CN107789631B

CN107789631B - Anti-human ErbB2 double-epitope antibody-drug conjugate and application thereof

Info

Publication number: CN107789631B
Application number: CN201711069107.1A
Authority: CN
Inventors: 程联胜; 赵群; 方晴; 刘雯婷; 王凤荣; 孟李凯
Original assignee: Hefei Hankemab Biotechnology Co ltd
Current assignee: Hefei Hankemab Biotechnology Co ltd
Priority date: 2017-11-03
Filing date: 2017-11-03
Publication date: 2021-03-16
Anticipated expiration: 2037-11-03
Also published as: CN107789631A

Abstract

The invention discloses an anti-human ErbB2 bi-epitope antibody-drug conjugate and application thereof. The anti-human ErbB2 bi-epitope antibody-drug conjugate provided by the invention comprises a bispecific anti-HER 2 antibody and a drug part coupled with the bispecific anti-HER 2 antibody; the medicine part contains one, two or more than two medicines. The anti-human ErbB2 bispecific antibody-drug conjugate provided by the invention has a good anti-tumor effect, and in vitro experimental results show that compared with the T-DM1 (trade name Kadcyla) which is already on the market and is used for treating HER-2 positive advanced metastatic breast cancer, in a breast cancer cell line, the anti-human ErbB2 bispecific antibody-drug conjugate provided by the invention has better effect on inhibiting the growth of tumor cells than the Kadcyla, so that the anti-human ErbB2 bispecific antibody-drug conjugate has a good medical application prospect.

Description

Anti-human ErbB2 double-epitope antibody-drug conjugate and application thereof

Technical Field

The invention belongs to the technical field of biotechnology and chemistry, and relates to an anti-human ErbB2 bi-epitopic antibody-drug conjugate and application thereof, in particular to an anti-HER 2 binding molecule (such as an antibody and an antigen binding fragment thereof), a derivative HER2 binding molecule (such as a bispecific anti-HER 2 antibody) and an Antibody Drug Conjugate (ADC) which binds to an extracellular domain of a HER2 receptor. In addition, the invention also relates to a preparation method of the antibody-small molecule drug conjugate and application of the antibody-small molecule drug conjugate in treating diseases associated with HER 2-mediated signal transduction.

Background

The ErbB2 receptor, also known as HER2 or P185, is a glycoprotein belonging to the epidermal growth factor receptor (ErbB/HER) family of the type I receptor tyrosine kinase family, which also possesses the following members: ErbB1/HER1/EGFR, ErbB3/HER3 and ErbB4/HER 4. HER family members play important regulatory roles in cellular physiological processes, mediating signal transduction through ligand binding or formation of dimers among each other. The ErbB2 receptor is closely related to the occurrence and development of tumors, and the phenomenon of amplification or protein overexpression of the ErbB2 protooncogene can be detected in most cancers such as breast cancer, lung cancer, pancreatic cancer, ovarian cancer and the like. Meanwhile, the ErbB2 is also frequently used as an important prognostic indicator for some tumors, such as breast cancer, and tumors with over-expression of ErbB2 are often poor in prognostic effect. Therefore, ErbB2 has become an important target for tumor-targeted therapy.

The medicines for targeted ErbB2 treatment mainly comprise small molecule tyrosine protein kinase inhibitors such as Lapatinib and various forms of antibody medicines, wherein the antibody medicines become hot spots for research and development of anti-tumor biological medicines due to the advantages of high specificity, high affinity, low in vivo metabolic rate, low toxicity, various inhibition mechanisms and the like. With the advent and development of humanized antibody technology, in recent years, there have been commercially available humanized antibodies against human ErbB2, of which two types of humanized antibodies, Trastuzumab (trade name Herceptin, english name Trastuzumab/Herceptin) and Pertuzumab (trade name Perjeta, english name Pertuzumab) have been more successful. Nevertheless, due to primary or secondary tumor resistance and tumor heterogeneity, it has become increasingly difficult to rely on single epitope antibodies and the physiological effects of the antibodies themselves to achieve inhibition of tumor growth.

Antibody-conjugated drug (ADC) is a novel targeted drug therapy method, and is formed by coupling an antibody and a small molecular chemical drug with strong cytotoxicity, and has strong lethality of the small molecular drug and high targeting of a monoclonal antibody, so that the antibody-conjugated drug (ADC) becomes a research and development hotspot of tumor targeted therapy. An ADC generally comprises three parts connected in some way: antibodies, linkers (linkers) and small molecule chemicals. The ADC is targeted from an antibody part, the toxicity is mainly from small molecule chemical drugs, and the antibody part can also have toxicity. After the antibody part is combined with the surface antigen of the tumor cell, the antibody part is endocytosed into the cell, and then the ADC drug is decomposed in lysosomes to release active chemical drug poisons, destroy DNA or prevent the tumor cell from dividing, and finally kill the tumor cell. ADC has the following characteristics over other therapeutic modalities: the treatment efficacy is strong; the tumor cell specificity is high, the false killing rate is low, and the treatment safety window is larger; the immunogenicity is weak, and the drug resistance is not easy to generate; long circulation time in serum (shorter than naked antibody); weak cytotoxicity to non-target point.

The concept of ADC began first with the Magic bulletin concept of Paul Erlich, nobel medical awarded by german chemists in 1913. The earliest approved ADC drug by the FDA was the pfeiffer Mylotarg, marketed in 2000 for the treatment of Acute Myeloid Leukemia (AML), and released from the market in 2016 at 6 months due to clinical trials showing that this drug instead increases mortality. The ADC drugs currently on the market are only two, Ado-trastuzumab-emtansine by Genentech (T-DM 1) and Adcetris (Brentuximab vector) by Seattle Genetics, Luo. In the case of kadcyl, kadcyl (Ado-Trastuzumab-emtansine; T-DM1) is used mainly for treating HER2 positive advanced breast cancer patients, in which Trastuzumab (trade name Herceptin, english name Trastuzumab/Herceptin) is linked to DM1 (a maytansine derivative, a microtubule inhibitor) via a thioether linker (MCC). Clinical results showed that T-DM1 conjugated to chemotherapeutics almost doubled survival relative to the primary naked anti-Herceptin, although the clinical conditions were not the same. However, T-DM1 has a black box warning when approved by the FDA alerting patients and health care professionals that the drug may cause hepatotoxicity, cardiotoxicity and death. This is mainly caused by the release of small DM1 molecules due to the degradation of the shed DM1 and T-DM1 in vivo.

Accordingly, those skilled in the art are constantly working to find new, more effective antibody-drug conjugates.

Disclosure of Invention

In order to solve the above problems, the present invention provides a novel antibody-drug conjugate.

The antibody-drug conjugate provided by the invention comprises a bispecific anti-HER 2 antibody and a drug moiety conjugated to the bispecific anti-HER 2 antibody; the medicine part contains one, two or more than two medicines.

Wherein the bispecific anti-HER 2 antibody comprises a first immunoglobulin antigen-binding domain and a second immunoglobulin antigen-binding domain. The first and second immunoglobulin antigen binding domains are capable of binding to different HER2 antibody binding sites on the HER2 antigen. The first immunoglobulin antigen binding domain is capable of binding to a first HER2 antibody binding site on the HER2 antigen, the first HER2 antibody binding site is an epitope on the HER2 antigen located at domain I, and the first HER2 antibody binding site is distinct from the antibody binding site of pertuzumab.

Further, the first immunoglobulin antigen-binding domain comprises a light chain variable region (VL) and a heavy chain variable region (VH); the light chain variable region (VL) can be the light chain variable region (VL) of the HuA21 antibody and the heavy chain variable region (VH) can be the heavy chain variable region (VH) of the HuA21 antibody.

Specifically, the amino acid sequence of the light chain variable region (VL) of the HuA21 antibody is shown as 21-134 of SEQ ID No.1, and the amino acid sequence of the heavy chain variable region (VH) of the HuA21 antibody is shown as 155-274 of SEQ ID No. 1.

Further, the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may each comprise or consist of: (a) a heavy chain variable region (VH) and a light chain variable region (VL); (ii) a (b) Single chain antibodies (scFv); (c) a diabody; (d) a small antibody; (e) f (ab') 2; (f) f (ab).

Further, the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain may each be a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an affinity-optimized antibody.

Further, the first and second immunoglobulin antigen binding domains specifically bind to different HER2 epitopes.

Further, the second immunoglobulin antigen-binding domain is capable of binding to the same epitope on the HER2 antigen as the antibody binding site of trastuzumab.

Further, in the bispecific anti-HER 2 antibody, the linkage relationship of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is (a1) or (a2) as follows:

(a1) the first immunoglobulin antigen-binding domain is covalently linked to the amino terminus of the heavy chain of the second immunoglobulin antigen-binding domain;

(a2) the second immunoglobulin antigen-binding domain is covalently linked to the amino terminus of the heavy chain of the first immunoglobulin antigen-binding domain.

In (a1), the heavy chain of the second immunoglobulin antigen-binding domain comprises a constant region comprising an Fc domain; in (a2), the heavy chain of the first immunoglobulin antigen-binding domain comprises a constant region comprising an Fc domain.

Still further, the bispecific anti-HER 2 antibody consists of a first polypeptide chain and a second polypeptide chain which are associated with each other.

Wherein the structure of the first polypeptide chain (heavy chain) can be selected from any one of (b1) - (b6) as follows:

(b1)[AscFv]-[L]-[BVH]-[BCH]-[Fcx]

(b2)[AscFv]-[L]-[L]-[BVH]-[BCH]-[Fcx]

(b3)[AscFv]-[L]-[L]-[L]-[BVH]-[BCH]-[Fcx]

(b4)[BscFv]-[L]-[AVH]-[ACH]-[Fcx]

(b5)[BscFv]-[L]-[L]-[AVH]-[ACH]-[Fcx]

(b6)[BscFv]-[L]-[L]-[L]-[AVH]-[ACH]-[Fcx]

[ AscFv ] represents the first immunoglobulin antigen-binding domain in scFv form;

[ BscFv ] represents the second immunoglobulin antigen-binding domain in the form of an scFv;

[ AVH ] and [ ACH ] represent the heavy chain variable region (VH) and heavy chain constant region 1(CH1), respectively, of the first immunoglobulin antigen-binding domain;

[ BVH ] and [ BCH ] represent the heavy chain variable region (VH) and heavy chain constant region 1(CH1), respectively, of the second immunoglobulin antigen binding domain;

[ L ] represents a linker peptide;

[ Fcx ] denotes an Fc domain.

The structure of the second polypeptide chain (light chain) can be selected from (c1) or (c2) as follows:

(c1)[AVL]-[CL]；

(c2)[BVL]-[CL]；

[ AVL ] - [ CL ] represents the light chain variable region (VL) and the light chain constant region (CL) of the first immunoglobulin antigen-binding domain;

[ BVL ] - [ CL ] denotes the light chain variable region (VL) and the light chain constant region (CL) of the second immunoglobulin antigen binding domain.

The light chain constant region (CL) is selected from: human kappa constant region, human lambda constant region.

The first polypeptide chain (heavy chain) and the second polypeptide chain (light chain) are linked by a disulfide bond.

Still further, the first immunoglobulin antigen binding domain is an scFv comprising: the variable region of the light chain (VH region) of the HuA21 antibody shown at positions 21-134 of SEQ ID No.1 and the variable region of the heavy chain (VL region) of the HuA21 antibody shown at positions 155-274 of SEQ ID No. 1. The second immunoglobulin antigen-binding domain is capable of specifically binding to the same HER2 epitope as trastuzumab.

In a first embodiment of the invention, said bispecific anti-HER 2 antibody is in particular obtained by linking a single chain antibody against HER2 at the N-terminus of the heavy chain of trastuzumab via a linking peptide;

the heavy chain variable region in the single chain antibody is the heavy chain variable region of the HuA21 antibody; the light chain variable region in the single-chain antibody is the light chain variable region of the HuA21 antibody; the amino acid sequence of the connecting peptide is 275 th-289 position of SEQ ID No. 1.

More specifically, the amino acid sequence of the heavy chain of the bispecific anti-HER 2 antibody is specifically shown as SEQ ID No.1, and the amino acid sequence of the light chain is specifically shown as SEQ ID No. 2.

The bispecific anti-HER 2 antibody can be prepared according to a method comprising the steps of:

(1) the coding gene (SEQ ID No.3) of the heavy chain of the bispecific anti-HER 2 antibody and the coding gene (SEQ ID No.4) of the light chain of the anti-human ErbB2 bispecific antibody are cloned into pcDNA3.4 vector to obtain two recombinant plasmids (namely the two recombinant plasmids pcDNA3.4 (A) described above_3LHVHCH) and pcDNA3.4 (A)_3LHVLCL))。

(2) Co-transfecting the two recombinant plasmids obtained in the step (1) into a receptor cell to obtain a recombinant cell, and culturing the recombinant cell to obtain the bispecific anti-HER 2 antibody.

In such a method, the bispecific anti-HER 2 antibody can be isolated and purified by affinity chromatography, whereby the bispecific antibody can be purified to a substantially homogeneous mass, e.g., as a single band on SDS-PAGE.

In the present invention, the above-mentioned recipient cell may be a mammalian cell or an insect cell. The mammalian cell is specifically human kidney epithelial cell line HEK 293F.

In the antibody-drug conjugate, the drug may be selected from the following: cytotoxins, radioisotopes, immunomodulators, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factors, hormones, hormone antagonists, enzymes, oligonucleotides, DNA, RNA, siRNA, RNAi, microrna, photoactive therapeutic agents, anti-angiogenic agents, pro-apoptotic agents, peptides, lipids, carbohydrates, chelating agents, or combinations thereof;

further, the cytotoxin may be a maytansine derivative (e.g., DM1, DM4), an auristatin, a dolastatin, a tubulysin, or a Pyrrolobenzodiazepine (PBD).

In an embodiment of the invention, the drug is specifically any one of the following three: monomethyl Auristatin D (MMAD), Monomethyl auristatin E (MMAE) and maytansine derivative DM 1.

In the present invention, the bispecific anti-HER 2 antibody is coupled to MMAD via PY-VC-PAB.

In the present invention, the bispecific anti-HER 2 antibody is coupled to MMAE via MC-VC-PAB.

In the present invention, the bispecific anti-HER 2 antibody is coupled to DM1 via MAL-EBE-MAL.

More specifically, in the present invention, the antibody-drug conjugate is prepared according to a method comprising the steps of: the bispecific anti-HER 2 antibody was first reduced with tris (2-carboxyethyl) phosphine (TCEP); then, MAL-EBE-MAL-DM1, MC-VC-PAB-MMAE or PY-VC-PAB-MMAD was added to the treated bispecific anti-HER 2 antibody to carry out a reaction (e.g., a reaction with stirring at 25 ℃ for 2 hours).

The application of the antibody-drug conjugate in preparing an anti-tumor drug or a tumor cell proliferation inhibitor also belongs to the protection scope of the invention.

Wherein, the tumor cell can be breast cancer cell, ovarian cancer cell or gastric cancer cell.

Furthermore, the tumor cells not only comprise cell strains with high HER2 expression, such as NCI-N87, but also comprise cell strains with low HER2 expression, such as MDA-MB-453, and particularly show good effect of inhibiting the growth of the tumor cells in Herceptin resistant cell strains, such as JIMT-1.

More specifically, in one embodiment of the invention, the tumor cell is selected from the group consisting of: SKBR3, SKOV3, NCI-N87, MDA-MB-361, MDA-MB-453 and JIMT-1.

The anti-human ErbB2 bispecific antibody-drug conjugates provided by the present invention are capable of inducing internalization upon binding to a HER2 target, promoting efficient lysosomal trafficking after internalization, inducing HER2 target degradation, blocking ligand-induced AKT phosphorylation in low HER2 expressing cancer cells, disrupting ligand-induced HER2: HER3 dimerization.

The anti-human ErbB2 bispecific antibody-drug conjugate provided by the invention has a good anti-tumor effect, and in vitro experimental results show that compared with the T-DM1 (trade name Kadcyla) which is already on the market and is used for treating HER-2 positive advanced metastatic breast cancer, in a breast cancer cell line, the anti-human ErbB2 bispecific antibody-drug conjugate provided by the invention has better effect on inhibiting the growth of tumor cells than the Kadcyla, so that the anti-human ErbB2 bispecific antibody-drug conjugate has a good medical application prospect.

Drawings

Figure 1 is a schematic representation of the structure of a bispecific antibody.

FIG. 2 is a SDS-PAGE pattern of the bispecific antibody. The left is SDS-PAGE under reducing conditions, and the right is SDS-PAGE under non-reducing conditions.

FIG. 3 is a graph showing the determination of the binding properties of bispecific antibody to antigen by ELISA method.

FIG. 4 shows the detection of bispecific antibody binding to tumor cells and endocytosis by immunofluorescence assay.

FIG. 5 shows the measurement of the binding property of the antibody-small molecule drug conjugate to antigen by ELISA method.

FIG. 6 shows the results of a competitive assay of antibody-small molecule drug conjugates against antigen. The left picture is A_3LH、A_3LH-DM1、A_3LH-MMAE、A_3LThe result of a competition experiment of H-MMAD on the Herceptin binding site; the right picture is A_3LH、A_3LH-DM1、A_3LH-MMAE、A_3LResults of competition experiments for the binding site of HuA21 by H-MMAD.

FIG. 7 shows the internalization efficiency of cell surface antigens detected by flow cytometry. In the figure, ADC-DM1, ADC-MMAE, and ADC-MMAD respectively represent A_3LH-DM1、A_3LH-MMAE、A_3LH-MMAD。

FIG. 8 shows the effect of antibody-small molecule drug conjugate on inhibiting tumor cell proliferation measured by CCK-8 method. A: a. the_3LH-DM1、A_3LH-MMAE、A_3LThe proliferation inhibition experiment result of the H-MMAD on the SKBR3 breast cancer cell line; b: a. the_3LH-DM1、A_3LH-MMAE、A_3LThe proliferation inhibition experiment result of the H-MMAD on the SKOV3 ovarian cancer cell line; c: a. the_3LH-DM1、A_3LH-MMAE、A_3LThe proliferation inhibition experiment result of the H-MMAD on the NCI-N87 gastric cancer cell line; d: a. the_3LH-DM1、A_3LH-MMAE、A_3LThe proliferation inhibition experiment result of the H-MMAD on MDA-MB-361 breast cancer cell strain; e: a. the_3LH-DM1、A_3LH-MMAE、A_3LProliferation inhibition experiment results of H-MMAD breast cancer cell lines; f: a. the_3LH-DM1、A_3LH-MMAE、A_3LThe result of an experiment for inhibiting the proliferation of JIMT-1 breast cancer cell lines by H-MMAD. In the figure, ADC-DM1, ADC-MMAE, and ADC-MMAD respectively represent A_3LH-DM1、A_3LH-MMAE、A_3LH-MMAD。

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and related definitions and terms may be found, for example, in Current Protocols in Molecular Biology (Ausubel). The abbreviations used therein are all standard three-letter or single-letter designations in the literature and represent one of the common L-amino acids in 20.

As used herein, the terms "antibody drug conjugate," "antibody-drug conjugate," "immunoconjugate," "antibody-small molecule drug conjugate" are interchangeable and refer to a conjugate of an antibody or active fragment thereof and a drug.

As used herein, the terms "antibody drug conjugate of the invention", "antibody of the invention and drug conjugate" or "ADC of the invention" are used interchangeably and refer to a conjugate of an antibody of the invention or an active fragment thereof that targets human HER2 and a drug.

ErbB2 and HER2 can be used as such, both of which represent the native sequence of the human HER2 protein (Genebank accession number X03363, see, e.g., Semba et al, 1985, PNAS, 82: 6497-.

Preferred HER2 in the present invention is human HER2 of natural origin.

The terms "bispecific antibody" and "bi-epitopic antibody" are used interchangeably and are conjugates formed from a first antibody (fragment) and a second antibody (fragment) via a coupling arm, which conjugates retain the activity and antigen-recognition epitopes of the respective antibodies and thus are bifunctional and bispecific.

Example 1 construction of recombinant vectors for expression of anti-human ErbB2 bispecific antibodies

The sequence of anti-human ErbB2 monoclonal antibody Trastuzumab (IgG1, kappa) is from patent US5821337 of Genentech, the sequence of anti-human ErbB2 monoclonal antibody HuA21 is self-created by Hemati Kemibo Biotechnology Limited, the sequence is from patent ZL201410489895X, and the following six bispecific antibodies are respectively constructed according to the series sequence between the two antibodies and the difference of the number of the amino acids of the connecting short peptide: a. the_1LH，A_2LH，A_3LH and H_1LA，H_2LA and H_3LA。

A_1LH，A_2LH，A_3LH is an ScFv formed by connecting the light-heavy chain variable regions of HuA21 in series to the N end of the heavy chain of Herceptin through linkers with different lengths. In the same way, H_1LA，H_2LA and H_3LA is the N-terminal of the heavy chain of HuA21 to which ScFv formed by linking the light and heavy chain variable regions of Herceptin is connected in series by a linker of different lengths. Simply speaking, the ScFv formed by connecting the light and heavy chain variable regions of the X antibody is connected in series to the N end of the heavy chain of the Y antibody through linkers with different lengths, wherein the X and Y antibodies refer to Herceptin and HuA21, the amino acid sequence of the ScFv connected with the light and heavy chain variable region is GGGGSGGGGSGGGGSGGGGS, and the amino acid sequence of the linker connected between the ScFv and the other antibody heavy chain is GGGGS (1L), GGSGGGGS (2L) or GGGGGGSGGGGSGGGGS (3L). A schematic representation of the structure of the bispecific antibody is shown in FIG. 1.

Below with A_3LH, an example, details the construction process of the recombinant vector for expressing the bispecific antibody:

according to the literature, the nucleotide sequence of the Herceptin heavy chain (shown as position 868-2223 of SEQ ID No.3) was directly synthesized (general-purpose organism), and the nucleotide sequence of ScFv in which the light-heavy chain variable region of HuA21 was linked (shown as position 61-822 of SEQ ID No.3 (general-purpose organism)) was directly synthesized based on the nucleotide sequence of HuA 21. Design of Forward primer 5'-GAGGTGCAGCTGGTCGAGAG-3' and reverse primer 5 ' -CCG based on the nucleotide sequences of the 5 ' and 3 ' ends of the Herceptin heavy chainAAGCTTTCACTTCCCGGGGCTCAGGCTCAGG-3', a Hind III cleavage site (Universal organism) was introduced into the reverse primer. Design of forward primer 5 ' -AAT based on the nucleotide sequences of 5 ' and 3 ' ends of HuA21-ScFvGGATCCACTGGTGACATCGTTTTGACTCAATCTCC-3 ' and the reverse primer 5'-GCTCTCGACCAGCTGCACCTCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCTGAAGAAACAGTAACCAAAGTAC-3', which incorporates a BamHI cleavage site (Universal organism) in the forward primer. Furthermore, a complementary sequence was introduced into the forward primer of the former and the reverse primer of the latter, and the heavy chains of HuA21-ScFv and Herceptin were connected in tandem by Overlap PCR using this complementary sequence to obtain A_3LHVH is double digested with BamH I and Hind III and connected to pcDNA3.4(Life company) vector, and the PCR DNA3.4 (A) is obtained after the correctness of sequencing verification_3LHVHCH). The PCR reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃; 30s at 59 ℃; 2min 10 s at 72 ℃ for 30 cycles; 10min at 72 ℃.

Artificially synthesizing Herceptin light chain gene and adding BamHI and HindIII recognition sequences at two ends, wherein the specific sequence is "GGATCC+SEQ ID No.4+AAGCTT"(general purpose organism), after double enzyme digestion with BamHI and Hind III, cloned to pcDNA3.4 vector (Life company) by the same double enzyme digestion, after the correctness of sequencing verification, pcDNA3.4 (A) is obtained_3LHVLCL)。

SEQ ID No.3 encodes the heavy chain of the bispecific anti-HER 2 antibody shown in SEQ ID No.1, and SEQ ID No.4 encodes the light chain of the bispecific anti-HER 2 antibody shown in SEQ ID No. 2.

The recombinant vector construction method for expressing other five bispecific antibodies is the same as A_3LH。

Example 2 expression and purification of bispecific antibodies

With A_3LH for example, pcDNA3.4 (A)_3LHVHCH) and pcDNA3.4 (A)_3LHVLCL) were simultaneously transiently transferred to human kidney epithelial cell line HEK293F (ATCC us cell bank), and after three days of culture, antibody proteins were purified from the culture supernatant using Protein a affinity chromatography column. The purified antibody protein was quantified by the BCA method. The purified antibody was checked for molecular weight and purity using polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. In addition, A was detected by capillary electrophoresis_3LIsoelectric point of H.

The results showed that under reducing conditions (10%) the heavy chains of the six bispecific antibodies were all 75-80kD, and the light chains were slightly different in size, but also in the range of 25-30kD (FIG. 2). The result of isoelectric point measurement by capillary electrophoresis shows that_3LThe isoelectric point of H is around 8.5 (Table 1).

TABLE 1A_3LIsoelectric Point results of H

Example 3 determination of bispecific antibody and antigen binding Properties by ELISA method

T6-17 cell (professor Mark I Greene of university of Pennsylvania medical school, granted by Wang, Xiexiang, Cao Liang, etc., expression of tumor protein P185 in culture supernatant of T6-17 cell, J.Immunol of cell and molecule, 2008,24(10):1018 and 1019, publicly available from the applicant and only used in duplicate experiments) is a stable cell strain with cell membrane transformed from mouse epithelial fibroblast NIH3T3 into human Her2/ErbB2 gene, and the cell membrane of the stable cell strain is a cell strain with cell membraneThe surface of the recombinant human HER2 protein is highly expressed. Resuspending T6-17 cells in logarithmic growth phase in pancreatin digestion medium, centrifuging at 1000rpm/min for 5min, collecting supernatant, and concentrating at 1 × 10⁷Cell lysate (formulation: 50mM Tris-HCl pH7.5, 150mM NaCl, volume fraction 1% Triton-100, 4mM protease inhibitor complete (Roche)) was added at a ratio of individual cells/mL and resuspended, lysed on ice for 20min, and then centrifuged at 12000rpm/min for 15min, and the supernatant was collected. Lysate with 50mM NaHCO₃The pH9.6 solution was diluted at a volume ratio of 1:1000, 100. mu.l per well was coated with an ELISA plate (Nunc Co.), and incubated overnight at 4 ℃; PBST (PBS + 0.1% Tween 20,% representing volume percent) containing 1% (10g/L) BSA was added and blocked at 37 ℃ for 1 h; the test antibodies (six bispecific antibodies prepared above) and the control antibodies (HuA21 and Herceptin) were diluted to 1, 0.25, 0.0625, 0.015625, 0.003906, 0.000977, 0.000244, 0.000061 μ g/mL for 8 gradients, 2 parallel wells per gradient, 100 μ l per well was added to the ELISA plate and incubated for 1h at room temperature with shaking; adding 100 μ l of horse radish peroxidase-labeled goat anti-human IgG (Thermo Co.) diluted 1:8000 (volume ratio) into each well, and incubating for 1h at room temperature with shaking; adding OPD substrate, developing for 3-5min, and finally using 1M H₂SO₄The reaction was stopped, and OD 490 was measured by using a BIO-TEK ELX-800 microplate reader. The plots were plotted according to antibody and antigen response curves using a 4 parameter Logistic fit method.

The results show that the affinity of all six bispecific antibodies is reduced relative to HuA21, where H is_2LThe attenuation of a is more pronounced and there is no significant difference between other bispecific antibodies. And with respect to Herceptin, except H_2LExcept for A, the affinity of other antibodies to the antigen has not been obviously changed. See in particular fig. 3.

Example 4 detection of bispecific antibody binding to tumor cells and endocytosis by immunofluorescence

SKBR3 or BT474 breast cancer cells (Shanghai cell bank) were cultured in vitro to the logarithmic growth phase, and were transferred to 24-well plates (Nunc Corp.) for overnight culture after trypsinization, and the wells were previously plated with round cell culture plates. The next day, the original medium was aspirated and the test antibody (prepared above) containing 10. mu.g/mL was added separatelySix bispecific antibodies of (1) or a control antibody (HuA21, Herceptin and Pertuzumab), and treated for 4 hours, after which the cells were treated according to the following procedure: washing with ice-cold PBS for 5min for 2 times; 3.7% Formaldehyde in PTEM (formulation: 100mM Pipes, pH 6.8, 10mM EGTA, 1mM MgCl)₂0.2% Triton X-100) for 10 min; washing with PBS twice, each for 5 min; blocking overnight at 4 ℃ with PBST (formulation: PBS + 0.1% Tween 20,% by volume) containing 1% (10g/L) BSA; adding 10 μ g/mL FITC labeled goat anti-human IgG, reacting at room temperature for 40min-1h, and washing with PBS for 5min for four times; adding DAPI for reaction for 5min, and washing with PBS for 5min for three times; the cell culture plates were removed from the 24-well plate and inverted onto a glass slide with anti-quencher added, mounted with nail polish, and finally photographed in a fluorescent microscope (Zeiss).

The results of the immunofluorescence experiments are shown in FIG. 4. H_3LA and A_3LH can well bind to an ErbB2 receptor on the surface of a tumor cell, and at 4H, a large amount of antibody enters the interior of the cell to form a phagosome, and only a small amount of antibody exists on the cell membrane. However, the endocytosis capacity of HuA21, Herceptin and Pertuzumab is relatively weak, and at 4h, a large amount of antibody remains only on the cell surface and is rarely endocytosed into the cell. The experimental results show that the bispecific antibody provided by the invention has good application value in the research and development of antibody-targeted drugs (such as antibody-chemical drug conjugate ADC).

Example 5 preparation of antibody-drug conjugates

Firstly, preparing a reducing agent and a protective agent, and diluting the reducing agent and the protective agent by using a PBS buffer solution, wherein the formula is as follows: 1-20mmol/L TCEP (tris (2-carboxyethyl) phosphine), 1-20mmol/L DTPA (diethyltriaminepentaacetic acid) stock solution. According to the required drug-antibody ratio, a certain amount of the stock solution is mixed with the antibody at a certain concentration (for example, 5-30mg/ml) and a certain volume ratio (for example, 1:1), so that the molar ratio of TCEP to antibody in the final reaction system is (0.5-6.0): 1. Then the reaction system was stirred at 25 ℃ for 2 hours. After the reaction was borrowed, the concentration of free thiol was detected with Ellman's reagent DTNB (2-nitrobenzoic acid) at a wavelength of 412nm, and the molar ratio between free thiol and antibody was calculated. The reduction with TCEP is very reproducible and gives free thiol groups in amounts of from 1.0 to 8.0.

After reduction of TCEP, the antibody can be used directly for the subsequent conjugation reaction. A certain concentration (10mM) of small molecule drug (MAL-EBE-MAL-DM1, MC-VC-PAB-MMAE, PY-VC-PAB-MMAD) (Haoshaoyi scientific Co., Ltd.) was diluted with 25% DMSO (dimethyl sulfoxide) and then the molar ratio of drug to sulfhydryl (0.3-2.8): 1 is slowly added to the previously prepared A_3LThe H antibody is removed, and then the reaction is carried out for 2H at 25 ℃, and the stirring is required during the reaction. After the reaction, the concentration of free thiol groups (close to zero) was also determined by Ellman's reagent DTNB (2-nitrobenzoic acid) at a wavelength of 412nm, residual unreacted drug and other small molecules (e.g., DMSO) were removed by purification on Sephadex column G-25, and the coupling was determined by SDS-PAGE (polyacrylamide gel electrophoresis), R-HPLC (reverse phase high performance liquid chromatography) and hydrophobic interaction chromatography (HIC-HPLC).

Example 6 determination of affinity of antibody-drug conjugates

The T6-17 cell lysate (same as example 3) was treated with 50mM NaHCO₃The pH9.6 solution was diluted at a volume ratio of 1:1000, 100. mu.l per well was coated with an ELISA plate (Nunc Co.), and incubated overnight at 4 ℃; PBST (PBS + 0.1% Tween 20,% representing volume percent) containing 1% (10g/L) BSA was added and blocked at 37 ℃ for 1 h; the antibody-drug conjugates to be tested [ the 3 antibody-drug conjugates prepared above, respectively, are A_3LH-MAL-EBE-MAL-DM1 (A for short)_3LH-DM1)，A_3LH-MC-VC-PAB-MMAE (A for short)_3LH-MMAE)，A_3LH-PY-VC-PAB-MMAD (short for A)_3LH-MMAD) and control antibody (A)_3LH) Diluted to 10, 2.5, 0.625, 0.15625, 0.03906, 0.00977, 0.00244, 0.00061, 0.00015 and 0.000038 μ g/mL for 10 gradients, each gradient comprising 2 parallel wells, 100 μ l of each well was added to the ELISA plate, and incubated for 1h at room temperature with shaking; adding horseradish peroxidase label diluted by 1:8000 (volume ratio)Goat anti-human IgG (Thermo Co.) was added at 100. mu.l/well and incubated at room temperature for 1 hour with shaking; adding OPD substrate, developing for 3-5min, and finally using 1M H₂SO₄The reaction was stopped, and OD 490 was measured by using a BIO-TEK ELX-800 microplate reader. EC50 was calculated from the antibody and antigen response curves using a 4-parameter Logistic fit method, as shown in fig. 5 and table 2. This result indicates that the binding activity between the antibody itself and the antigen is not significantly affected after drug conjugation.

TABLE 2 EC50 values for the relative affinities of the respective antibody-drug conjugates for antigen

	A_3LH	A_3LH-DM1	A_3LH-MMAE	A_3LH-MMAD
					EC5(μg/mL)	0.05092	0.08477	0.06095	0.07664

Example 7 competitive binding assay for antibody-drug conjugates

The T6-17 cell lysate (same as example 3) was treated with 50mM NaHCO₃The pH9.6 solution was diluted at a volume ratio of 1:1000 and 100. mu.l of each well was coated with an ELISA plate(Nunc Corp.); incubated overnight at 4 ℃ and blocked by adding PBST (PBS + 0.1% Tween 20,% expressed as% by volume) containing 1% (10g/L) BSA at 37 ℃ for 1 h; the antibody-drug conjugates to be tested [ the 3 antibody-drug conjugates prepared above, respectively, are A_3LH-MAL-EBE-MAL-DM1 (A for short)_3LH-DM1)，A_3LH-MC-VC-PAB-MMAE (A for short)_3LH-MMAE)，A_3LH-PY-VC-PAB-MMAD (short for A)_3LH-MMAD) and control antibody (A)_3LH) Diluting to 200, 66.6667, 22.2222, 7.4074, 2.4691, 0.8230, 0.2743, 0.0914, 0.03048 and 0.01016 mu g/ml, and adding 2 parallel wells of each gradient, adding a certain concentration of biotin-labeled competitive antibody (biotin-labeled HuA210.25 mu g/ml or biotin-labeled Herceptin 2 mu g/ml) into each well, mixing well, adding 100 mu l of each well into an ELISA plate, and incubating for 1h at room temperature with shaking; diluting horseradish peroxidase-labeled avidin (ThermoFisher Co.) at a volume ratio of 1:8000, adding 100 μ l per well, and incubating at room temperature for 1h under shaking; adding OPD substrate, developing for 3-5min, and performing color development with 1M H₂SO₄The reaction was stopped and the OD 490 value was measured with a BIO-TEK ELX-800 microplate reader. The plots were plotted according to antibody and antigen response curves using a 4 parameter Logistic fit method.

The experimental result is shown in fig. 6, and the result shows that the small molecule chemical drug labeling does not obviously affect the affinity of the original double-epitope antibody A3LH for each antibody binding site of human HER 2.

Example 8 detection of internalization efficiency of cell surface antigens by flow cytometry

BT474 breast cancer cells in log phase (shanghai cell bank) were resuspended in tryptic digest medium and collected in conical-bottomed centrifuge tubes. Centrifuging at 4 deg.C and 1000rpm for 5min to precipitate cells, adding culture medium to resuspend cells to make the cell concentration after resuspension 2 × 10⁷Cells/ml, the cell suspension was dispensed into each centrifuge tube at 50. mu.l per tube.

Diluting to 100 μ g/ml with culture medium to obtain antibody-drug conjugates to be tested [ 3 kinds of antibody-drug conjugates prepared above, respectively, are A_3LH-MAL-EBE-MAL-DM1 (A for short)_3LH-DM1)，A_3LH-MC-VC-PAB-MMAE (A for short)_3LH-MMAE)，A_3LH-PY-VC-PAB-MMAD (short for A)_3LH-MMAD) and control antibody (A)_3LH) And the cell suspension were mixed to a final volume of 100. mu.l (50. mu.l antibody sample and 50. mu.l cell suspension). After mixing, ice bath for 60min, flicking at intervals to prevent long-term cell precipitation, centrifuging at 4 deg.C and 1000rpm for 5min to precipitate cells, removing supernatant, washing twice with 1ml RPMI1640 medium to remove residual antibody, adding 500 μ L of fixing solution (1% paraformaldehyde solution: 5g of paraformaldehyde, 250 μ L of 1mol/L sodium hydroxide solution, adding 10 XPBS 50ml, adding water to a constant volume of 500ml) into one tube of each group of antibody, fixing on ice for 20min, centrifuging at 1000rpm for 5min, and discarding supernatant.

Add 200. mu.l of medium per tube and incubate at 37 ℃ for 30min to 4h for various periods (30min, 60min, 120min, 240min) to internalize the antibody bound to the cell surface. At each time point, the 37 ℃ incubation tube corresponding to that time point was immediately transferred to an ice bath and centrifuged at 4 ℃ and 1000rpm for 5min to pellet the cells, 500. mu.l of fixative was added and fixed on ice for 20min, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Mu.l of 10. mu.g/ml FITC-labeled goat anti-human IgG was added to each tube, and after incubation at 4 ℃ for 30min in the dark, 1ml PBS was added to wash the cells, and the procedure was repeated twice, and finally the cells were resuspended in 500. mu.l PBS and flow-observed.

The results are shown in FIG. 7, from which it can be seen that for these three antibody-drug conjugates A_3LH-DM1，A_3LH-MMAE and A_3LH-MMAD, the mark of small molecule chemical drug does not affect the endocytosis effect, and the anti-A gene still has the effect of naked antibody_3LH is equivalent to endocytosis, which just meets the requirement of the ADC drug on endocytosis, and also shows that the potential of A3LH as a carrier for coupling a small molecule chemical drug to prepare the ADC drug is huge.

Example 9 CCK-8 assay of inhibition of tumor cell proliferation by antibody-drug conjugates

The following cell lines are involved in this experiment in total, but are not limited to:

TABLE 3 Table of the tumor cell lines used in the experiment

After each cell line cultured in vitro was resuspended in trypsinization medium, the cell line was inoculated into a 96-well cell culture plate at 100. mu.l/well in CO₂After 24h of incubation in an incubator, the cells containing the antibody-drug conjugate to be tested [ 3 antibody-drug conjugates prepared above, respectively A_3LH-MAL-EBE-MAL-DM1 (A for short)_3LH-DM1)，A_3LH-MC-VC-PAB-MMAE (A for short)_3LH-MMAE)，A_3LH-PY-VC-PAB-MMAD (short for A)_3LH-MMAD) ] and control antibody-drug conjugate (T-DM1, trade name Kadcylla, Roche) at a maximum concentration of 20. mu.g/ml, diluted in 3-fold gradient at 10 concentrations, 3 parallel wells were removed for further culture for 72H, the medium was discarded, 100. mu.l of medium containing CCK-8 reagent (Dongren's chemical) (CCK-8 chromogenic reagent diluted 1:10 by volume) was added to each well, and 5% CO was added at 37 ℃ with a concentration of 5%₂The color development is carried out under the condition that the OD value is measured by a BIO-TEK ELX-800 enzyme-linked immunosorbent assay at 490nm, and the cell growth inhibition rate is calculated by the following formula:

inhibition ═ OD value (OD value vs control well-OD value dosing well)/OD value control well x 100%

And (5) processing the data by using GraphPad software and adopting a four-parameter regression method.

As shown in FIG. 8 and Table 4, the results of the experiments were shown for four cell lines, SKBR3, NCI-N87, MDA-MB-361 and MDA-MB-453, A_3LThe inhibition effect of H-DM1 on the growth of tumor cells is poorer than that of T-DM1, and the semi-effective inhibition concentration (IC50) is slightly higher than that of T-DM 1. However, for cell lines other than NCI-N87 and MDA-MB-453, A_3LH-MMAE showed no worse than T-DM1, while A_3LThe H-MMAD is obviously better than T-DM1, especially for the Herceptin resistant cell strain JIMT-1, which shows that A is_3LApplication of H-MMAD in preparation of antitumor drugsHas good application prospect.

TABLE 4 IC50 values for each antibody-drug conjugate for each HER2 expressing cell line

<110> Sakuaimaibo Biotechnology Limited

<120> anti-human ErbB2 double-epitope antibody-drug conjugate and application thereof

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Claims

1. An antibody-drug conjugate comprising a bispecific anti-HER 2 antibody and a drug moiety conjugated to the bispecific anti-HER 2 antibody; the medicine part contains one, two or more medicines;

the bispecific anti-HER 2 antibody contains a first immunoglobulin antigen-binding domain and a second immunoglobulin antigen-binding domain; the first and second immunoglobulin antigen binding domains are capable of binding to different HER2 antibody binding sites on the HER2 antigen; the first immunoglobulin antigen binding domain is capable of binding to a first HER2 antibody binding site on the HER2 antigen, the first HER2 antibody binding site is an epitope on the HER2 antigen located at domain I, and the first HER2 antibody binding site is distinct from the antibody binding site of pertuzumab;

the first immunoglobulin antigen binding domain comprises a light chain variable region and a heavy chain variable region; the light chain variable region is the light chain variable region of the HuA21 antibody, and the heavy chain variable region is the heavy chain variable region of the HuA21 antibody;

the amino acid sequence of the light chain variable region of the HuA21 antibody is shown as 21 st-134 th position of SEQ ID No.1, and the amino acid sequence of the heavy chain variable region of the HuA21 antibody is shown as 155 nd-274 th position of SEQ ID No. 1; the sequence of the connecting peptide between the light chain variable region of the HuA21 antibody and the heavy chain variable region of the HuA21 antibody is shown as 135-154 of SEQ ID No. 1;

the second immunoglobulin antigen-binding domain is capable of binding to the same epitope on the HER2 antigen as the antibody binding site of trastuzumab; the second immunoglobulin antigen-binding domain comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as the 290-740 th position of SEQ ID No.1, and the amino acid sequence of the light chain is shown as the SEQ ID No. 2;

in the bispecific anti-HER 2 antibody, the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain are linked in a relationship: the first immunoglobulin antigen-binding domain is covalently linked to the amino terminus of the heavy chain of the second immunoglobulin antigen-binding domain;

the bispecific anti-HER 2 antibody consists of a first polypeptide chain and a second polypeptide chain that are associated with each other;

the first polypeptide chain has the structure:

[AscFv]-[L]-[L]-[L]-[BVH]-[BCH]-[Fcx]

wherein [ AscFv ] represents the first immunoglobulin antigen-binding domain in scFv form;

[ BVH ] and [ BCH ] represent the heavy chain variable region and heavy chain constant region 1, respectively, of the second immunoglobulin antigen binding domain;

[ L ] represents a linker peptide having the amino acid sequence GGGGS;

[ Fcx ] represents an Fc domain;

the second polypeptide chain has the structure:

[BVL]-[CL]；

wherein [ BVL ] - [ CL ] represents the light chain variable region and the light chain constant region of the second immunoglobulin antigen binding domain.

2. The antibody-drug conjugate of claim 1, wherein: the amino acid sequence of the heavy chain of the bispecific anti-HER 2 antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain is shown in SEQ ID No. 2.

3. The antibody-drug conjugate of claim 1 or 2, characterized in that: the drug is selected from the following: cytotoxins, radioisotopes, immunomodulators, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factors, hormones, hormone antagonists, enzymes, oligonucleotides, DNA, RNA, siRNA, RNAi, microrna, photoactive therapeutic agents, anti-angiogenic agents, pro-apoptotic agents, peptides, lipids, carbohydrates, chelating agents, or combinations thereof.

4. The antibody-drug conjugate of claim 3, wherein: the cytotoxin is a maytansine derivative, an auristatin, a dolastatin, a tubulysin or a pyrrolobenzodiazepine.

5. The antibody-drug conjugate of claim 3, wherein: the drug is monomethyl auristatin D, monomethyl auristatin E or maytansine derivative DM 1.

6. Use of the antibody-drug conjugate according to any one of claims 1 to 5 for the preparation of an antitumor drug or a tumor cell proliferation inhibitor.