CN119816522B - 5T4 antibody-drug conjugate and its application - Google Patents

Abstract

The invention relates to a 5T4 antibody drug conjugate and application thereof, and particularly provides an antibody drug conjugate, or pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody drug conjugate has a structure shown in a formula I, and Ab is an anti-5T 4 antibody. The antibody drug conjugate has good activity of inhibiting the growth of tumor cells in vivo and in vitro and has good application prospect. Ab- (L-D) p formula I.

Description

5T4 antibody drug conjugate and application thereof

Cross Reference to Related Applications

The present application is based on and claims priority from CN application number 202311048671.0, day 2023, month 8, 18, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to the field of biological medicine, in particular to a 5T4 antibody drug conjugate and application thereof.

Background

Oncofetal 5T4, also known as trophoblast glycoprotein (Trophoblast glycoprotein, TPBG), wnt activation inhibitor 1 (WAIF 1), is a transmembrane glycoprotein encoded by the TPBG gene, highly glycosylated at the N-terminus. The TPBG protein has a molecular weight of about 72kDa and comprises 420 amino acids, consisting of a 310 amino acid extracellular domain, a 20 amino acid transmembrane domain, and a 44 amino acid cytoplasmic domain, which can affect cytosolic remodeling and intercellular integrity by binding to proteins comprising the PDZ domain. Studies have shown that 5T4 is closely related to different physiological and pathological processes, such as intercellular junctions, cell morphology and movement, cell adhesion capacity, cell membrane integrity, and the like. 5T4 is relatively widely expressed during embryonic development, whereas normal tissue is present only in certain specific epithelium, such as basal lamina squamous epithelium, glandular and ductal epithelium, as well as retinal secondary neurons and olfactory bulb.

In contrast, 5T4 is highly expressed in many malignant solid tumors, including pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, non-small cell lung cancer, breast cancer, head and neck cancer, cervical cancer, renal cancer, gastric cancer, colorectal cancer, and the like. There is evidence in colon, gastric or ovarian cancer that the expression level of 5T4 is associated with a low cure rate of cancer. In tissues of non-small cell lung, kidney or pancreas cancer, 5T4 expression is up to 95% or more.

Existing tumor-related studies indicate that 5T4 may act through three mechanisms, 1) epithelial-to-mesenchymal transition (EMT), 2) modulation of CXCL12/CXCR4 biological axes, and 3) inhibition of Wnt signaling.

In conclusion, 5T4 is highly expressed in various solid tumor tissues, but rarely exists in normal mature tissues, and is related to tumor metastasis and poor prognosis, so that it becomes one of ideal targets of tumor drugs. Aiming at the target, the clinical medicine is researched and related to various immunotherapies including monoclonal antibodies, bispecific antibodies, trispecific antibodies, ADC, tumor vaccines, CAR-raNK and the like. The ADC drugs which enter clinical research at present are PF-06263507 (A1-mcMAF) developed by the schduff, ASN-004 developed by Asana BioSciences and SYD-1875 developed by Byondis BV. PF-06263507 (A1-mcMAF) consisted of the 5T 4-specific humanized antibody PF-06281192, a non-cleavable maleimidohexyl (mc) linker and the tubulin inhibitor monomethyl auristatin F (MMAF). ASN-004 consists of 5T4 specific single chain scFv-Fc antibody, dolaflexin drug linker (Mersana Therapeutics), tubulin inhibitor Austrastatin derivative (AF-HPA). SYD-1875 consists of antibody HCP41C, linker payload vc-seco-DUBA, which is a 5T 4-specific engineered cysteine residue. No objective relief was observed for PF-06263507, and Dose Limiting Toxicity (DLT) was observed, which was predominantly ocular toxicity, as reported, with the clinical development of PF-06263507 being terminated at the point of appearance. In addition, SYD-1875 had no information for further development of the clinical trial after completion of phase 1 clinical trial. Therefore, there is a need to further develop new, safe, more effective antibody drug conjugates targeting 5T 4.

Disclosure of Invention

The present inventors have made a new anti-5T 4 antibody drug conjugate (antibody drug conjugate, ADC) through a lot of experiments and creative efforts, and confirmed that it has good biological activity, thereby completing the present application.

To this end, in a first aspect of the invention, the invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of such a salt thereof, the antibody drug conjugate having a structure as shown in formula I,

Ab-(L-D)_p

I is a kind of

Wherein:

Ab is an anti-5T 4 antibody or antigen-binding fragment thereof, said anti-5T 4 antibody comprising a heavy chain variable region and a light chain variable region,

The heavy chain variable region CDR1 comprises the sequence shown in SEQ ID NO. 3,

The heavy chain variable region CDR2 comprises the sequence shown in SEQ ID NO. 4,

The heavy chain variable region CDR3 comprises the sequence shown in SEQ ID NO. 5,

The light chain variable region CDR1 comprises the sequence shown in SEQ ID NO. 6,

The light chain variable region CDR2 comprises the sequence shown as SEQ ID NO. 7;

the light chain variable region CDR3 comprises the sequence shown in SEQ ID NO. 8;

l is a linker;

D is a cytotoxin;

p is any number between 1 and 10 (e.g., 1、1.5、2、2.5、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9 or 10, or e.g., 1-1.5、1.5-2、2-2.5、2.5-3、3-3.5、3.5-4、4-4.5、4.5-5、5-5.5、5.5-6、6-6.5、6.5-7、7-7.5、7.5-8、8-8.5、8.5-9、9-9.5 or 9.5-10).

In formula I, L-D represents a linker linked to the cytotoxin by a covalent bond to form an L-D molecule, and Ab- (L-D) _p represents p L-D molecules coupled to Ab by covalent bonds.

In the present invention, the drug-to-antibody ratio (drug antibody ratio, DAR) refers to the number of drug molecules coupled to an antibody (e.g., p in formula I). The number of drug molecules included in the antibody drug conjugates described herein may be an integer or may be a fraction. Whether integer or fractional, refers to the average number of drug molecules coupled per antibody. "p is any number between 1 and 10", which means that p may be any integer selected from 1 to 10 (inclusive of endpoints 1 and 10), or may be any fraction selected from 1 to 10. Meanwhile, it will be understood by those skilled in the art that the DAR values of the antibody drug conjugates prepared in different batches are not necessarily identical even though the same preparation method is employed, and may float within a range of up and down to not more than 0.5, for example.

The drug-to-antibody ratio (DAR) can be determined by conventional means such as mass spectrometry, ELISA assays, HIC and HPLC. Quantitative distribution of the ADC in p can also be determined. In some cases, separation, purification and validation of homogeneous ADCs with p being a certain value from ADCs with other drug loading may be achieved by means such as HIC, reverse phase HPLC or electrophoresis.

In some embodiments, the linker is selected from the group consisting of MC-AAN, MCC-AAQ, 6-Maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), maleimidopropionyl (MP), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), 4- (N-maleimidomethyl) -cyclohexane-1-formyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).

In some embodiments, the linker is selected from the group consisting of MC-AAN, MCC-AAQ, 6-Maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

Wherein, the structural formula of MC, MCC, MC-vc-PAB is shown as follows:

In some embodiments, the cytotoxin is selected from the group consisting of topoisomerase inhibitors, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, gemcitabine (Gemcitabine), maytansinoids (e.g., MAYTANSINE DM1, MAYTANSINE DM 4), calicheamicin (calicheamicin), MGBA (e.g., duocarmycin), doxorubicin (doxorubicin), ricin, diphtheria toxin, duocarmycin SA (Duocarmycin SA), I131, interleukins, tumor necrosis factor, chemokines, and nanoparticles.

In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor. Exemplary topoisomerase I inhibitors include camptothecins and derivatives thereof. Exemplary camptothecin derivatives include irinotecan (Exatecan), belotecan (Belotecan), SN-38, topotecan (Topotecan), irinotecan (Irinotecan; cpt-11), delutetecan (Deruxtecan), and the like.

In some embodiments, the cytotoxin is selected from the group consisting of irinotecan (Exatecan, abbreviated Exa), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF).

In some embodiments, L-D is selected from MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF.

In some embodiments, the structural formula of L-D is as follows:

When the four L-Ds are coupled to Ab by covalent bonds, the four L-Ds are formed by coupling the succinimide at the ends of the L-D with the thiol groups in the antibody. For example, MC-AAN-Exa when coupled to Ab by a covalent bond, the structural formula of the formed ADC is shown below:

In the ADC formed as described above, the antibody Ab is linked to the carbon atom of the succinimide at the L-D terminus via-S-which is not a thiol group of Ab introduced separately, but a thiol group contained in the antibody Ab itself after the disulfide bond is opened by reduction of the antibody Ab.

In some embodiments, the anti-5T 4 antibody has a heavy chain variable region CDR1 sequence as shown in SEQ ID NO. 3, a heavy chain variable region CDR2 sequence as shown in SEQ ID NO. 4, a heavy chain variable region CDR3 sequence as shown in SEQ ID NO. 5, a light chain variable region CDR1 sequence as shown in SEQ ID NO. 6, a light chain variable region CDR2 sequence as shown in SEQ ID NO. 7, and a light chain variable region CDR3 sequence as shown in SEQ ID NO. 8.

In some embodiments, the sequence of the heavy chain variable region of the anti-5T 4 antibody is shown in SEQ ID NO. 1 and the sequence of the light chain variable region of the anti-5T 4 antibody is shown in SEQ ID NO. 2.

In some embodiments, the sequence of the heavy chain variable region of the anti-5T 4 antibody is shown in SEQ ID NO. 9 and the sequence of the light chain variable region of the anti-5T 4 antibody is shown in SEQ ID NO. 10.

In some embodiments, the sequence of the heavy chain variable region of the anti-5T 4 antibody is shown in SEQ ID NO. 11 and the sequence of the light chain variable region of the anti-5T 4 antibody is shown in SEQ ID NO. 10.

In some embodiments, the heavy chain constant region of the anti-5T 4 antibody is selected from a human-derived IgG, igM, igA, igD, igE constant region or a mutant of the above constant region. In some embodiments, the IgG is selected from the group consisting of IgG1, igG2, igG3, and IgG4.

In some embodiments, the light chain constant region of the anti-5T 4 antibody is selected from a human lambda constant region, a kappa constant region, or a mutant of the above constant regions.

In some embodiments, p is any number between 2 and 8.

In some embodiments, p is any number between 3 and 8 (e.g., 3.8, 4.1, 4.2, 7.9, or 8.0).

In a second aspect of the invention, the invention provides a pharmaceutical composition comprising an antibody drug conjugate as described previously, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof.

In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.

In some embodiments, the chemotherapeutic agent is, for example, doxorubicin (Adriamycin), cyclophosphamide, a taxane such as Taxol (Taxol), docetaxel (Taxotere), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (navelbin), tamoxifen, an aromatase inhibitor (ryoder, freon, minoxin), 5-FU garcinia acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, and the like, or a combination thereof.

In some embodiments, the immunotherapeutic agent is, for example, PD-1 mab (e.g., palbociclib mab, nal Wu Liyou mab), PD-L1 mab (e.g., atezolizumab), TIGIT mab, 4-1BB mab, VEGFR2 mab (e.g., ramucirumab, apatinib), HER2 mab (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), or the like, or a combination thereof.

In some embodiments, the immunosuppressant is selected from (1) glucocorticoids such as cortisone and prednisone, (2) microbial metabolites such as cyclosporine and tacrolimus, etc., (3) antimetabolites such as azathioprine and 6-mercaptopurine, etc., (4) polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3, etc., (5) alkylating agents such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, praecox, cenipecotr, sully, cyclosporine, tacrolimus, rapamycin, mycophenolate, mizoribine, cyclophosphamide, fingolimod, and the like.

In a third aspect of the invention, the invention provides the use of an antibody drug conjugate as hereinbefore described, or a pharmaceutically acceptable salt, solvate or solvate of such a salt or a pharmaceutical composition as hereinbefore described, in the manufacture of a medicament for the prophylaxis and/or treatment of a disease associated with 5T 4.

In a fourth aspect of the invention, the invention provides an antibody drug conjugate as defined above, or a pharmaceutically acceptable salt, solvate or solvate of such a salt or a pharmaceutical composition as defined above, for use in the prevention and/or treatment of a 5T 4-related disorder.

In a fifth aspect of the invention, the invention provides a method of treating and/or preventing a 5T 4-associated disease comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of an antibody drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of such a salt or a pharmaceutical composition as described above.

In some embodiments, the 5T 4-associated disease is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (e.g., non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, renal cancer, gastric cancer, colorectal cancer (e.g., colon cancer), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

In some embodiments, the 5T 4-associated disease is breast cancer, non-small cell lung cancer, colorectal cancer.

In a sixth aspect of the invention there is provided a method of non-therapeutically inhibiting angiogenesis, delaying progression, growth or proliferation of a tumour cell in vitro comprising contacting the tumour cell with an antibody drug conjugate as hereinbefore described, or a pharmaceutically acceptable salt, solvate or solvate of said salt or a pharmaceutical composition as hereinbefore described, said tumour being 5T4 expressing.

In some embodiments, the tumor of 5T4 expression is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (e.g., non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, renal cancer, gastric cancer, colorectal cancer (e.g., colon cancer), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

In some embodiments, the 5T4 expressing tumor is selected from breast cancer, non-small cell lung cancer, colorectal cancer.

Advantageous effects

1. The newly developed 5T4-ADC of the invention has stronger cell killing effect on various tumor (such as breast cancer and non-small cell lung cancer) cells.

2. The newly developed 5T4-ADC of the invention shows remarkable drug effect for inhibiting the growth of tumor cells in various tumor models (such as a CRC#047PDX model of a naked mouse with human colorectal cancer).

Drawings

FIG. 1-1 shows the binding activity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus 5T4-His protein;

FIGS. 1-2 show the binding activity of chimeric antibody 14G12 to CHOK1-hu5T4 cells;

FIGS. 1-3 show the activity of humanized antibodies 14G12z28 and 14G12z43 binding to human 5T4-His protein and to CHOK1-hu5T4 cells, and the activity of humanized antibody 14G12z28 binding to MCF-7 cells;

FIG. 2 shows a 14G12-vcmMAE hydrophobic interaction chromatogram;

FIG. 3 shows a 14G12-MC-MMAF hydrophobic interaction chromatogram;

FIG. 4 shows a 14G12z28-MC-MMAF hydrophobic interaction chromatogram;

FIG. 5 shows a 14G12z43-MC-MMAF hydrophobic interaction chromatogram;

FIG. 6 shows a 14G12z28-MC-AAN-Exa hydrophobic interaction chromatogram;

FIG. 7 shows a 14G12z28-MCC-AAQ-Exa hydrophobic interaction chromatogram;

FIG. 8 shows the binding affinity of each test substance for tumor cells NCI-H1975;

FIG. 9 shows the binding affinity of each test substance for tumor cell HCT 116;

FIG. 10 shows the internalization results of each test on HCT116 cells;

FIG. 11 shows a representative graph of the killing of tumor cells MCF7 by different 5T 4-ADCs;

FIG. 12 shows a representative graph of killing of tumor cells NCI-H1975 by various 5T 4-ADCs;

FIG. 13 shows a representative graph of killing of tumor cells MDA-MB-468 by different 5T 4-ADCs;

FIG. 14 shows in vivo antitumor activity of different 5T 4-ADCs in a CRC #047PDX mouse model;

FIG. 15 shows the effect of different 5T 4-ADCs on body weight of a CRC #047PDX mouse model.

Detailed Description

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

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

In the present invention, unless otherwise indicated, any numerical range should be understood to include any value or any subrange within the range.

In the present invention, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chains of antibodies can be classified into two classes, kappa and lambda. Heavy chains can be categorized as mu, delta, gamma, alpha or epsilon, and antibodies can be categorized as IgM, igD, igG, igA and IgE depending on the heavy chain. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V _H) and a heavy chain constant region (C _H). The heavy chain constant region consists of 3 domains (C _H1、C_H and C _H). Each light chain consists of a light chain variable region (V _L) and a light chain constant region (C _L). The light chain constant region consists of one domain C _L. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and component C1q of the complement system. The V _H and V _L regions can also be subdivided into regions of high denaturation, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each of V _H and V _L consists of 3 CDRs and 4 FRs arranged from amino-terminus to carboxyl-terminus of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions (V _H and V _L) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows the definition of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.

In the present invention, a "humanized" antibody refers to a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., fv, fab, fab ', F (ab') 2 or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the Complementarity Determining Regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

Furthermore, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations, such as PCR-mediated mutations, can be introduced, and their effect on antibody binding or other functional properties can be assessed using in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions or deletions. In addition, mutations within the CDRs typically do not exceed one or two.

In the present invention, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Generally, see, fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, RAVEN PRESS, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') ₂, fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies, single domain antibodies (singledomain antibody), chimeric antibodies, linear antibodies, nanobodies (techniques from Domanis), probody and such polypeptides, comprising an antibody sufficient to confer specific antigen binding capacity to the polypeptide engineered antibody variants are reviewed in Holliger et al, 2005;Nat Biotechnol,23:1126-1136.

In the present invention, the term "Fd" means an antibody fragment composed of VH and CH1 domains, the term "Fab fragment" means an antibody fragment composed of VL, VH, CL and CH1 domains, the term "F (ab ') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and the term "Fab ' fragment" means a fragment obtained after reduction of disulfide bonds linking two heavy chain fragments in the F (ab ') ₂ fragment, consisting of one complete light and heavy chain Fd fragment (composed of VH and CH1 domains).

In the present invention, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

In the present invention, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); pluckaphin, the Pharmacology of Monoclonal Antibodies, vol. 113, volume Roseburg and Moore, springer-Verlag, new York, pp 269-315 (1994)). Such scFv molecules may have the general structure NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ may be used, but variants thereof may also be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56, and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv. In certain embodiments of the invention, an scFv may form a di-scFv, which refers to two or more individual scFv in tandem to form an antibody. In certain embodiments of the invention, an scFv may form (scFv) ₂, which refers to two or more individual scFv that are connected in parallel to form an antibody.

In the present invention, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but that a linker is used that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); poljak R.J. Et al, structure 2:1121-1123 (1994)).

In the present invention, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which a full-length antibody binds. Single domain antibodies are also known as nanobodies (nanobodies).

In the present invention, the term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antigen-binding fragments in the same manner as used for intact antibodies.

The antigen binding fragments of the invention may be obtained by hydrolysis of the intact antibody molecule (see Morimoto et al, J. Biochem. Biophys. Methods 24:107-117 (1992); brennan et al, science 229:81 (1985)). Alternatively, these antigen binding fragments can be produced directly from recombinant host cells (see Hudson, curr. Opin. Immunol.11:548-557 (1999); little et al, immunol. Today,21:364-370 (2000)). For example, fab ' fragments can be obtained directly from host cells, and the Fab ' fragments can be chemically coupled to form the F (ab ') ₂ fragment (Carter et al, bio/Technology,10:163-167 (1992)). Alternatively, fv, fab or F (ab') ₂ fragments may be isolated directly from recombinant host cell culture broth. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

In the present invention, algorithms for determining sequence homology and percent sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1977) Nucl. Acid. Res.25:3389-3402 and Altschul et al (1990) J.mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used to determine the percent amino acid sequence homology of the invention using, for example, the parameters described in the literature or default. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI).

In the present invention, a mutant of the amino acid sequence refers to a sequence having homology of more than 70%, for example, more than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the amino acid sequence, for example, a sequence having 3, 2 or 1 substitutions, deletions or additions of amino acids. Preferably, the amino acids substituted, added or deleted are not more than 3 amino acids. More preferably, the amino acids substituted, added or deleted are not more than 2 amino acids. Most preferably, the amino acids substituted, added or deleted are not more than 1 amino acid.

"Substitution type" variants are those in which at least one amino acid residue in the natural sequence has been removed and a different amino acid has been inserted at the same position. The substitution may be single, wherein only one amino acid in the molecule is substituted, or multiple, wherein two or more amino acids in the same molecule are substituted. Multiple substitutions may be located at successive positions. Also, an amino acid may be substituted with multiple residues, where such variants include both substitutions and insertions. An "insertion-type" (or "additive") variant is a variant in which one or more amino acids are inserted at a particular position immediately adjacent to a segment of the native sequence. By immediately adjacent amino acid is meant a linkage to the alpha-carboxyl or alpha-amino functionality of the amino acid. A "deleted" variant is a variant in which one or more amino acids in the natural amino acid sequence have been removed. Typically, a deletion variant has one or two amino acids deleted in a particular region of its molecule.

In certain embodiments, less than the theoretical maximum of drug moieties are conjugated to the antibody in the conjugation reaction. In general, antibodies do not contain many free and reactive cysteine thiol groups, which can be attached to a drug moiety, and in fact, most of the cysteine thiol groups in antibodies exist as disulfide bridges. In certain embodiments, the antibody may be reduced with a reducing agent such as Dithiothreitol (DTT) or tricarbonyl ethyl phosphine (TCEP) under partially or fully reducing conditions to produce a reactive cysteine thiol group.

In the present invention, the term "pharmaceutically acceptable salt" refers to salts of (i) acidic functional groups present in the conjugates provided herein with suitable inorganic or organic cations (bases) and includes, but is not limited to, alkali metal salts, such as sodium, potassium, lithium salts, etc., alkaline earth metal salts, such as calcium, magnesium salts, etc., other metal salts, such as aluminum, iron, zinc, copper, nickel, cobalt salts, etc., inorganic base salts, such as ammonium salts, organic base salts, such as tertiary octyl amine salts, dibenzyl amine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N' -dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethyl amine salts, piperazine salts, tetramethyl amine salts, tris (hydroxymethyl) aminomethane salts. And (ii) salts of basic functional groups present in the conjugates provided herein with suitable inorganic or organic anions (acids) and include, but are not limited to, hydrohalates such as hydrofluoric acid salts, hydrochloric acid salts, hydrobromic acid salts, hydroiodic acid salts and the like, inorganic acid salts such as nitrate salts, perchlorate salts, sulfate salts, phosphate salts and the like, lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate and the like, aryl sulfonates such as benzenesulfonate, p-benzenesulfonate and the like, organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate and the like, amino acid salts such as glycinate, trimethylglycinate, arginate, ornithine, glutamate, aspartate and the like.

Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic material with a suitable acid that provides a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic material with a suitable base that provides a pharmaceutically acceptable cation.

In the present invention, solvates represent those forms of the antibody drug conjugate of the present invention that are complexes in solid or liquid form formed by complexation with solvent molecules. Hydrates are a specific form of solvates, with coordinated water molecules. In the present invention, the hydrate is a preferred solvate.

Methods of preparing various pharmaceutical compositions containing certain amounts of the active ingredient are known or will be apparent to those of skill in the art in light of the present disclosure. As described in REMINGTON' S PHARMACEUTICAL SCIENCES, martin, E.W., ed., mack Publishing Company,19th ed. (1995), the method of preparing the pharmaceutical composition involves the incorporation of suitable pharmaceutical excipients, carriers, diluents, and the like, which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed.

In the present invention, the pharmaceutical excipients refer to excipients and additives used in the production of medicines and formulation prescriptions, and refer to substances which have been reasonably evaluated in terms of safety except for active ingredients and are contained in pharmaceutical preparations. The pharmaceutical excipients not only form, serve as carriers and improve stability, but also have important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which can influence the quality, safety and effectiveness of the medicine. Natural, semisynthetic and fully synthetic can be classified according to their origin. According to their actions and uses, solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusters, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, permeation enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarders, and the like, and may be classified by their routes of administration into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, ocular administration, and the like. The same medicinal auxiliary material can be used for medicinal preparations with different administration routes, and has different effects and uses.

In the present invention, the pharmaceutical composition may be formulated into various suitable dosage forms according to the administration route. Such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder mists, sprays and the like. Wherein the pharmaceutical composition or suitable dosage form may contain 0.01mg to 1000mg of the antibody drug conjugate of the invention, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof.

The term "treatment" as used herein generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic according to the complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic according to the partial or complete stabilization or cure of the disease and/or side effects due to the disease. As used herein, "treatment" encompasses any treatment of a disease in a patient, including (a) preventing a disease or condition that is susceptible to infection but has not yet been diagnosed in a patient suffering from the disease or condition, (b) inhibiting the symptoms of the disease, i.e., preventing its progression, or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or condition.

In the present invention, "subject" refers to a vertebrate. In certain embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

In the present invention, an "effective amount" refers to an amount effective to achieve the desired therapeutic or prophylactic effect at the necessary dosages and times. The "therapeutically effective amount" of a substance/molecule of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual, the ability of the substance/molecule to elicit a desired response in the individual, and the like. A therapeutically effective amount also encompasses an amount of the substance/molecule that has a therapeutic benefit over any toxic or detrimental effect. "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic effect at the dosages and for the time necessary. Generally, but not necessarily, since the prophylactic dose is for the subject prior to the onset of the disease or early in the disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells, reduce the tumor volume, inhibit (i.e., slow, preferably stop) infiltration of cancer cells into surrounding organs, inhibit (i.e., slow, preferably stop) tumor metastasis, inhibit tumor growth to a degree, and/or reduce to a degree one or more symptoms associated with the cancer.

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

The invention will be further illustrated with reference to specific examples, which are not intended to limit the scope of the invention.

Example 1 preparation of anti-5T 4 chimeric antibodies, humanized modifications and related Performance detection

1. Anti-human 5T4 monoclonal antibody immunoscreening

To obtain monoclonal antibodies targeting the human 5T4 protein, a primary immunization with the human 5T4-His protein was first performed in SJL mice, balb/C mice, C57BL/6 mice, followed by booster immunization in immunized mice with the human 5T4-His protein or CHO-K1 cells overexpressing human 5T4 (CHOK 1-hu5T 4). Antibody titers against human 5T4-his protein in the serum of immunized mice were detected by ELISA experiments and FACS experiments. And selecting an immunized mouse with high serum antibody titer to prepare hybridoma cells. The binding activity of the antibodies in the hybridoma supernatants was detected by ELISA and FACS experiments, and hybridoma cells capable of binding to both human 5T4-His protein (human 5T 4) and cynomolgus 5T4-His protein (cyno 5T 4) and specifically binding to CHOK1-hu5T4 cells were selected. The hybridoma clone 14G12 antibody obtained by screening was subjected to sequencing analysis to obtain the variable region amino acid sequence shown in Table 1-1.

TABLE 1-1:14G12 antibody variable region sequences

2. Binding Activity of chimeric antibody 14G12

Chimeric antibody 14G12 was constructed by ligating the variable region sequence of the 14G12 antibody to a human IgG 1/C.kappa.backbone.

The affinity profile of chimeric antibody 14G12 binding to human 5T4-His protein was detected by the SPR method. Chimeric antibody 14G12 was immobilized on a Protein a chip and the affinity of chimeric antibody 14G12 for binding to human 5T4-His Protein was detected by fitting binding curves under different antigen concentration conditions, as shown in tables 1-4.

The binding activity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus 5T4-His protein was examined by ELISA assay. After the 96-well plate is coated with the human 5T4-His protein or the cynomolgus 5T4-His protein, the chimeric antibody 14G12 with gradient dilution is added, and the mixture is incubated for 60 minutes at room temperature. After PBST washing, mouse-anti-human IgG Fc antibody conjugated with HRP was added and incubated for 30min at room temperature. After PBST washing, TMB substrate was added and OD 450nm absorbance was analyzed. The binding affinities of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus 5T4-His protein are shown in tables 1-2 and FIGS. 1-1. The chimeric antibody 14G12 had comparable human 5T4-His binding activity (fig. 1-1A) and significantly better cynomolgus monkey 5T4-His binding activity (fig. 1-1B) than the control antibody napgummomab.

TABLE 1-2 chimeric antibody 14G12 binding Activity

The binding activity of chimeric antibody 14G12 to CHOK1-hu5T4 cells or MCF-7 cells was examined by FACS experiments. Log phase grown CHOK1-hu5T4 cells or MCF-7 cells were collected. After centrifugation, cells were resuspended in FACS buffer and split into 96-well U-bottom cell culture plates. Antibodies were added and incubated for 30min at 4 ℃ with FACS buffer gradient. After washing cells in wells with FACS buffer, PE Goat anti-Human IgG Fc Secondary Antibody (eBioscience ^TM, invitrogen) diluted with FACS buffer was added and incubated at 4 ℃ for 30min in the dark. After FACS buffer washing of cells in wells, fluorescence signal analysis was performed on cell samples using MACSQuant Analyzer flow cytometer (Miltenyi). The experimental results are shown in FIGS. 1-2, where chimeric antibody 14G12 has good binding affinity for CHOK1-hu5T4 cells.

3. Humanized engineering of chimeric antibody 14G12

By comparing the variable region framework sequence of the 14G12 antibody with the framework sequence of the existing human IgG variable region, the human sequence with high matching degree is screened. And (3) transplanting the CDR sequence of the 14G12 antibody into a selected human IgG variable region framework sequence, and carrying out reverse mutation transformation to obtain a series of 14G12 humanized antibodies.

Humanized antibodies 14G12z28 (also known as Hu14G 12-28) and 14G12z43 (also known as Hu14G 12-43) with optimal binding activity were selected by SPR method, ELISA experiment and FACS experiment (detailed method is the same), and the variable region amino acid sequences are shown in tables 1-3.

TABLE 1-3 humanized antibody variable region sequences

As shown in tables 1-4, humanized antibodies 14G12z28 and 14G12z43 bind human 5T4-His protein with slightly weaker affinity than chimeric antibody 14G12.

TABLE 1-4 affinity of chimeric and humanized antibodies

As shown in FIGS. 1-3, the humanized antibodies 14G12z28 and 14G12z43 bind human 5T4-His protein with activity comparable to that of chimeric antibody 14G12 (FIGS. 1-3A, B), and with activity slightly better than that of chimeric antibody 14G12 (FIGS. 1-3C, D) for CHOK1-hu5T4 cells. In addition, the humanized antibody 14G12z28 had good binding affinity for MCF-7 cells (FIGS. 1-3E).

Example 2 preparation of linker drug conjugate (linker-payload)

1. Preparation of MC-AAN-Exatecan

1. Synthesis of intermediate one

Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-L-alanine, CAS number: 35661-39-3) was activated with HOSu (N-hydroxysuccinimide, CAS number: 6066-82-6) and reacted with L-Ala (L-alanine, CAS number: 56-41-7) to give intermediate one, which was prepared by adding Fmoc-Ala-OH (3 g,1.0 eq) and HOSu (1.45 g,1.3 eq) to a reaction flask, adding 21mL of THF, controlling the temperature at room temperature, slowly adding DCC (2.59 g,1.3 eq) with stirring, reacting at room temperature, monitoring by HPLC, filtering the reaction solution after the reaction, and rinsing the filter cake with THF (6 mL). Purified water (15 mL) was added to the filtrate, followed by L-Ala (1.12 g,1.3 eq), sodium bicarbonate solid (0.81 g,1.0 eq), stirred at room temperature, monitored by HPLC, citric acid (2.02 g,1.0 eq) was added after the reaction was completed and stirred, then the reaction solution was extracted with ethyl acetate, the organic phase was concentrated, DMF (12 mL) was added to dissolve the product, filtered and then prep-HPLC (preparative high performance liquid chromatography) was used to concentrate the preparation until no obvious droplets flowed out, extracted with ethyl acetate, and the ethyl acetate phase was concentrated to dryness to afford intermediate one, the reaction scheme was as follows:

2. Synthesis of intermediate II

The intermediate I (N- [ fluorenylmethoxycarbonyl ] -L-alanyl-L-alanine, CAS number: 87512-31-0) was activated by HOSu (N-hydroxysuccinimide, CAS number: 6066-82-6) and reacted with L-Asn (L-asparagine, CAS number: 70-47-3) to give intermediate II, which was obtained by adding intermediate I (0.87 g,1.0 eq), HOSu (0.34 g,1.3 eq) and THF (9 mL) to a reaction flask, controlling the temperature at room temperature, slowly adding DCC (0.61 g,1.3 eq) under stirring, reacting at room temperature, monitoring by HPLC, filtering the reaction solution after the reaction, and rinsing the filter cake with THF (2 mL). Purified water (10 mL) was added to the filtrate, L-Asn (0.34 g,1.1 eq) was added, sodium bicarbonate solid (0.19 g,1.0 eq) was stirred at room temperature for reaction, monitored by HPLC, citric acid monohydrate (0.48 g,1.0 eq) was added after the reaction was completed and stirred, the reaction solution was concentrated to remove most of the solvent to prepare a purified residue, the preparation solution was concentrated until no obvious droplets flowed out, extracted with ethyl acetate, and the organic phase was concentrated to dryness to afford intermediate two, the reaction scheme was as follows:

3. synthesis of intermediate III

Adding DEA (diethylamine, CAS number: 109-89-7) into a reaction bottle to remove Fmoc and obtain an intermediate III, adding the intermediate II (100 mg) and DMF (1.5 mL) into the reaction bottle, controlling the temperature at room temperature, dropwise adding DEA (300 mu L), performing reaction at room temperature, monitoring by HPLC until the intermediate II is free, concentrating to remove DMF, adding DCM (4 mL) and purified water (4 mL), separating the liquid after stirring, and concentrating the aqueous phase until the aqueous phase is dried to obtain the intermediate III, wherein the reaction formula is as follows:

4. synthesis of intermediate IV

Intermediate III and 6- (maleimide group) caproic acid succinimidyl ester (CAS number: 55750-63-5) react to obtain an intermediate IV, wherein the specific steps are that the intermediate III (92 mg,1.0 eq), 6- (maleimide group) caproic acid succinimidyl ester (135 mg,1.3 eq), DMF (1.5 mL) and DIPEA (0.059 mL,1.0 eq) are added into a reaction bottle, the HPLC monitoring is carried out, and after the reaction is completed, the preparation solution is prepared and purified, and concentrated to obtain an intermediate IV, wherein the reaction formula is as follows:

5. Synthesis of products

The product is obtained by condensation reaction of the intermediate IV and the Eptification Kang Jia sulfonate (CAS number: 169869-90-3), and the specific steps are that under the condition of room temperature, the intermediate IV (26 mg,1.0 eq) is added into a reaction bottle, DMF (1.5 mL) is added, and the Eptification Kang Jia sulfonate (29.6 mg,1.0 eq) and EEDQ (20.7 mg,

1.5 Eq), HATU (31.8 mg,1.5 eq), DMAP (0.7 mg,0.1 eq), DIPEA (29.2 μl,3.0 eq), and the reaction was carried out at room temperature. After the reaction is finished, the HPLC monitoring is carried out, the preparation and purification are carried out, and the preparation liquid is concentrated to obtain the product, wherein the reaction formula is as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.22-8.29(d,J＝2.0Hz,1H),7.94-8.05(d,J＝2.0Hz,3H),7.83-7.88(d,J＝2.0Hz,1H),7.75-7.82(d,J＝2.0Hz,1H),7.34-7.40(m,1H),7.28-7.33(m,1H),6.96-7.02(d,J＝8.0Hz,2H),6.87-6.94(m,1H),5.42-5.55(m,1H),5.41-5.46(m,2H),5.21-5.26(m,1H),4.41-4.49(m,1H),4.00-4.12(m,2H),3.32-3.40(m,2H),3.12-3.17(m,2H),2.86-2.96(m,2H),2.71-2.76(m,2H),2.36-2.43(m,2H),2.14-2.24(m,1H),1.97-2.07(m,3H),1.81-1.93(m,2H),1.36-1.52(m,4H),1.05-1.25(m,8H),0.83-0.93(m,3H).

2. Preparation of MCC-AAQ-Exatecan

1. Synthesis of Fmoc-Gln-Exatecan of intermediate one

Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg1.1 eq.) and Exatecan (100 mg1.0 eq.) were added to the reaction flask, DMF (1 ml), DIEA (29 mg 1.5 eq.), TBTU (72.5 mg1.1 eq.) were added, and the reaction was carried out at room temperature, monitored by HPLC, with no starting material remaining. After the reaction was completed, the mixture was purified by passing through a medium pressure column (DCM/MeoH). The product was collected and concentrated to dryness to afford intermediate one.

The reaction formula is as follows:

2. Synthesis of intermediate two Gln-Exatecan

Intermediate-Fmoc-Gln-Exatecan (130 mg 1.0 eq.) was added to the reaction flask, DCM (2 ml) was added and stirred at room temperature. DEA (0.5 ml) was added, reacted at room temperature, monitored by HPLC, and no starting material remained. The reaction was stopped, MTBE (10 ml) was slowly added, a large amount of solid was precipitated, and stirred for 30min. Filtration and MTBE washing gave an off-white solid which was dried to afford intermediate two.

The reaction formula is as follows:

3. synthesis of Fmoc-Ala-Ala-Gln-Exatecan for intermediate III

Intermediate di Gln-Exatecan (100 mg 1.0 eq.) was added to the reaction flask, DMF (1 mL), fmoc-Ala-Ala-OH (67.8 mg 1.0 eq.), TBTU (68.4 mg 1.2 eq.) DIEA (34.4 mg 1.5 eq.) and the reaction was monitored at room temperature and HPLC until the reaction ended. Purification by passing through a column under medium pressure (DCM/MeOH) and collecting the product gives intermediate three.

The reaction formula is as follows:

4. synthesis of Ala-Ala-Gln-Exatecan of intermediate IV

The intermediate tri Fmoc-Ala-Ala-Gln-Exatecan (140 mg 1.0 eq.) was added to the reaction flask, DCM (2 ml) was added and stirred at room temperature. DEA (0.5 ml) was added, reacted at room temperature and monitored by HPLC until the reaction was completed. MTBE (10 ml) was slowly added and a large amount of solid was precipitated and stirred for 30min. Filtration and MTBE washing gave an off-white solid which was dried to afford intermediate four.

The reaction formula is as follows:

5. product synthesis MCC-Ala-Ala-Gln-Exatecan

The intermediate tetra Ala-gin-Exatecan (50 mg 1.0 eq.) was added to the reaction flask, DMF (1 mL), then MCC (18 mg 1.1 eq.), DIEA (12.1 mg 1.5 eq.) was added, the reaction was allowed to proceed at room temperature, HPLC monitored until no intermediate four remained, and the reaction was stopped. Purifying by Prep-HPLC, collecting the product, concentrating and drying to obtain the product.

The reaction formula is as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.17-8.26(d,J＝2.0Hz,1H),7.83-7.91(d,J＝2.0Hz,2H),7.65-7.74(d,J＝2.0Hz,2H),7.21-7.28(d,J＝8.0Hz,2H),6.98-7.04(m,2H),5.43-5.50(m,1H),5.36-5.43(m,2H),5.15-5.24(m,1H),5.01-5.11(m,1H),4.14-4.24(m,1H),3.97-4.08(m,1H),3.47-3.57(m,1H),3.20-3.27(m,1H),3.05-3.16(m,2H),2.30-2.36(m,2H),2.00-2.15(m,4H),1.77-1.92(m,3H),1.65-2.00(m,5H),1.45-1.64(m,4H),1.09-1.24(m,5H),0.95-1.07(m,4H),0.79-0.94(m,5H).

example 3 preparation of 5T4-ADC

1. Preparation of antibody conjugate 5T4 antibody-vcMAE and 5T4 antibody-MC-MMAF

A. the total amount of protein was calculated by taking 5T4 antibody (e.g., 14G12z28 or 14G12z 43), adjusting the pH of the antibody to about 7.5 with Tris-EDTA solution, detecting the protein concentration with Nanodrop, and weighing the net weight of the antibody solution. Adding TCEP solution into the antibody, placing on a 3D shaking table, reacting for more than 120min at room temperature, and continuously and uniformly mixing to reduce disulfide bonds among the chains of the antibody.

B. Adding excessive MC-vc-PAB-MMAE solution (manufactured by Shanghai Meiya Kennel Biotechnology Co., ltd., dissolving in DMSO) or MC-MMAF solution (purchased from Borui medical organism (Suzhou) Co., ltd., dissolving in DMSO) into the reduced antibody solution, mixing, placing on a 3D shaking table, reacting at room temperature for more than 30min, and continuously mixing. After the reaction is finished, adding excessive N-acetylcysteine solution into the reaction solution, placing the solution on a 3D shaking table, reacting for more than 30 minutes at room temperature, and continuously and uniformly mixing.

C. The coupled product was purified using a 30KD ultrafiltration centrifuge tube and replaced into stock (10mM Histidine,pH5.5 or so) with a fold greater than 1000 fold. Then filtering with a 0.22um sterilizing filter to obtain antibody drug conjugates 14G 12-vcMAE, 14G12-MC-MMAF, 14G12z28-MC-MMAF and 14G12z43-MC-MMAF, and storing at 4deg.C.

D. Protein concentration of the antibody drug conjugate was detected by UV/BCA, DAR detection was performed by HIC (as shown in FIGS. 2-5), DAR was 3.8, 4.2 and 4.1, respectively, and SEC was performed for purity detection.

2. Preparation of antibody conjugate 5T4 antibody-MC-AAN-Exatecan and 5T4 antibody-MCC-AAQ-Exatecan

A. The method comprises the steps of taking a 5T4 antibody such as 14G12z28, adjusting the pH of the antibody to about 7.5 by using a Tris-EDTA solution, detecting the protein concentration by using Nanodrop, weighing the net weight of the antibody solution, and calculating the total protein. Adding TCEP solution into the antibody, placing on a 3D shaking table, reacting for more than 120min at room temperature, and continuously and uniformly mixing to reduce disulfide bonds among the chains of the antibody.

B. Adding excessive MC-AAN-Exatecan solution or MCC-AAQ-Exatecan (dissolved in DMSO) into the reduced antibody solution, mixing, placing on a 3D shaking table, reacting at room temperature for more than 30min, and continuously mixing. After the reaction is finished, adding excessive N-acetylcysteine solution into the reaction solution, placing the solution on a 3D shaking table, reacting for more than 30 minutes at room temperature, and continuously and uniformly mixing.

C. The coupled product was purified using a 30KD ultrafiltration centrifuge tube and replaced into stock (10mM Histidine,pH5.5 or so) with a fold greater than 1000 fold. Then filtering with a 0.22um sterilizing filter to obtain the antibody drug conjugate 14G12z28-MC-AAN-Exa and 14G12z28-MCC-AAQ-Exa ℃ for preservation.

D. protein concentration of the antibody drug conjugate was detected by UV/BCA, DAR detection was performed by HIC (as shown in FIGS. 6-7), DAR was 8.0 and 8.0, respectively, and purity was detected by SEC.

EXAMPLE 4 5T4-ADC pharmacological pharmacodynamics study

1. Cell binding Activity of 5T 4-ADCs

It was confirmed by FACS experiments that the cell binding activity of ADC to target antigen expression was not substantially affected before and after antibody conjugation.

NCI-H1975 lung cancer cells or HCT116 colorectal cancer cells grown in log phase were collected. After centrifugation, cells were resuspended in FACS buffer (pbs+3% FBS), cell density was adjusted, and cells were separated into 96-well U-bottom cell culture plates, containing 200,000 to 500,000 cells per well. Antibody or ADC diluted 4-fold in FACS buffer was added and mixed to give final initial final concentrations of 10. Mu.g/mL for each sample. And placing the 96-well plate at 4 ℃ for incubation for 45-90 min. Cells in the wells were washed thoroughly with FACS buffer to remove unbound antibodies or ADCs. Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, alexa Fluor 488 or 647 (Invitrogen, # A-11013 or #A-21445) diluted with FACS buffer 1:1,000 were added and incubated at 4℃for 30-60 min in the absence of light. Cells in the wells were washed thoroughly with FACS buffer to remove unbound secondary antibody. The cell samples were analyzed for fluorescent signals using CytoFLEX flow cytometer (Beckman Coulter). The experimental results are shown in fig. 8-9, and the binding affinity of the chimeric antibody 14G12 and the humanized antibody 14G12z28 to the NCI-H1975 or HCT116 cells expressing 5T4 before and after the cytotoxin is coupled does not show significant changes, indicating that the cell binding activity of the antibodies is not substantially affected before and after the antibody is coupled with the toxin. The binding affinities of each test substance to tumor cells are shown in table 2 and fig. 8 to 9.

TABLE 2 binding of 5T4 antibodies and ADC to tumor cells (EC ₅₀)

2. Internalization of antibodies in 5T 4-ADCs

The internalization ability of 5T 4-targeted mab and its ADC was confirmed.

HCT116 colorectal cells grown in log phase were collected. After centrifugation, the cells are washed once, resuspended in a pre-chilled 3% FBS-containing cell culture medium, the cell density adjusted, and the cells are separated into 96-well U-bottom cell culture plates, containing 500,000-800,000 cells per well. Antibodies or ADCs diluted with DMEM medium containing 3% fbs were added and mixed to give a final sample concentration of 10 μg/mL. The 96-well plates were incubated on ice for 60min. Cells in wells were washed thoroughly with pre-chilled FACS buffer (pbs+3% FBS) to remove unbound antibodies or ADCs. Cells in the wells were equally divided into 4 96-well U-plates, centrifuged and the cells resuspended in cell culture medium. 1 96-well plate was placed on ice and the other 3 were incubated in a 37 ℃ cell incubator. After 0.5h, 1h and 2h, 1 plate at 37 ℃ was transferred onto ice, respectively. After the 3 rd culture plate is transferred onto ice for 5-10 min, diluted Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, alexa Fluor 647 (Invitrogen, #A-21445) are added and incubated on ice for 30-60 min in the absence of light. Cells in the wells were washed with pre-chilled FACS buffer to remove unbound secondary antibody. The cell samples were subjected to fluorescent signal analysis using CytoFLEX flow cytometer (Beckman Coulter), indicated by MFI (GeoMean fluorescence intensity). Except for 37 ℃ incubation, the whole experimental procedure was kept on ice or at 4 ℃.

The percent reduction of cell surface molecules was calculated as follows:

Cell surface molecules were reduced by% = (MFI _{On ice} -MFI_37℃)/MFI _{On ice} x 100%.

The experimental results are shown in table 3, and the humanized antibody 14G12z28 targeting 5T4 did not change the internalization ability of HCT116 cells before and after conjugation of the cytotoxin, indicating that the conjugated toxin had no significant effect on the internalization of the antibody. The results of internalization (expressed as a percentage of reduction of cell surface molecules) of each test species on HCT116 cells are shown in table 3 and fig. 10.

TABLE 3 internalization of 5T4 antibodies and ADCs on HCT116 cells

EXAMPLE 5 in vitro pharmacodynamic Studies

Cell killing activity of 5T4 targeting ADC molecules coupled to various linker-payload was evaluated.

Tumor cells grown in log phase were collected. After centrifugation, the cells were resuspended in fresh medium and counted. Cells were seeded in 96-well bottom-permeabilized black cell culture plates (Costar) and cultured overnight in a cell incubator. The next day, ADC molecules were subjected to 4-fold gradient dilutions with medium, carefully transferring the dilutions into black plates, to give final initial final concentrations of 200ng/mL or 10,000ng/mL for each sample. Placed in a cell incubator for 4 days (MMAE or MMAF ADC) or 6 days (Exatecan ADC), then 1/10 of the well inner volume of PrestonBlue reagent (Invitrogen) was added and incubated for 1h. The fluorescent signal was read with a SpectraMax M5 reader, and the excitation and emission wavelengths of the instrument were set to 560nm and 590nm, respectively. The resulting fluorescent signal data were analyzed using SoftMax Pro 6.5 software.

1. Experimental reagents and sources:

TABLE 4 test drug

TABLE 5 cell lines used in cell killing Activity experiments

2. Experimental results:

the average value of EC ₅₀ for each 5T4-ADC cell killing activity is shown in Table 6. FIGS. 11-13 are representative graphs of tumor cell killing by different 5T 4-ADCs.

TABLE 6 EC ₅₀ values for cell killing Activity of different 5T 4-ADCs in tumor cells

As can be seen from the results of Table 6 and FIGS. 11-13, the ADC of the present invention formed by the toxin-conjugated 5T4 antibody shows a strong cell killing activity.

EXAMPLE 6 in vivo pharmacodynamics Studies

The in vivo antitumor activity of the different 5T 4-ADCs was tested in a CRC #047PDX mouse model.

The CRC #047PDX model demonstrates 5T4 positive expression of cells from this model by FACS analysis. The establishment process of the CRC#047PDX model of the human colorectal cancer nude mouse comprises the step of transplanting tumor tissues with the volume of about 30mm ³ under the skin on the right back side of the BALB/c nude mouse. When the tumor volume reached 200-300mm ³, the groups were grouped by random block method, and the group was recorded as Day 0. Each group of 6 mice was given a balance of tumor volume and weight between groups, 4 groups, including vehicle group, non-binding-MC-AAN-Exa (10 mg/kg) control dosing group and 14G12z28-MC-AAN-Exa (10 and 3 mg/kg) dosing group. At Day 0 and Day 7, each tail vein administration was performed once. Wherein Non-binding indicates that the antibody is a Non-binding human IgG1 isotype control, and does not bind to a target on the surface of a tumor cell used in the experiment, and is used as a negative control antibody.

Analysis of data tumor volumes were determined twice a week during the experiment. The calculation formula of the Tumor Volume (TV) is tv=l×w ²/2. Wherein l and w represent tumor measurement length and width, respectively. From the measurements, relative tumor volumes (relative tumor volume, RTV), rtv=v _f/V₀ were calculated. Where V ₀ is the tumor volume measured at the time of group administration (i.e., day 0) and V _f is the tumor volume measured the last Day. Relative tumor proliferation rate T/C (%) = (RTV in dosing group/RTV in Vehicle group) ×100%. When T/C (%) is less than or equal to 40%, and P is less than 0.05, the test product is considered to have a remarkable inhibition effect on tumor growth.

The experimental results are shown in fig. 14 to 15. At Day 28, the relative tumor proliferation rate T/C (%) of the 14G12z28-MC-AAN-Exa (10 mg/kg) administration group was 10.02% (P < 0.0001), the relative tumor proliferation rate T/C (%) of the 14G12z28-MC-AAN-Exa (3 mg/kg) administration group was 13.28% (P < 0.0001), and the relative tumor proliferation rate T/C (%) of the Non-binding-MC-AAN-Exa (10 mg/kg) control administration group was 55.54% (P > 0.05). The average weight change of mice in Day 28 was in the range of 3.74% -5.21% compared to Day 0.

Experimental results show that when the administration doses are 3mg/kg and 10mg/kg, the 14G12z28-MC-AAN-Exa has remarkable inhibition effect on tumor growth. Tumor-bearing mice have good tolerance to all test samples.

Claims (14)

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate has the structure shown in Formula I, Ab-(LD) _p Formula I in: Ab is an anti-5T4 antibody or an antigen-binding fragment thereof, wherein the anti-5T4 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, The sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 3, The sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 4, The sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 5, The sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 6, The sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 7, The sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 8; L-D is selected from: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF; p is any value between 1 and 10. 2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the anti-5T4 antibody has one technical feature selected from the following (i)-(ii): (i) the heavy chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 9, and the light chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 10; (ii) the heavy chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 11, and the light chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 10. 3. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the anti-5T4 antibody has one or more technical features selected from the following (i)-(ii): (i) the heavy chain constant region of the anti-5T4 antibody is selected from human IgG, IgM, IgA, IgD, and IgE constant regions; (ii) The light chain constant region of the anti-5T4 antibody is selected from a human lambda constant region and a kappa constant region. 4. The antibody-drug conjugate according to claim 3, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the IgG is selected from IgG1, IgG2, IgG3 and IgG4. 5. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein p is any value between 2 and 8. 6. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein p is any value between 3 and 8. 7. The antibody drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein p is 3.8, 4.1, 4.2, 7.9 or 8.0. 8. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof. 9. The pharmaceutical composition of claim 8, further comprising at least one pharmaceutical excipient. 10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug and an immunosuppressant for treating tumors. 11. Use of the antibody-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the pharmaceutical composition according to any one of claims 8 to 10 in the preparation of a medicament for preventing and/or treating a disease associated with 5T4; The disease associated with 5T4 is selected from non-small cell lung cancer, breast cancer, and colorectal cancer. 12. The use according to claim 11, wherein the colorectal cancer is colon cancer. 13. An in vitro non-therapeutic method for delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation, comprising: contacting tumor cells with the antibody drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the pharmaceutical composition according to any one of claims 8 to 10, wherein the tumor is a tumor expressing 5T4; The 5T4-expressing tumor is selected from non-small cell lung cancer, breast cancer, and colorectal cancer. 14. The method of claim 13, wherein the colorectal cancer is colon cancer.