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CN118126181B - Human and monkey cross species anti-CCR 8 membrane protein antibodies - Google Patents

Human and monkey cross species anti-CCR 8 membrane protein antibodies Download PDF

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CN118126181B
CN118126181B CN202410136386.2A CN202410136386A CN118126181B CN 118126181 B CN118126181 B CN 118126181B CN 202410136386 A CN202410136386 A CN 202410136386A CN 118126181 B CN118126181 B CN 118126181B
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antibody
antigen
binding fragment
ccr8
ccr
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CN118126181A (en
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徐万熙
程智
陈诗诚
封赵杰
何怀瑜
冉晓园
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Kaikai Biotechnology Shanghai Co ltd
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Abstract

The invention provides a human and monkey cross species anti-CCR 8 membrane protein antibody, and also provides a preparation method and application of the antibody. According to the invention, the N-terminal 1-35AA epitope of the CCR8 protein is displayed in a non-envelope VLP multivalent mode for the first time, and is subjected to expression by a mammalian cell to obtain post-translational modification with important functions, and the post-translational modification is used as an antigen protein for immunization, so that a monoclonal antibody is obtained through screening. The antibody has the binding activity of cross species of human and monkey, has high-level binding affinity, and has potential for antibody drug development after humanized transformation.

Description

Human and monkey cross species anti-CCR 8 membrane protein antibodies
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-CCR 8 membrane protein antibody of cross species of human and monkey, and a preparation method and application of the antibody.
Background
CCR8 consists of 355 amino acids (Uniprot ID: P51685), is a seven transmembrane G-protein coupled receptor (GPCR), and the ligand for CCR8 includes four chemokines CCL1, CCL8, CCL16 and CCL18. Chemokines can be divided into four subfamilies, CXC, CC, C (or XC) and CX3C, based on the arrangement of the N-terminal cysteines of the proteins. Chemokine receptor GPCRs are also correspondingly divided into four subfamilies: CXCR, CCR, CR, CX3CR. Chemokines and their receptors are critical for tumor cell migration, invasion and metastasis. CCR8 is specifically highly expressed as a C-C subfamily chemokine receptor in tumor-infiltrated Tregs, as well as on Th2 cells and tumor cells, while it is not substantially expressed in peripheral blood Tregs or normal tissues. CCR8 promotes immune escape of tumor cells by mediating recruitment of Treg and Th2 cells to inflammation and tumor sites through interactions with its ligands. CCL1 is the primary ligand for CCR8, CCR8 being the only receptor for CCL 1. CCL1 secreted by tumor stem cells plays a role by acting on CCR8 receptors on the surfaces of different cells, CCL1 activates the CCR8 receptors on cancer cells, and proliferation, apoptosis resistance and migration of the cancer cells are promoted; CCL1 promotes tumor angiogenesis by activating CCR8 receptors on endothelial cells; CCL1 mediates the conversion of cd4+ T cells to tregs.
CCR8 is a tumor immune target with great potential, and the medicine targeting CCR8 can achieve the tumor treatment effect by depleting FOXp3+/CCR8+ Treg cells infiltrated by tumors or blocking CCL1/CCR8 pathways. There are many drug enterprises at home and abroad which are designed to target CCR8 monoclonal antibody drugs. However, it is very difficult to obtain sufficient amounts of GPCRs, especially CCR8 membrane proteins, for immunization and screening. Displaying full-length membrane proteins as enveloped virus-like particles (VLPs), or displaying membrane protein epitopes as non-enveloped VLPs, is an effective way of preparing membrane proteins or other antigens. VLP particles display multivalent membrane protein molecules or epitopes and VLPs are themselves capable of producing the effect of immunoadjuvants and are therefore widely used in the vaccine field [1].
The literature indicates that the N-terminus of the extracellular region of CCR8 (1-35 AA) and 3 ECL loops are involved in ligand binding, especially post-translational modifications of the N-terminal sequence, including the sulfoation of tyrosine residues (Tyr), and the glycosylation of asparagine (Asn) both affect the binding activity of ligand CCL1 [2,3]. The N-terminal (1-35 AA) sequence of CCR8 is therefore also an important epitope for antibody targeting.
In view of the many difficulties faced in developing CCR8 antibodies at present, there is a need in the art to develop efficient methods for developing CCR8 antibodies to obtain CCR8 antibodies with better performance for development of antibody drugs.
Disclosure of Invention
The invention aims at providing an antibody for resisting CCR8 membrane protein, a method for preparing the antibody and application of the antibody.
In a first aspect of the invention there is provided an anti-CCR 8 antibody or antigen binding fragment thereof, said antibody comprising a heavy chain variable region comprising the following 3 CDRs:
H-CDR1 shown in SEQ ID NO. 7;
H-CDR2 shown in SEQ ID NO. 8;
H-CDR3 shown in SEQ ID NO 9; and
The light chain variable region comprises the following 3 CDRs:
L-CDR1 shown in SEQ ID NO. 13;
L-CDR2 with sequence shown as KVS;
The L-CDR3 shown in SEQ ID NO. 14.
In another preferred embodiment, the heavy chain variable region and the light chain variable region further comprise an FR region.
In another preferred embodiment, the heavy chain variable region comprises an FR region of murine or human origin and/or the light chain variable region comprises an FR region of murine or human origin.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown as SEQ ID NO. 6, or an amino acid sequence having at least 90% (preferably at least 95%,96%,97%,98%, 99%) sequence identity thereto.
In another preferred embodiment, the light chain variable region of the antibody comprises the amino acid sequence shown as SEQ ID NO. 12, or an amino acid sequence having at least 90% (preferably at least 95%,96%,97%,98%, 99%) sequence identity thereto.
In another preferred embodiment, the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 6 and/or a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 12.
In another preferred embodiment, the antibody further comprises a heavy chain constant region and/or a light chain constant region.
In another preferred embodiment, the heavy chain constant region and/or the light chain constant region is murine or human.
In another preferred embodiment, the heavy chain constant region is derived from a mouse heavy chain IgG1 and/or the light chain constant region is derived from a mouse kappa (kappa) chain.
In another preferred embodiment, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 10.
In another preferred embodiment, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 15.
In another preferred embodiment, the antibody comprises: monoclonal antibodies, polyclonal antibodies, diabodies, single chain antibodies (scFv), fab ', F (ab') 2 antibodies.
In another preferred embodiment, the antibody is a recombinant antibody.
In another preferred embodiment, the antibody is a recombinant monoclonal antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO. 1 and a light chain having an amino acid sequence as shown in SEQ ID NO. 2.
In another preferred embodiment, the antibodies include animal-derived antibodies (e.g., murine antibodies of different subtypes), chimeric antibodies (e.g., human-murine chimeric antibodies), humanized antibodies.
In another preferred embodiment, the antibody specifically binds to human CCR8 and cynomolgus CCR8, having human and monkey cross-species binding activity.
In a second aspect of the present invention, there is provided a recombinant antibody having:
(i) The sequence of an anti-CCR 8 antibody or antigen binding fragment thereof according to the first aspect of the invention; and
(Ii) Optionally a signal peptide that facilitates secretory expression of the antibody and/or a tag sequence for purification and detection.
In another preferred embodiment, the recombinant antibody has a signal peptide that promotes secretory expression of the antibody.
In another preferred embodiment, the signal peptide has the amino acid sequence shown in SEQ ID NO. 5 or 11.
In another preferred embodiment, the tag sequence is selected from the group consisting of: FLAG, myc, his tags, etc.
In another preferred embodiment, the recombinant antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO. 1 and a light chain having an amino acid sequence as shown in SEQ ID NO. 2.
In a third aspect of the invention there is provided a polynucleotide molecule encoding a polypeptide selected from the group consisting of:
(1) An anti-CCR 8 antibody or antigen-binding fragment thereof according to the first aspect of the invention; or (b)
(2) The recombinant antibody according to the second aspect of the invention.
In another preferred embodiment, the polynucleotide molecule comprises a nucleotide sequence as set forth in SEQ ID NO. 3 and/or SEQ ID NO. 4.
In a fourth aspect of the invention there is provided an expression vector comprising a polynucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the vector comprises a eukaryotic cell expression vector, a prokaryotic cell expression vector.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.
In a fifth aspect of the invention there is provided a host cell comprising a vector according to the fourth aspect of the invention, or having incorporated into its genome a polynucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the host cell comprises a eukaryotic cell (e.g., a mammalian cell), a prokaryotic cell.
In a sixth aspect of the invention, there is provided an antibody conjugate comprising:
(a) An anti-CCR 8 antibody or antigen-binding fragment thereof according to the first aspect of the invention or a recombinant antibody according to the second aspect of the invention; and
(B) A coupling moiety selected from the group consisting of: detectable labels, enzymes, drugs, toxins, cytokines, radionuclides, nanoparticles/nanorods.
In another preferred embodiment, the detectable label comprises a fluorescent label or a chemiluminescent label.
In another preferred embodiment, the radionuclide comprises: isotopes for diagnosis; and/or therapeutic isotopes.
In another preferred embodiment, the coupling moiety is a drug or a toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
In another preferred embodiment, the antibody conjugate is an Antibody Drug Conjugate (ADC).
In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(a) An anti-CCR 8 antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant antibody according to the second aspect of the invention, or an antibody conjugate according to the sixth aspect of the invention; and
(B) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of an injection.
In another preferred embodiment, the pharmaceutical composition is used for the preparation of a medicament for the prevention and/or treatment of a disease or disorder associated with CCR 8.
In another preferred embodiment, the CCR8 related disease or disorder is cancer or tumor.
In another preferred embodiment, the cancer or tumor is a solid tumor.
In another preferred embodiment, the cancer or tumor includes, but is not limited to: colorectal cancer, cervical cancer, gastric cancer, and other solid tumors.
In an eighth aspect of the invention there is provided the use of an anti-CCR 8 antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant antibody according to the second aspect of the invention, or an antibody conjugate according to the sixth aspect of the invention for the preparation of:
(1) A medicament for the prevention and/or treatment of a CCR8 related disease or disorder;
(2) A detection reagent, a detection plate or a detection kit for detecting human or cynomolgus monkey CCR8 molecules.
In another preferred embodiment, the CCR8 related disease or disorder is cancer or tumor.
In another preferred embodiment, the cancer or tumor is a solid tumor.
In another preferred embodiment, the cancer or tumor includes, but is not limited to: colorectal cancer, cervical cancer, gastric cancer, and other solid tumors.
In another preferred embodiment, the assay comprises a flow assay, a cellular immunofluorescence assay, an ELISA assay.
In another preferred embodiment, the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
In a ninth aspect of the invention there is provided a method of producing an anti-CCR 8 antibody or antigen binding fragment thereof as described in the first aspect of the invention, or a recombinant antibody as described in the second aspect of the invention, comprising the steps of:
(s 1) culturing a host cell according to the fifth aspect of the invention under conditions suitable for the production of antibodies, thereby obtaining a culture comprising said antibodies;
(s 2) isolating or recovering the antibody from the culture; and
(S 3) optionally purifying and/or modifying the antibody obtained in step (b).
In a tenth aspect of the present invention, there is provided a method of detecting CCR8 protein in a sample, said method comprising the steps of:
(1) Contacting the sample with an anti-CCR 8 antibody or antigen-binding fragment thereof according to the first aspect of the invention, a recombinant antibody according to the second aspect of the invention, or an antibody conjugate according to the sixth aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CCR8 protein in the sample.
In another preferred embodiment, the sample comprises: an ex vivo tissue sample, a cell sample.
In another preferred embodiment, the aspect is an in vitro method.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
In an eleventh aspect of the invention, there is provided a method of preventing and/or treating a CCR8 related disease or disorder, the method comprising: administering to a subject in need thereof an anti-CCR 8 antibody or antigen binding fragment thereof as set forth in the first aspect of the invention, a recombinant antibody as set forth in the second aspect of the invention, or an antibody conjugate as set forth in the sixth aspect of the invention, or a pharmaceutical composition as set forth in the seventh aspect of the invention.
In another preferred embodiment, the subject in need thereof comprises a human or non-human mammal.
In another preferred embodiment, the CCR8 related disease or disorder is cancer or tumor.
In another preferred embodiment, the cancer or tumor is a solid tumor.
In another preferred embodiment, the cancer or tumor includes, but is not limited to: colorectal cancer, cervical cancer, gastric cancer, and other solid tumors.
In a twelfth aspect of the invention, there is provided a method of screening for an anti-CCR 8 antibody comprising the steps of:
(S1) expressing the extracellular region N-terminal 1-35AA epitope (hCCR 8 NTD) of human CCR8 in non-enveloped VLP multivalent display with mammalian cells, thereby obtaining the antigen protein hCCR8 NTD-VLP for immunization;
(S2) immunizing a test animal with the antigen protein hCCR8 NTD-VLP prepared in step (S1), and collecting antisera of the immunized animal;
(S3) detecting the binding of the collected antiserum to the antigen protein hCCR8 NTD-VLP, and if the binding is positive, taking the spleen of the immunized animal, and preparing hybridoma clones;
(S4) primary screening of hybridoma clones with the antigen protein hCCR8 NTD-VLP, thereby obtaining antigen-binding positive hybridoma clones;
(S5) further screening hybridoma clones binding thereto using a fusion protein hCR 8 NTD-hFc of hCR 8 NTD and human Fc fragment, thereby obtaining positive hybridoma clones producing an anti-CCR 8 antibody.
In another preferred embodiment, the method further comprises the steps of:
(S6) screening a monoclonal antibody having a CCR 8-positive expression cell binding function by detecting binding of the monoclonal antibody produced by the positive hybridoma clone to CCR 8-expressing cells; and
(S7) utilizing a biological membrane interference technology (BLI) to characterize the binding of monoclonal antibodies generated by positive hybridoma clones to human CCR8 and cynomolgus monkey CCR8 full-length protein nano-phospholipid disks (nanodisc), thereby screening monoclonal antibodies with human and monkey cross-species binding activity.
In another preferred embodiment, the amino acid sequence of hCR 8 NTD is shown in SEQ ID NO. 16.
In another preferred embodiment, the mammalian cell is selected from the group consisting of: HEK293 cells, CHO cells.
In another preferred embodiment, the subject animal is selected from the group consisting of: mice, rats, rabbits, goats, monkeys, etc.
In another preferred embodiment, the detection in step (S3) is an ELISA detection.
In another preferred embodiment, the screening in steps (S4) and (S5) is performed by indirect ELISA.
In another preferred embodiment, the CCR8 expressing cells in step (S6) may be selected from CCR8 expressing tumor cells, such as HuT-78 cells.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the results of cross-binding of the hCR 8 NTD-VLP1 antigen expressed in mammals and the immune antisera expressed in E.coli.
FIG. 2 shows ELISA screening of hybrid rumen polyclonal cell lines using human CCR8 NTD-Fc fusion protein as antigen.
FIG. 3 shows that 5C hybrid rumen polyclonal antibodies bind human CCR8 NTD-hFc and cynomolgus CCR8 NTD-hFc fusion proteins test species cross-binding.
FIG. 4 shows FACS diagram of recombinant monoclonal antibody 5C10 binding to HuT-78 tumor cell line.
FIG. 5 shows the cross-binding of BLI-characterized recombinant monoclonal antibody 5C10 to the species of human and monkey full-length CCR8 nanodisc. Wherein a and B show binding of recombinant monoclonal antibody 5C10 and control antibody 4a19 to human full length CCR8 nanodisc; c and D show binding of recombinant monoclonal antibody 5C10 and control antibody 4a19 to cynomolgus full length CCR8 nanodisc.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies, screening and preparing an anti-CCR 8 antibody having human and monkey cross-binding activity. According to the invention, the N-terminal 1-35AA epitope of the CCR8 protein is displayed in a non-envelope VLP multivalent mode for the first time, and the N-terminal 1-35AA epitope is subjected to post-translational modification with important functions through mammalian cell expression. The antigen protein is used for immunization. Screening to obtain a monoclonal antibody, named 5C10, which has cross-species binding activity on human and cynomolgus monkey full-length membrane protein CCR8 nano-phospholipid discs (nanodisc) and has high-level binding affinity. The 5C10 antibody and the humanized modified antibody have potential for developing antibody medicaments. On this basis, the present invention has been completed.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that there may be, but need not be, 1,2, or 3 antibody heavy chain variable regions of a particular sequence.
"Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%.
As used herein, the term "non-enveloped VLP multivalent display" refers to the structural fusion of an epitope to a VLP (Virus LIKE PARTICLE) capsid monomer subunit, the self-assembly of VLP monomers into VLP particles and the display of the epitope to the VLP surface upon expression by mammalian cells.
As used herein, the term "nanophospholipid disk (nanodisc)" refers to the stabilization of full-length membrane proteins and phospholipid molecules via encapsulation of amphiphilic polymers or scaffold proteins around the transmembrane region of the membrane protein, such that the full-length membrane protein forms particles of about 20nm in diameter and is capable of stable presence in aqueous solution.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer formed from two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH) linked to a heavy chain constant region. Each light chain is N-terminal to a variable region (VL) that is linked to a light chain constant region.
As used herein, the term "variable" means that the variable regions in an antibody differ in a particular sequence, resulting in the affinity and specificity of a particular antibody for binding to a particular antigen. Antibody variable regions include Complementarity Determining Regions (CDRs) or hypervariable regions (CDRs) and Framework Regions (FRs) that are more conserved in sequence. The primary sequence of the heavy and light chain variable region consists of 4 FR sequences and 3 CDR sequences arranged at intervals (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The sequence and spatial structural conformation of the heavy and light chain variable regions determine the specific binding of antibodies to epitopes. The antibody constant region does not directly participate in the binding of the antibody to the antigen, but has an effect on the performance of the capture antibody and the detection antibody.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of kappa and lambda based on the amino acid sequence of its constant region. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant region, mainly 5 classes of immunoglobulins: igA, igD, igE, igG and IgM, and antibody subtypes (isotypes), such as mouse IgG includes IgG1, igG2a, igG2b subtypes. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical. Monoclonal antibodies are highly specific for a single antigenic determinant (epitope). The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-CCR 8 monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-CCR 8 monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain comprising a variable region (complementarity determining region, CDR) provided that the variable region is identical or has at least 90% homology, preferably at least 95% homology, to the variable regions of the light chain and heavy chain of the invention.
As known to those skilled in the art, antibody conjugates and fusion expression products include: fluorescent or luminescent labels, radioactive labels, enzymes capable of producing a detectable product, gold nanoparticles/nanorods, and other molecules available for detection, bound to said anti-CCR 8 antibody or antigen binding fragment thereof; or a conjugate formed by combining a therapeutic molecule such as a therapeutic drug, a toxin, a radionuclide, or the like with the anti-CCR 8 antibody or antigen-binding fragment thereof.
The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond on the longer chain region; (iii) scFv fragments consisting of VH and VL domains of a single arm of an antibody.
The invention includes not only intact monoclonal antibodies, but also antibody fragments having binding activity, such as Fab or (Fab') 2 fragments; antibody heavy chain; antibody light chain or scFv.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen.
As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, the antibody includes murine antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art. The term "murine antibody" is herein a monoclonal antibody against CCR8 membrane protein prepared according to the knowledge and skill in the art.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH". The term "light chain variable region" is used interchangeably with "VL".
The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface.
In one aspect of the invention, an anti-CCR 8 antibody or antigen binding fragment thereof is provided. The invention obtains the cross-binding antibody of human and monkey species resisting CCR8 through mouse immune screening, and the clone number is 5C10.
Heavy chain amino acid sequence of 5C10 recombinant antibody (SEQ ID NO: 1):
wherein the bold underlined portion of the N-terminus is the signal peptide sequence (SEQ ID NO: 5); the middle part is the heavy chain variable region (SEQ ID NO: 6), and the underlined parts are the CDRs of the heavy chain variable region in order: H-CDR1 (SEQ ID NO: 7), H-CDR2 (SEQ ID NO: 8) and H-CDR3 (SEQ ID NO: 9), according to the IMGT rules; the heavy chain constant region (mouse IgG1, SEQ ID NO: 10) is italicized and bolded.
Light chain amino acid sequence of 5C10 recombinant antibody (SEQ ID NO: 2):
Wherein the bold underlined portion of the N-terminus is the signal peptide sequence (SEQ ID NO: 11); the middle part is the light chain variable region (SEQ ID NO: 12), and the underlined parts are the CDRs of the light chain variable region in order: L-CDR1 (SEQ ID NO: 13), L-CDR2 (KVS) and L-CDR3 (SEQ ID NO: 14), according to the IMGT rule; the italic bold portion is the light chain constant region (mouse Ig kappa, SEQ ID NO: 15).
The function of the antibodies of the invention is determined by the antibody light and heavy chain variable region sequences and the antibody structural conformation, and can specifically bind to CCR8 membrane proteins. Using the antibody variable region gene or Complementarity Determining Region (CDR) gene, different forms of engineered antibodies can be engineered and produced in any expression system utilizing prokaryotic and eukaryotic cells.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to an antibody that specifically binds to a CCR8 membrane protein, such as a protein or polypeptide having a heavy chain (the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3) and/or a light chain (the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4).
"Fragment", "derivative" and "analog" of an antibody refer to a polypeptide that retains substantially the same biological function or activity of an antibody of the invention. The polypeptide fragments, derivatives or analogues of the invention may be (i) polypeptides having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent in one or more amino acid residues, or (iii) polypeptides formed by coupling a mature polypeptide with another compound, such as a compound for chemiluminescence, for example an acridine ester, or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a tag protein sequence or other fusion protein sequence for purification or detection of the polypeptide. Such fragments, derivatives and analogs are within the purview of one skilled in the art.
The antibody of the present invention refers to a polypeptide having CCR8 membrane protein binding activity and comprising the CDR regions described above. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained using antisera raised against the antibodies of the invention.
The invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
Polynucleotide molecules, vectors and host cells
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence set forth in SEQ ID NO. 3 or 4 or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence encoding a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differing from the coding region sequences set forth in SEQ ID NO. 3 or 4.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences. The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. In addition, the heavy or light chain may be fused to a protein or tag sequence (e.g., fluorescent protein, flag tag) to form a fusion protein.
Once the antibody sequences are obtained, recombinant antibody production can be performed using bioengineering methods. Typically, the gene encoding the antibody sequence is cloned into a vector, the expression vector is transferred into cells for expression, the cells or the expression supernatant is harvested, and the recombinant antibody is obtained by purification. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence may then be introduced into various existing plasmids (or other expression vectors) known in the art. In addition, mutations can be introduced into the antibody sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
The host cell is transformed with the recombinant DNA and the recombinant expression of the antibody is a routine technique well known to those skilled in the art. The recombinantly expressed antibodies may be isolated and purified by conventional techniques well known to those skilled in the art and are not described in detail herein.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof, or fusion protein thereof, or immunoconjugate thereof, as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration.
The pharmaceutical compositions of the invention can be used directly to bind CCR8 protein molecules and thus can be used to treat CCR8 related diseases, such as cancer and the like. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. Furthermore, the antibodies or immunoconjugates of the invention may also be used with other therapeutic agents.
In one embodiment of the invention, where a pharmaceutical composition is used, a safe and effective amount of an antibody or immunoconjugate of the invention is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Application of
The antibody of the invention has wide biological application value and clinical application value, and the application thereof relates to a plurality of fields of diagnosis and treatment, basic medical research, biological research and the like of diseases related to CCR 8. One preferred application is for clinical diagnosis and targeted therapy against CCR8, such as treatment and diagnosis against CCR 8-highly expressed cancers or tumors, especially CCR 8-highly expressed solid tumors including but not limited to colorectal, cervical, gastric, and other solid tumors, and the like.
The invention has the main advantages that:
(1) The invention adopts VLP to display antigen epitope hCR 8 NTD (a 35AA polypeptide sequence), and displays hCR 8 NTD as non-envelope VLP, and the VLP has the effect of immunological adjuvant, so that mice can generate stronger immunological response.
(2) The hCR 8 NTD-VLP antigen protein is expressed by using mammalian cells, so that the hCR 8 NTD can obtain posttranslational modification with important functions, and the antibody obtained by adopting the antigen protein for postimmunization screening can effectively bind to CCR8 membrane protein expressed on tumor cells.
(3) The anti-CCR 8 antibodies of the present invention have cross species binding activity to CCR8 full length membrane protein nanodisc of humans and cynomolgus monkeys and thus can be used for animal experiments in the development of antibody drugs.
(4) The anti-CCR 8 antibody has high affinity with full-length CCR8 nanodisc of human and cynomolgus monkey, which is characterized by biological membrane interference (BLI), and reaches 16.2nM and 36.2nM respectively.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. Experiments in which specific conditions are not noted in the examples or test cases of the present invention are generally conducted under conventional conditions or under conditions recommended by the raw material/commodity manufacturer; the reagents of specific origin are not noted and are commercially available conventional reagents.
EXAMPLE 1 hCR 8 NTD-VLP immunization and antiserum detection
The N-terminal 1-35AA epitope (hCR 8NTD, SEQ ID NO: 16) of the extracellular region of human CCR8 is displayed in a non-envelope VLP multivalent manner, antigen proteins are constructed, and are expressed by mammalian cells and escherichia coli respectively, so that hCR 8NTD-VLP1 and hCR 8NTD-VLP 2 are obtained.
The sequence of the extracellular region N-terminal 1-35AA epitope of human CCR8 (hCR 8 NTD, SEQ ID NO: 16):
MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK
1.1 immunization of mammalian cell expressed non-enveloped hCCR8 NTD-VLP 1: primary immunization was performed with 100ul of hCR 8 NTD-VLP1 (estimated total protein concentration 0.2 mg/ml) mixed with an equal volume of aluminum hydroxide adjuvant and muscle multi-injected into 6 week female Balb/C mice. The booster immunization was the same as the primary immunization dose method, and the immunization was performed at two-week intervals for a total of five times. Antisera anti-serum1 generated after immunization of mice were collected.
1.2 Immunization of E.coli expressed non-enveloped hCR 8 NTD-VLP 2: primary immunization was performed using 50ul hCR 8 NTD-VLP2 (estimated total protein concentration 0.4 mg/ml) mixed with an equal volume of aluminum hydroxide adjuvant and muscle multi-injected into 6 week female Balb/C mice. The booster immunization was the same as the primary immunization dose method, and the immunization was performed at two-week intervals for a total of five times. Antisera anti-serum2 generated after immunization of mice were collected.
1.3 Antiserum potency assay: the antisera anti-ser 2 and anti-ser 1 were cross-conjugated with hCCR8 NTD-VLP1 and hCCR8 NTD-VLP2 antigen-coated plates, respectively. Antisera were diluted 100-fold and added to ELISA plate binding antigen at 3-fold gradient dilution. The ELISA binding signal was derived from antibodies in serum that specifically bound hCCR8 NTD epitope, but not VLP, because of the different species and sequences of VLP1 and VLP2 that assembled the above antigen proteins. As shown in FIG. 1, immunization of anti-serum1, i.e., hCR 8 NTD-VLP1 from mammalian cell expression, produced antibodies that bound the hCR 8 NTD epitope, whereas immune antisera anti-serum2 from E.coli expression bound hCR 8 NTD with hCR 8 NTD. The mice producing anti-serum1 are selected for further hybridoma cell fusion and clone screening.
Example 2 selection of hybridoma cell lines
Mice were immunized with non-enveloped hCCR8 NTD-VLP1 expressed by mammalian cells in example 1, and boosted by intraperitoneal injection using 100ul hCCR8-NTD-VLP1 diluted to 300ul with 1 x PBS prior to cell fusion. The spleen of the mouse is taken to be subjected to cell fusion with PEG1500, after the fused cells are cultured for 10 days, hCR 8-NTD-VLP1 ul/hole is coated on a polyvinyl chloride plate, and antigen binding positive hybrid rumen polyclonal is initially screened by an indirect ELISA. Further using human CCR8 NTD-hFc fusion protein to test binding to hybridoma polyclonal supernatants, FIG. 2 shows polyclonal cell lines [1#,2#,3#,4#,5#,6#,5C ] and anti-ser 1 positive controls, capable of binding to human CCR8 NTD-hFc fusion protein, wherein 5C has the highest binding activity. FIG. 3 shows that the antigen binding activity of polyclonal cell line 5C was tested by indirect ELISA method by coating polyvinyl chloride plates with 1ug/ml of human CCR8 NTD-hFc and cynomolgus monkey CCR8 NTD-hFc fusion proteins as antigens (wherein the amino acid sequence of cynomolgus monkey CCR8 NTD is shown as SEQ ID NO: 17), indicating that polyclonal cell line 5C has human and cynomolgus monkey cross-species binding activity.
The extracellular region N-terminal 1-35AA epitope (cynoCCR NTD, SEQ ID NO: 17) sequence of cynomolgus monkey CCR 8:
MDYTLDPSMTTMTDYYYPDSLSSPCDGELIQRNDK
5C polyclonal hybridoma cells are subjected to expansion culture and frozen storage after limited dilution to a monoclonal state. The expanded monoclonal cell supernatants were again validated by ELISA to confirm that the 5C10 monoclonal cell lines had cross-binding activity to human and monkey species. The antibody subtype heavy chain was determined to be mouse IgG1 and the light chain was determined to be mouse Ig kappa using Wu Hansan hawk biotechnology Co., ltd subtype identification kit.
EXAMPLE 3 sequencing of hybridoma monoclonal antibodies and purification of recombinant antibody expression
The 5C10 hybridoma cell line was cultured to a cell density of 1E+07cell/ml and the hybridoma monoclonal antibodies were sequenced (Sony Biotech Co., ltd.). Gene synthesis encodes signal peptide, variable region and constant region antibody heavy chain and light chain gene sequences (SEQ ID NO:3 and SEQ ID NO: 4), and constructs into mammalian cell expression vectors respectively, wherein the expression vectors can be selected from any mammalian cell expression commercialized vectors such as pTT5, pCDNA3.1 and the like. Recombinant plasmid transfects an Expi293 mammalian cell, secretes and expresses an antibody molecule, and cell expression supernatant is subjected to protein A affinity purification to obtain a 5C10 recombinant antibody.
Heavy chain nucleotide sequence of 5C10 recombinant antibody (SEQ ID NO: 3):
ATGAAGTGTAGTTGGGTTATCTTCTTTCTCATGGCCGTGGTAACAGGAGTGAACTCAGAAGTTCAGCTTCACCAGAGCGGAGCGGAACTTGTTAAGCCCGGAGCTAGCGTGAACCTGTCATGTACCGCCTCTGGGTTCAACATAAAGGACACGTATATGCAGTGGATTAAGCAGCGGCCAGAACAGGGTCTGGAATGGATAGGCAGGATTGCCCCAGCAAACGGAAACCCCAAGTACGATCCCAAATTCCAGGGCAAGGCAACAATCATACCAGACACTAGTTCCAACACCGCTTACCTGCAGCTGAACAGCCTGACTAGCGACGACACAGCCGTTTATTACTGCGCGTCTTTCTTCCCCGGCTCATGGTTTGCTTGCTGGGGCCAGGGCACCTTGGTGACAGTGTCTGCCGCCAAGACCACCCCTCCTAGCGTGTACCCCCTGGCCCCTGGATCCGCCGCTCAGACAAACTCTATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACAGTCACATGGAACTCCGGCAGCCTGTCTAGCGGCGTGCACACCTTCCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAGCGTTACCGTGCCTAGCAGCACATGGCCCTCCGAGACAGTGACATGTAATGTGGCCCACCCAGCCTCCTCCACCAAGGTGGACAAGAAGATCGTGCCTAGGGACTGCGGCTGCAAGCCCTGTATCTGTACAGTGCCTGAGGTGTCCTCCGTGTTCATCTTCCCACCTAAGCCTAAGGATGTGCTGACCATCACACTGACACCTAAGGTGACATGTGTGGTGGTGGATATCTCCAAGGACGATCCTGAGGTGCAGTTTAGCTGGTTTGTGGACGACGTGGAGGTGCACACCGCCCAGACCCAGCCCAGAGAGGAGCAGTTCAATTCCACATTCAGGAGCGTGAGCGAGCTGCCTATCATGCACCAGGATTGGCTGAATGGCAAGGAGTTCAAGTGTAGAGTGAACAGCGCCGCCTTCCCAGCTCCTATCGAGAAGACCATCAGCAAGACAAAGGGCAGACCTAAGGCTCCTCAGGTGTACACCATCCCACCTCCTAAGGAGCAGATGGCCAAGGACAAGGTGAGCCTGACCTGTATGATCACCGATTTCTTCCCAGAGGATATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAATTACAAGAACACCCAGCCCATCATGGATACAGACGGCTCCTACTTTGTGTACTCCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAATACATTCACCTGCTCCGTGCTGCACGAGGGCCTGCACAATCACCACACCGAGAAGAGCCTGTCCCACAGCCCAGGCAAG
5C10 recombinant antibody light chain nucleotide sequence (SEQ ID NO: 4):
ATGAAACTGCCCGTCCGACTCCTTGTGCTTATGTTCTGGATCCCAGCCTCATCTAGCGACGTCGTGATGACTCAGACGCCCCTGAGCTTGCCCGTATCTTTGGGCGACCAAGCATCTATTAGCTGTCGCAGCAGTCAGAGCCTGGTTCACTCTAACGGCAATACCTACCTGCATTGGTACCTGCAGAAGCCAGGCCAGCCACCAAAGGTGCTGATATACAAGGTGTCAAACCGGTTCTCAGGAGTCCCTGATCGGTTCAGCGGGAGTGGCAGCGGGACTGATTTCACACTGAAGATCAGCCGGGTTGAAGCCGAAGACCTTGGAGTCTACTTCTGTAGCCAGGGGACTCATGTGCCCTATACCTTTGGAGGCGGCACCAAGCTGGAGATTAAAAGGGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC
EXAMPLE 4 FACS detection of recombinant antibodies of 5C10 binding to HuT-78 cells
4.1 Cell preparation
Logarithmic growth of HuT-78 cells (CCR 8 expressing tumor cells) in T75 flasks was digested with trypsin at 37 ℃ for 3 min, stopped with medium, gently blown down and passed through a 70um cell sieve. Cells were prepared as single cell suspensions and cell densities were adjusted to 2E+06 cells/ml.
4.2 Cell closure
The prepared cells were collected by centrifugation, resuspended in 1 XPBS containing 3% FBS as blocking solution, and blocked by standing at 4℃for 30 minutes.
4.3 First antibody incubation
The 5C10 recombinant antibody and the control antibody 4A19 (the cross-species antibody binding to the N-terminal CCR8 epitope in patent CN 115768792A) were diluted to 10nM and the blocked cells were incubated separately. The blocked cells were incubated with PBS, mouse IgG1, FITC goat anti-mouse antibody as negative control, isotype control, and secondary antibody control, respectively, and allowed to stand at 4℃for 1 hour.
4.4 Secondary antibody incubation
Cells were washed three times with 1 XPBS, incubated with goat anti-mouse FITC (corresponding to 5C10 recombinant antibody) and goat anti-human FITC (corresponding to control 4A19 antibody) respectively, and allowed to stand at 4℃for 30 minutes.
4.5FACS detection
Cells were washed three times with 1×pbs and gently blown down to uniform single cells for FACS flow-on-machine analysis. FIG. 4 shows that the 5C10 recombinant antibody and the control 4A19 antibody bind to HuT-78 cells.
Example 5BLI characterization of binding of 5C10 recombinant antibodies to human and monkey full length CCR8 membrane protein Nanodisc GPCR membrane protein nanodisc employs as a stabilizer a substance having both hydrophobic and hydrophilic properties, the hydrophobic face of which towards the inner phospholipid layer can stabilize the transmembrane region of the membrane protein, the hydrophilic face of which enables stable presence of membrane protein nanodisc in aqueous solution. The extracellular and intracellular domains of GPCRs are located on either side of the phospholipid plate, respectively, and remain exposed to solution, maintaining the native conformation of the full-length membrane protein. This example uses the biological membrane interferometry technique (BLI, gator Bio) to characterize the binding of human and monkey full-length CCR8 membrane protein (Uniprot ID: O97665) Nanodisc to the 5C10 recombinant antibody. The C-terminal of the intracellular region of CCR8 nanodisc is fused with His tag, and a BLI Anti-His (His) probe can capture CCR8 nanodisc, and then a 5C10 recombinant antibody and a 4A19 control antibody are respectively used as analytes to characterize the combination of the antibodies and extracellular functional regions of CCR8 membrane proteins.
FIGS. 5A and B show capture of full-length human CCR8 membrane protein Nanodisc with Anti-His (His) probe, where antibodies were used as analytes, concentration gradients were set at 200, 100, 50, 25, 12.5, 6.25, 3.125nM. FIG. 5A shows the binding results with a 5C10 recombinant antibody as the analyte; b shows the binding results with the 4a19 control antibody as analyte. FIGS. 5C and D show capture of full length cynomolgus monkey CCR8 membrane protein Nanodisc with Anti-His (His) probe, where C shows binding with 5C10 recombinant antibody as analyte, concentration gradients set at 1000, 500, 250, 125, 62.5, 31.25, 15.625nM; d shows binding results with the 4A19 control antibody as analyte, concentration gradients set at 200, 100, 50, 25, 12.5, 6.25, 3.125nM. The results showed that the 5C10 recombinant antibody bound with a binding affinity (KD) level of about 16.2nM to full length human CCR8 nanodisc and about 36.2nM to full length monkey CCR8 nanodisc.
Reference to the literature
1.Mohsen,MO.et al.Virus-l ike particle vaccinology,from bench to bedside.Cel lular&Molecular Immunology(2022)19:993–1011
2.Gutiérrez,J.et al.Analysis of Post-translational CCR8Modifications and Their Influence on Receptor Activity.The Journal Of Biological Chemistry(2004)279,14726–14733
3.Sun,D.et al.Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.Nature Communications(2023)14:7940
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (20)

1. An anti-CCR 8 antibody or antigen-binding fragment thereof, characterized in that, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the following 3 CDRs:
H-CDR1 shown in SEQ ID NO. 7;
H-CDR2 shown in SEQ ID NO. 8;
H-CDR3 shown in SEQ ID NO 9; and
The light chain variable region comprises the following 3 CDRs:
L-CDR1 shown in SEQ ID NO. 13;
L-CDR2 with sequence shown as KVS;
the L-CDR3 shown in SEQ ID NO. 14.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 6, or an amino acid sequence having at least 90% sequence identity thereto.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 12, or an amino acid sequence having at least 90% sequence identity thereto.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a murine antibody, a chimeric antibody.
5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a humanized antibody.
6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody specifically binds to human CCR8 and cynomolgus CCR8, and has human and monkey cross-species binding activity.
7. A recombinant antibody, said recombinant antibody comprising:
(i) The sequence of the anti-CCR 8 antibody or antigen binding fragment thereof of any one of claims 1-6; and
(Ii) Optionally a signal peptide that facilitates secretory expression of the antibody and/or a tag sequence for purification and detection.
8. The recombinant antibody according to claim 7, wherein the signal peptide has an amino acid sequence as shown in SEQ ID NO. 5 or 11.
9. The recombinant antibody according to claim 7, wherein the recombinant antibody comprises a heavy chain having an amino acid sequence shown in SEQ ID NO. 1 and a light chain having an amino acid sequence shown in SEQ ID NO. 2.
10. A polynucleotide molecule encoding a polypeptide selected from the group consisting of:
(1) The anti-CCR 8 antibody or antigen binding fragment thereof of any one of claims 1-6; or (b)
(2) The recombinant antibody of claim 7.
11. An expression vector comprising the polynucleotide molecule of claim 10.
12. A host cell comprising the vector of claim 11, or having integrated into its genome the polynucleotide molecule of claim 10.
13. An antibody conjugate, characterized in that, the antibody conjugate comprises:
(a) The anti-CCR 8 antibody or antigen-binding fragment thereof of any one of claims 1-6 or the recombinant antibody of claim 7; and
(B) A coupling moiety selected from the group consisting of: detectable labels, radionuclides, nanoparticles/nanorods;
wherein the detectable label is selected from a fluorescent label or a chemiluminescent label.
14. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises:
(a) The anti-CCR 8 antibody or antigen binding fragment thereof of any one of claims 1-6, the recombinant antibody of claim 7, or the antibody conjugate of claim 13; and
(B) A pharmaceutically acceptable carrier.
15. Use of an anti-CCR 8 antibody or antigen binding fragment thereof of any one of claims 1-6, a recombinant antibody of claim 7, or an antibody conjugate of claim 13 in the preparation of a detection reagent for detecting human or cynomolgus monkey CCR8 molecules.
16. Use of an anti-CCR 8 antibody or antigen binding fragment thereof of any one of claims 1-6, a recombinant antibody of claim 7, or an antibody conjugate of claim 13 for the preparation of a detection plate for detecting human or cynomolgus monkey CCR8 molecules.
17. Use of an anti-CCR 8 antibody or antigen binding fragment thereof of any one of claims 1-6, a recombinant antibody of claim 7, or an antibody conjugate of claim 13 in the preparation of a detection kit for detecting human or cynomolgus monkey CCR8 molecules.
18. The use according to any one of claims 15 to 17, wherein the assay comprises a flow assay, a cellular immunofluorescence assay, an ELISA assay.
19. A method of producing the anti-CCR 8 antibody or antigen-binding fragment thereof of any one of claims 1-6, or the recombinant antibody of claim 7, comprising the steps of:
(s 1) culturing the host cell of claim 12 under conditions suitable for the production of an antibody or antigen-binding fragment thereof, thereby obtaining a culture comprising the antibody or antigen-binding fragment thereof;
(s 2) isolating or recovering the antibody or antigen-binding fragment thereof from the culture; and
(S 3) purifying the antibody or antigen-binding fragment thereof obtained in step (s 2).
20. A method for non-diagnostic and non-therapeutic in vitro detection of CCR8 protein in a sample, said method comprising the steps of:
(1) Contacting a sample with the anti-CCR 8 antibody or antigen-binding fragment thereof of any one of claims 1-6, the recombinant antibody of claim 7, or the antibody conjugate of claim 13;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CCR8 protein in the sample.
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