CN117750981A

CN117750981A - Peptides comprising N-formyl-halomethionine residues and engineered antibody-peptide conjugates thereof

Info

Publication number: CN117750981A
Application number: CN202280041692.7A
Authority: CN
Inventors: 谢默斯·帕特里克·布伦南; 马修·大卫·林尼克; 弗朗西斯科·阿尔西德斯·瓦伦苏埃拉
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2021-06-11
Filing date: 2022-06-07
Publication date: 2024-03-22

Abstract

Peptides are provided comprising N-formylmethionine in which the methyl group of the side chain of methionine has been substituted with one or more halogens such as fluorine. N-formyl-halogen substituted methionine exhibits oxidation resistance. Peptides comprising N-formyl-halogen substituted methionine may be used as agonists of formyl peptide receptor-1 (FPR-1) and may be conjugated to antibodies or antigen binding fragments thereof. The antibody conjugates thus prepared are useful for targeting cells and attracting and activating immune cells comprising FPR-1 directed against the target cells.

Description

Peptides comprising N-formyl-halomethionine residues and engineered antibody-peptide conjugates thereof

Cross reference to related applications

The present application claims priority benefits from U.S. provisional patent application Ser. No.63/209,762 filed on day 11 6 of 2021 and U.S. provisional patent application Ser. No.63/210,292 filed on day 14 of 2021, the disclosures of which are incorporated herein by reference in their entireties.

Sequence listing

The present application is accompanied by a sequence listing submitted as a sequence listing ASCII text file entitled "083389_01624_st25.Txt", which file size was 162,084 bytes and was created at 2022, 6, 7. The sequence listing is submitted electronically with the present application via EFS-Web and is incorporated herein by reference in its entirety.

Background

The field of the invention relates to peptides comprising an N-formyl-halomethionine residue and engineered antibody conjugates comprising the peptides. The peptides and antibody conjugates are useful in methods of treating diseases and disorders, such as cell proliferative diseases and disorders.

Antibodies and antigen binding fragments thereof may be conjugated to a variety of payload molecules, including therapeutic, cytotoxic, and diagnostic peptides or other small molecules, for in vivo and in vitro applications. In particular, natural or engineered free cysteine sulfhydryl groups generated on the surface of immunoglobulin heavy or light chain residues can be used as reactive nucleophiles to synthesize antibody conjugates by forming stable chemical bonds with payload molecules via sulfhydryl conjugation either directly or via various linkers. Antibodies and antigen binding fragments thereof for conjugation to payload molecules are known in the art.

Antibodies so engineered to include payload molecules may be particularly useful in cancer immunotherapy. Cancer immunotherapy attacks cancer cells using the body's immune system and is a viable area in oncology drug discovery and development. Thus, cancer immunotherapy represents a paradigm shift in which the host immune system is involved in recognizing and destroying tumor cells, as compared to therapies based on the use of tumoricidal agents (e.g., targeted tumoricidal agents) that may exhibit off-target toxicity. Two successful cancer immunotherapy strategies are: (1) Inhibition of the immune system so as to be able to activate the adaptive and/or innate immune system, particularly tumor-directed cytotoxic T cells (i.e., immune checkpoint blockade), and (2) antibody modification designed to participate in and/or enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

Successful clinical results have recently been achieved with immune checkpoint modulators designed to modify the interaction between T cell surface receptors (such as PD-1 and CTLA-4) and cognate ligands in a manner that results in T cell activation and results in T cell mediated tumor cell destruction. PD-1 targeted cancer immunotherapy (e.g., nivolumabAnd pembrolizumab->) And CTLA-4-targeted cancer immunotherapy (e.g., ipilimumab) Has been FDA approved for the treatment of cancers such as squamous non-small cell lung cancer and metastatic melanoma.

ADCC involves the interaction of an antibody Fc domain of a targeted antibody with a receptor (e.g., fcγ receptor IIIa) located on the surface of an immune system cell (e.g., a natural killer cell or "NK" cell) such that cytolysin is released from the immune cell, followed by destruction of the targeted tumor cell. Bulk antibody therapies exhibiting ADCC include(rituximab), ->(Aofatuzumab),>(trastuzumab) and +.>(alemtuzumab). Efforts to design antibodies with improved ADCC activity by enhanced Fc receptor binding engineering are effective in patients in which antibodies with similar target specificity and less ADCC activation are ineffective or no longer sufficiently effective in the disease (e.g., Trastuzumab)).

Despite advances in current cancer immunotherapy, there remains a need for alternative methods of engaging the immune system in the treatment of cancer. For example, the percentage of patients responding to T cell directed immunotherapy varies and there is a lack of reliable prognostic assays to determine which patients will respond. Furthermore, treatment-induced autoimmune diseases are serious side effects associated with immune checkpoint inhibitor therapies. The appearance of autoimmune diseases with immune checkpoint inhibitors may be related to their mechanism of action, as they are designed to eliminate the inhibition of T cell repertoires so that tumor-specific T cells can appear, proliferate and be activated. Thus, they are relatively non-specific, and one consequence of this lack of specificity is that they enable autoreactive T cells to break tolerance and induce autoimmune disease, which is not necessarily reversible after treatment ceases. Enhanced ADCC methods are designed to involve NK cells in tumor cell killing. However, NK cells constitute only about 5% of the total number of leukocytes in blood.

Targeting polymorphonuclear cells (PMNs) of the innate immune system to participate in tumor cell killing represents an alternative approach to cancer immunotherapy. PMNs constitute greater than 50% of the total number of leukocytes and are the primary defense against pathogens, including commensal bacteria and foreign bacteria. During the innate immune response, pathogen-associated molecular patterns (PAMPs) presented by pathogens are recognized by Pattern Recognition Receptors (PRRs) located on the surface of immune cells, such as neutrophils. One such PRR is formyl peptide receptor 1 (FPR-1), a membrane-bound G protein-coupled receptor expressed on the surface of neutrophils. FPR-1 detects proteins and peptides with N-formyl-methionine, including those produced and released by bacteria after infection. Conjugation of FPR-1 on the surface of neutrophils to N-formyl-methionine containing peptides triggers motility/chemotaxis of neutrophils towards the site of infection. Activation of FPR-1 by formyl peptides also triggers pathogen killing mechanisms such as degranulation to release cytotoxic molecules, production of Reactive Oxygen Species (ROS), and phagocytosis to destroy pathogens. Natural and non-natural FPR-1 agonists are widely described in the literature. (see, e.g., he HQ and Ye RD, molecules.2017, 3, 13; 22 (3) pii: E455.Doi:10.3390/molecules22030455; hwang TL et al, org Biomol chem.2013, 6 th month 14; 11 (22): 3742-55.doi:10.1039/C3ob40215k; cavicchioni G et al, biorg chem.2006, 34 (5): 298-318; higgins JD et al, JMed chem.1996, 1 month 39 (5): 1013-5; vergeli C et al, drug Dev Res.2017, 2 month 78 (1): 49-62.doi:10.1002/ddr.21370; kirpoina et al, mol Phacol.2010, 77 (2): 159-70.doi: 10.1124/mol.673; cibrizi A et al, J2009.8:9/Mol 6, 50:52 (5): 6 m.35, 9:52.1021, 50.52.9).

Tumor-targeted therapeutic antibodies capable of promoting the involvement of PMN neutrophils of the innate immune system in tumor cell destruction may also provide advantages over current cancer immunotherapy. For example, such therapeutic antibodies may enhance T cell responses to tumors, and may not require the presence of tumor-specific T cells to drive tumor cell killing. The involvement of PMN neutrophils in anti-tumor activity depends on the presence of FPR (e.g., FPR-1) naturally expressed on neutrophils in all patients. Furthermore, agents capable of participating PMN neutrophils in tumor cell killing would benefit from a robust, continuous supply of tumor killing cells, since it has been estimated that 1x10 is produced daily ¹¹ Neutrophils. Can make neutrophil participated in tumorCell killing tumor-targeted antibodies may have safety advantages over immune checkpoint modulators. Unlike checkpoint modulators, neutrophil targeted therapies do not induce or require proliferation of immune cells, as circulating neutrophils are short lived. In addition, when neutrophils kill target tumor cells to which the antibody is attached, the tumor-targeting antibody is eliminated to provide a negative feedback loop that reduces immune stimulation as the therapeutic antibody is consumed by the target effector cells.

Another way that tumor-targeted therapeutic antibodies that are able to bind to FPR-1 positive innate immune cells in tumor cells may prove useful is for the treatment of cold tumors (codtumors) that have low mutational burden and are therefore not readily recognized by the immune system. Attracting and activating neutrophil-mediated killing of tumor cells can result in local production of neoantigens in a cytokine-rich environment, such that cells of the adaptive immune system acquire the ability to recognize the tumor and target the tumor for elimination.

Tumor-targeted antibodies capable of engaging neutrophils in tumor cell killing may also be advantageous over toxic agent-based Antibody Drug Conjugates (ADCs) that are typically designed to release toxic payloads after internalization into tumor cells. Like ADC, tumor-targeted antibodies that are capable of causing neutrophils to participate in tumor cell killing should recognize antigens that have high expression on tumor cells and low expression on normal tissues. However, unlike ADC, tumor-targeted antibodies capable of engaging 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 with relatively less internalizing potential.

Antibodies conjugated to N-formyl peptides are disclosed in the art and may be referred to as "bacterial antibodies" based on the presence of N-formyl peptides in bacteria (see, e.g., WO2018/232088, the contents of which are incorporated herein by reference in their entirety). One difficulty in using antibodies conjugated to peptides containing N-formyl-methionine as targeting agents and agonists for attracting and activating cells containing the FPR-1 receptor is that oxidation of N-formyl-methionine occurs in vivo with the formation of sulfur atoms and methyl sulfoxide or the formation of Met (O). Oxidation of the sulfur atom of the methionine residue results in a significant decrease in the efficacy of N-formylmethionine as an FPR-1 agonist. Thus, there is a need for N-formylmethionine and peptides containing N-formylmethionine that are antioxidant and act as FPR-1 agonists.

SUMMARY

Peptides comprising N-formylmethionine are disclosed wherein the methyl group of the side chain of methionine may be substituted with one or more halogens such as fluorine. N-formyl-halogen substituted methionine exhibits oxidation resistance. Peptides comprising N-formyl-halogen substituted methionine may be used as agonists of Formyl Peptide Receptor (FPR) and may be conjugated to antibodies or antigen binding fragments thereof. The antibody conjugates thus prepared can be used to target cells and attract and activate immune cells comprising FPR against the target cells.

Brief Description of Drawings

FIG. 1 oxidation of N-formylmethionine to N-formylmethionine S-oxide. N-formylmethionine (CF) ₃ ) And S-oxidation resistance.

FIG. 2 illustrates an exemplary synthetic peptide-linker. frm is formyl; MIFL: met-Ile-Phe-Leu; peg is polyethylene glycol monomer; m (CF) ₃ ) Trifluoromethyl methionine; dpg di-n-propylglycine; 2Nal 2-naphthylalanine; alpha MeF alpha-methyl-phenylalanine; nle is norleucine; γe, glutamic acid residues linked via their side chain γcarboxyl groups; epsilon K is a lysine residue linked via its side chain epsilon amino group.

FIG. 3 illustrates an exemplary synthetic peptide-linker. frm is formyl; MIFL: met-Ile-Phe-Leu; peg is polyethylene glycol monomer; mal maleimide; MLF: met-Leu-Phe; dpg di-n-propylglycine; 2Nal 2-naphthylalanine; alpha MeF alpha-methyl-phenylalanine; nle is norleucine; D-Nle, D-norleucine; γe, glutamic acid residues linked via their side chain γcarboxyl groups; m (O) oxidized methionine (control); m (CF) ₃ ) Trifluoromethyl methionine. The formula provided is FRM-046 (FRM-047 w/o Mal).

FIG. 4 illustrates an exemplary synthetic peptide-linker. frm is formyl; MIFL: met-Ile-Phe-Leu; peg polyethylene glycol monomerThe method comprises the steps of carrying out a first treatment on the surface of the Mal maleimide; MLF: met-Leu-Phe; dpg di-n-propylglycine; 2Nal 2-naphthylalanine; alpha MeF alpha-methyl-phenylalanine; nle is norleucine; D-Nle, D-norleucine; γe, glutamic acid residues linked via their side chain γcarboxyl groups; m (O) oxidized methionine (control); m (CF) ₃ ) Trifluoromethyl methionine; 4-Pal 4-pyridylalanine.

FIG. 5. Chemistry for preparing Fmoc-L-trifluoromethanel from Fmoc-S-trityl-L-homocysteine.

FIG. 6 Reactive Oxygen Species (ROS) production in neutrophils activated with various peptides.

FIG. 7 Reactive Oxygen Species (ROS) production in neutrophils activated with various peptides.

Figure 8 neutrophil chemotaxis following peptide exposure.

Fig. 9 Reactive Oxygen Species (ROS) generation in neutrophils activated with peptides conjugated to trastuzumab.

Fig. 10A. Pharmacokinetics of trastuzumab.

Fig. 10B. Pharmacokinetics of trastuzumab conjugated to peptide FRM 047.

Fig. 10C table data of the results in fig. 9A and 9B.

Fig. 10D. PK parameters of the results in fig. 9A and 9B using the two-compartment model.

FIG. 11 trastuzumab parent antibody and anti-tumor antibody with frm-Met (CF ₃ ) The exposure curve of frauzumab anti-bacterial antibody (bacterial) of FRM-058 shows similar exposure between bacterial antibody (Tmab bacterial) and Tmab parent.

Detailed description of the preferred embodiments

The invention is described herein using several definitions as set forth below and throughout the application.

The terms "a" and "an" mean "one or more" unless the context indicates otherwise or indicated. For example, "a peptide", "a linker" and "an antibody" should be interpreted to mean "one or more peptides", "one or more linkers" and "one or more antibodies", respectively.

As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and vary to some extent in the context in which they are used. If the use of these terms is not clear to one of ordinary skill in the art depending on the context in which they are used, "about" and "approximately" refer to the addition or subtraction of 10% or less of a particular term, while "substantially" and "significantly" refer to the addition or subtraction of >10% of a particular term.

As used herein, the term "comprising" has the same meaning as the term "comprising," i.e., the terms are "open" transitional terms that do not limit the claims to only the elements listed after such transitional terms. The term "consisting of …" while encompassed by the term "comprising" should be construed as a "closed" transitional term that limits the claims to only the elements listed after such transitional term. The term "consisting essentially of …" should be interpreted as a "partially closed" transitional term, although encompassed by the term "comprising" that allows additional elements following this transitional term, provided that such additional elements do not materially affect the basic and novel characteristics of the claims.

As used herein, a "subject in need thereof" refers to a human or non-human mammal, more preferably a human, that has been diagnosed as having a condition or disorder suitable for treatment or management with the peptides and conjugates disclosed herein.

As used herein, a "subject in need thereof" may include a subject suffering from or at risk of developing a disease or disorder that may be treated and/or prevented by modulating an immune response in the subject. As disclosed herein, "modulation" may include inducing and/or enhancing an immune response in a subject.

A subject in need thereof may include a subject suffering from or at risk of developing a cell proliferative disease or disorder. Cell proliferative diseases and disorders may include, but are not limited to, cancers such as breast, lung, prostate, skin, colorectal, bladder, kidney, liver, thyroid, endometrial, muscle, bone, mesothelial, vascular, fibrous, leukemia or lymphoma.

As used herein, the term "effective amount" refers to the amount or dose of the conjugated antibody compounds of the invention that, upon single or multiple dose administration to a patient, provides the desired pharmacological effect to the patient. An effective amount can be readily determined by the attending diagnostician as one of skill in the art by considering a number of factors, such as the species of mammal; its body shape, age and general health; the specific disease or surgical procedure involved; the extent or severity of the disease or aeipathia (malady); response of the individual patient; the particular compound or composition being administered; mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant medication.

The disclosed subject matter relates to peptides and polypeptides, which may include fusion polypeptides and conjugates. As used herein, the terms "peptide" or "polypeptide" or "protein" are used interchangeably to refer to a polymer of amino acids. In general, a "polypeptide" or "protein" is defined as a longer amino acid polymer that is typically greater than 50, 60, 70, 80, 90, or 100 amino acids in length. A "peptide" is generally defined as a short polymer of amino acids, typically 50, 40, 30, 20 or less amino acids in length.

A "polypeptide," "protein" or "peptide" as contemplated herein generally comprises a polymer of protein amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) or non-protein amino acids as contemplated herein.

The term "protein amino acids" refers to those amino acids found in naturally occurring proteins, and may be referred to as "encoded amino acids". The term "non-protein amino acids" refers to those amino acids not found in naturally occurring proteins, and may be referred to as "non-coding amino acids". Thus, the term "non-protein amino acid" refers to amino acids other than alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

The term "fusion" refers to a polypeptide sequence comprising an exogenous amino acid sequence fused to a natural amino acid sequence. The exogenous sequence may be fused to the N-terminus of the native amino acid sequence, the C-terminus of the native amino acid sequence, or within the native amino acid sequence such that the fusion protein comprises the N-terminal portion of the native amino acid sequence, the exogenous amino acid sequence, and the C-terminal portion of the native amino acid sequence.

The term "conjugate" refers to a molecule in which two non-native covalently bound components are covalently bound directly or via a linking group. Conjugates may include peptides or polypeptides that have been covalently bound to an antibody or antigen binding fragment thereof. The disclosed conjugates can be covalently bound through a bond formed between a reactive group present on the peptide or polypeptide and a reactive group present on the antibody or antigen binding fragment thereof. In some embodiments, a bond may be formed between an electrophilic reactive group present on a peptide or polypeptide and a nucleophilic reactive group present on an antibody or antigen-binding fragment thereof. Electrophilic reactive groups may include, but are not limited to, maleimide groups, maleimide-diaminopropionate groups, iodoacetamide groups, or vinyl sulfone groups. Nucleophilic reactive groups may include, but are not limited to, free sulfhydryl groups (i.e., reduced disulfide bonds).

The disclosed subject matter relates to antibodies and antigen binding fragments thereof. The term "antibody" refers to an immunoglobulin molecule comprising two heavy and two light chains interconnected by disulfide bonds, unless otherwise specified. The amino-terminal portion of each chain comprises a variable region of about 100 to about 110 amino acids, which is primarily responsible for antigen recognition by the Complementarity Determining Regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

As used herein, the term "antigen-binding fragment" refers to any antibody fragment that retains the ability to bind to its antigen. Such "antigen binding fragments" may include, but are not limited to Fv, scFv、Fab、F(ab') ₂ Fab', scFv-Fc fragments and diabodies. The antigen binding fragment of an antibody typically comprises at least one variable region. Preferably, the antigen binding fragment comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR). More preferably, the antigen binding fragments as used herein comprise HCVR and LCVR that confer antigen binding specificity for an epitope of a target antigen.

As used herein, the term "Light Chain Variable Region (LCVR)" refers to a portion of the LC of an antibody molecule that includes the amino acid sequences of the complementarity determining regions (CDRs; i.e., LCDR1, LCDR2, and LCDR 3) and the Framework Regions (FR).

As used herein, the term "Heavy Chain Variable Region (HCVR)" refers to a portion of an HC of an antibody molecule that includes the amino acid sequences of complementarity determining regions (CDRs; i.e., HCDR1, HCDR2, and HCDR 3) and Framework Regions (FR).

As used herein, the terms "complementarity determining regions" and "CDRs" refer to non-contiguous antigen binding sites found within the variable regions of LC and HC polypeptides of an antibody or antigen binding fragment thereof.

CDRs are interspersed with regions that are more conserved, known as framework regions ("FR"). Each LCVR and HCVR consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: PRE CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3", and the three CDRs of HC are referred to as "HCDR1, HCDR2 and HCDR3". CDRs contain most of the residues that interact specifically with the antigen. Numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is according to known practices.

Common numbering conventions include the "kabat numbering" and "EU IndexNumbering" systems. As used herein, "Kabat Numbering" or "Kabat Numbering system" refers to the Numbering system for amino acid residues in the variable and constant domains designed and set forth by the authors in Kabat et al, sequences ofProteins ofImmunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991) for specifying heavy and light chains of antibodies. As used herein, "EU Index Numbering" or "EU Index Numbering system" refers to the numbering convention used to designate amino acid residues in the antibody heavy chain constant domain and is also set forth in Kabat et al (1991). Other conventions that include a correct or alternative numbering system for the 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 (HoneggerA, pluckthunA (2001) Jmol Biol 309:657-670). All references to immunoglobulin heavy chain constant regions c| -1, hinges, c| -2, and c| -3 amino acid residues (i.e., numbers) presented in the specification, examples, and claims are based on EU Index Numbering unless explicitly stated otherwise herein.

The general structure of "IgG antibodies" is well known. Wild-type (WT) antibodies of the IgG type are heterotetramers of four polypeptide chains (two identical heavy chains and two identical light chains) that are cross-linked by intra-and inter-chain disulfide bonds. Each Heavy Chain (HC) consists of an N-terminal heavy chain variable region ("V) _H ") and heavy chain constant region (" C) _H ") composition. The heavy chain constant region consists of three domains (C _H 1、C _H 2 and C _H 3) At C _H 1 and C _H The hinge region between domains 2 ("hinge"). Each Light Chain (LC) consists of an N-terminal light chain variable region ("V) _L ") and light chain constant region (" C) _L ") composition. V (V) _L And C _L The region may be of the kappa ("kappa") or lambda ("lambda") isoforms (respectively "C _κ "OR" C _λ "). Each heavy chain is linked by an interface (V _H /V _L Interface) and heavy chain constant domain C _H 1 and the constant domain of the light chain (C _H 1/C _L Interface) associated with a light chain. Each V _H -C _H 1 and V _L -C _L The association between fragments forms two identical antigen binding fragments (Fab) that direct the antibody to bind to the same antigen target or epitope. Each heavy chain passing through hinge-C of each heavy chain _H 2-C _H The interface between the 3 fragments associates with another heavy chain, two of which C _H 2-C _H The association between the 3 fragments forms the Fc region of the antibody. Each Fab and Fc together form the characteristic "Y-shaped" architecture of IgG antibodies Wherein each Fab represents an "arm" of "Y". IgG antibodies can be further divided into subclasses, such as IgG1, igG2, igG3, and IgG4, which differ in the length of the hinge region, the number and position of inter-and intra-chain disulfide bonds, and the amino acid sequence of the respective HC constant regions.

The variable regions of each heavy chain-light chain pair associate to form a binding site. Heavy chain variable region (V) _H ) And a light chain variable region (V _L ) May be subdivided into hypervariable regions of complementarity determining regions ("CDRs") interspersed with regions that are more conserved, i.e., framework regions ("FR"). Each V _H And V _L Consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs of the heavy chain may be referred to as "CDRH1, CDRH2, and CDRH3", while 3 CDRs of the light chain may be referred to as "CDRL1, CDRL2, and CDRL3". The FRs of the heavy chain may be referred to as HFR1, HFR2, HFR3, and HFR4, while the FRs of the light chain may be referred to as LFR1, LFR2, LFR3, and LFR4. CDRs contain most of the residues that interact specifically with the antigen.

Antibodies and antigen binding fragments thereof for the disclosed conjugates can be produced using techniques well known in the art, such as recombinant expression in mammalian or yeast cells. In particular, the methods and procedures of the embodiments herein may be readily employed. Furthermore, the antibodies and antigen binding fragments of the invention may be further engineered to comprise framework regions derived from a fully human framework. A variety of different human framework sequences may be used to carry out embodiments of the present invention. As a particular embodiment, the framework regions used in the antibodies and antigen binding fragments of the conjugates of the invention are of human or substantially human origin (at least 95%, 97% or 99% are of human origin). The sequence of the framework regions of human origin is known in the art and can be obtained from The Immunoglobulin Factsbook, marie-Paule Lefranc, gerard Lefranc, academic Press 2001,ISBN 012441351.

Expression vectors capable of directing the expression of genes operably linked thereto are well known in the art. The expression vector contains appropriate control sequences such as promoter sequences and replication initiation sites. They may also encode suitable selectable markers and signal peptides that facilitate secretion of the desired polypeptide product by the host cell. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide. Nucleic acids encoding a desired polypeptide, such as the HC and LC components of the conjugated IgG antibodies of the invention, may be expressed independently using different promoters operably linked thereto in a single vector, or nucleic acids encoding a desired product may be expressed independently using different promoters operably linked thereto in separate vectors. Standard methods can be used to prepare single expression vectors encoding the HC and LC components of the cysteine engineered IgG antibodies of the invention.

As used herein, "host cell" refers to a cell that is stably or transiently transfected, transformed, transduced or infected with a nucleotide sequence encoding one or more desired polypeptide products. Creation and isolation of host cell lines producing IgG antibodies for use in the present invention can be accomplished using standard techniques known in the art. Mammalian cells are preferred host cells for expressing cysteine engineered IgG antibodies according to the invention. Specific mammalian cells include HEK293, NSO, DG-44 and CHO cells. Preferably, the assembled protein is secreted into the medium in which the host cells are cultured, from which the protein can be recovered and isolated. The medium into which the protein has been secreted may be purified by conventional techniques. For example, the medium may be applied to and eluted from the protein a or G column using conventional methods. Soluble aggregates and multimers 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, for example at-70 ℃, or may be lyophilized. As will be appreciated by those skilled in the art, antibodies, when expressed in certain biological systems, such as mammalian cell lines, glycosylate in the Fc region, unless mutations are introduced in the Fc to reduce glycosylation. In addition, antibodies may also be glycosylated at other positions.

Disclosed herein are novel chemical entities and uses of chemical entities, for example in the form of peptides and conjugates of the disclosed peptides. Chemical entities may be described using terms known in the art and discussed further below.

The term "alkyl" as considered herein includes all isomeric forms of straight or branched chain alkyl groups thereof, such as straight or branched chain groups of 1 to 12, 1 to 10 or 1 to 6 carbon atoms, referred to herein as C1-C12 alkyl, C1-C10-alkyl and C1-C6-alkyl, respectively.

The term "alkylene" refers to a divalent group that is a straight or branched chain alkyl group (i.e., straight or branched C ₁ Divalent radical of a C6 alkyl group). Exemplary alkylene groups include, but are not limited to, -CH ₂ -、-CH ₂ CH ₂ -、-CH ₂ CH ₂ CH ₂ -、-CH(CH ₃ )CH ₂ -、-CH ₂ CH(CH ₃ )CH ₂ -、-CH(CH ₂ CH ₃ )CH ₂ -and the like.

The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH ₂ F、-CHF ₂ 、-CF ₃ 、-CH ₂ CF ₃ 、-CF ₂ CF ₃ Etc.

The term "heteroalkyl" as used herein refers to an "alkyl" in which at least one carbon atom has been replaced with a heteroatom (e.g., O, N or S atom). One type of heteroalkyl is "alkoxy".

The term "alkenyl" as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched chain group of 2-12, 2-10 or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl and C2-C6-alkenyl, respectively.

The term "alkynyl" as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched chain group of 2-12, 2-10 or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl and C2-C6-alkynyl, respectively.

The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic or bridged (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein as, for example, "C4-8-cycloalkyl", which is derived from a cycloalkane. Unless otherwise indicated, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate (phosphonato), phosphinate (phosphonato), sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl groups. In certain embodiments, the cycloalkyl is unsubstituted, i.e., it is unsubstituted.

The term "cycloheteroalkyl" refers to a monovalent saturated cyclic, bicyclic or bridged hydrocarbon radical of 3-12, 3-8, 4-8, or 4-6 carbons, wherein at least one carbon of the cycloalkane is replaced by a heteroatom, such as N, O and/or S.

The term "cycloalkenyl" refers to cycloalkyl groups that are unsaturated at one or more ring bonds.

The term "partially unsaturated carbocyclyl" refers to a monovalent cyclic hydrocarbon containing at least one double bond between ring atoms, wherein at least one ring of the carbocyclyl is not aromatic. Partially unsaturated carbocyclyl groups may be characterized by the number of ring carbon atoms. For example, a partially unsaturated carbocyclyl group may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms and is therefore referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl group, respectively. The partially unsaturated carbocyclyl group may be in the form of a monocyclic carbocycle, a bicyclic carbocycle, a tricyclic carbocycle, a bridged carbocycle, a spiro carbocycle, or other carbocyclic ring system. Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl and partially unsaturated bicyclic carbocyclyl. Unless otherwise indicated, a partially unsaturated carbocyclic group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxy, imino, ketone, nitro, phosphate, phosphonate (phosphinato), phosphinate (phosphinato), sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the partially unsaturated carbocyclyl is unsubstituted, i.e., it is unsubstituted.

The term "aryl" is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjacent rings (which rings are "fused rings"), wherein at least one ring is aromatic, and for example the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and/or aryl. Unless otherwise indicated, an aromatic ring may be substituted at one or more ring positions with, for example, halo, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido or carboxyamido, carboxylic acid, -C (O) alkyl, -CO ₂ Alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF ₃ -CN, etc. In certain embodiments, the aromatic ring is substituted with halogen, alkyl, hydroxy, or alkoxy at one or more ring positions. In certain other embodiments, the aromatic ring is unsubstituted, i.e., it is unsubstituted. In certain embodiments, aryl is a 6-10 membered ring structure.

The terms "heterocyclyl" and "heterocyclic group" are art-recognized and refer to a saturated, partially unsaturated, or aromatic 3-to 10-membered ring structure, or 3-to 7-membered ring, which ring structure includes 1 to 4 heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl can be specified using the 5Cx-Cx nomenclature, wherein x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3-to 7-membered ring structure containing 1 to 4 heteroatoms, such as nitrogen, oxygen and sulfur. The designation "C3-C7" means that the heterocycle contains a total of 3 to 7 ring atoms, including any heteroatoms that occupy the ring atom position.

The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines (e.g., mono-or di-substituted amines), wherein the substituents can include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

The term "alkoxy" is art-recognized and refers to an alkyl group as defined above having an oxygen group attached thereto. Representative alkoxy groups include methoxy, ethoxy, t-butoxy, and the like.

An "ether" is two hydrocarbons covalently linked through an oxygen or sulfur atom. Thus, the substituent of the alkyl group that makes the alkyl group an ether is or is analogous to an alkoxy group or a thiol.

The term "carbonyl" as used herein refers to the group-C (O) -.

The term "oxo" refers to a divalent oxygen atom-O-.

The term "carboxamido" as used herein refers to the group-C (O) NRR ', where R and R' may be the same or different. R and R' may, for example, independently be hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl or heterocyclyl.

The term "carboxy" as used herein refers to the group-COOH or its corresponding salt, such as-COONa or the like.

As used herein, the term "amide" or "amido (amidyl)" refers to-R ¹ C(O)N(R ² )-、-R ¹ C(O)N(R ² )R ³ -、-C(O)NR ² R ³ or-C (O) NH ₂ A group of the form, wherein R ¹ 、R ² And R is ³ For example, each independently is hydrogen, alkyl, alkoxy, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxy, ketone, or nitro.

The compounds of the present disclosure (e.g., peptides and conjugates thereof) may contain one or more chiral centers and/or double bonds, and thus exist as stereoisomers, such as geometric isomers, enantiomers, or diastereomers. As used herein, the term "stereoisomer" consists of all geometric isomers, enantiomers or diastereomers. These compounds may be represented by the symbols "R" or "S" or "+" or "-" depending on the configuration of substituents around the steric carbon atom and/or the optical activity observed. The present invention includes various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated "(±)" in nomenclature, but the skilled artisan will recognize that the structure may imply a chiral center. It is to be understood that the graphic description of a chemical structure (e.g., a general chemical structure) encompasses all stereoisomeric forms of the specified compounds, unless the context clearly dictates otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiomerically pure compound, which compositions may comprise, consist essentially of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of the R enantiomer of the given compound).

When read from left to right, the polypeptide chains described herein may be depicted by their amino acid sequence from the N-terminus to the C-terminus, wherein each amino acid is represented by its single letter or three letter amino acid abbreviation. Unless otherwise indicated herein, all amino acids used in the preparation of the polypeptides of the invention are L-amino acids and stereoisomers thereof are D-amino acids. The "N-terminus" (or amino terminus) of an amino acid or polypeptide chain refers to the free amine group on the amino acid, or on the first amino acid residue of the polypeptide chain. Furthermore, the term "N-terminal amino acid" refers to the first amino acid in a polypeptide chain. Likewise, the "C-terminal" (or carboxyl-terminal) of an amino acid or polypeptide chain refers to the free carboxyl group on the amino acid, or on the last amino acid residue of the polypeptide chain. Furthermore, the term "C-terminal amino acid" refers to the last amino acid in a polypeptide chain.

As used herein, the phrase "substituted at a residue..the term" amino acid name "in reference to a heavy or light chain polypeptide refers to substitution of a parent amino acid with a specified amino acid. For example, a heavy chain comprising "alanine substituted at residue 235" refers to a heavy chain in which the parent amino acid sequence has been mutated to contain alanine in place of the parent amino acid at residue number 235. Such mutations can also be indicated by indicating specific amino acid residue numbers, preceded by a parent amino acid, followed by a replacement amino acid. For example, "F235A" refers to substitution of phenylalanine at residue 235 with alanine. Similarly, "235A" refers to the substitution of alanine for the parent amino acid. "engineering" a cysteine refers to substituting a parent amino acid with a cysteine.

As used herein, "N-formyl-methionine peptide" refers to a peptide in which the N-terminal amino acid is formylated methionine. The N-formyl-methionine residues of the disclosed peptides may comprise one or more halogen substitutions. Thus, an N-formyl-halogen substituted methionine residue may have the formula:

wherein R is ¹ 、R ² And R is ³ Independently selected from hydrogen and halogen (e.g., F, cl, br or I), and R ¹ 、R ² And R is ³ At least one of (C), preferably R ¹ 、R ² And R is ³ At least two of (a) are halogen, more preferably R ¹ 、R ² And R is ³ Each is halogen (e.g., wherein the methionine residue comprises C (halogen) at the end of the side chain ₃ ). As used herein, "N-formyl-CF ₃ By methionine peptide "is meant a peptide wherein the N-terminal amino acid is formylated methionine comprising a trifluoro-substituted methyl group at the end of the methionine side chain.

As used herein, "linker" refers to a structure that connects two or more additional structures. Examples of linkers include peptide linkers, protein linkers, PEG linkers, and combinations thereof. As used herein, "maleimide-PEG linker" is meant to include the formula "- (O-CH") ₂ -CH ₂ ) n- "polyethylene glycol (PEG) polymer and chemical moiety of derivatized maleimide functional group, wherein" n "is 3-24, wherein the linker can form a covalent linkage to the antibody or antigen-binding fragment thereof via a thioether bond between the maleimide functional group and a cysteine residue in the antibody or antigen-binding fragment Covalent linkages to the N-formyl-methionine peptide may be made by an amide bond to the epsilon amino side chain of the C-terminal lysine of the N-formyl-methionine peptide or to the gamma carboxyl group of the C-terminal glutamic acid of the N-formyl-methionine peptide.

As shown, the disclosed peptides and conjugates may include one or more polyethylene glycol (PEG) polymer attachment sequences, and may be considered pegylated. As will be appreciated by those skilled in the art, pegylation reagents are generally described with reference to the molecular weight (in daltons or kilodaltons) of the PEG polymer moiety of the PEG-containing compound in the reagent. In addition, many commercial PEG-containing reagents typically have a degree of polydispersity, which means that the number of repeating ethylene glycol monomer units ("n") contained within the reagent varies within a range, typically within a narrow range. Thus, references to the molecular weight of a PEG polymer in a reagent generally refer to the average molecular weight of the PEG polymer contained within the reagent. Ethoxy monomer- (O-CH) for reagents for preparing conjugated antibody compounds of the invention ₂ -CH ₂ ) Having a molecular weight of about 44g/mol or 44 daltons. Thus, when using a pegylation reagent represented by its average molecular weight, one skilled in the art can readily determine the value of "n", and as such, the value of "n" in the resulting conjugated antibody compound.

As used herein, a formyl group consists of a carbonyl group bonded to a hydrogen bond, and is represented by formula CH (O) -or the following formula.

The N-terminal methionine residue may include a formyl group (i.e., an N-formyl substitution on the N-terminal nitrogen atom).

Maleimide refers to the following structure:

/>

maleimide-containing moieties useful for conjugation to the molecules disclosed herein may include maleimide-diaminopropionic acid having the structure:

and

maleimidopropionic acid having the structure:

The disclosed subject matter relates to peptides comprising N-formylmethionine in which the methyl group of the methionine side chain has been substituted with one or more halogens such as fluorine. N-formyl-halogen substituted methionine exhibits oxidation resistance. Peptides comprising N-formyl-halogen substituted methionine are useful as agonists of formyl peptide receptor (FPR-1) and may be conjugated to antibodies or antigen binding fragments thereof. The conjugates thus prepared are useful for targeting cells and attracting and activating immune cells comprising FPR-1 directed against the target cells.

In some embodiments, the disclosed subject matter relates to conjugated antibodies or antigen binding fragments thereof. The conjugate comprises an antibody or antigen-binding fragment thereof conjugated to a peptide comprising an N-formyl-halogen substituted methionine residue at the N-terminus of the peptide. Suitable N-formyl-halomethionine residues may include, but are not limited to, N-formyl-methionine trifluoride. N-formyl-fluoromethionine can be prepared using methods disclosed in the art. (see, e.g., houston et al, biorg & medical. Chem. Lett ", vol.7, no.23, pages 3007-3012, 1997, the contents of which are incorporated herein by reference in their entirety).

The peptide and antibody or antigen binding fragment thereof may be directly conjugated via a reactive group in the peptide and a reactive group in the antibody or antigen binding fragment. Alternatively, the peptide and antibody or antigen binding fragment thereof may be conjugated via a linker having a reactive group for conjugation to the peptide and having a reactive group for conjugation to the antibody or antigen binding fragment thereof. In some embodiments, the conjugate may have the formula:

/>

the components of the disclosed conjugates, i.e., the peptide, optional linker, and antibody or antigen binding fragment thereof, may be conjugated via bonds formed between any suitable reactive groups. In some embodiments, the peptide comprises a C-terminal glutamic acid residue, and the peptide is conjugated to the linker via an amide bond formed between the gamma carboxyl group of glutamic acid and the amino group of the linker. In other embodiments, the peptide comprises a C-terminal lysine residue and the peptide is conjugated to the linker via an amide bond formed between the epsilon amino group of the lysine and the carboxyl group of the linker.

The disclosed conjugates can comprise a plurality of peptides, a plurality of linkers, and/or a plurality of antibodies or antigen binding fragments thereof. In some embodiments, the conjugate comprises at least two peptides and a linker, and may form a branched structure. In some embodiments, the conjugate has the formula:

Which may be characterized as branched structures.

The peptides disclosed herein generally include an N-terminal, N-formyl, halogen substituted methionine residue. The peptide generally comprises additional amino acids, and in some embodiments, the peptide may comprise 2-50 amino acids (or 2-40, 2-30, 2-20, or 2-10 amino acids) bonded via peptide bonds formed between amino and carboxyl groups in the backbone or side chains of the amino acids. Preferably, the disclosed peptides are resistant to cleavage by endopeptidases (e.g., neutrophil-associated endopeptidases, particularly integral membrane endopeptidases). In some embodiments, the disclosed peptides are resistant to endopeptidase 24.11 (EP 24.11; e.c.3.4.24.11, also known as enkephalinase neutral endopeptidase, CALLA, CD10 or enkephalinase); and/or endopeptidase 24.15 (EP 24.15; E.C.3.4.24.15, a metallopeptidase found in alveolar macrophages, monocytes, T lymphocytes and B lymphocytes); and/or CD 13/aminopeptidase N (CD 13/APN); and/or BP-1/6C 3/aminopeptidase A (BP-1/6C 3/APA); and cleavage by CD 26/dipeptidyl peptidase IV (CD 26/DPPIV).

In some embodiments, the disclosed peptides and conjugates thereof comprise one or more non-protein amino acids at the N-terminus, including N-formyl-halogen substituted methionine, and optionally one or more non-protein amino acids other than N-formyl-halogen substituted methionine. Preferably, the non-protein amino acids and/or the bonds formed between the non-protein amino acids render the peptide resistant to cleavage by endopeptidases as disclosed herein.

As understood in the art, a non-protein amino acid is an amino acid that is not encoded in an organism and is not observed to be naturally present in a protein. Protein amino acids include L-amino acid forms of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Thus, a non-protein amino acid may be defined as an amino acid that is not any of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine (i.e., a molecule comprising a free amino group and a free carboxyl group bonded to an alpha-carbon atom). For example, the non-protein amino acid may have the formula NH ₂ -C (R) -COOH, wherein R is not any side chain encoding an amino acid of a protein.

In some embodiments, the disclosed peptides and conjugates thereof comprise one or more non-protein amino acids selected from the group consisting of D-amino acids. Suitable D-amino acids may include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and/or valine.

In some embodiments, the disclosed peptides comprise a non-protein amino acid that is a homolog of the encoded amino acid that lacks one or more methylene groups (-CH) between the alpha-carbon and side chains of the amino acid ₂ -). Suitable homologs of non-protein amino acids for use as the disclosed peptides and conjugates thereof may include, but are not limited to, 2-aminoisobutyric acid, 2-amino-2-glycolic acid, 2α -methyl-2-hydroxy-glycine, 2-amino-2-methylbutanoic acid (i.e., isovaline), methylcysteine, azetidine-2-carboxylic acid, phenylglycine, 4-hydroxyphenylglycine, 3-indolylglycine, aminomalonic acid, 2, 3-diamino-3-oxopropionic acid, 2-amino-2- (1H-imidazol-5-yl) acetic acid, ornithine, 2, 4-diaminobutyric acid, 2, 3-diaminopropionic acid, and 2-amino-4- (diaminomethyleneamino) butyric acid.

In some embodiments, the disclosed peptides comprise a non-protein amino acid that is a homolog of the encoded amino acid having one or more additional methylene groups (-CH) between the alpha-carbon and the side chain ₂ (-) (e.g., having a single additional methylene (-CH) ₂ (-) homoamino acids, having two additional methylene groups (-CH) ₂ -CH ₂ Double high amino acids (bishomo-amino acids), etc.). Suitable homologs of non-protein amino acids for use as disclosed peptides and conjugates thereof may include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine homoamino acids such as homoalanine, homoarginine, homoglutamine, homoglutamic acid, homoisoleucine, homoleucine, homolysine, homomethionine, homophenylalanine, homoproline (i.e., piperidine-2-carboxylic acid), homoserine, homothreonine, homotryptophan, and homotyrosine. By using Suitable homologs of non-protein amino acids for the disclosed peptides and conjugates thereof can include, but are not limited to, the dihomo amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

In some embodiments, the disclosed peptides comprise non-protein amino acids that are substituted with an alkyl group on the alpha-carbon (e.g., C ₁ -C ₆ Alkyl substituted, such as methyl). Suitable alkyl-substituted amino acids may include alpha-carboalkyl-substituted alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, such as 2-methyl-serine (i.e., alpha-methyl-serine), 2-methyl-threonine (i.e., alpha 0-methyl-threonine), alpha 1-methyl-valine, alpha 2-methyl-leucine, 2-amino-2, 3-dimethyl-pentanoic acid (i.e., alpha 3-methyl-isoleucine), alpha 4-methyl-methionine, alpha 5-methyl-cysteine, 2-methyl-proline, alpha 6-methyl-phenylalanine, alpha-methyl-tyrosine, alpha-methyl-tryptophan, 2-methyl-aspartic acid, 2-methyl-glutamic acid, 2, 4-diamino-2-methyl-4-oxobutyric acid (i.e., alpha-methyl-asparagine), 2, 5-diamino-2-methyl-5-oxopentanoic acid (i.e., alpha 3-methyl-isoleucine), alpha 4-methyl-methionine, alpha 5-methyl-cysteine, alpha-methyl-phenylalanine, alpha-methyl-alpha-2-methyl-lysine, alpha-methyl-lysine, and lysine in some embodiments, the disclosed peptides comprise non-protein amino acids that are substituted with a dialkyl group on the alpha-carbon (e.g., C ₁ -C ₆ Dialkyl substituted, such as dimethyl). Suitable dialkyl substituted amino acids may comprise alpha-carbodialkyl substituted glycine, such as di-n-propyl glycine (Dpg).

In some embodiments, the disclosed peptides comprise non-protein amino acids that are amino-substituted (e.g., C ₁ -C ₆ Alkyl-substituted, e.g. methyl) alkylated amino groupsAnd (3) acid. Suitable N-alkylated amino acids may include N-alkylated alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (e.g., N-methyl-alanine, N-methyl-arginine, N-methyl-asparagine, N-methyl-aspartic acid, N-methyl-cysteine, N-methyl-glutamic acid, N-methyl-glutamine, N-methyl-glycine, N-methyl-histidine, N-methyl-isoleucine, N-methyl-leucine, N-methyl-lysine, N-methyl-methionine, N-methyl-phenylalanine, N-methyl-proline, N-methyl-serine, N-methyl-threonine, N-methyl-tryptophan, N-methyl-tyrosine and N-methyl-valine).

In some embodiments, the disclosed peptides comprise a non-protein amino acid selected from phenylalanine, tyrosine, tryptophan, histidine, proline, naphthylalanine, which optionally includes a peptide selected from C ₁ -C ₆ Ring substitutions of alkyl substitutions, halogen substitutions and cyano substitutions. Suitable non-protein amino acids may include 2-fluoro-phenylalanine, 2-methyl-tyrosine, and 2-naphthylalanine.

In some embodiments, the disclosed peptides comprise non-protein amino acids selected from the group consisting of n-amino acids (nor-amino acids) and/or linear core amino acids (linear core amino acids). Suitable noramino acids and/or linear core amino acids may include, but are not limited to, norleucine (Nle), norvaline (Nva), 12-amino-dodecanoic acid, 8-amino-octanoic acid, 7-amino-heptanoic acid, 6-amino-hexanoic acid, and 5-amino-pentanoic acid.

In some embodiments, the disclosed peptides comprise non-protein amino acids, which are encoded protein amino acids substituted with substituents. Suitable non-protein amino acids may include alanine substituted with a substituent selected from alkynyl (e.g., propargylglycine), azido (e.g., 4-azido-homoalanine), phenylthio, thienyl (e.g., 3- (2-thienyl) -alanine), pyridyl (e.g., 3- (4-pyridyl) -alanine (4-Pal)), anthracenyl, cycloalkyl, diphenyl, furyl, and naphthyl (e.g., 2-naphthylalanine).

In some embodiments, the disclosed peptides comprise non-protein amino acids containing ethyleneoxy moieties. Suitable non-protein amino acids may include those having the formula NH ₂ -CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _n -COOH amino acids, wherein n is selected from 1-24.

In some embodiments, the disclosed peptides comprise non-protein amino acids that are not alpha-amino acids. Suitable non-alpha-amino acids may include beta-amino acids, gamma-amino acids, delta-amino acids, epsilon-amino acids, and zeta-amino acids (e.g., beta-, gamma-, delta-, epsilon-and zeta-amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

In some embodiments, the disclosed peptides comprise non-protein amino acids that are cyclic amino acids (e.g., non-proline cyclic amino acids). A cyclic amino acid is an amino acid that contains a cyclic group formed from a nitrogen atom and a carboxyl group. Suitable cyclic amino acids may include, but are not limited to, aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, azepane-2-carboxylic acid, cyclic leucine, homocyclic leucine (homocyclic), 1-piperidine-4-carboxylic acid, piperidine-3-carboxylic acid, 1-piperazine acetic acid, 4-piperidine acetic acid, and 1-piperidine acetic acid.

In some embodiments, the disclosed peptides comprise protein amino acids and/or non-protein amino acids optionally including amino protecting groups. Suitable amino protecting groups may include, but are not limited to, allyloxycarbonyl (Alloc), 9-fluorenylmethylcarbonyl (Fmoc), t-Butylcarbonyl (BOC), and benzylcarbonyl (Cbz).

The disclosed peptides can be conjugated directly to an antibody or antigen binding fragment thereof. In other embodiments, the disclosed peptides can be indirectly conjugated to an antibody or antigen binding fragment thereof via a linker. In some embodiments, the linker has a selected linear length. Suitable selected lengths may include at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 angstroms or longer, or ranges defined by any of these values (e.g., 5-10 angstroms, 10-20 angstroms, 15-25 angstroms, 20-35 angstroms, 30-40 angstroms, 35-50 angstroms, and 40-50 angstroms).

In some embodiments, the disclosed joints have spacer arms that can provide the joint with a selected length. For example, the disclosed linkers can have the formula:

suitable spacer arms may comprise a polymeric moiety, such as polyethylene glycol.

In some embodiments, the linker has a formula selected from the group consisting of:

Wherein n is an integer selected from 3-24.

As a particular embodiment, the linker may include a maleimide moiety and a PEG moiety, and may be referred to as a "maleimide-PEG linker" that will N-formyl-CF ₃ Methionine conjugated to an antibody. Exemplary conjugates may have a formula selected from the group consisting of:

in some embodiments, the spacer comprises a peptide sequence of 1-20 amino acids. In some embodiments, the spacer is a peptide sequence comprising an amino acid selected from glycine, serine, and alanine (e.g. (G ₄ S) _m Wherein m is an integer selected from 1-5).

The disclosed linkers may comprise polyethylene glycol moieties, for example as spacer arms or otherwise. In some embodiments, the linker comprises a polyethylene glycol (PEG) moiety (i.e., (-O-CH 2-CH 2) _1-24 )。

In some embodimentsThe disclosed linkers may comprise two or more PEG moieties separated by a non-PEG moiety such as an amino acid moiety. In some embodiments, the disclosed linker comprises a polypeptide represented as- ((PEG) _1-24 )-(AA) _1-2 -((PEG) _1-24 ) -a separate polyethylene glycol moiety wherein AA is glutamic acid bonded via gamma aminoacylation or lysine bonded via epsilon aminoacylation.

Suitable peptide-linkers as disclosed herein may have a formula selected from the group consisting of:

frm-M(CF ₃ )-Ile-Phe-Leu-Peg12-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Leu-Phe-Peg12-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg6-γE-γE-Peg6-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg6-εK-εK-Peg6-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y

frm-M(CF ₃ )-Dpg-2Nal-αMeF-γE-Peg12-NH-(CH ₂ ) ₂ -Y,

frm-M(CF ₃ )-Dpg-4Pal-αMeF-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y,

Wherein Y comprises an amino or cysteine reactive moiety for conjugating the peptide-linker to the antibody. Suitable cysteine reactive moieties may include, but are not limited to, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone.

In some embodiments of the disclosed conjugates, the linker comprises a maleimide moiety, and the linker is conjugated to the antibody or antigen-binding fragment thereof via a thioether bond formed between the maleimide moiety and a cysteine residue of the antibody.

Suitable amino acid residues for conjugation to the disclosed peptides and linkers may include cysteine residues. Suitable cysteine residues may be engineered in the antibody or antigen-binding fragment thereof, wherein a cysteine residue is a native cysteine residue of the antibody or antigen-binding fragment thereof, or a non-native cysteine residue of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG heavy chain constant region and a light chain region, wherein the cysteine residue is selected from C based on EU Numbering Index _H 1 domain residues 124, C _H Residue 378 of 3 domain, or C _H Residues 124 and C of the 1 domain _H 3 domain residues 378. In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG heavy chain constant region comprising isoleucine substituted for cysteine at residue 247, glutamic acid substituted for cysteine at residue 332, or both isoleucine substituted for cysteine at residue 247 and glutamic acid substituted for cysteine at residue 332, based on EUNumbering Index. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53, 54, 55, 56 or 57.

Suitable antibodies for use in the disclosed conjugates and methods may include human antibodies. Other suitable antibodies may include mouse, rat, or rabbit antibodies. Suitable therapeutic antibodies may include human antibodies, chimeric or hybrid antibodies, and humanized antibodies.

Suitable antibodies may include IgG isotypes. Suitable IgG isotypes can include isotypes having an IgG heavy chain constant region selected from a human IgG1 isotype or a human IgG4 isotype.

Suitable antibodies may include monoclonal antibodies. Suitable antibodies may include monospecific antibodies and bispecific antibodies.

The disclosed peptides and optional linkers can be used to prepare conjugates of antibodies or antigen binding fragments thereof known in the art. For example, the disclosed peptides and optional linkers can be conjugated to existing cancer therapeutic antibodies to make N-methyl acyl-Met (CF) ₃ ) Peptide conjugated immunotherapeutic agents.

Exemplary cancer therapeutics for the preparation of peptide conjugates can include IgG1 therapeutic antibodies (i.e., igG1 antibodies, such as trastuzumab and pertuzumab) that target solid tumors, including HER-2 expressing tumors, igG1 therapeutic antibodies (i.e., igG1 and IgG1 enhanced ADCC antibodies, such as rituximab, ofatuzumab, octuzumab and AME133 v) that target liquid tumors that express CD20, and antibodies (i.e., emituzumab) that target c-Met expressing tumors.

N-formyl-Met (CF) as disclosed herein ₃ ) The peptide may be conjugated to a therapeutic antibody comprising a cytotoxic agent to act as an additional therapeutic agent. Alternatively, an N-formyl-Met (CF) ₃ ) Peptides can replace cytotoxic agents in therapeutic antibodies to create novel therapeutic antibodies that target antigens that are overexpressed in cancer cells. Target antigens and representative therapeutic antibodies may include, but are not limited to, GPNMB (vitamin-GmbH (glembatumumab vedotin)), CD56 (moxacin-Luo Wo Tuozhu mab (lorvotuzumab mertansine) (IMGN-901)), TACSTD2 (TROP 2; go Sha Tuozhu mab (sacituzumab govitecan), (IMMU-132)), CEACAM5 (La Bei Zhushan anti-labtuzumab) SN-38), folate receptor-a (Soxhlet-Mituximab (mirvetuximab soravtansine) (IMGN-853), vintakolide), mucin 1, sialoglycorope) CA6; SAR-566658, STEAP1 (vitamin-Wandouzumab (vandortuzumab vedotin) (RG-7450)), mesothelin (DMOT 4039A, lei Xing-anetuzumab (anetumab ravtansine) (BAY-94-9343), BMS-986148), connexin 4 (enrolment mab (enfortumab vedotin) (ASG-22M 6E); ASC-22 CE), ENPP3 (AGS-16M 8F), guanylate cyclase C (statin-infliximab (indusatumab vedotin) (MLN-0264)), SLC44A4 (ASG-5 ME), naPi2b (statin-infliximab (lifastuzumab vedotin)), CD70 (TNFSF 7; DNIB0600A, AMG-172, MDX-1243, statin-Wo Setuo bead mab (vorsetuzumab mafodotin) (SGN-75)), CA9 carbonic anhydrase (BAY 79-4620), 5T4 (TPBG; PF 06263507), SLTRK6 (ASG-15 ME), SC-16 (anti-Fyn 3; SC16LD6.5), tissue factor (HuMax-TF-ADC (TF-011-MMAE)), LIV-1 (ZIP 6; SGN-LIV 1A), P-cadherin (PCA), C9 carbonic anhydrase (BAY 79-4620), PSMA (MLN 2704, PSMA-acd), fibronectin Extra domain B (Extra-domain B) (human mabs L19 and F8), endothelin receptor ETB (RG-7636), VEGFR2 (CD 309; anti-VEGFR-2 ScFv-As 203-invisible nanoparticles (stealth nanoparticles)), tenascin c (anti-TnC-A1 antibody SIP (F16)), periostin (periostin) (anti-periostin antibody), DLL3 (lovastatin B), soravtan sine), HER2 (T-DM 1, ARX788, SYD 985), EGFR (ABT-414, IMGN289, AMG-595), CD30 (rituximab (brentuximab vedotin), rituximab (iraumab-060)), CD22 (oxybutyzumab (Inotuzumab ozogamicin) (544), vitamin-pinacob (pinatuzumab vedotin), epalruzumab (CD 38), CD19 (CD 35), sguzumab (CD 35), sg35 (CD-4639), sg35 (sg35) and CD-4639 (CD-4639), sg35 (sg35) and (CD 35). (see, e.g., thomas et al, lancet Oncol.2016, month 6; 17 (6) e254-62 and Diamond and Banerji, brit. Journ. Cancer,2016;114,362-367, the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the antibody or antigen binding fragment thereof of the disclosed conjugates comprises one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a known antibody optionally selected from the group consisting of vildagliclazide, moxacin-Wo Tuozhu mab (IMGN-901), TROP2; golian Sha Tuozhu mab, (IMMU-132), la Bei Zhushan anti-SN-38, sorcin-rituximab (IMGN-853), vintakolide, salivary glycoepitope CA6; SAR-566658, enrolment mAb (ASG-22M 6E), ASC-22 CE), ZIP6, SGN-LIV1A, DMOT4039A, lei Xing-anetuzumab (BAY-94-9343), BMS-986148, statin-Sofostuzumab (sofituzumab vedotin), sorrow-midtuximab (IMGN-853), vintaflolide, la Bei Zhushan anti-SN-38, statin-glituzumab, mortieren-Wo Tuozhu mAb (IMGN-901), statin-wandopitumomab (RG-7450), pharmaceutical compositions containing the same, and pharmaceutical compositions containing the same AGS-16M8F, vitin-British tozumab (MLN-0264), ASG-5ME, vitin-Lifastuzumab, TNFSF7, DNIB0600A, AMG-172, MDX-1243, martin-Wo Setuo bead mAb (SGN-75), BAY79-4620, TPBG, PF 06263507, SLTRK6 (ASG-15 ME), anti-Fyn 3, SC16LD6.5), huMax-TF-ADC (TF-011-MMAE), PCA062, human mAb L19, human mAb F8, RG-7636, CD309; anti-VEGFR-2 ScFv-As2O 3-invisible nanoparticles, anti-TnC-A1 antibody SIP (F16), anti-periostin antibody, lovastatin soravtansine, ABT-414, IMGN289 AMG-595, bentuximab, itumomab MDX-060, oxintuzumab (CMC-544), statin-pinatuzumab, epaizumab SN38, poloxamer, lei Xing-cootuximab, SAR-3419, SGN-CD19A, lei Ying totuzumab, milatuzumab doxorubicin, IMGN-529, gemtuzumab ozogamicin, IMGN779, SGN CD 33A, and IGN523.

The peptides and conjugates thereof may be formulated as pharmaceutical compositions. In some embodiments, the disclosed pharmaceutical compositions comprise: (i) a conjugated antibody or antigen fragment thereof as disclosed herein; and (ii) one or more pharmaceutically acceptable carriers, diluents or excipients.

The disclosed peptides, conjugates, and pharmaceutical compositions thereof are useful in methods of treating diseases and disorders in a subject in need thereof. In some embodiments, the disclosed methods include methods of treating solid cancer or liquid tumors comprising administering to a patient in need thereof an effective amount of a conjugated antibody as disclosed herein or a pharmaceutical composition thereof. Suitable cancers for treatment using the disclosed peptides, conjugates, pharmaceutical compositions and methods can include, but are not limited to, breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia or lymphoma.

The disclosed peptides, conjugates, and pharmaceutical compositions are useful for treating a subject in need thereof. In some embodiments, the disclosed peptides, conjugates, and pharmaceutical compositions are useful for treating solid or liquid tumors, optionally selected from breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia, or lymphoma.

Also disclosed herein are methods of activating neutrophils, particularly stimulating Reactive Oxygen Species (ROS) production in neutrophils in vivo or in vitro. The method comprises contacting neutrophils with a conjugate as disclosed herein under conditions such that the conjugate stimulates ROS production in the neutrophils. In some embodiments, the conjugate comprises a spacer of suitable length for inducing ROS production in neutrophils. For example, the conjugate may comprise a polyethylene glycol (PEG) spacer of suitable length for inducing ROS production (e.g., a PEG spacer comprising at least 12 monomers).

Also disclosed herein are compounds, which may otherwise be referred to herein as peptides. In some embodiments, the compound has the formula:

R-P ¹ -P ² -P ³ -NH-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y, or a salt thereof, wherein:

r is HC (=O)

P ¹ Met (C (halogen) _m Wherein m is 1-3 (e.g., met (CF) ₃ ) Or Met (CHF) ₂ ) Or Met (CH) ₂ F))；

P ² Is bonded to P via peptide bond ¹ And 1-6 proteinogenic or non-proteinogenic amino acids bonded to each other;

P ³ is composed of a compound containing a-COOH moiety (e.g., glutamic acid or aspartic acid) or-NH ₂ Amino acids of part (e.g. lysine) of the side chain, of glutamic acid residues optionally linked via its side chain gamma-carboxyl group or of lysine residues linked via its side chain epsilon-amino group, and P ³ Bonding to P via peptide bond ² ；

n is an integer selected from 3-24; and

y comprises an amino or cysteine reactive moiety, optionally wherein Y is selected from maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone.

In other embodiments, the disclosed compounds have the formula (i):

r is HC (=O)

P ¹ Met or Met (C (halogen) _m Wherein m is 1-3 (e.g., met (CF) ₃ ) Or Met (CHF) ₂ ) Or Met (CH) ₂ F))；

n is an integer selected from 3-24; and

The disclosed peptides and conjugates are preferably agonists of one or more members of the formyl peptide receptor family. Preferably, the disclosed peptides and conjugates are agonists of formyl peptide receptor 1 (FPR-1). Preferably, the disclosed peptides and conjugates bind to one or more members of the formyl peptide receptor family. Preferably, the disclosed peptides and conjugates bind to one or more members of the formyl peptide receptor family present on the surface of neutrophils. Preferably, the disclosed peptides and conjugates bind to one or more members of the formyl peptide receptor family with a Kd of at least about 10uM, 1uM, 100nM, 50nM, 10nM or less. Thus, the disclosed peptides and conjugates can be used in a method of agonizing a formyl peptide receptor comprising contacting the formyl peptide receptor with the peptide or conjugate.

Examples

The following examples are illustrative and should not be construed as limiting the scope of the claimed subject matter.

Nail for preparing conjugatesacyl-Met (CF 3) and PEG peptides

Abstract

The oxidation of the sulphur atom of the methionine residue and the formation of methyl sulfoxide or the formation of met (O) occurs in vivo for the natural formyl-methionine peptide. (see FIG. 1). Oxidation of the sulfur atom of the methionine residue results in a significant reduction (e.g., up to 10×) in formyl peptide receptor 1 (FPR-1) agonist activity. Thus, oxidation-resistant formyl-trifluoro-methionine-based peptides (i.e., frm-Met-Containing (CF) ₃ ) Peptide of (c). Peptides containing additional non-protein amino acids and polyethylene linkers/spacers were also prepared. These peptides were tested to determine if they could act as FPR-1 agonists to determine if these peptides were suitable for use in the preparation of antibody bioconjugates to promote FPR-1 mediated and targeted cell killing by innate immune cells.

It was also observed that the fraction containing frm-Met (CF ₃ ) Is capable of activating the human formyl peptide receptor (FPR-1) on neutrophils, which makes them suitable for modifying antibodies and creating antibody conjugates that exhibit FPR-1 mediated and targeted cell killing. It was further observed that the linker length relative to the polyethylene linker was important for inducing free Radical Oxygen (ROS) production in neutrophils. Finally, it was found that the composition contained frm-Met (CF ₃ ) The peptide-conjugated antibodies of (2) exhibit similar clearance properties to unconjugated antibodies (clearance profile).

Background

Bacterial antibodies (bacillabodes) are antibody bioconjugates that promote participation of the innate immune system in targeted cell killing. They consist of antibodies targeting specific cells conjugated to pathogen-associated molecular patterns (PAMPs) that can activate innate immune cells to kill target cells.

The formyl peptide provides a PAMP that can be conjugated to an antibody to make a bacterial antibody (bacterial antibody). Two well-characterized formyl peptides are formyl-Met-Leu-Phe (fMLF) and formyl-Met-Ile-Phe-Leu (fMIFL). fMLF and fmifel are Formyl Peptide Receptor (FPR) agonists and FPR-1 receptor is an activating receptor present on innate immune cells. fMLF is an agonist that is effective at human FPR-1, while fmifel is an agonist that is effective at both human and mouse FPR-1 receptors.

One concern with the in vivo activity of formyl-methionine based FPR-1 agonists is that oxidation of methionine also reduces the activity of the frm-Met peptide. (see FIG. 1). Furthermore, while frm-Met peptides with natural amino acids perform well in vitro, they degrade rapidly in vivo, possibly by natural endopeptidases present on the surface of cells carrying FPR-1 or by circulating endopeptidases. To address metabolic stability, modified FPR-1 agonist peptides containing non-protein orthoacids that are stable for in vivo use were created. Finally, although the frm-Met peptide itself acts as an agonist, this study shows that the frm-Met peptide must be presented via a linker to obtain maximum activity.

Described herein are trifluoro-modified peptides with methionine that eliminate methionine oxidation potential while retaining FPR-1 agonism. The disclosed peptides also include non-protein amino acids to inhibit digestion by endopeptidases. The disclosed peptides also include a PEG-containing linker, which the inventors have shown to be important for maximum agonist activity.

Results and observations

Creation of peptides.As shown in FIGS. 2, 3 and 4, the present inventors prepared a group of compounds having frm-Met (CF ₃ ) Non-protein amino acids and PEG-conjugated peptides.

frm-Met(CF ₃ ) The synthetic chemistry of (2) is shown in figure 5. Fmoc-S-trityl-L-homocysteine (1, 1.695g, 2.719 mmol) was dissolved in DCM (25 mL) and triisopropylsilane (4 mL,19.5 mmol) was added at 21℃followed by TFA (15 mL,198.4 mmol) and the reaction mixture was stirred for 1 h. Concentrated in vacuo and co-distilled with MeOH to give2(0.973 g,2.72 mmol). Will be2Dissolve in DCM (25 mL) and cool the solution to-78 ℃. To be dissolved in DCM3(1.00 g,2.94 mmol) was added to the reaction mixture and stirred at-78℃for 30 minutes. The reaction mixture was adsorbed onto celite and purified by reverse phase chromatography (50% -70% water/acetonitrile containing 0.1% fa) to give Fmoc-S- trifluoromethyl-L-homocysteine (4, 153mg,0.33mmol, 12.2%). ¹ H NMR(500HMz,DMSO)δ12.7(bs,1H),7.89(d,2H),7.73-7.70(m,3H),7.42(t,2H),7.33(t,2H),4.31-4.30(m,2H),4.24-4.23(m,1H),4.12-4.08(m,1H),2.80-2.70(m,2H),2.16-1.95(m,2H)； ¹⁹ F(352MHz,DMSO)δ-40(s,3F)；MS[M ⁺ +Na]448 (448); purity 90%, based on HPLC-UV (300 nm).

Briefly, FRM-023 and FRM063 comprise the human peptide MIFL, except that FRM-063 comprises FRM-Met (CF ₃ ) Whereas FRM-023 contains FRM-Met.

Similarly, FRM-050 and FRM-054 contained the mouse peptide MFL, except that FRM-054 contained FRM-Met (CF ₃ ) Whereas FRM-50 comprises FRM-Met.

FRM-052 represents oxidized control peptide.

FRM-059 represents FRM-Met (CF) of FRM-047 ₃ ) A derivative.

FRM-055 represents a D-Nle derivative of FRM-047.

FRM-060 and FRM-061 include two "split" PEG6 linkers to reduce the flexibility of the PEG 12 linker present in FRM-059.

FRM-041 and FRM-051 are forms of FRM-023 and FRM-050, respectively, with terminal maleimides.

FRM-053 is a form of FRM-052 with terminal maleimide.

FRM-058 is a form of FRM-055 with terminal maleimide.

FRM-048 and FRM-049 are branched versions of FRM-047 that contain two peptides and lack terminal maleimide (FRM-048) or have terminal maleimide (FRM-049).

FRM-057 is a derivative of FRM-047 having an N-terminal methoxin residue (i.e., methionine in which the sulfur atom is replaced with an oxygen atom).

FRM-056 is a polymer having FRM-Met (CF) ₃ ) And lacks FRM-047 derivatives of Nle.

FRM-062 is a catalyst having FRM-Met (CF) ₃ ) And FRM-047 derivatives of 4-Pal.

In vitro.frm-Met peptides and frm-Met (CF) ₃ ) C-terminal polyethylene glycol of peptideAlcoholization enhances FPR-1 mediated Reactive Oxygen Species (ROS) production from primary human neutrophils. As shown in FIGS. 6 and 9, frm-Met peptide and frm-Met (CF ₃ ) C-terminal PEGylation of peptides enhances ROS production activity in neutrophils. With frm-Met (CF) having a protein amino acid ₃ ) In comparison with the peptide (plus PEG), frm-Met (CF) with non-protein amino acids ₃ ) Peptides (plus PEG) also enhance ROS production from primary human neutrophils.

As shown in FIG. 8, migration of primary human neutrophils was in frm-Met peptide with protein amino acid (MLF plus PEG) and frm-Met with protein amino acid (CF) ₃ ) Peptide (frm-M (CF) ₃ ) LF plus PEG).

The inventors also tested conjugated peptides. As shown in fig. 9, the generation of ROS from primary human neutrophils by peptides conjugated to specific eCys sites on trastuzumab through maleimide requires a linker/spacer (e.g., PEG linker/spacer). frm-Met peptide conjugated to trastuzumab and lacking a spacer failed to stimulate ROS production from human neutrophils.

In vivo.Trastuzumab bacterial antibodies (bacillabodes) with frm-Met peptides and non-protein amino acids cleared faster than trastuzumab parent antibodies, so as to be less exposed to bacterial antibodies (bacillabods). As shown in fig. 10Avs, fig. 10B (as well as fig. 10C and 10D), conjugated peptide FRM-047 resulted in higher clearance of conjugated antibodies than the unconjugated parent antibody. Estimated exposure based on AUC0- ≡showed that the exposure of the Tmab parent was 3.8 times higher compared to the Tmab bacterial antibody (bacillabody) with FRM-047 peptide. However, when trastuzumab (Tmab) was conjugated to the peptide FRM-058 (which was a peptide with FRM-Met (CF) ₃ ) Partial peptide FRM-047 derivative) clearance is similar to that of the parent trastuzumab. (see FIG. 11). Thus, frm-Met (CF) ₃ ) The above trifluoro substitution provides reduced clearance for trastuzumab anti-bacterial antibody (bacterial) compared to the same bacterial antibody (bacterial) with frm-Met peptide, and clearance is similar to that of the Tmab parent antibody.

Method

ROS production.Human neutrophils were purified from fresh blood collection as described above. Reactive oxygen species production from primary human neutrophils was measured using luminol-enhanced chemiluminescence. After separation, the PMNs were separated at 1X10 ⁶ Individual cells/ml were suspended in HBSS (Gibco # 14025-092) containing calcium and magnesium supplemented with 0.25% human serum albumin (Gemini Bio product # 800-120) and 50 μm luminol (Sigma-Aldrich # 123072-2.5G). Then 100. Mu.l of the cell suspension (1X 10 ⁵ Individual total cells) were dispensed into each well of a 96-well plate (Greiner # 655098) suitable for fluorescence measurement, and the temperature was equilibrated to 37 ℃ for 5 minutes. After equilibration, 10x agonist solution was applied to the wells to reach a final concentration of 1 x. Immediately after the addition of the agonist, the chemiluminescent signal was recorded by photometer. Area Under Curve (AUC) scores were calculated using the luminescence signals from the first 5 minutes of each run. The values are shown as relative units of luminescence.

Chemotaxis.Chemotaxis of neutrophils across the transwell membrane (Corning # 3415) to agonists was measured in a modified Boyden chamber assay. About 2-4x10 from neutrophil enriched formulation ⁵ The individual cells were seeded in the upper transwell chamber on a membrane with 3.0 μm pores. The lower transwell chamber contains buffer with or without a test agent. After inoculation in transwells, the cells were placed in a humidified incubator at 37 ℃. After 1 hour, any cells in the upper chamber were removed and CellTiter-Glo was used ^tm (Promega #G7571) the percentage of cells successfully migrated to the lower chamber was quantified according to the manufacturer-specified procedure. The percentage of successful migration relative to the maximum cell input value was determined using a standard curve. Values are shown as a percentage of the initial cell input value. A U-shaped dose response curve is expected. The migration from the upper chamber to the lower chamber requires a concentration gradient, and as the peptide concentration increases, the enhanced diffusion of the peptide to the upper chamber results in a degradation of the concentration gradient required to drive the migration from the upper chamber to the lower chamber.

Conclusion(s)

frm-Met peptides and frm-Met (CF) ₃ ) Pegylation of peptides enhances FPR-1 mediated Reactive Oxygen Species (ROS) production from primary human neutrophils. With non-proteinsfrm-Met (CF) of amino acid ₃ ) Peptide ratio frm-Met (CF) with natural amino acids ₃ ) The peptides more effectively drive FPR-1 mediated ROS production from primary human neutrophils.

In the foregoing description, it will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been described in terms of specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

A number of patent and non-patent references may be cited herein. The cited references are incorporated herein by reference in their entirety. In the case where there is an inconsistency between the definitions of terms in the present specification and those of the cited references, the terms should be interpreted based on the definitions in the present specification.

Some embodiments are as follows, numbered 1-53:

(1) A conjugated antibody or antigen-binding fragment thereof comprising an antibody or antigen-binding fragment thereof conjugated to a peptide comprising an N-formyl-halomethionine residue at the N-terminus of the peptide.

(2) The conjugated antibody of embodiment 1, wherein said N-formyl-halomethionine residue is N-formyl-methionine trifluoride.

(3) The conjugated antibody of embodiment 1 or 2, wherein the antibody is conjugated to the peptide via a linker.

(4) The conjugated antibody of any of the preceding embodiments, wherein the conjugated antibody has the formula:

(5) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises a C-terminal glutamic acid residue and the peptide is conjugated to the linker via an amide bond formed between the gamma carboxyl group of glutamic acid and the amino group of the linker.

(6) The conjugated antibody of any of embodiments 1-5, wherein said peptide comprises a C-terminal lysine residue and said peptide is conjugated to a linker via an amide bond formed between the epsilon amino group of lysine and the carboxyl group of the linker.

(7) The conjugated antibody of any of the preceding embodiments, wherein the conjugated antibody has the formula:

(8) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids and comprise a halogen substituted amino acid (e.g., N-formyl-methionine trifluoride at the N-terminus).

(9) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of D-amino acids (e.g., D-amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

(10) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from amino acid homologs lacking one or more methylene groups between the a-carbon and the side chain or amino acid homologs having additional methylene groups between the a-carbon and the side chain (e.g., homoamino acids, dihomoamino acids, etc.).

(11) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which are optionally non-proteinogenic amino acids selected from the group consisting of amino acid homologs lacking one or more methylene groups between the a-carbon and side chains (e.g., 2-aminoisobutyric acid, 2-amino-2-glycolic acid, 2 a-methyl-2-hydroxy-glycine, 2-amino-2-methylbutanoic acid (i.e., isovaline), methylcysteine, azetidine-2-carboxylic acid, phenylglycine, 4-hydroxyphenylglycine, 3-indolylglycine, aminomalonic acid, 2, 3-diamino-3-oxopropionic acid, 2-amino-2- (1H-imidazol-5-yl) acetic acid, ornithine, 2, 4-diaminobutyric acid, 2, 3-diaminopropionic acid, and 2-amino-4- (diaminomethyleneamino) butyric acid.

(12) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of homoamino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, such as homoalanine, homoarginine, homoglutamine, homoglutamic acid, homoisoleucine, homoleucine, homolysine, homomethionine, homophenylalanine, homoproline (i.e., piperidine-2-carboxylic acid), homoserine, homothreonine, homotryptophan, and homotyrosine).

(13) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of dihomo amino acids (e.g., dihomo amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

(14) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino groupsAn acid, optionally a non-protein amino acid selected from the group consisting of amino acids comprising an alkyl substitution on the alpha-carbon (e.g., C ₁ -C ₆ Alkyl substituted) alkylated amino acids (e.g., alpha-carboalkyl substituted alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, such as 2-methyl-serine (i.e., alpha-methyl-serine), 2-methyl-threonine (i.e., alpha 0-methyl-threonine), alpha 1-methyl-valine, alpha 2-methyl-leucine, 2-amino-2, 3-dimethyl-pentanoic acid (i.e., alpha 3-methyl-isoleucine), alpha 4-methyl-methionine, alpha 5-methyl-cysteine, 2-methyl-proline, alpha-methyl-phenylalanine, alpha-methyl-tyrosine, alpha-methyl-tryptophan, 2-methyl-aspartic acid, 2-methyl-glutamic acid, 2, 4-diamino-2-methyl-4-oxobutyric acid (i.e., alpha-methyl-asparagine), 2, 5-diamino-2-methyl-5-oxopentanoic acid (i.e., alpha 3-methyl-isoleucine), alpha 4-methyl-methionine, alpha 5-methyl-cysteine, alpha-methyl-phenylalanine, alpha-methyl-2-methyl-arginine and alpha-lysine, alpha-methyl-lysine)).

(15) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of amino acids comprising dialkyl substitutions on the alpha-carbon (e.g., C ₁ -C ₆ Alkyl substituted) dialkylated amino acids (e.g., alpha-carbodialkyl substituted glycine, such as dipropylglycine (Dpg)).

(16) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of amino groups comprising alkyl substitutions (e.g., C ₁ -C ₆ Alkyl substituted) alkylated amino acids (e.g., N-alkylated alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, such as N-methyl-alanine, N-methyl-arginineAmino-asparagine, N-methyl-aspartic acid, N-methyl-cysteine, N-methyl-glutamic acid, N-methyl-glutamine, N-methyl-glycine, N-methyl-histidine, N-methyl-isoleucine, N-methyl-leucine, N-methyl-lysine, N-methyl-methionine, N-methyl-phenylalanine, N-methyl-proline, N-methyl-serine, N-methyl-threonine, N-methyl-tryptophan, N-methyl-tyrosine and N-methyl-valine.

(17) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, optionally non-protein amino acids selected from the group consisting of phenylalanine, tyrosine, tryptophan, histidine, proline, naphthylalanine, optionally comprising a peptide selected from the group consisting of C ₁ -C ₆ One or more ring substitutions of alkyl substitution, halogen substitution, and cyano substitution (e.g., 2-fluoro-phenylalanine, 2-methyl-tyrosine, and 2-naphthylalanine).

(18) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from noramino acids (nor-amino acids) and/or linear core amino acids (linear core amino acids) such as norleucine (Nle), norvaline (Nva), 12-amino-dodecanoic acid, 8-amino-octanoic acid, 7-amino-heptanoic acid, 6-amino-hexanoic acid, and 5-amino-pentanoic acid.

(19) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of: amino acids (e.g., alanine) comprising a substituent selected from the group consisting of alkynyl (e.g., propargylglycine), azido (e.g., 4-azido-homoalanine), phenylthio, thienyl (e.g., 3- (2-thienyl) -alanine), pyridyl (e.g., 3- (4-pyridyl) -alanine (4-Pal)), anthracenyl, cycloalkyl, diphenyl, furyl, and naphthyl.

(20) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of having the formula NH ₂ -CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _n -COOH amino acid, wherein n is selected from1-24。

(21) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of β -amino acids, γ -amino acids, δ -amino acids, ε -amino acids, and ζ -amino acids (e.g., β -, γ -, δ -, ε -and ζ -amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

(22) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which are optionally non-protein amino acids selected from the group consisting of cyclic amino acids (e.g., aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, azepane-2-carboxylic acid, cyclic leucine, homocyclic leucine (homocyclic leucine), 1-piperidine-4-carboxylic acid, piperidine-3-carboxylic acid, 1-piperazine acetic acid, 4-piperidine acetic acid, and 1-piperidine acetic acid).

(23) The conjugated antibody of any of the preceding embodiments, wherein said peptide comprises an amino protecting group (-N-optionally selected from allyloxycarbonyl (Alloc), 9-fluorenylmethylcarbonyl (Fmoc), t-Butylcarbonyl (BOC), and benzylcarbonyl (Cbz)).

(24) The conjugated antibody of any of the preceding embodiments, wherein said linker comprises a spacer arm having a length of about 10-50 angstroms (or 10-40 angstroms, or 10-30 angstroms, or 10-20 angstroms).

(25) The conjugated antibody of any of the preceding embodiments, wherein the linker comprises a spacer and the linker has a formula selected from the group consisting of:

(26) The conjugated antibody of embodiment 24 or 25, wherein said linker has a formula selected from the group consisting of

Wherein n is an integer selected from 3-24.

(27) The conjugated antibody of embodiment 24 or 25, wherein said spacer is a peptide sequence comprising an amino acid selected from the group consisting of glycine, serine, and alanine (e.g. (G ₄ S) _m Wherein m is an integer selected from 1-5).

(28) The conjugated antibody of any of the preceding embodiments, wherein said linker comprises a polyethylene glycol (Peg) moiety (i.e., (-O-CH 2) _1-24 )。

(29) The conjugated antibody of any of the preceding embodiments, wherein said linker comprises a moiety expressed as- ((Peg) _1-24 )-(AA) _1-2 -((Peg) _1-24 ) -a separate polyethylene glycol moiety wherein AA is a glutamic acid residue linked via its side chain γ carboxyl group or a lysine residue linked via its side chain epsilon amino group.

(30) The conjugated antibody of any of the preceding embodiments, wherein said peptide-linker has a formula selected from the group consisting of: frm-M (CF) ₃ )-Ile-Phe-Leu-Peg12-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Leu-Phe-Peg12-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg6-γE-γE-Peg6-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg6-εK-εK-Peg6-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y、frm-M(CF ₃ )-Dpg-2Nal-αMeF-γE-Peg12-NH-(CH ₂ ) ₂ Y and frm-M (CF) ₃ )-Dpg-4Pal-αMeF-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y, wherein Y comprises an amino or cysteine reactive moiety for conjugating the peptide-linker to the antibody.

(31) The conjugated antibody of embodiment 30, wherein said cysteine-reactive moiety is selected from maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone.

(32) The conjugated antibody of any of the preceding embodiments, wherein the linker comprises a maleimide moiety, and the linker is conjugated to the antibody via a thioether bond formed between the maleimide moiety and a cysteine residue of the antibody.

(33) The conjugated antibody of embodiment 32, wherein said antibody comprises an IgG heavy chain constant region and a light chain region, wherein said cysteine residue is selected from the group consisting of C _H 1 domain residues 124, C _H Residue 378 of 3 domain, or C _H Residues 124 and C of the 1 domain _H 3 domain residues 378.

(34) The conjugated antibody of any of the preceding embodiments, wherein the antibody is a human antibody, chimeric or hybrid antibody, or a humanized antibody.

(35) The conjugated antibody of any of the preceding embodiments, wherein said antibody comprises an IgG heavy chain constant region selected from the group consisting of a human IgG1 isotype or a human IgG4 isotype.

(36) The conjugated antibody of any of the preceding embodiments, wherein the antibody comprises an IgG heavy chain constant region comprising isoleucine substituted at residue 247, glutamic acid substituted at residue 332, or both isoleucine substituted at residue 247 and glutamic acid substituted at residue 332, wherein numbering is based on EU Index Numbering.

(37) The conjugated antibody of any of the preceding embodiments, wherein said antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NOs 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53, 54, 55, 56 or 57.

(38) The conjugated antibody of any of the preceding embodiments, wherein said antibody is a monoclonal antibody.

(39) The conjugated antibody of any of the preceding embodiments, wherein said antibody is a bispecific antibody.

(40) The conjugated antibody of any of the preceding embodiments, wherein said antibody binds to an antigen selected from the group consisting of HER2, PSMA, TROP2, MUC-1, connexin 4, LIV-1, mesothelin, MUC-16, folate receptor-R1, CEACAM5, GPNMB, CD56, STEAP1, ENPP3, guanylate cyclase C, SLC A4, naPi2B, CD70, CA9 carbonic anhydrase, 5T4, SC-16, tissue factor, P-cadherin, fibronectin extra domain B, endothelin receptor ETB, VEGFR2, tenascin c, periostin (peristin), DLL3, EGFR, CD30, CD22, CD79B, CD19, CD138, CD74, CD37, CD33, and CD 98.

(41) The conjugated antibody of any of the preceding embodiments, wherein the antibody comprises one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody selected from the group consisting of: T-DM1, ARX788, SYD985, MLN2704, PSMA-aDC, TACSTD2, ago Sha Tuozhu mab, (IMMU-132), mucin 1, sialoglycoepitope CA6; SAR-566658, enrolment mAb (ASG-22M 6E), ASC-22 CE), ZIP6, SGN-LIV1A, DMOT4039A, lei Xing-anetuzumab (BAY-94-9343), BMS-986148), viltin-solituzumab, sorrow-mitoximab (IMGN-853), vintakolide, la Bei Zhushan anti-SN-38, viltin-gabexamab, morcin-lox Wo Tuozhu mAb (IMGN-901), viltin-vorituximab (RG-7450), AGS-16M8F, viltin-infltuzumab (MLN-0264), ASG-5ME, viltin-rituximab, TNF 7, DNIB0600A, AMG-172, MDFSX-1243, martin-Wo Setuo bead mAb (SGN-75), BAY79-4620, TPBG, PF 06263507, SLG 6, FG-15), SCYNAF-16M 8, and MMF-0628, MMF-062-309, and MMF-062-011; anti-VEGFR-2 ScFv-As2O 3-invisible nanoparticles, anti-TnC-A1 antibody SIP (F16), anti-periostin antibody, lovatuzumab, soravtansine, ABT-414, IMGN289 AMG-595, bentuximab, itumumab MDX-060, oxtuzumab (CMC-544), veterinatuzumab-pinacouzumab, epaizumab SN38, poloxamer, lei Xing-cootuximab, SAR-3419, SGN-CD19A, lei Ying tuximab, milatuzumab doxorubicin, IMGN-529, gemtuzumab ozogamicin, IMGN779, SGN CD 33A and IGN523.

(42) A pharmaceutical composition comprising the conjugated antibody of any of the preceding embodiments and one or more pharmaceutically acceptable carriers, diluents or excipients.

(43) A method of treating a solid cancer or a liquid tumor comprising administering to a patient in need thereof an effective amount of a conjugated antibody as described in any one of embodiments 1-41 or a pharmaceutical composition thereof as described in embodiment 42.

(44) The method of embodiment 43, 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, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia, or lymphoma.

(45) The conjugated antibody of any of embodiments 1-41 for use in therapy.

(46) The conjugated antibody of any of embodiments 1-41 for use in the treatment of solid cancer or liquid tumors.

(47) The conjugated antibody of embodiment 46, 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, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia, or lymphoma.

(48) Use of the antibody of any one of embodiments 1-47 for the manufacture of a medicament for the treatment of solid or liquid cancer.

(49) The use of embodiment 48, wherein the solid or liquid tumor is selected from the group consisting 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, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia, or lymphoma.

(50) A method of stimulating Reactive Oxygen Species (ROS) production in neutrophils, the method comprising contacting neutrophils with the conjugated antibody of any of embodiments 1-41 under conditions such that the conjugated antibody stimulates ROS production in neutrophils.

(51) The method of embodiment 48, wherein the linker of the conjugated antibody comprises a polyethylene glycol spacer comprising at least 12 monomers.

(52) Having the formula R-P ¹ -P ² -P ³ -NH-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -a compound of Y or a salt thereof, wherein: r is HC (=o) -; p (P) ¹ Met (C (halogen) _m Wherein m is 1-3 (e.g. Met(CF ₃ ) Or Met (CHF) ₂ ) Or Met (CH) ₂ F)；P ² Is bonded to P via peptide bond ¹ And 1-6 proteinogenic or non-proteinogenic amino acids bonded to each other; p (P) ³ Is composed of a compound containing a-COOH moiety (e.g., glutamic acid or aspartic acid) or-NH ₂ Amino acids of part (e.g. lysine) of the side chain, of glutamic acid residues optionally linked via its side chain gamma-carboxyl group or of lysine residues linked via its side chain epsilon-amino group, and P ³ Bonding to P via peptide bond ² The method comprises the steps of carrying out a first treatment on the surface of the n is an integer selected from 3-24; and Y comprises an amino or cysteine reactive moiety, optionally wherein Y is selected from maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone.

(53) Having the formula R-P ¹ -P ² -P ³ -NH-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -a compound of Y or a salt thereof, wherein: r is HC (=o) -; p (P) ¹ Met or Met (C (halogen) _m Wherein m is 1-3 (e.g., met (CF) ₃ ) Or Met (CHF) ₂ ) Or Met (CH) ₂ F)；P ² Is bonded to P via peptide bond ¹ And 1-6 proteinogenic or non-proteinogenic amino acids bonded to each other; p (P) ³ Is composed of a compound containing a-COOH moiety (e.g., glutamic acid or aspartic acid) or-NH ₂ Amino acids of part (e.g. lysine) of the side chain, of glutamic acid residues optionally linked via its side chain gamma-carboxyl group or of lysine residues linked via its side chain epsilon-amino group, and P ³ Bonding to P via peptide bond ² The method comprises the steps of carrying out a first treatment on the surface of the n is an integer selected from 3-24; and Y comprises an amino or cysteine reactive moiety, optionally wherein Y is selected from maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone.

Sequence listing

<110> Eli Lilly and Company

<120> peptide comprising N-formyl-halomethionine residue and engineered antibody-peptide conjugate thereof

<130> 083389.01624

<150> 63/209,762

<151> 2021-06-11

<150> 63/210,292

<151> 2021-06-14

<160> 59

<170> PatentIn version 3.5

<210> 1

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (373)..(373)

<223> Xaa at position 373 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 2

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (122)..(122)

<223> Xaa at position 122 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 2

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 3

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (122)..(122)

<220>

<221> THIOETH

<222> (373)..(373)

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 4

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (122)..(122)

<220>

<221> THIOETH

<222> (370)..(370)

<223> Xaa at position 370 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 4

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Xaa Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 5

<211> 215

<212> PRT

<213> Chile person

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 6

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (127)..(127)

<223> Xaa at position 127 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<220>

<221> THIOETH

<222> (381)..(381)

<223> Xaa at position 381 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Xaa Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 7

<211> 214

<212> PRT

<213> Chile person

<400> 7

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 8

<211> 450

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (128)..(128)

<223> Xaa at position 128 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<220>

<221> THIOETH

<222> (382)..(382)

<223> Xaa at position 382 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 8

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Arg Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Leu Thr Gly Asp Thr Ser Tyr Asn Gln Lys Ser

50 55 60

Lys Leu Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Val Gly Gly Asp Trp Gln Phe Asp Val Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Ile Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Gln Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Xaa Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 9

<211> 213

<212> PRT

<213> Chile person

<400> 9

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Pro Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ala Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Leu Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 10

<211> 330

<212> PRT

<213> Chile person

<400> 10

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 11

<211> 326

<212> PRT

<213> Chile person

<400> 11

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 12

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<223> Xaa in position 7 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 12

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 13

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (258)..(258)

<223> Xaa at position 258 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 13

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 14

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (255)..(255)

<223> Xaa at position 255 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Xaa Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 15

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (258)..(258)

<400> 15

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 16

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (255)..(255)

<400> 16

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Xaa Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 17

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<400> 17

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 18

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (261)..(261)

<223> Xaa at position 261 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 18

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 19

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (258)..(258)

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Xaa Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 20

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (261)..(261)

<400> 20

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 21

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (258)..(258)

<400> 21

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Xaa Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 22

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa in position 4 is a lysine residue modified by amide bond formation with maleimide-PEG linker

<400> 22

Met Leu Phe Xaa

1

<210> 23

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<400> 23

Met Leu Phe Lys

1

<210> 24

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> peptide with maleimide-PEG linker

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<400> 24

Met Leu Phe Xaa

1

<210> 25

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formylated peptide

<400> 25

Met Leu Phe Lys

1

<210> 26

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (410)..(410)

<223> Xaa at position 410 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Xaa Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 27

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (157)..(157)

<223> Xaa at position 157 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Xaa Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 28

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (172)..(172)

<223> Xaa at position 172 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Xaa Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 29

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (192)..(192)

<223> Xaa at position 192 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Xaa

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 30

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (194)..(194)

<223> Xaa at position 194 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Xaa Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 31

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (203)..(203)

<223> Xaa at position 203 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Xaa Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 32

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (209)..(209)

<223> Xaa at position 209 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Xaa Phe Asn Arg Gly Glu Cys

210 215

<210> 33

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (127)..(127)

<220>

<221> THIOETH

<222> (160)..(160)

<223> Xaa at position 160 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Xaa

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 34

<211> 448

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (126)..(126)

<223> Xaa at position 126 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<220>

<221> THIOETH

<222> (380)..(380)

<223> Xaa at position 380 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Thr Met Asp Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Glu Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val Ala Thr Gly Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Xaa Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 35

<211> 450

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (128)..(128)

<220>

<221> THIOETH

<222> (382)..(382)

<223> cysteine residue modified by forming a thioether linkage with maleimide-PEG linker at Xaa at position 382

<400> 35

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20 25 30

Trp Met His Trp Val Arg Tyr Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Met Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Xaa Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 36

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide having maleimide-PEG linker

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa in position 5 is a lysine residue side chain modified by formation of an ε -amide bond with a hydrolyzed maleimide-PEG linker

<400> 36

Met Ile Phe Leu Xaa

1 5

<210> 37

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa in position 2 is diethyl glycine

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa at position 4 is a leucine residue C-terminally attached to a PEG linker by amide bond formation

<400> 37

Met Xaa Phe Xaa

1

<210> 38

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa in position 2 is dipropylglycine

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<400> 38

Met Xaa Phe Xaa

1

<210> 39

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa in position 3 is a phenylalanine residue which is C-terminally attached to the PEG linker by amide bond formation

<400> 39

Met Ile Xaa

1

<210> 40

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<400> 40

Met Ile Phe Xaa

1

<210> 41

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa in position 4 is a leucine residue modified by amide bond formation with maleimide-PEG linker

<400> 41

Met Ile Phe Xaa

1

<210> 42

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> N-formyl peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> formylation

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa at position 1 is norleucine

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa in position 3 is phenylalanine which is C-terminally attached to the maleimide-PEG linker by amide bond formation

<400> 42

Xaa Leu Xaa

1

<210> 43

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<400> 43

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 44

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (155)..(155)

<223> Xaa at position 155 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 44

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Xaa Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 45

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (160)..(160)

<400> 45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Xaa

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 46

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (257)..(257)

<223> Xaa at position 257 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 46

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Xaa Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 47

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (370)..(370)

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Xaa Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 48

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (392)..(392)

<223> Xaa at position 392 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 48

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Xaa Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 49

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> MISC_FEATURE

<222> (122)..(122)

<220>

<221> MISC_FEATURE

<222> (155)..(155)

<220>

<221> MISC_FEATURE

<222> (373)..(373)

<400> 49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Xaa Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 50

<211> 441

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (122)..(122)

<220>

<221> THIOETH

<222> (160)..(160)

<220>

<221> THIOETH

<222> (373)..(373)

<400> 50

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Xaa

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 51

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (127)..(127)

<220>

<221> THIOETH

<222> (381)..(381)

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Xaa Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Ile Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Gln Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Xaa Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 52

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (40)..(40)

<223> Xaa at position 40 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 52

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Xaa Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 53

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (40)..(40)

<400> 53

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Xaa Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 54

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (40)..(40)

<400> 54

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Xaa Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 55

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (45)..(45)

<223> Xaa at position 45 is a cysteine residue modified by the formation of a thioether bond with a maleimide-PEG linker

<400> 55

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Xaa Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 56

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (40)..(40)

<220>

<221> THIOETH

<222> (258)..(258)

<400> 56

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Xaa Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 57

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> modified human antibody chain

<220>

<221> THIOETH

<222> (7)..(7)

<220>

<221> THIOETH

<222> (45)..(45)

<220>

<221> THIOETH

<222> (258)..(258)

<400> 57

Ala Ser Thr Lys Gly Pro Xaa Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Xaa Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 58

<211> 212

<212> PRT

<213> Chile person

<400> 58

Arg Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly

20 25 30

Val Ala Trp Tyr Gln Asp Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Tyr Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Trp

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys

210

<210> 59

<211> 213

<212> PRT

<213> Chile person

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met

20 25 30

Tyr Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser His Ile Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

Claims

1. A conjugated antibody or antigen-binding fragment thereof comprising an antibody or antigen-binding fragment thereof conjugated to a peptide comprising an N-formyl-halomethionine residue at the N-terminus of the peptide.

2. The conjugated antibody of claim 1, wherein said N-formyl-halomethionine residue is N-formyl-methionine trifluoride.

3. The conjugated antibody of claim 1, wherein the antibody is conjugated to the peptide via a linker.

4. The conjugated antibody of claim 3, wherein the conjugated antibody has the formula:

5. the conjugated antibody of claim 3, wherein the peptide comprises a C-terminal glutamic acid residue and the peptide is conjugated to the linker via an amide bond formed between the gamma carboxyl group of glutamic acid and the amino group of the linker.

6. The conjugated antibody of claim 3, wherein the peptide comprises a C-terminal lysine residue and the peptide is conjugated to the linker via an amide bond formed between the epsilon amino group of the lysine and the carboxyl group of the linker.

7. The conjugated antibody of claim 3, wherein the conjugated antibody has the formula:

8. the conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids.

9. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of D-amino acids.

10. The conjugated antibody of claim 1, wherein the peptide comprises 2-10 amino acids selected from amino acid homologs lacking one or more methylene groups between the a-carbon and the side chain or amino acid homologs having additional methylene groups between the a-carbon and the side chain.

11. The conjugated antibody of claim 10, wherein said peptide comprises 2-10 amino acids selected from amino acid homologs lacking one or more methylene groups between the alpha-carbon and the side chain.

12. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of high amino acids.

13. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of dihomo amino acids.

14. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of alkylated amino acids comprising an alkyl substitution on the alpha-carbon.

15. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of dialkylated amino acids comprising dialkyl substitutions on the a-carbon.

16. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of alkylated amino acids comprising alkyl substitution on the amino group.

17. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of phenylalanine, tyrosine, tryptophan, histidine, proline, naphthylalanine.

18. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of an n-amino acid and a linear core amino acid.

19. The conjugated antibody of claim 1, wherein the peptide comprises 2-10 amino acids selected from the group consisting of amino acids comprising substitutions selected from the group consisting of alkynyl, azido, thiophenyl, thienyl, pyridyl, anthracenyl, cycloalkyl, diphenyl, furyl, and naphthyl.

20. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of having the formula NH ₂ -CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _n -COOH amino acids, wherein n is selected from 1-24.

21. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of β -amino acids, γ -amino acids, δ -amino acids, epsilon-amino acids, and ζ -amino acids.

22. The conjugated antibody of claim 1, wherein said peptide comprises 2-10 amino acids selected from the group consisting of cyclic amino acids.

23. The conjugated antibody of claim 1, wherein said peptide comprises an amino protecting group.

24. The conjugated antibody of claim 3, wherein said linker comprises spacer arms having a length of about 10-50 angstroms.

25. The conjugated antibody of claim 3, wherein the linker comprises a spacer and the linker has a formula selected from the group consisting of:

26. the conjugated antibody of claim 25, wherein said linker has a formula selected from the group consisting of

Wherein n is an integer selected from 3-24.

27. The conjugated antibody of claim 24, wherein said spacer is a peptide sequence comprising an amino acid selected from the group consisting of glycine, serine, and alanine.

28. The conjugated antibody of claim 3, wherein said linker comprises a polyethylene glycol (Peg) moiety (i.e., (-O-CH 2) _1-24 )。

29. The conjugated antibody of claim 3, wherein said linker comprises a moiety expressed as- ((Peg) _1-24 )-(AA) _1-2 -((Peg) _1-24 ) -a separate polyethylene glycol moiety wherein AA is a glutamic acid residue linked via its side chain γ carboxyl group or a lysine residue linked via its side chain epsilon amino group.

30. The conjugated antibody of claim 3, wherein said peptide-linker has a formula selected from the group consisting of:

frm-M(CF ₃ )-Ile-Phe-Leu-Peg12-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Leu-Phe-Peg12-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg6-γE-γE-Peg6-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Dpg-2Nal-αMeF-Nle-γE-Peg6-εK-εK-Peg6-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y，

frm-M(CF ₃ )-Dpg-2Nal-αMeF-γE-Peg12-NH-(CH ₂ ) ₂ -Y and

frm-M(CF ₃ )-Dpg-4Pal-αMeF-Nle-γE-Peg12-NH-(CH ₂ ) ₂ -Y，

wherein Y comprises an amino or cysteine reactive moiety for conjugating the peptide-linker to the antibody.

31. The conjugated antibody of claim 30, wherein said cysteine-reactive moiety is selected from the group consisting of maleimide, maleimide-diaminopropionic acid, iodoacetamide, or vinyl sulfone.

32. The conjugated antibody of claim 3, wherein the linker comprises a maleimide moiety and the linker is conjugated to the antibody via a thioether bond formed between the maleimide moiety and a cysteine residue of the antibody.

33. The conjugated antibody of claim 32, wherein said antibody comprises an IgG heavy chain constant region and a light chain region, wherein said cysteine residue is selected from the group consisting of C _H 1 domain residues 124, C _H Residue 378 of 3 domain, or C _H Residues 124 and C of the 1 domain _H 3 domain residues 378.

34. The conjugated antibody of claim 1, wherein the antibody is a human antibody, chimeric or hybrid antibody, or a humanized antibody.

35. The conjugated antibody of claim 1, wherein said antibody comprises an IgG heavy chain constant region selected from the group consisting of a human IgG1 isotype or a human IgG4 isotype.

36. The conjugated antibody of claim 35, wherein the antibody comprises an IgG heavy chain constant region comprising isoleucine substituted at residue 247, glutamic acid substituted at residue 332, or both isoleucine substituted at residue 247 and glutamic acid substituted at residue 332, wherein numbering is based on EU Index Numbering.

37. The conjugated antibody of claim 36, wherein said antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NOs 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53, 54, 55, 56 or 57.

38. The conjugated antibody of claim 1, wherein the antibody is a monoclonal antibody.

39. The conjugated antibody of claim 1, wherein the antibody is a bispecific antibody.

40. The conjugated antibody of claim 1, wherein said antibody binds to an antigen selected from the group consisting of HER2, PSMA, TROP2, MUC-1, connexin 4, LIV-1, mesothelin, MUC-16, folate receptor-R1, CEACAM5, GPNMB, CD56, STEAP1, ENPP3, guanylate cyclase C, SLC A4, naPi2B, CD70, CA9 carbonic anhydrase, 5T4, SC-16, tissue factor, P-cadherin, fibronectin extra domain B, endothelin receptor ETB, VEGFR2, tenascin c, periostin, DLL3, EGFR, CD30, CD22, CD79B, CD19, CD138, CD74, CD37, CD33, and CD 98.

41. The conjugated antibody of claim 1, wherein the antibody comprises one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of an antibody selected from the group consisting of: T-DM1, ARX788, SYD985, MLN2704, PSMA-aDC, TACSTD2, go Sha Tuozhu mAb, (IMMU-132), SAR-566658, enromumab (ASG-22M 6E), ASC-22CE, ZIP6, SGN-LIV1A, DMOT4039A, lei Xing-anetomab (BAY-94-9343), BMS-986148, viltin-sorafestuzumab, sorcin-midothuzumab (IMGN-853), vintaflolide, la Bei Zhushan anti-SN-38, viltin-glistuzumab, morcin-Wo Tuozhu mAb (IMGN-901), viltin-vandoluzumab (RG-7450), AGS-16M8F, viltin-dulcituzumab (MLN-0264), ASG-5ME, viltin-lizumab, TNF7; DNIB0600A, AMG-172, MDX-1243, martin-Wo Setuo bead mAb (SGN-75), BAY79-4620, TPBG, PF 06263507, SLTRK6 (ASG-15 ME), anti-Fyn 3, SC16LD6.5, huMax-TF-ADC (TF-011-MMAE), PCA062, human mAb L19, human mAb F8, RG-7636, anti-VEGFR-2 ScFv-As2O 3-invisible nanoparticles, anti-TnC-A1 antibody SIP (F16), anti-periostin antibody, lovatuzumab soravtansine, ABT-414, IMGN289 AMG-595, rituximab, itumumab MDX-060, oxtrastuzumab (CMC-544), vitamin-pinnacizumab, epaizumab SN38, potuzumab, lei Xing-cootuzumab, SAR-3419, SGN-CD19A, lei Ying toximab, milatuzumab doxorubicin, IMGN-529, gemtuzumab ozogamicin, IMGN779, SGN CD 33A and IGN523.

42. A pharmaceutical composition comprising the conjugated antibody of claim 1 and one or more pharmaceutically acceptable carriers, diluents or excipients.

43. A method of treating solid or liquid cancer comprising administering to a patient in need thereof an effective amount of a conjugated antibody as set forth in claim 1 or a pharmaceutical composition thereof as set forth in claim 42.

44. The method of claim 43, 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, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia, or lymphoma.

45. The conjugated antibody of claim 1 for use in therapy.

46. The conjugated antibody of claim 1 for use in the treatment of solid cancer or liquid tumors.

47. The conjugated antibody of claim 46, 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, mesothelial cancer, vascular cancer, fibrotic cancer, leukemia or lymphoma.

48. Use of the antibody of claim 1 for the manufacture of a medicament for the treatment of solid or liquid cancers.

49. The use of claim 48, wherein said solid or liquid tumor is selected from the group consisting of breast, lung, prostate, skin, colorectal, bladder, kidney, liver, thyroid, endometrial, muscle, bone, mesothelial, vascular, fibrous, leukemia, or lymphoma.

50. A method of stimulating Reactive Oxygen Species (ROS) production in neutrophils, the method comprising contacting neutrophils with the conjugated antibody of claim 1 under conditions such that the conjugated antibody stimulates ROS production in neutrophils.

51. The method of claim 48, wherein the linker of the conjugated antibody comprises a polyethylene glycol spacer comprising at least 12 monomers.

52. Having the formula R-P ¹ -P ² -P ³ -NH-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -a compound of Y or a salt thereof, wherein:

r is HC (=o) -;

P ¹ met (C (halogen) _m Wherein m is 1-3;

P ³ is composed of a-COOH-containing moietyor-NH ₂ Amino acids of side chains of part, and P ³ Bonding to P via peptide bond ² ；

n is an integer selected from 3-24; and

y comprises an amino or cysteine reactive moiety.

53. Having the formula R-P ¹ -P ² -P ³ -NH-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -a compound of Y or a salt thereof, wherein:

R is HC (=o) -;

P ¹ met or Met (C (halogen) _m Wherein m is 1-3;

P ³ is an amino acid comprising a side chain containing a-COOH moiety, and P ³ Bonding to P via peptide bond ² ；

n is an integer selected from 3-24; and

y comprises an amino or cysteine reactive moiety.