Engineered antibody with site-specific conjugation, antibody-drug conjugate, and application thereof

Abstract

Disclosed are an engineered antibody with site-specific conjugation, an antibody-drug conjugate, and an application thereof. The engineered antibody with site-specific conjugation is an IgG antibody, wherein a cysteine at a position corresponding to an interchain disulfide bond-forming site in a wild-type IgG antibody is substituted and / or deleted; and the IgG antibody does not have a disulfide bond or a free sulfhydryl group between heavy and light chains. The antibody of the present invention can be site-specifically conjugated to DAR4 ADC, and the resulting ADC has a uniform DAR value, high homogeneity, low batch-to-batch variability, and is suitable for large-scale production.

Description

Site-directed conjugate antibodies, antibody-drug conjugates and their applications in engineering modification

[0001] This application claims priority to Chinese patent application 2024115933132, filed on 2024 / 11 / 8. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of medicine, specifically relating to an engineered site-conjugated antibody, an antibody-drug conjugate, and their applications. Background Technology

[0003] Antibodies are key immune molecules that fight against foreign pathogens. The development of monoclonal antibody (mAb) technology has enabled the widespread application of monoclonal antibodies in disease research, diagnosis, and treatment. First-generation mAbs (mostly monospecific or bivalent mAbs) have achieved success in treating a variety of diseases, including cancer, autoimmune diseases, and infectious diseases. However, many diseases, such as solid tumors, have proven to exhibit considerable resistance to antibody-based therapies.

[0004] Antibody-drug conjugates (ADCs) are small-molecule active drugs chemically linked to monoclonal antibodies, giving them both the specific targeting ability of monoclonal antibodies and the cancer-killing ability of cytotoxic drugs. The ability to select specific monoclonal antibody-drug combinations and advancements in monoclonal antibody-drug conjugation have opened new possibilities for targeting cancer while minimizing exposure to healthy tissue. As of 2022, 15 ADCs had been approved by the FDA, including Roche's Kadcyla. TM and Polivy TM Pfizer's Mylotarg TM and Besponsa TM AstraZeneca's Lumoxiti TM Daiichi Sankyo / AstraZeneca Enhertu TM And Rongchang Bio's VidiCetuximab, among others. Besides the ADC drugs that have already been approved for marketing, a large number of ADC drugs are currently under clinical development.

[0005] The conjugation method is a crucial factor affecting the uniformity of drug-to-antibody ratio (DAR) in ADCs. Traditional methods of drug-antibody conjugation often result in heterogeneous ADC-drug mixtures. For example, cytotoxic drugs are typically conjugated to antibodies via surface-exposed lysine or cysteine ​​residues obtained by reducing interchain disulfide bonds, producing different types of ADCs with varying attachment sites and drug-to-antibody ratios (DARs) on the antibody. The amino group of lysine is linked to an activated carboxylic acid ester linker via an amide bond, while the thiol group of cysteine ​​reacts with a maleimide group. An antibody molecule has approximately 80 lysine residues, and conjugation can occur randomly at multiple different lysine residues; breaking interchain disulfide bonds yields multiple cysteine ​​residues, simultaneously disrupting the integrity of the antibody molecule. Therefore, traditional ADCs are highly heterogeneous mixtures with poor homogeneity and low stability. Non-specific site conjugation poses challenges to ADC conjugation process control, product characterization, and quality control. Furthermore, different types of ADCs may exhibit very different safety and efficacy characteristics. Since the disulfide bond between the heavy chain of the IgG1 antibody and its paired light chain is likely to be the preferred bond to be reduced during the coupling process, the resulting drug conjugates with light chains may be less stable and may lose their light chains during cycling.

[0006] Traditional antibody-cysteine ​​conjugation, with optimized conjugation conditions, can achieve an average DAR value of 3-4, distributed between 0-7. However, due to its high heterogeneity and poor uniformity, and the generally wide distribution of DAR leading to reduced efficiency, strict control of the distribution is necessary. Commercially available ADCs prepared by conjugation with cysteine ​​include Adcetris, Polivy, Padcev, Enhertu, Blenrep, Trodelvy, Zynlonta, and RC48. Due to a lack of site specificity, these ADCs are typically generated as random mixtures, with an average DAR between 3-4. However, there are two exceptions: Enhertu and Trodelvy are generated through near-quantitative binding of all interchain cysteine ​​residues, with DARs of 7.7 and 7.6, respectively, thus exhibiting site specificity.

[0007] In recent years, several site-directed conjugation techniques have been developed to obtain more uniform ADCs. These techniques involve designing tags, such as certain amino acids like cysteine, non-natural amino acid residues like p-acetylphenylalanine (pAcF), or short peptides that can be recognized and modified by enzymes, at specific sites in the antibody's amino acid sequence. Some studies have also attempted sugar-mediated conjugation using glycans attached to the antibody heavy chain. Currently, site-directed conjugation techniques mainly focus on DAR2 or DAR8 ADCs. DAR4 ADC site-directed conjugation techniques are primarily achieved through thiol bridging, where each bifunctional drug linker can capture two free cysteine ​​thiol groups, resulting in the complete reduction of all eight interchain disulfide bonds and conjugation into a DAR4 ADC with low heterogeneity. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an engineered site-conjugated antibody, an antibody-drug conjugate, and their applications, wherein the engineered site-conjugated antibody is an IgG antibody.

[0009] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0010] A first aspect of the present invention provides an IgG antibody in which cysteine ​​residues corresponding to the interchain disulfide bonds formed by wild-type IgG antibodies are substituted and / or deleted; the IgG antibody lacks interchain disulfide bonds and free thiol groups.

[0011] In some embodiments, the IgG antibody comprises a light chain constant region and a heavy chain constant region, wherein the heavy chain constant region comprises a hinge region.

[0012] In some implementations, the first cysteine ​​residue in the hinge region is substituted or absent.

[0013] In some implementations, the last cysteine ​​residue in the constant region of the light chain is substituted or absent.

[0014] In some specific embodiments, the first cysteine ​​residue in the hinge region is substituted, and the last cysteine ​​residue in the light chain constant region is substituted; or, the first cysteine ​​residue in the hinge region is substituted, and the last cysteine ​​residue in the light chain constant region is missing; or, the first cysteine ​​residue in the hinge region is missing, and the last cysteine ​​residue in the light chain constant region is substituted; or, the first cysteine ​​residue in the hinge region is missing, and the last cysteine ​​residue in the light chain constant region is missing.

[0015] In some implementations, the IgG antibody is a human IgG antibody.

[0016] In some embodiments, the substitution is any amino acid substitution other than cysteine.

[0017] In some specific embodiments, any amino acid that is not cysteine ​​is selected from amino acids without side chains, amino acids with short side chains, and their derivatives; the side chain of the amino acid with short side chains contains 1-2 carbon atoms.

[0018] In some embodiments, any amino acid of the noncysteine ​​is selected from glycine, alanine, serine, threonine and their derivatives.

[0019] In some specific embodiments, any amino acid of the non-cysteine ​​is serine or a serine derivative.

[0020] In some specific embodiments, the hinge region comprises the amino acid sequence EPKSX1DKTHTCPPCP (SEQ ID NO:13), wherein X1 is any amino acid residue other than cysteine ​​or none.

[0021] In some specific implementations, when the light chain constant region is a kappa chain, the light chain constant region corresponds to any non-cysteine ​​amino acid residue or deletion at C235 of the wild-type kappa chain; when the light chain constant region is a λ chain, the light chain constant region corresponds to any non-cysteine ​​amino acid or deletion at C234 of the wild-type λ chain.

[0022] In some specific implementations, the light chain constant region and the heavy chain constant region use IMGT numbering or EU numbering rules.

[0023] In some specific embodiments, the hinge region contains an amino acid sequence as shown in SEQ ID NO:14 or 15.

[0024] In some specific implementations, the C235 or C234 of the light chain constant region is mutated to serine or absent.

[0025] In some implementations, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 or IgG4.

[0026] In some specific implementations, the heavy chain constant region is the heavy chain constant region of IgG1.

[0027] In some specific embodiments, the light chain constant region contains an amino acid sequence as shown in SEQ ID NO:18 or 19.

[0028] In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in any one of SEQ ID NO:16-17 or 36.

[0029] In some embodiments, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:18 or 19.

[0030] In some specific embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:16, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:16, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19.

[0031] In some embodiments, the IgG antibody further comprises an Fv region that specifically binds to an antigen selected from the group consisting of: CEACAM5, DR5, ROR1, CDH17, EGFR, HER2, HER3, BCMA, B7-H3, CEA, CEACAM6, and claudin. 18.2, c-MET, folate receptor, CD3, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD48, CD56, CD70, CD73, CD74, CD79b, CD98, CD138, CD309 (VEGFR2), collagen IV, endothelin receptor ETB, ENPP3, fibronectin extradomain B, GCC, GPNMB, LIV-1 (ZIP6), MUC1, MUC16, mesothelin, NaPi2b, cohesin 4, p-cadherin, periostealin, PSMA, SC-16 (anti-Fyn3), SLC44A4, SLTRK6, STEAP1, tendinin c, tissue factor, Trop2 and 5T4 (TPBG).

[0032] In some specific implementations, the antigen is HER2 or B7-H3.

[0033] In some specific embodiments, the Fv region comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:20-22, and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23-25; or, the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:26-28, and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:29-31; the HCDR and LCDR are defined according to Kabat numbering.

[0034] In some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:32 or having at least 80% sequence identity with SEQ ID NO:32, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:33 or having at least 80% sequence identity with SEQ ID NO:33; or, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:34 or having at least 80% sequence identity with SEQ ID NO:34, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:35 or having at least 80% sequence identity with SEQ ID NO:35.

[0035] In some embodiments, the heavy chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1; and the light chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0036] In some embodiments, the heavy chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3; and the light chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0037] In some embodiments, the heavy chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5; and the light chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6.

[0038] In some embodiments, the heavy chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and the light chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0039] In some embodiments, the heavy chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9; and the light chain of the IgG antibody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10. In some specific embodiments, the heavy chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:1; and the light chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:2.

[0040] In some specific embodiments, the heavy chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:3; and the light chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:4.

[0041] In some specific embodiments, the heavy chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:5; and the light chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:6.

[0042] In some specific embodiments, the heavy chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:7; and the light chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:8.

[0043] In some specific embodiments, the heavy chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:9; and the light chain of the IgG antibody comprises the amino acid sequence shown in SEQ ID NO:10.

[0044] A second aspect of the present invention provides a nucleic acid molecule that encodes the IgG antibody described in the first aspect.

[0045] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0046] In some implementations, the recombinant expression vector is selected from viral vectors and non-viral vectors.

[0047] In some specific implementations, the non-viral vector is selected from plasmids, linear DNA fragments, and RNA.

[0048] In some specific implementations, the recombinant expression vector is a plasmid.

[0049] A fourth aspect of the invention provides a transformant comprising, in a host cell, a nucleic acid molecule as described in the second aspect and / or a recombinant expression vector as described in the third aspect.

[0050] In some implementations, the host cell is a eukaryotic cell or a prokaryotic cell.

[0051] In some embodiments, the eukaryotic cells are yeast cells or mammalian cells.

[0052] In some specific implementations, the mammalian cells are HEK293 cells.

[0053] A fifth aspect of the present invention provides a method for preparing IgG antibodies, comprising culturing a transformant as described in the fourth aspect and obtaining IgG antibodies from the culture.

[0054] In some embodiments, the method comprises substituting and / or deleting cysteine ​​residues that form interchain disulfide bonds in wild-type IgG antibodies to form IgG antibodies as described in the first aspect.

[0055] A sixth aspect of the invention provides an antibody-drug conjugate, a solvate thereof, or a pharmaceutically acceptable salt thereof, said antibody-drug conjugate comprising an IgG antibody as described in the first aspect, and a cytotoxic drug or tag conjugated to said IgG antibody.

[0056] In some embodiments, the cytotoxic drug is selected from DNA synthesis inhibitors, RNA synthesis inhibitors, structural protein inhibitors such as microtubule inhibitors, immunomodulators, and DNA damaging agents.

[0057] In some embodiments, the DNA synthesis inhibitor is selected from DNA alkylating agents, topoisomerase I inhibitors, and topoisomerase II inhibitors; the RNA synthesis inhibitor is selected from RNApoIII inhibitors; the tubulin inhibitor is selected from microtubule destabilizers, tubulin polymerization inhibitors, and tubulin elongation inhibitors; the immunomodulator is a cytokine; and the DNA damaging agent is a radionuclide.

[0058] In some specific embodiments, the DNA alkylating agent is selected from chachiin, ducamycin, amprolium-like PBD, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, and nerabine; the topoisomerase I inhibitor is selected from camptothecin and its derivatives, eczemacium EXD and its derivatives, and DXD; the camptothecin and its derivatives are preferably camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, and rubotecan; the topoisomerase II inhibitor is selected from PNU-159682, actinomycetamide, and other similar products. The following are listed: amycin D, doxorubicin, doxorubicin, docalamicin, daunorubicin, mitoxantrone, podophyllotoxin, and etoposide; the RNApoIII inhibitor is selected from α-amanitin; the microtubule destabilizer is selected from saurus toxins such as MMAE and MMAF, maytansine such as DM1, DM4, and tubulysin; the microtubule polymerization inhibitor is selected from vinca alkaloids such as vincristine and vinblastine, paclitaxel, docetaxel, and carbazide; the microtubule elongation inhibitor is selected from spongiform alkaloids and eribulin; the cytokines are selected from interleukins, tumor necrosis factor, and chemokines; the radionuclides are selected from... 18 F, 32 P, 47 Sc、 55 Co、 60 Cu、 61 Cu、 62 Cu、 64 Cu、 66 Ga、 67 Cu、 67 Ga、 68 Ga、 82 Rb、 86 Y、 87 Y、 89 Sr, 89Zr, 90 Y、 99 mTc, 105 Rh、 111 Ag、 111 In、 117 mSn, 124 I, 125 I, 131 I, 149 Pm, 153 Sm、 166 Ho、 169 Er、 177 Lu、 186 Re、 188 Re、 201 Tl、 211 At、 212 Bi、 223 Ra、225 Ac and 227 Th.

[0059] In some embodiments, the IgG antibody is conjugated to the cytotoxic drug via a chemical linker comprising a component selected from the group consisting of: 6-maleimide hexanoyl (MC), methanesulfonylpyrimidine, maleimide propionyl (MP), valine-citrulline (Val-Cit), valine-alanine (Val-Ala), GGFG, alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), 6-maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PAB), and Mal-PEG. n -Val-Cit-PAB (n=1-20), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP and perfluorophenyl 3-(pyridin-2-yldithio)propionic acid.

[0060] In some specific embodiments, the antibody-drug conjugate has the following structure:

[0061] Ab-LD;

[0062] The Ab is an IgG antibody, which comprises a heavy chain and a light chain.

[0063] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:1; the light chain comprises the amino acid sequence shown in SEQ ID NO:2; or

[0064] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:3; the light chain comprises the amino acid sequence shown in SEQ ID NO:4; or

[0065] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:5; the light chain comprises the amino acid sequence shown in SEQ ID NO:6; or

[0066] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:7; the light chain comprises the amino acid sequence shown in SEQ ID NO:8; or

[0067] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:9; the light chain comprises the amino acid sequence shown in SEQ ID NO:10;

[0068] D is a cytotoxic drug, and the cytotoxic drug is MMAE, DM1, or EXD;

[0069] L is a connector, and the connector is MC-VC or MC-GGFG.

[0070] A seventh aspect of the invention provides a formulation comprising an antibody-drug conjugate as described in the sixth aspect, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the number of molecules of the antibody-drug conjugate having a drug-antibody ratio (DAR) of 4 accounts for at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total number of molecules.

[0071] An eighth aspect of the present invention provides a method for producing antibody-drug conjugates, their solvates, or pharmaceutically acceptable salts thereof, wherein at least 90% of the antibody-drug conjugates have a DAR of 4; the method comprises:

[0072] The interchain disulfide bonds in the IgG antibody as described in the first aspect are completely reduced, and the drug molecule is linked through the free thiol groups after reduction.

[0073] A ninth aspect of the present invention provides a pharmaceutical composition or a kit containing the same, said pharmaceutical composition or kit containing the same comprising an IgG antibody as described in the first aspect, or an antibody-drug conjugate, a solvate thereof, or a pharmaceutically acceptable salt thereof as described in the sixth aspect, and a pharmaceutically acceptable carrier.

[0074] The tenth aspect of the present invention provides the use of IgG antibodies as described in the first aspect, antibody-drug conjugates as described in the sixth aspect, solvates thereof or pharmaceutically acceptable salts thereof, or pharmaceutical compositions or cassettes containing such conjugates as described in the ninth aspect, in the preparation of medicaments for the prevention and / or treatment of diseases.

[0075] In some implementations, the disease is a tumor.

[0076] In some specific implementations, the tumor is a hematoma or a solid tumor.

[0077] In some specific implementations, the solid tumor is gastric cancer.

[0078] The eleventh aspect of the present invention provides IgG antibodies as described in the first aspect, antibody-drug conjugates as described in the sixth aspect, solvates thereof or pharmaceutically acceptable salts thereof, or pharmaceutical compositions or kits containing the same as described in the ninth aspect, for the prevention and / or treatment of diseases.

[0079] In some implementations, the disease is a tumor.

[0080] In some specific implementations, the tumor is a hematoma or a solid tumor.

[0081] In some specific implementations, the solid tumor is gastric cancer.

[0082] The twelfth aspect of the present invention provides a method for preventing and / or treating a disease, the method comprising administering to a subject in need an effective amount of an IgG antibody as described in the first aspect, an antibody-drug conjugate as described in the sixth aspect, a solvate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the ninth aspect, or a cassette containing such a composition.

[0083] In some implementations, the disease is a tumor.

[0084] In some specific implementations, the tumor is a hematoma or a solid tumor.

[0085] In some specific implementations, the solid tumor is gastric cancer.

[0086] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0087] The reagents and raw materials used in this invention are all commercially available.

[0088] The significant advancements of this invention lie in its design of an antibody structure capable of site-specific conjugation into a uniform DAR4 ADC. Compared to traditional conjugation methods that suffer from ADC heterogeneity, site-specific conjugation technology ensures uniform DAR values, high homogeneity, and minimal batch-to-batch variation, making it suitable for large-scale production. Attached Figure Description

[0089] Figure 1 is a schematic diagram of the engineered antibody structure.

[0090] Figures 2-6 show the SEC-HPLC chromatograms of recombinant proteins 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4. Figure 2 shows 27B4-01; Figure 3 shows TLS4; Figure 4 shows TLS4-2; Figure 5 shows TLS4-3; and Figure 6 shows TLS4-4.

[0091] Figures 7-12 are HIC spectra of the ADCs prepared from the engineered antibodies of this invention, wherein a1: 27B4-01, a2: 27B4-01-MMAE, a3: 27B4-01-DM1, a4: 27B4-01-EXD; b1: TLS4, b2: TLS4-MMAE, b3: TLS4-DM1, b4: TLS4-EXD; c1: TLS4-2, c2: TLS4-2-MMAE, c3: TLS4-2-DM1, c4: TLS4-2-EXD; d1: TLS4-3, d2: TLS4-3-MMAE, d3: TLS4-3-DM1, d4: TLS4-3-EXD; e1: TL S4-4, e2: TLS4-4-MMAE, e3: TLS4-4-DM1, e4: TLS4-4-EXD; f1: trastuzumab, f2: trastuzumab-MMAE.

[0092] Figure 13 shows the binding curves of different engineered antibodies and control antibodies with HER2 according to embodiments of the present invention.

[0093] Figure 14 shows the cytotoxicity curve of the engineered ADC against NCI-N87 human gastric cancer cells according to an embodiment of the present invention. Detailed Implementation

[0094] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0095] As used herein, the term "antibody" is used in the broadest sense, encompassing monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv), conventional antibodies (tetrapeptide chains consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), and antibodies with antigen-binding activity such as Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, and domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies). Conventional antibodies (also called "full-length antibodies" or "complete antibodies") are typically heterotetraglycoproteins of approximately 150,000 Daltons, consisting of a tetrapeptide chain consisting of two identical light chains (L) and two identical heavy chains (H) linked by interchain disulfide bonds. Each heavy chain of a full-length antibody consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are divided into α, δ, ε, γ, and μ heavy chains. Mammalian light chains are divided into λ or kappa light chains. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, the fourth constant region) linked together, with disulfide bonds (interchain) formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain that binds to a single light chain and a first constant region (CH1). The "Fc" region consists of two heavy chain segments containing the CH2 and CH3 domains of the antibody, held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0096] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are efficiently linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), also serve equivalent functions.

[0097] The term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0098] The term "host cell" refers to a cell in which the vector can proliferate and whose DNA can be expressed; said cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the tested host cell. It should be understood that not all progeny are identical to the parent cell, as mutations can occur during replication; such progeny are included.

[0099] The term "antibody-drug conjugate (ADC)" refers to a targeted biologic agent that conjugates a highly specific and affinity monoclonal antibody to a highly cytotoxic drug via a specific linker. This structural design allows ADCs to efficiently deliver the cytotoxic drug to target tumor cells, thereby exerting an anti-tumor effect. ADCs typically contain three main components: an antibody (Ab), a linker, and a cytotoxic drug. The antibody acts as a carrier, specifically recognizing and binding to antigens on the surface of tumor cells. The linker is responsible for linking the antibody and the cytotoxic drug; it needs to be stable after reaching tumor cells but can be cleaved inside the cells by specific enzymes or conditions (such as low pH) to release the cytotoxic drug. The cytotoxic drug is responsible for killing tumor cells and is usually a small molecule drug with high cytotoxicity.

[0100] The term "linker" refers to the molecular part that forms a covalent link between an antibody and a small molecule payload (cytotoxic drug), and is a key element with design properties in targeted drug therapy. The main function of the linker is to remain stable in the bloodstream to avoid premature release of the cytotoxic drug, thereby reducing damage to normal cells; at the same time, after reaching tumor cells, the linker needs to be able to be cleaved by specific enzymes or conditions (such as low pH) to release the cytotoxic drug and exert a therapeutic effect. Among them, linkers can be divided into two categories according to their release mechanism: (1) Cleavable linkers: These linkers are designed to be cleaved by specific intracellular conditions (such as low pH, enzymes, or glutathione) after reaching the target cells, thereby releasing the cytotoxic drug. The cleavage of cleavable linkers is usually triggered by proteases, glutathione reduction, or acidic pH. This design allows for the release of drugs within tumor cells while reducing the impact on normal cells. The advantage of cleavable linkers is that they can improve drug selectivity, but they may have poor stability in the bloodstream; (2) Uncleavable linkers: These linkers are very stable in the bloodstream and need to enter the cell through endocytosis and then be degraded in lysosomes to release the active molecules. The advantage of uncleavable linkers is that they have a longer half-life and lower off-target toxicity in the blood, but they may require higher internalization efficiency to ensure effective drug release.

[0101] The term "DAR" refers to the Drug-to-Antibody Ratio, a key quality attribute of antibody-drug conjugates (ADCs). DAR is defined as the average number of drug molecules linked to an antibody molecule. This ratio directly impacts the efficacy and safety of ADCs. A high DAR value means each antibody carries more drug molecules, typically enhancing cell-killing effects. However, excessively high DAR can lead to ADC molecule instability, increasing the risk of drug detachment and potentially causing side effects. A low DAR value means each antibody carries fewer drug molecules, potentially requiring a higher dose to achieve the same efficacy, but generally improving ADC stability and safety. Methods for determining DAR include liquid chromatography-mass spectrometry (LC-MS), gel filtration chromatography (GPC), and ultraviolet-visible spectroscopy (UV-Vis Spectroscopy). These techniques are used to accurately measure the number of drug molecules linked to antibody molecules or to infer the DAR value through absorption at specific wavelengths. In the development of antibody-drug conjugates (ADCs), a reasonable drug absorption rate (DAR) plays a crucial role in the drug's efficacy, toxicity, pharmacokinetic properties, and clinical application. An appropriate DAR ensures that the ADC releases sufficient drug molecules into specific target cells while maintaining antibody stability, thereby reducing damage to healthy cells and minimizing side effects.

[0102] The term "hinge region" refers to a region in an immunoglobulin molecule located between the CH1 and CH2 functional regions of the heavy chain. The hinge region is rich in proline, thus possessing high flexibility, allowing the two Fab segments (antigen-binding fragments) of an antibody to have some mobility when binding to the antigen, thereby enabling better complementary binding to the two antigenic epitopes.

[0103] The term "derivative" refers to a new compound obtained by altering the chemical structure of the original compound. This alteration can involve adding, removing, or replacing atoms or groups of atoms in the original molecule.

[0104] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0105] Example 1: Design and production of recombinant proteins 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4

[0106] The schematic diagram of the engineered antibody structure of this invention is shown in Figure 1. Plasmids (pcDNA3.4 plasmids) encoding recombinant proteins 27B4-01 (amino acid sequences of the heavy and light chains are shown in SEQ ID NO:1 and 2, respectively), TLS4 (amino acid sequences of the heavy and light chains are shown in SEQ ID NO:3 and 4, respectively), TLS4-2 (amino acid sequences of the heavy and light chains are shown in SEQ ID NO:5 and 6, respectively), TLS4-3 (amino acid sequences of the heavy and light chains are shown in SEQ ID NO:7 and 8, respectively), and TLS4-4 (amino acid sequences of the heavy and light chains are shown in SEQ ID NO:9 and 10, respectively) were constructed and expressed. HEK293 cells (Gibco) were transiently transfected with the plasmids. One-step purification using a Protein A affinity column yielded sufficient quantities of recombinant proteins 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4. The antibodies prepared in this form showed good expression and high yield, and can be purified to high purity using a one-step Protein A purification process. The purity and yield of recombinant proteins 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4 were detected and identified by SEC-HPLC (Thermo Fisher, Vanquish F) and NanoDrop (Merinton, SMA4000), and the results are shown in Figures 2-6 and Table 1. It can be seen that the recombinant proteins have high stability, and stable and homogeneous recombinant proteins can be obtained according to conventional methods.

[0107] Table 1. Purity and yield of recombinant proteins 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4

[0108] Example 2: Coupling to generate ADC

[0109] 1. Production of DAR4 ADC

[0110] Antibodies 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4 were subjected to ultrafiltration replacement. The buffer was replaced with EDTA / DPBS (Sigma), and TCEP reducing agent (Sigma) was added. The reaction was carried out at room temperature for 3-4 hours. DM1 (Lianning) containing a maleimide (MC) linker, MMAE (MCE) containing an MC-VC linker, or EXD (MCE) containing an MC-GGFG (SEQ ID NO:37) linker were added. The reaction was carried out at room temperature for 1 hour. The buffer was replaced 5 times using a 30KD ultrafiltration tube (Amicon) to remove excess small molecules. The resulting product was the ADC (DAR4).

[0111] 2. Coupling Result Analysis

[0112] The coupling results were analyzed using HIC (Thermo Fisher, model Q Exactive Plus).

[0113] As shown in Figures 7-12, after 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4 were conjugated with small molecule toxins, the elution times of the antibodies and ADCs differed. The elution times of the ADCs were all later than those of the corresponding antibodies, proving that the antibodies were conjugated with small molecule toxins. Moreover, compared with the average DAR4, 27B4-01, TLS4, TLS4-2, TLS4-3, and TLS4-4, after being conjugated with small molecule toxins, could form a uniform DAR4 ADC.

[0114] Example 3: Identification of ADC coupling sites and DAR values

[0115] The coupling sites and DAR values ​​of the above ADCs were identified using LC-MS.

[0116] ADC samples were added to denaturing buffer, denatured with DTT (Sigma), and then alkylated with IAM (Sigma). The buffer was replaced with enzymatic digestion buffer using a NAP-5 desalting column (GE Healthcare). A suitable amount of Trypsin (Promega) was added, and the enzyme was digested at 37°C for 4 hours. The reaction was terminated by acidification with TFA (Sigma). The peptide samples after enzyme digestion were analyzed using liquid chromatography-mass spectrometry (Tables 2 and 3).

[0117] The analysis results show that, by using the method of producing DAR4 ADC in Example 2, ADCs with a uniform DAR value of 4 were obtained.

[0118] Table 2 ADC Coupling DAR Value Results

[0119] Table 3. Results of ADC coupling site identification

[0120] The positions of each point are numbered according to the amino acid numbering system of IMGT, referring to human IgG1 (kappa) (Figure 1).

[0121] Example 4: Combining activity detection

[0122] Human HER2 protein (1000 ng / mL) (Acro) was coated onto a 96-well plate and incubated overnight at 4°C. Antibodies such as TLS4 and their ADC samples were serially diluted 5-fold starting at 5000 ng / mL, for a total of 8 dilutions. The diluted samples were then transferred to the HER2-coated plate and incubated at room temperature for 2 hours. A colorimetric reaction was performed using HRP-labeled anti-human IgG Fc (Sigma) as the detection antibody and TMB (Sino Biological Inc). Absorbance at 450 / 650 nm was read using a microplate reader (Molecular Devices, SpectraMax i3X), and a dose-response curve was fitted using a four-parameter equation for data analysis.

[0123] As shown in Figure 13, antibodies such as TLS4 and their ADCs can bind to human HER2 protein, and the conjugation of small molecules does not affect the binding activity of the antibodies to human HER2 protein.

[0124] Example 5: Cytotoxicity Detection

[0125] NCI-N87 human gastric cancer cells (from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were seeded into 96-well cell culture plates. Serially diluted TLS4 and other ADCs, along with trastuzumab-conjugated MMAE to form the mean DAR4 ADC, were added. The plates were cultured at 37°C and 5% CO2 for 5 days. Then, CCK-8 reagent (Baiying Biotechnology) was added, and absorbance at 450 / 650 nm was measured using a SpectraMax microplate reader. The cytotoxicity of the TLS4 and other ADCs combined with the trastuzumab-conjugated mean DAR4 ADC on tumor cells was assessed by detecting the number of viable cells.

[0126] As shown in Figure 14, the average DAR4 ADC conjugated with TLS4 and other ADCs and trastuzumab was cytotoxic to NCI-N87 human gastric cancer cells, and the cytotoxicity results were almost identical, indicating that conjugating small molecule toxins with modified antibodies such as TLS4 does not change the tumor-suppressive activity of ADCs.

[0127] The partial sequences used in this invention are as follows:

[0128] 27B4-01:

[0129] Heavy chain

[0130] Light chain

[0131] TLS4:

[0132] Heavy chain

[0133] Light chain

[0134] TLS4-2:

[0135] Heavy chain

[0136] Light chain

[0137] TLS4-3:

[0138] Heavy chain

[0139] Light chain

[0140] TLS4-4:

[0141] Heavy chain

[0142] Light chain

[0143] Trastuzumab:

[0144] Heavy chain

[0145] Light chain

[0146] Hinge area

[0147] EPKSX1DKTHTCPPCP (SEQ ID NO:13), where X1 is any amino acid residue other than cysteine ​​or none.

[0148]

[0149] 27B4-01 Heavy Chain Constant Region

[0150] TLS4-2 and TLS4-3 relink constant regions

[0151] TLS4-3 and TLS4-4 light chain constant zone

[0152] 27B4-01, TLS4, TLS4-2 light chain constant zone

[0153] 27B4-01 HCDR 1-3

[0154] 27B4-01 LCDR 1-3

[0155] TLS4 / TLS4-1 / TLS4-2 / TLS4-3 / TLS4-4 HCDR 1-3

[0156] TLS4 / TLS4-1 / TLS4-2 / TLS4-3 / TLS4-4 HCDR 1-3

[0157] 27B4-01 Heavy Chain Variable Region

[0158] 27B4-01 Light Chain Variable Region

[0159] TLS4-TLS4-4 heavy chain variable region

[0160] TLS4-TLS4-4 light chain variable zone

[0161] TLS4, TLS4-4 heavy chain constant zone

[0162] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An IgG antibody, characterized in that, The cysteine ​​residues that form interchain disulfide bonds in the IgG antibody corresponding to those in the wild-type IgG antibody are substituted and / or missing; the IgG antibody lacks interchain disulfide bonds and free thiol groups.

2. The IgG antibody as described in claim 1, characterized in that, The IgG antibody comprises a light chain constant region and a heavy chain constant region, wherein the heavy chain constant region comprises a hinge region; The first cysteine ​​residue in the hinge region is substituted or missing; and / or, the last cysteine ​​residue in the light chain constant region is substituted or missing. Preferably, the first cysteine ​​residue in the hinge region is substituted, and the last cysteine ​​residue in the light chain constant region is substituted; or, the first cysteine ​​residue in the hinge region is substituted, and the last cysteine ​​residue in the light chain constant region is missing; or, the first cysteine ​​residue in the hinge region is missing, and the last cysteine ​​residue in the light chain constant region is substituted; or, the first cysteine ​​residue in the hinge region is missing, and the last cysteine ​​residue in the light chain constant region is missing.

3. The IgG antibody as described in claim 1 or 2, characterized in that, The IgG antibody is a human-derived IgG antibody; And / or, the substitution is any amino acid substitution other than cysteine; Preferably, any amino acid that is not cysteine ​​is selected from amino acids without side chains, amino acids with short side chains, and their derivatives; the side chain of the amino acid with short side chains contains 1-2 C atoms. More preferably, any amino acid of the noncysteine ​​is selected from glycine, alanine, serine, threonine and their derivatives; for example, any amino acid of the noncysteine ​​is serine or a serine derivative.

4. The IgG antibody according to any one of claims 1-3, characterized in that, The hinge region contains the amino acid sequence EPKSX1DKTHTCPPCP (SEQ ID NO:13), wherein X1 is any non-cysteine ​​amino acid residue or none; When the light chain constant region is a kappa chain, the light chain constant region corresponds to any non-cysteine ​​amino acid residue or deletion at C235 of the wild-type kappa chain; when the light chain constant region is a λ chain, the light chain constant region corresponds to any non-cysteine ​​amino acid or deletion at C234 of the wild-type λ chain. The light chain constant region and the heavy chain constant region are designated by IMGT.

5. The IgG antibody as described in claim 4, characterized in that, The hinge region contains an amino acid sequence as shown in SEQ ID NO:14 or 15; And / or, the C235 or C234 of the light chain constant region is mutated to serine or absent.

6. The IgG antibody according to any one of claims 1-3, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1; and / or, the light chain constant region contains an amino acid sequence as shown in SEQ ID NO:18 or 19; Preferably, the heavy chain constant region comprises an amino acid sequence as shown in any one of SEQ ID NO:16-17 or 36; More preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:16, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:16, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18; or, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

19.

7. The IgG antibody according to any one of claims 1-6, wherein the IgG antibody further comprises an Fv region, the Fv region specifically binding to an antigen, wherein the antigen is selected from the group consisting of: CEACAM5, DR5, ROR1, CDH17, EGFR, HER2, HER3, BCMA, B7-H3, CEA, CEACAM6, and claudin. 18.2, c-MET, folate receptor, CD3, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD48, CD56, CD70, CD73, CD74, CD79b, CD98, CD138, CD309 (VEGFR2), collagen IV, endothelin receptor ETB, ENPP3, fibronectin extradomain B, GCC, GPNMB, LIV-1 (ZIP6), MUC1, MUC16, mesothelin, NaPi2b, cohesin 4, p-cadherin, periostealin, PSMA, SC-16 (anti-Fyn3), SLC44A4, SLTRK6, STEAP1, tendinin c, tissue factor, Trop2 and 5T4 (TPBG); Preferably, the antigen is B7-H3 or HER2.

8. The IgG antibody as described in claim 7, characterized in that, The Fv region comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:20-22, and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23-25; or, the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:26-28, and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:29-31; HCDR and LCDR are defined according to Kabat numbering. Preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:32 or having at least 80% sequence identity with SEQ ID NO:32, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:33 or having at least 80% sequence identity with SEQ ID NO:33; or, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:34 or having at least 80% sequence identity with SEQ ID NO:34, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:35 or having at least 80% sequence identity with SEQ ID NO:35; More preferably, the IgG antibody comprises a heavy chain and a light chain, wherein, The heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1; the light chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:2; or The heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3; the light chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4; or The heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5; the light chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6; or The heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7; the light chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8; or The heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9; the light chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10; More preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:1; the light chain comprises the amino acid sequence shown in SEQ ID NO:2; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:3; the light chain comprises the amino acid sequence shown in SEQ ID NO:4; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:5; the light chain comprises the amino acid sequence shown in SEQ ID NO:6; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:7; the light chain comprises the amino acid sequence shown in SEQ ID NO:8; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:9; the light chain comprises the amino acid sequence shown in SEQ ID NO:

10.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the IgG antibody as described in any one of claims 1-8.

10. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 9; Preferably, the recombinant expression vector is selected from: viral vectors and non-viral vectors; More preferably, the non-viral vector is selected from: plasmids, linear DNA fragments, and RNA; The recombinant expression vector is, for example, a plasmid.

11. A transformant, characterized in that, The host cell contains the nucleic acid molecule as described in claim 9 and / or the recombinant expression vector as described in claim 10; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; More preferably, the eukaryotic cell is a yeast cell or a mammalian cell; The mammalian cells mentioned are, for example, HEK293 cells.

12. A method for preparing IgG antibodies, characterized in that, The process includes culturing the transformant as described in claim 11 and obtaining IgG antibodies from the culture; or The cysteine ​​residues that form interchain disulfide bonds in wild-type IgG antibodies are substituted and / or deleted to form IgG antibodies as described in any one of claims 1-8.

13. An antibody-drug conjugate, its solvate, or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate comprises an IgG antibody as described in any one of claims 1-8, and a cytotoxic drug or tag conjugated to the IgG antibody.

14. The antibody-drug conjugate, its solvate, or a pharmaceutically acceptable salt thereof as described in claim 13, characterized in that, The cytotoxic drugs are selected from DNA synthesis inhibitors, RNA synthesis inhibitors, structural protein inhibitors such as microtubule inhibitors, immunomodulators, and DNA damage agents. Preferably, the DNA synthesis inhibitor is selected from DNA alkylating agents, topoisomerase I inhibitors, and topoisomerase II inhibitors; the RNA synthesis inhibitor is selected from RNApoIII inhibitors; the tubulin inhibitor is selected from microtubule destabilizers, tubulin polymerization inhibitors, and tubulin elongation inhibitors; the immunomodulator is a cytokine; and the DNA damaging agent is a radionuclide. More preferably, the DNA alkylating agent is selected from chachiin, ducamycin, atrazodone-type PBD, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, and nerabine; the topoisomerase I inhibitor is selected from camptothecin and its derivatives, eczemacium excreta and its derivatives, and DXD; the camptothecin and its derivatives are preferably selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, and rubotecan; the topoisomerase II inhibitor is selected from PNU-159682, actinomycin D, Doxorubicin, doxorubicin, docalimicin, daunorubicin, mitoxantrone, podophyllotoxin, and etoposide; the RNApoIII inhibitor is selected from α-amanitin; the microtubule destabilizer is selected from saurus toxins such as MMAE and MMAF, maytansine such as DM1, DM4, and tubulysin; the microtubule polymerization inhibitor is selected from vinca alkaloids such as vincristine and vinblastine, paclitaxel, docetaxel, and carbazide; the microtubule elongation inhibitor is selected from spongiform alkaloids and eribulin; the cytokines are selected from interleukins, tumor necrosis factor, and chemokines; the radionuclides are selected from... 18 F, 32 P, 47 Sc、 55 Co、 60 Cu、 61 Cu、 62 Cu、 64 Cu、 66 Ga、 67 Cu、 67 Ga、 68 Ga、 82 Rb、 86 Y、 87 Y、 89 Sr, 89Zr, 90 Y、 99 mTc, 105 Rh、 111 Ag、 111 In、 117 mSn, 124 I, 125 I, 131 I, 149 Pm, 153 Sm、 166 Ho、 169 Er、 177 Lu、 186 Re、 188 Re、 201 Tl、 211 At、 212 Bi、 223 Ra、 225 Ac and 227 Th.

15. The antibody-drug conjugate, its solvate, or a pharmaceutically acceptable salt thereof as described in claim 13 or 14, characterized in that, The IgG antibody is conjugated to the cytotoxic drug via a chemical linker, the chemical linker comprising a group selected from the group consisting of: 6-maleimide hexanoyl (MC), methanesulfonylpyrimidine, maleimide propionyl (MP), valine-citrulline (Val-Cit), valine-alanine (Val-Ala), GGFG, alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), 6-maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG. n -Val-Cit-PAB (n=1-20), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP and perfluorophenyl 3-(pyridin-2-yldithio)propionic acid.

16. The antibody-drug conjugate, its solvate, or a pharmaceutically acceptable salt thereof as described in any one of claims 13-15, wherein, The antibody-drug conjugate has the following structure: Ab-LD; The Ab is an IgG antibody, which comprises a heavy chain and a light chain. The heavy chain comprises the amino acid sequence shown in SEQ ID NO:1; the light chain comprises the amino acid sequence shown in SEQ ID NO:2; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:3; the light chain comprises the amino acid sequence shown in SEQ ID NO:4; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:5; the light chain comprises the amino acid sequence shown in SEQ ID NO:6; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:7; the light chain comprises the amino acid sequence shown in SEQ ID NO:8; or The heavy chain comprises the amino acid sequence shown in SEQ ID NO:9; the light chain comprises the amino acid sequence shown in SEQ ID NO:10; D is a cytotoxic drug, and the cytotoxic drug is MMAE, DM1, or EXD; L is a connector, and the connector is MC-VC or MC-GGFG.

17. A formulation comprising the antibody-drug conjugate, its solvate, or a pharmaceutically acceptable salt thereof as described in any one of claims 13-16, wherein, The number of molecules with a drug-antibody ratio (DAR) of 4 in the antibody-drug conjugate accounts for at least 80% of the total number of molecules; preferably, the number of molecules with a DAR of 4 in the antibody-drug conjugate accounts for at least 90% of the total number of molecules; more preferably, the number of molecules with a DAR of 4 in the antibody-drug conjugate accounts for at least 95% of the total number of molecules.

18. A method for producing antibody-drug conjugates, their solvates, or pharmaceutically acceptable salts thereof, wherein, At least 90% of the antibody-drug conjugates have a DAR of 4; the method comprises: Completely reduce the interchain disulfide bonds in the IgG antibody as described in any one of claims 1-8, and link drug molecules through the free thiol groups after reduction.

19. A pharmaceutical composition or a medicine box containing the same, characterized in that, The pharmaceutical composition or the kit containing it comprises an IgG antibody as described in any one of claims 1-8, or an antibody-drug conjugate, a solvate thereof, or a pharmaceutically acceptable salt thereof as described in any one of claims 13-16, and a pharmaceutically acceptable carrier.

20. The use of the IgG antibody as described in any one of claims 1-8, the antibody-drug conjugate as described in any one of claims 13-16, its solvate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in claim 19, or a cassette containing the same, in the preparation of a medicament for the prevention and / or treatment of a disease; Preferably, the disease is a tumor; more preferably, the tumor is a hematologic malignancy or a solid tumor.

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