CN118440196A

CN118440196A - Anti-IL 1R2 antibodies, antigen binding fragments thereof and uses thereof

Info

Publication number: CN118440196A
Application number: CN202310970667.3A
Authority: CN
Inventors: 杨衡; 蔡巧梅; 张雨瑞
Original assignee: Suzhou Institute Of Systems Medicine
Current assignee: Suzhou Institute Of Systems Medicine
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2024-08-06

Abstract

The present disclosure relates to antibodies against IL1R2, antigen binding fragments thereof and uses thereof, belonging to the biotechnology field. In particular, the disclosure relates to a monoclonal antibody, and also provides phage displaying the monoclonal antibody on the surface, a preparation method of the monoclonal antibody and a pharmaceutical composition containing the monoclonal antibody. The monoclonal antibody can specifically bind to IL1R2 protein, especially IL1R2 protein of human and mouse sources, and has good inhibition effect on tumor growth.

Description

Anti-IL 1R2 antibodies, antigen binding fragments thereof and uses thereof

Technical Field

The invention relates to an anti-IL 1R2 antibody, an antigen binding fragment and application thereof, belonging to the technical field of biology.

Background

Regulatory T cells (regulatory T cells, treg) are a class of T cell subpopulations ^[1] with significant immunosuppressive effects, expressing Foxp3, CD25, CD4 as a phenotypic feature of the cells. Treg cells can play a negative regulation role on the immune response of an organism through intercellular contact and secretion of inhibitory cytokines in various immune cell subsets, and inhibit the immune response of the organism and tolerance to autoantigens, thereby avoiding ^[2-4] the occurrence of autoimmune diseases. Also, in some tumors, treg cells can cause the body to generate antigen tolerance to tumor cells through immunosuppression, so that the tumor cells can generate immune escape, thereby indirectly accelerating the proliferation of the tumor cells and enhancing the infiltration capacity ^[5] of the tumor cells. Therefore, how to reduce the function of Treg cells is of great importance in tumor therapy. Cytotoxic T lymphocyte-associated antigen 4 (cytotoxin T-lymphocyte-associated protein, CTLA-4) expressed at the surface of Treg cells is thought to be able to competitively bind to B7 expressed by antigen presenting cells (ANTIGEN PRESENTING CELL, APC) thereby preventing its stimulation of naive T cells ^[6,7]. In fact, it has been proposed that CTLA-4 expressed on tregs can physically remove B7 molecules on the surface of APCs, thereby eliminating their costimulatory activity. Thus, inhibitors against CTLA-4 have been developed by many pharmaceutical companies. The U.S. Food and Drug Administration (FDA) approved ipilimumab (Ipilimumab), commonly known as the Y drug, passing BMS in 2011, was the first immune checkpoint inhibitor worldwide and was the only anti-CTLA-4 mab ^[8] currently marketed. With the intensive research, researchers believe that CTLA-4 inhibitors do not exert anti-tumor efficacy by blocking the action between CTLA-4 and B7, but effectively remove tregs which infiltrate into tumor tissues with high expression of CTLA-4 through antibody Fc receptor-mediated cell-mediated cytotoxicity (ADCC) effect, thereby eliminating the immunosuppressive effect of tregs and achieving anti-tumor effect ^[7].

Although immune checkpoint inhibitor (immune checkpoint inhibitor, ICI) therapies represented by CTLA-4 have significant clinical efficacy as an important means for tumor immunotherapy, development of new targets has become particularly important ^[9] because of low overall rate of ICI remission and extremely high toxic and side effects due to immunosuppression in the malignant tumor microenvironment (tumor microenvironment, TME).

The interleukin 1 type II receptor (interleukin 1receptor type II,IL1R2), also known as CD121b (cluster of Differentiation 121 b), is one of the members of the interleukin I receptor family (IL-1R) that includes 10 structurally similar soluble proteins ^[10,11]. IL1R2 binds to IL1 and inhibits its interaction with activated IL1R1, and therefore IL1R2 is considered a decoy receptor, thereby neutralizing the pro-inflammatory function ^[10,11] of IL 1. Numerous studies have shown that IL1R2 is highly expressed on the cell membrane of Treg cells, but not ^[12,13] on CD4 ⁺ conventional T cells (Tconv). In addition, IL1R2 has higher expression level in tumor invasive Treg cells such as human breast cancer, colorectal cancer, lung cancer and the like. Therefore, it is possible to develop antibodies against IL1R2 to enhance the anti-tumor immunity of the body.

Disclosure of Invention

Problems to be solved by the invention

Recent studies have shown that while tregs play an important role in maintaining peripheral tolerance and preventing autoimmunity, etc., they also suppress the anti-tumor immune effects within the tumor microenvironment. Thus inhibiting or eliminating the function or number of Treg cells infiltrated by the tumor microenvironment becomes critical for anti-tumor immunotherapy. As a target point of the Treg cells, the CTLA-4 targeted inhibitor can promote the generation of more immune cells, can remove the infiltrated Treg cells in tumor tissues, and plays a role in enhancing immunity ^[16]. Although monoclonal antibody drugs targeting CTLA-4 molecules show better overall survival rate in clinical experimental stages and have obvious clinical efficacy improvement compared with control groups, when the antibody drugs are clinically applied, side effects such as rash, diarrhea, fatigue, pruritus, headache, weight loss, nausea and the like are common, and some patients die ^[17] due to excessive activation of immune systems. Therefore, there is a need to find new targets for Treg cells in an effort to remedy the drawbacks of targeted CTLA-4 inhibitors.

Solution for solving the problem

Phage display technology (PHAGE DISPLAY technology) is a biotechnology ^[14,15] in which a DNA sequence of a foreign protein or polypeptide is inserted into a suitable position of a phage coat protein structural gene, so that the foreign gene is expressed with expression of the coat protein, and simultaneously the foreign protein is displayed on the phage surface with reassembly of the phage. In the disclosure, a phage humanized antibody library is constructed by human scFv, and specific antibodies aiming at IL1R2 are screened out through specific binding with human IL1R2 protein, and the anti-tumor effect of the antibodies is evaluated.

The technical scheme of the present disclosure is as follows:

[1] an anti-IL 1R2 antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from any one of (a 1) to (a 4) below:

(a1) LCDR1 as shown in SEQ ID NO. 9, LCDR2 as shown in sequence RNN and LCDR3 as shown in SEQ ID NO. 10;

(a2) LCDR1 as shown in SEQ ID NO. 11, LCDR2 as shown in sequence RNN and LCDR3 as shown in SEQ ID NO. 12;

(a3) LCDR1 as shown in SEQ ID NO. 13, LCDR2 as shown in sequence KVS and LCDR3 as shown in SEQ ID NO. 14; or (b)

(A4) LCDR1 as shown in SEQ ID NO. 15, LCDR2 as shown in sequence GNS and LCDR3 as shown in SEQ ID NO. 16;

the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from any one of (b 1) to (b 4) below:

(b1) HCDR1 as shown in SEQ ID NO. 17, HCDR2 as shown in SEQ ID NO. 18 and HCDR3 as shown in SEQ ID NO. 19;

(b2) HCDR1 as shown in SEQ ID NO. 20, HCDR2 as shown in SEQ ID NO. 21 and HCDR3 as shown in SEQ ID NO. 22;

(b3) HCDR1 as shown in SEQ ID NO. 23, HCDR2 as shown in SEQ ID NO. 24 and HCDR3 as shown in SEQ ID NO. 25; or (b)

(B4) HCDR1 as shown in SEQ ID NO. 26, HCDR2 as shown in SEQ ID NO. 27 and HCDR3 as shown in SEQ ID NO. 28.

[2] An anti-IL 1R2 antibody or antigen-binding fragment thereof according to [1], comprising a heavy chain variable region as shown in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or more sequence identity to any one of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7; and/or the number of the groups of groups,

Comprising a light chain variable region as set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or more sequence identity to any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8.

[3] The anti-IL 1R2 antibody or antigen-binding fragment thereof according to [1] or [2], wherein the anti-IL 1R2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3 or IgG4 or a variant thereof; and/or the number of the groups of groups,

The anti-IL 1R2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, lambda chain, or variant thereof.

[4] The antibody or antigen-binding fragment thereof against IL1R2 of any one of [1] to [3], which comprises a human antibody; and/or the number of the groups of groups,

The anti-IL 1R2 antibody or antigen binding fragment thereof is selected from at least one of full-length antibody, fab fragment, fab 'fragment, F (ab') 2 fragment, fv fragment, single chain antibody and multispecific antibody.

[5] An isolated polynucleotide, wherein the polynucleotide encodes the anti-IL 1R2 antibody or antigen-binding fragment thereof of any one of [1] to [4 ].

[6] An expression cassette, wherein the expression cassette comprises the polynucleotide of [5 ].

[7] A recombinant vector, wherein the recombinant vector comprises the polynucleotide of [5] or the expression cassette of [6 ].

[8] A host cell, wherein the host cell comprises the polynucleotide of [5], the expression cassette of [6], or the recombinant vector of [7 ].

[9] The host cell according to [8], wherein the host cell is a microorganism or a mammalian cell;

optionally, the microorganism is selected from bacteria and/or yeasts;

Optionally, the mammalian cell is selected from HEK293T cells, HEK293F cells, HEK293 cells and/or CHO cells.

[10] A phage, wherein the phage surface displays the anti-IL 1R2 antibody or antigen binding fragment thereof of any one of [1] to [4 ].

[11] A method of producing the anti-IL 1R2 antibody or antigen-binding fragment thereof of any one of [1] to [4], which comprises culturing the host cell of [8] or [9] under conditions allowing production of the anti-IL 1R2 antibody or antigen-binding fragment thereof, and recovering and isolating the anti-IL 1R2 antibody or antigen-binding fragment thereof.

[12] A pharmaceutical composition comprising: the anti-IL 1R2 antibody or antigen-binding fragment thereof of any one of [1] to [4], the polynucleotide of [5], the expression cassette of [6], the recombinant vector of [7], or the host cell of [8] or [9 ]; and

Optionally, pharmaceutically acceptable carriers and/or excipients.

[13] Use of an anti-IL 1R2 antibody or antigen-binding fragment thereof according to any one of [1] to [4], a polynucleotide according to [5], an expression cassette according to [6], a recombinant vector according to [7], a host cell according to [8] or [9], or a pharmaceutical composition according to [12] in the manufacture of a medicament for the treatment of an IL1R2 mediated disease or disorder.

ADVANTAGEOUS EFFECTS OF INVENTION

Four clones 3999-R3P1-B1, 3999-R3P1-H1, 3999-R3P1-E4 and 3999-R3P1-E11 were selected by phage display technology and subjected to antibody expression purification. Purified antibodies were subjected to SDS-PAGE qualitative and quantitative analysis, with quality control. The purified antibodies were then subjected to ELISA experiments.

(1) In one embodiment, the antibodies of the present disclosure have good binding capacity for human-derived IL1R2 proteins, while also being capable of binding mouse-derived IL1R2 proteins.

(2) In another embodiment, the antibodies of the present disclosure are effective in inhibiting tumor growth in mice and increasing overall survival in mice. Meanwhile, a new target spot and a new research direction are provided for the immunotherapy of tumors.

Drawings

FIG. 1 is a Coomassie brilliant blue staining pattern of purified antibodies; MW: protein standards (unit: kDa), lanes 1-4 are 3999-R3P1-B1, 3999-R3P1-H1, 3999-R3P1-E4 and 3999-R3P1-E11, respectively.

FIG. 2 is a graph showing ELISA results for screening antibodies.

Fig. 3 is a graph showing tumor growth in mice after 3 antibody injections.

Fig. 4 is a graph showing tumor growth in mice after 6 antibody injections.

FIG. 5 is a graph of survival of mice.

FIG. 6 is a Coomassie brilliant blue staining pattern of IL1R2 protein; MW: protein standard (unit: kDa).

Detailed Description

Various exemplary embodiments, features and aspects of the invention are described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention.

Unless otherwise indicated, all units used in this specification are units of international standard, and numerical values, ranges of values, etc. appearing in the present invention are understood to include systematic errors unavoidable in industrial production.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably 1%.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In this specification, the term "and/or" when used to connect two or more selectable items is understood to mean any one of the selectable items or any two or more of the selectable items.

In this specification, the term "interleukin 1 type II receptor" or "IL12R" is a decoy receptor that neutralizes interleukin-1 (IL 1) and possesses 3 immunoglobulin extracellular domains and a bolt transmembrane region, which captures and sequesters IL-1, thereby blocking the IL-1 signaling pathway and down regulating IL-1. It is expressed mainly on neutrophils, B cells, monocytes and macrophages, and can also be induced on keratin cells and endothelial cells.

In the present disclosure, the terms "polypeptide," "protein," "peptide," and "peptides" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a similar peptide backbone.

In the present disclosure, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNAs (mrnas), transfer RNAs, ribosomal RNAs, ribozymes, cdnas, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNAs of any sequence, nucleic acid probes and primers. The nucleic acid molecule may be linear or circular.

In the present disclosure, the amino acid three-letter code and the one-letter code used are as described in j.biol. Chem,243, p3558 (1968).

In the present disclosure, the term "antibody" as described in the present disclosure refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

In the present disclosure, the antibody light chain may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin, or variants thereof.

In the present disclosure, the antibody heavy chain may further comprise a heavy chain constant region comprising an IgG1, igG2, igG3, igG4 or variant thereof of human or murine origin.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain are CDRL1, CDRL2, and CDRL3; the 3 CDR regions of the heavy chain are CDRH1, CDRH2 and CDRH3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature 342:877-883, al-Lazikani et al ,"Standard conformations for the canonical structures of immunoglobulins",Journal of Molecular Biology,273,927-948(1997)), based on Kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition ,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London), International ImMunoGeneTics database (IMGT) (on the world Wide Web.) on the basis of the three-dimensional structure of the antibody and topology of the CDR loops, and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) utilizing a large number of crystal structures.

The term "human antibody" or "humanized antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

In the present disclosure, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, domain antibodies, single domain antibodies or nanobodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson,2005, nat. Biotechnol.23:1126-1136.

The term "Fab fragment" comprises a light chain and a heavy chain CH1 and variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The term "Fab' fragment" contains a light chain and a heavy chain portion or fragment containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains.

The term "F (ab') 2 fragment" contains two light chains and two heavy chains, the heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. The F (ab ') 2 fragment thus consists of two Fab' fragments, which are linked together by a disulfide bond between the two heavy chains.

The term "Fv fragment" comprises variable regions from the heavy and light chains, but lacks constant regions.

In the present disclosure, the term "single chain antibody", "single chain Fv" or "scFv", i.e. "single chain variable region fragment", is an antibody fragment consisting of a variable region of the heavy chain and a variable region of the light chain of an antibody by means of a short peptide of 15-20 amino acids, wherein these domains are present as a single polypeptide chain, belonging to a small molecule genetically engineered antibody.

The term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The intensity or affinity of an immunological binding interaction may be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunological binding properties between the two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) referring to a particular antibody-antigen interaction can be calculated from the concentration and the actual rate of association and dissociation, see MALMQVIST M,1993, nature,361:186-187. The Kd/Ka ratio is equal to the dissociation constant KD, see Davies DR et al 1990,Annual Rev Biochem, 59:439-473. KD, ka and KD values can be measured by any effective method. In a preferred embodiment, the dissociation constant is measured using bioluminescence interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., biacore) or KinExa.

The term "phage display library" refers to a "library" of phages on the surface of which exogenous peptides or proteins are expressed. The foreign peptide or polypeptide is displayed on the outer surface of the phage capsid. The foreign peptide may be displayed as a recombinant fusion protein incorporated as part of a phage coat protein; typically not phage coat proteins, but can become recombinant fusion proteins incorporated into the outer surface of the capsid; or a protein or peptide that becomes covalently or non-covalently linked to the protein. This is achieved by inserting an exogenous nucleic acid sequence into a nucleic acid that can be packaged into phage particles. The exogenous nucleic acid sequence may, for example, be inserted into the coding sequence of a bacteriophage coat protein gene. If the foreign sequence is an in-frame clone, the protein it encodes will be expressed as part of the coat protein. Thus, a library of nucleic acid sequences, such as a library of antibody lineages made from gene segments encoding the entire B cell lineage of one or more individuals, can be inserted into phage in such a manner as to produce a "phage library". A "peptide display library" is generated when those peptides and proteins representing the peptides and proteins encoded by the nucleic acid library are displayed by phage. Although a variety of phages are used in the library construction, filamentous phages are generally used (Dunn (1996) Curr. Opin. Biotechnol. 7:547-553). The antibody library is generally classified into a natural antibody library (naive antibody library), an immune antibody library (immune antibody library), a synthetic antibody library (SYNTHETIC ANTIBODY LIBRARY), a semisynthetic antibody library (semi-SYNTHETIC ANTIBODY LIBRARY), and the like.

The term "individual," "patient," or "subject" as used in the context of the present disclosure includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

In the present disclosure, the term "antigen binding site" refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment of the present disclosure.

In the present disclosure, "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the maximum percent identity.

In the present disclosure, the term "expression cassette" is a recombinant expression element comprising a gene of interest, and a regulatory element that regulates the expression of the gene of interest. In some embodiments, the target gene is a gene encoding a polypeptide of interest. In some embodiments, the regulatory element is a promoter element for initiating transcription of the coding gene. In some embodiments, regulatory elements may also include enhancers, silencers, insulators, and the like.

In the present disclosure, a "vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes a cis-acting component sufficient for expression; other components for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide is introduced.

In the present disclosure, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such designations include offspring. Thus, the terms "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell.

In the present disclosure, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the enterobacteriaceae (enterobacteriaceae), such as strains of escherichia coli (ESCHERICHIA COLI) or Salmonella (Salmonella); the family of Bacillaceae (baciliaceae) such as bacillus subtilis (Bacillus subtilis); pneumococci (Pneumococcus); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

"Administering," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic agent, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering an internally or externally used therapeutic agent, such as any antibody comprising the present disclosure, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom.

The term "diagnosis" includes detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will have a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or the likely progression or regression thereof) of a subject with a disease or condition, and determining the effect of treatment on a subject with a disease or condition.

In the present disclosure, an "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

In this disclosure, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or to delay or minimize one or more symptoms associated with a disorder. A therapeutically effective amount refers to an amount of a therapeutic agent alone or in combination with other therapies that provides a therapeutic benefit in the treatment of a disorder. The term "therapeutically effective amount" may include improving overall therapy; reducing or avoiding symptoms, signs, or causes of the disorder; and/or an amount that enhances the therapeutic efficacy of another therapeutic agent.

In the present disclosure, a "prophylactically effective amount" is an amount sufficient to prevent a disorder or one or more symptoms associated with a disorder or prevent recurrence thereof. A prophylactically effective amount refers to an amount of a therapeutic agent alone or in combination with other agents that provides a prophylactic benefit in preventing a disorder. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

The term "subject" refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young, middle-aged, or elderly)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), a commercially relevant mammal (e.g., cow, pig, horse, sheep, goat, cat, or dog), or a bird.

"Pharmaceutical composition" means a composition comprising one or more of the antibodies described herein, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

In the present disclosure, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to an animal or human, as appropriate.

< Antibody against IL1R2 or antigen-binding fragment thereof >

Treg cells are an important component of tumor immune tolerance, and research shows that IL1R2 (CD 121 b) is highly expressed in the Treg cells in tumor microenvironment, and IL1R2 becomes a potential new intervention target in tumor immunotherapy.

The phage antibody library technology can perform in-vitro library construction screening on B cell antibody libraries of humans and other animals, avoids the steps of immunization, cell fusion and the like, shortens the experimental period, increases the stability, and realizes screening of the fully human antibody sequences with higher affinity in a short period. In the present disclosure, phage humanized antibody library is constructed, and humanized anti-IL 1R2 antigen monoclonal antibody is screened out.

A first aspect of the present disclosure provides an anti-IL 1R2 antibody, or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein,

In some specific embodiments, an anti-IL 1R2 antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (a 1) LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in sequence RNN, and LCDR3 as shown in SEQ ID NO: 10; the heavy chain variable region comprises (b 1) an HCDR1 as shown in SEQ ID NO. 17, an HCDR2 as shown in SEQ ID NO. 18, and an HCDR3 as shown in SEQ ID NO. 19; and/or the number of the groups of groups,

The light chain variable region comprises (a 2) LCDR1 as shown in SEQ ID NO. 11, LCDR2 as shown in sequence RNN, and LCDR3 as shown in SEQ ID NO. 12; the heavy chain variable region comprises (b 2) an HCDR1 as shown in SEQ ID NO. 20, an HCDR2 as shown in SEQ ID NO. 21 and an HCDR3 as shown in SEQ ID NO. 22; and/or the number of the groups of groups,

The light chain variable region comprises (a 3) LCDR1 as shown in SEQ ID NO. 13, LCDR2 as shown in sequence KVS, and LCDR3 as shown in SEQ ID NO. 14; the heavy chain variable region comprises (b 3) HCDR1 as shown in SEQ ID NO. 23, HCDR2 as shown in SEQ ID NO. 24 and HCDR3 as shown in SEQ ID NO. 25; and/or the number of the groups of groups,

The light chain variable region comprises (a 4) LCDR1 as shown in SEQ ID NO. 15, LCDR2 as shown in sequence GNS and LCDR3 as shown in SEQ ID NO. 16; the heavy chain variable region comprises (b 4) HCDR1 as shown in SEQ ID NO:26, HCDR2 as shown in SEQ ID NO:27, and HCDR3 as shown in SEQ ID NO: 28.

In some embodiments, the anti-IL 1R2 antibody or antigen binding fragment thereof, which comprises a heavy chain variable region as shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or more sequence identity to any one of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7; and/or comprising a light chain variable region as set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or more sequence identity to any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8.

In some specific embodiments, the anti-IL 1R2 antibody or antigen binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO.1 and a light chain variable region as shown in SEQ ID NO. 2.

In some specific embodiments, the anti-IL 1R2 antibody or antigen binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO. 3 and a light chain variable region as shown in SEQ ID NO. 4.

In some specific embodiments, the anti-IL 1R2 antibody or antigen binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO. 5 and a light chain variable region as shown in SEQ ID NO. 6.

In some specific embodiments, the anti-IL 1R2 antibody or antigen binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO. 7 and a light chain variable region as shown in SEQ ID NO. 8.

In some embodiments, the anti-IL 1R2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3, or IgG4, or a variant thereof; and/or the anti-IL 1R2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, lambda chain, or variant thereof.

In some embodiments, the heavy chain constant region sequence is set forth in SEQ ID NO. 37 and the light chain constant region sequence is set forth in SEQ ID NO. 38.

In some embodiments, the anti-IL 1R2 antibody or antigen-binding fragment thereof comprises a human antibody; and/or the anti-IL 1R2 antibody or antigen binding fragment thereof is selected from at least one of full length antibody, fab fragment, fab 'fragment, F (ab') 2 fragment, fv fragment, single chain antibody, multispecific antibody.

In addition, other amino acid sequences which do not substantially affect the activity, expression level and stability of the anti-IL 1R2 antibody or antigen-binding fragment thereof of the present invention may be added to the amino-or carboxy-terminus of the anti-IL 1R2 antibody or antigen-binding fragment thereof. Preferably, these added amino acid sequences facilitate expression (e.g., signal peptide), facilitate purification (e.g., 6 XHis sequence), or other sequences that promote activity, expression, or stability of the anti-IL 1R2 antibody or antigen-binding fragment thereof.

Furthermore, in order to further increase the expression level of the host cell, the coding sequence of the antibody of the present invention may be modified, for example, by using codons preferred by the host cell, to eliminate sequences detrimental to transcription and translation of genes.

In some embodiments, after obtaining the DNA sequences encoding the antibodies or antigen-binding fragments thereof of the disclosure against IL1R2, they are cloned into a suitable expression vector and transferred into a suitable host cell. Finally, culturing the transformed host cells, and obtaining the anti-IL 1R2 antibody or antigen binding fragment thereof of the disclosure through separation and purification.

< Polynucleotide >

The present disclosure provides an isolated polynucleotide encoding an anti-IL 1R2 antibody or antigen-binding fragment thereof as described above.

In some embodiments, the polynucleotide may be in DNA form or RNA form. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded, preferably double-stranded DNA. The DNA may be a coding strand or a non-coding strand. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

< Expression cassette, recombinant vector, host cell >

Based on the polynucleotides obtained by the present disclosure, the present disclosure provides expression cassettes comprising the polynucleotides as described in the present disclosure.

In other embodiments, the present disclosure provides recombinant vectors comprising a polynucleotide or expression cassette as described in the present disclosure.

In other embodiments, the present disclosure provides host cells comprising a polynucleotide, expression cassette, or recombinant vector as described in the present disclosure.

In some embodiments, the host cell is a bacterium; illustratively, the host cell is, but not limited to, E.coli, B.subtilis.

In other embodiments, the host cell is a yeast, illustratively, but not limited to, pichia pastoris, saccharomyces cerevisiae.

In other specific embodiments, the host cell is a mammalian cell, illustratively, but not limited to, a mammalian cell line, such as HEK293T cells, HEK293F cells, HEK293 cells, CHO cells.

< Phage >

The present disclosure provides a phage displaying on its surface the above-described anti-IL 1R2 antibody or antigen-binding fragment thereof.

In some embodiments, phage display technology is employed in the construction of a library of human scFv antibodies, the DNA sequence of a foreign protein or polypeptide is incorporated into the phage coat protein structural gene in place, allowing expression of the foreign gene with expression of the coat protein, while the foreign protein is displayed to the phage surface with reassembly of the phage.

< Pharmaceutical composition >

The present disclosure provides a pharmaceutical composition comprising an anti-IL 1R2 antibody or antigen-binding fragment thereof, a polynucleotide, an expression cassette, a recombinant vector, or a host cell as described above.

Further, the pharmaceutical composition can also comprise pharmaceutically acceptable carriers and/or auxiliary materials. In some embodiments, the pharmaceutically acceptable carrier and/or adjuvant is described in detail in Remington "s Pharmaceutical Sciences (19 th ed., 1995), which is used as needed to aid stability of the formulation or to aid in improving bioavailability of the active substance or to create an acceptable mouthfeel or odor in the case of oral administration, and the pharmaceutical composition so formulated may be administered by any suitable means known to those skilled in the art as needed.

In some embodiments, the pharmaceutical composition may be prepared in any pharmaceutically conventional dosage form.

< Therapeutic use >

The present disclosure provides the use of an anti-IL 1R2 antibody or antigen-binding fragment thereof, polynucleotide, expression cassette, recombinant vector, host cell, or pharmaceutical composition as described herein in the manufacture of a medicament for the treatment of an IL1R 2-mediated disease or disorder.

In some embodiments, the IL1R 2-mediated disease or disorder includes urothelial cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, skin cancer, non-small cell lung cancer, osteosarcoma, lymphoma, liver cancer, adrenocortical cancer, and the like.

< Treatment method >

The present disclosure provides antibodies, or antigen-binding fragments thereof, polynucleotides, expression cassettes, recombinant vectors, host cells, or pharmaceutical compositions, against IL1R2 as described in the present disclosure, for use in the prevention and/or treatment of IL1R 2-mediated diseases or conditions.

The present disclosure provides a method of preventing and/or treating an IL1R 2-mediated disease or disorder comprising administering to a subject a prophylactically or therapeutically effective amount of an anti-IL 1R2 antibody, or antigen-binding fragment thereof, polynucleotide, expression cassette, recombinant vector, host cell, or pharmaceutical composition as described in the present disclosure.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

1. Screening and Synthesis of antibodies

(1) Construction of humanized phage display antibody libraries in the form of human scFv

① PCR amplifying antibody variable region sequence by using cDNA obtained by reverse transcription of human lymphocyte RNA as a template, wherein the cDNA comprises a heavy chain variable region VH and a light chain variable region VL; the target fragment with the size of 300-500bp is recovered after 1% agarose gel electrophoresis.

② Joining the heavy chain variable region and the light chain variable region by overlap extension PCR method with a linker peptide linker (GGGGSGGGGSGGGGSGGGGAS, SEQ ID NO: 41); recovering the target fragment with the size of 700-800bp by using the gel.

③ The recovered target fragment and a vector pCANTAB5E (Amersham biosciences Co.) are digested with SfiI (Thermo Scientific, cat# FD 1824) and NotI (Thermo Scientific, cat# FD 0595), and then are subjected to ligation at 16 ℃ overnight under the action of T4 ligase (NEB, cat# M0202S); transferring the ligation product into TG1 electrotransformation competent cells (company: lucigen, cat# 60502), spreading on 2YT-Amp-Glucose (2 XYT medium, 2% Glucose and 100ug/ml ampicillin) solid medium (under the same formula), and culturing at 37deg.C overnight;

④ Scraping bacterial colonies on a culture medium, uniformly mixing, taking part of bacterial liquid, inoculating the bacterial liquid into a2 XYT-Amp-Glucose culture medium, and culturing at 37 ℃ until OD ₆₀₀ = 0.4-0.6; adding auxiliary phage, and culturing at 37 ℃ for 1h; centrifuging the bacterial liquid at 5000rpm, discarding the supernatant, and re-suspending the bacterial liquid by using a 2YT-Amp-Kan culture medium (50 ug/ml kanamycin and 100ug/ml ampicillin are added into the 2 XYT culture medium) with the same volume; culturing overnight at 30deg.C; the next day, centrifugate and get the supernatant; adding 1/4 volume of 5 XPEG/NaCl solution into the supernatant, mixing thoroughly, standing on ice for 1-2h; the supernatant was centrifuged off and the pellet was resuspended in PBS to finally obtain a single-chain ScFv phage library. Preserving at 20 ℃;

primers required for library construction:

Heavy chain amplification primers:

5′L-VH 1：ACAGGTGCCCACTCCCAGGTGCAG(SEQ ID NO:42)，

5′L-VH 3：AAGGTGTCCAGTGTGARGTGCAG(SEQ ID NO:43)，

5′L-VH 4/6：CCCAGATGGGTCCTGTCCCAGGTGCAG(SEQ ID NO:44)，

5′L-VH 5/7：CAAGGAGTCTGTTCCGAGGTGCAG(SEQ ID NO:45)，

Reverse primer:

VH_R1：ACTCGAGACGGTGACCAGGGTGCC(SEQ ID NO:46)，

VH_R2：ACTCGAGACGGTGACCAGGGTTCC(SEQ ID NO:47)；

Kappa light chain amplification primers:

5′L VK 1/2：ATGAGGSTCCCYGCTCAGCTGCTGG(SEQ ID NO:48)，

5′L VK 3：CTCTTCCTCCTGCTACTCTGGCTCCCAG(SEQ ID NO:49)，

5′L VK 4/5：ATTTCTCTGTTGCTCTGGATCTCTG(SEQ ID NO:50)，

3′CK：TGCTGTCCTTGCTGTCCTGCT(SEQ ID NO:51)；

Lambda light chain amplification primers:

5′L Vλ1：GGTCCTGGGCCCAGTCTGTGCTG(SEQ ID NO:52)，

5′L Vλ2：GGTCCTGGGCCCAGTCTGCCCTG(SEQ ID NO:53)，

5′L Vλ3：GCTCTGTGACCTCCTATGAGCTG(SEQ ID NO:54)，

5′L Vλ4/5：GGTCTCTCTCSCAGCYTGTGCTG(SEQ ID NO:55)，

5′L Vλ6：GTTCTTGGGCCAATTTTATGCTG(SEQ ID NO:56)，

5′L Vλ7：GGTCCAATTCYCAGGCTGTGGTG(SEQ ID NO:57)，

5′L Vλ8/9/10：GAGTGGATTCTCAGACTGTGGTG(SEQ ID NO:58)，

3′Cλ：CACCAGTGTGGCCTTGTTGGCTTG(SEQ ID NO:59)。

(2) Tubes were coated with 50ug/mL of human IL1R2 protein (amino acid sequence shown in SEQ ID NO: 39) (experimental group) and 0ug/mL of IL1R2 protein (control group) and shaken overnight at 4 ℃.

The synthesis procedure for human IL1R2 protein is as follows:

① Two digestion sites of EcoRI and NotI are respectively introduced at two ends of cDNA (SEQ ID NO: 40) of human IL1R2, and digestion is carried out by using the two enzymes;

② The expression vector pATX (Co., ltd.: proteoGenix) was digested with EcoRI (Co., ltd.: thermo Scientific, cat. No. FD 0274) and NotI (Co., ltd.: thermo Scientific, cat. FD 0595) to linearize the two enzymes;

③ Connecting the fragment subjected to enzyme digestion with the vector subjected to enzyme digestion, and converting to obtain an expression vector with IL1R2 cDNA;

④ The vector was transfected into HEK293T for expression purification, and then IL1R2 protein was purified by His tag and verified by SDS-PAGE, and the results are shown in FIG. 6.

(3) The coated tube from step (2) was removed and washed three times with 0.05% (v/v) PBST.

(4) 5Ml of 5% skim milk or 1% (m/v) casein was added to the tube and blocked at 37℃for 2h;

meanwhile, the phage library was diluted with 1% (m/v) milk or 0.2% (m/m) casein, and the diluted phage library was incubated at 25℃for 2 hours.

(5) After the incubation, the tubes blocked in step (4) were washed once with 0.05% (v/v) PBST.

(6) Placing the diluted phage display library into the tube sealed in the step (5), and incubating at 37 ℃ for 2 hours.

(7) After the incubation, the incubation was completed, washed three times with 0.05% PBST, followed by two washes with PBS.

(8) The conjugate was eluted with 1mL Glycine buffer (Glycine-HCl) and the eluate was neutralized with Glycine-HCl until pH 7.0.

2. Phage titer detection

(1) E.coli TG1 (company: lucigen, cat# 60502) was cultured until OD ₆₀₀ = 0.4-0.6.

(2) Diluting the liquid eluted in the step (8) in the step of "1. Antibody screening and Synthesis", and adding 10. Mu.L of the diluted liquid to 180. Mu.L of E.coli TG1 to obtain a culture.

(3) The culture obtained in the step (2) is placed in a 37 ℃ incubator for 30 minutes, then poured into a2 XYT-A (Amp 100 ug/mL) culture dish, evenly coated and dried. The plates were placed in an incubator at 37℃overnight for incubation and the titer of the eluted phage was determined.

3. Amplification of eluted phages

(1) Mu.L of E.coli TG1 was added to 800. Mu.L of 2 XYT liquid medium and cultured in a shaker at 37℃until OD ₆₀₀ = 0.4-0.6.

(2) TG1 cultured to logarithmic phase was transferred to 10mL of 2 XYT-G liquid medium (final concentration of glucose is 2%) and cultured on a shaker at 37℃until OD ₆₀₀ = 0.4-0.6.

(3) The product eluted in step (8) of "screening and Synthesis of antibodies" was added to TG1 of step (2), incubated for 30 minutes at 37℃in an incubator, followed by shaking for 30 minutes at 37℃in a shaking table.

(4) 30ML of 2 XYT-AG liquid medium (final concentration of Amp: 0.1% and final concentration of glucose: 2%) was added to the system of step (3), and the mixture was cultured on a shaker at 37℃for 1 hour.

(5) M13KO7 (company: NEB, cat# N0315S, M13KO7: TG 1=20:1) was added, incubated in an incubator at 37℃for 30 minutes, and then shaken on a shaker at 37℃for 30 minutes.

(6) The bacterial solution obtained in step (5) above was centrifuged at 5000rpm for 5 minutes, the supernatant was discarded, and then resuspended in 40mL of 2YT-AK and incubated overnight on a shaker at 30 ℃.

(7) Centrifuging the culture obtained in the step (6), transferring the supernatant to a sterile tube, adding 1/5 (volume ratio) of PEG/NaCl solution to the supernatant, mixing uniformly, and incubating the mixture on ice for 2 hours.

(8) Centrifuging the mixture after incubation on ice in step (7), re-suspending the pellet with 1mL PBS, re-centrifuging, and transferring the supernatant into a sterile tube, wherein the substance in the sterile tube is amplified phage, and the phage can be used for the next biopanning.

4. Detection and screening of amplified phage titer

(1) E.coli TG1 was cultured until OD ₆₀₀ = 0.4-0.6.

(2) The amplified phage obtained in step (8) in the above "amplification of eluted phage" was diluted, and 10. Mu.L of the diluted solution was added to 180. Mu.L of E.coli TG1.

(3) The above culture was incubated in an incubator at 37℃for 30 minutes, then poured into a 2 XYT-A (Amp 100 ug/mL) dish, spread evenly, and air-dried. The plates were placed in an incubator at 37℃overnight for incubation.

5. The above steps are repeated until phage are screened out by panning. Table 1 illustrates that the desired results were obtained with three rounds of elutriation.

TABLE 1 elutriation results

6. Polyclonal phage ELISA detection

(1) Coating: the assay plates were coated with antigen (4. Mu.g/ml IL1R2 protein in the experimental group, PBS in the control group) and overnight at 4 ℃.

(2) Cleaning: antigen coated plates were washed twice with 0.05% (v/v) PBST and then once with PBS.

(3) Closing: the plates were blocked with 300 μl of 5% skim milk at 30deg.C for 2h.

(4) Incubation: 100. Mu.L of the amplified phage after gradient dilution was added and incubated at 37℃for 2 hours.

(5) Cleaning: plates were washed three times with 0.05% (v/v) PBST.

(6) Secondary antibody incubation: adding diluted anti-M13-HRP antibody (SinoBiological, 11973-MM 05T-H) (1:6000, dilution with blocking solution); mu.L of each well was added and incubated at 37℃for 1 hour.

(7) Cleaning: plates were washed with 0.05% (v/v) PBST and a total of three washes were performed.

(8) Color development: 100 mu L of TMB color development liquid is added into each hole, and the reaction is carried out at room temperature and in a dark place for 20 minutes; then each well was terminated by adding 100. Mu.L of 2M HCl stop solution.

(9) Reading: absorbance at 450nm was measured on a microplate reader.

Table 2 shows ELISA results of the polyclonal phage library. As can be seen from the table, the ideal results were obtained by three rounds of screening.

TABLE 2 polyclonal phage ELISA assay results (OD ₄₅₀)

Note that: ag in table 2 is the experimental group and NC is the control group.

7. Monoclonal phage ELISA detection

(1) 96 Single colony clones were selected from the bacterial plates and incubated on a shaker at 37℃ (shaker speed 250 rpm) until OD ₆₀₀ = 0.4-0.6.

(2) M13KO7 infection broth (moi=20:1), incubated for 30min at 37 ℃, then shaken for 30min on a shaking table at 37 ℃. Centrifugation was followed by discarding the supernatant, adding an equal volume of 2 XYT-AK medium (Amp 100. Mu.g/mL, kan 100. Mu.g/mL) and culturing overnight on a shaker at 37 ℃.

(3) Centrifuging the culture solution obtained in the step (2), and using the supernatant for ELISA detection.

(4) Coating: the assay plates were coated with antigen (4. Mu.g/mL IL1R2 protein in the experimental group, PBS in the control group) and overnight at 4 ℃.

(5) Cleaning: antigen coated plates were washed twice with 0.05% (v/v) PBST and then once with PBS.

(6) Closing: the washed plates were blocked with 300. Mu.L of 5% skimmed milk at 30℃for 2h.

(7) Incubation: 100. Mu.L of amplified phage after gradient dilution was added to the blocked plates and incubated at 37℃for 2 hours.

(8) Cleaning: plates were washed three times with 0.05% (v/v) PBST.

(9) Secondary antibody incubation: adding diluted anti-M13-HRP antibody (1:6000, diluting with blocking solution); mu.L of each well was added and incubated at 37℃for 1 hour.

(10) Cleaning: plates were washed three times in total with 0.05% (v/v) PBST.

(11) Color development: 100 μLTMB of color development was added to each well and reacted at room temperature for 20 minutes in the dark. Then each well was terminated by adding 100. Mu.L of 2M HCl stop solution.

(12) Reading: absorbance at 450nm was measured on a microplate reader.

Tables 3 and 4 show the results of the ELISA experiments with the monoclonal phages of the experimental and control groups, respectively. Based on the results, we sent positive clones in the table (underlined clones) for sequencing. Finally, 4 different sequences were obtained and these positive clones were verified a second time.

TABLE 3 Experimental group monoclonal phage ELISA results (Ag: IL1R2 protein, OD ₄₅₀)

TABLE 4 control monoclonal phage ELISA results (NC: PBS, OD ₄₅₀)

8. ELISA detection of Positive clones

(1) 50. Mu.L of the positive clone detected in the above "7. Monoclonal phage ELISA test" was added to 500. Mu.L of 2 XYT-AG medium for cultivation until OD ₆₀₀ = 0.4-0.6.

(2) M13KO7 infection broth (moi=20:1), incubated for 30min at 37 ℃, then shaken for 30min on a shaking table at 37 ℃; centrifugation was followed by discarding the supernatant, adding an equal volume of 2 XYT-AK medium (Amp 100. Mu.g/mL, kan 100. Mu.g/mL) and culturing overnight on a shaker at 37 ℃.

(6) Closing: the plates were blocked with 300 μl of 5% skim milk at 30deg.C for 2h.

(7) Incubation: 100. Mu.L of the amplified phage after gradient dilution was added and incubated at 37℃for 2 hours.

(8) Cleaning: wash three times with 0.05% (v/v) PBST.

(10) Cleaning: plates were washed three times in total with 0.05% (v/v) PBST.

(11) Color development: 100 mu L of TMB color development liquid is added into each hole, and the reaction is carried out at room temperature and in a dark place for 20 minutes; then each well was terminated by adding 100. Mu.L of 2M HCl stop solution.

ELISA results for 4 positive clones are shown in Table 5, and the variable region heavy and light chain amino acid sequences of the resulting antibodies are shown in Table 6 (the amino acid sequences of the CDRs are determined according to the IMGT definition rules).

TABLE 5 Positive clone ELISA results

Note that: in table 5, ag1 is an experimental group and NC is a control group.

TABLE 6 variable region amino acid sequence of positive clones

TABLE 7 CDR amino acid sequences of positive clones

Example two

Recombinant expression of antibodies

Four clones 3999-R3P1-B1, 3999-R3P1-H1, 3999-R3P1-E4 and 3999-R3P1-E11 were selected by phage display technology and subjected to antibody expression purification.

Subcloning the screened sequence onto PATX carrier to obtain full-length antibody light chain and heavy chain:

The coding sequences of the heavy chain and the light chain of 3999-R3P1-B1 are respectively shown as SEQ ID NO. 29 and SEQ ID NO. 33;

the coding sequences of the heavy chain and the light chain of 3999-R3P1-H1 are respectively shown as SEQ ID NO. 30 and SEQ ID NO. 34;

the coding sequences of the heavy chain and the light chain of 3999-R3P1-E4 are respectively shown as SEQ ID NO. 31 and SEQ ID NO. 35;

The heavy and light chain coding sequences of 3999-R3P1-E11 are shown in SEQ ID NO. 32 and SEQ ID NO. 36, respectively.

The prepared endotoxin-free DNA was then transfected into XtenCHO cells (specific transfection protocols are shown in table 8). After waiting 14 days for transfection, the cell culture medium was collected and protein purified, and the purified antibodies were subjected to SDS-PAGE qualitative and quantitative analysis, and the results are shown in FIG. 1.

TABLE 8 transfection patterns of antibody plasmids

Example III

ELISA detection of antibodies

(1) Antigen coating

Mu.l of 5. Mu.g/mL antigen (the antigen is hIL1R2 protein of human origin) was added to each well of the 96-well plate, and incubated at 37℃for 2h.

After coating was completed, the coating was washed twice with 0.05% (v/v) PBST, followed by one wash with PBS.

(2) Closure

The coated plates were blocked with 300. Mu.L of 3% (v/v) BSA-PBS blocking solution at 37℃for 1.5h per well.

(3) Cleaning

Plates were dried and washed three times with 0.05% (v/v) PBST.

(4) Incubation with primary antibody

The screened antibodies were diluted with a concentration gradient of 100. Mu.L per well at 37℃for 1h.

(5) Cleaning

The plates were dried and washed three times with PBST.

(6) Second antibody incubation

Goat anti-human (H+L) antibody (company: cwbio, cat# CW 0169S) was administered according to a 1:10000, adding 100 mu l diluted antibody into each hole of the plate in the step (5), and incubating at 37 ℃ for 0.5h; plates were then dried and washed three times with PBST.

(7) TMB color development

TMB substrate (company: thermo Scientific, cat# TK 2666052) was added, 100. Mu.L per well, and the reaction was carried out at 37℃for 10min in the absence of light; then adding 50 mu L of 2M HCl stop solution into each well to stop; finally, the absorbance at 450nm is read on an microplate reader.

The results are shown in FIG. 2: the screened antibodies bind well to human IL1R2 protein.

Example IV

Evaluation of antitumor Effect of antibodies

To evaluate whether our screened antibodies had a certain antitumor effect, we transplanted MC38 tumor cells (derived from ATCC, colorectal cancer cell line of mice) subcutaneously in C57BL/6N mice, and injected the screened antibodies (3 mg/kg) intraperitoneally into mice 5 days after tumor transplantation (at this time, tumors were approximately 5×5mm ²), with PBS as a control group, once every other day for a total of three injections, starting tumor measurement on day 5, stopping measurement when tumor size exceeded 300mm ², and measuring total of 37 days. Three of the antibodies selected (3999-R3P 1-B1, 3999-R3P1-E4, 3999-R3P 1-E11) were found to have some antitumor effect (as shown in FIG. 3).

Then we repeated the above experiments with the three antibodies of 3999-R3P1-B1, 3999-R3P1-E4 and 3999-R3P1-E11 screened, and injected intraperitoneally 6 times each mouse, and the tumor measurement was started every 5 th day, and stopped every other day or 2 days when the tumor size exceeded 300mm ², and the two antibodies 3999-R3P1-B1 and 3999-R3P1-E4 were detected on the 35 th day together (as shown in FIG. 4), and the survival rate of the mouse was significantly improved (as shown in FIG. 5).

3999-R3P1-B1-H (SEQ ID NO:29, variable region underlined, constant region in the remainder)

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCATCAGTGCTGGTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTAGGACATACTACAGGTCCAAGTGGAATTATGATTATGCACCTTCTGTGAAAAGTCGAACAACCATCGACCCAGACACATCCAAGAACCAGTTCTCCCTGCATCTGAACTCTGTGACTCCCGAGGACACCGCTGTGTATTACTGTGTAAGATCGACCTCGGCTCGTCTGTTTGACCGCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGA

3999-R3P1-H1-H (SEQ ID NO:30, variable region underlined, constant region in the remainder)

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGGCATCTCCGGGGACAGTGTCTCTAGCAACACTGCTGTTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCACGTGGCATCATGATTATCCATTATCTGTGAAAAGTCGAATCACCATCAACCCAGACACATCCAACAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAGGACGCGGCTGTTTATTACTGTGCAAGAGGGACTCTTCGCTTCGGTATGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGA

3999-R3P1-E4-H (SEQ ID NO:31, variable region underlined, constant region in the remainder)

CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGAGGGTTCCCGAAGGGGGTACCAGCTGCTATCTCGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGTGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGA

3999-R3P1-E11-H (SEQ ID NO:32, variable region underlined, constant region in the remainder)

CAGGTGCAGCTGGTGCAATCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAACCATAGCAGTGGCTGGTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGA

3999-R3P1-B1-L (SEQ ID NO: variable region underlined), the remainder being constant region

TCCTATGAGCTGACACAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGA

3999-R3P1-H1-L (SEQ ID NO:34, variable region underlined, constant region in the remainder)

CAGCTTGTGCTGACTCAATCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTATGTCTTCGGAACTGGCACCAAGGTGACCGTCCTCCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGA

3999-R3P1-E4-L (SEQ ID NO:35, variable region underlined, constant region in the remainder)

GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCCTACACAGTGATGGAAACACCTACTTGAATTGGTTTCACCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTCTAAGGTTTCTAACCGGGTCGCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGACACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACAAACTGGCACCGGACGTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGA

3999-R3P1-E11-L (SEQ ID NO:36, variable region underlined, constant region in the remainder)

CAGTCTGTGTTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTTTTGTTCCGGAAGCAGCTCCAACATCGGAAGTAATTATGTCCACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGGGTGCTTGGGTGTTCGGCGGAGGCACCAAGGTGACCGTCCTCCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGA

Heavy chain constant region (SEQ ID NO: 37)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain constant region (SEQ ID NO: 38)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Human IL1R2 protein amino acid sequence (SEQ ID NO: 39):

MLRLYVLVMGVSAFTLQPAAHTGAARSCRFRGRHYKREFRLEGEPVALRCPQVPYWLWASVSPRINLTWHKNDSARTVPGEEETRMWAQDGALWLLPALQEDSGTYVCTTRNASYCDKMSIELRVFENTDAFLPFISYPQILTLSTSGVLVCPDLSEFTRDKTDVKIQWYKDSLLLDKDNEKFLSVRGTTHLLVHDVALEDAGYYRCVLTFAHEGQQYNITRSIELRIKKKKEETIPVIISPLKTISASLGSRLTIPCKVFLGTGTPLTTMLWWTANDTHIESAYPGGRVTEGPRQEYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVKEGS

nucleotide sequence of human IL1R2 (SEQ ID NO: 40)

ATGTTGCGCTTGTACGTGTTGGTAATGGGAGTTTCTGCCTTCACCCTTCAGCCTGCGGCACACACAGGGGCTGCCAGAAGCTGCCGGTTTCGTGGGAGGCATTACAAGCGGGAGTTCAGGCTGGAAGGGGAGCCTGTAGCCCTGAGGTGCCCCCAGGTGCCCTACTGGTTGTGGGCCTCTGTCAGCCCCCGCATCAACCTGACATGGCATAAAAATGACTCTGCTAGGACGGTCCCAGGAGAAGAAGAGACACGGATGTGGGCCCAGGACGGTGCTCTGTGGCTTCTGCCAGCCTTGCAGGAGGACTCTGGCACCTACGTCTGCACTACTAGAAATGCTTCTTACTGTGACAAAATGTCCATTGAGCTCAGAGTTTTTGAGAATACAGATGCTTTCCTGCCGTTCATCTCATACCCGCAAATTTTAACCTTGTCAACCTCTGGGGTATTAGTATGCCCTGACCTGAGTGAATTCACCCGTGACAAAACTGACGTGAAGATTCAATGGTACAAGGATTCTCTTCTTTTGGATAAAGACAATGAGAAATTTCTAAGTGTGAGGGGGACCACTCACTTACTCGTACACGATGTGGCCCTGGAAGATGCTGGCTATTACCGCTGTGTCCTGACATTTGCCCATGAAGGCCAGCAATACAACATCACTAGGAGTATTGAGCTACGCATCAAGAAAAAAAAAGAAGAGACCATTCCTGTGATCATTTCCCCCCTCAAGACCATATCAGCTTCTCTGGGGTCAAGACTGACAATCCCGTGTAAGGTGTTTCTGGGAACCGGCACACCCTTAACCACCATGCTGTGGTGGACGGCCAATGACACCCACATAGAGAGCGCCTACCCGGGAGGCCGCGTGACCGAGGGGCCACGCCAGGAATATTCAGAAAATAATGAGAACTACATTGAAGTGCCATTGATTTTTGATCCTGTCACAAGAGAGGATTTGCACATGGATTTTAAATGTGTTGTCCATAATACCCTGAGTTTTCAGACACTACGCACCACAGTCAAGGAAGGTAGTCA

Reference is made to:

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It should be noted that, although the technical solution of the present invention is described in specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the technical improvements in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. An anti-IL 1R2 antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein,

2. The anti-IL 1R2 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region as set forth in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or more sequence identity to any one of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7; and/or the number of the groups of groups,

3. The anti-IL 1R2 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the anti-IL 1R2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3 or IgG4 or a variant thereof; and/or the number of the groups of groups,

4. The anti-IL 1R2 antibody or antigen-binding fragment thereof according to any one of claims 1-3, which comprises a human antibody; and/or the number of the groups of groups,

5. An isolated polynucleotide, wherein the polynucleotide encodes the anti-IL 1R2 antibody or antigen-binding fragment thereof of any one of claims 1-4.

6. An expression cassette, wherein the expression cassette comprises the polynucleotide of claim 5.

7. A recombinant vector, wherein the recombinant vector comprises the polynucleotide of claim 5 or the expression cassette of claim 6.

8. A host cell, wherein the host cell comprises the polynucleotide of claim 5, the expression cassette of claim 6, or the recombinant vector of claim 7.

9. The host cell of claim 8, wherein the host cell is a microorganism or a mammalian cell;

optionally, the microorganism is selected from bacteria and/or yeasts;

10. A phage wherein the phage surface displays an anti-IL 1R2 antibody or antigen-binding fragment thereof according to any one of claims 1-4.

11. A method of making the anti-IL 1R2 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising culturing the host cell of claim 8 or 9 under conditions that allow production of the anti-IL 1R2 antibody or antigen-binding fragment thereof, and recovering and isolating the anti-IL 1R2 antibody or antigen-binding fragment thereof.

12. A pharmaceutical composition comprising: an anti-IL 1R2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, a polynucleotide according to claim 5, an expression cassette according to claim 6, a recombinant vector according to claim 7, or a host cell according to claim 8 or 9; and

Optionally, pharmaceutically acceptable carriers and/or excipients.

13. Use of an anti-IL 1R2 antibody or antigen-binding fragment thereof according to any one of claims 1-4, a polynucleotide according to claim 5, an expression cassette according to claim 6, a recombinant vector according to claim 7, a host cell according to claim 8 or 9, or a pharmaceutical composition according to claim 12 in the manufacture of a medicament for the treatment of an IL1R2 mediated disease or disorder.