CN119409812A

CN119409812A - ICAM-1 antibody and its application

Info

Publication number: CN119409812A
Application number: CN202411490127.6A
Authority: CN
Inventors: 张守涛; 程文慧; 黄一博
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2024-10-24
Filing date: 2024-10-24
Publication date: 2025-02-11

Abstract

The invention provides a monoclonal antibody or an antigen binding portion thereof, which can bind ICAM-1 and comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of CDR1-3 regions of the heavy chain variable region are shown as SEQ ID NOs 1-3 in sequence, and the amino acid sequences of CDR1-3 regions of the light chain variable region are shown as SEQ ID NOs 4-6 in sequence. The invention also provides application of the novel compound. The invention can specifically inhibit LFA-1/ICAM-1 interaction, especially as a diagnostic reagent for preventing or treating related tumors and inflammations.

Description

ICAM-1 antibodies and uses thereof

Technical Field

The present invention relates to an antibody and uses thereof.

Background

LFA-1 is a member of the integrin β2 family, expressed on the leukocyte cell surface, and consists of the αl (CD 11 a) subunit and the β2 (CD 18) subunit. ICAM-1 is mainly involved in leukocyte trafficking and immune cell recruitment, and up-regulated in expression upon inflammatory factor stimulation. LFA-1/ICAM-1 interactions play a role in the T cell activation of lymph nodes, T cell migration, and potential secondary activation of ocular surface T cells. ICAM-1 plays an important role in promoting adhesion at sites of inflammation, controlling tumor progression and metastasis, and regulating immune response in the body. It enhances the adhesion between leucocytes, inflammatory cells, tumor cells and the like and endothelial cells through the specific combination with the receptor thereof, promotes the activation of the endothelial cells and makes them easier to penetrate the endothelium. Intercellular adhesion molecules on endothelial cells mediate cell-to-cell or cell-to-matrix interactions and binding, thereby participating in physiological and pathological processes such as cell signaling and activation, cell tissue growth and differentiation, immune response, inflammatory response, angiogenesis, and tumor metastasis. Studies have shown that effector T cells reach the ocular surface along blood vessels, secreting various cytokines to promote the production of pro-inflammatory mediators by ocular tissues, causing ocular surface damage. Inhibiting LFA-1/ICAM-1 interactions, thereby disrupting various key steps of T cell mediated inflammation, can inhibit inflammatory pathways of tumors such as solid tumors or Dry Eye (DED).

Disclosure of Invention

The present invention provides an antibody or antigen binding portion thereof that binds ICAM-1, and specifically inhibits LFA-1/ICAM-1 interactions, treating malignant tumors or related inflammatory conditions such as DED.

Specifically, the invention includes the following aspects:

The first aspect of the present invention relates to an anti-ICAM-1 monoclonal antibody or an antigen-binding portion thereof, wherein the amino acid sequences of CDR1-3 regions of a heavy chain variable region VH are shown in SEQ ID NOs 1-3 in sequence (NOs represent a parallel list of sequence numbers in the art, namely, sequence numbers are 1/2/3), and the amino acid sequences of CDR1-3 regions of a light chain variable region VL are shown in SEQ ID NOs 4-6 in sequence. Optionally, the VH amino acid sequence is SEQ ID NO. 7, or the VL sequence is SEQ ID NO. 8. Optionally, the monoclonal antibody or antigen binding portion thereof is a full antibody, a bispecific antibody, scFv, fab, fab ', F (ab') 2 or Fv, such as scFv, in which case the VH, VL may be coupled via a flexible linker, which may be a short peptide such as (GGGS) nC, (GGSC) N, (GGGGS) N or (GGG) N, where n=1-10, preferably n=3, the linking position of the VH, VL may be either the N-terminus of the heavy chain linked to the C-terminus of the light chain or the N-terminus of the light chain linked to the C-terminus of the heavy chain. The constant region-containing scheme of the invention comprises the following steps:

In one embodiment, the heavy chain constant region of the anti-ICAM-1 monoclonal antibody or antigen-binding portion thereof is selected from the group consisting of IgG, igM, igE, igD and IgA derived from human.

In one embodiment of the present invention, the heavy chain constant region of the anti-ICAM-1 monoclonal antibody or antigen-binding portion thereof is selected from the group consisting of IgG1, igG2, igG3, and IgG4 derived from human.

In a specific embodiment of the present invention, the heavy chain constant region of the anti-ICAM-1 monoclonal antibody or antigen-binding portion thereof is derived from human IgG1.

In one embodiment of the invention, the light chain constant region of the anti-ICAM-1 monoclonal antibody or antigen binding portion thereof is kappa or lambda derived from human.

In a specific embodiment of the present invention, the light chain constant region of the anti-ICAM-1 monoclonal antibody or antigen-binding portion thereof is derived from human kappa.

The present invention also relates to a nucleic acid molecule comprising a sequence encoding an anti-ICAM-1 monoclonal antibody or antigen binding portion thereof according to any of the first aspects of the invention.

In one embodiment of the invention, the nucleic acid molecule comprises the sequences h and/or l:

sequence(s) h: CAGGTCAAACTGCAGCAGTCAGGGGCTGACCTGGTGAAGCCTGGGGCTTCAGTGATGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAGATCCTCCAGCACAGCATACATGCAACTCAGCAATCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACAGGCTGGTTACTACGGATACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Sequence l: GACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAGTCAGCAGTGTAGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG, comprises, for example:

CAGGTCAAACTGCAGCAGTCAGGGGCTGACCTGGTGAAGCCTGGGGCTTCAGTGATGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAGATCCTCCAGCACAGCATACATGCAACTCAGCAATCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACAGGCTGGTTACTACGGATACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAGTCAGCAGTGTAGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG.

the invention also relates to recombinant vectors comprising a nucleic acid molecule according to any one of the above-described aspects of the invention.

In the present invention, the vector may be a cloning vector or an expression vector. The vector contains the nucleic acid molecule of any one of the present invention, which can be obtained, for example, by inserting the above-mentioned nucleic acid molecule into a cloning vector or an expression vector, or can be obtained by artificial synthesis.

In the present invention, the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector, a phage vector or a viral vector. Wherein the prokaryotic expression vector is, for example, a PET vector, a PGEX vector, the eukaryotic expression vector is, for example, pcDNA3.1, pEGFP-C1, pPIC9K, the phage vector is, for example, a lambda phage vector λgt, λgt- λB, and the viral vector is, for example, a retrovirus, lentivirus, adenovirus or adeno-associated virus vector.

The invention also relates to recombinant cells comprising the recombinant vector of any one of the invention.

In one embodiment of the invention, the cell is a mammalian cell, or a bacterium such as E.coli, or a fungus such as yeast, the mammalian cell being a cell suitable for expression of antibodies, e.g.a mammalian cell of human, murine or monkey origin, and in a more specific embodiment the mammalian cell is a CHO cell, e.g.a CHO-K1 cell.

The invention also relates to a composition comprising an anti-ICAM-1 monoclonal antibody or antigen-binding portion thereof of any of the invention, a nucleic acid molecule of any of the invention, a recombinant vector of any of the invention, or a recombinant cell of any of the invention, and optionally a pharmaceutically acceptable carrier or excipient.

The present invention also relates to a method for producing an anti-ICAM-1 monoclonal antibody or an antigen-binding portion thereof of any one of the present invention, which comprises the step of using the nucleic acid molecule of any one of the present invention, the recombinant vector of any one of the present invention, or the recombinant cell of any one of the present invention.

In one embodiment of the invention, it specifically comprises the steps of:

1) Cloning the nucleotide sequence of the nucleic acid molecule of any one of the invention into an expression vector to obtain a recombinant expression vector;

2) Transferring the recombinant expression vector into a host cell to obtain a recombinant cell;

3) Culturing the recombinant cells obtained in the step 2) in a target culture medium to obtain a cell strain capable of expressing the antibody;

4) Gradually amplifying and culturing the cell strain obtained in the step 3), and harvesting culture supernatant;

5) Purifying the culture supernatant obtained in step 4) to obtain the anti-ICAM-1 monoclonal antibody or an antigen-binding portion thereof according to any one of the present invention.

In one embodiment of the invention, the cell is a mammalian cell, which is a cell suitable for expressing antibodies, e.g., a mammalian cell of human, murine or monkey origin. In a specific embodiment of the invention, the mammalian cell is a CHO cell, for example a CHO-K1 cell.

The invention also relates to the use of an anti-ICAM-1 monoclonal antibody or antigen binding portion thereof according to any one of the invention for specifically inhibiting LFA-1/ICAM-1 interactions, in particular as a diagnostic agent, for preventing or treating related tumors, including solid tumors such as breast cancer, thyroid cancer, etc., an example of which is DED.

The invention is further described below:

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

In the present invention, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V _H) and a heavy chain constant region (C _H). The heavy chain constant region consists of 3 domains (C _H1、C_H and C _H). Each light chain consists of a light chain variable region (V _L) and a light chain constant region (C _L). The light chain constant region consists of one domain C _L. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The V _H and V _L regions can also be subdivided into regions of high denaturation, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each of V _H and V _L consists of 3 CDRs and 4 FRs arranged from amino-terminus to carboxyl-terminus of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions (V _H and V _L) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol. Biol. 196:901-917; chothia et al (1989) Nature 342:878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. the antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

In the present invention, the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., PCSK 9) to which the antibody binds, competing with the intact antibody for specific binding to the antigen. In some cases, the antigen binding portion includes Fab, fab ', F (ab') _2、 Fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

In the present invention, the term "Fd fragment" means an antibody fragment consisting of the V _H and C _H 1 domains, the term "Fv fragment" means an antibody fragment consisting of the V _L and V _H domains of a single arm of an antibody, the term "dAb fragment" means an antibody fragment consisting of the V _H domain (Ward et al Nature 341:544-546 (1989)), the term "Fab fragment" means an antibody fragment consisting of the V _L、V_H、C_L and C _H 1 domains, and the term "F (ab') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

In some cases, the antigen-binding portion of the antibody is a single chain antibody (e.g., scFv), in which the V _L and V _H domains form monovalent molecules by pairing through a linker that enables production as a single polypeptide chain (see, e.g., bird et al, science 242:423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules may have the general structure NH ₂-V_L -linker-V _H -COOH or NH ₂-V_H -linker-V _L -COOH. Suitable prior art linkers (connecting peptides) consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ may be used, but variants thereof may also be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56, and Roovers et al (2001), cancer Immunol. In an embodiment of the invention, the sequence of the connecting peptide is (GGGGS) ₃.

In some cases, the antibody is a bispecific antibody capable of binding to two antigens or antigen epitopes, respectively, comprising a light chain, heavy chain, or antigen binding portion thereof of an antibody that specifically binds a first antigen, and a light chain, heavy chain, or antigen binding portion thereof of an antibody that specifically binds a second antigen. In embodiments of the invention, the light chain, heavy chain or antigen binding portion thereof of the bispecific antibody that binds a first antigen may be an antibody or antigen binding portion thereof of any one of the invention, and the light chain, heavy chain or antigen binding portion thereof of the antibody that specifically binds a second antigen may be another anti-ICAM-1 antibody or antigen binding portion thereof or an antibody or antigen binding portion thereof directed against another antigen.

In some cases, the antibody is a diabody, i.e., a diabody, in which the V _H and V _L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, forcing the domains to pair with the complementary domain of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R. J. Et al, structures 2:1121-1123 (1994)).

The antigen-binding portion of an antibody (e.g., the antibody fragment described above) can be obtained from a given antibody (e.g., monoclonal antibody 2E 12) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antigen-binding portions of the antibody in the same manner as for an intact antibody.

In the present invention, the antigen-binding portion includes single chain antibodies (scFv), chimeric antibodies, diabodies, scFv-Fc bivalent molecules, dAbs and Complementarity Determining Region (CDR) fragments, fab fragments, fd fragments, fab 'fragments, fv and F (ab') ₂ fragments.

In the present invention, the lambda light chain constant region includes various allotypes such as lambda I, lambda II, lambda III, lambda VI. In an embodiment of the invention, the lambda light chain constant region is of lambda II-type.

The antibody nucleic acid molecules of the invention may also be obtained using conventional genetic engineering recombinant techniques or chemical synthesis methods. In one aspect, the invention relates to an antibody nucleic acid molecule comprising the heavy chain variable region of an anti-ICAM-1 antibody or a portion of the nucleic acid sequence of an antibody molecule. In another aspect, the sequences of the antibody nucleic acid molecules to which the present invention relates also include the light chain variable region of an anti-ICAM-1 antibody or a portion of the nucleic acid sequence of an antibody molecule. In another aspect, the invention relates to a sequence of an antibody nucleic acid molecule further comprising CDR sequences of the heavy or light chain variable regions. The complementarity determining regions (complementary determinant region, CDRs) are the sites for binding to the epitope, and the CDR sequences in the present invention are determined by IMGT/V-QUEST (http:// IMGT. Cines. Fr/textes/vquest /). However, the CDR sequences obtained by the different partitioning methods are slightly different.

The present invention relates to recombinant expression vectors containing said nucleic acid molecules and to host cells transformed with these molecules. Furthermore, the present invention relates to a method for culturing and isolating the antibody of the invention under specific conditions using host cells comprising said nucleic acid molecules.

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

In the present invention, the term "host cell" refers to a cell into which a vector is introduced, and includes many cell types such as prokaryotic cells such as E.coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

The antibody fragments of the invention may be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). alternatively, the antibody fragments may be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000))., e.g., fab 'fragments may be obtained directly from E.coli cells or chemically conjugated to form F (ab') 2 fragments (Carter et al, bio/Technology, 10:163-167 (1992)). For another example, F (ab ') ₂ fragments may be obtained by ligation with leucine zipper GCN 4. In addition, fv, fab or F (ab') ₂ fragments may also be isolated directly from recombinant host cell culture broth. Other techniques for preparing antibody fragments are well known to those of ordinary skill in the art.

In the present invention, "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen from which the antibody is produced. Here, the binding affinity of an antibody that binds a first antigen to a second antigen is undetectable or weak. In certain embodiments, an antigen-specific antibody is one that binds the antigen with an affinity (KD). Ltoreq.10 ^-5 M (e.g., 10 ^-6 M、10^-7 M、10^-8 M、10^-9 M、10^-10 M, etc.), wherein KD refers to the ratio of dissociation rate to binding rate (koff/kon), which can be determined by methods familiar to those skilled in the art.

In the present invention, 20 conventional amino acids and abbreviations thereof follow conventional usage. See Immunology-A SYNTHESIS (2 nd edition, e.s. Golub and d.r. Gren, eds., sinauer Associates, sunderland, mass. (1991)), which is incorporated herein by reference.

Drawings

FIG. 1 shows the results of the activity detection of the antibody of example 1 of the present invention.

Detailed Description

EXAMPLE 1 construction, screening and identification of anti-ICAM-1 phage Single-chain antibody library

1. The phagemid vector pCANTAB5E and the helper phage M13K07 are provided by the laboratory, the extracellular sequence of the transmembrane protein is found according to the known ICAM-1 sequence, the expression and purification of the ICAM-1 extracellular protein are realized, the ICAM-1 ECD protein expressed and purified is immunized with a BALB/c mouse, after the successful immunization is confirmed, the spleen of the mouse is isolated, the total RNA is extracted, the total RNA is reversely transcribed into cDNA, the specific degenerate primer is used for amplifying antibody light chain genes and heavy chain genes by PCR, then a single-chain antibody phage library is constructed, an anti-ICAM-1 phage scFv antibody is obtained through multiple rounds of screening, the sequence of the antibody gene is ：CAGGTCAAACTGCAGCAGTCAGGGGCTGACCTGGTGAAGCCTGGGGCTTCAGTGATGTTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAGATCCTCCAGCACAGCATACATGCAACTCAGCAATCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACAGGCTGGTTACTACGGATACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAGTCAGCAGTGTAGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG,, the chain composition form is V _H-linker-V_L,V_H, the mark of the sequence is QVKLQQSGADLVKPGASVMLSCKASGYTFTSYYMYWVKQRPGQGLEWIGEINPSNGGTNFNEKFKTKATLTVDRSSSTAYMQLSNLTSEDSAVYYCTRQAGYYGYYAMDYWGQGTTVTVSS（, the mark of the sequence represents CDR1-3, and then the mark is identical), the sequence of the V _L is DIELTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKLLIYRTSNLASGVPARFSGSGSGTSYSLTVSSVEAEDAATYYCQQYSGYPLFTFGSGTKLEIKR, connecting peptide linker is GGGGSGGGGSGGGGS;

Using the above scFv antibody gene, an expression vector was constructed, expressed in E.coli, purified, and the Western Blot indicated successful expression of the recombinant protein, and used for further activity confirmation below.

2. Elisa for detecting affinity activity of anti-ICAM-1 single-chain antibody with antigen

(1) Coating plate ICAM-1 ECD protein was diluted with coating solution (pH=9.6), and 100. Mu.L of protein solution was added to each well of the ELISA plate at 4℃overnight.

(2) Washing, namely, removing the coating liquid, adding 200 mu L of PBST into each hole, washing 5 min, and washing 3 times.

(3) Blocking, 200. Mu.L of blocking solution was added to each well and placed at 37℃for 2 h.

(4) Repeating the step (2).

(5) The primary antibody was incubated by adding 100. Mu.L of diluted protein per well of the gradient diluted scFv antibody and leaving it at 37℃for 2 h.

(6) Repeating the step (2).

(7) Incubation of secondary antibody 100 μl of HRP-labeled mouse anti-His antibody was added to each well and placed at 37 ℃ for 1 h.

(8) Repeating the step (2).

(9) Color development 100. Mu.L TMB color developing solution was added to each well and left at 37℃for 15-30 min. A further 100. Mu.L of Elisa stop solution was added to each well.

(10) Reading, namely opening the multifunctional enzyme label instrument in advance, setting parameters, and recording the value of the OD450, wherein the result is shown in figure 1.

ICAM-1 ECD antigen concentration is 1 u g/mL and 5 u g/mL, and IC50 substituted into the affinity constant calculation formula, through calculation of the scFv affinity constant is 1.02x ⁶ L/mol.

Claims

1. A monoclonal antibody or antigen-binding portion thereof, which is capable of binding ICAM-1 and comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of CDR1-3 regions of the heavy chain variable region are shown as SEQ ID NOs 1-3 in sequence, and the amino acid sequences of CDR1-3 regions of the light chain variable region are shown as SEQ ID NOs 4-6 in sequence.

2. The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region amino acid sequence is shown in SEQ ID No. 7.

3. The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the light chain variable region amino acid sequence is shown in SEQ ID No. 8.

4. The monoclonal antibody, or antigen-binding portion thereof, of any one of claims 1-3, which is a full antibody, a bispecific antibody, scFv, fab, fab ', F (ab') ₂, or Fv.

5. The monoclonal antibody or antigen binding portion thereof according to the preceding claim, which is an scFv, the connecting peptide of the heavy chain variable region and the light chain variable region of which is (GGGS) _nC、（GGGGS）_n、（GGSC）_n or (GGG) _n, wherein n = 1-10, such as 3.

6. The monoclonal antibody or antigen binding portion thereof according to the preceding claim, wherein the C-terminal of the heavy chain variable region is linked to the N-terminal of the light chain variable region by means of said linking peptide.

7. A nucleic acid molecule comprising a sequence encoding the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6.

8. Recombinant cell comprising the nucleic acid molecule of the preceding claim, preferably said cell is a mammalian cell or a bacterial, fungal, e.g. Ecoli, yeast or CHO cell.

9. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, or the recombinant cell of claim 8.

10. Use of a monoclonal antibody or antigen binding portion thereof according to any preceding claim in the manufacture of a diagnostic agent or a medicament for the treatment of solid tumors or dry eye.