CN113801231A

CN113801231A - anti-DOG-1 antibodies or antigen-binding fragments thereof and uses thereof

Info

Publication number: CN113801231A
Application number: CN202110962262.6A
Authority: CN
Inventors: 陈向征; 伍洋平; 李文婷; 杨金亮; 李为民
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-12-17
Anticipated expiration: 2041-08-20
Also published as: CN113801231B

Abstract

The present invention provides antibodies, methods, compositions and articles of manufacture having improved efficacy, which are broadly applicable in the field of antibody therapy and diagnosis, and which can be used in combination with antibodies capable of reacting with various targets. The invention provides nucleic acid molecules that bind to anti-DOG-1 antibodies or antigen-binding fragments thereof, encode anti-DOG-1 antibodies, vectors and host cells for expressing anti-4-1 BB antibodies. The invention further provides application of the anti-DOG-1 antibody disclosed by the invention in preparation of a medicament and/or a detection reagent for detecting, treating and/or preventing tumors.

Description

anti-DOG-1 antibodies or antigen-binding fragments thereof and uses thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an anti-DOG-1 antibody or an antigen binding fragment thereof and application thereof.

Background

DOG1(Discovered on gastric structural tumor-1), a calcium ion activated chloride channel protein, is also known as ANO1, TAOS2, ORAOV2 and TMEM16A, and is involved in physiological processes such as cell membrane potential, body fluid secretion, olfactory formation, nerve impulse conduction and smooth muscle contraction. In tumors, DOG1 was abnormally highly expressed. Research has shown that DOG1 is generally highly expressed in Gastrointestinal stromal tumors (GIST), with a positive rate as high as 99%. DOG1 is also highly expressed in various digestive tract tumors, including esophageal squamous cell carcinoma, gastric adenocarcinoma, lung adenocarcinoma, leiomyoma and the like, and the expression level in normal tissues is extremely low.

In addition, a large sample clinical trial consisting of 1840 patients with GIST demonstrated that DOG1 has more accuracy than the conventional c-KIT target as a diagnostic molecular target for GIST, and DOG1 has been widely used in the diagnosis of GIST.

Gastrointestinal stromal tumors GIST are the most common tumors of gastrointestinal stromal origin. GIST is thought to originate from Interstitial Cells of Cajal (ICC) or stem cell precursors. Most patients have a KIT mutation and a small percentage have a PDGFRA mutation. Such mutations result in sustained activation of ligand independent Receptor Tyrosine Kinases (RTKs), leading to rapid tumor progression. There are also wild-type tumors without the above mutations, whose occurrence and proliferation mechanisms are unclear. Gastrointestinal stromal tumors are not susceptible to chemotherapy, and surgical resection is the current treatment of choice. In recent years, with the development of targeted drugs, targeted drug therapy can be used for patients with large volume and difficult surgery. Imatinib (Imatinib mesylate, Gleevec) is a small molecule inhibitor, can effectively inhibit KIT tyrosine kinase receptor, has activity of resisting KIT and PDFGFRA, and is a first-line clinical targeted drug for treating unresectable GIST in the existing operation. Other small molecule targeted drugs such as Sunitinib (Sutent) and the like are currently used as alternative therapeutic drugs for GIST patients after imatinib treatment failure. However, the drugs can quickly generate drug resistance in clinical application. In conclusion, in the process of targeted therapy of colon cancer and interstitial tumor, the tumor has the phenomenon of rapid drug resistance, so that the continuous development of novel targeted drugs has great significance.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide an anti-DOG-1 antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the anti-DOG-1 antibody, a vector and a host cell for expressing the anti-DOG-1 antibody. The invention also provides application of the anti-DOG-1 antibody in preparation of a medicament and/or a detection reagent for detecting, treating and/or preventing tumors.

To achieve the above object, the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to DOG-1, the antibody or the antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

(i) VH CDR1 having the amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (for example, substitution, deletion, or addition of 1, 2, or 3 amino acids) compared with the sequence of CDR1 contained in the VH;

(ii) VH CDR2 having the amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (for example, substitution, deletion, or addition of 1, 2, or 3 amino acids) compared with the sequence of CDR2 contained in the VH; and

(iii) VH CDR3 having the amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having substitution, deletion, or addition of one or several amino acids (for example, substitution, deletion, or addition of 1, 2, or 3 amino acids) compared with the sequence of CDR3 contained in the VH;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) VL CDR1 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared with the sequence of CDR1 contained in the VL;

(v) VL CDR2 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared with the sequence of CDR2 contained in the VL; and

(vi) VL CDR3 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion, or addition of one or several amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared with the sequence of CDR3 contained in the VL.

In certain preferred embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution.

In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system.

In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof alternatively comprises:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) a VH CDR1, consisting of the sequence: SEQ ID NO: 3, or a variant of SEQ ID NO: 3 with one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3 or 4 amino acid substitutions);

(ii) a VH CDR2, consisting of the sequence: SEQ ID NO: 4, or a sequence identical to SEQ ID NO: 4 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5 or 6 amino acid substitutions); and

(iii) a VH CDR3, consisting of the sequence: SEQ ID NO: 5, or a variant of SEQ ID NO: 5 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions);

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) a VL CDR1, consisting of the sequence: SEQ ID NO: 6, or a variant of SEQ ID NO: 6 with one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions);

(v) a VL CDR2, consisting of the sequence: SEQ ID NO: 7, or a variant of SEQ ID NO: 7 with one or several amino acid substitutions, deletions or additions (e.g. 1 or 2 amino acid substitutions); and

(vi) a VL CDR3, consisting of the sequence: SEQ ID NO: 8, or a variant of SEQ ID NO: 8 with one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5 or 6 amino acid substitutions);

wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise a Framework Region (FR) derived from a mammalian (e.g., murine or human) immunoglobulin.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) Framework Region (FR) derived from a murine immunoglobulin, and/or the VL of said antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a murine immunoglobulin.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are humanized.

In certain preferred embodiments, the antibodies or antigen binding fragments thereof of the invention are humanized to a degree of at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) Framework Region (FR) derived from a human immunoglobulin, and/or the VL of said antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a human immunoglobulin.

In such embodiments, the heavy chain variable region FR and/or the light chain variable region FR of the antibody or antigen binding fragment thereof of the invention may comprise one or more non-human (e.g., murine) amino acid residues, e.g., the heavy chain framework region FR and/or the light chain framework region FR may comprise one or more amino acid back mutations in which there is a corresponding murine amino acid residue.

In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a framework region of a human immunoglobulin (e.g., a framework region comprised in an amino acid sequence encoded by a human germline antibody gene), which optionally comprises one or more mutations from human residues to murine residues.

In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises either:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 1;

(ii) and SEQ ID NO: 1 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions);

or

(iii) And SEQ ID NO: 1, having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%;

and/or

(b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 2;

(v) and SEQ ID NO: 2 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions);

or

(vi) And SEQ ID NO: 2, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived. In certain preferred embodiments, the variant has conservative substitutions of one or more amino acids compared to the sequence from which it is derived.

In certain preferred embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived;

and/or

The light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In certain preferred embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

In certain preferred embodiments, the heavy chain constant region is a murine IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

In certain preferred embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, preferably the heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.

In certain preferred embodiments, the light chain constant region is a kappa light chain constant region, preferably, a murine kappa light chain constant region or a human kappa light chain constant region.

In certain preferred embodiments, the antibodies of the invention are chimeric or humanized antibodies.

In certain preferred embodiments, the antigen-binding fragment of the invention is selected from the group consisting of scFv, Fa b ', (Fa b') 2, Fv fragments, diabodies (diabodies).

In other aspects, the invention also provides a conjugate comprising an antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

In certain preferred embodiments, the conjugate is an antibody-drug conjugate (ADC).

In certain preferred embodiments, the therapeutic agent is selected from a cytotoxin or a radioisotope.

Non-limiting examples of suitable therapeutic agents in the present invention include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents.

In certain preferred embodiments, the antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof comprises an anti-DOG-1 antibody and one or more small molecule toxins, including, but not limited to, small molecule drugs such as camptothecin derivatives, calicheamicin (calicheamicin), maytansine (maytansine) or derivatives thereof, maytansinoids (maytansinoids), dolastatins (dolastatins), auristatins, trichothecenes (trichothecenes), and CC1065, and fragments of these drugs having cytotoxic activity.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is conjugated to the therapeutic agent, optionally through a linker.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are conjugated to the therapeutic agent via a cleavable linker (e.g., a peptide linker, disulfide, or hydrazone linker).

In other aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region and/or a light chain variable region thereof.

In certain preferred embodiments, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and/or a light chain variable region thereof.

In certain preferred embodiments, the invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein,

the nucleic acid molecule encoding the variable region of the antibody heavy chain has a sequence selected from the group consisting of:

(a) as shown in SEQ ID NO: 9, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, as compared to the nucleotide sequence of (a));

the nucleic acid molecule encoding the variable region of the antibody light chain has a sequence selected from the group consisting of: (a) as shown in SEQ ID NO: 50 or 52, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, as compared to the nucleotide sequence of (a)).

In certain preferred embodiments, the nucleic acid molecule encoding the variable region of the antibody heavy chain has the amino acid sequence as set forth in SEQ ID NO: 9, and the nucleic acid molecule encoding the variable region of the antibody light chain has the nucleotide sequence shown in SEQ ID NO: 10, or a nucleotide sequence shown in the figure.

In certain preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence as set forth in SEQ id no: 9, and/or a nucleic acid molecule as set forth in SEQ ID NO: 10, a nucleic acid molecule encoding an antibody light chain variable region.

In other aspects, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention.

In certain preferred embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

In certain preferred embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (e.g., a mammal, e.g., a human).

In other aspects, the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention. Such host cells include, but are not limited to, prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In certain preferred embodiments, the host cell of the invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHOS, CHO DG 44).

In other aspects, methods of making an antibody or antigen-binding fragment thereof of the invention are provided, comprising culturing a host cell of the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

In other aspects, the invention provides methods for making an anti-DOG-1 antibody, or antigen-binding portion thereof, comprising expressing the antibody, or antigen-binding portion thereof, in a host cell and isolating the antibody, or antigen-binding portion, from the host cell.

In other aspects, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention or a conjugate of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

In certain preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as an additional immune checkpoint inhibitor, an oncolytic virus, a chemotherapeutic agent, an anti-angiogenic drug, an anti-metabolite drug, a tumor-targeting drug, an immunostimulant, and the like.

In certain preferred embodiments, the additional pharmaceutically active agent is a drug for treating an infection, such as an antiviral agent, an antifungal agent, an antibacterial agent, an immunostimulant, and the like.

In other aspects, the invention provides a kit comprising an antibody or antigen-binding fragment thereof of the invention.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are detectably labeled.

In certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the invention. Preferably, the second antibody further comprises a detectable label.

In other aspects, the invention provides the use of an anti-DOG-1 antibody, or antigen-binding portion thereof, as disclosed herein, in the preparation of a medicament for the treatment or prevention of a tumor, an infectious disease, and an autoimmune disease.

In other aspects, the invention provides the use of an anti-DOG-1 antibody, or antigen-binding portion thereof, as disclosed herein, in the preparation of a diagnostic agent for the diagnosis of a tumor, an infectious disease, and an autoimmune disease.

In certain preferred embodiments, the tumor is selected from a solid tumor.

In certain preferred embodiments, the tumor is selected from a tumor of the digestive tract.

In certain preferred embodiments, the tumor is a DOG-1 positive tumor.

In certain preferred embodiments, the tumor is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or a metastatic, refractory, or recurrent lesion of the cancer.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings presented herein. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a surface plasmon resonance method for detecting the affinity of HX-DOG1001 antibody to human DOG-1;

FIG. 2 flow cytometry detection of the binding of HX-DOG1001 antibodies to various tumor cell lines;

FIG. 3 flow cytometry compares the binding of HX-DOG1001 antibody to the commercial antibody SP31 to human gastrointestinal stromal tumor cells GIST 882;

FIG. 4 comparison of the staining effect of HX-DOG1001 antibody on paraffin-embedded normal tissue and different tumor tissues.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Unless otherwise indicated, the molecular biological experimental methods and immunoassays used in the present invention are essentially described by reference to j.sam brook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

EXAMPLE 1 production of anti-DOG-1 antibodies

1.1 screening for anti-DOG-1 antibodies

(1) 50. mu.g of human DOG-1 ectodomain antigen (polypeptide fragment 1-301 of the sequence shown in Genbank: NP-060513.5, prepared by XXX company) was fully emulsified with Freund's complete adjuvant, and then male Balb/C mice were immunized by a multi-point immunization method with an immunization cycle of 2-3 weeks.

(2) On day 10 after the third immunization, tail vein blood was taken, plasma anti-DOG-1 antibody titer was measured by ELISA, and then mice producing the highest anti-DOG-1 antibody titer were boosted once 3 days before fusion.

(3) The mice were sacrificed 3 days later and their spleens were removed and fused with a mouse myeloma Sp2/0 cell line.

(4) 2X 108 Sp2/0 cells were pooled and 2X 108 splenocytes were fused in a 50% polyethylene glycol (MW 1450) and 5% dimethyl sulfoxide (DMSO) solution.

(5) Spleen cells were adjusted to 5X 105/mL using HAT selection medium (DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 0.1mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.g thymidine), added to wells of a 96-well plate (0.3 mL per well), and cultured in a 5% CO2 incubator at 37 ℃.

(6) After 10 days of culture, clones with high affinity binding of the antibody and the DOG-1 antigen in the supernatant are detected by a high-throughput ELISA screening method.

(7) The fusion cells in the wells of the monoclonal antibody are then subcloned.

(8) And screening out positive holes for competitive binding to DOG-1 by a competitive ELISA method to obtain the hybridoma cell strain.

(9) The culture of the specific antibody producing clones is continued. When the cell density reached about 5X 105 cells/mL, the medium was replaced with serum-free medium. After three days of culture, the cultured medium was centrifuged to collect the culture supernatant for further purification of the antibody. Finally obtaining the monoclonal antibody corresponding to the purified hybridoma cell strain to be tested.

1.2 sequence analysis of HX-DOG1001 antibody

The antibody specifically binding to DOG-1 was prepared as described above for 1 strain and named HX-DOG 1001. The VH and VL sequences of the antibodies are shown in table 1. The CDR Sequences of the HX-DOG1001 antibodies obtained by the screening were further determined using the method described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, public health Service, national institutes of health, Besserda, Md. (1991), p.647-.

Table 1: HX-DOG1001 antibody light and heavy chain variable region amino acid sequence

Table 2: HX-DOG1001 antibody CDR sequence

Example 2 detection of antigen affinity Activity of antibody by Surface Plasmon Resonance (SPR)

Antigen affinity determination of the antibody was performed using a Biacore T200 instrument (GE Healthcare).

1. Calculation of antigen coupling Ru

According to the Rmax (MWanalyte/MWligand) multiplied by RL multiplied by Sm, the actual coupling amount is 1.5 times of RL. Calculating the antigen coupling amount of 1557Ru suitable for detecting the HX-DOG1001 antibody;

2. coupling operation: the coupling Buffer was buffered to 1 XHBS-EP Buffer, 50mM NaOH as wash. Her2 protein was formulated to 10. mu.g/ml using sodium acetate solution at pH 4.5. Selecting amino coupling, inputting 1557Ru into Target level, placing an antigen sample, NaOH, ethanolamine, EDC, NSH and an empty test tube into corresponding positions in a sample disc according to prompts, and starting by clicking;

3. sample detection

Samples were diluted to 32nM, 16nM, 8nM, 4nM, 2nM, 1nM, 0.5nM, 0.25nM with 1 XHBS-EP Buffer. 13,000rpm/min, centrifuging for 3 min; all samples were set up in one replicate.

Selecting 2-1 for Flow pat in the Kinetics/Affinity options; regeneration option 2. Clicking the next step, wherein the Contact time is 180 s; dissociation time was set to 4800 s; regeneration conditions are as follows: 1: gly 2.015 s; 2: gly 2.015 s. The samples tested in duplicate were spaced apart to set three 0 concentrations, and 3 start ups.

Selecting a suitable concentration corresponding to a response unit change curve, and using the HX-DOG1001 antibody as a test sample by using a 1: 1 calculating the model to fit. Kinetic analysis was performed.

The results of detection of HX-DOG1001 antibody are shown in Table 3, and the affinity detection profile is shown in FIG. 1.

Table 3 full kinetic binding affinity of DOG1 antibody to DOG1 protein using surface plasmon resonance

Name of antibody	Ka(1/Ms)	Kd(1/s)	KD(M)
				HX-DOG1001	7.266E+9	22.44	3.088E-9

The result shows that the HX-DOG1001 antibody prepared by the invention has the affinity of 3.088 multiplied by 10 < -9 > M to human DOG-1 and can well bind to the human DOG-1 antigen.

Example 3 flow cytometry detection of antibody binding to tumor cell surface DOG-1

(1) Respectively recovering a human gastrointestinal stromal tumor cell line GIST-882, human colon cancer cell lines HT29 and HCT116, a human liver cancer cell line HepG2 and HCC LM3, human esophageal cancer cell lines Kyse180 and Eca109 and human gastric cancer cell lines MGC803 and NCI-N87;

(2) digesting the cells in the logarithmic growth phase with pancreatin, adding a serum-containing culture medium to terminate, centrifuging at 1000rpm/min for 3min, and collecting the cells;

(3) dividing each cell line into 2 groups, namely a negative control group and an experimental group respectively, wherein the number of cells in each group is 106;

(4) after each group of cells was washed three times with 1 XPBS, it was centrifuged at 1000rpm/min for 3min, and the supernatant was discarded.

(5) Adding PBS 100 μ L into negative control group, adding HX-DOG 10011 μ g/100 μ L PBS into experimental group, and incubating for 30min on ice; washing the cells with 1 × PBS for 3 times, and centrifuging at 1000rpm/min for 3 min; the negative control group and the experimental group were divided into 1: 100 percent of diluted secondary antibody (fluorescein (FITC) -conjugated affinity Goat Anti-Human IgG (H + L), SA00003-12, Proteintech), 100 μ L/group, incubated on ice for 30 min; washing the cells with 1 × PBS for 3 times, and centrifuging at 1000rpm/min for 3 min; finally, 500. mu.L of PBS was added to resuspend the cells and examined by flow cytometry.

The results of the flow cytometry for HX-DOG1 and DOG-1 on the surface of various tumor cells are shown in fig. 2, where black is the negative control group and grey is the experimental group. The result shows that the HX-DOG1001 antibody prepared by the method can be combined with DOG-1 on the surfaces of various tumor cells including gastrointestinal stromal tumors, colon cancer, liver cancer, esophageal cancer, gastric cancer and the like, and has great potential for detecting and treating various cancers.

Example 4 flow cytometry comparison of binding of HX-DOG1001 antibody to a commercial antibody

Using human gastrointestinal stromal tumor cell GIST882 as an example, the binding force difference between HX-DOG1001 antibody prepared in this application and the commercial antibody SP31 was compared.

Recovering cells, digesting with pancreatin, centrifuging at 1000rpm/min for 3min, and collecting cells in logarithmic phase; dividing the cells into 3 groups, namely a negative control group, a positive control group and an experimental group, wherein the number of the cells in each group is 106; after each group of cells was washed three times with 1 XPBS, it was centrifuged at 1000rpm/min for 3min, and the supernatant was discarded. Adding PBS 100 μ L to the negative control group, adding commercial DOG-1 antibody SP31(Abcam, cat number ab64085)1 μ g/100 μ L PBS to the positive control group, adding HX-DOG 10010011 μ g/100 μ L PBS to the experimental group, and incubating for 30min on ice; washing the cells with 1 × PBS for 3 times, and centrifuging at 1000rpm/min for 3 min; adding PBS 100 μ L into the negative control group, and incubating for 30min on ice; the positive control group and experimental group were divided into 1: 100 (FITC-conjugated affinity Goat Anti-Rabbit IgG (H + L), cat # SA00003-2, Proteintech; Fluorescein (FITC) -conjugated affinity Goat Anti-Human IgG (H + L), cat # SA00003-12, Proteintech), 100 μ L/group, incubating on ice for 30 min; the cells were washed 3 times with 1 XPBS and centrifuged at 1000rpm/min for 3 min. Finally, 500. mu.L PBS was added to resuspend the cells and examined by flow cytometry.

The results of comparison of HX-DOG1001 and SP31 binding to DOG1 expressing GIST882 cells as detected by flow cytometry are shown in figure 3, where figure 3 is the results of detection of SP31 antibody and HX-DOG001 antibody. The gate M2 was set at a standard of < 1% for the control group, and as shown in table 4, the positive rates for the commercial antibody SP31 and the HX-DOG001 antibody of the present application were 21.67% and 19.62%, respectively.

Table 4 comparison of binding of the antibodies of the present application and commercial antibodies to DOG1 using flow cytometry

The above results indicate that HX-DOG1001 has a good binding potency to cell surface DOG-1 of GIST882, comparable to that of the commercial antibody SP 31.

EXAMPLE 5 immunohistochemical assay for detection of DOG-1 in Paraffin-embedded tissues with DOG-1 antibody

1. Sample preparation

Selecting cancer tissues such as gastrointestinal stromal tumor, colon stromal tumor, esophageal squamous cell carcinoma, colon cancer, esophageal adenocarcinoma and the like and normal liver, colon and stomach tissues as objects to carry out immunohistochemical experiments.

2. Tissue chip staining

2.1 Paraffin tissue chip dewaxing: the tissue chip was placed in an oven at 65 ℃ and wax was baked for 1 hour. And taking out the tissue chip from the oven after the tissue chip is baked, and dewaxing.

The dewaxing process was as follows:

immersing the tissue chip in xylene for 10min, and repeating for 3 times;

immersing the tissue chip in absolute ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol and 60% ethyl alcohol in sequence for 5 minutes respectively;

the tissue chip was rinsed 3 times with purified water for 3min each time.

2.2 antigen retrieval

Immersing the tissue chip in the citric acid repair liquid, and putting the tissue chip into a microwave oven;

heating with microwave oven on middle fire for 10 min;

stopping heating after the time is up, continuously immersing the tissue chip in the citric acid repair liquid, and cooling for 35 min;

2.3 endogenous peroxide blocking

The tissue chip is washed 3 times with TBST buffer solution, 3min each time;

immersing the sheet in a peroxide blocking agent for 10 min;

2.4 Primary antibody incubation

The tissue chip is washed 3 times with TBST buffer solution, 3min each time;

washing the tissue chip with pure water for 3 times, each time for 3 min;

blocking the tissue chip with 10% normal serum (TBST diluted) for 2 hours;

adding the following components in percentage by weight of 1: 100 diluted primary antibody (0.8mg/mL HX-DOG1001TBST dilution), put into a wet box, put in a refrigerator at 4 ℃ for overnight incubation;

2.5 color development

Taking out the wet box from the refrigerator, standing at room temperature for 30min to restore the tissue chip in the wet box to room temperature, taking out the tissue chip, and washing the tissue chip with TBST buffer solution for 3 times, each time for 3 min;

adding the following components in percentage by weight of 1: 100 diluted secondary antibodies (goat anti-human IgG-HRP, SE101, Solarbio, TBST dilution) were incubated for 1h at room temperature; the tissue chip is washed 3 times with TBST buffer solution, 3min each time;

taking out the DAB kit from the refrigerator, and preparing according to 1mL of DAB buffer solution and 1mL of DAB coloring stock solution;

dropwise adding diluted DAB on the slices, and incubating for 5-10 minutes (according to actual conditions, dyeing is changed into brown, and the dyeing is not deepened any more);

washing with pure water for 5 min;

2.6 Nuclear staining

Immersing the tissue chip in hematoxylin for 1 min;

then immersing the tissue chip in 1% hydrochloric acid alcohol solution for 10 seconds; immediately washing the tissue chip with pure water for 2 min;

2.7 drying and fixing

Putting the slices into a 65 ℃ oven for airing until the slices have no moisture;

encapsulating with neutral gum.

3. And (4) judging a result:

the positive cell criteria are divided according to the degree of color development:

negative (0): no specific brown staining of tumor cells;

weak positive (1 +): the tumor cell membrane and plasma are stained with light brown specificity;

moderate positive (2 +): the tumor cell membrane and plasma have deep brown-yellow specific staining;

strong positive (3 +): a large amount of dark brown specific staining is seen in tumor cell membranes and plasma.

FIG. 4 shows HX-DOG1001 as a primary antibody to immunohistochemical staining of DOG-1 in tissues tested. The results show that the stomach interstitial tumor, colon interstitial tumor, esophageal squamous cell carcinoma, colon adenocarcinoma, hepatocellular carcinoma and esophageal adenocarcinoma are moderate to strong positive staining, and normal liver, colon and stomach tissues are negative to weak positive staining. The antibody of the invention has good specificity and strong positive signal, and can be better applied to the detection of cancers.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. An anti-DOG-1 antibody or antigen-binding fragment thereof, wherein the anti-DOG-1 antibody or antigen-binding fragment thereof is capable of specifically binding to DOG-1, and wherein the anti-DOG-1 antibody or antigen-binding fragment thereof comprises:

(a) complementarity determining regions of the following 3 heavy chain variable regions:

(i) VH CDR1 having the amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence of CDR1 contained in the VH;

(ii) VH CDR2 having the amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence of CDR2 contained in the VH;

(iii) VH CDR3 having the amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence of CDR3 contained in the VH;

and/or

(b) Complementarity determining regions of the following 3 light chain variable regions:

(iv) VL CDR1 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence of CDR1 contained in the VL;

(v) VL CDR2 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence of CDR2 contained in the VL;

(vi) VL CDR3 having the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence of CDR3 contained in the VL;

(i) the substitution of any one of (1) to (vi) is a conservative substitution.

2. The anti-DOG-1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-DOG-1 antibody or antigen-binding fragment thereof either comprises:

(i) a VH CDR1, consisting of the sequence: SEQ ID NO: 3, or a variant of SEQ ID NO: 3 sequences having one or several amino acid substitutions, deletions or additions compared to the corresponding sequence;

(ii) a VH CDR2, consisting of the sequence: SEQ ID NO: 4, or a sequence identical to SEQ ID NO: 4 compared to a sequence having one or several amino acid substitutions, deletions or additions;

(iii) a VH CDR3, consisting of the sequence: SEQ ID NO: 5, or a variant of SEQ ID NO: 5 sequences having one or several amino acid substitutions, deletions or additions;

and/or

(b) The following 3 light chain variable region (VL) Complementarity Determining Regions (CDRs):

(iv) a VL CDR1, consisting of the sequence: SEQ ID NO: 6, or a variant of SEQ ID NO: 6 sequences having one or several amino acid substitutions, deletions or additions;

(v) a VL CDR2, consisting of the sequence: SEQ ID NO: 7, or a variant of SEQ ID NO: 7 sequences having one or several amino acid substitutions, deletions or additions compared to the corresponding sequence;

(vi) a VL CDR3, consisting of the sequence: SEQ ID NO: 8, or a variant of SEQ ID NO: 8 sequences having one or several amino acid substitutions, deletions or additions;

(i) the substitution described in any of (1) to (vi) is a conservative substitution.

3. The anti-DOG-1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-DOG-1 antibody or antigen-binding fragment thereof either comprises:

(a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of:

(i) SEQ ID NO: 1; or

(ii) And SEQ ID NO: 1 compared with a sequence with one or more amino acid substitutions, deletions or additions; or

(iii) And SEQ ID NOs: 1, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

and/or

(iv) SEQ ID NO: 2; or

(v) And SEQ ID NO: 2 compared with the sequence shown in the sequence 2, wherein one or more amino acids are replaced, deleted or added; or

(vi) And SEQ ID NOs: 2, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) the substitutions in (a) or (v) are conservative substitutions.

4. An anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein said anti-DOG-1 antibody is a chimeric or humanized antibody.

5. An anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-DOG-1 antibody is a full-length antibody comprising human antibody constant regions selected from the group consisting of human IgG1, IgG2, IgG3, IgG4 constant regions and conventional variants thereof, and light chain constant regions selected from the group consisting of human kappa or lambda chain constant and conventional variants thereof.

6. An anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-DOG-1 antibody or antigen-binding fragment thereof is selected from the group consisting of ScFv, Fab ', (Fab') 2, Fv fragments, disulfide linked Fv (dsFv), diabodies (diabodies), bispecific antibodies and multispecific antibodies.

7. An anti-DOG-1 antibody or antigen-binding fragment thereof according to any of claims 1 to 3, wherein said anti-DOG-1 antibody or antigen-binding fragment thereof carries a detectable label, said detectable label being an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin.

8. An isolated nucleic acid molecule encoding the anti-DOG-1 antibody or antigen-binding fragment thereof, the heavy and/or light chain thereof, or the heavy chain variable region and/or the light chain variable region thereof of any one of claims 1-7;

the nucleic acid molecule encoding the variable region of the antibody heavy chain has a sequence selected from the group consisting of: (a) as shown in SEQ ID NO: 9; or (b) a sequence substantially identical to the nucleotide sequence of (a);

the nucleic acid molecule encoding the variable region of the antibody light chain has a sequence selected from the group consisting of: (a) as shown in SEQ ID NO: 10; or (b) a sequence substantially identical to the nucleotide sequence of (a);

the nucleic acid molecule for encoding the heavy chain variable region of the antibody has the nucleotide sequence shown in SEQ ID NO: 9;

the nucleic acid molecule for encoding the variable region of the antibody light chain has the nucleotide sequence shown in SEQ ID NO: 10, or a nucleotide sequence shown in the figure.

9. A vector comprising the nucleic acid molecule of claim 8, wherein the vector is a cloning vector or an expression vector.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A method of making an anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1-7, comprising culturing the host cell of claim 10 under conditions that allow expression of the anti-DOG-1 antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

12. A pharmaceutical composition comprising an anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or a vector according to claim 9, or a host cell according to claim 10, and a pharmaceutically acceptable carrier and/or excipient.

13. A conjugate comprising an anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1-7 and a therapeutic agent linked to the anti-DOG-1 antibody or antigen-binding fragment thereof; the therapeutic agent is selected from a cytotoxin or a radioisotope; the cytotoxin is selected from camptothecin derivatives, calicheamicin, maytansine or derivatives thereof, maytansinoids, dolastatin, auristatin, and trichothecene.

14. Use of an anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1-7 or a nucleic acid molecule according to claim 8 or a pharmaceutical composition according to claim 12 or a conjugate according to claim 13 for inhibiting tumor cell growth, metastasis in a subject.

15. Use of an anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1-7 or a nucleic acid molecule according to claim 8 or a pharmaceutical composition according to claim 12 or a conjugate according to claim 13 for the manufacture of a medicament for the treatment or prevention of tumors, infectious diseases and autoimmune diseases.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the nucleic acid molecule of claim 8 or the pharmaceutical composition of claim 12 or the conjugate of claim 13 for the preparation of a medicament for the diagnosis of tumors, infectious diseases and autoimmune diseases.

17. The use of any one of claims 14-16, wherein the tumor is a DOG-1 positive tumor.

18. The use of any one of claims 14-16, wherein the tumor is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer, liver cancer, gastric cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, melanoma, mesothelioma, interstitial tumor, lymphoma, myeloma, sarcoma, glioblastoma, leukemia, or a metastatic, refractory, or recurrent lesion of the cancer.

19. A kit comprising an anti-DOG-1 antibody or antigen-binding fragment thereof according to any one of claims 1-7 or a pharmaceutical composition according to claim 12 or a conjugate according to claim 13.