CN110950959B - EpCAM-targeted antibody and preparation and application thereof - Google Patents

Abstract

The invention discloses an antibody targeting EpCAM and a preparation method and application thereof. The EpCAM-targeting antibody comprises a VL and/or a VH comprising VH CDR1 as set forth in the amino acid sequence of SEQ ID No. 5, VH CDR2 as set forth in the amino acid sequence of SEQ ID No. 6, and/or VH CDR3 as set forth in the amino acid sequence of SEQ ID No. 7; the VL comprises VL CDR1 as shown in the amino acid sequence of SEQ ID NO. 8, VL CDR2 as shown in the amino acid sequence of SEQ ID NO. 9, and/or VL CDR3 as shown in the amino acid sequence of SEQ ID NO. 10. The invention overcomes the defects of an antibody targeting EpCAM in the field, and provides an antibody which has a fully human sequence and high affinity and is capable of being combined with cynomolgus monkey antigen in a cross-species manner, and a preparation method and application thereof.

Description

EpCAM-targeted antibody and preparation and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to an anti-EpCAM antibody and preparation and application thereof.

Background

Epithelial cell adhesion molecules (EpCAM, CD326), also known as Trop-1, 17-1A, ESA, EGP314, AUA1, EGP40, GA733-2, and the like, are approximately 40 kD transmembrane glycoproteins consisting of 314 amino acids, consisting of a 242 amino acid large extracellular domain (N-terminal), a 23 amino acid single transmembrane domain, and a 26 amino acid short cytoplasmic domain (C-terminal).

EpCAM also transduces 3 independent cellular signaling pathways, including the nPKC-dependent pathway, Wnt and eRAS/RAC/AKT pathway, the intracellular domain (EpICD) of EpCAM can also translocate to the nucleus after cleavage and induce c-myc and E-FABP expression in a FHL-2 and β -catenin dependent manner.

The amino acid sequence of human EpCAM (NCBI accession number: P16422) and Macaque (Rhesuus Macaque or Macaque)Macaca mulatta) EpCAM (NCBI accession number: q1WER 1) was 93%, as compared to mouse EpCAM (NCBI accession number: q99JW 5) was 81%, with Cynomolgus Monkey (Cynomolgus Monkey or Cynomolgus Monkey)Macaca fascicularis) EpCAM (NCBI accession number: XP — 005576740.1, predicted) was 84%.

EpCAM is specifically expressed in different types of epithelial cells. In healthy adult tissues, EpCAM is expressed on the basolateral cell membrane of simple, pseudostratified and transitional epithelium. EpCAM is also highly expressed in major types of human malignancies, such as colon, lung, prostate, liver, kidney, pancreas, breast, cervix and ovary. EpCAM has become a hot target in cancer therapy including vaccines, murine or human monoclonal antibodies, and antibodies that bind bacterial toxins or chemotherapeutic drugs, such as EpCAM-specific antibody I G-1, adalimumab, eculizumab, and the like.

There are many antibodies against human EpCAM, of which NR-LU-10 (nonfetumomab merpentan) is a radioisotope conjugate, and is used for diagnosis of lung cancer and other EpCAM-highly expressed cancers and metastases and/or relapses; removab (cataxomab) as the first double-antibody drug on the market, the drug is used for abdominal cavity metastasis, but the drug has been released from the market due to a series of problems such as side effects and murine antibodies; currently, only panorex (edrecolomab) is studied in clinical stage III for indications of breast and colorectal cancer, and other antibodies are in clinical stage I/II or preclinical study. Since edrecolomb is a human-mouse chimeric antibody, there is a risk of immunogenicity, and the affinity is not very good, and it cannot cross-react with cynomolgus monkey species antigen, increasing the potential risk of toxicity. Therefore, there is a lack in the art of high affinity antibodies targeting the human EpCAM antigen, particularly antibodies that are fully human and cross-reactive with cynomolgus monkey antigen, as well as antibodies with better ADCC activity, endocytic activity, etc.

Disclosure of Invention

In order to solve the technical problem that the affinity, ADCC activity and endocytosis activity of the EpCAM-targeted fully-humanized antibody are insufficient in the field, the invention provides an EpCAM-targeted antibody and a preparation method and application thereof.

In order to solve the above technical problems, a first aspect of the present invention provides an antibody targeting EpCAM, comprising a light chain variable region (VL) and/or a heavy chain variable region (VH), wherein,

the VH comprises the following VH complementarity determining regions (HCDR): VHCDR1 as shown in the amino acid sequence of SEQ ID NO. 5, VH CDR2 as shown in the amino acid sequence of SEQ ID NO. 6, and/or VH CDR3 as shown in the amino acid sequence of SEQ ID NO. 7;

the VL comprises the following VL complementarity determining regions (LCDRs): LCDR1 as shown in the amino acid sequence of SEQ ID NO. 8, LCDR2 as shown in the amino acid sequence of SEQ ID NO. 9, and/or LCDR3 as shown in the amino acid sequence of SEQ ID NO. 10;

alternatively, the VH comprises the CDR sequences: a variant of HCDR1 having 3, 2 or1 amino acid mutations, respectively, on the basis of the amino acid sequence shown in SEQ ID NO. 5, a variant of HCDR2 having 3, 2 or1 amino acid mutations, respectively, on the basis of the amino acid sequence shown in SEQ ID NO. 6, and/or a variant of HCDR3 having 3, 2 or1 amino acid mutations, respectively, on the basis of the amino acid sequence shown in SEQ ID NO. 7; the VL comprises the CDR sequences: LCDR1 variants having 3, 2 or1 amino acid mutations, respectively, on the basis of the amino acid sequence shown as SEQ ID NO. 8, LCDR2 variants having 3, 2 or1 amino acid mutations, respectively, on the basis of the amino acid sequence shown as SEQ ID NO. 9, and/or LCDR3 variants having 3, 2 or1 amino acid mutations, respectively, on the basis of the amino acid sequence shown as SEQ ID NO. 10.

Preferably, the VH further comprises a heavy chain variable region framework region (HFWR), and/or the VL further comprises a light chain variable region framework region (LFWR); wherein the HFWR is the heavy chain variable region framework region of a human antibody and the LFWR is the light chain variable region framework region of a human antibody;

more preferably, the HFWR comprises HFWR1 as shown in the amino acid sequence of SEQ ID NO. 11, HFWR2 as shown in the amino acid sequence of SEQ ID NO. 12, HFWR3 as shown in the amino acid sequence of SEQ ID NO. 13, HFWR4 as shown in the amino acid sequence of SEQ ID NO. 14;

the LFWR includes LFWR1 shown in the amino acid sequence of SEQ ID NO. 15, LFWR2 shown in the amino acid sequence of SEQ ID NO. 16, LFWR3 shown in the amino acid sequence of SEQ ID NO. 17, and LFWR4 shown in the amino acid sequence of SEQ ID NO. 18.

Even more preferably, the amino acid sequence of the VH is the amino acid sequence shown as SEQ ID NO. 3 or a mutation thereof; and/or the amino acid sequence of the VL is the amino acid sequence shown as SEQ ID NO. 4 or mutation thereof; the mutation is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the VH and/or VL, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of the VH and/or VL and retains or improves binding of the antibody to EpCAM; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.

In the present application, the amino acid sequences of the above-listed CDRs are shown in accordance with the Chothia definition rules. However, it is well known to those skilled in the art that the CDRs of an antibody can be defined in the art by a variety of methods, such as the Kabat definition rule based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institutes of health, Besseda, Md. (1991)) and the Chothia definition rule based on the position of the structural loop region (see JMolBiol 273:927-48, 1997). In the present application, the rules of combinatorial definition, including the Kabat definition and the Chothia definition, can also be used to determine amino acid residues in variable domain sequences. The Combined definition rule combines the Kabat definition with the Chothia definition to obtain a larger range of amino acid residues, as detailed in Table 1. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any of the above-described known schemes described by the present invention. Although the EpCAM-targeting antibodies claimed herein are based on the sequences shown by the Chothia definition rules, amino acid sequences corresponding to other CDR definition rules should fall within the scope of the invention.

Preferably, the EpCAM-targeted antibody further comprises an antibody heavy chain constant region and an antibody light chain constant region;

more preferably, the heavy chain constant region is selected from human IgG1 (hIgG 1), hIgG2, hIgG3 or hIgG4 or a mutation thereof, and the light chain constant region is selected from a human antibody light chain kappa chain or lambda chain or a mutation thereof;

even more preferably, the heavy chain constant region is hIgG1 and the light chain constant region is a light chain kappa chain of a human antibody.

Preferably, the EpCAM-targeting antibody is a full-length antibody, Fab ', F (ab')₂Fv, scFv (single chain antibody), bispecific antibody, multispecific antibody, single domain antibody or single domain antibody, or monoclonal antibody or polyclonal antibody made from the above antibodies. The monoclonal antibody can be developed by various means and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the monoclonal antibody is prepared from wild-type or transgenic mice by the hybridoma technology in the mainstream.

Preferably, the EpCAM-targeting antibody is a full-length antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID No. 1 or a mutation thereof and a light chain; and/or the amino acid sequence of the light chain is the amino acid sequence shown as SEQ ID NO. 2 or mutation thereof; the mutation is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the VL and/or VH, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of the VL and/or VH and retains or improves binding of the antibody to EpCAM; said at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most preferably at least 99% sequence identity.

In order to solve the above technical problem, the second aspect of the present invention provides an isolated nucleic acid encoding the EpCAM-targeting antibody.

In order to solve the above technical problems, the third aspect of the present invention provides an expression vector comprising the isolated nucleic acid.

In order to solve the above technical problems, a fourth aspect of the present invention provides a host cell comprising the expression vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell. The host cell may be prepared by methods conventional in the art, for example: the expression vector is transformed into a host cell. The host cell is any host cell conventionally used in the art, so long as it is sufficient that the expression vector is stably self-replicating and the nucleic acid carried thereby can be efficiently expressed. Preferably, the host cell isE.coliTG1 or BL21 cells (expressing single chain antibodies or Fab antibodies), or CHO-K1 cells (expressing full length IgG antibodies). Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.

In order to solve the above technical problems, a fifth aspect of the present invention provides a method for producing an EpCAM-targeting antibody, comprising culturing the host cell, and obtaining the EpCAM-targeting antibody from the culture.

In order to solve the above technical problems, a sixth aspect of the present invention provides an immunoconjugate comprising a cytotoxic agent, and the EpCAM-targeting antibody.

In order to solve the technical problems, the seventh aspect of the present invention provides a pharmaceutical composition comprising the EpCAM-targeting antibody or the antibody drug conjugate.

In order to solve the technical problems, the eighth aspect of the present invention provides the use of the EpCAM-targeting antibody, the immunoconjugate and the pharmaceutical composition in the preparation of a medicament for the treatment and/or prevention of cancer; preferably, the cancer is colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer or ovarian cancer.

In order to solve the above technical problem, a ninth aspect of the present invention provides: providing a kit combination comprising kit a and kit B; the kit a comprises an EpCAM-targeting antibody according to the first aspect of the invention, a host cell according to the fourth aspect, an immunoconjugate according to the sixth aspect and a pharmaceutical composition according to the seventh aspect; the kit B comprises further antibodies, bispecific antibodies, genetically modified cells or pharmaceutical compositions targeting CD3, B7H4, ROR1 or other targets. The medicine box A and the medicine box B are not used in sequence, or the medicine box A is used firstly and then the medicine box B is used, or the medicine box B is used firstly and then the medicine box A is used.

The EpCAM-targeting antibody, the immunoconjugate and the pharmaceutical composition or the kit combination of the invention can be administered to a patient for the treatment of a tumor of interest.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The three letter and one letter codes for amino acids used in the present invention are known to those skilled in the art or described in j. biol. chem, 243, p3558 (1968).

As used herein, the terms "comprising" or "including" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements, but, as the context dictates, also include the case of "consisting of … …".

The term "antibody" as used herein includes immunoglobulins (igs), which are tetrapeptide chains composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds, and the constant regions of the heavy chains of the immunoglobulins have different amino acid compositions and sequences and thus different antigenicities, and accordingly, the immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains thereof are μ chains, δ chains, γ chains, α chains, and epsilon chains, and the same class of igs can be classified into different subclasses according to differences in the amino acid composition of the hinge region and the number and positions of disulfide bonds of the heavy chains, e.g., igs can be classified into IgG1, IgG2, IgG3, and IgG 4.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human kappa, lambda chain or a variant thereof. In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human IgG1, 2, 3, 4 or a variant thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 Complementarity Determining Regions (CDRs) and 4 framework regions (FWRs), which are arranged in the order from amino terminus to carboxyl terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR 4. 3 CDRs of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDRs of the heavy chain refer to HCDR1, HCDR2 and HCDR 3.

The term "mutation" includes substitution, addition and/or deletion of an amino acid or a nucleotide, and "substitution of an amino acid" is a substitution in which an amino acid residue is substituted with another amino acid residue and with an amino acid residue having a similar side chain. In the present invention, the mutation may include mutations that are currently known to those skilled in the art, for example, mutations that may be made to the antibody during the production or use of the antibody, for example, mutations at a site that may be present, particularly, post-transcriptional modifications (PTMs) of CDRs, including related mutations such as aggregation, deamidation sensitivity (site (NG, NS, and/or NH, etc.), aspartic acid isomerization (DG, DP) sensitivity site, N-glycosylation (N- { P } S/T) sensitivity site, and oxidation sensitivity site of the antibody.

"amino acid mutation" in "having 3, 2 or1 amino acid mutations" means that there is a mutation of an amino acid in the sequence of a variant as compared with the original amino acid sequence, including substitution, addition and/or deletion of an amino acid based on the original amino acid sequence. An exemplary explanation is that the mutation of the Complementarity Determining Regions (CDRs) may comprise 3, 2 or1 amino acid mutations, and that the same or different number of amino acid residues may optionally be selected between the CDRs for mutation, e.g., 1 amino acid mutation of CDR1, and no amino acid mutations of CDR2 and CDR 3.

The term "vector" or "expression vector" as used herein is a composition that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics (biolistics).

As used herein, the term EC₅₀Means the half-maximum effect concentration (concentration for 50% of maximum effect), i.e. the concentration that causes 50% of the maximum effect.

The immunoconjugates can be prepared by methods conventional in the art, preferably by methods described in Doronina, 2006, Bioconjugate chem.17, 114-124. Preferably, the preparation method produces an antibody drug conjugate with a minimal Low Conjugate Fraction (LCF) of less than 10%. The immunoconjugate can be present in any physical form known in the art, preferably a clear solution.

As used herein, the terms "cancer", "tumor" are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. Examples include, but are not limited to, solid tumors, hematologic cancers, soft tissue tumors, and metastatic lesions.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention overcomes the defects of an antibody targeting EpCAM in the field, and provides an antibody which has a fully human sequence, high affinity, cross-species binding to cynomolgus monkey antigen and high ADCC activity and endocytosis activity, a preparation method and application thereof.

Drawings

Figure 1 shows the results of testing the binding strength of the antibody molecules of the present invention to EpCAM-expressing cell lines. a. The binding strength test result of the antibody molecule and the 293T stably-transfected cell line over-expressing the human EpCAM molecule shows that compared with a control antibody, EC50 of PR001081 is obviously superior to that of the control antibody 1, and MFI value is obviously superior to that of the control antibody 1 and the control antibody 2; b. PR001081 and CHO-K1-cynoEpCAM combined strength test result, PR001801 shows significant cross-binding activity with the cynoEpCAM cell line, and combined MFI is better than that of control antibody 1 and control antibody 2; the result of the test of the binding strength of PR001081 and the Capan-2 tumor cell line shows that the binding activity of PR001081 and the tumor cell is remarkably superior to that of the existing control antibody.

FIG. 2 shows the result of measurement of KD value of the recombinant antibody by SPR method. a. Determination of KD value for binding of control antibody 1 to antigen; b. determination of KD value for control antibody 2 binding to antigen; c. determining the KD value of the recombinant antibody PR001081 combined with the antigen; among them, PR001081 has a Ka value superior to control antibody 2, a Kd value significantly superior to control antibody 1 and control antibody 2, and a Kd value significantly superior to control antibody 1 and control antibody 2.

FIG. 3 shows the determination of the epitope bound by the recombinant antibody by the octet method. a. Determining the overlapping degree of the binding sites of the three antibodies by taking the control antibody 1 as a primary antibody; b. determining the overlapping degree of the binding sites of the three antibodies by taking the control antibody 2 as a primary antibody; c. PR001081 is used as a primary antibody, and the overlapping degree of the binding sites of the three antibodies is measured; PR001081 binds to the same epitope as control antibody 2, and is close to, but not identical to, control antibody 1.

FIG. 4 is a graph showing the determination of the endocytosis effect of the antibody of the present invention on the tumor cell Capan-2; the endocytosis effect of PR001081 is obviously better than that of the control antibody 1, and is the same as that of the control antibody 2.

FIG. 5 is a graph of the detection of ADCC induction by antibodies in the tumor cell line Capan-2. a. Assay results in a commercial reporter cell system; b. assay results in a self-established reporter cell system; of these, PR001081 induced ADCC levels significantly better than control antibody 1.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 antigen preparation, mouse immunization and hybridoma preparation

a. Antigen preparation

Human EpCAM (abbreviated huEpCAM)/TROP 1 Protein, Fc Tag, Gln24-Lys265-Fc, available from Acrobiosystems, Cat #: the EPM-H5254 is,

HuEpCAM-ECD-His tag, i.e., Recombinant Human EpCAM/TROP1/CD326 (C-6His), Gln24-Lys265-6 × His, available from novoprotein, Cat #: C339.

Cynomolgus EpCAM Protein, recombinant (His tag), Met1-Lys265, C-terminal coupling of multiple His tags, purchased from Silobiological, Cat #: 90299-C08H, batch number: the LC11JU2102 was set to zero,

Rhesus macaqueEpCAM/TROP1 Protein, His Tag, Gln24-Lys265, C-terminal coupled multiple His tags, purchased from Acrobiosystems, Cat #: EPM-C5227, batch number: 557-34A51-4A,

TABLE 1 EpCAM antigen information from different sources for use in the present invention

Species of species	NCBI accession number
		Human being	P16422
Kiwi fruit	Q1WER1
		Mouse source	Q99JW5
Macaca fascicularis	XP_005576740.1

b. Immunization

Fully human anti-EpCAM antibodies were identified from hybridomas generated from H2L2 mice immunized with the huEpCAM-ECD-Fc protein (and platinum medicine, granted patent EP2379727B 1). 50 μ g of the above fusion protein was injected for the first time, immunized with CFA as an immunoadjuvant, and then boosted with 25 μ g of protein and Ribi Adjuvant (Sigma-Aldrich; Sigma Adjuvant System; Cat #: S6322) 7 more times on days 15, 29, 43, 57, 71 and 86. Binding affinities of mouse sera were tested by FACS using HEK293T cells or tumor cell lines expressing human EpCAM (HEK 293T-huEpCAM, purchased from kyinno; Capan-2 cells, purchased from ATCC) in parallel with ELISA using the huEpCAM-ECD-His tag protein on day 50, 78 and 107 blood sampling tests. According to the detection result of the serum titer of the immunized mice, the mice are selected for hybridoma fusion, and the last strengthening is carried out 3 days before the fusion, namely 132 days, by 25 mu g of protein and Ribi adjuvant.

c. Fusion

Hybridomas were produced and cloned by conventional methods, i.e., by electrofusion, spleen and lymph nodes from mice were extracted, single cells were triturated, lysed, and washed to mix with sp2/0 cells. And (3) placing the cell suspension into an electric fusion tank for electric shock fusion, standing, replacing 20% FBS HT medium, and then replacing HAT medium for culture.

The collected spleen B cells were mixed with a mouse myeloma cell line Sp2/0 at a ratio of 2:1 (cell number ratio), the mixed cells were subjected to cell fusion using an electrofusion apparatus (BTX ECM 2001), the fused cells were plated on 96-well cell culture plates, and after culturing at 37 ℃ for 10 days in a carbon dioxide incubator, preliminary screening of hybridomas was performed. After overnight reconstitution, the fused cells were plated in 96-well plates by limiting dilution and screened with hypoxanthine-aminopterin-thymidine. Hybridoma culture supernatants were tested for the presence of anti-EpCAM antibodies by ELISA assay and flow cytometry.

Example 2 antibody screening and sequencing

a. ELISA screening

At 4 deg.C, 1. mu.g/ml of freshly prepared huEpCAM-ECD-His tag protein was added to a 96-well plate (corning 9018) overnight, and the supernatant was discarded and washed 3 times with PBST. Plates were blocked with 5% milk for 2 hours at room temperature and washed 3 times with PBST. Add 100 u l/hole hybridoma supernatant, room temperature incubation for 1 hours, then PBST washing 3 times. 100 μ l/well of secondary antibody was added and incubated for 1 hour at room temperature after washing. Then 100. mu.l/well of TMB was added to the plate, incubated at room temperature for 15 minutes, and then the reaction was stopped and the results read. Screening and picking positive hybridomas by ELISA, transferring the hybridomas from a 96-hole culture plate to a 24-hole culture plate for expanded culture, rescreening the supernatant in the holes of the 24-hole culture plate after 5 days, and rescreening by using a FACS method.

b. FACS screening

For flow cytometry screening, adherent cells were digested with TypLE for 3 minutes, then the digestion was stopped with complete medium containing 10% FBS, the cells were washed with FACS buffer and counted, then diluted to 3-5 × 10⁶Density per ml. Cells were added to a 96-well plate (Corning 3894) at 100. mu.l/well. After blocking for 3-4 minutes, 100. mu.l/well hybridoma supernatant was added and incubated at 4 ℃ for 1 hour. After washing, secondary antibody was added and incubated at 4 ℃ for 1 hour. Cells were then washed and FACS analyzed.

c. Subcloning and screening

Positive hybridoma clones were subcloned into 96-well plates by limiting dilution as described above, and positive monoclonals were identified and selected by FACS screening (same procedure as above).

d. Sequencing

And selecting positive monoclonals, and extracting total RNA. RT-PCR produced cDNA, followed by PCR amplification of heavy and light chains, respectively (RT-PCR from SuperScript first strand synthesis system from Saimerfi, Cat #: 11904018, see product Specification for specific procedures PCR used high fidelity DNA polymerase from NEB, Cat #: M0530L, see product Specification for specific procedures). Then, the PCR product was constructed on a T-vector, and sequencing (Shanghai division, Biotech, Inc., Beijing Optimalaceae) was performed while antibody subtype determination was performed.

e. Antibody sequence information

The numbering system commonly used for antibodies is Kabat and Chothia et al, and the numbering of the antibodies of the invention is detailed in Table 2 below. Because of the misalignment of HCDR1 and HCDR2 in the two numbering systems, the present invention specifically combines (i.e., Combined) the two numbering systems to obtain the amino acid residue positions of HCDR1 and HCDR2 in the heavy chain.

TABLE 2 location of CDRs of the invention in antibody light or heavy chains under different numbering systems

CDR \ numbering system	Kabat	Chothia	Combined
				LCDR1	L24 - L34	L24 - L34	L24 - L34
LDR2	L50 - L56	L50 - L56	L50 - L56
				LDR3	L89 - L97	L89 - L97	L89 - L97
HCDR1	H31 - H35	H26 - H32	H26 - H35
				HCDR2	H50 - H65	H52 - H56	H50 - H65
HCDR3	H95 - H102	H95 - H102	H95 - H102

TABLE 3 amino acid sequences of CDRs of antibodies of the invention (based on the Chothia system)

HCDR1

HCDR2

HCDR3

LCDR1

LCDR2

LCDR3

Antibodies of the invention

GFTFSRY

WFDGSN

EMAAAGFYL

RASQSVSSYLA

DASNRAT

QQRSNWPPIT

Sequence numbering

SEQ ID NO: 5

SEQ ID NO: 6

SEQ ID NO: 7

SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 10

Wherein the control antibody 1 is edrecolomab, trade name Panorex, a murine monoclonal antibody; for specific information on control antibody 2 see WO2017157305 (anti-EpCAM Ab hIgG 1).

f. Framework region sequences of antibody variable regions

Table 4 shows the combination of control antibodies and preferred framework region sequences of the present invention.

TABLE 4 framework region sequence combinations

Framework region/antibody	Sequence of H2L2 antibody PR001081	Sequence numbering
			HFWR1	QVQLVESGGGVVQPGRSLRLSCAAS	SEQ ID NO: 11
HFWR2	WVRQAPGKGLEWVA	SEQ ID NO: 12
			HFWR3	RFAISRDNSKNTLYLQMNSLRAEDTAVYYCAR	SEQ ID NO: 13
HFWR4	WGRGTLVTVSS	SEQ ID NO: 14
			LFWR1	EIVLTQSPATLSLSPGERATLSC	SEQ ID NO: 15
LFWR2	WYQQKPGQAPRLLIY	SEQ ID NO: 16
			LFWR3	GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC	SEQ ID NO: 17
LFWR4	FGQGTRLEIK	SEQ ID NO: 18

The variable regions of the light chain and the heavy chain of the antibodies obtained according to the present invention and the control antibody are shown in table 5 below.

TABLE 5 amino acid sequences of light and heavy chain variable regions

The light chain variable region sequence of the H2L2 antibody PR001081 of the invention is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIK（SEQ ID NO: 4）

the heavy chain variable region sequence of the antibody of the invention is as follows:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYDMNWVRQAPGKGLEWVAVIWFDGSNKYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCAREMAAAGFYLWGRGTLVTVSS（SEQ ID NO: 3）

example 3 antibody production, purification and validation

a. Antibody light chain, heavy chain sequence

After obtaining the light and heavy chain variable domain sequences encoding the antibody molecules, the light and heavy chain variable domain sequences and the corresponding human antibody light and heavy chain constant domain sequences can be subjected to fusion expression by adopting the conventional recombinant DNA technology to obtain the recombinant antibody molecules. The heavy chain constant region of the antibody of the invention may be selected from one of hIgG1, hIgG2, hIgG3 or hIgG 4. In this example, antibody heavy chain variable domain sequences (VH) were genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding human IgG1 antibody heavy chain constant domain sequences to encode the full-length heavy chain that produced IgG1 antibody. Antibody light chain variable domain sequences (VL) were genetically synthesized and cloned into mammalian cell expression plasmid vectors encoding human antibody Ig kappa light chain constant domain sequences to encode full-length light chains that produce antibodies. In this example, since the sequence of the variable domain of the monoclonal antibody molecule obtained from the immunized and platinum medical H2L2 mouse was a human antibody sequence, the example also obtained a fully human anti-EpCAM recombinant IgG1 antibody.

For example, the light chain sequence of an antibody of the invention is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQID NO: 2）

for example, the heavy chain sequence of an antibody of the invention is:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYDMNWVRQAPGKGLEWVAVIWFDGSNKYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCAREMAAAGFYLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 1）

b. antibody production and purification

Specifically, HEK293 cells are expanded and cultured in FreeStyle F17 Expression Medium (Thermo, Cat #: A1383504), before transient transfection, the cell concentration is adjusted to 6-8 × 10⁵Cells/ml, 8% CO at 37 ℃₂Culturing in shaking bed for 24 hr to obtain cell concentration of 1.2 × 10⁶Cells/ml. 30 ml of cultured cells were prepared. The above plasmid encoding the antibody heavy chain and plasmid encoding the antibody light chain were mixed at a ratio of 2:3 (mass ratio) and 30. mu.g of the total plasmid was dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, Cat #: 31985088) and sterilized by filtration through a 0.22. mu.m filter. Then 1.5 ml of Opti-MEM was dissolved in 120. mu.l of PEI (Polysciences, Cat #: 23966-2) at 1mg/ml and left to stand for 5 minutes. Slowly adding PEI into the plasmid, incubating for 10 min at room temperature, slowly dropping the mixed solution of the plasmid PEI while shaking the culture flask, and adding 8% CO at 37 DEG C₂Cultured in a shaker for 5 days. Cell viability was observed after 5 days. Collecting the culture, centrifuging at 3300g for 10 min, and collecting the supernatant; the supernatant was then centrifuged at high speed to remove impurities. Gravity columns (Bio-Rad, Cat #: 7311550) containing MabSelect ™ antibodies (GE Healthcare Life Science, Cat #: 71-5020-91 AE) were equilibrated with PBS (pH 7.4) and washed 2-5 column volumes. Passing the supernatant sample through a column; washing the column with 5-10 column volumes of PBS, eluting the desired protein with 0.1M glycine pH 3.5, followed by T pH 8.0The ris-HCl was adjusted to neutral, and finally the solution was concentrated and changed to PBS buffer using an ultrafiltration tube (Millipore, Cat #: UFC 901024) to obtain a purified antibody solution. Finally, the concentration is measured by a NanoDrop (Thermo Scientific. NanoDrop. loop), and the cells are subpackaged and stored for later use.

A proper amount of the purified samples are respectively loaded on an analytical SEC column TSKgel G3000SWxl (HPLC instrument model: Agilent 1260 Infinity II) to detect the purity of the samples and ensure that the purity of the uniform samples is more than 95 percent, the mobile phase of the method is1 × PBS, the pH value is 7.4 (raw product, Cat #: E607016), the room temperature, the flow rate is 1.0 ml/min, the sample concentration is 1mg/ml, the sample injection volume is 20 mu l, the detection wavelength is 280nm, and after collection, a chromatogram is integrated by using ChemStation software and relevant data are calculated.

c. Recombinant antibody validation

FACS verification: the recombinant antibodies were diluted at a semilogarithmic dilution of 1: 3.16 at concentrations of 100 nM, 31.6nM, 10 nM, 3.16 nM, 1nM, 0.316 nM, 0.1 nM, 0.0316 nM, 0.01nM and 0nM, respectively. And were verified after incubation for 1 hour in 293T-huEpCAM, CHO-K1-CynEpCAM and Capan-2 cell lines, and addition of secondary antibody for a further 1 hour incubation.

Among them, 293T-huEpCAM was purchased from Kangyuan Bo Chuang Biotech (Beijing) Ltd, Cat #: KC-0994. CHO-K1-cynoEpCAM was prepared in the laboratory by the following specific method: inoculation in 6-well plates 10⁵CHO-K1 cells per well were supplemented with F-12K (21127-022, Gibco) medium to 2ml and incubated overnight at 37 ℃. After overnight, 1E6 units of cynoEpCAM virus (Cat #: 42579-1, Kjeldahl gene) and polybrene (Cat #: H9286, sigma) at a final concentration of 4. mu.g/ml were added to the wells, incubated at 37 ℃ for 8 hours, the medium was changed, and the culture was continued for 72 hours. After 72 hours, cells were digested with TrpLE (12605036, Gibco) and counted, diluted to 5/ml with F-12K medium containing 8. mu.g/ml puromycin (A1113803, Gibco) and inoculated into 10 96 well plates at 100. mu.l/well, and after 10 days of culture at 37 ℃ single clones were picked up, transferred to 24 well plates for 5 days and confirmed with EpCAM antibody, and the single clones with the highest expression were selected for expansion culture and frozen.

Capan-2 was purchased from ATCC, Cat #: HTB-80. Virus subscription from the jenky gene, virus-related information: polybrene, Cat #: REVG0001, 10mg/ml, 20. mu.l. Virus: LV-EPCAM, Cat #: 42579-1, titer 2e9/ml, 50. mu.l/via. The preparation method of the stable transformant comprises the following steps: inoculating 2 ml/well CHO-K1 cell suspension into 6-well plate at density of 50000/ml, incubating overnight, adding polybrene to make final concentration 4 μ g/ml, mixing, adding 10 μ l virus, mixing, incubating at 37 deg.C for 8 hr, removing supernatant, replacing with fresh culture medium, and culturing at 37 deg.C. After 24 hours, the resuspended cells were digested, diluted to 5/ml in complete medium containing 8. mu.g/ml puromycin, cultured in 10 96 well plates at 37 ℃ for 10 days at 100. mu.l/well, single clones were picked for FACS validation and the validated clones were expanded and frozen.

The results of the verification of example 3b are shown in fig. 1 and table 6.

TABLE 6 EC binding of antibodies to different cell lines₅₀Value and MFI value

ND: no binding activity was measured

In a of fig. 1, it is shown that after binding PR001081 to the 293T-huEpCAM stable cell line, the maximum MFI value (72712) is significantly better than the control antibody (27434 for control antibody 1, no binding activity at monovalent; 29157 for control antibody 2, binding activity at monovalent);

in fig. 1 b, PR001081 binds to CHO-K1-cynoEpCAM, PR001081 shows cross-binding activity to the cynoEpCAM cell line with an MFI value (25970) significantly higher than that of control antibody 2 (18184), control antibody 1 has no cross-binding activity at all with cynomolgus or cynomolgus derived EpCAM.

The binding of PR001081 to the Capan-2 tumor cell line is shown in c of FIG. 1. PR001081 shows binding activity to tumor cells with a significantly higher MFI value (115407) than control antibody 1 (42444) and control antibody 2 (81875), with EC₅₀(2.371 nM) was also significantly higher than control antibody 1.

Example 4 BIACORE detection of the affinity of PR001081 to human EpCAM recombinant protein

The entire test used HBS-EP + (10 mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20, pH7.4, GE Healthcare, Cat #: BR-1006-69) as the running buffer, series S CM5 (GE Healthcare, Cat #: BR-1005-30) as the experimental chip, and the human EpCAM-His information see above.

a. Conjugation of antigens

Setting a flow rate of 10 mu l/min, coupling 4 antigens on 4 channels of two CM5, 1) setting an injection time of 300s, freshly mixing 50mM NHS and 200mM EDC in a volume ratio of 1: 1, and injecting the mixture into the 4 channels, 2) diluting the human EpCAM-His recombinant protein to 1ug/ml by using sodium acetate (Cat #: BR-1003-50) with pH4.5, injecting the diluted protein into 2 channels of a chip I, 2 channels of a chip II and 4 channels of a chip I respectively, 3) injecting 1M ethanolamine (pH8.5) for 300s to block residual active carboxyl on the surface of the chip, and then continuing to balance the instrument for two hours by using 1 × HBS-EP + buffer solution after blocking.

b. Affinity assay

A multi-cycle kinetic pattern was set, each cycle comprising binding of control antibody 1, control antibody 2 or PR001081 and regeneration of the chip. Two-fold gradient dilution of antibody was injected into the four channels at a flow rate of 30 μ l/min, setting the binding time to 180s, and the dissociation time to 400s or 600 s. Finally, 10mM glycine-hydrochloric acid pH1.5 (GE LifeSciences, Cat #: BR-1003-54) was injected at the same flow rate for 60s to regenerate the chip.

And analyzing the experimental result by using Biacore T200 analysis software, deducting the 1 channel serving as a reference channel, and selecting a 1: 1 kinetic fitting model as an analysis model.

FIG. 2 is an affinity assay for antibody binding to EpCAM-His. A, b and c of fig. 2 are affinity assays for control antibody 1, control antibody 2 and PR001081, respectively. The affinity (KD = 6.372E-9) of PR001081 is significantly higher than control antibody 1 (2.477E-7) and control antibody 2 (6.527E-7). The specific results are shown in Table 7 below.

TABLE 7 affinity determination of antibodies

Example 5 determination of epitope Competition of antigen binding proteins binding to EpCAM Using the BLI method

Epitope competition experiments were performed on antigen binding protein PR001081, control antibody 1 and control antibody 2 using an Octet Red96e instrument (fortiebo). Firstly, capturing human EpCAM protein (Novoprotein, Cat #: C339) with His label by using HIS1K sensor, wherein the capture height is 0.2 nm; the sensor was then immersed in the first antibody (100 nM) for 180 seconds and the signal at 180 seconds was recorded as the 100% signal of the antibody; the sensor was then immersed in a mixture of primary and secondary antibodies (both antibodies at a final concentration of 100 nM) for 180 seconds and the final signal recorded as the signal of the secondary antibody. The inhibition rate was calculated by the following formula,

inhibition ratio (%) = (a-B)/a 100

(Note: A: 100% signal of an antibody, B: signal of a second antibody)

If the inhibition rate is greater than 80%, it means that the two antibodies have very similar epitopes; if the inhibition rate is between 40-80%, it means that the two antibodies have epitopes that are relatively close but do not completely overlap; if the inhibition rate is less than 40%, it means that the two antibodies have non-overlapping epitopes.

The results show that antigen binding protein PR001081 and control antibody 1 have relatively close but not completely overlapping epitopes, and PR001081 and control antibody 2 have very similar epitopes, see table 8.

Table 8 epitopes compete for experimental signal and inhibition rates.

TABLE 8-1 nM bound secondary antibody when binding was detected by Octet

TABLE 8-2 epitope Competition test signals

	Control antibody 1	Control antibody 2	PR001081
				100% Signal (nM)	0.2479667	0.4177667	0.4561000

TABLE 8-3 inhibition

FIG. 3 shows the detection of the binding epitope of the antibody of the invention and a control antibody against the human EpCAM-His antigen.

In the a, b and c shown in the figure 3, three antibodies, namely a control antibody 1, a control antibody 2 and PR001081, are taken as primary antibodies respectively, and the epitope overlapping degree of other antibodies and the primary antibodies is detected. PR001081, which is identical in epitope to control antibody 2 and close to control antibody 1, affects binding partially, but not completely.

Example 6 CTG assay of the Effect of PR001081 endocytosis in the Capan-2 cell line

The ability of EpCAM antibody to induce cell killing by co-culture with MMAF-coupled anti-human IgG antibody (Moradec, Cat #: AH-102-AF) was tested against Capan-2 cells using the CellTiter-Glo luminometric cell viability assay kit (Promega, Cat #: G7573). cell Capan-2 was centrifuged at 300G for 5 minutes and then resuspended in McCoy's 5A (Capan-2 cell medium, Gibco, Cat #: 16600108) + 10% FBS medium, the cell density was adjusted to 2 × 10⁴Cells/ml. Add 90. mu.l of cell suspension to each well of 96-well plate and incubate at 37 ℃Overnight. EpCAM antibody was diluted to different concentrations in culture medium and 10 μ l of antibody dilution was added to each well of a 96-well plate. The MMAF-coupled anti-human IgG antibody was diluted in medium, and 2. mu.l of the antibody dilution was added to each well of a 96-well plate to a final concentration of 6.6 nM. Cells were incubated with antibody at 37 ℃ for 5 days. The 96-well plate was allowed to stand at room temperature for 30 minutes, and 100. mu.l/well of room-temperature CellTiter-Glo color developing solution was added. After that, the sample was incubated for 10 minutes at normal temperature in the dark. The plate was read with PE Enspire. Cell viability (%) = [ (fluorescent sample)/(fluorescent mock control)]× 100 Control antibody 1 and Control antibody 2 served as positive controls, and humanIso IgG1 (collectively referred to as Human IgG1 Isotype Control, CrownBio, Cat #: C0001-4) antibody served as a negative Control FIG. 4 shows the survival of target cells when the antibody was co-cultured with MMAF-coupled anti-Human IgG antibody PR001081, when co-cultured with MMAF-coupled anti-Human IgG antibody, produced a more potent cytotoxic effect on the Capan-2 cells than Control antibody 1 in a dose-dependent manner, but was similar to Control antibody 2.

Example 7 ADCC Activity of EpCAM antibodies

Activity of EpCAM antibodies to elicit ADCC Effect against the EpCAM endogenously expressing tumor cell line, Capan-2, was tested using the OneGlo kit (Promega, Cat #: E6120) target cells were adjusted to 4 × 10⁵PerkinElmer (PerkinElmer, Cat #: 6005181) in 50. mu.l/well in 96-well bottom-penetrating plates and cultured overnight at 37 ℃ cell density was adjusted to 3 × 10 using commercial Jurkat-CD16a-NFAT (Vazyme, Cat #: DD 1301-01) or self-established Jurkat-CD16a-NFAT reporter cell lines⁶Perml, 50. mu.l per well was seeded on target cell plates. The test antibody was diluted to different concentrations in culture medium and 50. mu.l of each well was added to the cell plate. After incubation of the cell plates at 37 ℃ for 6 hours, 75. mu.l of OneGlo working solution was added to each well, and after equilibration at room temperature for 10 minutes, chemiluminescence detection was performed on Envision. Figure 5 shows that PR001081 antibody is significantly superior to control antibody 1 in both commercial reporter (a) and self-established reporter (b) systems.

We have immunized and obtained a conjugate with higher MFI than baseline, showing a potential binding affinity suitable for the CAR-T approach. The antibody also has good binding activity to cynoEpCAM, which facilitates further toxicity assessment, and thus can be developed for a variety of uses, including ADCs and BiTEs. The molecule has excellent ADC and ADCC effects at the same time.

SEQUENCE LISTING

<110> and platinum medicine (Shanghai) Limited liability company

<120> EpCAM-targeted antibody, preparation and application thereof

<130>P19012750C

<160>22

<170>PatentIn version 3.5

<210>1

<211>448

<212>PRT

<213>Artificial Sequence

<220>

<223> PR001081 heavy chain

<400>1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Phe Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Met Ala Ala Ala Gly Phe Tyr Leu Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>2

<211>215

<212>PRT

<213>Artificial Sequence

<220>

<223> PR001081 light chain

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 7580

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210>3

<211>118

<212>PRT

<213>Artificial Sequence

<220>

<223> PR001081 heavy chain variable region

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Phe Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Met Ala Ala Ala Gly Phe Tyr Leu Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>4

<211>108

<212>PRT

<213>Artificial Sequence

<220>

<223> PR001081 light chain variable region

<400>4

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210>5

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>HCDR1

<400>5

Gly Phe Thr Phe Ser Arg Tyr

1 5

<210>6

<211>6

<212>PRT

<213>Artificial Sequence

<220>

<223>HCDR2

<400>6

Trp Phe Asp Gly Ser Asn

1 5

<210>7

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>HCDR3

<400>7

Glu Met Ala Ala Ala Gly Phe Tyr Leu

1 5

<210>8

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223>LCDR1

<400>8

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210>9

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>LCDR2

<400>9

Asp Ala Ser Asn Arg Ala Thr

1 5

<210>10

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>LCDR3

<400>10

Gln Gln Arg Ser Asn Trp Pro Pro Ile Thr

1 5 10

<210>11

<211>25

<212>PRT

<213>Artificial Sequence

<220>

<223>HFWR1

<400>11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>12

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>HFWR2

<400>12

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210>13

<211>32

<212>PRT

<213>Artificial Sequence

<220>

<223>HFWR3

<400>13

Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>14

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223>HFWR4

<400>14

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210>15

<211>23

<212>PRT

<213>Artificial Sequence

<220>

<223>LFWR1

<400>15

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

15 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>16

<211>15

<212>PRT

<213>Artificial Sequence

<220>

<223>LFWR2

<400>16

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210>17

<211>32

<212>PRT

<213>Artificial Sequence

<220>

<223>LFWR3

<400>17

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210>18

<211>10

<212>PRT

<213>Artificial Sequence

<220>

<223>LFWR4

<400>18

Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

1 5 10

<210>19

<211>116

<212>PRT

<213>Artificial Sequence

<220>

<223> control antibody 1 heavy chain variable region

<400>19

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210>20

<211>107

<212>PRT

<213>Artificial Sequence

<220>

<223> control antibody 1 light chain variable region

<400>20

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>21

<211>118

<212>PRT

<213>Artificial Sequence

<220>

<223> control antibody 2 heavy chain variable region

<400>21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Pro Ser Trp Glu Gln Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210>22

<211>111

<212>PRT

<213>Artificial Sequence

<220>

<223> control antibody 2 light chain variable region

<400>22

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Tyr

20 25 30

Tyr Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Asp Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Lys Gly

85 90 95

Phe Gly His Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

Claims (15)

1. An antibody targeting EpCAM, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein,

the VH comprises HCDR1 shown as the amino acid sequence of SEQ ID NO. 5, HCDR2 shown as the amino acid sequence of SEQ ID NO. 6, and HCDR3 shown as the amino acid sequence of SEQ ID NO. 7;

the VL comprises LCDR1 shown as the amino acid sequence of SEQ ID NO. 8, LCDR2 shown as the amino acid sequence of SEQ ID NO. 9, and LCDR3 shown as the amino acid sequence of SEQ ID NO. 10;

the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.

2. The EpCAM-targeting antibody of claim 1, wherein said heavy chain variable region further comprises a heavy chain variable region framework region (HFWR), and/or said light chain variable region further comprises a light chain variable region framework region (LFWR), wherein said HFWR is the heavy chain variable region framework region of a human antibody and said LFWR is the light chain variable region framework region of a human antibody.

3. The EpCAM-targeting antibody of claim 2,

the HFWR comprises HFWR1 shown in an amino acid sequence shown as SEQ ID NO. 11, HFWR2 shown in an amino acid sequence shown as SEQ ID NO. 12, HFWR3 shown in an amino acid sequence shown as SEQ ID NO. 13, and HFWR4 shown in an amino acid sequence shown as SEQ ID NO. 14;

the LFWR includes LFWR1 shown in the amino acid sequence of SEQ ID NO. 15, LFWR2 shown in the amino acid sequence of SEQ ID NO. 16, LFWR3 shown in the amino acid sequence of SEQ ID NO. 17, and LFWR4 shown in the amino acid sequence of SEQ ID NO. 18.

4. The EpCAM-targeting antibody of claim 1, wherein said VH comprises the amino acid sequence set forth in SEQ ID No. 3 and said VL comprises the amino acid sequence set forth in SEQ ID No. 4.

5. The EpCAM-targeting antibody of claim 1, further comprising a heavy chain constant region and/or a light chain constant region.

6. The EpCAM-targeting antibody of claim 5, wherein the heavy chain constant region is selected from the group consisting of hIgG1, hIgG2, hIgG3, and hIgG4, and the light chain constant region is selected from the group consisting of a kappa chain and a lambda chain.

7. The EpCAM-targeting antibody of claim 6, wherein the heavy chain constant region is hIgG1 and the light chain constant region is a kappa chain of a human antibody.

8. The EpCAM-targeting antibody of claim 1, which is a full-length antibody, Fab ', F (ab')₂Fv, scFv, bispecific or multispecific antibodies, or monoclonal antibodies made from such antibodies.

9. The EpCAM-targeting antibody of claim 8, which is a full length antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID No. 1 and a light chain comprising the amino acid sequence set forth in SEQ ID No. 2.

10. An isolated nucleic acid encoding the EpCAM-targeted antibody of any one of claims 1-9.

11. An expression vector comprising the isolated nucleic acid of claim 10.

12. A host cell comprising the expression vector of claim 11, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

13. A method of producing an antibody targeting EpCAM, comprising the steps of: culturing a host cell as claimed in claim 12, and obtaining an antibody targeting EpCAM from the culture.

14. A pharmaceutical composition comprising an EpCAM-targeting antibody according to any one of claims 1 to 9.

15. Use of an EpCAM-targeted antibody according to any one of claims 1 to 9 and a pharmaceutical composition according to claim 14 for the preparation of a medicament for the treatment and/or prevention of cancer, which is pancreatic cancer.

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