Recombinant human GPC3 protein (Shanghai primary energy cell medical technology Co., ltd., amino acid sequence shown as SEQ ID NO: 74) is taken as antigen, and phage natural humanized antibody library (Shanghai primary energy cell medical technology Co., ltd.) is sorted, and four rounds of sorting are carried out together. Antigen GPC3 protein 1ml was coated in ELISA using CBS buffer at GPC3 concentration of 20. Mu.g/ml (first, second round) or 10. Mu.g/ml (third, fourth round) overnight at 4 ℃; the immune tube is sealed by 2ml of PBS buffer solution containing 10% of skimmed milk powder for the next time; 1ml of blocked phage (phage) was added to the immune tube and incubated for 1h at room temperature; PBST wash 10 times (first, second) or 15 times (third, fourth); adding 800. Mu.l of Gly-HCl buffer solution with pH of 2.2, eluting, immediately adding 400. Mu.l of Tris-HCl buffer solution with pH of 8.0 for neutralization; adding into 20ml of E.coli strain E.coli SS320 in logarithmic growth phase (optical density value OD about 0.8), mixing, and standing at 37deg.C for 1 hr; 500 μl was removed for phage titer determination and glycerol for bacteriostasis; coating the rest bacterial liquid on a flat plate, and culturing overnight at 37 ℃ in an incubator; the next day, scraping the flat plate bacteria, inoculating the flat plate bacteria into 80ml of 2YT-Amp culture medium according to a certain proportion, enabling the OD to be equal to 0.2, adding 160 mu l of helper phage (helper phage) when the OD reaches 0.8 after a plurality of hours of culture, and then uniformly mixing and standing for 1h at 37 ℃; adding isopropyl thiogalactoside (IPTG) and Kan antibiotics, shaking and culturing at 250rpm and 30 ℃ overnight; collecting the supernatant, precipitating phage with PEG/NaCl solution, and re-suspending the precipitated phage in 1.5ml PBS buffer; the resuspended phage was used for the next round of enrichment screening, after 4 rounds of panning, significant enrichment was observed. The phage antibody clone obtained by panning is identified by ELISA, and the specific method is as follows: human GPC3 protein (Shanghai Primary energy cell medicine technologies Co., ltd.) was coated on 96-well ELISA plates at a concentration of 1. Mu.g/ml overnight at 4 ℃. The non-specific binding sites were then blocked with 10% nonfat milk powder and after extensive washing, the monoclonal phage supernatant was added to a 96-well plate and incubated for 2 hours at 37 ℃. After extensive washing, anti-M13-HPR (GE healthcare, 27-9421-01) was added, reacted for 45min at 37℃and developed by adding TMB after extensive washing, reacted for 5-10min at room temperature, and finally the reaction was stopped with sulfuric acid, and the OD of each well was measured at 450 nm.
4 phage antibody clones (i.e., the isolated antigen binding proteins described herein) that specifically bind human GPC3 were obtained by ELISA identification, and after sequencing VH and VL gene sequences were obtained, these 4 phage antibody clones were designated 204A, L H2, L1H6, L2H6, respectively. SEQ ID NO. 62 and SEQ ID NO. 46 show the nucleic acid sequence and the amino acid sequence, respectively, of the light chain variable region VL of phage antibody clone 204A. SEQ ID NO. 65 and SEQ ID NO. 50 show the nucleic acid sequence and the amino acid sequence, respectively, of the heavy chain variable region VH of phage antibody clone 204A. SEQ ID NO. 63 and SEQ ID NO. 47 show the nucleic acid sequence and the amino acid sequence, respectively, of the light chain variable region VL of phage antibody clone L1H 2. SEQ ID NO. 66 and SEQ ID NO. 51 show the nucleic acid sequence and the amino acid sequence, respectively, of the VH of the heavy chain variable region of phage antibody clone L1H 2. SEQ ID NO. 63 and SEQ ID NO. 47 show the nucleic acid sequence and the amino acid sequence, respectively, of the light chain variable region VL of phage antibody clone L1H 6.SEQ ID NO. 67 and SEQ ID NO. 52 show the nucleic acid sequence and the amino acid sequence, respectively, of the VH of the heavy chain variable region of phage antibody clone L1H 6.SEQ ID NO. 64 and SEQ ID NO. 48 show the nucleic acid sequence and the amino acid sequence, respectively, of the light chain variable region VL of phage antibody clone L2H6.SEQ ID NO. 67 and SEQ ID NO. 52 show the nucleic acid sequence and the amino acid sequence, respectively, of the VH of the heavy chain variable region of the phage antibody clone L2H6.
EXAMPLE 2 expression and purification of anti-GPC 3 Whole antibodies
Phage antibody clone 204A, L H2, L1H6, L2H6 were redesigned to be intact IgG1, kappa antibody (i.e., the isolated antigen binding protein described herein) by: designing a primer to carry out PCR amplification on the VH cloned by the phage antibody, and cloning a PCR product to a pCMV-IgG1NDL vector subjected to AgeI and SalI double enzyme digestion through recombination; primers were designed to PCR amplify VL of phage antibody clones and the PCR products were cloned by recombination into the AgeI and BsiWI double digested pCMV-kappa vector. After the sequencing is correct, the heavy chain expression vector and the light chain expression vector are transfected into 293F cells for transient expression, and purified by a ProteinA column to obtain 204A, L H2, L1H6 and L2H6 complete IgG1 and kappa antibodies. SEQ ID NO. 68 and SEQ ID NO. 56 show the nucleic acid sequence and amino acid sequence of the 204A whole antibody light chain, and SEQ ID NO. 71 and SEQ ID NO. 59 show the nucleic acid sequence and amino acid sequence of the 204A whole antibody heavy chain. SEQ ID NO. 69 and SEQ ID NO. 57 show the nucleic acid sequence and amino acid sequence of the L1H2 complete antibody light chain, and SEQ ID NO. 72 and SEQ ID NO. 60 show the nucleic acid sequence and amino acid sequence of the L1H2 complete antibody heavy chain. SEQ ID NO. 69 and SEQ ID NO. 57 show the nucleic acid sequence and amino acid sequence of the L1H6 complete antibody light chain, and SEQ ID NO. 73 and SEQ ID NO. 61 show the nucleic acid sequence and amino acid sequence of the L1H6 complete antibody heavy chain. SEQ ID NO. 70 and SEQ ID NO. 58 show the nucleic acid sequence and amino acid sequence of the L2H6 complete antibody light chain, and SEQ ID NO. 73 and SEQ ID NO. 61 show the nucleic acid sequence and amino acid sequence of the L2H6 complete antibody heavy chain.
Example 3 detection of binding affinity of intact antibodies against GPC3
The binding affinity of 204A, L H2, L1H6, L2H6 whole antibodies obtained in example 2 against recombinant human GPC3 protein was measured using an Octet RED384 instrument (Pall ForteBio). First, recombinant human GPC3 protein (i.e., antigen) was labeled with Biotin (EZ-Link Sulfo-NHS-LC-Biotin, pierce, 21327). Binding kinetics analysis between antigen and antibody was performed by biofilm interference (BLI) technique using a molecular interaction analyzer fortebioctetred 384 (sall) (PBS buffer with 0.1% bsa and 0.02% tween 20 for both antigen and antibody dilutions). Immobilized with SA sensor using biotin-conjugated antigen at a concentration of 50nM, 1500rpm/min, 10min; then re-diluted with a para-diluted antibody (i.e.204A obtained in example 2)L1H2, L1H6, L2H6 whole antibody) solution was bound for 10min at 1500rpm/min. Finally, dissociation was carried out for 10 minutes at 1500rpm/min. The remaining antibody will be regenerated by glycine pulses. The obtained results were subjected to data analysis by OctetDataAnalysis9.0 software (fortebio) to calculate the binding strength of antigen and antibody to obtain K D Values, ka (1/Ms) and Kd (1/s) values. The anti-GPC 3 intact antibodies 204A, L H2, L1H6, L2H6 were all capable of high affinity binding to recombinant human GPC3 protein (as shown in table 1).
TABLE 1 binding affinity of anti-GPC 3 intact antibodies to human GPC3 protein
Figure BDA0003772909260000331
In table 1, ka: a binding rate constant; kd: dissociation rate constant; k (K) D : affinity constant, equal to Kd/Ka.
Example 4 epitope analysis of intact antibodies against GPC3
Specific positions of epitopes of L1H2, L1H6 and L2H6 antibodies were determined, and the sequence of GPC3-His protein was subjected to site-directed mutagenesis by PCR site-directed mutagenesis (Tiangen, KM 101). The anti-GPC 3 antibody and the murine GPC3 antibody do not have cross reaction, and different sites of wild-type human GPC3 protein and murine GPC3 protein are mutated into amino acids corresponding to the murine one by one, respectively, and 11 sites are used as shown in FIG. 1. These 11 mutated GPC3-His muteins and GPC-His eukaryotic expression were then each carried out to obtain the corresponding proteins. The baseline for the above 12 proteins was matched using Anti-6 XHis-HRP (Abcam, ab 1187). Then coating 1ug/ml of each of the 12 proteins in equal amount, and overnight at 4 ℃; after the next day of blocking with 5% nonfat milk powder (Bio, A600669), the primary antibodies were added to L1H2, L1H6, L2H6 and GC33 antibodies (4 ug/ml initial concentration, 4-fold gradient dilution, total 7 wells, last well with PBS), reacted for one hour, and the secondary antibodies were used with goat anti-human IgG HRP (Abcam, ab 97225), 1:8000. Wherein GC33 represents a control antibody (see patent document titled anti-glypican 3 antibody, bulletin number CN 1842540B). After the reaction is carried out for 45min at 37 ℃, TMB is added for color development after full washing, and after the reaction is carried out for 5-10min at room temperature, the reaction is stopped by sulfuric acid, and OD value of each hole is measured at 450 nm.
The statistical results are shown in FIGS. 2A-2B. The results showed that when wild-type human GPC3 protein (human Wildtype human GPC 3) was used, L1H2 bound slightly more strongly than GC33 antibody. When phenylalanine of human GPC3 was mutated to serine (as shown in mutant 7), L1H2 and L2H6 binding to GPC-His muteins was greatly reduced, while L1H6 and GC33 were less affected. When human GPC3 was mutated to glutamine (as shown in mutant 5), L2H6 binding to GPC-His mutein was attenuated, with the other three antibodies being less affected. It was shown that the epitope for L1H2 is located in the vicinity of phenylalanine, while the epitope for GC33 is located within the sequence of KDNEIST from the C-terminus of GPC 3.
EXAMPLE 5 detection of binding of anti-GPC 3 intact antibodies to cell surface GPC3
Cell surface target antigen (GPC 3) binding affinity detection with antibodies (L1H 2, L1H6, L2H6 whole antibodies obtained in example 2) was performed by flow cytometry using an iQue screen flow machine (purchased from intelllicyt corporation) using PBS containing 0.1% bsa as buffer, as follows:
1. concentration was 1 x 10 using buffer 6 Target cells of cells/ml (i.e., hepG2 hepatoma cells, huh7 hepatoma cells or L02 hepatoma cells) were added to a 96 Kong Jian bottom plate (burning 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 a 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 analytical results of the flow affinity binding experiments are shown in table 2, in which GC33 represents a control antibody (see patent document titled anti-glypican 3 antibody, publication No. CN 1842540B), and the expression purification method is the same as in example 2.
TABLE 2 binding results of anti-GPC 3 intact antibodies to cell surface GPC3 proteins
Figure BDA0003772909260000351
As can be seen from Table 2, the streaming binding capacity of L1H2, L2H6 and L1H6 intact antibodies on the surface of HepG2 liver cancer cells expressing high GPC3 and Huh7 liver cancer cells expressing medium GPC3 was higher than that of GC33 antibody.
EXAMPLE 6 ADCC Activity detection of anti-GPC 3 Whole antibody
ADCC activity of anti-GPC 3 intact antibodies (i.e., L1H2, L1H6 and L2H6 intact antibodies) against GPC 3-highly expressed hepatoma cell line HepG2 was demonstrated by
Figure BDA0003772909260000358
Luciferase assay System (+)>
Figure BDA0003772909260000359
Luciferase Assay system) are tested.
The specific treatment process of the cell and antibody sample is as follows:
1. HepG2 cells (HepG 2-luc) stably expressing luciferase (luciferases) were cultured and expanded in DMEM (GIBICO) complete medium containing 10% FBS (GIBICO), and 1.95x10 after digestion of the cells with 0.25% pancreatin (GIBICO) 6 Cells were resuspended to 13ml with X-VIVO (Lonza) medium to form 1.5X10 5 The cell suspension was kept in suspension/ml for use.
2. 2ml of the single blood was diluted three times to 6ml with PBS, spread on top of 5ml of ficoll (GE) separating liquid, centrifuged at 500g for 30 minutes, and the white film was taken, washed 2 times with 10ml of PBS and centrifuged at 500g for 5 minutes each. Take 5x10 7 The obtained PBMC cells were resuspended to 6.9ml with X-VIVO (Lonza) medium to form 7.2X10 6 The cell suspension was kept in suspension/ml for use.
3. The L1H2, L1H6 and L2H6 whole antibodies were diluted to 30. Mu.g/ml with X-VIVO (Lonza) medium, followed by 10-fold dilution in sequence, yielding 3. Mu.g/ml, 0.3. Mu.g/ml, 0.03. Mu.g/ml antibody solutions for use.
4. Plating (96 well V bottom plate (Thermo)) was performed with 70. Mu.l HepG2-luc cell suspension per well and 70. Mu.l PBMC cell suspension per well and 70. Mu.l each antibody solution per well, two sub-wells per group, CO at 37 ℃ 2 Incubate in incubator for 5 hours.
5.70. Mu.l HepG2-luc cell suspension plus 70. Mu.l PBMC cell suspension and 70. Mu. l X-VIVO Medium wells and 210. Mu. l X-VIVO Medium wells were used as non-killing control wells and bottom wells, two auxiliary wells per group, CO at 37℃respectively 2 Incubate in incubator for 5 hours.
Figure BDA0003772909260000352
Luciferase assay System (+)>
Figure BDA0003772909260000353
Luciferase Assay system) the specific detection process is as follows:
1. equilibrate the reagent to room temperature before use
Figure BDA0003772909260000354
Luciferase assay buffer (+.>
Figure BDA0003772909260000355
Luciferase Assay Buffer) resuspension->
Figure BDA0003772909260000356
Luciferase assay substrate (+)>
Figure BDA0003772909260000357
Luciferase Assay Substrate)。
2. The resuspended detection reagent was plated at 100. Mu.l/well in 96-well flat bottom white plates (thermo).
3. The experimental well plate was prepared from CO at 37 DEG C 2 The mixture was taken out of the incubator and allowed to stand for 10 minutes to equilibrate to room temperature.
4. The cells resuspended in each well were thoroughly blown with a lance to a uniform cell suspension.
5. Immediately taking 100 μl of the cell suspension into a whiteboard containing detection reagent with a row gun, and blowing and mixing
6. The mixture was left to stand in the dark for 10 minutes, and read by a Luminescence (Luminescence) detection program of a TECAN SPARK 10M enzyme-labeled detector.
7. The read values were analyzed:
killing% = 100X (no killing control Kong antibody experimental well)/no killing control
As shown in fig. 3, it can be seen that the L1H2, L1H6 and L2H6 complete antibodies are all capable of effectively mediating the effective killing of liver cancer cell line HepG2 with high expression of GPC3, i.e. all have significant ADCC activity.
EXAMPLE 7 anti-tumor Activity of L1H2 antibody against mouse model transplanted with human hepatoma
Huh7 cells were grown at 1X 10 7 A total of 20 BALB/c nude mice were inoculated subcutaneously on the right side at a concentration of 0.2mL, and groups of 16 random groups of 2 were selected according to tumor volume, 8 groups each, on day 6 of tumor inoculation. The administration route is tail vein injection, and Day0, 7, 14 and 28 are administered for 4 times after grouping, and the drug effect project data is Day 41. L1H2 antibody group was given 10mg/kg of L1H2 by tail vein, and the control group was given the same volume of PBS by tail vein. The body weight and tumor volume of the mice were measured 2-3 times per week during dosing and observation, and the measurements were recorded. At the end of the experiment, animals were euthanized and tumor volume changes were calculated.
In this experiment, the anti-tumor effect of the L1H2 antibody on a mouse model transplanted with human hepatoma was evaluated by the change of tumor volume with time. Tumor volumes were calculated by the following formula.
Tumor volume= (major axis) × (minor axis)/2
As shown in FIG. 4, the inhibition rate of tumor growth in mice was observed to be 59.67% in the L1H2 antibody group (L1H 2 10mg/kg IV) compared to the control group. Thus, the antibodies of the present application are shown to have anti-cancer effects on mouse models transplanted with human hepatomas.
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.

Claims (10)

1. An isolated antigen binding protein capable of binding to a GPC3 protein, the antigen binding protein comprising an antibody light chain or fragment thereof comprising LCDR1, LCDR2 and LCDR3 and an antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from the group consisting of:
(1)HCDR1：SEQ ID NO:12、HCDR2：SEQ ID NO:13、HCDR3：SEQ ID NO:14、LCDR1：SEQ ID NO:7、LCDR2：SEQ ID NO:8、LCDR3：SEQ ID NO:9；
(2) HCDR1: SEQ ID NO. 12, HCDR2: SEQ ID NO. 13, HCDR3: SEQ ID NO. 14, LCDR1: SEQ ID NO. 7, LCDR2: SEQ ID NO. 15, LCDR3: SEQ ID NO. 9; and
(3)HCDR1：SEQ ID NO:4、HCDR2：SEQ ID NO:5、HCDR3：SEQ ID NO:6、LCDR1：SEQ ID NO:1、LCDR2：SEQ ID NO:2、LCDR3：SEQ ID NO:3。
2. the isolated antigen binding protein of claim 1, wherein the antibody light chain or fragment thereof comprises a light chain variable region VL, and wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs 46-48.
3. The isolated antigen binding protein of any one of claims 1-2, wherein the antibody light chain comprises the amino acid sequence set forth in any one of SEQ ID NOs 56-58.
4. The isolated antigen binding protein of any one of claims 1-3, wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID No. 50 or 52.
5. The isolated antigen binding protein of any one of claims 1-4, wherein the antibody heavy chain comprises the amino acid sequence set forth in SEQ ID NO 59 or 61.
6. An isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-5.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.
9. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-5, the nucleic acid molecule of claim 6, the vector of claim 7 and/or the cell of claim 8, and optionally a pharmaceutically acceptable adjuvant.
10. Use of the isolated antigen binding protein of any one of claims 1-5, the nucleic acid molecule of claim 6, the vector of claim 7, the cell of claim 8 and/or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the prevention, alleviation and/or treatment of a tumor.
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CN108659129B (en) * 2018-05-17 2021-08-03 新疆大学 Nanometer antibody for resisting GPC3 protein, and preparation method and application thereof

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