Engineered antibodies, antibody-drug conjugates and uses thereof

Abstract

The present invention provides an isolated IgG antibody comprising two identical heavy chains, each comprising a hinge region having an amino acid sequence including an additional cysteine ​​upstream of the two cysteines in the CPPCP sequence of a native IgG hinge region, and a CH1 domain including a cysteine ​​at position 142 according to the IMGT numbering scheme. The present invention also provides an ADC based on an antibody having this structure.

Description

Detailed Description of the Invention

[0001] <CROSS REFERENCE TO RELATED APPLICATIONS> This application claims priority to International Application PCT / CN2020 / 130409, filed November 20, 2020. This application incorporates by reference the entire disclosure of the above patent application.

[0002] [Background technology] Antibodies are important immune molecules against foreign pathogens. Advances in monoclonal antibody (mAb) technology have led to their widespread use in disease research, diagnosis, and treatment. Therapeutic use of first-generation mAbs (mainly monospecific bivalent mAbs) has been successful in treating a variety of diseases, including cancer, autoimmune diseases, and infectious diseases. However, many diseases, such as solid tumors, have demonstrated great resistance to antibody-based therapies.

[0003] Antibody Drug Conjugates (ADCs) are mAbs chemically linked to an active cytotoxic drug, and therefore offer the targeting specificity of mAbs and the cancer-killing potential of cytotoxic drugs. The ability to select specific mAb-cytotoxic drug combinations, and advances in mAb-drug conjugation, have offered new possibilities to target cancer while minimizing exposure of healthy tissues. As of 2019, Ado-trastuzumab emtansine (ADC), Kadcyla TM ), brentuximab vedotin (Adcetris TM ), inotuzumab ozogamicin (Besponsa TM ), gemtuzumab ozogamicin, Mylotarg TM ), polatuzumab vedotin-piiq (Polivy TM ), Enfortumab vedotin (PadcevTM ), and trastuzumab deruxtecan (Enhertu TM A total of seven ADCs have been approved by the FDA: 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) 26) 27) 28) 29) 30)

[0004] Conventional methods of conjugating drugs to antibodies can usually result in a heterogeneous mixture of ADCs. For example, cytotoxic drugs are usually conjugated to antibodies via surface-exposed lysines or cysteines (obtained by reducing interchain disulfide bonds) to generate different types of ADCs with different binding sites on the antibody and different drug-to-antibody ratios (DARs). Non-specific conjugation poses challenges in terms of conjugation process control, product characterization, and quality control of ADC products. Furthermore, different types of ADCs can have significantly different safety and efficacy profiles. Because the disulfide bond between the heavy chain of an IgG1 antibody and its paired light chain may be the preferred bond to be reduced in the conjugation process, the resulting types of ADCs with light chain-drug conjugates may be less stable and more prone to losing their light chains during in vivo circulation.

[0005] In recent years, several site-specific ADC technologies have been developed to obtain more homogeneous ADCs. For example, specific amino acids (e.g., cysteine), unnatural amino acid residues (e.g., p-acetylphenylalanine (pAcF)), and short peptide tags that can be recognized and modified by enzymes have been engineered into selected sites of the antibody amino acid sequence. Others have attempted glycan-mediated conjugation using glycans attached to the heavy chain of the antibody.

[0006] Summary of the Invention One aspect of the invention provides an isolated IgG antibody comprising two identical heavy chains, each heavy chain comprising (a) a hinge region comprising the amino acid sequence -(X1)-C-(X2)-CPPCP-, where X1 is a polypeptide fragment having 0 to 7 amino acid residues, each independently selected from any non-cysteine ​​amino acid residue, and X2 is a polypeptide fragment having 2 to 7 amino acid residues, each independently selected from any non-cysteine ​​amino acid residue, and (b) a CH1 domain located upstream of and connected to the hinge region, the CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme. The antibody may further comprise two identical kappa light chains, each paired with a heavy chain.

[0007] The CH1 domain of the IgG antibody may have a sequence identical to that of a naturally occurring human IgG2, IgG3, or IgG4 subclass antibody, or the sequence of the CH1 domain of the IgG antibody may be identical to that of a naturally occurring human IgG1 antibody having an S142C mutation.

[0008] Each of the heavy chains may further comprise a CH2-CH3 domain located downstream of and connected to the hinge region of a native human IgG1, IgG2, IgG3, or IgG4 subclass antibody, the CH2-CH3 domain optionally comprising one or more mutations.

[0009] In some embodiments, the amino acid sequence of the hinge region is: (a) CKTHTCPPCP (SEQ ID NO: 1); (b) DKTCHTCPPCP (SEQ ID NO: 2); (c) ERKSCVECPPCP (SEQ ID NO: 3), (d) EPKSCDKTHTCPPCP (SEQ ID NO: 4), (e) EPKSDCKTHTCPPCP (SEQ ID NO: 5), (f) EPKSDKCTHTCPPCP (SEQ ID NO: 6), (g) EPKSDCKTHTVECPPCP (SEQ ID NO: 7), (h) EPKSDCKTVECPPCP (SEQ ID NO: 8), (i) EPKSDKCTHTVECPPCP (SEQ ID NO: 9), (j) EPPKSCDKTHTVECPPCP (SEQ ID NO: 10), (k) EPPKSDCKTHTVECPPCP (SEQ ID NO: 11), (l) EPPPKSCDKTHTVECPPCP (SEQ ID NO: 12), and (m) EPPPKSCDKTHTVECPPCP (SEQ ID NO: 13), and (n) EPPKSDCKTKTVECPPCP (SEQ ID NO: 28).

[0010] In some embodiments, the IgG antibody further comprises a Fab domain that specifically binds to an antigen, the antigen being EGFR, HER2, HER3, BCMA, B7H3, CEA, CEACAM6, 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 extra-domain B, or EGFR. B), GCC, GPNMB, LIV-1 (ZIP6), MUC1, MUC16, mesothelin, NaPi2b, nectin 4, p-Cadherin, periostin, PSMA, SC-16 (anti-Fyn3), SLC44A4, SLTRK6, STEAP1, tenascin c, tissue factor, Trop2 and 5T4 (TPBG).

[0011] In some embodiments, the IgG antibody is (a) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:14, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:23; (b) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 15, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 23; (c) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 16, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 17, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 23; (e) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 18, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 24; (f) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 19, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 25; (g) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:20, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:26; (h) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:21, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:25; (I) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:22, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:27; (j) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:29, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:31; and (k) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 30, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 32; The structure has one of the following structures:

[0012] The present disclosure further provides, in a further aspect, an antibody drug conjugate (ADC) comprising an antibody according to the invention conjugated to a cytotoxic drug via a chemical linker, or a pharma- ceutically acceptable salt thereof. In some embodiments, the cytotoxic agent is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, anthracycline, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, and the like. A), uncialamycin, amanitin, ricin, diphtheria toxin, eribulin (CAS), 131 I, interleukin, tumor necrosis factor, chemokine, irinotecan (SN38), exatecan, exatecan derivatives, and nanoparticles.

[0013] The chemical linker linking the antibody moiety and the cytotoxic agent may be cleavable or non-cleavable. In some embodiments, the linker comprises a PEG spacer region, where n is 1-20 (i.e., (CH2CHO) having 1-20 repeating units). In some embodiments, the chemical linker further comprises a linker fragment connected to the PEG spacer region. In some embodiments, the chemical linker comprises a linker fragment that does not include a PEG spacer region. In some embodiments, the linker fragment is 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG, n -Val-Cit-PAB (n=1 to 20), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridine-2-yldisulfanyl)propanoate, or a combination thereof.

[0014] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 21, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 25, and the cytotoxic agent is eribulin or MMAE.

[0015] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 19, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 25, and the cytotoxic agent is eribulin or MMAE.

[0016] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 20, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 26, and the cytotoxic agent is eribulin or MMAE.

[0017] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:29, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:31, and the cytotoxic agent is eribulin or MMAE.

[0018] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 30, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 32, and the cytotoxic agent is eribulin or MMAE.

[0019] In the formulation of the ADC or pharma- ceutically acceptable salt thereof, at least 80% of the ADC is conjugated to a chemical linker via a cysteine ​​on the heavy chain of the antibody. In some embodiments of the formulation, ADC molecules having a drug-to-antibody ratio (DAR) of 2 account for 60% or more of the total amount of ADC molecules.

[0020] A further aspect of the present disclosure provides an antibody drug conjugate (ADC) or a pharma- ceutically acceptable salt thereof comprising the reaction product of an IgG antibody according to the invention that has been subjected to at least partial reduction, such that at least some of the HH disulfide bonds between corresponding cysteines in the hinge region of the antibody are reduced to free sulfhydryls, and a chemical linker comprising a terminal thiol-reactive group, attached to a cytotoxic drug molecule.

[0021] A further aspect of the present disclosure provides a method for preparing an antibody drug conjugate (ADC) or a pharma- ceutically acceptable salt thereof, comprising at least partially reducing the HH disulfide bonds between corresponding cysteines in the hinge region of an IgG antibody according to the present invention, thereby obtaining free sulfhydryls, and reacting the sulfhydryls of the cysteines in the hinge region with a chemical linker comprising a terminal thiol-reactive group, thereby conjugating the antibody to one or more cytotoxic drug molecules via the chemical linker.

[0022] Description of the drawings FIG. 1 shows a schematic diagram of the amino acid numbering system of antibodies used in this application. (a) IMGT-based amino acid numbering system for human IgG1(κ). (b) Numbering system for mutants (in the case of the IgG1 hinge region).

[0023] Figure 2. Part A shows a schematic diagram of an engineered antibody according to an embodiment of the invention. Part B shows disulfide bonds in an IgG antibody according to an embodiment of the invention.

[0024] [Figure 3] Characteristics of the antibody of the embodiment of the present invention. (a) Purity and yield of the antibody produced in HEK293 cells. (b) SDS-PAGE analysis of reduced (R) and non-reduced (NR) antibodies. (c) SEC-HPLC analysis of the purified antibody.

[0025] Figure 4 shows HIC profiles of ADCs made from native human IgG1(κ), IgG2(κ) and engineered antibodies of the examples of the present invention. a: IgG1(κ)-MMAE, b: IgG2(κ)-MMAE, c: BB0500-2a-MMAE, d: BB0500-2b-MMAE, e: BB0500-2g-MMAE, f: BB0500-2n-MMAE, g: BR0301-MMAE and h: BR0302-MMAE.

[0026] Figure 5 shows CE-SDS profiles of ADCs of certain examples of the invention: a: IgG1(κ)-MMAE, b: IgG2(κ)-MMAE, c: BB0500-2a-MMAE, and d: BB0500-2g-MMAE.

[0027] FIG. 6 shows binding curves of different engineered antibodies according to embodiments of the invention and a control (wt) antibody to the proteins EGFR (a), HER3 (b), and HER2 (c).

[0028] FIG. 7 shows cytotoxicity curves of engineered ADCs according to embodiments of the invention against EGFR-expressing cancer cells (a: A431 cells, b: NUGC3 cells) and cytotoxicity curves of other engineered ADCs according to embodiments of the invention against HER2-expressing cancer cells (c: NCI-N87 (high HER2 expression), and d: A431 (low HER2 expression)).

[0029] [Specific embodiment] One aspect of the disclosure provides novel engineered antibodies, whose heavy chains comprise a CH1 domain with a cysteine ​​residue at or near amino acid 142, and whose hinge region comprises a sequence arranged in a three cysteine ​​pattern that can be considered as a sequence that includes a third or additional cysteine ​​residue located upstream of the two unique cysteines in the human IgG1 hinge region, said third cysteine ​​residue being referred to herein as the "HG3 cysteine."

[0030] Antibodies comprising this form of heavy chain preferably form an HH interchain disulfide bond between C142 (or a cysteine ​​at or near position 142, the site being defined as per the IMGT numbering system further described below) of the CH1 domain and the last cysteine ​​residue of the light chain. A cysteine ​​residue in the hinge region upstream of the native CPPCP sequence forms a third HH interchain disulfide bond. The cysteine ​​at or near amino acid position 142 of the CH1 domain may be introduced by mutation or insertion of a single amino acid of the IgG1 subtype, or may be a native cysteine ​​residue from the CH1 domain of the IgG2, IgG3, or IgG4 subtype. Compared to the native HH disulfide bond in IgG1 (located between the cysteine ​​in the hinge region of the IgG1 antibody heavy chain and the end of the light chain that pairs with it), this form of HH disulfide bond is more stable and can remain intact under reducing conditions during the drug conjugation process to the antibody. This greatly reduces the possibility of obtaining an ADC that contains a light chain drug conjugate.

[0031] The exact location of the third cysteine ​​in the hinge region and the amino acid sequence surrounding it may vary. The third inter-HH chain disulfide bond is more susceptible to reduction than the intrinsic inter-HH chain disulfide bond or the inter-HL chain disulfide bond. The reducible nature of the disulfide bond can facilitate site-specific drug conjugation. Selected reduction and drug conjugation conditions can be used to obtain site-specific drug conjugation primarily to the third cysteine ​​"HG3 cysteine".

[0032] The IMGT numbering system for the immunoglobulin superfamily is used herein to simplify the numbering system, where the VH or VL domain comprises amino acid residues 1-128, respectively. Thus, the amino acid numbering in the CH1 domain is aa 129-226, the kappa domain is aa 129-235, the hinge region is aa 227-241 (according to IgG1), CH2 is aa 242-351, and CH3 is aa 352-456 (see part a in FIG. 1). Based on this numbering system, the HL interchain disulfide bond in wild-type IgG1(κ) is formed between H(C231)-L(C235), whereas in IgG2(κ) (or IgG3(κ) or IgG4(κ)), it may be formed between H(C142)-L(C235). An IgG1 mutant in which the serine at position 230 of the heavy chain is changed to a cysteine ​​is designated IgG1(S230C), an IgG1 mutant in which C231 is deleted is designated IgG1(Δ231), an IgG1 mutant in which one lysine is inserted after C231 is designated K231.1, and an IgG1 mutant in which two amino acids, KL, are inserted after C231 is designated K231.1L231.2 (see part b of FIG. 1 for some symbolic examples of mutations in the hinge region of IgG1).

[0033] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. An isolated antibody that specifically binds to an antigen is substantially free of antibodies that do not bind to that antigen.

[0034] The term "monoclonal antibody" as used herein refers to a preparation having a population of antibody molecules consisting of substantially homogeneous molecules, where the individual antibodies within the population of antibody molecules are identical except for naturally occurring mutations that may be present in minor amounts.

[0035] In some embodiments, the disclosure provides an isolated IgG antibody comprising two identical heavy chains, each heavy chain comprising: (a) a hinge region comprising the amino acid sequence -(X1)-C-(X2)-CPPCP-, where X1 is a polypeptide fragment having 0 to 7 amino acid residues, each independently selected from any non-cysteine ​​amino acid residue, and X2 is a polypeptide fragment having 2 to 7 amino acid residues, each independently selected from any non-cysteine ​​amino acid residue; and (b) a CH1 domain upstream of and connected to the hinge region, the CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme.

[0036] The IgG antibody may further comprise two identical kappa light chains, the C-terminal cysteine ​​of which can form a disulfide bond with CH1 C142.

[0037] In some embodiments of the IgG antibody, the CH1 domain of the IgG antibody has a sequence identical to the CH1 domain of a native human IgG2, IgG3, or IgG4 subclass antibody.

[0038] In some embodiments of the IgG antibody, the sequence of the CH1 domain of the IgG antibody is identical to the sequence of the CH1 domain of a native human IgG1 antibody having an S142C mutation.

[0039] In some embodiments of the IgG antibodies, each of the heavy chains may further comprise the amino acid sequence of a CH2-CH3 domain located downstream and connected to the hinge region of a native human IgG1, IgG2, IgG3, or IgG4 subclass antibody.

[0040] In some embodiments, the amino acid sequence of the hinge region is: (a) CKTHTCPPCP (SEQ ID NO: 1); (b) DKTCHTCPPCP (SEQ ID NO: 2); (c) ERKSCVECPPCP (SEQ ID NO: 3); (d) EPKSCDKTHTCPPCP (SEQ ID NO: 4); (e) EPKSDCKTHTCPPCP (SEQ ID NO: 5); (f) EPKSDKCTHTCPPCP (SEQ ID NO: 6); (g) EPKSDCKTHTVECPPCP (SEQ ID NO: 7); (h) EPKSDC KTVECPPCP (SEQ ID NO: 8), (i) EPKSDKCTHTVECPPCP (SEQ ID NO: 9), (j) EPPKSCDKTHTVECPPCP (SEQ ID NO: 10), (k) EPPKSDCKTHTVECPPCP (SEQ ID NO: 11), (l) EPPPKSCDKTHTVECPPCP (SEQ ID NO: 12), (m) EPPPKSCDKTHTVECPPCP (SEQ ID NO: 13), and (n) EPPKSDCKTKTVECPPCP (SEQ ID NO: 28).

[0041] The antibody may further comprise a Fab domain that specifically binds to an antigen, the antigen being selected from the group consisting of EGFR, HER2, HER3, BCMA, B7H3, CEA, CEACAM6, 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 extra-domain B, GCC, GPNMB, LIV-1 (ZIP6), MUC1, MUC16, mesothelin, NaPi2b, nectin 4, and the like. 4), p-Cadherin, periostin, PSMA, SC-16 (anti-Fyn3), SLC44A4, SLTRK6, STEAP1, tenascin c, tissue factor, Trop2, and 5T4 (TPBG).

[0042] Examples of the sequences of the IgG antibodies include: (a) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:14, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:23; (b) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 15, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 23; (c) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 16, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 17, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 23; (e) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 18, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 24; (f) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 19, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 25; (g) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:20, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:26; (h) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:21, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:25; (I) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:22, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:27; (j) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:29, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:31; and (k) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO: 30, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO: 32; Includes:

[0043] DNA encoding amino acid sequence mutants of the starting polypeptide can be produced by a variety of methods known in the art. These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of the DNA encoding the initially produced polypeptide. Mutants of recombinant antibodies can also be constructed by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers code for cysteine ​​codon substitutions. Standard mutagenesis techniques can be employed to generate DNA encoding such mutant engineered antibodies.

[0044] A still further aspect of the present disclosure provides a nucleic acid molecule encoding an antibody according to the invention or an antigen-binding portion of any said antibody. Host cells (e.g., CHO cells, lymphocytic cells, human embryonic kidney cells or microorganisms (e.g., fungi such as E. coli and yeast) containing expression vectors comprising the nucleic acid molecules can be used to produce antibodies, preferably monoclonal antibodies, of the disclosure. In one embodiment, DNA encoding partial or full-length antibodies of the disclosure can be obtained by standard molecular biology techniques, where the gene is inserted into one or more expression vectors such that the gene is operably linked to transcriptional and translational control sequences. The term "operatively linked" is intended to mean that the antibody gene is linked to a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. See, e.g., Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) such control sequences are described. Preferred control sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)) and polyoma virus. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or the β-globin promoter.In addition, regulatory elements have been composed of sequences from different sources, such as the SV40 early promoter and the SRα promoter system, which contains sequences from the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected that are compatible with the expression host cell used.

[0045] DNA encoding an antibody can be inserted into the expression vector. The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The DNA encoding the antibody can be cloned into the vector such that the signal peptide is linked in-frame to the DNA encoding the amino terminus of the antibody. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin source).

[0046] The present disclosure further provides, in a further aspect, an antibody drug conjugate (ADC) comprising an antibody according to the invention conjugated to a cytotoxic drug via a chemical linker, or a pharma- ceutically acceptable salt thereof. The cytotoxic drugs include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, anthracycline, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, and the like. A), uncialamycin, amanitin, ricin, diphtheria toxin, eribulin, 131 I, interleukin, tumor necrosis factor, chemokine, irinotecan (SN38), exatecan, exatecan derivatives and nanoparticles.

[0047] The chemical linker linking the antibody moiety and the cytotoxic agent may be cleavable or non-cleavable. In some embodiments, the linker comprises a PEG spacer region, where n is 1-20 (i.e., (CH2CHO) having 1-20 repeating units). In some embodiments, the chemical linker further comprises a linker fragment connected to the PEG spacer region. In some embodiments, the chemical linker comprises a linker fragment that does not include a PEG spacer region. In some embodiments, the linker fragment is 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG, n -Val-Cit-PAB (n=1 to 20), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridine-2-yldisulfanyl)propanoate, or a combination thereof.

[0048] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 21, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 25, and the cytotoxic agent is eribulin or MMAE.

[0049] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 19, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 25, and the cytotoxic agent is eribulin or MMAE.

[0050] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 20, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 26, and the cytotoxic agent is eribulin or MMAE.

[0051] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:29, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:31, and the cytotoxic agent is eribulin or MMAE.

[0052] In a particular embodiment of the ADC, the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 30, the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 32, and the cytotoxic agent is eribulin or MMAE.

[0053] In this disclosure, pharma- ceutically acceptable salts of ADCs include acid addition salts of inorganic acids, carboxylic acids, and sulfonic acids, such as the salts of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid, and benzoic acid.

[0054] Pharmaceutically acceptable salts of the antibody-drug conjugates of the present disclosure further include salts of conventional bases, such as alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and ammonium salts derived from ammonia or organic amines containing from 1 to 16 carbon atoms, including, for example, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, diamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine, and 1,2-ethylenediamine.

[0055] ADC as used herein refers to a molecule that includes both a drug molecule and an antibody (or antigen-binding portion thereof), and it should be understood that the drug and antibody (or antigen-binding portion thereof) are covalently linked by a linker. An "ADC formulation" herein refers to a collection or population of ADC molecules that may differ in structure due to possibly different attachment sites of the chemical linker to the antibody (or antigen-binding portion thereof). In some embodiments, the chemical linker is conjugated primarily or predominantly (e.g., ≧80%, ≧85%, ≧90%, ≧95%, or ≧98%) to cysteines on the heavy chain, resulting in an ADC formulation that is substantially free of light chain conjugates. In some embodiments, the chemical linker is conjugated to the antibody primarily through cysteines in the hinge region of the antibody heavy chain. In certain embodiments, ADC molecules with a drug-antibody ratio (DAR) of 2 account for at least 60%, at least 70%, at least 80%, at least 85%, or at least 90% of the total amount of ADC molecules.

[0056] In a further embodiment, the ADC or a pharma- ceutically acceptable salt thereof is or comprises the reaction product of an antibody according to the invention that has been at least partially reduced, such that at least some of the HH disulfide bonds between corresponding cysteines in the hinge region of the antibody are reduced to free sulfhydryls, and a chemical linker that comprises a terminal thiol-reactive group attached to a cytotoxic drug molecule.

[0057] A further aspect of the present disclosure provides a method for preparing said ADC or a pharma- ceutically acceptable salt thereof, comprising at least partially reducing the HH disulfide bonds between corresponding cysteines in the hinge region of an antibody according to the invention, thereby obtaining free sulfhydryls, and reacting the sulfhydryls of the cysteines in the hinge region with a chemical linker comprising a terminal thiol-reactive group, thereby conjugating the antibody to one or more cytotoxic drug molecules via the chemical linker.

[0058] In a further aspect of the disclosure, a pharmaceutical composition is provided comprising one or more antibodies, one or more ADCs or pharma- ceutically acceptable salts thereof of the present invention, and a pharma- ceutically acceptable carrier. As used herein, "pharma- ceutically acceptable carrier" includes pharma- ceutical acceptable carriers, excipients, or stabilizers. These include, but are not limited to, physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending aids, solubilizing agents, colorants, flavoring agents, disintegrating agents, lubricants, sweeteners, preservatives, and isotonic agents. The selection of a suitable carrier is within the knowledge of one of ordinary skill in the art.

[0059] The composition may include one or more additional active pharmaceutical ingredients, such as another antibody and a drug (e.g., a cytotoxic or antitumor agent). The pharmaceutical compositions of the invention may also be administered in combination therapy, such as with another anti-cancer agent and another anti-inflammatory agent.

[0060] The pharmaceutical compositions may be applied for intravenous, intramuscular, subcutaneous, parenteral, epidermal, and other routes of administration. Depending on the route of administration, the active ingredient may be coated on a material or otherwise carried on a material or structure to protect it from the action of acids and other natural conditions which may inactivate it. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, compositions of the invention can be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration (eg, intranasal, oral, vaginal, rectal, sublingual or topical administration).

[0061] Working Example 1. Design of Engineered Antibodies The antibodies in Table 1 are designed and manufactured, where the CH1 domain of these antibodies contains a cysteine ​​residue at amino acid position 142. In some antibodies, the cysteine ​​at amino acid position 142 in the CH1 domain is introduced by a single amino acid mutation or insertion (S142C in Table 1 is a cysteine ​​replacing serine at position 142), and in other antibodies, C142 is derived from a natural cysteine ​​residue in the CH1 domain of the IgG2 subtype (designated G2CH1 in Table 1). Each antibody contains an artificial hinge sequence that contains a third cysteine ​​residue (HG3 cysteine) upstream of the CPPCP sequence in place of the natural hinge region. The amino acid sequence around the HG3 cysteine ​​residue can vary depending on the engineered antibody. By introducing a cysteine ​​residue at position 142 in CH1 of the engineered antibody, a HL interchain disulfide bond can be formed between C142 of the CH1 domain and C235 of the light chain. The HG3 cysteine ​​can pair with a cysteine ​​at the same position in another H chain, forming an extra HH interchain disulfide bond in addition to the intrinsic HH interchain disulfide bond (Figure 2).

[0062] [Table 1] JPEG0007627518000002.jpg223169JPEG0007627518000003.jpg11169

[0063] The structures and sequences of the engineered antibodies in the examples are set forth as follows: 1. BB0500-3 is produced in the form of an IgG2K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:3)-CH2CH3(IgG2) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-3 is shown in SEQ ID NO:14. The sequence of the light chain of BB0500-3 is shown in SEQ ID NO:23.

[0064] 2. BB0500-5 is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(S142C)-hinge(SEQ ID NO:4)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-5 is shown in SEQ ID NO:15. The sequence of the light chain of BB0500-5 is shown in SEQ ID NO:23.

[0065] 3. BB0500-2a is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:4)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-2a is shown in SEQ ID NO:16. The sequence of the light chain of BB0500-2a is shown in SEQ ID NO:23.

[0066] 4. BB0500-2b is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:5)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-2b is shown in SEQ ID NO:17. The sequence of the light chain of BB0500-2b is shown in SEQ ID NO:23.

[0067] 5. BB0500-6a is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:6)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-6a is shown in SEQ ID NO:18. The sequence of the light chain of BB0500-6a is shown in SEQ ID NO:24.

[0068] 6. BB0500-2f is produced in the form of an IgG2K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:9)-CH2CH3(IgG2) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-2f is shown in SEQ ID NO:19. The sequence of the light chain of BB0500-2f is shown in SEQ ID NO:25.

[0069] 7. BB0500-2g is produced in the form of an IgG2K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:10)-CH2CH3(IgG2) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-2g is shown in SEQ ID NO:20. The sequence of the light chain of BB0500-2g is shown in SEQ ID NO:26.

[0070] 8. BB0500-2n is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-EGFR)-CH1(IgG2)-hinge(SEQ ID NO:11)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0500-2n is shown in SEQ ID NO:21. The sequence of the light chain of BB0500-2n is shown in SEQ ID NO:25.

[0071] 9. BB0802 is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-HER3)-CH1(S142C)-hinge(SEQ ID NO:12)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BB0802 is shown in SEQ ID NO:22. The sequence of the light chain of BB0802 is shown in SEQ ID NO:27.

[0072] 10.BR0301 is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-HER2)-CH1(IgG2)-hinge(SEQ ID NO:28)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BR0301 is shown in SEQ ID NO:29. The sequence of the light chain of BR0301 is shown in SEQ ID NO:31.

[0073] 11.BR0302 is produced in the form of an IgG1K isotype, with its heavy chain comprising VH(anti-HER2)-CH1(IgG2)-hinge(SEQ ID NO:28)-CH2CH3(IgG1) and its light chain comprising VL-CL(K). The sequence of the heavy chain of BR0302 is shown in SEQ ID NO:30. The sequence of the light chain of BR0302 is shown in SEQ ID NO:32.

[0074] 2. Expression and Purification of Engineered Antibodies Codon optimization and gene synthesis were performed for the expression of the engineered antibodies. Specific full-length heavy and full-length light chain DNAs were cloned into separate pcDNA3 plasmids. The paired plasmids were used to transiently transfect HEK293 cells and one-step Protein A purification was performed to produce sufficient protein for detection. The antibodies produced in this manner showed good expression, high yields, and could be purified to high purity by a one-step Protein A purification process (parts a, b, and c in Figure 3).

[0075] 3. Profiles of antibody-drug conjugates made with native IgG1(κ) and IgG2(κ) Under mild reducing conditions (tris(2-chloroethyl)phosphate (TCEP):mAb=1-3, neutral pH, room temperature for <240 min), the interchain disulfide bonds of IgG antibodies can be partially reduced and conjugated to drug-linkers to form ADCs. Purified antibodies were conjugated to cytotoxic drugs via linkers (e.g., MMAE, DM1, or eribulin). Purified antibodies in phosphate buffer at neutral pH were partially reduced with TCEP to generate drug conjugates. Drug-linkers (MC-Val-Cit-PAB-MMAE or Mal-PEG2-Val-Cit-PAB-Eribulin) were added to the DMA and reacted with the antibody to obtain the desired drug-to-antibody ratio (DAR). For antibody and ADC characterization, hydrophobic interaction chromatography (HIC) was used to assess drug distribution and drug to antibody molar ratios in the ADC. CE-SDS was also performed on non-reduced ADCs to assess the proportion of non-covalently bound components in the ADC preparations, including free light chains (L), free heavy chains (H), half antibodies (HL), intact antibodies (LHHL), and antibodies lacking one or two light chains (HHL or HH).

[0076] The HIC profile of the IgG1(κ) ADC shows that very little naked anti-antibody (DAR0) remains at the end of the reaction period, and the resulting ADC product is a mixture of DAR2-8 species (Figure 4, part a). CE-SDS results of the non-reduced ADC product show the presence of a high percentage of free light chains (L, 18%), and structures lacking one or two light chains (20% + 18%), with a high percentage of conjugates occurring at cysteine ​​residues that form H-L interchain disulfide bonds (Figure 5, part a).

[0077] Conversely, under similar reduction and conjugation conditions, IgG2(κ) is more resistant to the reaction. The rate of opening of HL or HH interchain disulfide bonds is very low, and the majority of IgG2 molecules remain at DAR0 at the end of the reaction period (part b of Figure 4). The CE-SDS profile shows that there is a high percentage of intact antibody (LHHL, 89%) in the IgG2(κ) ADC product, and the level of free light chain (L, ~1%, representing light chain drug conjugate) is very low (part b of Figure 5). These results suggest that the disulfide bonds in IgG2(κ) molecules are much more stable than those in IgG1 antibodies.

[0078] 4. Conjugates of drugs and engineered antibodies The HIC profiles show that the ADC products based on the engineered antibodies (e.g., BB0500-2a and 2b in parts c and d of Figure 4) consist of a mixture of DAR0-10 species, where the DAR2 species accounts for the highest percentage (35-50%), followed by DAR0 (note that DAR0 is the antibody (or part thereof) that is not converted to an ADC in the conjugation process), DAR4, and DAR6 species (in the range of 10-25%). The DAR8-10 species account for a very small percentage in the mixture (<5%) (parts c and d of Figure 4). The CE-SDS results verified the overall reduction resistance of the engineered antibodies compared to IgG1(κ), especially the HL interchain disulfide bond of the free light chain (representing the bound light chain), which accounts for only ~2% (part c of Figure 5). Thus, the obtained ADC products consist mainly of conjugates containing only heavy chains.

[0079] 5. Site-selective drug conjugates ADCs produced from certain engineered antibodies of the present disclosure are predominantly DAR2 ADC species (approximately 60-70% as shown in parts e and f of FIG. 4, where the antibodies in the ADC are BB0500-2g and BB0500-2n, respectively, and approximately 70-80% as shown in parts g and h of FIG. 4, where the antibodies in the ADC are BR0301 and BR0302, respectively). It is believed that the sequence of the hinge region of these clones makes the HH interchain disulfide bond between the HG3 cysteines more amenable to reduction than the remaining interchain disulfide bonds, making the HG3 cysteine ​​the preferred or primary drug conjugation site. Antibodies containing different hinge amino acid sequences surrounding the HG3 cysteine ​​require different reduction and conjugation conditions to obtain predominantly DAR2 ADC products. The CE-SDS profiles show that very low levels (~1%) of free light chains (representing light chain-drug conjugates) are present in these ADC products (Figure 5, part d).

[0080] The disulfide bond pattern of the engineered antibody was assessed by trypsin digestion under non-reducing conditions using LC-MS and LC-MS / MS analysis. All disulfide bond linked peptides were identified by their molecular weight and sequence. The total sequence coverage was 100% when LC-UV and LC-MS detection were combined. The disulfide bond between the light and heavy chains, including Cys142, and the extra disulfide bond between the two heavy chains (derived from an additional Cys upstream of the CPPCP sequence in the hinge region) were verified. The other 14 disulfide bond linkages are consistent with typical IgG1-type disulfide bond linkages.

[0081] After conjugation, the conjugation site was determined by the characteristics of the late eluting peak in peptide mapping. The distribution of the peaks was based on the mass and MS / MS spectra of the observed conjugated peptides. The major conjugation site was determined to be an additional Cys residue upstream of the CPPCP sequence in the heavy chain hinge region. A low level of conjugation of the Cys residue in the CPPCP sequence in the hinge region was also detected. This is consistent with the predicted binding site of the interchain disulfide bond between the two heavy chains in the hinge region.

[0082] 6. Binding activity of different engineered antibodies against proteins EGFR, HER2 or HER3 ELISA method was used to detect and compare the EGFR, HER2 or HER3 binding ability between engineered antibodies and control wild type (wt) antibodies. Human EGFR, HER2 and HER3 proteins were coated on 96-well plates and the plates were incubated overnight. Engineered antibody samples or corresponding control antibodies were diluted. The diluted samples were then transferred to the protein-coated plates and incubated at room temperature. HRP-labeled goat anti-human IgG Fc antibody (Sigma) was used as the detection reagent, colorimetric reaction was performed with TMB, absorbance on the enzyme-labeled plate was read using a microplate reader (Molecular Devices, SpectraMax 190) at 450 / 650 nm, and data analysis was performed using a four parameter logistic model in the form of a dose-response curve.

[0083] The results in Figure 6 show that the engineered antibodies of different targets have similar binding activities compared to the corresponding controls, suggesting that the novel engineered antibodies do not affect the activity of the engineered antibodies against the target proteins.

[0084] 7. Cytotoxicity of ADCs made from engineered antibodies against EGFR high / low expressing cells To investigate the cytotoxicity of ADCs derived from engineered antibodies, in vitro cytotoxicity against target-expressing cancer cells was evaluated with a colorimetric-based cytotoxicity assay. To perform the assay, target cells were seeded into 96-well flat-bottom tissue culture plates at cell densities optimized for each cell line and incubated overnight (16-20 h) at 37 °C, 5% CO2. Serially diluted ADC samples were transferred to the cell plates, and the assay plates were incubated for a specific period (3-5 days, depending on the cell line) to achieve optimal killing effects. Data analysis was performed using a four-parameter logistic model of dose-response curves. The results in Figure 7 show that eribulin-containing EGFR-targeted ADCs produced from the engineered antibody moieties of BB0500-2f, 2g, or 2n exert effective cytotoxicity against EGFR-high expressing cells (A431 cells, part a of Figure 7) and EGFR-low expressing cells (NUGC3 cells, part b of Figure 7). Similarly, the results of MMAE-containing HER2-targeting ADCs containing the antibody moiety of BB0301 or BB0302 show that they exert effective cytotoxicity against HER2-high expressing cells (NCI-N87 cells, part c of Figure 7) and HER2-low expressing cells (A431 cells, part d of Figure 7).

[0085] Sequence List SEQ ID NOs: 1-13 and 28 are provided in Table 1.

[0086] SEQ ID NO:14 (BB0500-3 heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQ GTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:15 (BB0500-5 heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG TMVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:16 (BB0500-2a heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:17 (BB0500-2b heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSDCK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:18 (BB0500-6a heavy chain): QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSDDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSDKC THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:19 (BB0500-2f heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNANYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSDKCT HTVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:20 (BB0500-2g heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVAGAFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPPKSCDK THTVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:21 (BB0500-2n heavy chain): QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNANYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPPKSDCKT HTVECPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:22 (BB0802 heavy chain): EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMGWVRQAPGKGLEWVSYISPSGGHTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLETGLVDAFDIWGQ GTMVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPPPKSCD KTHTVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:23 (light chain): DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:24 (light chain): DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:25 (light chain): DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:26 (light chain): DIQMTQSPSSLSASVGDRVTITCQASQDISNALNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:27 (light chain): QYELTQPPSVSVYPGQTASITCSGDQLGSKFVSWYQQRPGQSPVLVMYKDKRRPSEIPERFSGSNSGNTATLTISGTQAIDEADYYCQAWDSSTYVFGTGTKVTVL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:29 (BR0301 heavy chain): EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPPKSDCKT KTVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:30 (BR0302 heavy chain): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPPKSDCK TKTVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:31 (BR0301 light chain): DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:32 (BR0302 light chain): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Brief description of the drawings]

[0087] [Figure 1] Figure 1 shows a schematic diagram of the amino acid numbering system of antibodies used in this application: (a) IMGT-based amino acid numbering system for human IgG1(κ); (b) numbering system for mutants (for IgG1 hinge region). [Diagram 2] Part A shows a schematic diagram of an engineered antibody according to an embodiment of the invention. Part B shows disulfide bonds in an IgG antibody according to an embodiment of the invention. [Diagram 3] 1 shows the characteristics of the antibody of the present invention. (a) Purity and yield of the antibody produced in HEK293 cells. (b) SDS-PAGE analysis of reduced (R) and non-reduced (NR) antibodies. (c) SEC-HPLC analysis of the purified antibody. [Figure 4]Figure 1 shows HIC profiles of ADCs made from native human IgG1(κ), IgG2(κ) and engineered antibodies of the examples of the invention: a: IgG1(κ)-MMAE, b: IgG2(κ)-MMAE, c: BB0500-2a-MMAE, d: BB0500-2b-MMAE, e: BB0500-2g-MMAE, f: BB0500-2n-MMAE, g: BR0301-MMAE and h: BR0302-MMAE. [Diagram 5] Figure 2 shows CE-SDS profiles of ADCs of certain examples of the invention: a: IgG1(κ)-MMAE, b: IgG2(κ)-MMAE, c: BB0500-2a-MMAE and d: BB0500-2g-MMAE. [Figure 6] 1 shows binding curves of different engineered antibodies according to embodiments of the invention and a control (wt) antibody to the proteins EGFR (a), HER3 (b), and HER2 (c). [Figure 7] 4 shows cytotoxicity curves of engineered ADCs according to embodiments of the invention against EGFR-expressing cancer cells (a: A431 cells, b: NUGC3 cells) and cytotoxicity curves of other engineered ADCs according to embodiments of the invention against HER2-expressing cancer cells (c: NCI-N87 (high HER2 expression), and d: A431 (low HER2 expression)).

Claims

1. 1. An isolated IgG antibody comprising two identical heavy chains, Each heavy chain is (a) a hinge region, the hinge region comprising an amino acid sequence (i) EPKSDCKTHTVECPPCP (SEQ ID NO: 7); (ii) EPKSDKCTHTVECPCP (SEQ ID NO:9); (iii) EPPKSCDKTHTVECPPCP (SEQ ID NO: 10); (iv) EPPKSDCKTHTVECPPCP (SEQ ID NO: 11); (v) EPPPKSCDKTHTVECPPCP (SEQ ID NO: 12); (vi) EPPPPKSCDKTHTVECPPCP (SEQ ID NO: 13); and (vii) EPPKSDCKTKTVECPPCP (SEQ ID NO:28); A hinge region selected from the group consisting of: (b) a CH1 domain located upstream of the hinge region and connected to the hinge region; Including, the CH1 domain of said IgG antibody has the sequence of the CH1 domain of a native human IgG1 antibody having an S142C mutation, or An isolated IgG antibody, wherein the CH1 domain of the IgG antibody has a sequence identical to the CH1 domain of a native human IgG2, IgG3, or IgG4 subclass antibody.

2. The IgG antibody of claim 1 , further comprising two identical kappa light chains.

3. 2. The IgG antibody of claim 1, wherein each of the heavy chains further comprises a CH2-CH3 domain located downstream of and connected to the hinge region of a native human IgG1, IgG2, IgG3, or IgG4 subclass antibody, the CH2-CH3 domains optionally comprising one or more mutations.

4. The IgG antibody of claim 1 , wherein the amino acid sequence contained in the hinge region is EPPKSDCKTKTVECPPCP (sequence number 28).

5. The IgG antibody described in claim 1, wherein a disulfide bond is formed between the first cysteine ​​residues from the N-terminus to the C-terminus of the two heavy chains in the hinge region.

6. The IgG antibody further comprises a Fab domain specifically binding to an antigen, the antigen being selected from the group consisting of EGFR, HER2, HER3, BCMA, B7H3, CEA, CEACAM6, 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 extra domain B, GCC, The IgG antibody of any one of claims 1 to 5, which is selected from the group consisting of GPNMB, LIV-1 (ZIP6), MUC1, MUC16, mesothelin, NaPi2b, nectin4, p-cadherin, periostin, PSMA, SC-16 (anti-Fyn3), SLC44A4, SLTRK6, STEAP1, tenascin-c, tissue factor, Trop2, and 5T4 (TPBG). (a) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:19, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:25; (b) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:20, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:26; (c) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:21, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:25; (d) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:22, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:27; (e) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:29, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:31; and (f) two identical heavy chains, each comprising the amino acid sequence set forth in SEQ ID NO:30, and two light chains, each comprising the amino acid sequence set forth in SEQ ID NO:32; An isolated IgG antibody comprising one of the following:

8. 8. An antibody-drug conjugate (ADC) or a pharma- ceutically acceptable salt thereof comprising the IgG antibody of any one of claims 1 to 7 conjugated to a cytotoxic drug via a chemical linker.

9. The cytotoxic drugs include monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin E, auristatin F, maytansines DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, tylanstatin A, uncialamicin, amanitin, ricin, diphtheria toxin, eribulin, １３１ 9. The ADC or pharma- ceutically acceptable salt thereof of claim 8, wherein the ADC is selected from the group consisting of I, interleukins, tumor necrosis factors, chemokines, irinotecan (SN38), exatecan, and exatecan derivatives.

10. The moieties included in the chemical linker include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), p-aminobenzyloxycarbonyl (PAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG, ｎ 9. The ADC or a pharma- ceutically acceptable salt thereof of claim 8, wherein the ADC is selected from the group consisting of -Val-Cit-PAB (n=1 to 20), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridin-2-yldisulfanyl)propanoate.

11. The ADC or a pharma- ceutically acceptable salt thereof of any one of claims 8 to 10, wherein the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:21, and the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:25, and wherein the cytotoxic drug is eribulin or MMAE.

12. The ADC or a pharma- ceutically acceptable salt thereof of any one of claims 8 to 10, wherein the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 19, and the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 25, and wherein the cytotoxic drug is eribulin or MMAE.

13. The ADC or a pharma- ceutically acceptable salt thereof of any one of claims 8 to 10, wherein the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:20, and the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:26, and wherein the cytotoxic drug is eribulin or MMAE.

14. The ADC or a pharma- ceutically acceptable salt thereof of any one of claims 8 to 10, wherein the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:29, and the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO:31, and wherein the cytotoxic drug is eribulin or MMAE.

15. The ADC or a pharma- ceutically acceptable salt thereof of any one of claims 8 to 10, wherein the two heavy chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 30, and the two light chains of the antibody each comprise the amino acid sequence set forth in SEQ ID NO: 32, and wherein the cytotoxic drug is eribulin or MMAE.

16. 11. The formulation of any one of claims 8 to 10, wherein at least 80% of the ADC is conjugated to a chemical linker via a cysteine ​​on the heavy chain of the antibody, or a pharma- ceutically acceptable salt thereof.

17. 11. The formulation of any one of claims 8 to 10, wherein ADC molecules having a drug-to-antibody ratio (DAR) of 2 account for 60% or more of the total amount of ADC molecules.

18. An IgG antibody according to any one of claims 1 to 7, which has been at least partially reduced such that at least some of the H-H disulfide bonds between corresponding cysteines in the hinge region of the antibody are reduced to free sulfhydryls; and a chemical linker comprising a terminal thiol reactive group attached to a cytotoxic drug molecule, or a pharma- ceutically acceptable salt thereof.

19. reducing the H-H disulfide bond between the first cysteine ​​residues from the N-terminus to the C-terminus of the two heavy chains in the hinge region of the IgG antibody according to any one of claims 1 to 7, thereby obtaining free sulfhydryls; and reacting a chemical linker comprising a terminal thiol reactive group, thereby conjugating each of the two heavy chains of the antibody to one cytotoxic drug molecule via the chemical linker.

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