Manipulated antibody compounds and their conjugates

Abstract

This invention provides novel antibody compounds and methods for using them. [Solution] Manipulated antibody compounds and their conjugates are provided, which are useful as agents for cancer immunotherapy.

Description

[Technical Field]

[0001] This invention relates to a novel antibody compound and a method for using the same. [Background technology]

[0002] Antibodies, and their cleaved fragments, can be conjugated with various payloads, including therapeutic, cytotoxic, and diagnostic peptides or other small molecules, for in vivo and in vitro applications. Antibody conjugates can be synthesized using free cysteine ​​sulfhydryl groups generated on the surface of immunoglobulin heavy or light chain residues as reactive nucleophiles, and can form stable chemical bonds with payloads via various linkers. However, conventional thiol bonds after reduction of interchain disulfide bonds result in heterogeneous antibody-drug conjugate mixtures depending on the reaction conditions. Even carefully controlled reactions will result in a distribution of conjugate-to-antibody ratios (CR). Conjugate mixtures with high CRs exhibit different chemical and biophysical characteristics compared to conjugate mixtures with low CRs. The addition of payloads to antibodies can also alter the pharmacological properties of the antibodies, including potentially affecting target binding and Fc receptor interactions. Therefore, it is desirable to obtain conjugates with a more uniform and targeted distribution of conjugate-to-antibody ratios.

[0003] To enable a more homogeneous and targeted distribution of payload-conjugated antibodies, cysteine ​​residues have been incorporated into parent mAbs to promote site-specific binding of drug payloads via thiol linkages (e.g., U.S. Patent Nos. 7,521,541). However, mutations to cysteine ​​in the parent surface amino acid residues can affect the biophysical properties and expression of the mAb. For example, engineered cysteine ​​residues could disrupt the native disulfide, which is crucial for proper protein folding. Furthermore, the resulting unpaired cysteine ​​could form intermolecular disulfides, potentially leading to higher-order aggregates. Therefore, there remains a need for further IgG mAbs containing alternative engineered cysteine ​​residues. There also remains a need for such antibodies in compounds involved in immune system cells.

[0004] Cancer immunotherapy, which utilizes the body's immune system to attack cancer cells, is a dynamic field in drug discovery and development in oncology. In contrast to therapies based on the use of antitumor drugs, this therapeutic approach represents a paradigm shift in involving the host's immune system to recognize and destroy tumor cells. Two successful cancer immunotherapy strategies are antibody modifications designed to suppress immune system suppression and enable the activation of the adaptive and / or innate immune systems, particularly tumor-specific cytotoxic T cells (i.e., immune checkpoint blockade), and antibody modifications designed to engage in and / or enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0005] Successful clinical outcomes have recently been achieved using immune checkpoint modulators designed to modify the interaction between T cell surface receptors such as PD-1 and CTLA-4 and their congeneral ligands, resulting in T cell activation and consequently T cell-mediated tumor cell destruction. Cancer immunotherapies targeting PD-1 (e.g., nivolumab (Opdivo®) and pembrolizumab (Keytruda®)) and CTLA-4 (e.g., ipilimumab (Yervoy®)) have been approved by the FDA for the treatment of cancers such as squamous non-small cell lung cancer and metastatic melanoma.

[0006] ADCC involves the interaction between the antibody Fc domain and receptors on the surface of immune system cells (e.g., natural killer or "NK" cells) (e.g., Fc gamma receptor IIIa), resulting in the subsequent destruction of targeted tumor cells along with the release of cytolytic proteins from immune cells. Approved antibody therapies presenting ADCC include Rituxin® (rituximab), Alzera® (ofatumumab), Herceptin® (trastuzumab), and Campas® (aremtuzumab). Efforts to manipulate antibodies with improved ADCC activity via enhanced Fc receptor binding have been effective in patients where antibodies with similar target specificity and low ADCC activation are either ineffective or no longer sufficiently effective for the disease (e.g., Gazyva® (obinutuzumab)).

[0007] Despite advances in current cancer immunotherapy, the need for alternative approaches involving the immune system in treating cancer remains. For example, the proportion of patients who respond to T-cell specific immunotherapy varies, and there is a lack of reliable prognostic assays to identify which patients will respond. Furthermore, therapy-induced autoimmune diseases are a serious side effect associated with immune checkpoint inhibitor therapy. The emergence of autoimmune diseases with immune checkpoint inhibitors is likely related to the mechanism of action of immune checkpoint inhibitors, as they are designed to remove the suppression of the T-cell repertoire so that tumor-specific T cells can emerge, proliferate, and be activated. Thus, immune checkpoint inhibitors are relatively nonspecific, and one consequence of this lack of specificity is that autoreactive T cells break tolerance and induce autoimmune diseases that are not necessarily reversible upon discontinuation of therapy. Enhanced ADCC approaches are designed to involve NK cells for killing tumor cells. However, NK cells make up only about 5% of the total white blood cell count in the blood.

[0008] Targeting polymorphonuclear cells (PMNs) of the innate immune system to participate in the killing of tumor cells represents an alternative approach to cancer immunotherapy. PMNs comprise over 50% of the total white blood cell count and are the primary line of defense against pathogens, including commensal and exogenous bacteria. During the innate immune response, pathogen-associated molecular patterns (PAMPs) presented by pathogens are recognized by pattern recognition receptors (PRRs) on the surface of immune cells such as neutrophils. One such PRR is formylpeptide receptor 1 (FPR1), a membrane-bound G protein-coupled receptor expressed on the surface of neutrophil cells. FPR1 detects N-formyl-methionine-containing proteins and peptides, including those produced and released by bacteria after infection. Binding of FPR1 on the surface of neutrophils to N-formyl-methionine-containing peptides, particularly peptides presenting the N-formyl-methionine-leucine-phenylalanine (fMLF) residue, induces neutrophil motility / chemotaxis to the site of infection. Activation of FPR1 by formyl peptide also triggers pathogen-killing mechanisms such as degranulation to release cytotoxic molecules, production of reactive oxygen species, and phagocytosis in order to destroy the pathogen.The following is a detailed description of the FPR-1 missile system (He HQ and Ye). RD,Molecules.2017 Mar 13;22(3).pii:E455.doi:10.3390 / molecules22030455、Hwang TL et al.,Org BiomolChem.2013 Jun 14;11(22):3742-55.doi:10.1039 / c3ob40215k;Cavicchioni G et al.,BioorgChem.2006 Oct;34(5):298-318;Higgins JD et al.,J MedChem.1996 Mar 1;39(5):1013-5;Vergelli C et al.,Drug Dev Res.2017 Feb;78(1):49-62.doi:10.1002 / ddr.21370;Kirpotina LN et al.,Mol Pharmacol.2010 Feb;77(2):159-70.doi:10.1124 / mol.109.060673;Cilibrizzi A et al.,J MedChem.2009 Aug 27;52(16):5044-57.doi:10.1021 / jm900592h.

[0009] Previous attempts to attract macrophages using fMLF bioconjugates (antibodies conjugated to a peptide) to kill tumor cells had several limitations. Obrist and Sandberg used the chemistry of carbodiimide to link the peptide to free lysine, thereby conjugating fMLF to a polyclonal rabbit antitumor antibody. This nonspecific conjugation of fMLF to the polyclonal antibody resulted in a significant reduction in affinity, a 100-fold reduction in the potency of fMLF in promoting macrophage chemotaxis, and a significant decrease in the antibody's ability to induce complement-dependent 51Cr release from pre-labeled hepatocellular carcinoma cells using normal rabbit serum as the complement source (Obrist and Sandberg, Clin. Immun. Immunopathology, 25;91-102 (1982)). These data are consistent with the possibility that nonspecific addition of fMLF to the antibody via the chemistry of lysine reduces antigen-binding affinity, FPR-1 receptor involvement, and Fc receptor involvement.

[0010] Obrist et al. demonstrated that coupling fMLF with a mouse monoclonal antibody using the chemical properties of carbodiimide preserved affinity for human ovarian cancer cells, but this binding reduced the chemotactic response to human peripheral blood mononuclear cells. The effect of binding on complement fixation had not been reported. (Obrist et al., Int. J. Immunopharmac., 5(4); 307-314 (1983)). Similar findings (preservation of binding and reduction of chemotaxis) were also reported when fMLF was directly bound to melanoma mAb 9.2.27 via the chemical properties of carbodiimide (Obrist et al., Caner Immunol. Immunother., 32; 406-08 (1991)). The antibody-binding compound of the present invention can attract and activate human neutrophils in addition to mononuclear cells and macrophages, although previous literature findings have been almost exclusively directed towards mononuclear cells and macrophages. This may have significant therapeutic relevance because neutrophils represent a larger proportion of the total white blood cell population in human circulation, are produced at a higher rate than all other white blood cell populations, can easily migrate into tissues, and, when activated, are highly effective in eliminating target bacteria.

[0011] The most common methods of antibody-drug conjugation are alkylation of reduced interchain disulfides, acylation of lysine residues, and alkylation of genetically modified cysteine ​​residues. This invention aims to demonstrate that all common methods for generating antibody conjugates would be effective in generating antibody conjugates capable of activating FPR-1 on neutrophils and innate immune system cells.

[0012] Tumor-targeted therapeutic antibodies that can involve PMN neutrophils of the innate immune system in tumor cell destruction may also offer advantages over current cancer immunotherapies. For example, such therapeutic antibodies may enhance the T cell response against tumors and may not require the presence of tumor-specific T cells to drive tumor cell killing. The involvement of PMN neutrophils in antitumor activity would depend on the presence of FPR (e.g., FPR1), which would naturally be expressed in neutrophils in all patients. Furthermore, drugs that can involve PMN neutrophils in tumor cell killing could potentially provide 1 × 10⁶ doses per day. 11 Since it has been estimated that a certain number of neutrophils are produced, we would benefit from a potent and sustained supply of tumor-killing cells. Tumor-targeted antibodies that can involve neutrophils in killing tumor cells may have a safety advantage over immune checkpoint modulators. Unlike checkpoint modulators, neutrophil-targeted therapies would not induce or require the proliferation of immune cells because circulating neutrophils are short-lived. Furthermore, when neutrophils kill target tumor cells using attached antibodies, the tumor-targeted antibodies are eliminated, providing a negative feedback loop that reduces immune stimulation as therapeutic antibodies are consumed by target effector cells.

[0013] Another way to demonstrate the usefulness of tumor-targeted therapeutic antibodies that can involve FPR-1-positive innate immune cells within tumor cells is for the treatment of inactive tumors that have low mutational loads and are therefore not readily recognized by the immune system. By inducing and activating neutrophil-mediated tumor cell killing, novel antigens are subsequently produced locally in a cytokine-rich environment, thereby enabling cells of the adaptive immune system to recognize the tumor and target tumor cells for elimination.

[0014] Tumor-targeted antibodies that can involve neutrophils in tumor cell killing may also have advantages over toxicist-based antibody-drug conjugates (ADCs) that are typically designed to release a toxic payload after internalization into tumor cells. Similar to ADCs, tumor-targeted antibodies that can involve neutrophils in tumor cell killing should recognize antigens that are highly expressed in tumor cells and low in normal tissues. However, unlike ADCs, tumor-targeted antibodies that can involve neutrophils in tumor cell killing require agonist exposure to receptors on the surface of the innate immune system and are therefore expected to function better with target antigens that are relatively less likely to be internalized. [Overview of the project]

[0015] Conjugated antibodies can be produced by reducing interchain disulfides to generate reactive thiols or by utilizing surface lysine for binding; however, these conventional binding methods can result in antibody instability or loss of binding affinity. Therefore, the present invention provides an antibody peptide conjugate to which site-specific addition(s) of an N-formyl-methionine peptide modulus to an engineered cysteine ​​residue provides one or more of the following advantages: (i) site-specific addition enables homogeneous binding properties required for the potency and maximum efficacy of the N-formyl-methionine peptide bioconjugate; (ii) the use of a spacer allows the potency of the N-formyl-methionine peptide bioconjugate to be preserved for the migration and activation of human neutrophils when bound to the antibody; (iii) site-specific addition preserves Fc receptor interactions in the IgG1 construct which can contribute to tumor cell killing; (iv) site-specific addition allows the antibody to preserve antigen-binding affinity, which has already been achieved in some, though not all, examples in the prior literature; and (v) site-specific binding maintains antibody stability which can be a significant advantage in the manufacture of drug substances and the stability of drug products.

[0016] The present invention also provides IgG antibodies comprising engineered cysteine residues for use in the generation of antibody-conjugated compounds (also referred to as bioconjugates). More particularly, the present invention provides therapeutic compounds comprising tumor-targeting antibodies consisting of engineered cysteine residues conjugated to peptides or peptidomimetics that can activate FPR-1 on cells of the innate immune system. In embodiments, the antibody binds to a peptide or peptidomimetic that can activate FPR-1. In some specific embodiments, the peptide or peptidomimetic is a compound of one of the following formulas: Formula I. R-P1―P2-P3-NH(CH2CH2O) n CH2CH2-Y (wherein R is HC(=O)- or R 1 NHC(=O)NH-, R 1 is optionally substituted C5-C 10 aryl, P1 is Met or Nle, P2 is a peptide or peptidomimetic, P3 is epsilon-aminoacylated lysine, n is an integer from 6 to 24, Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide or vinyl sulfone), or a salt thereof. Formula II. R-P1-P2-NH(CH2CH2O) n CH2CH2-P3-Y (wherein R is HC(=O)- or R 1 NHC(=O)NH-, R 1 is optionally substituted C5-C 10 aryl, P1 is Met or Nle, P2 is a peptide or peptidomimetic, ​n is an integer between 6 and 24. Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. or its salt. Formula III.R-Met-X1―X2―X3―X4-NH(CH2CH2O) n CH2CH2--X5-Y (In the formula, R is either HC(=O)- or R 1 NHC(=O)NH- R 1 These are phenyl, 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, aryl, substituted aryl, or 2-allyl. X1 is Leu, Ile, Nle, diethylglycine, or dipropylglycine. X2 is Phe, α-Me-Phe, DPhe, 4-F-Phe, 2-Nal, or 1-Nal. X3 is Glu, Leu, Nle, α-Me-Leu, DLeu, or absent. X4 is either Glu, DGlu, γGlu, Gla, or absent. X5 is C2~C 10 It is a diaminoalkyl, and Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. or its salt.

[0017] In some other detailed embodiments, the peptide is one of the compounds of the following formula: Formula IV.[R-P1-P2-NH(CH2CH2O) n CH2CH2-]2-QXY (In the formula, R is either HC(=O)- or R 1 NHC(=O)NH- R 1 C5~C may be substituted or not substituted. 10 It is Ariel, P1 is either Met or Nle, P2 is a peptide or peptide mimetic. n is an integer between 6 and 24. Q is an amino difunctional residue that can be acylated at the alpha-amino group and the side-chain amino group. X is C2~C 10 It is a diaminoalkyl, and Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. or its salt. Formula V.[[R-P1-P2-NH(CH2CH2O) n CH2CH2-]4- (Q)2-QXY (In the formula, R is either HC(=O)- or R 1 NHC(=O)NH- R 1 C5~C may be substituted or not substituted. 10 It is Ariel, P1 is either Met or Nle, P2 is a peptide or peptide mimetic. n is an integer between 6 and 24. Q is an amino difunctional residue that can be acylated at the alpha-amino group and the side-chain amino group. X is C2~C 10 It is a diaminoalkyl, and Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. or its salt. Formula VI.[[[R-P1-P2-NH(CH2CH2O) n CH2CH2-]8-(Q)4-(Q)2-QXY (In the formula, R is either HC(=O)- or R 1 NHC(=O)NH- R 1 C5~C may be substituted or not substituted. 10 It is Ariel, P1 is either Met or Nle, P2 is a peptide or peptide mimetic. n is an integer between 6 and 24. Q is an amino difunctional residue that can be acylated at the alpha-amino group and the side-chain amino group. X is C2~C 10 It is a diaminoalkyl, and Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. or its salt.

[0018] The compounds of formulas IV to VI contain two or more chemoattractants linked together via an amino difunctional residue (represented by "Q"). In some embodiments, Q is Lys, Orn, Dap, or Dab. In preferred embodiments, the difunctional residue is a lysine residue or an ornithine residue. The difunctional residue can be linked to two additional amino difunctional residues via each amino group, thereby increasing the number of chemoattractants to four. The additional difunctional residues allow for an additional number of chemoattractants. In preferred embodiments, the number of chemoattractants is eight or less. For example, if Q2 is a repeating lysine branched residue, the structure is as follows: [ka]

[0019] The present invention provides one compound from formulas I to VI, where P2 is given by X1-X2-X3-X4, and X1 is Leu, Ile, Nle, diethylglycine, or dipropylglycine. X2 is Phe, α-Me-Phe, DPhe, 4-F-Phe, 2-Nal, or 1-Nal. X3 is Glu, Leu, Nle, α-Me-Leu, DLeu, or absent, and X4 is either Glu, DGlu, γGlu, Gla, or absent.

[0020] In some embodiments, any one of the compounds of formulas I, II, III, IV, V, or VI can activate formyl peptide receptor 1 and form a covalent bond with the protein. In some embodiments, any one of the compounds of formulas I, II, III, IV, V, or VI is conjugated to an antibody via a linker. In some detailed embodiments, the compound is conjugated via a maleimide-PEG linker as described herein. In some detailed embodiments, the PEG linker is bound to a diaminoalkyl of X. In some detailed embodiments, the PEG linker is absent, and any one of the compounds of formulas I, II, III, IV, V, or VI is directly bound to the diaminoalkyl of X. In some such embodiments, the compounds derived from any one of the compounds of formulas I, II, III, IV, V, or VI can activate formyl peptide receptors on the surface of innate immune cells such as neutrophils.

[0021] Embodiments of the present invention are also useful in non-tumor contexts to involve innate immune cells in the specific elimination of target cells of interest, which has utility beyond cancer therapy. For example, in situations where the elimination of normal cells is desirable in hypertrophied tissue, restricted access tissue, or virus-infected cells, antibodies that specifically target cells of interest, which can also activate cells of the innate immune system against the killing of the target cells, would be useful in eliminating the target tissue or infected cells.

[0022] The present invention envisions a series of linkers for attaching an FPR-1 agonist to an engineered cysteine ​​residue (Yao et al., Int J Mol Sci. 2016 Feb 2;17(2).pii:E194.doi:10.3390 / ijms17020194). Examples provided include a maleimide-based linker for forming a thioether bond to cysteine. The use of other linkers, such as haloacetyl linkers, can also be used for antibody conjugation.

[0023] Accordingly, the present invention provides an antibody comprising an IgG heavy chain and a light chain constant region, the constant region comprising at least one cysteine. In one embodiment, the constant region comprises an unpaired free cysteine ​​on its surface. In another embodiment, the constant region comprises an engineered cysteine. In some detailed embodiments, the constant region comprises the following residues, namely C H Residue 124 in one domain, C H Residue 157 in one domain, C H 162 residues in one domain, C H Residue 262 in the 2 domain, C H Residue 375 in domain 3, C H Residue 373 in the 3 domain, C H Residues 397 in the 3 domains, C H Residue 415 in the 3 domains, C カッパ Residue 156 in the domain, C カッパ Residue 171 in the domain, C カッパ Residue 191 in the domain, C カッパ Residue 193 in the domain, C カッパ Residue 202 in the domain, or C カッパ One of the 208 residues in the domain contains at least one manipulated cysteine.

[0024] The present invention also provides an antibody containing an IgG heavy chain constant region, wherein the constant region is C H The cysteine ​​at residue 124 in one domain, and C H Residues 157 and 162 in one domain, and C HThe 3 domains include cysteine ​​at one but not all of the residues 375 and 378. In a more detailed embodiment, the IgG heavy chain constant region is a human, mouse, rat, or rabbit IgG constant region. More specifically, the IgG heavy chain constant region is a human IgG1, human IgG2, or human IgG4 isotype, and more specifically, human IgG1 or human IgG4. In a further detailed embodiment, the IgG heavy chain constant region is a human IgG1 isotype, certified by the amino acid sequence of SEQ ID NOs: 17, 18, 19, or 52, and more specifically, the amino acid sequence of SEQ ID NOs: 20, 21, or 53. In a further detailed embodiment of the above-described antibody containing a human IgG1 heavy chain constant region, the constant region further includes isoleucine substituted at residue 247 and glutamine substituted at residue 339. In another embodiment, the constant region includes isoleucine substituted at residue 247, glutamine substituted at residue 339, and glutamic acid substituted at residue 332. As an alternative detailed embodiment, the IgG heavy chain constant region is a human IgG4 isotype and is affixed by the amino acid sequence of SEQ ID NOs: 12, 13, 14, 54, or 55, and more specifically by the amino acid sequence of SEQ ID NOs: 15, 16, 56, or 57. As a further detailed embodiment to the above-described antibody containing a human IgG4 heavy chain constant region, the constant region further comprises proline substituted at residue 228, alanine substituted at residue 234, and alanine substituted at residue 235.

[0025] The present invention further provides an antibody comprising two heavy-chain IgG constant regions, each IgG constant region comprising at least one cysteine. In embodiments, each IgG constant region comprises the following residue, namely C H Residue 124 in one domain, C H Residue 157 in one domain, C H 162 residues in one domain, C H Residue 375 in the 3 domains, and C H The present invention contains one cysteine ​​among 378 residues in the 3 domains. In this invention, each IgG constant region is C H124 cysteine ​​residues in one domain, and C H Residues 157 and 162 in one domain, and C H We also provide any of the antibodies described above that include two heavy chain IgG constant regions containing cysteine ​​at one but not all of the residues 375 and 378 in the 3 domain. More specifically, each IgG constant region is human, mouse, rat, or rabbit IgG, more specifically human IgG1, human IgG2, or human IgG4 isotype, more specifically human IgG1 or human IgG4. In a further detailed embodiment, each IgG heavy chain constant region is a human IgG1 isotype and is given by the amino acid sequence of SEQ ID NOs: 17, 18, 19, or 52, more specifically the amino acid sequence of SEQ ID NOs: 20, 21, or 53. In a further detailed embodiment of the antibody described above that includes two human IgG1 heavy chain constant regions, the constant region further includes isoleucine substituted at residue 247 and glutamine substituted at residue 339. In another embodiment, the constant region includes isoleucine substituted at residue 247, glutamine substituted at residue 339, and glutamic acid substituted at residue 332. As an alternative detailed embodiment, each IgG heavy chain constant region is a human IgG4 isotype and is given by the amino acid sequence of SEQ ID NOs: 12, 13, 14, 54, or 55, and more specifically by the amino acid sequence of SEQ ID NOs: 15, 16, 56, or 57. As a further detailed embodiment to the above antibody comprising two human IgG4 heavy chain constant regions, the constant region further comprises proline substituted at residue 228, alanine substituted at residue 234, and alanine substituted at residue 235.

[0026] The present invention is C H Residue 124 in one domain, C H Residue 157 in one domain, C H 162 residues in one domain, C H Residue 262 in the 2 domain, C H Residue 375 in domain 3, C H Residue 373 in the 3 domain, C H Residues 397 in the 3 domains, CH Residue 415 in the 3 domains, C カッパ Residue 156 in the domain, C カッパ Residue 171 in the domain, C カッパ Residue 191 in the domain, C カッパ Residue 193 in the domain, C カッパ Residue 202 in the domain, or C カッパ The present invention further provides any of the above antibodies in which each cysteine ​​residue 208 in the domain is conjugated to a chemoattractant. In embodiments, the chemoattractant is an f-Met peptide, a small molecule FPR-1 agonist, a PRR agonist, a peptide mimetic, an N-ureido peptide, or a bacterial sugar. In detailed embodiments, the chemoattractant is an N-formyl-methionine peptide. In some embodiments, the chemoattractant is conjugated to the antibody cysteine ​​via a maleimide linker, where the linker comprises a maleimide functional group and cysteine ​​(C H Residue 124 in one domain, C H Residue 157 in one domain, C H 162 residues in one domain, C H Residue 262 in the 2 domain, C H Residue 375 in domain 3, C H Residue 373 in the 3 domain, C H Residues 397 in the 3 domains, C H Residue 415 in the 3 domains, C カッパ Residue 156 in the domain, C カッパ Residue 171 in the domain, C カッパ Residue 191 in the domain, C カッパ Residue 193 in the domain, C カッパ Residue 202 in the domain, or C カッパLocated at residue 208 in the domain, a thioether bond is formed between the IgG heavy chain and light chain constant region, and a covalent bond is also formed to the N-formyl-methionine peptide via an amide bond to the epsilon-amino side chain of the C-terminal lysine of the N-formyl-methionine peptide. In embodiments, the present invention provides any of the above antibodies in which each cysteine ​​referred to herein is conjugated to an N-formyl-methionine peptide via a maleimide linker, the linker forming a covalent bond to the IgG heavy chain constant region via a thioether bond between the maleimide functional group and the cysteine, and a covalent bond is also formed to the N-formyl-methionine peptide via an amide bond to the epsilon-amino side chain of the C-terminal lysine of the N-formyl-methionine peptide. In detailed embodiments, the present invention provides each IgG constant region being C H 124 cysteine ​​residues in one domain, and C H Residues 157 and 162 in one domain, and C H Further providing antibody compounds containing two heavy chain IgG constant regions, each containing a cysteine ​​residue at one of but not all of residues 375 and 378 in the 3 domain, where each C H Each of the 124 cysteine ​​residues in one domain, as well as C H Residues 157 or 162 in one domain, each C H Each cysteine ​​at 375 or 378 of the three domains is conjugated to the N-formyl-methionine peptide via a maleimide linker, which is covalently bonded to the antibody via a thioether linkage between the maleimide functional group and the cysteine ​​at residues 124, 157, or 162 and 375 or 378 of the IgG constant region, and via an amide linkage to the N-formyl-methionine peptide's C-terminal lysine side chain. More specifically for the conjugated antibody described above, the maleimide linker is of formula [ka] The formula has n=1 to 24, more specifically n=6 to 24, and even more specifically n=12. More specifically, the N-formyl-methionine peptide is N-formyl-methionine-leucine-phenylalanine-X (SEQ ID NO: 22), where X is lysine modified by amide bond formation to the maleimide linker. More specifically, each IgG constant region of the above-mentioned conjugated antibody compound is a human IgG1 or human IgG4 isotype, and more specifically, each IgG heavy chain constant region is a human IgG1 isotype and further comprises isoleucine substituted at residue 247 and glutamine substituted at residue 339, or each IgG heavy chain constant region is a human IgG4 isotype and further comprises proline substituted at residue 228, alanine substituted at residue 234 and alanine substituted at residue 235.

[0027] The manipulated cysteine ​​residues of the present invention can be incorporated into the IgG constant region of existing cancer therapeutic antibodies to facilitate the generation of alternative N-formyl-methionine peptide-conjugated immunotherapies. Alternatively, the heavy chain CDR or variable domain of existing cancer therapeutic antibodies can be combined with the IgG constant region containing the manipulated cysteine ​​residues of the present invention to generate conjugated immunotherapies. Exemplary cancer therapies for these applications include IgG1 therapeutic antibodies targeting solid tumors, including HER-2 expressing tumors (i.e., IgG1 antibodies such as trastuzumab and pertuzumab), IgG1 therapeutic antibodies targeting humoral tumors, including CD20 expressing humoral tumors (i.e., IgG1 and IgG1-enhanced ADCC antibodies such as rituximab, ofatumumab, obinutuzumab, and AME133v), and antibodies targeting c-Met expressing tumors (i.e., emibetuzumab).

[0028] The N-formylmethionine peptide-conjugated antibodies disclosed herein can serve as a platform for further conjugation with cytotoxic agents to achieve higher efficacy, or as a substitute for drug conjugates in antibody-drug conjugates targeting antigens overexpressed in cancer cells. Examples of target antigens containing antibody-drug conjugates include GPNMB (grembatumumab vedotin), CD56 (lorbotuzumab meltansine (IMGN-901)), TACSTD2 (TROP2, sacituzumab govitecan (IMMU-132)), CEACAM5 (rabetuzumab SN-38), folate receptor-α (milbetuximab sorabtansine (IMGN-853), vintafolide), mucin 1 (sialoglycotope CA6, SAR-566658), STEAP1 (bundutuzumab vedotin (RG-7450)), mesothelin (DMOT4039A, anetumablubutensin (BAY-94-9343), BMS) -986148), Nectin 4 (Enfortumab Vedotin (ASG-22M6E), ASC-22CE), ENPP3 (AGS-16M8F), Guanylyl Cyclase C (Indazumab Vedotin (MLN-0264)), SLC44A4 (ASG-5ME), NaPi2b (Rifasutuzumab Vedotin), CD70 (TNFSF7, DNIB0600A, AMG-172, MDX-1243, Borsetuzumab Vedotin (SGN-75)), CA9 Carbonic Anhydrase (BAY79-4620), 5T4 (TPBG, PF06263507), SLTRK6 (ASG-15ME), SC-16 (Anti-Fyn3, SC16LD6.5) Tissue factor (HuMax-TF-ADC (TF-011-MMAE)), LIV-1 (ZIP6, SGN-LIV1A), P-cadherin (PCA062), PSMA (MLN2704, PSMA-ADC), fibronectin extra domain B (human mAb L19 and F8), endothelin receptor ETB (RG-7636), VEGFR2 (CD309, anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles), tenascin c (anti-TnC-A1 antibody SIP (F16)), periostin (anti-periostin antibody), DLL3 (robatumumab sorabtansine), HER2 (T-DM1, ARX788, SYD985), EGFR (ABT-414, IMGN289 AMG-595), CD30 (brentuximab vedotin, iratumumab MDX-060), CD22 (inotuzumab ozogamicin (CMC-544), pinatuzumab vedotin, epratuzumab SN38), CD79b (polatuzumab vedotin), CD19 (coltuximab rabutansine, SAR-3419, SGN-CD19A), CD138 (indatuximab rabutansine), CD74 (miratuzumab doxorubicin), CD37 (IMGN-529), CD33 (gemtuzumab ozogamicin, IMGN779, SGN CD33 A), and CD98 (IGN523) are included, but not limited to these. (See, for example, Thomas et al, Lancet Oncol. 2016 Jun;17(6)e254-62 and Diamantis and Banerji, Brit. Journ. Cancer, 2016;114, 362-367).

[0029] Therefore, the present invention further provides an IgG antibody comprising any of the heavy chain CDRs and light chain CDRs of the above-mentioned cancer therapeutic antibodies, and each IgG constant region is C H the cysteine at residue 124 in the C H 1 domain and residues 157 and 162 in the C HIt contains cysteine residues that are in one but not all of residues 375 and 378 in the 3 domain. Further, the present invention provides any of the antibodies in which the cysteine at residue 124 of each IgG constant region and the cysteine at each of residues 157, 162, 375 or 378 of each IgG constant region are conjugated to an N-formyl-methionine peptide via a maleimide-PEG linker, as all are described herein.

[0030] The present invention provides a compound which is an antibody containing at least one cysteine bound, optionally via a linker, to a chemoattractant capable of attracting and / or activating one or more cells of the immune system, the agent being bound to the antibody at one or more cysteine residues within the antibody. In some embodiments, the antibody comprises an IgG heavy chain constant region, the constant region having the following residues, namely C H residue 124 in the 1 domain, C H residue 157 in the 1 domain, C H residue 162 in the 1 domain, C H residue 262 in the 2 domain, C H residue 375 in the 3 domain, C H residue 373 in the 3 domain, C H residue 397 in the 3 domain, C H residue 415 in the 3 domain, C カッパ residue 156 in the domain, C カッパ residue 171 in the domain, C カッパ residue 191 in the domain, C カッパ residue 193 in the domain, C カッパ residue 202 in the domain, or C カッパThe domain contains at least one cysteine ​​among the 208 residues. In some embodiments, the cysteine ​​is engineered cysteine. In further embodiments, the number of engineered cysteines on each heavy and / or light chain is 1 to 3. In other embodiments, the antibody is conjugated to a chemoattractor via a linker. In some embodiments, the linker is a maleimide-PEG linker or a Mal-Dap linker. In other embodiments, the chemoattractor is an f-Met peptide, a small molecule FPR-1 agonist, a PRR agonist, a peptide mimetic, an N-ureido-peptide, or a bacterial sugar.

[0031] The present invention provides a compound which is an antibody containing at least one cysteine ​​optionally conjugated via a linker to a chemotactic, which can attract and / or activate one or more cells of the immune system, wherein the agent is conjugated to one or more cysteine ​​residues within the antibody, and the chemotactic is any one of the compounds of formulas I, II, III, IV, V, or VI as described herein. In some embodiments, the compound can attract and activate one or more cells of the immune system. In some detailed embodiments, the compound can attract and activate one or more cells of the innate immune system. In preferred embodiments, a linker is present.

[0032] Furthermore, the present invention also provides any of the antibodies, their IgG heavy chain constant region, and N-formylmethionine peptide conjugates, each as specifically illustrated herein. In further embodiments, the present invention provides any of the antibodies, their IgG heavy chain constant region, conjugated antibodies, or nucleic acids encoding one of these in an “isolated” form. As used herein, the term “isolated” refers to a protein, polypeptide, or nucleic acid that does not contain, or substantially contains, other macromolecular species found in the cellular environment.

[0033] The present invention further provides a pharmaceutical composition comprising any of the N-formylmethionine peptide conjugated antibodies described herein and a pharmaceutically acceptable carrier or excipient. Furthermore, the present invention further provides a method for treating solid tumors including breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone mesodermal carcinoma, vascular cancer, and fibrocarcinoma and associated metastases, and humoral tumors including leukemia and lymphoma, comprising administering to a patient in need of such treatment an effective amount of the N-formyl-methionine peptide conjugated antibody or the pharmaceutical composition thereof, as described herein. Furthermore, the present invention further provides any of the N-formyl-methionine peptide conjugated antibodies or the pharmaceutical compositions thereof described herein for use in therapy. In particular, the present invention provides any of the N-formyl-methionine peptide conjugated antibodies described herein, and a pharmaceutical composition thereof, for use in the treatment of breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone mesodermal carcinoma, vascular cancer and fibrocarcinoma, leukemia, and lymphoma. In detailed embodiments of the methods, uses, and compositions described herein, the N-formylated methionine peptide is N-formyl-Met-Leu-Phe-Lys-OH. [Modes for carrying out the invention]

[0034] definition The general structure of "IgG antibodies" is very well known. Wild-type (WT) IgG antibodies are heterotetramers consisting of four polypeptide chains (two identical "heavy" chains and two identical "light" chains) crosslinked via intrachain and interchain disulfide bonds. Each heavy chain (HC) has an N-terminal heavy chain variable region ("V"). H It consists of a heavy chain constant region and three domains (C H 1, C H 2, and C H 3) and C H 1 domain and C HIt consists of a hinge region ("hinge") between two domains. Each light chain (LC) has an N-terminal light chain variable region ("V"). L ) and light chain constant region ("C L It consists of ''). L and C L The region may be of the kappa ("κ") or lambda ("λ") isotype ("Cκ" or "Cλ", respectively). Each heavy chain has an interface (V) between the heavy chain variable domain and the light chain variable domain. H / V L Interface), and heavy chain steady C H 1 and light chain constant domain (C H 1 / C L (Bonds to one light chain via an interface). V H -C H 1 and V L -C L The binding between each segment forms two identical antigen-binding fragments (Fabs) that direct antibody binding to the same antigen target or epitope. Each heavy chain has a hinge-C of each heavy chain. H 2-C H The three segments bond to other heavy chains via the interface, and two C H 2-C H The binding between the three segments forms the Fc region of the antibody. Together, each Fab and Fc forms the characteristic "Y-shaped" architecture of the IgG antibody, with each Fab representing an "arm" of the "Y". IgG antibodies can be divided into subtypes, such as IgG1, IgG2, IgG3, and IgG4, which differ in the length of the hinge region, the number and position of interchain and intrachain disulfide bonds, and the amino acid sequence of their respective HC constant regions.

[0035] The variable regions of each heavy-light chain pair bind together to form a binding site. Heavy chain variable region (V H ) and light chain variable region (V L The first region (V) can be further divided into a hyper-variable region called the complementarity-determining region (CDR), which is sandwiched between more conserved regions called the framework region ("FR"). H and V LThe CDR consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain CDRs are sometimes called "CDRH1, CDRH2, and CDRH3," and the three light chain CDRs are sometimes called "CDRL1, CDRL2, and CDRL3." The heavy chain FRs are sometimes referred to as HFR1, HFR2, HFR3, and HFR4, while the light chain FRs are sometimes called LFR1, LFR2, LFR3, and LFR4. The CDRs contain most of the residues that form specific interactions with the antigen.

[0036] The compounds and methods of the present invention involve designed amino acid modifications to specific residues within the constant region of a heavy chain polypeptide. As those skilled in the art will understand, various numbering rules can be employed to designate specific amino acid residues within the constant and variable region sequences of IgG. Commonly used numbering rules include the "Kabat numbering" and "EU index numbering" systems. "Kabat numbering" or "Kabat numbering system" as used herein refers to the numbering system devised and revealed by the authors in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD (1991), for designating amino acid residues in both the variable and constant domains of the heavy and light chains of an antibody. "EU index numbering" or "EU index numbering system" as used herein refers to the numbering rules for designating amino acid residues in the constant domain of an antibody heavy chain, also revealed in Kabat et al (1991). Other rules, including modified or alternative numbering systems for variable domains, include Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196:901-917, Chothia, et al. (1989), Nature 342:877-883), IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27:55-77), and AHo (Honegger A, Pluckthun A (2001), J Mol Biol 309:657-670). Unless otherwise specified herein, the constant region of immunoglobulin heavy chain C is represented in the specification, examples and claims. H 1. Hinge, C H 2, and C HAll references (i.e., numbers) to three amino acid residues are based on EU index numbering. With knowledge of residue numbers using EU index numbering, a person skilled in the art can apply the teachings to identify amino acid sequence modifications within the present invention according to some commonly used numbering rules. While the specification, examples, and claims of the present invention employ EU index numbering to identify specific amino acid residues, it is understood that the sequence numbers represented in the examples and sequence listings attached to this application provide sequential numbering of amino acids within a given polypeptide, as resulting from Patent In Version 3.5, and therefore do not correspond to the corresponding amino acid residue numbers provided by EU index numbering.

[0037] The polypeptide chains described herein are represented by an amino acid sequence from the N-terminus to the C-terminus, read from left to right, with each amino acid represented by either a one-letter or three-letter amino acid abbreviation. Unless otherwise specified herein, all amino acids used in the preparation of the polypeptides of the present invention are L-amino acids. The “N-terminus” (or amino-terminus) of an amino acid or polypeptide chain refers to a free amine group on the amino acid, or a free amine group on the first amino acid residue of the polypeptide chain. Furthermore, the term “N-terminal amino acid” refers to the first amino acid in the polypeptide chain. Similarly, the “C-terminus” (or carboxy-terminus) of an amino acid or polypeptide chain refers to a free carboxyl group on the amino acid, or a free carboxyl group on the last amino acid residue of the polypeptide chain. Furthermore, the term “C-terminal amino acid” refers to the last amino acid in the polypeptide chain.

[0038] As used herein, the phrase “[amino acid name] substituted with residue …” refers to the substitution of a parent amino acid at the indicated amino acid in a heavy-chain or light-chain polypeptide. For example, “alanine substituted at residue 235” refers to a heavy-chain in which the parent amino acid sequence has been mutated to contain alanine at residue number 235 instead of the parent amino acid. Such mutations can also be expressed by indicating a specific amino acid residue number, where the parent amino acid precedes the substitution amino acid. For example, “F235A” refers to the substitution of phenylalanine with alanine at residue 235. Similarly, “235A” refers to the substitution of a parent amino acid with alanine. “Engineered” cysteine ​​refers to the substitution of a parent amino acid with cysteine.

[0039] As used herein, "N-formyl-methionine peptide" refers to a peptide with a length of 4 to 10 amino acids, where the N-terminal amino acid is formylated methionine and the C-terminal amino acid is lysine. A specific N-formyl-methionine peptide is the peptide N-formyl-methionine-leucine-phenylalanine-lysine-OH ("fMLFK", SEQ ID NO: 23).

[0040] As used herein, "linker" refers to a structure that connects two or more additional structures. Examples of linkers include peptide linkers, protein linkers, and PEG linkers. As used herein, a "maleimide-PEG linker" is a structure of the formula "-(O-CH2-CH2)" where "n" is between 6 and 24. nThe term "-" refers to a chemical moiety comprising a polyethylene glycol (PEG) polymer and a derivatized maleimide functional group, where the linker forms a covalent bond to the IgG antibody heavy chain via a thioether bond between the maleimide functional group and a cysteine ​​residue in the heavy chain constant region, and also forms a covalent bond to the N-formyl-methionine peptide via an amide bond to the epsilon-amino side chain of the C-terminal lysine of the N-formyl-methionine peptide. In a detailed embodiment, the maleimide-PEG linker of the compound of the present invention has the following structure, where the dashed lines indicate the positions of covalent bonds to the IgG antibody heavy chain and the N-formyl-methionine peptide: [ka] In the formula, "n" is between 6 and 24, and more specifically, "n" is 12.

[0041] In this case, the reagent used to prepare the test compound adopted in the following examples (Mal-dPEG12-OH (QuantaBiodesign catalog number 10285, lot IH1-A1240-80)) is a monodisperse reagent, meaning it contains a discrete number of ethyl-oxymonomer (O-CH2-CH2) units. Similarly, using this reagent will produce a conjugated antibody compound containing a maleimide-PEGn linker with n=12 (O-CH2-CH2) units.

[0042] However, as those skilled in the art will understand, pegylation reagents are often described by reference to the molecular weight (in daltons or kilodaltons) of the PEG polymer portion of the PEG-containing compound in the reagent. Furthermore, many commercially available PEG-containing reagents generally have some degree of polydispersity, meaning that the number of repeating ethylene glycol monomer units ("n") contained in the reagent varies over a range, typically a narrow range. Thus, a reference to the molecular weight of the PEG polymer in a polydisperse reagent is typically a reference to the average molecular weight of the PEG polymer contained in the reagent. The ethyl oxymonomer (O-CH2-CH2) of the reagent used to prepare the conjugated antibody compound of the present invention has a molecular weight of about 44 g / mol or 44 daltons. Therefore, those skilled in the art can easily determine the value of "n" when using a polydisperse pegylation reagent indicated by its average molecular weight, and similarly, the value of "n" in the resulting conjugated antibody compound.

[0043] "R1 may be substituted or not substituted C5~C 10 The term "substituted" as used in the phrase "aryl" means, for example, in this specification, that one or more substituents may be present, selected from atoms and groups, and that their presence in the compounds of formulas II, III, IV, V or VI does not prevent the compound from functioning as a chemical attractant. Substitution C5~C 10 Examples of substituents that can be present in an aryl group include hydroxyls, halides (I, Cl, F, BR), alkoxy groups (MeO-, EtO-, Pro, or C1-C4) covalently bonded to the aryl structure, or alkyl groups (Me-, Et-, Pr, or C1-C4).

[0044] The term diaminoalkyl refers to the structure -NH(CH2) n It is given by NH-, where n = 2 to 10.

[0045] The formyl group consists of a carbonyl group bonded to a hydrogen atom and is represented by the following structure, namely CH(=O), or by the following formula. [ka] Maleimide-diaminopropionic acid is coupled to Y via an amide bond to a free amine, resulting in the following structure: [ka] Maleimide refers to 3-maleimidopropionic acid, which is given by coupling to Y via an amide bond to a free amine, through the following structure. [ka]

[0046] As used herein, the term “patient in need of it” means a human or non-human mammal, more preferably a human, that has been diagnosed with a condition or disorder for which treatment or administration of the compounds of the present invention is indicated.

[0047] As used herein, the term “effective dose” refers to the amount or dosage of the conjugated antibody compound of the present invention that provides the desired pharmacological effect in a patient in a single or multiple administration. The effective dose can be readily determined by a diagnostician skilled in the art by considering many factors, including the species of the mammal, the size, age, and overall health of the mammal, the specific disease or surgical procedure involved, the degree or severity of the disease or illness, the individual patient’s response, the specific compound or composition administered, the mode of administration, the bioavailability characteristics of the administered preparation, the chosen treatment plan, and the use of any concomitant medications.

[0048] Cysteine-modified IgG antibodies for use in the present invention can be produced using techniques well known in the art, such as recombinant expression in mammalian animal cells or yeast cells. In particular, the methods and procedures of the examples herein can be readily adopted. Furthermore, the IgG antibodies of the present invention may be further modified to include framework regions entirely derived from human frameworks. Various different human framework sequences can be used in carrying out embodiments of the present invention. In detailed embodiments, the framework regions employed in the IgG antibodies of the present invention are of human origin or substantially human (at least 95%, 97%, or 99% of human origin). Sequences of human-derived framework regions are known in the art and can be obtained from The Immunoglobulin Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.

[0049] Expression vectors capable of guiding the expression of manipulatively linked genes are well known in the art. Expression vectors contain appropriate regulatory sequences, such as promoter sequences and replication initiation sites. Expression vectors may also encode appropriate selection markers and signal peptides that promote the secretion of a desired polypeptide product(s) from host cells. The signal peptide(s) may be immunoglobulin signal peptides or heterologous signal peptides. Nucleic acids encoding a desired polypeptide, for example, the HC and LC components of the conjugated IgG antibody of the present invention, can be expressed independently using different promoters manipulatively linked in a single vector, or alternatively, the nucleic acids encoding a desired product can be expressed independently using different promoters manipulatively linked in separate vectors. A single expression vector encoding both the HC and LC components of the cysteine-manipulated IgG antibody of the present invention can be prepared using standard methods.

[0050] As used herein, “host cell” means a cell that has been stably or transiently transfected, transformed, transduced, or infected with a nucleotide sequence encoding one or more desired polypeptide products. The creation and isolation of host cell lines that produce IgG antibodies for use in the present invention can be achieved using standard techniques known in the art. Mammalian animal cells are preferred host cells for the expression of cysteine-manipulated IgG antibodies according to the present invention. Specific mammalian animal cells include HEK293, NS0, DG-44, and CHO cells. Preferably, the assembled protein is secreted into the culture medium in which the host cells are cultured, from which the protein can be recovered and isolated. The medium from which the protein has been secreted can be purified by conventional techniques. For example, the medium can be applied to a protein A or protein G column using conventional methods and eluted therefrom. Soluble aggregates and polymers can be effectively removed by common techniques, including size exclusion, hydrophobic interactions, ion exchange, hydroxyapatite, or mixed-mode chromatography. The recovered product may be immediately frozen at -70°C, for example, or freeze-dried. As those skilled in the art will understand, when expressed in a particular biological system, such as a mammalian cell line, the antibody is glycosylated at the Fc region unless a mutation is introduced in the Fc region to reduce glycosylation. Furthermore, the antibody may be similarly glycosylated at other positions.

[0051] As used herein, "bacterial sugar" refers to the polysaccharide found on the outer surface of bacteria. An example of a bacterial sugar is carrageenan.

[0052] As used herein, “mimetic” refers to a molecule that functions similarly to a naturally occurring molecule. For example, a peptide mimetic may be a peptide, a modified peptide, or any other molecule that biologically mimics the active ligand of a hormone, cytokine, enzyme substrate, virus, or other naturally occurring molecule.

[0053] As used herein, “chemoattractant” refers to a structure, such as a peptide, that can attract and / or activate cells of the immune system. In preferred embodiments, a chemoattractant is a structure that can attract and activate cells of the immune system. Examples of chemoattractants include f-Met peptides, small molecule FPR-1 agonists, PRR agonists, peptide mimes, N-ureido peptides, and bacterial sugars. More specific examples include any one compound of formulas I-IV and any one peptide of sequence numbers 22, 36-39.

[0054] The following embodiments further illustrate the present invention and provide typical methods and procedures for carrying out various detailed embodiments of the invention. However, it will be understood that the embodiments are illustrated for illustrative purposes and that various modifications may be made by those skilled in the art. [Examples]

[0055] Example 1: Design of an IgG heavy chain constant region containing manipulated cysteine ​​residues The IgG heavy chain constant domain residues are selected to allow mutations using cysteine ​​designs manipulated with parent mAbs that have diverse variables or antigen-binding domains. Simply put, valine, alanine, and serine residues within the constant domain that are not important to the antibody's secondary and tertiary structures are selected for initial mutations in silico. H Using the published crystal structures of 1-C kappa Fab (pdb: 4DTG) and IgG4 Fc (pdb: 4C55), we design multiple different antibody single-cysteine ​​manipulated constructs. The genes encoding each mutant design are constructed in human IgG4 heavy chain and kappa light chain plasmids, expressed intracellularly, and mAbs containing unbound manipulated cysteine ​​are characterized by expression levels and analytical properties. Constructs retaining essentially the same target binding affinity and expression levels (determined by ELISA) as the parental wild-type mAb are produced on a scale at the smallest high molecular weight aggregates (<10%) before binding and further characterized.

[0056] Next, more than 20 mAb constructs, each containing a single cysteine ​​mutation manipulated to its respective HC and LC constant domain, are expressed in HEK293 cells, purified, and conjugated via a linker to cytotoxic payloads such as monomethyl auristatin E (MMAE) and cryptophycin. Binding efficiency is monitored by standard procedures such as ESI-TOF mass spectrometry or hydrophobic index chromatography (HIC), and aggregation tendency is measured by analytical size exclusion chromatography. Constructs with a binding efficiency exceeding approximately 60% and less than approximately 10% high molecular weight aggregates after binding to both payloads are further investigated through ex vivo plasma and in vivo stability testing.

[0057] Simply put, the conjugate is incubated with plasma for several days and analyzed by mass spectrometry to confirm that the payload is still conjugated to the antibody. Conjugate constructs containing residue mutations at S124C, S157C, A162C, S375C, or A378C in each HC have been shown to have adequate stability. Combining the HC 124C mutation with any of 157C, 162C, 375C, or 378C can yield higher antibody-drug ratios. Furthermore, C H Heavy chain residues 124, 157, and 162 in one domain, C H Residue 262 in the 2 domain, C H Residues 375, 378, and 397 in the 3 domains, and light chain residues 156, 171, 191, 193, 202, and 208 in the C-kappa domain were generated for binding with various formyl peptides.

[0058] In addition to monovalent IgG antibodies containing engineered cysteine ​​conjugated with chemoattractants, bivalent antibody constructs can also be developed using engineered cysteine ​​conjugated with chemoattractants as disclosed herein. Bivalent antibody constructs having engineered cysteine ​​include, but are not limited to, IgG-scFv form (reported in PCT / US2015 / 058719) and bivalent IgG form (disclosed in US2018 / 0009908). According to such bivalent antibody constructs, site-specific engineered cysteine ​​includes surface-exposed cysteine ​​for the binding of chemoattractants to the bispecific antibody. According to a specific embodiment (a bispecific antibody having a bivalent IgG form with two HCs in SEQ ID NOs. 34, 35 and two LCs in SEQ ID NOs. 58, 59), cysteine ​​at heavy chain residues 124 and 378 is engineered for the binding of chemoattractants. Expression and assembly of such exemplary constructs remained unchanged, but binding to the test peptide resulted in a CR comparable to that of monospecific antibodies.

[0059] Example 2: Synthesis of pegylated fMLFK peptide Example 2(A): Synthesis of formyl-Met-Leu-Phe-Lys(Mal-PEG12)-OH ("peptide-'183") (SEQ ID NO: 22). [ka] Peptide-'183, which has a hydrolyzed maleimide group and is used as an unbound peptide. [ka]

[0060] The chemotactic peptide formyl-Met-Leu-Phe-Lys-OH (SEQ ID NO: 23) is synthesized and purified as an HCl salt. This material is used as a substrate for further derivatization at the ε-amino group of lysine.

[0061] Peptides were produced via manual solid-phase peptide synthesis using standard Fmoc / tBu chemistry on a 0.3 mmol scale in a 100 mL frit glass manual reaction vessel from Ace Glassware Inc. The solid support used for synthesis was Fmoc-Lys(Boc)-Wang resin (NovaBiochem, catalog no. 8.56013, lot S6696713-529), 100-200 mesh, with a substitution of 0.57 meq / g. The standard amino acids used were Fmoc-Phe-OH (NovaBiochem, catalog no. 04-12-1030, lot A21653), Fmoc-Leu-OH (NovaBiochem, catalog no. 04-12-1025, lot A25917), and Fmoc-Met-OH (MidWest Biotech, catalog no. 12400, lot OP12240). Prior to each coupling step, the Fmoc group is removed by treatment with 20% piperidine in DMF (2 × 10 mins). All coupling is carried out for 6 hours using equiproportional amounts of Fmoc amino acids, diisopropylcarbodiimide (Sigma-Aldrich, catalog no. DI25407, lot 80896APV), and HOAt (AK Scientific, catalog no. D046, lot 1188G50I) in a 3-fold molar excess exceeding the theoretical peptide resin substitution of approximately 0.2 M final concentration in DMF. After coupling the last amino acid and removing the N-terminal Fmoc group, the mixture is formylated by treatment with a 6-fold excess of 2,4,6-trichlorophenyl formic acid (TCI, catalog no. T3121, lot P8AFA-PE) dissolved in 200 μL of diisopropylethylamine-containing DMF, and reacted at room temperature for 3 hours. Next, the resin is washed with DCM and diethyl ether and completely dried by applying vacuum suction to the reaction vessel for 5 minutes. The dry resin is treated with 25 mL of a cutting cocktail (TFA:anisole:water:triisopropylsilane = 88:5:5:1 (v / v)) at room temperature for 2 hours. The resin is filtered off, washed twice with 5 mL of anhydrous TFA, and the combined filtrate is treated with 50 mL of cold diethyl ether to precipitate the crude peptide.Next, the peptide / ether suspension was centrifuged at 4000 rpm for 4 minutes to form a solid pellet, the ether was decanted, the solid pellet was further pulverized twice with ether, and dried under vacuum for 30 minutes. The crude peptide was solubilized in 20% acetonitrile / water and purified by reverse-phase HPLC on a C18 preparative column (Phenomenex, Luna Phenyl-Hexyl, 21 × 250 mm) with a linear gradient of acetonitrile in 0.1% HCl-containing water to obtain the lyophilized peptide as an HCl salt (125 mg, 73% yield based on starting resin substitution). Purity was evaluated using analytical reverse-phase HPLC and found to be over 99%. Molecular weight was determined by analytical electrospray MS. Calculated value: 565.7 Da, measured value: 565.3 Da (average molecular weight). The following ion was observed: 566.3 (M+1H).

[0062] The ε-amino group of lysine is acylated as follows: Approximately 50 mg (approximately 0.088 mmol) of lyophilized peptide is dissolved in 5 mL of anhydrous DMF using an sonicator. In a separate scintillation vial, 74 mg (1.1 equivalents) of Mal-dPEG12-OH (Quanta Biodesign catalog no. 10285, lot IH1-A1240-80) is activated at room temperature for 25 minutes with 29 mg (1.1 equivalents) of TSTU (OakWood Chemicals, catalog no. 024891, lot 024891) and 61 μL (4 equivalents) of DIPEA in 1 mL of anhydrous DMF. The activated Mal-PEG12-OH is added dropwise to the peptide solubilized in DMF (1 mL), and 62 μL (5 equivalents) of triethylamine is added. The reaction is mixed at room temperature. After 1 hour, the reaction is stopped by adding cold diethyl ether. Next, the solution was separated and transferred to two 50 mL conical tubes, and a larger volume of cold ether was added to further precipitate the peptide. Then, the peptide / ether suspension was centrifuged at 4000 rpm for 4 minutes to form a solid pellet, the ether was decanted, the solid pellet was pulverized twice more with ether, and dried under vacuum for 30 minutes. The combined crude peptide pellet was solubilized in 20% acetonitrile / water and purified by reverse-phase HPLC using a C18 preparative column (Phenomenex, Luna Phenyl Hexyl 21×250 mm) with a linear gradient of acetonitrile in 0.1% TFA-containing water to obtain lyophilized peptide as TFA salt (44.4 mg, 38% yield based on starting material). Purity was evaluated using analytical reverse-phase HPLC and found to be over 96%. Molecular weight was determined by analytical electrospray MS. Calculated value: 1316.6 Da, Measured value: 1316.2 Da (average molecular weight). The following ions were observed: 659.0 (M+2H) and 1317.2 (M+1H). Next, this peptide (formyl-Met-Leu-Phe-Lys(Mal-PEG12)-OH) can be conjugated to an antibody as described in Example 3 below.

[0063] For the unbound peptide used in the following examples, the maleimide group was further hydrolyzed by incubating 20 mg of the product from Step 1 overnight at room temperature in 2 mL of 40 mM Tris-HCl buffer (pH 8.0). After 18 hours, the solution was diluted with 10 mL of 20% acetonitrile / water and purified by reverse-phase HPLC on a C18 preparative column (Phenomenex, Luna Phenyl Hexyl 21 × 250 mm) using a linear gradient of acetonitrile in 0.1% TFA-containing water to obtain the lyophilized peptide as a TFA salt (6.4 mg, 32% yield based on starting material). Purity was assessed using analytical reverse-phase HPLC and found to be over 94%. Molecular weight was determined by analytical electrospray MS: calculated: 1334.6 Da, measured: 1334.4 Da (average molecular weight). The following ions were observed: 668.0 (M+2H) and 1335.8 (M+1H).

[0064] Example 2(B): Synthesis of H-Met-Leu-Phe-Lys(Mal-PEG12-OH("peptide-'844"))(SEQ ID NO: 24). [ka] Peptide-'844, which has a hydrolyzed maleimide group and is used as an unbound peptide. [ka]

[0065] The formylated negative control peptide ((H-Met-LeuPhe-Lys-OH))(SEQ ID NO: 25) is produced by manual solid-phase peptide synthesis using the chemical properties of a standard fluorenyl methoxycarbonyl (Fmoc) / tertiary butyl group (tBu) on a 0.3 mmol scale. Peptide assembly is performed in a 100 mL frit glass manual reaction vessel from Ace Glassware Inc. The solid support used for synthesis is Fmoc-Lys(Mtt)-Wang resin (NovaBiochem, catalog number 8.56021, lot S6692621). 503), 100-200 mesh, 0.57 meq / g substitution. The standard amino acids used are Fmoc-Phe-OH (NovaBiochem, catalog number 04-12-1030, lot A21653), Fmoc-Leu-OH (NovaBiochem, catalog number 04-12-1025, lot A25917), and Fmoc-Met-OH (MidWest Biotech, catalog number 12400, lot OP12240).

[0066] The Fmoc group is removed prior to each coupling step by treatment with 20% piperidine in DMF (2 × 10 mins). All couplings are performed with equimwise Fmoc amino acids, diisopropylcarbodiimide (Sigma-Aldrich, catalog no. DI25407, lot 80896APV), and HOAt (AK Scientific, catalog no. D046, lot 1188G50I) at a 3x molar excess exceeding the theoretical peptide resin substitution level, and at a final concentration of approximately 0.2 M in DMF for 6 hours.

[0067] After coupling the last amino acid and removing the N-terminal Fmoc group, the peptidyl resin was protected with a Boc (butyloxycarbonyl) group by treatment with a 6-fold excess of Boc2O (NovaBiochem, catalog no. 01-63-0007, lot A25675) dissolved in 200 μL of diisoprolylethylamine-containing dimethylformamide (DMF), and reacted at room temperature for 3 hours. Next, the resin was washed eight times with dichloromethane (DCM), and the Mtt (4-methyltrityl) protecting group on the Lys residue was selectively removed by three consecutive treatments with 20% hexafluoroisopropanol (Oakwood Chemicals, catalog no. 003409) in DCM (2 × 10 min and 1 × 45 min) to expose the Lys residue to free epsilonamine for further reaction. The subsequent coupling of Fmoc PEG12-OH (BroadPharm, catalog number BP-22241) and 3-maleimidopropionic acid (Bachem, catalog number Q-2620) is carried out in the same manner as standard amino acid residues.

[0068] After synthesis is complete, the peptidyl resin is washed with DCM and diethyl ether and completely dried by vacuum suction of the reaction vessel for 5 minutes. The dried resin is treated with 25 mL of cutting cocktail (trifluoroacetic acid (TFA): anisole: water: triisopropylsilane = 88:5:5:1 (v / v)) at room temperature for 2 hours. The resin is filtered off, washed twice with 5 mL of anhydrous TFA, and the combined filtrate is treated with 50 mL of cold diethyl ether to precipitate the crude peptide. Next, the peptide / ether suspension is centrifuged at 4000 rpm for 4 minutes to form a solid pellet, the ether is decanted, the solid pellet is further pulverized twice with ether, and dried under vacuum for 30 minutes.

[0069] The crude peptide was solubilized in 20% acetonitrile / water and purified by RP-HPLC on a C18 preparative column (Phenomenex, Luna Phenyl-Hexyl, 21 × 250 mm) using a linear gradient of acetonitrile in 0.1% TFA-containing water to obtain the lyophilized peptide as a TFA salt (38.8 mg, 10% yield based on starting resin substitution). Purity was evaluated using analytical reverse-phase HPLC and found to be over 96%. Molecular weight was determined by analytical electrospray MS. Calculated value: 1288.5 Da, measured value: 1288.4 Da (average molecular weight). The following ions were observed: 645.0 (M+2H) and 1289.7 (M+1H). This peptide (H-Met-Leu-Phe-Lys(Mal-PEG12-OH)) can then be conjugated to an antibody as described in Example 3 below.

[0070] For the unbound peptide used in the following examples, the maleimide group was further hydrolyzed by incubating 20 mg of the product from step 1 overnight at room temperature in 2 mL of 40 mM Tris-HCl buffer (pH 8.0). After 18 hours, the solution was diluted with 10 mL of 20% acetonitrile / water and purified by reverse-phase HPLC on a C18 preparative column (Phenomenex, Luna Phenyl Hexyl 21 × 250 mm) using a linear gradient of acetonitrile in 0.1% TFA-containing water to obtain the lyophilized peptide as the TFA salt (5.2 mg, yield 26% based on starting material). Purity was assessed using analytical reverse-phase HPLC and found to be over 96%. Molecular weight was determined by analytical electrospray MS. Calculated value: 1306.6 Da, measured value: 1306.4 Da (average molecular weight). The following ions were observed: 654.0 (M+2H) and 1307.7 (M+1H).

[0071] Example 2(c): Synthesis of formyl-Nle-Leu-Phe-PEG12-Lys(maleimide-propionyl)-OH ("fNle", SEQ ID NO: 42) [ka]

[0072] The chemotactic peptide formyl-Nle-Leu-Phe-PEG12-Lys-OH is synthesized as its HCl salt (Peptides International) and used as a substrate for derivatization without further modification.

[0073] The acylation of the ε-amino group of lysine is carried out as follows: Using an ultrasonic device, approximately 50 mg (approximately 0.044 mmol) of lyophilized peptide is dissolved in 5 mL of anhydrous DMF. In a separate scintillation vial, 8.1 mg (1.1 equivalents) of maleimidopropionic acid (Bachem, catalog no. Q-2620, lot 0564230) is activated at room temperature for 25 minutes using 1 mL of anhydrous DMF with 14.5 mg (1.1 equivalents) of TSTU (OakWood Chemicals, catalog no. 024891, lot 024891) and 33.4 μL (4 equivalents) of DIPEA. The activated maleimidopropionic acid is added dropwise to the solubilized peptide in DMF (1 mL), and then 30 μL (5 equivalents) of triethylamine is added, and the reaction is mixed at room temperature. After 1 hour, the reaction is stopped by adding cold diethyl ether. Next, the solution was separated and transferred to two 50 mL conical tubes, and a larger volume of cold ether was added to further precipitate the peptide. Then, the peptide / ether suspension was centrifuged at 4000 rpm for 4 minutes to form a solid pellet, the ether was decanted, the solid pellet was pulverized twice more with ether, and dried under vacuum for 30 minutes. The combined crude peptide pellet was solubilized in 20% acetonitrile / water and purified by reverse-phase HPLC on a C18 preparative column (Phenomenex, Luna Phenyl Hexyl 21 × 250 mm) with a linear gradient of acetonitrile in 0.1% TFA-containing water to obtain lyophilized peptide as TFA salt (8.6 mg, 15.1% yield based on starting material). Purity was evaluated using analytical reverse-phase HPLC and found to be over 97%. Molecular weight was determined by analytical electrospray MS. Calculated value: 1298.5 Da, Measured value: 1298.8 Da (average molecular weight). The following ions were observed: 650.0 (M+2H) and 1299.8 (M+1H). Next, this peptide can be conjugated to an antibody as described in Example 3 below.

[0074] Example 3: Binding of IgG antibody to peptide Antibody-peptide bioconjugation can be prepared as follows: The parent antibody containing the manipulated cysteine ​​residue is buffered using a Zeba® spin desalting column (40K MWCO) to 50 mM tris(hydroxymethyl)aminomethane (Tris-HCl), 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5, to a final concentration of 5 mg / ml. Freshly prepared 100 mM dithiothreitol (DTT), solubilized in MilliQ water, is added to the antibody in a 40-fold molar excess. The reaction mixture is incubated at room temperature for 16 hours. After incubation, the reaction mixture is buffered using a Zeba spin desalting column to 50 mM tris(hydroxymethyl)aminomethane (Tris-HCl), 150 mM sodium chloride (NaCl), pH 7.5, to remove excess unreacted DTT.

[0075] 100 mM dehydroascorbic acid (dHAA) in freshly prepared dimethylacetamide is added to the antibody in a 30-fold molar excess and incubated at room temperature for 3 hours. After incubation, 4, 8, or 12-fold molar excesses of formyl-Met-Leu-Phe-Lys(Mal-PEG12)-OH (SEQ ID NO: 22), H-Met-Leu-Phe-Lys(Mal-PEG12)-OH (SEQ ID NO: 24), or formyl-Nle-Leu-Phe-PEG12-Lys(maleimide-propionyl)-OH (synthesized as described in Examples 2(A), 2(B), and 2(C), respectively) are added (dissolved in molecular-grade water) to antibodies having 1, 2, or 3 manipulated cysteine ​​residues, respectively, to obtain bioconjugates in ratios of 2, 4, or 6. This reaction mixture is incubated at room temperature for 1 hour. After incubation, the sample is buffer-changed to the desired buffer, and excess unbound peptides are removed using a desalting column, preparative size exclusion chromatography (pSEC), or dialysis.

[0076] Table 1 provides conjugated and unconjugated IgG antibody constructs to be tested in the following assays, which are prepared essentially as described herein and previously and include antibody HC and LC sequences and pegylated peptides used for conjugation. As used herein, “emibetuzumab,” “TMab” (trastuzumab), and “AME133” refer to antibody constructs containing the variable regions of the indicated antibodies. [Table 1-1] [Table 1-2]

[0077] Example 4: Determination of the bond ratio The binding ratios of peptide-183 on the cysteine-operated heavy chains of TMab ("trastuzumab"), AME133, and emibetuzumab constructs are determined by untreated mass spectrometry using a weighted average of conjugate additions. Untreated mass measurements are collected using an Agilent 1290HPLC in combination with an Agilent 6230 ESI-TOF mass spectrometer. The sample (2 ug) is analyzed using a PLRP-S reversed-phase column (Agilent) with water / 0.2% formic acid as mobile phase A and acetonitrile / 0.2% formic acid as mobile phase B, at a flow rate of 0.3 ml / min over 4 minutes using a 20-70% B gradient elution. The Agilent 6230 TOF is operated with 4000V cation mode, 65V skimmer, 300V fragmenter, 350°C gas temperature, 12psi dry gas, and 40psi atomizer gas. MS scanning is performed at 1 scan / second from 600m / z to 5000m / z. Data is collected from 2 to 15 minutes, and the molecular weight of proteins is determined by summing the TIC peak spectra and then summing the deconvolutions using Agilent Mass Hunter and Bioconfirm version 7.0. Deconvolutions of non-reducing samples are 50000–190000 Da., with a peak width of 1.0 Da., and consist of 20 repeats and a 1 Da. step. [Table 2]

[0078] Samples for serum stability were prepared by adding 50 μl of 1 mg / ml antibody conjugate to mouse serum and incubating at 37°C for 0.5–48 hours with shaking at 300 RPM. All in vivo or serum stability samples required extraction from the biological matrix prior to determining the binding ratio. The biological fluid was centrifuged at 13,000 RPM for 10 minutes and then applied to a human Fc selective affinity column using a stepped gradient. The bound antibody was captured in mobile phase A (PBS, pH 7.4) and eluted with 0.2% (V / V) formic acid. Sample fractions were collected manually and dried to 50–100 μl using vacuum centrifugation at low heat. The off-target rate indicated the attachment of the biological conjugate to sites other than the intended cysteine. The following data were obtained following the procedure described above. [Table 3] [Table 4]

[0079] These data demonstrate that the binding of monoclonal antibodies to formylated peptide constructs at manipulated cysteine ​​sites 124, 157, 375, and / or 378, via the maleimide chemistry, results in a peptide-to-antibody binding ratio predicted by the number of cysteines attached to the antibody, as demonstrated by the off-target rate.

[0080] Example 5: TMab bioconjugate that binds human HER2 The binding of TMab to human HER2 was determined by ELISA using a 96-well cell culture plate coated with human HER2. The plate was exposed to the conjugating antibody for 80 minutes, washed to remove the unconjugating antibody, and incubated with the secondary antibody for 50 minutes. After washing the plate, it was allowed to develop at 37°C for 25 minutes. Binding was measured using a 96-well plate reader at an optical density of 560. The following data were obtained by essentially following the procedure described above. [Table 5] The antibody constructs are named according to the same rules as described in Table 1 of Example 1 of this specification.

[0081] These data demonstrate that the binding of TMab to human Her2 is unaffected by heavy chain modification to introduce cysteine ​​at sites 124 and 378, and also by the binding of peptide-'183 to cysteine ​​residues at sites 124 and 378.

[0082] Example 6: PMN chemotaxis Chemotaxis is measured by observing the migration of primary human polymorphonuclear neutrophils (PMNs) to antibody conjugates via a Transwell membrane (Corning 3415) in a modified Boyden chamber assay. Approximately 2-4 × 10⁶ PMNs from neutrophil concentrates are observed. 5The cells are seeded into an upper transwell chamber on a membrane with pores of 3.0 μm. The lower transwell chamber contains a solution consisting of buffer, fMLF (N-formyl-Met-Leu-Phe peptide as a positive control), and an experimental antibody bioconjugate. Some experiments also included fMLFK(Mal[OH]-PEG12)-OH(hydrolyzed peptide-'183) and H-Met-Leu-Phe-Lys(Mal[OH]-PEG12-OH(hydrolyzed peptide-'844)) as positive controls. After seeding in Transwell, cells were placed in a humidified incubator at 37°C. After 1 hour, any cells in the upper chamber were removed, and the percentage of cells that successfully migrated to the lower chamber was quantified using CellTiter-Glo® (Promega G7571) according to the manufacturer's specified protocol. The migration rate was defined as (number of cells that migrated to the lower chamber / number of cells initially seeded). A standard curve was used to determine the cell count. All data were converted to a percentage of the maximum fMLF response for each individual experiment.

[0083] N-formyl modification is required to stimulate PMN chemotaxis. To determine the ability of N-formyl-modified peptides to induce PMN migration, primary human PMN was exposed to peptides with and without N-formyl modification, and the PMN migration response was measured. Following essentially the procedure described earlier, PMN responded maximally to fMLF, peptide-'183, and peptide-'844 at concentrations of 10 nM, 1 nM, and 1 μM, respectively (Table 4). Peptide-'844 was similar to peptide-'183 except for the lack of an N-formyl group, and was 1000-fold less potent in inducing PMN migration, as indicated by the dose-response difference between peptide-'183 and peptide-'844. The values ​​are given as the migration rate of PMN relative to 10 nM fMLF. [Table 6]

[0084] These data demonstrate that N-formyl modification of peptides is important for inducing PMN chemotaxis.

[0085] Formyl peptide mutants induce neutrophil chemotaxis. Primary human neutrophils are exposed to formyl peptide, and the PMN migration response is measured essentially as described above, except that raw migration values ​​are retained instead of being converted to cell counts. Following essentially the procedure described earlier, the following data are provided as a percentage of 100 nM fMLF. [Table 7]

[0086] These data demonstrate that modifications to the amino acid sequence and linker of formyl peptides can induce FPR1-mediated neutrophil migration. PEG-bound peptides [peptide-'183, FRM-021, FRM-029, FRM-030, and FRM-031] maximally induced neutrophil migration at exposure concentrations of 1–3 nM.

[0087] The role of N-formyl peptide amino acid sequence and binding site in driving PMN chemotaxis Human anti-MET IgG4 antibody (emibetuzumab) is modified to include a cysteine ​​residue at either CH1-S124 or CH3-A378 of each HC. The modified antibody binds to either peptide-'183 or f-Nle (formyl-Nle-Leu-Phe-PEG12-Lys(maleimide-propionyl)-OH) in a peptide-to-antibody ratio of approximately 2:1. Primary human PMNs are exposed to these different antibody conjugates, and the PMN migration response is measured.

[0088] The antibody-peptide bioconjugates are as follows: emibetuzumab-G4-fMLFK-HC-378C, emibetuzumab-G4-fNle-HC-378C, emibetuzumab-G4-fMLFK-HC-124C, and emibetuzumab-G4-fNle-HC-124C.

[0089] Essentially following the procedure described earlier, the fNle-conjugated antibody was less potent in stimulating PMN migration than the peptide-'183-conjugated antibody. Antibodies conjugated to peptide-'183 at sites A378 and S124 maximally induced PMN migration at 30 nM, resulting in migration responses equivalent to 99.1% and 117.8% of fMLF and the control, respectively. In contrast, the fNle antibody conjugate maximally induced PMN migration at 100 nM, resulting in migration responses equivalent to 71.7% and 76.5% of fMLF and the control, respectively. The values ​​in Table 5 below are given as PMN migration rates relative to 100 nM fMLF. [Table 8]

[0090] These data demonstrate that antibodies conjugated to peptide-'183 are significantly more potent than fNle antibody conjugates in inducing PMN migration. Both the A378 and S124 sites are suitable for N-formyl peptide conjugation.

[0091] A higher conjugation ratio between the peptide and the antibody increases the PMN migration response. Human anti-MET IgG4 antibodies (emibetuzumab) modified with amino acids at CH1-124C and 378C or 378C alone are conjugated to peptide-'183. Primary human PMNs are exposed to these antibody conjugates, and the PMN response is measured.

[0092] Following the essential procedures described earlier, emibetuzumab-G4-fMLFK-HC-124C-378C maximally induced migration at 12.5 nM, and emibetuzumab-G4-fMLFK-HC-378C maximally induced migration at 25 nM, inducing migration responses equivalent to 119.3 and 124.3 percent of the fMLF control, respectively (Table 6). Unconjugated antibodies did not induce PMN migration compared to conjugated antibodies. Values ​​are given as PMN migration rates relative to 3.12 nM fMLF. [Table 9]

[0093] These data demonstrate that increasing the ratio of peptide to antibody proportionally affects the relationship between PMN migration concentration and response.

[0094] TMab (trastuzumab) and AME133 antibody conjugate TMab-G1-fMLFK-HC-124C-378C, AME133-G1(IQ)-fMLFK-HC-124C-378C, and emibetuzumab-G4-UC-124C-378C were studied in essentially the PMN chemotactic assay described earlier. TMab-G1-fMLFK-HC-124C-378C and AME133-G1(IQ)-fMLFK-HC-124C-378C maximally induced PMN migration at 10 nM and 3 nM, respectively. Emibetuzumab-G4-UC-124C-378C did not induce PMN migration compared to the conjugated antibody. The values ​​are given in Table 7 below and represent the PMN migration rate relative to 30 nM fMLF. [Table 10]

[0095] These data demonstrate that TMab and AME133 antibodies conjugated to N-formyl peptide effectively induce PMN migration. Therefore, the conjugated antibodies of the present invention are considered useful for attacking cancer cells by utilizing the body's immune system.

[0096] Example 7: Production of reactive oxygen species (ROS) in PMN Polymorphonuclear neutrophils (PMNs) can produce reactive oxygen species (ROS) upon stimulation and possess ROS-producing enzymes such as myeloperoxidase. Stimulation of PMNs induces degranulation and releases pre-formed ROS and ROS-producing enzymes into the extracellular environment as a primary mechanism for responding to pathogens. Stimulation of ROS production by PMNs is sufficient to damage and kill a wide range of targets, from bacteria to eukaryotic cells. One of the most effective pathways for stimulating PMNs to produce ROS involves the involvement of formyl peptide receptor 1 (FPR1) on PMNs by N-formyl peptide. Involvement of Fc receptors by antibodies on PMNs is also an effective mechanism for inducing ROS production.

[0097] ROS production by human primary PMN is measured using luminol-amplified chemiluminescence. After isolation, PMN is added to HBSS containing calcium and magnesium (Gibco #14025-092) supplemented with 0.25% human serum albumin (Gemini Bio product #800-124) and 50 μM luminol (Sigma-Aldrich #123072-2.5G), with 1 × 10⁶ PMN added. 6 Suspend at a concentration of cells / ml. Next, add 100 μl of cell suspension (total cell count 1 × 10⁶). 5 Dispense the antibody conjugate into each well of a 96-well plate (Greiner #655098) suitable for fluorescence measurement, and equilibrate the temperature to 37°C for 5 minutes. After equilibration, apply a 10× solution of the antibody conjugate to each well to achieve the 1× final concentration.

[0098] Immediately after adding the antibody conjugate, the chemiluminescence signal was recorded using a luminometer maintained at 37°C (PerkinElmer EnVision Multilabel Plate Reader) with a residence time of 0.01 seconds per well, a total time between consecutive plate reads of 20 seconds, and a total trial time of 45 minutes. The area under the curve (AUC) score was calculated using the luminescence signal for the first 5 minutes of each trial, indicating the relative amplitude of the initial ROS burst for each exposure condition. Formyl-Met-Leu-Phe (fMLF) peptide was used as the positive control, and cyclosporine H was used as the FPR1 inhibitor. The value was expressed as the percentage of the fMLF control at the maximum exposure concentration ((AUC exposure condition / AUC fMLF) × 100).

[0099] Primary human polymorphonuclear neutrophils (PMNs) were exposed to peptides or bioconjugates, and ROS production was measured using luminol-amplified chemiluminescence, essentially as previously described. Following essentially the previously described procedure, N-formyl peptides conjugated to monoclonal antibodies with the indicated manipulated cysteine(s) effectively bound to the formyl peptide receptor expressed by primary human polymorphonuclear neutrophils, stimulating the production of cytotoxic reactive oxygen species. The stimulation of ROS production by the conjugated N-formyl peptide was primarily FPR1-dependent, as inhibition of FPR1 signaling by the FRP1 antagonist cyclosporine H significantly reduced PMN ROS production in response to the N-formyl peptide conjugated antibody. Examples of specific antibody conjugates are shown below.

[0100] N-formyl modification of peptides Primary human PMNs were exposed to peptides, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 8 below, and the data are reported as a percentage of 10 μM fMLF, using area under the curve calculation for luminescence recorded 5 minutes after exposure to the antibody conjugate. [Table 11]

[0101] These data demonstrate that PMNs exposed to peptide-'183 produced more ROS than observed for fMLF at concentrations of 10 nM to 10 uM. Peptide-'844-stimulated ROS production was substantially less than that observed for fMLF, indicating that peptide N-formyl modification is necessary for effective stimulation of ROS production by PMNs.

[0102] Formyl peptide mutants induce ROS production in neutrophils. Primary human neutrophils were exposed to formyl peptide mutants containing amino acid substitutions, including synthetic amino acids, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 8b below, and the data are reported as a percentage of 3000 nM fMLF using area under the curve calculation for luminescence recorded 5 minutes after exposure to the reagent. EC50 values ​​were calculated using the Best-Fit value from Graphpad PRISM. [Table 12]

[0103] These data demonstrate the efficacy of the exemplified formyl peptide variants for inducing ROS production. Incorporation of non-coding amino acids can improve peptide stability, and it is expected that incorporating non-coding amino acid variants will strengthen the binding between formyl peptide and FPR1, resulting in increased efficacy.

[0104] Mouse neutrophil FPR-1 is more sensitive to fMIFL peptides and antibody conjugates than to fMLF derivatives. Mouse neutrophils purified from bone marrow were exposed to formyl peptide or antibody conjugates, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 8c below, and the data are reported as a percentage of 10,000 nM fMLF, using area under the curve calculation for luminescence recorded 5 minutes after exposure to the reagent. [Table 13]

[0105] These data demonstrate that mouse neutrophils are significantly more sensitive to fMLF peptides and antibody conjugates than fMLF mutants. In humans, fMLF is one of the most potent FPR1 agonists, but its efficacy is significantly lower in mouse experiments. This relationship between mouse and human neutrophil FPR1 holds true regardless of whether the FPR1 agonist is a soluble peptide or conjugated to an antibody.

[0106] TMab bioconjugate Primary human PMNs were exposed to TMab bioconjugates, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 9 below, and the data are reported as a percentage of 1000 nM fMLF, using area under the curve calculation for luminescence recorded 5 minutes after exposure to the reagent. [Table 14]

[0107] These data demonstrate that PMNs exposed to 1000 nM TMab-G1-fMLFK-HC-124C-378C produced ROS at levels equivalent to 70.1% of fMLF controls, and significantly higher than those exposed to TMab-G1-UC-HC-124C-378C.

[0108] Emibetuzumab conjugate Primary human PMNs were exposed to emibetuzumab conjugates, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 10 below, and the data are reported as a percentage of 1000 nM fMLF using area under the curve calculation for luminescence recorded 5 minutes after exposure to the antibody conjugate. [Table 15]

[0109] These data demonstrate that PMNs exposed to 1000 nM emibetuzumab-G4-fMLFK-HC-124C-378C and emibetuzumab-G4-fMLFK-HC-378C produced ROS at levels equivalent to 62.2% and 48.9% of the 1000 nM fMLF control, respectively. Exposure to 1000 nM emibetuzumab-G4-UC-HC-124C-378C resulted in low ROS production, equivalent to only 32.2% of the control.

[0110] AME133 (anti-CD20) conjugate Primary human PMNs were exposed to AME133 antibody conjugates, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 11 below, and the data are reported as a percentage of 1000 nM fMLF, using area under the curve calculation for luminescence recorded 5 minutes after exposure to the antibody conjugate. [Table 16]

[0111] These data demonstrate that PMNs exposed to 1000 nM AME133-G1(IQ)-fMLFK-HC-124C-378C and AME133-UC produced ROS levels equivalent to 77.9% and 13.9% of the control group, respectively.

[0112] Inhibition of antibody conjugates and FPR1 signaling To determine whether conjugated antibodies induce more ROS production than unconjugated antibodies, ROS production is measured essentially as described earlier. All peptides are tested at a final concentration of 300 nM. PMN is pre-incubated with 1 uM cyclosporine H for 30 minutes before adding the peptides.

[0113] The buffer solution was HBSS (Gibco#14025-092) containing calcium and magnesium, supplemented with 0.25% human serum albumin (Gemini Bio product#800-124) and 50 μM luminol (SigmaAldric#123072-2.5G). The values ​​are reported in Table 12a below and are expressed as the percentage of the fMLF area under the curve for luminescence recorded 5 minutes after exposure to the antibody conjugate. [Table 17]

[0114] These data demonstrate that antibodies conjugated to fMLFK induce substantially greater ROS production from human PMN compared to unconjugated antibodies. The data also demonstrate that pretreatment of PMN with cyclosporine H, an FPR1 antagonist, results in a substantial reduction in ROS levels in antibody bioconjugates, but not in unconjugated controls.

[0115] Antibody mutations that enhance FcγR3 binding increase FPR1-mediated ROS production in response to N-formyl peptide bioconjugates. Primary human neutrophils were exposed to the Tmab N-formyl peptide conjugate with and without Fc region mutations that increase affinity for FcγR3 (247I, 339Q, ±332E mutations). ROS production was measured using luminol-amplified chemiluminescence, essentially as described earlier. The data are shown in Table 12b below, and the data are reported as a percentage of 1000 nM fMLF using area under the curve calculation of luminescence recorded 5 minutes after exposure to the reagent. EC for FPR1-mediated ROS production. 50 The value is calculated using the Best-Fit value in Graphpad PRISM. [Table 18]

[0116] These data demonstrate that N-formyl-Met bioconjugates can be manipulated to further enhance ROS production by optimizing neutrophil-mediated FcR involvement. Fc-optimized Tmab bioconjugates with IQ and IQE amino acid substitutions enhanced neutrophil-stimulated ROS production compared to wild-type Tmab IgG1 conjugates, and Tmab-G1-fMLFK-HC-124C-378C-IQ and Tmab-G1-fMLFK-HC-124C-378C-IQE mutants enhanced EC compared to Tmab-G1-fMLFK-HC-124C-378C, respectively. 50 This showed improvements of 2.98 and 14.9 times. Improved Fc manipulation in the activation of PMN cell killing mechanisms is expected to yield substantial benefits to neutrophil-mediated conjugation antibody-mediated cell killing.

[0117] Length of the compound linker Primary human neutrophils were exposed to N-formyl peptide Tmab conjugates with PEG linkers of various lengths, and ROS production was measured using luminol-amplified chemiluminescence, essentially as described above. The data are shown in Table 12c below, and the data are reported as a percentage of 3000 nM FRM-023 (SEQ ID NO: 40) using area under the curve calculation of luminescence recorded 5 minutes after exposure to the reagent. The EC50 value for ROS production via FPR1 was calculated using the Best-Fit value in Graphpad PRISM. [ka]

[0118] These data demonstrate that the N-formyl peptide conjugate maintains its functionality as an FPR1 agonist with PEG of various sizes.

[0119] Example 8: The antibody conjugate enables neutrophil-mediated tumor cell killing. This study aims to determine the ability of antibody compounds to target PMNs in tumors and to be involved in tumor cell killing. The ability of TMab, emibetuzumab, and AME133 antibody conjugates to involve PMNs in tumor cell killing will be evaluated in solid and humoral tumors.

[0120] Antibody-targeted killing of tumor cells by PMN is measured using the xCelligence Real-Time Cell Analysis System (ACEA Biosciences). This system monitors cell viability in real time by recording the electrical impedance between sensors on the growth surface of the culture plate. It reports a normalized cell index (NCI) normalized to control cells in parallel wells, allowing control over relative culture viability. The NCI is measured continuously for 24 hours at 15-minute intervals after incubation of tumor cultures with target antibodies, with human primary PMN added at a PMN-to-tumor cell ratio of 10:1. Prior to seeding tumor cells, the xCelligence 96-well E-Plate is calibrated for background signals. 50 μl of medium (RPMI + 10% FBS + antibiotic) is placed in each well, and the E-plate is equilibrated to 37°C in a humidified incubator equipped with an xCelligence plate reader.

[0121] After equilibration, the background change in the E-Plate wells is measured. Cultured tumor cell lines are isolated, counted, and 1 × 10⁶ cells are added to the culture medium. 5 Diluted to the final density of cells / ml, 100 μl of diluted tumor cells were seeded into E-Plate wells. The E-Plate was returned to the xCelligence reader, and the cell index was measured overnight at 15-minute intervals to establish a baseline.

[0122] The following day, PMN was isolated from fresh human blood samples, and the final density in the culture medium was 2 × 10⁻⁶. 6 Adjust to cells / ml. After recording overnight, remove the E-Plate from the xCelligence reader and add 22 μl of 10× antibody solution or buffer to the designated well. After 15 minutes, add 50 μl of diluted PMN (total cell count 1×10). 5The PMN (individual) or buffer was added to the designated wells. Immediately after adding PMN, the E-Plate was returned to the xCelligence reader, and the cell index was measured for up to 72 hours. After completion of this experiment, the cell index was normalized to the point immediately before antibody addition (NCI).

[0123] The percentage of NCIs is defined as ((NCIs of sample) / (NCIs of tumor cells alone) × 100). For non-adherent tumor cells (Daudi cells), tumor cells are fixed in E-Plate wells using the xCelligence Immunotherapy Kit-B Cell Killing Assay (ACEA#8100004) according to the manufacturer's protocol. After fixation and background acquisition, the protocol is carried out as previously described.

[0124] The data shown below demonstrates that antibodies conjugated to N-formyl peptide lead to PMN-mediated elimination of tumor cells.

[0125] N-formyl-Met-Leu-Phe peptide Two N-formylated peptides, f-Met-Leu-Phe and Peptide-'183, were assessed in the SKOV3 tumor cell killing assay to determine the effect of N-formylmethionine peptides on PMN-mediated tumor cell killing in the absence of monoclonal antibody targeting.

[0126] The NCI percentage value represents the relative viability of SKOV3 cells after 2 hours of exposure to the specified conditions. The value is given as the percentage of SKOV3 controls ± the normalized mean, with n=4 for all conditions. Statistical significance is determined by one-way ANOVA followed by post-Hock Dunnett's multiple comparison test and "+PMN". [ka]

[0127] These data demonstrate that in the absence of PMN, the peptides had no statistically significant effect on tumor cell viability. In the presence of PMN, these peptides caused a reduction in NCI only at the highest peptide concentrations.

[0128] TMab Adherent HER2(+)SKOV3 human adenocarcinoma tumor cells were seeded for approximately 24 hours and then incubated with TMab-G1-fMLFK-HC-124C-378C or TMab-G1-UC-HC-124C-378C, exposing them to primary human PMN at an effector-target-to-cell ratio of 10:1.

[0129] The NCI percentage value represents the relative viability of SKOV3 cells after 2 hours of exposure to the specified conditions. The values ​​are given in Table 14 below and are expressed as the normalized mean percentage ± standard deviation relative to the SKOV3 control. N=4 is used for all conditions. [ka]

[0130] These data demonstrate that after 2 hours, cells incubated with 10 nM TMab-G1-fMLFK-HC-124C-378C and exposed to PMN showed a decrease in normalized cellular index (NCI) equal to 63.5 ± 9.9% (p < 0.0001) of control cells, while cells exposed to 10 nM TMab-G1-UC-HC-124C-378C maintained an NCI of 103 ± 1.2% of control cells (not statistically significant). TMab-G1-fMLFK-HC-124C-378C did not reduce tumor cell viability after 2 hours in the absence of PMN, and the addition of PMN without antibody did not affect the viability of SKOV3 tumor cells.

[0131] Emibetuzumab Adherent MET(+) A549 human lung cancer cells were seeded for approximately 24 hours and then incubated with emibetuzumab-G4-fMLFK-HC-124C-375C or emibetuzumab-G4-UC-HC-124C-375C and exposed to primary human PMNs at an effector-to-target cell ratio of 10:1.

[0132] Following the procedure essentially as described previously, the following data were obtained and are shown in Table 15.

Table 19

[0133] The values of the percentage of NCI values represent the relative viability of A549 cells after exposure to the specified conditions for 2 hours. Values are given as the mean percentage normalized to the “+PMN” control ± standard deviation, with n = 4 for all conditions. Statistical significance was determined by one-way analysis of variance followed by post hoc Dunnett's multiple comparison test and “+PMN”. NCI: normalized cell index, PMN: primary human polymorphonuclear neutrophils, ns: not significant.

[0134] These data demonstrate that cultures exposed to 10 nM emibetuzumab-G4-fMLFK-HC-124C-375C in the presence of PMNs showed a reduction in NCI equal to 87.7 ± 0.9% of control cells after 2 hours of incubation, whereas emibetuzumab-G4-UC-HC-124C-375C-treated cells maintained an NCI of 102.5 ± 1.9% of control cells.

[0135] Example of AME133 Non-adherent CD20+ Daudi B lymphoblasts were fixed with the xCelligence immunotherapy kit (ACEA#8100004), and tumor cells were immobilized in E-Plate wells according to the manufacturer's protocol and exposed to the conditions shown in Table 16 below. The NCI percentage value represents the relative viability of DAUDI cells after 6 hours of exposure to the specified conditions. The value is given as the mean percentage ± standard deviation normalized to the "buffer control," with n=4 for all conditions. Statistical significance was determined by one-way ANOVA followed by post-Hock-Dunnett's multiple comparison test pair "+PMN." [Table 20]

[0136] These data showed that cultures exposed to 30nM AME133-G1(IQ)-fMLFK-124C-378C reduced NCI to 20±2.1% (p<0.0001) of control cells after 6 hours of incubation, while 30nM AME133-G1(IQ)-UC-124C-378C maintained 97.3±1.2% of control cell NCI. Neither AME133-G1(IQ)-fMLFK-124C-378C nor AME133-G1(IQ)-UC-124C-378C reduced tumor cell viability in the absence of PMN. However, exposure of Daudi cells to PMN in the absence of the antibody reduced tumor culture NCI to 66.9±5.2% of control cells (p<0.0001).

[0137] Conjugation of a single antibody conjugate with formyl peptides to multiple cysteine ​​molecules increases its potency. Primary human neutrophils were exposed to IgG4 antibody conjugates with varying numbers of manipulated cysteine-binding sites, and ROS production was measured using luminol-amplified chemiluminescence, essentially as previously described. The following data were obtained following the procedure essentially as previously described. [Table 21]

[0138] The data in Table 17 are reported as a percentage of 1000 nM fMLF, using area under the curve calculation for luminescence recorded 5 minutes after exposure to the reagent.

[0139] These data demonstrate that antibodies conjugated to fMLFK can become more potent at additional conjugation sites.

[0140] Practical Embodiments The following includes a list of exemplary embodiments of the Disclosure that represent various embodiments of the Disclosure.

[0141] These exemplary embodiments are not intended to be exhaustive or to limit the disclosure to the exact form disclosed, but rather are provided to help illustrate the disclosure further so that those skilled in the art can utilize these teachings.

[0142] 1. An antibody comprising an IgG heavy chain constant region and a light chain constant region, wherein the antibody contains the following residue, namely C H Residue 124 in one domain, C H Residue 157 in one domain, C H 162 residues in one domain, C H Residue 262 in the 2 domain, C H Residue 375 in domain 3, C H Residue 373 in the 3 domain, C H Residues 397 in the 3 domains, C H Residue 415 in the 3 domains, C カッパ Residue 156 in the domain, C カッパ Residue 171 in the domain, C カッパ Residue 191 in the domain, C カッパ Residue 193 in the domain, C カッパ Residue 202 in the domain, or C カッパ An antibody containing cysteine ​​in at least one of the 208 residues in its domain.

[0143] 2. The antibody is CH The antibody according to embodiment 1, comprising cysteine at residue 124 in the 1 domain, and the C H The antibody according to embodiment 1, comprising cysteine at residue 157 and residue 162 in the 1 domain, and the C H The antibody according to embodiment 1, further comprising cysteine at one but not all of residue 375 and residue 378 in the 3 domain.

[0144] 3. The antibody according to embodiment 1 or 2, wherein the antibody comprises cysteine at residue 157 in the CH1 domain.

[0145] 4. The antibody according to embodiment 2, wherein the antibody comprises cysteine at residue 375 in the CH3 domain.

[0146] 5. The antibody according to embodiment 2, wherein the antibody comprises cysteine at residue 378 in the CH3 domain.

[0147] 6. The antibody according to any one of embodiments 1 to 4, wherein the IgG heavy chain constant region is a human, mouse, rat, or rabbit IgG constant region.

[0148] 7. The antibody according to embodiment 5, wherein the IgG heavy chain constant region is a human IgG1 or human IgG4 isotype.

[0149] 8. The antibody according to embodiment 6, wherein the IgG heavy chain constant region is human IgG1.

[0150] 9. The antibody according to embodiment 1, wherein the heavy chain constant region is human IgG1 provided by the amino acid sequence of SEQ ID NO: 17, 18, 19, or 52.

[0151] 10. The antibody according to embodiment 2, wherein the heavy chain constant region is human IgG1 provided by the amino acid sequence of SEQ ID NO: 20, 21, or 53.

[0152] 11. The antibody according to any one of Embodiments 7 to 9, wherein the IgG1 heavy chain constant region further comprises isoleucine substituted at residue 247, glutamine substituted at residue 339, and optionally glutamic acid substituted at residue 332.

[0153] 12. The antibody according to Embodiment 6, wherein the constant region of the IgG heavy chain is human IgG4.

[0154] 13. The antibody according to Embodiment 1, wherein the heavy chain constant region is human IgG4 given by the amino acid sequence of SEQ ID NOs. 12, 13, 14, 54, or 55.

[0155] 14. The antibody according to Embodiment 2, wherein the heavy chain constant region is human IgG4 given by the amino acid sequence of SEQ ID NO: 15, 16, 56, or 57.

[0156] 15. The antibody according to any one of Embodiments 11 to 13, wherein the IgG 4 heavy chain constant region further comprises proline substituted at residue 228, alanine substituted at residue 234, alanine substituted at residue 235, and glutamine substituted at residue 339.

[0157] 16. It consists of two heavy chains and two light chains, and each heavy chain contains the following residue, namely C H Residue 124 in one domain, C H Residue 375 in the 3 domains, and C H The antibody according to Embodiment 1, comprising an IgG heavy chain constant region containing cysteine ​​in one of the 373 residues in the three domains.

[0158] 17. The antibody is the C of each heavy chain. H One domain contains cysteine ​​at residue 124, C H Residues 375 and 378 in the 3 domain, and C H The antibody according to Embodiment 15, wherein one of the 157 residues in one domain contains cysteine, but not all of them.

[0159] 18. The antibody contains the C of each heavy chain. H The antibody according to Embodiment 16, comprising cysteine ​​at residue 375 in the 3 domain.

[0160] 19. The antibody is the C of each heavy chain. H The antibody according to Embodiment 16, comprising cysteine ​​at residue 378 of the 3 domain.

[0161] 20. The antibody according to any one of embodiments 15 to 18, wherein each of the IgG heavy chain constant regions is a human, mouse, rat, or rabbit IgG constant region.

[0162] 21. The antibody according to Embodiment 19, wherein each of the constant regions of the IgG heavy chain is a human IgG1 or human IgG4 isotype.

[0163] 22. The antibody according to Embodiment 20, wherein each of the constant regions of the IgG heavy chain is human IgG1.

[0164] 23. The antibody according to Embodiment 15, wherein each of the heavy chain constant regions is human IgG1 given by the amino acid sequence of SEQ ID NO: 17, 18, 19, or 52.

[0165] 24. The antibody according to Embodiment 16, wherein each of the heavy chain constant regions is human IgG1 given by the amino acid sequence of SEQ ID NO: 20, 21, or 53.

[0166] 25. The antibody according to any one of Embodiments 21 to 23, wherein each of the IgG1 heavy chain constant regions further comprises isoleucine substituted at residue 247, glutamine substituted at residue 339, and optionally glutamic acid substituted at residue 332.

[0167] 26. The antibody according to Embodiment 20, wherein each of the constant regions of the IgG heavy chain is human IgG4.

[0168] 27. The antibody according to Embodiment 15, wherein each of the heavy chain constant regions is human IgG4 given by the amino acid sequence of SEQ ID NOs. 12, 13, 14, 54, or 55.

[0169] 28. The antibody according to Embodiment 16, wherein each of the heavy chain constant regions is human IgG4 given by the amino acid sequence of SEQ ID NO: 15, 16, 56, or 57.

[0170] 29. The antibody according to any one of Embodiments 25 to 27, wherein each of the constant regions of the IgG 4 heavy chain further comprises proline substituted at residue 228, alanine substituted at residue 234, alanine substituted at residue 235, and glutamine substituted at residue 339.

[0171] 30. The antibody according to any one of Embodiments 1 to 28, wherein each cysteine ​​residue 124, 157, 162, 375, or 378 of each IgG constant region is conjugated to an N-formyl-methionine peptide via a maleimide-PEG linker.

[0172] 31. Each IgG constant region contains cysteine ​​from residue 124 and cysteine ​​from one but not all of residues 157, 162, 375, and 378, wherein each IgG constant region residue 124 and each cysteine ​​from 157, 162, 375, or 378 are conjugated to an N-formyl-methionine peptide via a maleimide-PEG linker of the following formula: [ka] The conjugated antibody according to Embodiment 29, wherein the linker is covalently bonded to the antibody via thioether bonds to cysteine ​​residues 124 and 157, 162, 375, or 378 of the IgG constant region, and to the N-formyl-methionine peptide via an amide bond at the epsilon-amino group of the C-terminal lysine of the peptide, where n=6 to 24.

[0173] 32. The conjugated antibody according to Embodiment 30, wherein the cysteine ​​at residue 124 and residue 375 of each IgG constant region are conjugated to the N-formylmethionine peptide via the maleimide-PEG linker.

[0174] 33. The conjugated antibody according to Embodiment 30, wherein the cysteine ​​at residue 124 and residue 378 of each IgG constant region are conjugated to the N-formylmethionine peptide via the maleimide-PEG linker.

[0175] 34. A conjugated antibody according to any one of embodiments 30 to 32, wherein n=12.

[0176] 35. The conjugated antibody according to any one of Embodiments 29 to 33, wherein the N-formylmethionine peptide is given by SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

[0177] 36. A pharmaceutical composition comprising a conjugated antibody according to any one of embodiments 29 to 34 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0178] 37. A method for treating a solid tumor or a humoral tumor, comprising administering to a patient in need thereof an effective amount of a conjugated antibody or a pharmaceutical composition thereof as described in any one of embodiments 29 to 35.

[0179] 38. The method according to Embodiment 36 for treating breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelioma, vascular cancer, fibrocarcinoma, leukemia, or lymphoma.

[0180] 39. A conjugated antibody according to any one of embodiments 29 to 35 for use in therapy.

[0181] 40. A conjugated antibody according to any one of embodiments 29 to 35 for use in the treatment of solid tumors or humoral tumors.

[0182] 41. The conjugated antibody according to Embodiment 39 for use in the treatment of breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelioma, vascular cancer, fibrocarcinoma, leukemia, or lymphoma.

[0183] 42. A compound which is an antibody comprising at least one manipulated cysteine, wherein the antibody is conjugated by a linker to a chemoattract that can attract and / or activate one or more cells of the immune system, and the chemoattract is conjugated to the antibody by one or more cysteine ​​residues in the antibody.

[0184] 43. The compound according to Embodiment 42, wherein the antibody is a monoclonal antibody or a bispecific antibody.

[0185] 44. The compound according to Embodiment 42, wherein the antibody is a monoclonal antibody.

[0186] 45. The compound according to Embodiment 42, wherein the antibody is a bispecific antibody.

[0187] 46. ​​The compound according to any one of embodiments 42 to 45, wherein the cysteine ​​is an engineered cysteine ​​located within the antibody variable region.

[0188] 47. The compound according to any one of embodiments 42 to 45, wherein the cysteine ​​is an engineered cysteine ​​located within the constant region of the antibody.

[0189] 48. The compound according to any one of embodiments 42 to 45, wherein the cysteine ​​is an engineered cysteine ​​located within the CH1 or CH3 domain.

[0190] 49. The compound according to any one of Embodiments 42 to 48, wherein the cysteine ​​is manipulated to replace a natural serine, valine, alanine, glutamine, asparagine, threonine, or glycine.

[0191] 50. The compound according to Embodiment 49, wherein the cysteine ​​is manipulated at a position that replaces a natural serine, valine, or alanine.

[0192] 51. The compound according to any one of Embodiments 42 to 50, wherein the total number of manipulated cysteines is 2 to 6.

[0193] 52. The compound according to any one of embodiments 42 to 51, wherein the compound can attract and activate one or more cells of the immune system.

[0194] 53. The compound according to any one of Embodiments 42 to 52, wherein the immune system is an adaptive immune system.

[0195] 54. The compound according to any one of Embodiments 42 to 52, wherein the immune system is the innate immune system.

[0196] 55. The compound according to any one of embodiments 42 to 52, wherein the one or more cells of the immune system are neutrophils.

[0197] 56. The compound according to any one of embodiments 42 to 52, wherein one of the more numerous cells of the immune system is a macrophage.

[0198] 57. The compound according to any one of embodiments 42 to 56, wherein the linker is a PEG linker or a Mal-Dap linker.

[0199] 58. The compound according to Embodiment 57, wherein the linker is a PEG linker.

[0200] 59. The compound according to Embodiment 57, wherein the linker is a Mal-Dap linker.

[0201] 60. The antibody comprises an IgG heavy chain constant region and a light chain constant region, wherein the constant region consists of the following residues, namely C H Residue 124 in one domain, C HResidue 157 in one domain, C H 162 residues in one domain, C H Residue 262 in the 2 domain, C H Residue 375 in domain 3, C H Residue 373 in the 3 domain, C H Residues 397 in the 3 domains, C H Residue 415 in the 3 domains, C カッパ Residue 156 in the domain, C カッパ Residue 171 in the domain, C カッパ Residue 191 in the domain, C カッパ Residue 193 in the domain, C カッパ Residue 202 in the domain, or C カッパ A compound according to any one of embodiments 42 to 58, comprising an engineered cysteine ​​in at least one of the 208 residues in the domain.

[0202] 61. The antibody is C H One domain contains cysteine ​​at residue 124, C H Residues 157 and 162 in one domain, and C H The compound according to Embodiment 60, further comprising cysteine ​​in one but not all of the residues 375 and 378 in the 3 domains.

[0203] 62. The compound according to Embodiment 61, wherein the antibody contains cysteine ​​at residue 157 in the CH1 domain.

[0204] 63. The compound according to Embodiment 61, wherein the antibody contains cysteine ​​at residue 375 in the CH3 domain.

[0205] 64. The compound according to Embodiment 61, wherein the antibody contains cysteine ​​at residue 378 in the CH3 domain.

[0206] 65. The compound according to any one of embodiments 42 to 64, wherein the IgG heavy chain constant region is a human, mouse, rat, or rabbit IgG constant region.

[0207] 66. The compound according to embodiment 65, wherein the constant IgG heavy chain region is a human IgG1 or human IgG4 isotype.

[0208] 67. The compound according to embodiment 66, wherein the constant region of the IgG heavy chain is human IgG1.

[0209] 68. The compound according to Embodiment 67, wherein the heavy chain constant region is human IgG1 given by the amino acid sequence of SEQ ID NOs: 17, 18, 19, or 52.

[0210] 69. The compound according to Embodiment 67, wherein the heavy chain constant region is human IgG1 given by the amino acid sequence of SEQ ID NO: 20, 21, or 53.

[0211] 70. The compound according to any one of embodiments 66 to 69, wherein the IgG1 heavy chain constant region further comprises isoleucine substituted at residue 247, glutamine substituted at residue 339, and optionally glutamic acid substituted at residue 332.

[0212] 71. The compound according to Embodiment 66, wherein the constant region of the IgG heavy chain is human IgG4.

[0213] 72. The compound according to Embodiment 71, wherein the heavy chain constant region is human IgG4 given by the amino acid sequence of SEQ ID NOs: 12, 13, 14, 54, or 55.

[0214] 73. The compound according to Embodiment 71, wherein the heavy chain constant region is human IgG4 given by the amino acid sequence of SEQ ID NO: 15, 16, 56, or 57.

[0215] 74. The antibody according to any one of embodiments 71 to 73, wherein the IgG 4 heavy chain constant region further comprises proline substituted at residue 228, alanine substituted at residue 234, alanine substituted at residue 235, and glutamine substituted at residue 339.

[0216] 75. The compound according to any one of Embodiments 42 to 74, wherein the chemiactive attractant is an f-Met peptide, a small molecule FPR-1 agonist, a PRR agonist, a peptide mimetic, an N-ureido-peptide, or a bacterial sugar.

[0217] 76. The compound according to Embodiment 75, wherein the chemical attractant is N-formylmethionine peptide.

[0218] 77. The compound according to Embodiment 76, wherein the N-formyl peptide is given by SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

[0219] 78. The compound according to any one of embodiments 42 to 78, wherein the cysteine ​​is conjugated to a chemical attractant via a maleimide-PEG linker.

[0220] 79. The cysteine ​​is conjugated to a chemiactive attractant via a maleimide-PEG linker of the following formula: [ka] The compound according to Embodiment 78, wherein the linker is covalently bonded to the antibody via a thioether bond to the cysteine ​​and to the chemical attractant via an amide bond to the epsilon-amino group of the C-terminal lysine of the peptide, and in the formula, n=2 to 24.

[0221] 80. The compound according to embodiment 79, wherein n=12.

[0222] 81. A pharmaceutical composition comprising an antibody according to any one of embodiments 42 to 80 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0223] 82. A method for treating a solid tumor or a humoral tumor, comprising administering to a patient in need thereof an effective amount of a compound or a pharmaceutical composition thereof described in any one of embodiments 42 to 81.

[0224] 83. The method according to Embodiment 82 for treating breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelioma, vascular cancer, fibrocarcinoma, leukemia, or lymphoma.

[0225] 84. A compound or salt according to any one of embodiments 42 to 80 for use in therapy.

[0226] 85. A compound according to any one of embodiments 42 to 80 for use in the treatment of solid tumors or humoral tumors.

[0227] 86. A compound according to any one of Embodiments 42 to 80 for use in the treatment of breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelioma, vascular cancer, fibrocarcinoma, leukemia, or lymphoma.

[0228] 87.Compound R-P1-P2-P3-NH(CH2CH2O) n CH2CH2-Y, where in the formula, (i) R is either HC(=O)- or R 1 NHC(=O)NH- (ii)R 1 However, C5~C may be substituted or not substituted. 10 It is Ariel, (iii) P1 is either Met or Nle, (iv) P2 is a peptide or peptide mimetic, (v)P3 is epsilon-aminoacylated lysine, (vi)n is an integer between 6 and 24, (vii) Compound R-P1-P2-P3-NH(CH2CH2O) where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. n CH2CH2-Y, (viii) its salt.

[0229] 88.Compound R-P1-P2-NH(CH2CH2O) n CH2CH2-P3―Y, where in the formula, (i) R is either HC(=O)- or R 1 NHC(=O)NH- (ii)R 1 However, C5~C may be substituted or not substituted. 10 It is Ariel, (iii) P1 is either Met or Nle, (iv) P2 is a peptide or peptide mimetic, (v)P3 is epsilon-aminoacylated lysine, (vi)n is an integer between 6 and 24, (vii) Compound R-P1-P2-NH(CH2CH2O) where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. n CH2CH2-P3-Y, (viii) its salt.

[0230] 89.Compound R-Met-P2-NH(CH2CH2O) n CH2CH2-X5-Y, in the formula (i) R is either HC(=O)- or R 1 NHC(=O)NH- (ii)R 1 However, these are phenyl, 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, aryl, substituted aryl, or 2-allyl. (iii) P2 is a peptide or peptide mimetic, (iv) X5 is C2~C 10 It is a diaminoalkyl, and (v) Compound R-Met-P2-NH(CH2CH2O) where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. n CH2CH2-X5-Y, (xi) or a salt thereof.

[0231] 90. Compound [R-P1-P2-NH(CH2CH2O) n CH2CH2-]2-QXY, where in the formula, (i) R is either HC(=O)- or R 1 NHC(=O)NH- (ii)R 1 However, C5~C may be substituted or not substituted. 10 It is Ariel, (iii) P1 is either Met or Nle, (iv) P2 is a peptide or peptide mimetic, (v)n is an integer between 6 and 24, (vi) Q is Lys, Orn, Dap, Dab, or another amino difunctional residue that can be acylated at the alpha amino group and side chain amino group, (vii)X is C2~C 10 It is a diaminoalkyl, and (viii) Compound [R-P1-P2-NH(CH2CH2O)] where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone n CH2CH2-]2-QXY, (ix) or its salt.

[0232] 91. Compound [[R-P1-P2-NH(CH2CH2O) n CH2CH2-]4-(Q)2―QXY, where in the formula, (i) R is either HC(=O)- or R 1 NHC(=O)NH- (ii)R 1 However, C5~C may be substituted or not substituted. 10 It is Ariel, (iii) P1 is either Met or Nle, (iv) P2 is a peptide or peptide mimetic, (v)n is an integer between 6 and 24, (vi) Q may be Lys, Orn, Dap, Dab, or any other amino difunctional residue that can be acylated at an alpha amino group and a side-chain amino group. (vii)X is C2~C 10 It is a diaminoalkyl, and (viii) Compound [[R-P1-P2-NH(CH2CH2O) n CH2CH2-]4-(Q)2―QXY, (ix) or its salt.

[0233] 92. Compound [[[R-P1-P2-NH(CH2CH2O) n CH2CH2-]8-(Q)4-(Q)2-QXY, where in the formula, (i) R is either HC(=O)- or R 1 NHC(=O)NH- (ii)R 1 However, C5~C may be substituted or not substituted. 10 It is Ariel, (iii) P1 is either Met or Nle, (iv) P2 is a peptide or peptide mimetic, (v)n is an integer between 6 and 24, (vi) Q is Lys, Orn, Dap, Dab, or another amino difunctional residue that can be acylated with an alpha amino group and a side-chain amino group. (vii)X is C2~C 10 It is a diaminoalkyl, and (viii) Compound [[[R-P1-P2-NH(CH2CH2O) n CH2CH2-]8-(Q)4-(Q)2-QXY, (ix) or its salt.

[0234] 93. P2 is given by X1-X2-X3-X4, and (i) X1 is Leu, Ile, Nle, diethylglycine, or dipropylglycine, (ii) X2 is Phe, α-Me-Phe, DPhe, 4-F-Phe, 2-Nal, or 1-Nal, (iii) X3 is Glu, Leu, Nle, α-Me-Leu, DLeu, or absent, and (iv) The compound according to any one of Embodiments 87 to 92, wherein X4 is Glu, DGlu, γGlu, Gla, or absent.

[0235] 94. The compound according to any one of embodiments 87 to 93, wherein the compound can be covalently bonded to an antibody or antibody fragment via a thioether bond.

[0236] 95. The compound is C H 124 cysteine ​​residues in one domain, C H Residue 157 in one domain, C H 162 residues in one domain, C H Residue 262 in the 2 domain, C H Residue 375 in domain 3, C H Residue 373 in the 3 domain, C H Residues 397 in the 3 domains, C H Residue 415 in the 3 domains, C カッパ Residue 156 in the domain, C カッパ Residue 171 in the domain, C カッパ Residue 191 in the domain, C カッパ Residue 193 in the domain, C カッパ Residue 202 in the domain, or C カッパ The compound according to any one of embodiments 87 to 94, wherein the cysteine ​​residue at 208 in the domain can be covalently bound to an antibody or antibody fragment via a thioether bond.

[0237] 96. A compound which is an antibody containing at least one cysteine ​​conjugated by a linker to a compound according to any one of embodiments 87 to 95, which can attract and / or activate one or more cells of the immune system, wherein the agent is conjugated to the antibody by one or more cysteine ​​residues in the antibody.

[0238] array The antibody heavy chain of the emibetuzumab 378C conjugate (SEQ ID NO: 1) [Table 22] (X at position 373 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of emibetuzumab 124C conjugate (SEQ ID NO: 2) [Table 23] (X at position 122 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 124C-378C conjugate (SEQ ID NO: 3) [Table 24] (X at position 122 and X at position 373 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 124C-375C conjugate (SEQ ID NO: 4) [Table 25] (X at position 122 and X at position 370 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) Emibetuzumab conjugate antibody light chain (SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQVYSGYPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TMab124C~378C conjugate antibody heavy chain (SEQ ID NO: 6) [Table 26] (X at position 127 and X at position 381 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) TMab conjugate antibody light chain (SEQ ID NO: 7) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AME133 124C~378C conjugate antibody heavy chain (SEQ ID NO: 8) [Table 27] (X at position 128 and X at position 382 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) AME133 conjugate antibody light chain (SEQ ID NO: 9) EIVLTQSPGTLSLSPGERATLSCRASSSVPYIHWYQQKPGQAPRLLIYATSALASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Human IgG1 constant region (SEQ ID NO: 10) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG4 constant region (SEQ ID NO: 11) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG IgG4 124C conjugate antibody heavy chain constant region (SEQ ID NO: 12) [Table 28] (X at position 7 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) IgG4 378C conjugate antibody heavy chain constant region (SEQ ID NO: 13) [Table 29] (X at position 258 is a cysteine ​​residue modified by thioimide bond formation to the maleimide-PEG linker.) IgG4 375C conjugate antibody heavy chain constant region (SEQ ID NO: 14) [Table 30] (X at position 255 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) IgG4 124C-378C conjugate antibody heavy chain constant region (SEQ ID NO: 15) [Table 31] (X at position 7 and X at position 258 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) IgG4 124C-375C conjugate antibody heavy chain constant region (SEQ ID NO: 16) [Table 32] (X at position 7 and X at position 255 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) IgG1 124C conjugate antibody heavy chain constant region (SEQ ID NO: 17) [Table 33] (X at position 7 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) IgG1 378C conjugate antibody heavy chain constant region (SEQ ID NO: 18) [Table 34] (X at position 261 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) IgG1 375C conjugate antibody heavy chain constant region (SEQ ID NO: 19) [Table 35] (X at position 258 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) IgG1 124C-378C conjugate antibody heavy chain constant region (SEQ ID NO: 20) [Table 36] (X at position 7 and X at position 261 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) Antibody heavy chain constant region of IgG1 124C~375C conjugate (SEQ ID NO: 21) [Table 37] (X at position 7 and X at position 258 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) fMLFX (peptide-'183) (SEQ ID NO: 22) (Met at position 1 is formylated) (X at position 4 is a lysine residue modified by amide bond formation to the maleimide-PEG linker.) fMLFK (Sequence ID 23) (Met at position 1 is formylated) MLFX (Peptide-'844) (SEQ ID NO: 24) (X at position 4 is a lysine residue modified by amide bond formation to the maleimide-PEG linker.) MLFK (Sequence ID 25) MET415C antibody conjugate antibody heavy chain (SEQ ID NO: 26) [Table 38] (X at position 410 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) MET156C antibody conjugate antibody light chain (SEQ ID NO: 27) [Table 39] (X at position 157 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) MET171C antibody conjugate antibody light chain (SEQ ID NO: 28) [Table 40] (X at position 172 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) MET191C antibody conjugate antibody light chain (SEQ ID NO: 29) [Table 41] (X at position 192 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) MET193C antibody conjugate antibody light chain (SEQ ID NO: 30) [Table 42] (X at position 194 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) MET 202C antibody conjugate antibody light chain (SEQ ID NO: 31) [Table 43] (X at position 203 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) MET208C antibody conjugate antibody light chain (SEQ ID NO: 32) [Table 44] (X at position 209 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) Trastuzumab 124C-157C antibody conjugate antibody heavy chain (SEQ ID NO: 33) [Table 45] (X at position 127 and X at position 160 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) 124C~378C bispecific antibody I conjugate antibody heavy chain A (SEQ ID NO: 34) [Table 46] (X at position 126 and X at position 380 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) 124C~378C bispecific antibody I conjugate antibody heavy chain B (SEQ ID NO: 35) [Table 47] (X at position 128 and X at position 382 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) fMIFLX(FRM-021)(Sequence ID 36) (Met at position 1 is formylated) (X at position 5 is a lysine residue side chain modified via epsilonamide bond formation to the hydrolyzed maleimide-PEG linker.) fMXFX (FRM-029) (Sequence ID 37) (Met at position 1 is formylated) (X at position 2 is diethylglycine) (X at position 4 is a leucine residue that has been linked at the C-terminus by the formation of an amide bond to the PEG linker of formula (PEG6)2-NH-(CH2)2-NH2.) fMXFX (FRM-030) (Sequence ID 38) (Met at position 1 is formylated) (X at position 2 is dipropylglycine) (X at position 4 is a leucine residue that has been linked at the C-terminus by the formation of an amide bond to the PEG linker of formula (PEG6)2-NH-(CH2)2-NH2.) fMIX (FRM-031) (Sequence ID 39) (Met at position 1 is formylated) (X at position 3 is a phenylalanine residue attached at the C-terminus by amide bond formation to the PEG linker of formula PEG12-NH-(CH2)2-NH2) fMIFX(FRM-023)(Sequence ID 40) (Met at position 1 is formylated) (X at position 4 is a leucine residue that has been linked at the C-terminus by the formation of an amide bond to the PEG linker of the formula PEG12-NH-(CH2)-NH2.) fMIFX(FRM-032)(Sequence ID 41) (Met at position 1 is formylated) (X at position 4 is a leucine residue modified by the formation of an amide bond to the linker of formula NH-(CH2)-NH-[(Mal-Dap(NH2)]).) fNleLX(FRM-009)(Sequence ID 42) (Nle at position 1 is formylated) (X at position 3 is phenylalanine linked at the C-terminus by amide bond formation to the linker of formula PEG12-Lys(maleimide-propionyl)-OH) The antibody heavy chain of the emibetuzumab conjugate (SEQ ID NO: 43) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGRVNPNRRGTTYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARANWLDYWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG The antibody heavy chain of the emibetuzumab 157C antibody conjugate (SEQ ID NO: 44) [Table 48] (X at position 155 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 162C antibody conjugate (SEQ ID NO: 45) [Table 49] (X at position 160 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 262C antibody conjugate (SEQ ID NO: 46) [Table 50] (X at position 257 is a cysteine ​​residue modified by thioether bond formation to the maleimide PEG linker.) The antibody heavy chain of the emibetuzumab 375C antibody conjugate (SEQ ID NO: 47) [Table 51] (X at position 370 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 397C antibody conjugate (SEQ ID NO: 48) [Table 52] (X at position 392 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 124C-157C-378C antibody conjugate (SEQ ID NO: 49) [Table 53] (X at position 122, X at position 155, and X at position 373 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) The antibody heavy chain of the emibetuzumab 124C-162C-378C antibody conjugate (SEQ ID NO: 50) [Table 54] (X at position 122, X at position 160, and X at position 373 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) Tmab(IQE)124C-378C antibody conjugate antibody heavy chain (SEQ ID NO: 51) [Table 55] (X at position 127 and X at position 381 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) IgG1 157C conjugate antibody heavy chain constant region (SEQ ID NO: 52) [Table 56] (X at position 40 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) Antibody heavy chain constant region of IgG1 124C-157C conjugate (SEQ ID NO: 53) [Table 57] (X at position 7 and X at position 40 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) IgG4 157C conjugate antibody heavy chain constant region (SEQ ID NO: 54) [Table 58] (X at position 40 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) IgG4 162C conjugate antibody heavy chain constant region (SEQ ID NO: 55) [Table 59] (X at position 45 is a cysteine ​​residue modified by thioether bond formation to the maleimide-PEG linker.) Antibody heavy chain constant region of IgG4 124C-157C-373C conjugate (SEQ ID NO: 56) [Table 60] (X at position 7, X at position 40, and X at position 258 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) IgG4 124C-162C-373C conjugate antibody heavy chain constant region (SEQ ID NO: 57) [Table 61] (X at position 7, X at position 45, and X at position 258 are cysteine ​​residues modified by thioether bond formation to the maleimide-PEG linker.) Antibody light chain A of bispecific antibody I conjugate (SEQ ID NO: 58) RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQDKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEI KGQPKAAPSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAWSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC Antibody light chain B of bispecific antibody I conjugate (SEQ ID NO: 59) DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQRKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. Compound R-P １ -P ２ -P ３ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -Y, in the formula, (i) R is HC(=O)- or R １ NH C(=O)NH-, and (ii) R １ However, it is phenyl, 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, or aryl. (iii) P １ However, it is either Met or Nle. (iv) P ２ However, it is a peptide, (v) P ３ However, it is lysine, (vi)n is an integer between 6 and 24, (vii) Compound R-P where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. １ -P ２ -P ３ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -Y, (viiii) or its salt.

2. Compound R-P １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -P ３ -Y, in the formula, (i) R is HC(=O)- or R １ NHC(=O)NH-, (ii) R １ However, it is 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, or aryl, (iii) P １ However, it is either Met or Nle. (iv) P ２ However, it is a peptide, (v) P ３ However, it is lysine, (vi)n is an integer between 6 and 24, (vii) Compound R-P where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -P ３ -Y, (viiii) or its salt.

3. Compound R-Met-P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -X ５ -Y, in the formula (i) R is HC(=O)- or R １ NHC(=O)NH-, (ii) R １ However, it is phenyl, 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, or aryl. (iii) P ２ However, it is a peptide, (iv) X ５ However, C ２ ~C １０ It is a diaminoalkyl, (v) n is an integer between 6 and 24, and (vi) Compound R-Met-P, where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -X ５ -Y, (vii) or its salt.

4. Compound [R-P １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -] ２ -Q-X-Y, where in the formula, (i) R is HC(=O)- or R １ NHC(=O)NH-, (ii) R １ However, it is 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, or aryl, (iii) P １ However, it is either Met or Nle. (iv) P ２ However, it is a peptide, (v)n is an integer between 6 and 24, (vi) Q is Lys, Orn, Dap, or Dab, (vii) X is C ２ ~C １０ It is a diaminoalkyl, and (viiii) Compound [R-P] where Y is maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone. １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -] ２ -Q-X-Y, (ix) or its salt.

5. Compound [R-P １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -] ４ -(Q) ２ -Q-X-Y, wherein (i) R is HC(=O)- or R １ NHC(=O)NH-, (ii) R １ However, it is 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, or aryl, (iii) P １ However, it is either Met or Nle. (iv) P ２ is a peptide, (v)n is an integer between 6 and 24, (vi) Q is Lys, Orn, Dap, or Dab, (vii) X is C ２ ~C １０ It is a diaminoalkyl, and (viiii) Compound [[R-P １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -] ４ - (Q) ２ -Q-X-Y, (ix) or its salt.

6. Compound [R-P １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -] ８ - (Q) ４ - (Q) ２ -Q-X-Y, where in the formula, (i) R is HC(=O)- or R １ NHC(=O)NH-, (ii) R １ However, it is 4-chlorophenyl, 4-methoxyphenyl, p-tolyl, m-tolyl, or aryl, (iii) P １ However, it is either Met or Nle. (iv) P ２ However, it is a peptide, (v)n is an integer between 6 and 24, (vi) Q is Lys, Orn, Dap, or Dab (vii) X is C ２ ~C １０ It is a diaminoalkyl, and (viiii) Compound [[[R-P １ -P ２ -NH(CH ２ CH ２ O) ｎ CH ２ CH ２ -] ８ - (Q) ４ - (Q) ２ -Q-X-Y, (ix) or its salt.

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