Peptides containing N-formyl-halogenated methionine residues and engineered antibody-peptide conjugates thereof

Abstract

Peptides are provided that contain N-formylmethionine, in which the methyl group of the side chain of methionine is replaced with one or more halogens (e.g., fluorine). N-formyl-halogen-substituted methionine is resistant to oxidation. Peptides that contain N-formyl-halogen-substituted methionine are useful as agonists of formyl peptide receptor-1 (FPR-1) and may be conjugated to an antibody or an antigen-binding fragment thereof. The antibody conjugates prepared thereby may be useful for targeting cells and inducing and activating immune cells that contain FPR-1 against the targeted cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 209,762, filed June 11, 2021, and U.S. Provisional Patent Application No. 63 / 210,292, filed June 14, 2021, the entire disclosures of which are incorporated herein by reference.

[0002] Sequence Listing A Sequence Listing has been submitted with this application as an ASCII text file of the Sequence Listing referred to as "083389_01624_ST25.txt", which is 162,084 bytes in size and was created on June 7, 2022. Said Sequence Listing has been submitted electronically via EFS-Web with this application and is hereby incorporated by reference in its entirety. [Background technology]

[0003] The field of the invention relates to peptides containing N-formyl-halogenated methionine residues, and modified antibody conjugates containing said peptides, which can be used in methods for treating diseases and disorders, such as cell proliferative diseases and disorders.

[0004] Antibodies and antigen-binding fragments thereof can 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, antibody conjugates can be synthesized using natural or engineered free cysteine ​​sulfhydryl groups engineered on the surface of immunoglobulin heavy or light chain residues as reactive nucleophiles to form stable chemical bonds with payload molecules directly or through a variety of linkers by thiol conjugation.

[0005] Antibodies and their antigen-binding fragments for conjugation to payload molecules are known in the art. Therefore, antibodies modified with payload molecules can be particularly useful in cancer immunotherapy. Cancer immunotherapy utilizes the body's immune system to attack cancer cells and is a dynamic field in tumor drug discovery and development. Thus, cancer immunotherapy represents a paradigm shift in which the host's immune system is used to recognize and destroy tumor cells, in contrast 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) inhibiting the suppression of the immune system, particularly cytotoxic T cells against tumors (i.e., immune checkpoint disorder), which allows the activation of adaptive and / or innate immune systems, and (2) antibody modifications designed to engage and / or enhance antibody-dependent cellular cytotoxicity (ADCC).

[0006] Successful clinical outcomes have recently been achieved with immune checkpoint modulators designed to alter the interaction of T cell surface receptors (e.g., PD-1 and CTLA-4) with their cognate ligands to trigger T cell-mediated destruction of tumor cells. Cancer immunotherapies targeting PD-1 (e.g., nivolumab (Opdivo®) and pembrolizumab (Keytruda®)) and CTLA-4 (e.g., ipilimumab (Yervoy®)) have been approved by the FDA for the treatment of cancers such as squamous non-small cell lung cancer and metastatic melanoma.

[0007] ADCC involves the interaction of the antibody Fc domain of a targeting antibody with a receptor (e.g., Fc gamma receptor IIIa) located on the surface of immune system cells (e.g., natural killer or "NK" cells), which releases cytolytic proteins from the immune cells and subsequently results in the destruction of the targeted tumor cells. Approved antibody therapies that exhibit ADCC include Rituxan® (rituximab), Arzerra® (ofatumumab), Herceptin® (trastuzumab) and Campath® (alemtuzumab). Attempts to engineer antibodies with improved ADCC activity by increasing Fc receptor binding have been useful in patients for whom antibodies with similar target specificity and low ADCC activity are ineffective or no longer moderately effective against the disease (e.g., Gazyva® obinutuzumab).

[0008] Despite current advances in cancer immunotherapy, there is still a need for alternative methods that engage the immune system to treat cancer. For example, the percentage of patients who respond to immunotherapy directed against T cells varies, and there is a lack of reliable predictive assays to identify which patients will respond. In addition, treatment-induced autoimmune disease is a severe side effect associated with immune checkpoint inhibitor therapy. The emergence of autoimmune disease with immune checkpoint inhibitors is likely related to their mechanism of action, as tumor-specific T cells are designed to emerge, proliferate, and become activated by removing the suppression of the T cell repertoire. Thus, they are relatively nonspecific, and one consequence of this lack of specificity is that autoreactive T cells can break tolerance and induce autoimmune disease that is not necessarily reversible upon cessation of treatment. Methods to enhance ADCC have been designed to use NK cells to kill tumor cells. However, NK cells are only about 5% of the total white blood cell population in the blood.

[0009] Targeting the innate immune system involved in tumor cell killing, polymorphonuclear cells (PMNs), represents another avenue for cancer immunotherapy. PMNs comprise more than 50% of the total leukocyte population and are the main line of defense against pathogens, including commensal and foreign bacteria. During the innate immune response, pathogen-associated molecular patterns (PAMPs) presented by pathogens are recognized by pattern recognition receptors (PRRs) localized 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 cell surface of neutrophils. FPR-1 detects proteins and peptides with N-formyl-methionine, including those produced and released by bacteria following infection. Engagement of FPR-1 on the surface of neutrophils with peptides containing N-formyl-methionine triggers neutrophil motility / chemotaxis to the site of infection. Activation of FPR-1 by formyl peptides also triggers pathogen-killing mechanisms such as degranulation, which releases cytotoxic molecules, reactive oxygen species (ROS) generation, and phagocytosis to destroy pathogens.There are extensive descriptions of natural and non-natural FPR-1 agonists in the literature (e.g., He HQ and Ye RD, Molecules. 2017 Mar 13;22(3). pii: E455. doi: 10.3390 / molecules22030455; Hwang TL et al., Org Biomol Chem. 2013 Jun 14;1 1 (22): 3742-55. doi: 10.1039 / c3ob40215k; Cavicchioni G et al., Bioorg Chem. 2006 Oct;34(5):298-318; Higgins JD et al., J Med Chem. 1996 Mar 1 ;39(5): 1013-5; Vergelli C et al., Drug Dev Res. 2017 Feb;78(1):49-62. doi: 10.1002 / ddr.21370; Kirpotina LN et al., Mol Pharmacol. 2010 Feb;77(2): 159-70. doi: 10.1124 / mol.109.060673; Cilibrizzi A et al., J Med Chem. 2009 Aug 27;52(16):5044-57. doi: 10.1021 / jm900592h.).

[0010] Tumor-targeted therapeutic antibodies that can engage PMN neutrophil cells of the innate immune system involved in tumor cell destruction may also offer advantages in current cancer immunotherapy. For example, such therapeutic antibodies may boost T cell responses against tumors and may not require the presence of tumor-specific T cells to promote tumor cell killing. The engagement of antitumor activity by PMN neutrophils depends on the presence of FPRs (e.g., FPR-1) that are naturally expressed on neutrophils in all patients. Furthermore, agents that can engage PMN neutrophils in tumor cell killing may be used in combination with immunotherapy to treat tumors at a dose of up to 1x10 11Since it is estimated that 10 neutrophils are produced per day, tumors may benefit from a strong and continuous supply of tumor-killing cells. Tumor-targeting antibodies that can engage neutrophils in tumor cell killing may have safety advantages over immune checkpoint regulators. Unlike checkpoint regulators, neutrophil-targeting therapies do not induce or require immune cell proliferation because blood neutrophils are short-lived. Furthermore, tumor-targeting antibodies provide a negative feedback loop that reduces immune stimulation because neutrophils are eliminated to kill targeted tumor cells along with the bound antibodies, and therapeutic antibodies are consumed by targeted effector cells.

[0011] Another way in which tumor-targeting therapeutic antibodies capable of engaging FPR-1-positive innate immune cells in tumor cells may prove useful is for the treatment of cold tumors that have a low mutational burden and therefore are not easily recognized by the immune system. Induction and activation of neutrophil-mediated tumor cell killing can result in the local production of neoantigens in a cytokine-rich environment in which adaptive immune system cells acquire the ability to recognize the tumor and target it for elimination.

[0012] Tumor-targeting antibodies that can engage neutrophils in tumor cell killing may also have an advantage over antibody-drug conjugates (ADCs), which are typically based on toxic drugs designed to release a toxic payload after internalization into tumor cells. Similar to ADCs, tumor-targeting antibodies that can engage neutrophils in tumor cell killing must recognize antigens that are highly expressed in tumor cells and low expressed in normal tissues. However, unlike ADCs, tumor-targeting antibodies that can engage neutrophils in tumor cell killing require exposure of agonists to receptors on the surface of the innate immune system, which is expected to work better with target antigens that are less likely to be internalized.

[0013] Antibodies conjugated to n-formyl peptides are known in the art and may be referred to as "bactabodies" based on the presence of n-formyl peptides in bacteria (see, for example, WO2018 / 232088, the contents of which are incorporated herein by reference in their entirety). One of the difficulties in using antibodies conjugated to peptides containing N-formyl-methionine as targeting agents and agonists to induce and activate cells containing the FPR-1 receptor is that N-formyl-methionine is oxidized at the sulfur atom in vivo to form methyl-sulfoxide or generate Met(O). Oxidation of the sulfur atom of the methionine residue significantly reduces the effectiveness of N-formylmethionine as an agonist of FPR-1. Thus, there is a need for N-formyl-methionine and peptides containing N-formylmethionine that are resistant to oxidation and function as agonists of FPR-1. Summary of the Invention

[0014] Disclosed are peptides containing N-formylmethionine, in which the methyl group of the side chain of methionine can be substituted with one or more halogens (e.g., fluorine). N-formyl halogen-substituted methionine has been shown to be less susceptible to oxidation. Peptides containing N-formyl halogen-substituted methionine can be used as agonists of formyl peptide receptors (FPRs) and conjugated to antibodies or antigen-binding fragments thereof. The antibody conjugates prepared as above can be used to target cells and induce and activate immune cells containing FPRs against the targeted cells. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows the oxidation of N-formylmethionine to N-formylmethionine S-oxide. N-formylmethionine (CF3) is resistant to S-oxidation. [Diagram 2]Figure 2 shows typical synthetic peptide-linkers: frm: formyl; MIFL: Met-Ile-Phe-Leu; Peg: polyethylene glycol monomer; M(CF3): trifluoromethylmethionine; Dpg: di-n-propylglycine; 2Nal: 2-naphthylalanine; αMeF: alpha-methyl-phenylalanine; Nle: norleucine; γE: glutamic acid residue linked by its side chain gamma carboxyl group; εK: lysine residue linked by its side chain epsilon amino group. [Diagram 3] Figure 3 shows typical synthetic peptide-linkers. frm: formyl; MIFL: Met-Ile-Phe-Leu; Peg: polyethylene glycol monomer; Mal: ​​maleimide; MLF: Met-Leu-Phe; Dpg: di-n-propylglycine; 2Nal: 2-naphthylalanine; αMeF: alpha-methyl-phenylalanine; Nle: norleucine; D-Nle: D-norleucine; γE: glutamic acid residue linked by its side chain gamma carboxyl group; M(O): oxidized methionine (control); M(CF3): trifluoromethylmethionine. The formula provided is for FRM-046 (FRM-047 w / o Mal). [Figure 4] Figure 4 shows typical synthetic peptide-linkers: frm: formyl; MIFL: Met-Ile-Phe-Leu; Peg: polyethylene glycol monomer; Mal: ​​maleimide; MLF: Met-Leu-Phe; Dpg: di-n-propylglycine; 2Nal: 2-naphthylalanine; αMeF: alpha-methyl-phenylalanine; Nle: norleucine; D-Nle: D-norleucine; γE: glutamic acid residue attached by its side chain gamma carboxyl group; M(O): oxidized methionine (control); M(CF3): trifluoromethylmethionine; 4-Pal: 4-pyridyl-alanine. [Diagram 5] FIG. 5 shows the chemical reaction for preparing Fmoc-L-trifluoromethionine from Fmoc-S-trityl-L-homocysteine. [Figure 6]FIG. 6 shows the generation of reactive oxygen species (ROS) in neutrophils activated with various peptides. [Figure 7] FIG. 7 shows the generation of reactive oxygen species (ROS) in neutrophils activated with various peptides. [Figure 8] FIG. 8 shows chemotaxis of neutrophils following exposure to the peptides. [Figure 9] FIG. 9 shows the generation of reactive oxygen species (ROS) in neutrophils activated with peptides conjugated to trastuzumab. [Figure 10A] FIG. 10A shows the pharmacokinetics of trastuzumab. [Figure 10B] FIG. 10B shows the pharmacokinetics of trastuzumab conjugated to peptide FRM047. [Figure 10C] FIG. 10C shows tabular data of the results in FIGS. 9A and 9B. [Figure 10D] FIG. 10D shows the PK parameters using a two-compartment model of the results in FIG. 9A and FIG. 9B. [Figure 11] FIG. 11 depicts the exposure profile of the trastuzumab parent antibody and the trastuzumab bactabody with frm-Met(CF3)FRM-058 showing similar exposure for the Tmab bactabody compared to the Tmab parent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description The invention is described herein using several definitions, either below or set forth throughout the application.

[0017] Unless otherwise indicated or indicated by context, the terms "a," "an," and "the" mean "one or more." For example, "peptide," "linker," and "antibody" should be interpreted to mean "one or more peptides," "one or more linkers," and "one or more antibodies," respectively.

[0018] As used herein, "about," "approximately," "substantially," and "significantly" are understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If there are uses of these terms that are not clear to those of ordinary skill in the art given the context in which they are used, "about" and "approximately" mean plus or minus within 10% of the particular term, and "substantially" and "significantly" mean plus or minus greater than 10% of the particular term.

[0019] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising," and these latter terms are "open" transition terms that do not limit the claim to only the recited elements that follow the transition term. The term "consisting of" is inclusive like the term "comprising," but should be interpreted as a "closed" transition term that limits the claim to only the recited elements that follow the transition term. The term "consisting essentially of" is inclusive like the term "comprising," but should be interpreted as a "partially closed" transition term that allows for additional elements that follow the transition term, but only if those additional elements do not materially affect the basic and novel nature of the claim.

[0020] As used herein, a "subject in need thereof" means a human or non-human mammal, more preferably a human diagnosed as suffering from a disease or disorder for which treatment or administration of the peptides and conjugates disclosed herein is indicated.

[0021] As used herein, a "subject in need thereof" can include a subject suffering from or at risk of developing a disease or disorder that can be treated and / or prevented by modulating the immune response in the subject. As disclosed herein, "modulation" can include inducing and / or enhancing an immune response in a subject.

[0022] Subjects in need thereof may include those 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, cancer, such as breast cancer, lung cancer, prostate cancer, skin cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, uterine cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibroid tumors, leukemia, or lymphoma.

[0023] The term "effective amount" as used herein means the amount or dose of the conjugated antibody compound of the present invention that provides the desired pharmacological effect in a patient by administration to the patient in single or multiple doses.The effective amount can be easily determined by the attending diagnostician as one skilled in the art by considering many factors, such as the type of mammal; its size, age, and general health; the specific disease or associated surgical procedure; the extent or severity of the disease or condition; the responsiveness of each patient; the specific compound or composition administered; the mode of administration; the bioavailability characteristics of the administered pharmaceutical; the selected dosing regimen; and the use of any concomitant drugs.

[0024] The subject of the present disclosure relates to peptides and polypeptides, which may include fusion polypeptides and conjugates. As used herein, the terms "peptide", "polypeptide" or "protein" may be used interchangeably to mean a polymer of amino acids. Typically, a "polypeptide" or "protein" is defined as a longer polymer of amino acids, typically 50, 60, 70, 80, 90, or 100 amino acids or more in length. A "peptide" is typically defined as a short polymer of 50, 40, 30, 20 or less amino acids.

[0025] A "polypeptide", "protein" or "peptide" as intended herein typically comprises a polymer of proteinogenic 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-proteinogenic amino acids as intended herein.

[0026] The term "proteinogenic amino acid" refers to an amino acid found in naturally occurring proteins, which may be referred to as a "coded amino acid", and the term "non-proteinogenic amino acid" refers to an amino acid not found in naturally occurring proteins, which may be referred to as a "non-coded amino acid". Thus, the term "non-proteinogenic amino acid" refers to an amino acid 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.

[0027] The term "fusion" refers to a polypeptide sequence that includes an exogenous amino acid sequence fused to a native amino acid sequence. The exogenous sequence may be fused to the N-terminus of the native amino acid sequence, to the C-terminus of the native amino acid sequence, or internally within the native amino acid sequence, such that the fusion protein includes 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.

[0028] The term "conjugate" refers to a molecule in which two components that are not originally covalently linked are covalently linked, either directly or through a linking group. A conjugate may include a peptide or polypeptide covalently linked to an antibody or antigen-binding fragment thereof. The disclosed conjugates may be covalently linked by 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 certain embodiments, the bond may be formed between an electrophilic reactive group present on the peptide or polypeptide and a nucleophilic reactive group present on the antibody or antigen-binding fragment thereof. Electrophilic reactive groups may include, but are not limited to, maleimide groups, maleimide-diaminopropionate groups, iodoacetamide groups, or vinylsulfone groups. Nucleophilic reactive groups may include, but are not limited to, free thiol groups (i.e., reduced dithio bonds).

[0029] The subject of the present disclosure relates to antibodies and antigen-binding fragments thereof. Unless otherwise indicated, the term "antibody" refers to an immunoglobulin molecule comprising two heavy chains and two light chains linked together by disulfide bonds. The amino-terminal portion of each chain contains a variable region of about 100 to about 110 amino acids primarily responsible for antigen recognition through the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0030] As used herein, the term "antigen-binding fragment" refers to an 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. Antigen-binding fragments of antibodies typically contain at least one variable region. Preferably, the antigen-binding fragment contains a heavy chain variable region (HCVR) and a light chain variable region (LCVR). More preferably, the antigen-binding fragment used herein contains a HCVR and a LCVR that have antigen-binding specificity for an epitope or target antigen.

[0031] As used herein, the term "light chain variable region (LCVR)" refers to the LC portion of an antibody molecule comprising the amino acid sequences of the complementarity determining regions (CDRs; i.e., LCDR1, LCDR2, and LCDR3) and the framework regions (FR).

[0032] As used herein, the term "heavy chain variable region (HCVR)" refers to the HC portion of an antibody molecule that includes the amino acid sequences of the complementarity determining regions (CDRs; i.e., HCDR1, HCDR2, and HCDR3) and framework regions (FRs).

[0033] As used herein, the terms "complementarity determining region" and "CDR" refer to the non-contiguous antigen binding sites found within the variable regions of the LC and HC polypeptides of an antibody or antigen-binding fragment thereof.

[0034] The CDRs are interspersed with more conserved regions called framework regions ("FR"). Each LCVR and HCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-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 the HC are referred to as "HCDR1, HCDR2, and HCDR3". The CDRs contain the majority of the residues that form specific interactions with the antigen. The numbering and positioning of the CDR amino acid residues within the LCVR and HCVR regions are in accordance with known methods.

[0035] Commonly used numbering methods include the "Kabat numbering" and "EU index numbering" systems. "Kabat numbering" or "Kabat numbering system", as used herein, refers to the numbering system devised and described by the authors of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD (1991) for designating amino acid residues in both the variable and constant domains of the heavy and light chains of an antibody. "EU index numbering" or "EU index numbering system", as used herein, refers to the numbering method for designating amino acid residues in the heavy chain constant domain of an antibody, also described in Kabat et al (1991). Other methods, including offset or alternative numbering systems for variable domains, include Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196: 901-917; Chothia, et al. (1989), Nature 342: 877-883), IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27: 55-77), and AHo (AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309: 657-670). Unless expressly indicated otherwise herein, all references (i.e., numbers) to immunoglobulin heavy chain constant region C|-|1, hinge, C|-|2, and C|-|3 amino acid residues given in the specification, examples, and claims are based on EU index numbering.

[0036] The general structure of an "IgG antibody" is well known. Wild-type (WT) antibodies of the IgG type are hetero-tetramers of four polypeptide chains (two identical heavy chains and two identical light chains) cross-linked by inter- and intra-chain disulfide bonds. Each heavy chain (HC) contains an N-terminal heavy chain variable region ("V H ") and the heavy chain constant region ("C H The heavy chain constant region is composed of three domains (C H 1. C H 2, and C H 3), and C H Each light chain (LC) is composed of an N-terminal light chain variable region ("V L ") and the light chain constant region ("C L "). V L and C L The regions are designated as kappa ("κ") or lambda ("λ") isotypes ("C κ " or "C λ Each heavy chain can have a heavy chain and a light chain variable domain (V H / V L contact) and heavy chain constant C H 1 and the light chain constant domain (C H 1 / C L It is bound to one light chain by contact with the V H -C H 1 and V L -C L The binding of each of the fragments forms two identical antigen-binding fragments (Fab) of the antibody that bind to the same antigen target or epitope. Each heavy chain has two Cs that form the Fc region of the antibody. H 2-C H Hinge-C of each heavy chain that binds the three fragments H 2-C HThe Fab and Fc are bound to the other heavy chain by contact between the three fragments. Together, the Fab and Fc form the unique "Y-shaped" structure of the IgG antibody, with each Fab representing an "arm" of the "Y." IgG antibodies can be further classified into subtypes, such as IgG1, IgG2, IgG3, and IgG4, which differ by the length of the hinge region, the number and position of the intra- and inter-chain disulfide bonds, and the amino acid sequence of each HC constant region.

[0037] The variable regions of each heavy-light chain pair combine to form a binding site. H ) and the light chain variable region (V L ) can be divided into regions of hypervariability, the complementarity determining regions ("CDRs"), interspersed with regions of more conserved framework regions ("FRs"). H and V L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the heavy chain may be referred to as "CDRH1, CDRH2, and CDRH3," and the three 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, and the FRs of the light chain may be referred to as LFR1, LFR2, LFR3, and LFR4. The CDRs contain most of the residues which form specific interactions with antigen.

[0038] Antibodies and antigen-binding fragments thereof for use in 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 Examples herein can be readily used. Furthermore, the antibodies and antigen-binding fragments of the present invention can be further modified to include framework regions derived from full-length human frameworks. A variety of different human framework sequences can be used to implement embodiments of the present invention. In particular embodiments, the framework regions used in the antibodies and antigen-binding fragments of the conjugates of the present invention are of human origin or substantially human (at least 95%, 97% or 99% of human origin). The sequences of framework regions of human origin are known in the art and may be obtained from The Immunoglobulin Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.

[0039] Expression vectors capable of directing the expression of genes operably linked thereto are well known in the art. Expression vectors include appropriate regulatory sequences, such as promoter sequences and origins of replication. They may also encode appropriate selection markers and signal peptides that facilitate secretion of the desired polypeptide product from the host cell. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide. The nucleic acids encoding the desired polypeptides, for example, the HC and LC components of the conjugated IgG antibody of the present invention, may be expressed independently using different promoters operably linked in a single vector, or the nucleic acids encoding the desired products may be expressed independently using different promoters operably linked in separate vectors. A single expression vector encoding both the HC and LC components of the cysteine ​​engineered IgG antibody of the present invention may be prepared using standard methods.

[0040] As used herein, "host cell" refers to a cell that is stably or transiently transfected, transformed, transduced or infected with a nucleotide sequence encoding a desired polypeptide product. Creation and isolation of host cell lines that produce IgG antibodies for use in the present invention can be performed using standard techniques known in the art. Mammalian cells are preferred host cells for expression of cysteine ​​engineered IgG antibodies according to the present invention. Some mammalian cells include HEK293, NSO, DG-44, and CHO cells. Preferably, the constructed protein is secreted into a medium in which the host cells are cultured and the protein can be recovered and isolated. The medium into which the protein is secreted may be purified by conventional techniques. For example, the medium may be subjected to a protein A or G column and eluted using conventional methods. Soluble aggregates and multimers may be efficiently recovered by common techniques including size exclusion, hydrophobic interaction, ion exchange, hydroxyapatite, or mixed mode chromatography. The recovered product may be immediately frozen, for example at -70°C, or lyophilized. As one of skill in the art will appreciate, when expressed in a biological system, e.g., a mammalian cell line, the antibody will be glycosylated in the Fc region unless mutations are introduced into the Fc to reduce glycosylation, and furthermore, the antibody may be glycosylated at other positions as well.

[0041] Novel chemical entities and uses of chemical entities are disclosed herein, for example in the form of peptides and conjugates of the disclosed peptides, which are described using terms known in the art and may be further explained below.

[0042] As used herein, the term "alkyl" includes straight or branched chain alkyl groups in all their isomeric forms, e.g., C1-C 12 Alkyl, C1-C 10 These include straight or branched chain groups of 1 to 12, 1 to 10, or 1 to 6 carbon atoms, referred to herein, respectively, as C1-C6-alkyl, -alkyl, and C1-C6-alkyl.

[0043] The term "alkylene" refers to a diradical of a straight or branched chain alkyl group (i.e., a diradical of a straight or branched chain C1-C6 alkyl group). Typical alkylene groups include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)CH2-, -CH(CH2CH3)CH2-, and the like.

[0044] The term "haloalkyl" means an alkyl group substituted with at least one halogen, for example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0045] The term "heteroalkyl," as used herein, refers to an "alkyl" group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is the "alkoxy" group.

[0046] As used herein, the term "alkenyl" refers to an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon double bond, e.g., C2-C 12 -Alkenyl, C2-C 10 By these radicals is meant straight or branched chain groups of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, referred to herein as C2-C6-alkenyl, and C2-C6-alkenyl, respectively.

[0047] The term "alkynyl" as used herein refers to an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon triple bond, e.g., C-C 12 -Alkynyl, C2-C 10 "C2-C6-alkynyl" refers to straight or branched chain groups of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, referred to herein respectively as C2-C6-alkynyl, C2-C6-alkynyl, and C2-C6-alkynyl.

[0048] The term "cycloalkyl" refers to any group derived from a cycloalkane, e.g., "C 4-8"-cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3 to 12, 3 to 8, 4 to 8, or 4 to 6 carbons, referred to as "cycloalkyl." Unless otherwise specified, a cycloalkyl group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxamido, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl. In certain embodiments, a cycloalkyl group is unsubstituted, i.e., unsubstituted.

[0049] The term "cycloheteroalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3 to 12, 3 to 8, 4 to 8, or 4 to 6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom (such as, for example, N, O, and / or S).

[0050] The term "cycloalkylene" refers to a cycloalkyl group that is unsaturated at one or more ring bonds.

[0051] The term "partially unsaturated carbocycle" refers to a monovalent cyclic hydrocarbon containing at least one double bond between ring atoms, where at least one ring of the carbocycle is not aromatic. Partially unsaturated carbocycles may be characterized by the number of ring carbon atoms. For example, partially unsaturated carbocycles may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and thus may be referred to as 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocycles, respectively. Partially unsaturated carbocycles may be in the form of monocyclic carbocycles, bicyclic carbocycles, tricyclic carbocycles, bridged carbocycles, spirocyclic carbocycles, or other carbocyclic groups. Exemplary partially unsaturated carbocycle groups include cycloalkenyl groups and bicyclic carbocycle groups that are partially unsaturated. Unless otherwise specified, the partially unsaturated carbocyclic group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxamide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl. In some embodiments, the partially unsaturated carbocyclic ring is unsubstituted, i.e., unsubstituted.

[0052] 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 groups having two or more carbon rings where two or more carbons are common to two adjacent rings (said rings are "fused rings"), where at least one of the rings is aromatic and the other rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. Unless otherwise specified, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide or carboxamide, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF3, -CN, etc. In some embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In some other embodiments, the aromatic ring is unsubstituted, i.e., unsubstituted. In some embodiments, the aryl group is a 6-10 membered ring structure.

[0053] 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 a 3- to 7-membered ring, in which the ring structure contains from 1 to 4 heteroatoms (e.g., nitrogen, oxygen, and sulfur). The number of ring atoms in a heterocyclyl group can be specified using the five-point Cx-Cx notation, where x is an integer that specifies 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 that contains from 1 to 4 heteroatoms (e.g., nitrogen, oxygen, and sulfur). The notation "C3-C7" indicates that the heterocycle contains a total number of ring atoms from 3 to 7, including the heteroatoms occupying the ring atom positions.

[0054] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines (e.g., mono- or di-substituted amines), where the substituents can include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

[0055] The terms "alkoxy" or "alkoxyl" are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tert-butoxy, and the like.

[0056] An "ether" is two hydrocarbons covalently linked by an oxygen or sulfur atom. Thus, the substitution on an alkyl that renders it an ether is, or resembles, an alkoxyl or thiol.

[0057] As used herein, the term "carbonyl" refers to the group -C(O)-.

[0058] The term "oxo" refers to a divalent oxygen atom, --O--.

[0059] The term "carboxamide" as used herein refers to the group -C(O)NRR', where R and R' can be the same or different. R and R' can be, for example, independently, hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl. The term "carboxy" as used herein refers to the group -COOH or its corresponding salts, such as -COONa.

[0060] As used herein, the term "amide", "amido" or "amidyl" refers to a -R 1 C(O)N(R 2 )-, -R 1 C(O)N(R 2 )R 3-, -C(O)NR 2 R 3 or a group of —C(O)NH2, where R 1 , R 2 , and R 3 means, for example, a group which is, independently at each occurrence, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

[0061] The compounds of the present disclosure (e.g., peptides and conjugates thereof) may contain one or more chiral centers and / or double bonds and therefore may exist as stereoisomers, e.g., geometric isomers, enantiomers or diastereomers. The term "stereoisomers" as used herein 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 the substituents containing the stereogenic carbon atom and / or the observed optical rotation. The present invention encompasses the various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated (±)" for nomenclature purposes, although one of ordinary skill in the art will understand that the structure implicitly indicates a chiral center. Drawings of chemical structures, e.g., general chemical structures, are understood to encompass all stereoisomeric forms of the depicted compound, unless otherwise indicated. Compositions comprising, consisting essentially of, or consisting of enantiomerically pure compounds are also contemplated herein, which 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 a given compound).

[0062] Polypeptide chains described herein are designated by their sequence of amino acids from N-terminus to C-terminus when read from left to right, and each amino acid may be represented by its one-letter or three-letter amino acid abbreviation. Unless otherwise indicated herein, all amino acids used in the preparation of the polypeptides of the present invention are L-amino acids, the stereoisomer being D-amino acids. The "N-terminus" (or amino terminus) of an amino acid or polypeptide chain means the free amine group on the amino acid or the free amine group on the first amino acid residue of the polypeptide chain. Furthermore, the term "N-terminal amino acid" refers to the first amino acid in the polypeptide chain. Similarly, the "C-terminus" (or carboxy terminus) of an amino acid or polypeptide chain means the free carboxy group on the amino acid or the free carboxy group on the last amino acid residue of the polypeptide chain. Furthermore, the term "C-terminal amino acid" refers to the last amino acid in the polypeptide chain.

[0063] As used herein, the term "[amino acid name] substituted with residue ..." refers to the substitution of the parent amino acid with the indicated amino acid, with respect to a heavy or light chain polypeptide. As an example, a heavy chain containing "alanine substituted at residue 235" refers to a heavy chain in which the parent amino acid sequence has been mutated to contain an alanine in place of the parent amino acid at residue number 235. Such mutations may also be indicated by listing a particular amino acid residue number with the parent amino acid preceding it and the substituted amino acid following it. For example, "F235A" refers to the substitution of alanine for phenylalanine at residue 235. Similarly, "235A" refers to the substitution of alanine for the parent amino acid. An "altered" cysteine ​​refers to the substitution of cysteine ​​for the parent amino acid.

[0064] As used herein, "N-formyl-methionine peptide" refers to a peptide in which the N-terminal amino acid is a formylated methionine. The N-formyl-methionine residue of the peptides of the present disclosure may contain one or more halogen substituents. Thus, an N-formyl, halogen substituted methionine residue is: [ka] R 1 , R 2 , and R 3 is independently selected from hydrogen and halogen (e.g., F, Cl, Br, or I); R 1 , R 2 , and R 3 At least one of, preferably, R 1 , R 2 , and R 3 At least two of R are halogen, more preferably 1 , R 2 , and R 3 is a halogen (e.g., the methionine residue comprises a C(halogen)3 at the end of the side chain). As used herein, "N-formyl-CF3-methionine peptide" refers to a peptide in which the N-terminal amino acid is a formylated methionine that comprises a trifluoro-substituted methyl group at the end of the methionine side chain.

[0065] 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, a "maleimide-PEG linker" refers to a linker having the formula "-(O-CH2-CH2) n -" (where "n" is 3-24), and a chemical moiety comprising a derivatized maleimide functional group, which may be covalently attached to an 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 thereof, and / or may be covalently attached to an N-formyl-methionine peptide via an amide bond to the epsilon-amino side chain of the C-terminal lysine of the N-formyl-methionine peptide, or to the gamma-carboxyl group of the C-terminal glutamic acid of the N-formyl-methionine peptide.

[0066] As indicated, the peptides and conjugates of the present disclosure may include one or more polyethylene glycol (PEG) polymers linking the sequences and may be considered to be PEGylated. As one of skill in the art will appreciate, PEGylation reagents are often described in terms of the molecular weight (Daltons or kilodaltons) of the PEG polymer portion of the PEG-containing compound in the reagent. Furthermore, many commercially available PEG-containing reagents generally exhibit some degree of molecular weight dispersion, meaning that the number of repeating ethylene glycol monomer units ("n") contained within the reagent varies within a range, typically within a narrow range. Thus, the molecular weight of the PEG polymer in the reagent typically refers to the average molecular weight of the PEG polymer contained within the reagent. The ethyloxy monomer -(O-CH2-CH2)- of the reagent used to prepare the conjugated antibody compound of the present invention has a molecular weight of about 44 g / mol or 44 Daltons. Thus, one of skill in the art can easily determine the value of "n" when using a PEGylation reagent described by its average molecular weight, as well as the value of "n" in the resulting conjugated antibody compound.

[0067] As used herein, a formyl group consists of a carbonyl bonded to a hydrogen and has the formula CH(O)-, or [ka] As shown by:

[0068] The N-terminal methionine residue may include a formyl group (ie, an N-formyl substituent on the N-terminal nitrogen atom).

[0069] Maleimides have the structure: [ka] means.

[0070] Maleimide-containing moieties that can be used to conjugate the molecules described herein include those having the structure: [ka] Maleimidodiaminopropionic acid having the structure: [ka] Maleimidopropionic acid having the formula:

[0071] Peptides containing N-formyl-halogenated methionine residues and modified antibody-peptide conjugates thereof The main feature of the present disclosure relates to peptides comprising N-formylmethionine, in which the methyl group of the methionine side chain is replaced with one or more halogens (e.g., fluorine). N-formyl-halogen substituted methionine is resistant to oxidation. Peptides comprising N-formyl-halogen substituted methionine can be utilized as agonists of the formyl peptide receptor (FPR-1) and conjugated to an antibody or antigen-binding fragment thereof. The conjugates thus prepared can be used to target cells and induce and activate immune cells comprising FPR-1 against the targeted cells.

[0072] In certain embodiments, the main feature of the present disclosure relates to a conjugated antibody or antigen-binding fragment thereof. The conjugate includes 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-halogen methionine residues may include, but are not limited to, N-formyl-trifluorinated methionine. N-formyl, fluorine substituted methionine may be prepared using methods disclosed in the art (see, for example, Houston et al., Biorg & Medic. Chem. Lett," Vol. 7, No. 23, pp.3007-3012, 1997, the contents of which are incorporated herein by reference in their entirety).

[0073] The peptide and the antibody or antigen-binding fragment thereof may be directly conjugated via a reactive group on the peptide and a reactive group on the antibody or antigen-binding fragment thereof. Alternatively, the peptide and the antibody or antigen-binding fragment thereof may be conjugated via a linker having a reactive group for conjugating the peptide and a reactive group for conjugating the antibody or antigen-binding fragment thereof. In some embodiments, the conjugate is [ka] The compound may have a formula represented as:

[0074] The components of the conjugate of the present disclosure, namely, peptide, optional linker, and antibody or its antigen-binding fragment, may be conjugated through a bond formed between any suitable reactive groups.In one embodiment, the peptide comprises a C-terminal glutamic acid residue, and the peptide is conjugated to the linker through an amide bond formed between the gamma carboxyl group of glutamic acid and the amino group of the linker.In another embodiment, the peptide comprises a C-terminal lysine residue, and the peptide is conjugated to the linker through an amide bond formed between the epsilon amino group of lysine and the carboxyl group of the linker.

[0075] The conjugate of the present disclosure may comprise multiple peptides, multiple linkers, and / or multiple antibodies or antigen-binding fragments thereof. In some embodiments, the conjugate may comprise at least two peptides and a linker, forming a branched structure. In some embodiments, the conjugate may comprise: [ka] and may be characterized as a branched structure.

[0076] The peptides disclosed herein typically include an N-terminal, N-formyl, halogen-substituted methionine residue. The peptides typically include additional amino acids, and in certain embodiments, the peptides may include 2-50 amino acids (or 2-40, 2-30, 2-20, or 2-10 amino acids) linked by peptide bonds formed between amino and carboxyl groups in the backbone or side chains of the amino acids. Preferably, the peptides of the present disclosure are resistant to cleavage by endopeptidases, such as neutrophil-associated endopeptidases, particularly integral membrane endopeptidases. In certain embodiments, the peptides of the present disclosure are resistant to cleavage by endopeptidase 24.11 (EP24.11; EC 3.4.24.11 (also called enkephalinase neutral endopeptidase), CALLA, CD10, or neprilysin); and / or endopeptidase 24.15 (EP24.15; EC 3.4.24.15) (a metallopeptidase found in alveolar macrophages, monocytes, T lymphocytes, and B lymphocytes); and / or CD13 / aminopeptidase N (CD13 / APN); and / or BP-1 / 6C3 / aminopeptidase A (BP-1 / 6C3 / APA); and CD26 / dipeptidyl peptidase IV (CD26 / DPPIV).

[0077] In certain embodiments, the peptides of the present disclosure and conjugates thereof comprise one or more non-proteinogenic amino acids at the N-terminus, including N-formyl, halogen-substituted methionine, and optionally one or more non-proteinogenic amino acids other than N-formyl, halogen-substituted methionine. Preferably, the non-proteinogenic amino acids and / or the bonds formed between the non-proteinogenic amino acids render the peptide resistant to cleavage by endopeptidases as disclosed herein.

[0078] As understood in the art, non-proteinogenic amino acids are amino acids that are not encoded in vivo and are not found to occur naturally in proteins. Proteinogenic amino acids include the 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-proteinogenic amino acid may be defined as an amino acid (i.e., a molecule that contains a free amino group and a free carboxyl group attached to the α-carbon atom) that is not one 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. For example, a non-proteinogenic amino acid may have the formula: NH2-C(R)-COOH, where R is not the side chain of any of the encoded proteinogenic amino acids.

[0079] In some embodiments, the peptides and conjugates thereof of the present disclosure comprise one or more non-proteinogenic amino acids selected from D-amino acids.Suitable D-amino acids may include, but are not limited to, the 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 / or valine.

[0080] In some embodiments, the peptides of the present disclosure include non-proteinogenic amino acids that are homologs of the encoded amino acids that lack one or more methylene groups (-CH2-) between the α-carbon and the side chain of the amino acid. Suitable homologs for use as non-proteinogenic amino acids in the peptides of the present disclosure and their conjugates may include, but are not limited to, 2-aminoisobutyric acid, 2-amino-2-hydroxyacetic acid, 2α-methyl-2-hydroxy-glycine, 2-amino-2-methylbutyric acid (i.e., isovaline), methylcysteine, azetidine-2-carboxylic acid, phenylglycine, 4-hydroxyphenylglycine, 3-indolylglycine, aminomalonic acid, 2,3-diamino-3-oxopropanoic acid, 2-amino-2-(1H-imidazol-5-yl)acetic acid, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, and 2-amino-4-(diaminomethylideneamino)butanoic acid.

[0081] In certain embodiments, peptides of the disclosure include non-proteinogenic amino acids that are homologs of encoded amino acids that have one or more additional methylene groups (-CH2-) between the α-carbon and the side chain (e.g., homo-amino acids with a single additional methylene group (-CH2-), bishomo-amino acids with two additional methylene groups (-CH2-CH2-), etc.). Suitable homologs for use as non-proteinogenic amino acids in the peptides of the present disclosure and conjugates thereof can include, but are not limited to, homo-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, such as homo-alanine, homo-arginine, homo-glutamine, homo-glutamic acid, homo-isoleucine, homo-leucine, homo-lysine, homo-methionine, homo-phenylalanine, homo-proline (i.e., piperidine-2-carboxylic acid), homo-serine, homo-threonine, homo-tryptophan, and homo-tyrosine. Suitable homologs for use as non-proteinogenic amino acids in the peptides of the present disclosure and their conjugates can include, but are not limited to, the bishomo-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.

[0082] In some embodiments, the peptides of the present disclosure include non-proteinogenic amino acids that are alkylated amino acids that contain an alkyl substituent (e.g., a C1-C6 alkyl substituent such as methyl) on the α-carbon. Suitable alkyl-substituted amino acids include α-carbon, alkyl-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., α-methyl-serine), 2-methyl-threonine (i.e., α-methyl-threonine), α-methyl-valine, α-methyl-leucine, 2-amino-2,3-dimethyl-pentanoic acid (i.e., α-methyl- The non-proteinogenic amino acids may include 2-methyl-isoleucine, α-methyl-methionine, α-methyl-cysteine, 2-methyl-proline, α-methyl-phenylalanine, α-methyl-tyrosine, α-methyl-tryptophan, 2-methyl-aspartic acid, 2-methyl-glutamic acid, 2,4-diamino-2-methyl-4-oxobutanoic acid (i.e., α-methyl-asparagine), 2,5-diamino-2-methyl-5-oxopentanoic acid (i.e., α-methyl-glutamine), α-methyl-histidine, α-methyl-lysine, and 2-methyl-arginine (i.e., α-methyl-lysine). In certain embodiments, the peptides of the present disclosure include non-proteinogenic amino acids that are dialkylated amino acids that include a dialkyl substituent (e.g., a C1-C6 dialkyl substituent such as dimethyl) on the α-carbon. Suitable dialkylated substituted amino acids can include α-carbon, dialkyl substituted glycines, for example, di-n-propylglycine (Dpg).

[0083] In certain embodiments, the peptides of the present disclosure include non-proteinogenic amino acids that are alkylated amino acids that contain an alkyl substituent (e.g., a C1-C6 alkyl substituent such as methyl) on the amino group. Suitable N-alkylated amino acids 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).

[0084] In some embodiments, the peptide of the present disclosure comprises a non-proteinogenic amino acid selected from phenylalanine, tyrosine, tryptophan, histidine, proline, naphthylalanine, which may optionally comprise a ring substituent selected from C1-C6 alkyl, halogen, and cyano substituents.Suitable non-proteinogenic amino acids may include 2-fluoro-phenylalanine, 2-methyl-tyrosine, and 2-naphthylalanine.

[0085] In certain embodiments, the peptides of the present disclosure include non-proteinogenic amino acids selected from noramino acids and / or 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-caprylic acid, 7-amino-enanthic acid, 6-amino-caproic acid, and 5-amino-pentanoic acid.

[0086] In some embodiments, the peptides of the present disclosure include non-proteinogenic amino acids that are encoded proteinogenic amino acids that are substituted with a substituent.Suitable non-proteinogenic amino acids may include alanine that is substituted with a substituent selected from alkynyl (e.g., propargylglycine), azido (e.g., 4-azido-homo-alanine), thiophenyl, thienyl (e.g., 3-(2-thienyl)-alanine), pyridyl (e.g., 3-(4-pyridyl-alanine (4-Pal)), anthrenyl, cycloalkyl, diphenyl, furyl, and naphthyl (e.g., 2-naphthylalanine).

[0087] In certain embodiments, the peptides of the present disclosure include a non-proteinogenic amino acid that includes an ethylene-oxy moiety. Suitable non-proteinogenic amino acids include those of the formula NH2-CH2-CH2-(O-CH2-CH2) n It may contain an amino acid represented by -COOH (wherein n is selected from 1 to 24).

[0088] In some embodiments, the peptide of the present disclosure comprises a non-proteinogenic amino acid that is not an α-amino acid.Suitable non-α-amino acids may include 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).

[0089] In some embodiments, the peptide of the present disclosure comprises a non-proteinogenic amino acid that is a cycloamino acid (e.g., a cycloamino acid other than proline).A cycloamino acid is an amino acid that comprises a cyclic group formed by a nitrogen atom and a carboxyl group.Suitable cycloamino acids can include, but are not limited to, aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, azepane-2-carboxylic acid, cycloleucine, homocycloleucine, 1-piperidine-4-carboxylic acid, piperidine-3-carboxylic acid, 1-piperazineacetic acid, 4-piperidineacetic acid, and 1-piperidineacetic acid.

[0090] In certain embodiments, the peptides of the present disclosure include proteinogenic and / or non-proteinogenic amino acids that may optionally include 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).

[0091] The peptides of the present disclosure may be directly conjugated to an antibody or antigen-binding fragment thereof. In other embodiments, the peptides of the present disclosure may be indirectly conjugated to an antibody or antigen-binding fragment thereof via a linker. In certain embodiments, the linker has a selected linear distance. Suitable selected linear distances may include at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 angstroms or more, or ranges encompassed by these values ​​(e.g., 5-10 angstroms, 10-20 angstroms, 15-20 angstroms, 15-25 angstroms, 20-35 angstroms, 30-40 angstroms, 35-40 angstroms, 35-50 angstroms, and 40-50 angstroms).

[0092] In some embodiments, the linkers of the present disclosure have spacer arms that can provide a selected distance for the linker. For example, the linkers of the present disclosure include [ka] or [ka] The compound may have a formula represented as:

[0093] Suitable spacer arms may include polymer moieties such as polyethylene glycol.

[0094] In one embodiment, the linker is [ka] and [ka] [In the formula, n is an integer selected from 3 to 24] having a formula selected from:

[0095] In a particular embodiment, the linker may comprise a maleimide moiety and a PEG moiety, and may be referred to as a "maleimide-PEG linker" that conjugates N-formyl-CF3-methionine to the antibody. An exemplary conjugate is: [ka] and [ka] The compound may have a formula selected from:

[0096] In some embodiments, the spacer arm comprises a peptide sequence of 1 to 20 amino acids. In some embodiments, the spacer arm comprises a peptide sequence comprising amino acids selected from glycine, serine, and alanine (e.g., (G4S)m [wherein m is an integer selected from 1 to 5].

[0097] Linkers of the present disclosure may include a polyethylene glycol moiety, for example, as a spacer arm or other moiety. In some embodiments, the linker includes a polyethylene glycol (PEG) moiety (i.e., (-O-CH-CH) 1-24 ).

[0098] In some embodiments, the linkers of the present disclosure may include two or more PEG moieties separated by a non-PEG moiety, such as an amino acid moiety. In some embodiments, the linkers of the present disclosure include -((PEG) 1-24 )-(AA) 1-2 -((PEG) 1-24 )--wherein AA is glutamic acid attached by gamma aminoacylation or lysine attached by epsilon aminoacylation.

[0099] Suitable peptide-linkers disclosed herein include: frm-M(CF3)-Ile-Phe-Leu-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Leu-Phe-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-Nle-γE-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-Nle-γE-Peg6-γE-γE-Peg6-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-Nle-γE-Peg6-εK-εK-Peg6-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-γE-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-4Pal-αMeF-Nle-γE-Peg12-NH-(CH2)2-Y where Y comprises an amino group or a cysteine ​​reactive moiety for conjugating the peptide-linker to the antibody. Suitable cysteine ​​reactive moieties may include, but are not limited to, maleimide, maleimide-diaminopropione, iodoacetamide, or vinylsulfone.

[0100] In certain embodiments of the conjugates of the present disclosure, the linker comprises a maleimide moiety, and said linker is conjugated to the antibody or antigen-binding fragment thereof through a thioether bond formed between the maleimide moiety and a cysteine ​​residue of the antibody.

[0101] Suitable amino acid residues for conjugating the peptides and linkers of the present disclosure may include cysteine ​​residues. Suitable cysteine ​​residues may be engineered in the antibody or antigen-binding fragment thereof, and the cysteine ​​residue may be an endogenous cysteine ​​residue of the antibody or antigen-binding fragment thereof, or a non-endogenous 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, and the cysteine ​​residue is selected from the group consisting of C, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C31, C32, C43, C54, C65, C76, C87, C98, C11, C12, C13, C14, C15, C26, C16, C17, C28, C31, C43, C54, C65, C76, C87, C98, C12, C13, C21, C14, C15, C22, C31, C16, C17, C23, C32, C43, C54, C65, C76, C87, C98, C12, C13, C22, C31, C14, C15, C23, C32, C43, C16, C17, C24, C18, C25, C31, C19, C26, C32, C43, C12, C13, C22, C31, C14, C23, C32, C43, C15, C24, C16, C17, C2 H Residue 124 of domain 1, C H Residue 378 of domain 3, or C H Residues 124 and C of domain 1 HIn certain embodiments, the antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region comprising an isoleucine substituted for cysteine ​​at residue 247, a glutamic acid substituted for cysteine ​​at residue 332, or both an isoleucine substituted for cysteine ​​at residue 247 and a glutamic acid substituted for cysteine ​​at residue 332, based on the EU numbering index. In certain 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.

[0102] Suitable antibodies for the conjugates and methods of the present disclosure 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.

[0103] Suitable antibodies may include IgG isotypes. Suitable IgG isotypes may include isotypes having an IgG heavy chain constant region selected from a human IgG1 isotype or a human IgG4 isotype.

[0104] Suitable antibodies may include monoclonal antibodies. Suitable antibodies may include monospecific and bispecific antibodies.

[0105] The peptides of the present disclosure and any linker may be used to prepare conjugates of antibodies or antigen-binding fragments thereof known in the art.For example, the peptides of the present disclosure and any linker may be conjugated to existing cancer therapeutic antibodies to prepare N-formyl-Met(CF3) peptide-conjugate immunotherapeutic agents.

[0106] Exemplary cancer therapeutics used to prepare peptide conjugates may include IgG1 therapeutic antibodies targeting solid tumors, e.g., tumors expressing HER-2 (i.e., lgG1 antibodies such as trastuzumab and pertuzumab), liquid tumors, e.g., liquid tumors expressing CD20 (i.e., lgG1 and lgG1-enhancing ADCC antibodies, e.g., rituximab, ofatumumab, obinutuzumab, and AME133v), and antibodies targeting c-Met expressing tumors (i.e., erlotinib).

[0107] The N-formyl-Met(CF3)-peptides disclosed herein may be conjugated to therapeutic antibodies that contain cytotoxic agents to function as additional therapeutic agents. Alternatively, the N-formyl-Met(CF3)-peptides disclosed herein may replace the cytotoxic agents in therapeutic antibodies to create new therapeutic antibodies that target antigens that are overexpressed in cancer cells. Target antigens and representative therapeutic antibodies include, but are not limited to, GPNMB (glentuzumab vedotin), CD56 (lorvotuzumab mertansine (IMGN-901)), TACSTD2 (TROP2; sacituzumab govitecan, (IMMU-132)), CEACAM5 (labetuzumab SN-38), folate receptor-a (mirvetuximab soravtansine (IMGN-853), vintafolide), mucin 1, sialoglycotope CA6; SAR-566658, STEAP1 (bundutuzumab vedotin (RG-7450)), mesothelin (DMOT4039A, anetumab ravtansine (BAY-94-9343), BMS-986148), nectin 4 (enfortumab Vedotin (ASG-22M6E; ASC-22CE), ENPP3 (AGS-16M8F), guanylate cyclase C (indusatumab vedotin (MLN-0264)), SLC44A4 (ASG-5ME), NaPi2b (rifastuzumab vedotin), CD70 (TNFSF7; DNIB0600A, AMG-172, MDX-1243, borsetuzumab mafodotin (SGN-75)), CA9 carbonic anhydrase (BAY79-4620), 5T4 (TPBG; PF06263507) SLTRK6 (ASG-15ME), SC-16 (anti-Fyn3; SC16LD6.5), tissue factor (HuMax-TF-ADC(TF-011-MMAE)), LIV-1 (ZIP6; SGN-LIV1A), P-cadherin (PCA062), PSMA (MLN2704, PSMA-ADC), fibronectin extra domain B (human mAb L19 and F8), endothelin receptor ETB (RG-7636), VEGFR2 (CD309; anti-VEGFR-2ScFv-As203-stealth nanoparticles), tenascin-c (anti-TnC-A1 antibody SIP(F16)), periostin (anti-periostin antibody), DLL3 (rovalpituzumab, soravtansine), HER2 (T-DM1, ARX788, SYD985), EGFR (ABT-414, IMGN289 AMG-595), CD30 (brentuximab vedotin, iratumumab MDX-060), CD22 (inotuzumab ozogamicin (CMC-544), pinatuzumab vedotin, epratuzumab SN38), CD79b (polatuzumab vedotin), CD19 (cortuximab ravtansine, SAR-3419, SGN-CD19A), CD138 (indatuximab ravtansine), CD74 (milatuzumab doxorubicin), CD37 (IMGN-529), CD33 (gemtuzumab ozogamicin, IMGN779, SGN CD33A), and CD98 (IGN523) (see, e.g., Thomas et al, Lancet Oncol. 2016 Jun; 17(6)e254-62, the contents of which are incorporated herein by reference in their entirety). and Diamantis and Banerji, Brit. Journ. Cancer, 2016; 1 14, 362-367).

[0108] In certain embodiments, the antibody or antigen-binding fragment thereof of the conjugate of the present disclosure is selected from the group consisting of glembatumumab vedotin, lorvotuzumab mertansine (IMGN-901), TROP2; sacituzumab govitecan (IMMU-132), labetuzumab SN-38, mirvetuximab soravtansine (IMGN-853), vintafolide, sialoglycotope CA6; SAR-566658, enfortumab vedotin (ASG-22M6E), ASC-22CE), ZIP6, SGN-LIV1A, DMOT4039A, anetumab ravtansine (BAY-94-9343), BMS-986148, sofituzumab vedotin, mirvetuximab soravtansine (IMGN-853), vintafolide, labetuzumab SN-38, glenbatumumab vedotin, lorvotuzumab mertansine (IMGN-901), bundletuzumab vedotin (RG-7450), AGS-16M8F, indusatumab vedotin (MLN-0264), ASG-5ME, rifastuzumab vedotin, TNFSF7, DNIB0600A, AMG-172, MDX-1243, borsetuzumab mafodotin (SGN-75), BAY79-4620, TPBG, PF06263507, SLTRK6 (ASG-15ME), anti-Fyn3, SC16LD6.5), HuMax-TF-ADC (TF-011-MMAE), PCA062, human mAb L19, human mAb F8, RG-7636, CD309; anti-VEGFR-2ScFv-As2O3-stealth nanoparticles, anti-TnC-A1 antibody SIP (F16), anti-periostin antibody, rovalpituzumab soravtansine, ABT-414, IMGN289 AMG-595, brentuximab vedotin, iratumumab MDX-060, inotuzumab ozogamicin (CMC-544), pinatuzumab vedotin, epratuzumab SN38, polatuzumab vedotin, coltuximab ravtansine, SAR-3419, SGN-CD19A, indatuximab ravtansine, milatuzumab The antibody may include one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a known antibody, which may be appropriately selected from doxorubicin, IMGN-529, gemtuzumab ozogamicin, IMGN779, SGN CD33A, and IGN523.

[0109] The peptide and its conjugate may be formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition of the present disclosure comprises (i) the conjugated antibody or its antigen fragment disclosed herein; and (ii) one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0110] The peptides, conjugates, and pharmaceutical compositions thereof of the present disclosure can be used in methods for treating diseases and disorders in subjects in need of treatment.In some embodiments, the methods of the present disclosure include methods for treating solid or liquid tumors, comprising administering an effective amount of the conjugated antibody or pharmaceutical composition thereof disclosed herein to a patient in need of treatment.Appropriate cancers for treating with the peptides, conjugates, pharmaceutical compositions, and methods of the present disclosure can include, but are not limited to, breast cancer, lung cancer, prostate cancer, skin cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, uterine cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibroid tumor, leukemia, or lymphoma.

[0111] The peptide, conjugate and pharmaceutical composition of the present disclosure can be used to treat a subject in need of treatment.In some embodiments, the peptide, conjugate and pharmaceutical composition of the present disclosure can be used to treat solid or liquid tumors, which may be selected from breast cancer, lung cancer, prostate cancer, skin cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, uterine cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibroid tumor, leukemia or lymphoma.

[0112] Also disclosed herein is a method for activating neutrophils, particularly for activating the production of reactive oxygen species (ROS) in neutrophils in vivo or in vitro. The method comprises contacting neutrophils with the conjugate disclosed herein under conditions in which the conjugate activates ROS production in neutrophils. In some embodiments, the conjugate comprises a spacer of suitable length to induce ROS production in neutrophils. For example, the conjugate can comprise a polyethylene glycol (PEG) spacer of suitable length to induce ROS production (e.g., a PEG spacer comprising at least 12 monomers).

[0113] Disclosed herein are compounds, which may also be referred to herein as peptides. In some embodiments, the compounds have the formula: RP 1 -P 2 -P 3 -NH-(CH2CH2O) n -CH2CH2-Y [In formula: R is HC(=O)-; P 1 is Met(C(halogen) m ) wherein m is 1 to 3 (e.g., Met(CF3), Met(CHF2), or Met(CH2F)); P 2 , P 1 are 1 to 6 proteinogenic or non-proteinogenic amino acids linked together by peptide bonds; P 3 is an amino acid containing a side chain containing a -COOH moiety (e.g., glutamic acid or aspartic acid) or a -NH2 moiety (e.g., lysine), optionally containing a glutamic acid residue linked through its side chain gamma carboxyl group or a lysine residue linked through its side chain epsilon amino group; P 3 is a peptide bond. 2 Binds to; n is an integer selected from 3 to 24; and Y comprises an amino or cysteine ​​reactive moiety, and optionally Y is selected from maleimide, maleimide-diaminopropione, iodoacetamide, or vinylsulfone. or a salt thereof.

[0114] In other embodiments, the compounds of the present disclosure have the formula: RP 1 -P 2 -P 3 -NH-(CH2CH2O) n -CH2CH2-Y [In the formula, R is HC(=O)-; P 1 is Met or Met(C(halogen) m ) wherein m is 1 to 3 (e.g., Met(CF3), Met(CHF2), or Met(CH2F)); P 2 , P 1 are 1 to 6 proteinogenic or non-proteinogenic amino acids linked together by peptide bonds; P 3 is an amino acid containing a side chain containing a -COOH moiety (e.g., glutamic acid or aspartic acid) or a -NH2 moiety (e.g., lysine), optionally containing a glutamic acid residue linked through its side chain gamma carboxyl group or a lysine residue linked through its side chain epsilon amino group; P 3 is a peptide bond. 2 Binds to; n is an integer selected from 3 to 24; and Y comprises an amino or cysteine ​​reactive moiety, and optionally Y is selected from maleimide, maleimide-diaminopropione, iodoacetamide, or vinylsulfone. or a salt thereof.

[0115] The peptides and conjugates of the present disclosure are preferably agonists of one or more of the formyl peptide receptor family. Preferably, the peptides and conjugates of the present disclosure are agonists of formyl peptide receptor 1 (FPR-1). Preferably, the peptides and conjugates of the present disclosure bind to one or more of the formyl peptide receptor family. Preferably, the peptides and conjugates of the present disclosure bind to one or more of the formyl peptide receptor family present on the surface of neutrophils. Preferably, the peptides and conjugates of the present disclosure bind to one or more of the formyl peptide receptor family with a Kd of at least about 10uM, 1uM, 100nM, 50nM, 10nM or less. Therefore, the peptides and conjugates of the present disclosure can be used in a method of antagonizing formyl peptide receptor, comprising contacting the formyl peptide receptor with a peptide or conjugate. EXAMPLES

[0116] The following examples are illustrative and should not be construed as limiting the scope of the claimed features.

[0117] Formyl-Met (CF) for preparing the conjugate 3 ) and PEG peptides summary Natural formyl-methionine peptides are oxidized in vivo at the sulfur atom of the methionine residue to generate methyl-sulfoxide or met(O) (see FIG. 1). Oxidation of the sulfur atom of the methionine residue significantly reduces formyl peptide receptor 1 (FPR-1) agonist activity (e.g., by nearly 10×). Therefore, formyl-trifluoro-methionine-based peptides (i.e., frm-Met(CF3)-containing peptides) that are resistant to oxidation were prepared. Peptides containing additional non-proteinogenic amino acids and polyethylene linkers / spacers were also prepared. The peptides were tested to determine whether they could function as FPR-1 agonists to determine whether they are suitable for preparing antibody bioconjugates that promote FPR-1-mediated targeted cell killing by innate immune cells.

[0118] It was observed that frm-Met(CF3)-containing peptides can activate the human formyl peptide receptor (FPR-1) on neutrophils, making them suitable for modifying antibodies to create antibody conjugates that exhibit FPR-1-mediated targeted cell killing. It was further observed that the length of the linker with respect to the polyethylene linker is important for inducing reactive oxygen species (ROS) generation in neutrophils. Finally, it was found that antibodies conjugated to frm-Met(CF3)-containing peptides exhibited a similar clearance profile as unconjugated antibodies.

[0119] background Bactabodies are antibody bioconjugates that participate in the innate immune system in target cell killing. They consist of specific cell-targeting antibodies conjugated to pathogen-associated molecular patterns (PAMPs) that can activate innate immune cells to kill the target cells.

[0120] Formyl peptides provide PAMPs that can be conjugated to antibodies to prepare bactobodies. Formyl-Met-Leu-Phe (fMLF) and formyl-Met-Ile-Phe-Leu (fMIFL) are two well-characterized formyl peptides. fMLF and fMIFL are formyl peptide receptor (FPR) agonists, the FPR-1 receptor being an activating receptor present on innate immune cells. fMLF is a potent agonist of human FPR-1, and fMIFL is a potent agonist of both human and mouse FPR-1 receptors.

[0121] One concern for the in vivo activity of formylmethionine-based FPR-1 agonists is that oxidation of methionine also reduces the activity of the frm-Met peptide (see Figure 1). Also, frm-Met peptides with natural amino acids function well in vitro, but are degraded in vivo, presumably by endogenous endopeptidases present on the cell surface that produces FPR-1 or by endopeptidases in the blood. To address the metabolic stability, we prepared modified FPR-1 agonist peptides that contain non-proteinogenic amino acids that are stable for in vivo use. Thus, although the frm-Met peptide functions as an agonist by itself, this experiment shows that the frm-Met peptide needs to be presented via a linker for maximum activity.

[0122] Described herein is a peptide with trifluoro modification of methionine, which eliminates the possibility of methionine oxidation while maintaining FPR-1 antagonism.The peptide of the present disclosure also includes non-proteinogenic amino acids that inhibit digestion by endopeptidase.The peptide of the present disclosure also includes a linker that includes PEG, which the present inventors have shown to be important for maximum agonist activity.

[0123] Results and Discussion Peptide preparation As shown in Figures 2, 3, and 4, the present inventors prepared a set of peptides having frm-Met(CF3), a non-proteinogenic amino acid, and a PEG linker for in vitro and in vivo activity as agonists of FPR-1.

[0124] The synthesis chemistry of frm-Met(CF3) is shown in Figure 5. Fmoc-S-trityl-L-homocysteine ​​(compound 1, 1.695 g, 2.713 mmol) was dissolved in DCM (25 mL) and triisopropylsilane (4 mL, 19.5 mmol), followed by addition of TFA (15 mL, 198.4 mmol) at 21°C, and the reaction mixture was stirred for 1 h. Concentration in vacuum and co-distillation with MeOH gave compound 1. 2 The compound was obtained (0.973 g, 2.72 mmol). 2 was dissolved in DCM (25 mL) and the solution was cooled to -78°C. 3 (1.00 g, 2.94 mmol) was dissolved in DCM, the reaction mixture was added and stirred for 30 min at −78° C. The reaction mixture was adsorbed onto Celite and purified by reverse phase chromatography (50% to 70% 0.1% FA in water / acetonitrile) to give Fmoc-S-trifluoromethyl-L-homocysteine ​​(4,153 mg, 0.33 mmol, 12.2%). 1 H NMR (500 HMz, DMSO) δ 12.7 (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 (352 MHz, DMSO) δ -40 (s, 3F); MS [M + +Na] 448; purity 90% (by HPLC-UV (300 nm)).

[0125] Briefly, FRM-023 and FRM063 contain the human peptide MIFL, differing only in that FRM-063 contains frm-Met(CF3) and FRM-023 contains frm-Met.

[0126] Similarly, FRM-050 and FRM-054 contain the mouse peptide MFL, differing only in that FRM-054 contains frm-Met(CF3) and FRM-50 contains frm-Met.

[0127] FRM-052 represents an oxidized control peptide.

[0128] FRM-059 represents the frm-Met(CF3) derivative of FRM-047.

[0129] FRM-055 represents the D-Nle derivative of FRM-047.

[0130] FRM-060 and FRM-061 contain two "disconnected" PEG6 linkers to reduce the flexibility of the PEG12 linker present in FRM-059.

[0131] FRM-041 and FRM-051 are the terminal maleimide-bearing counterparts of FRM-023 and FRM-050, respectively.

[0132] FRM-053 is the counterpart of FRM-052 that has a terminal maleimide.

[0133] FRM-058 is the terminal maleimide-bearing counterpart of FRM-055.

[0134] FRM-048 and FRM-049 are branched versions of FRM-047 that contain two peptides, either lacking (FRM-048) or possessing (FRM-049) a terminal maleimide.

[0135] FRM-057 is a derivative of FRM-047 that has an N-terminal methionine residue (ie, a methionine with the sulfur atom replaced by an oxygen atom).

[0136] FRM-056 is a derivative of FRM-047 that has frm-Met(CF3) and lacks Nle.

[0137] FRM-062 is a derivative of FRM-047 that has frm-Met(CF3) and 4-Pal.

[0138] In vitro C-terminal PEGylation of frm-Met and frm-Met(CF3) peptides enhances FPR-1-mediated reactive oxygen species (ROS) generation from primary human neutrophils. As shown in Figure 6 and Figure 9, C-terminal PEGylation of frm-Met and frm-Met(CF3) peptides enhanced the activity of ROS generation in neutrophils. ROS generation from primary human neutrophils was also enhanced with frm-Met(CF3) peptide with non-proteinogenic amino acids (and PEG) compared to frm-Met(CF3) peptide with proteinogenic amino acids (and PEG).

[0139] As shown in Figure 8, migration of primary human neutrophils was similar for the frm-Met peptide with proteinogenic amino acids (MLF and PEG) and the frm-Met(CF3) peptide with proteinogenic amino acids (frm-M(CF3)LF and PEG).

[0140] We also tested conjugated peptides. As shown in Figure 9, ROS generation from primary human neutrophils by peptides conjugated to a specific eCys site on trastuzumab by maleimide requires a linker / spacer (e.g., PEG linker / spacer). The frm-Met peptide conjugated to trastuzumab and lacking a spacer could not activate ROS generation from human neutrophils.

[0141] In vivo Trastuzumab bactobody with frm-Met peptide and non-proteinogenic amino acids was cleared faster than trastuzumab parent antibody, resulting in lower exposure for bactobody. As shown in Figure 10A and Figure 10B (and Figure 10C and Figure 10D), conjugation of peptide FRM-047 resulted in higher clearance for conjugated antibody versus unconjugated parent antibody. Exposure calculated based on AUC0-∞ shows 3.8-fold higher exposure for Tmab parent compared to Tmab bactobody with FRM-047 peptide. However, when trastuzumab (Tmab) was conjugated to peptide FRM-058 (a peptide FRM-047 derivative with frm-Met(CF3) moiety), clearance was similar to parent trastuzumab (see Figure 11). Thus, the trifluoro substitution at frm-Met (CF3) of the trastuzumab bactobody confers reduced clearance relative to the equivalent bactobody bearing the frm-Met peptide, and clearance is similar to that of the Tmab parent antibody.

[0142] method ROS generation Human neutrophils were purified from fresh blood aspirates as previously described. Reactive oxygen species production by primary human neutrophils was measured using luminol-amplified chemiluminescence. After isolation, PMNs were cultured at 1x10 in HBSS containing calcium and magnesium (Gibco #14025-092) supplemented with 0.25% human serum albumin (Gemini Bio product #800-120) and 50 μM Luminol (Sigma-Aldrich #123072-2.5G). 6 Then, 100 μl of the cell suspension (total 1×10 5Cells) were dispensed into each well of a 96-well plate (Greiner #655098) suitable for luminescence measurement and the temperature was allowed to equilibrate for 5 minutes at 37°C. After equilibration, a 10x solution of agonist was added to the wells to give a 1x final concentration. Immediately after addition of agonist, the chemiluminescence signal was recorded by a luminometer. Area under the curve (AUC) values ​​were calculated using the luminescence signal from the first 5 minutes of each run. Values ​​are given in comparative luminescence units.

[0143] chemotaxis Neutrophil chemotaxis through Transwells (Corning #3415) to agonists was measured in a modified Boyden chamber assay. Approximately 2-4x10 cells from neutrophil-enriched preparations were 5 Cells were seeded in the upper Transwell chamber on a membrane with 3.0 □m pores. The lower Transwell chamber contained buffer with or without test agent. After seeding in the Transwell, cells were placed at 37°C in a humidified incubator. After 1 hour, the cells in the upper chamber were removed and the percentage of cells that successfully migrated to the lower chamber was quantified using CellTiter-Glo® (Promega #G7571) according to the manufacturer's specific protocol. 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 start of cell input. A U-shaped dose-response curve is expected. Migration from the upper chamber to the lower chamber requires a concentration gradient, and as peptide concentration increases, the rise of peptide into the upper chamber creates a decrease in the concentration gradient that requires migration from the upper chamber to the lower chamber.

[0144] conclusion PEGylation of frm-Met and frm-Met(CF3) peptides enhances FPR-1-mediated reactive oxygen species (ROS) generation from primary human neutrophils. The frm-Met(CF3) peptide with nonproteinogenic amino acids is more effective than the frm-Met(CF3) peptide with natural amino acids in promoting FPR-1-mediated ROS generation from primary human neutrophils.

[0145] In the above description, it will be readily understood by those skilled in the art that various substitutions and modifications may be made to the invention described herein without departing from the scope and spirit of the invention. The invention described herein by way of example may suitably be carried out without any elements or limitations not specifically disclosed herein. The terms and expressions used are used as descriptive terms and are not limiting, and the use of such terms and expressions other than any equivalents of the features or portions thereof shown and described is not intended, but it is recognized that various modifications are possible within the scope of the invention. Thus, while the invention has been illustrated by specific embodiments and optional features, it should be understood that modifications and / or variations of the disclosed concepts herein are possible by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0146] Numerous patent and non-patent literature citations may be made herein. The cited references are incorporated herein by reference in their entirety. If there is a discrepancy between the definition of a term in a cited reference and the definition of a term herein, the term shall be construed in accordance with the definition herein.

[0147] Particular embodiments are numbered 1 through 53 and are shown below:

[0148] (1) A conjugated antibody or antigen-binding fragment thereof, comprising an antibody or antigen-binding fragment thereof conjugated to a peptide containing an N-formyl-halogenated methionine residue at its N-terminus.

[0149] (2) The conjugated antibody described in embodiment 1, wherein the N-formyl-halogenated methionine residue is N-formyl-trifluorinated methionine.

[0150] (3) The conjugated antibody of embodiment 1 or 2, wherein the antibody is conjugated to the peptide via a linker.

[0151] (4) The conjugated antibody is [ka] 3. The conjugated antibody of any of the preceding embodiments, having the formula:

[0152] (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 by an amide bond formed between the gamma carboxyl group of the glutamic acid and an amino group of the linker.

[0153] (6) The conjugated antibody according to any one of embodiments 1 to 5, 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 a carboxyl group of the linker.

[0154] (7) The conjugated antibody is [ka] 3. The conjugated antibody of any of the preceding embodiments, having the formula:

[0155] (8) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids and include halogen-substituted amino acids (e.g., N-formyl-trifluoromethionine at the N-terminus).

[0156] (9) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from D-amino acids (e.g., the D-amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0157] (10) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids selected from homologs of amino acids lacking one or more methylene groups between the α-carbon and the side chain, or homologs of amino acids having an additional methylene group between the α-carbon and the side chain (e.g., homoamino acids, bishomoamino acids, etc.).

[0158] (11) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from amino acid homologs lacking one or more methylene groups between the α-carbon and the side chain, such as 2-aminoisobutyric acid, 2-amino-2-hydroxyacetic acid, 2α-methyl-2-hydroxy-glycine, 2-amino-2-methylbutyric acid (i.e., isovaline), methylcysteine, azetidine-2-carboxylic acid, phenylglycine, 4-hydroxyphenylglycine, 3-indolylglycine, aminomalonic acid, 2,3-diamino-3-oxopropanoic acid, 2-amino-2-(1H-imidazol-5-yl)acetic acid, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, and 2-amino-4-(diaminomethylideneamino)butanoic acid.

[0159] (12) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which may be non-proteinogenic amino acids selected from 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 homoamino acids, e.g., homoalanine, homoarginine, homoglutamine, homoglutamic acid, homoisoleucine, homoleucine, homolysine, homomethionine, homophenylalanine, homoproline (i.e., piperidine-2-carboxylic acid), homoserine, homothreonine, homotryptophan, and homotyrosine).

[0160] (13) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from bishomoamino acids (e.g., bishomoamino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0161] (14) The peptide is an α-carbon (e.g., α-carbon, alkyl-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., α-methyl-serine), 2-methyl-threonine (i.e., α-methyl-threonine), α-methyl-valine, α-methyl-leucine, 2-amino-2,3-dimethyl-pentanoic acid (i.e., α-methyl-isoleucine), α-methyl-methionine, α-methyl-cysteine, 2-methyl-proline, α-methyl The conjugated antibody of any of the preceding embodiments, comprising 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from alkylated amino acids comprising alkyl substituents (e.g., C1-C6 alkyl substituents) at: alpha-phenylalanine, alpha-methyl-tyrosine, alpha-methyl-tryptophan, 2-methyl-aspartic acid, 2-methyl-glutamic acid, 2,4-diamino-2-methyl-4-oxobutanoic acid (i.e., alpha-methyl-asparagine), 2,5-diamino-2-methyl-5-oxopentanoic acid (i.e., alpha-methyl-glutamine), alpha-methyl-histidine, alpha-methyl-lysine, and 2-methyl-arginine (i.e., alpha-methyl-lysine).

[0162] (15) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from dialkylated amino acids comprising a dialkyl substituent (e.g., a C1-C6 alkyl substituent) at the α-carbon (e.g., a dialkyl substituted glycine, e.g., dipropylglycine (Dpg)).

[0163] (16) The peptide is an amino acid that is substituted with an amino group (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, 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, 5. The conjugated antibody of any of the preceding embodiments, comprising 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from alkylated amino acids comprising alkyl substituents (e.g., C1-C6 alkyl substituents) at N-methyl-amino, 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.

[0164] (17) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which may be non-proteinogenic amino acids selected from phenylalanine, tyrosine, tryptophan, histidine, proline, naphthylalanine, optionally containing one or more ring substituents selected from C1-C6 alkyl substituents, halogen substituents, and cyano substituents (e.g., 2-fluoro-phenylalanine, 2-methyl-tyrosine, and 2-naphthylalanine).

[0165] (18) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2-10 amino acids, which may be noramino acids and / or linear core amino acids, e.g., non-proteinogenic amino acids selected from norleucine (Nle), norvaline (Nva), 12-amino-dodecanoic acid, 8-amino-caprylic acid, 7-amino-enanthic acid, 6-amino-carpoic acid, and 5-amino-pentanoic acid.

[0166] (19) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from amino acids (e.g., alanine) comprising a substituent selected from alkynyl (e.g., propargylglycine), azido (e.g., 4-azido-homoalanine), thiophenyl, thienyl (e.g., 3-(2-thienyl)-alanine), pyridyl (e.g., 3-(4-pyridyl-alanine (4-Pal)), anthrenyl, cycloalkyl, diphenyl, furyl, and naphthyl.

[0167] (20) The peptide has the formula: NH2-CH2-CH2-(O-CH2-CH2) n The conjugated antibody of any of the preceding embodiments, comprising 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from amino acids represented by the formula -COOH, where n is selected from 1 to 24.

[0168] (21) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from β-amino acids, γ-amino acids, δ-amino acids, ε-amino acids, and ζ-amino acids (e.g., the 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).

[0169] (22) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises 2 to 10 amino acids, which may be non-proteinogenic amino acids selected from cycloamino acids (e.g., aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, azepane-2-carboxylic acid, cycloleucine, homocycloleucine, 1-piperidine-4-carboxylic acid, piperidine-3-carboxylic acid, 1-piperazineacetic acid, 4-piperidineacetic acid, and 1-piperidineacetic acid).

[0170] (23) The conjugated antibody of any of the preceding embodiments, wherein the peptide comprises an amino protecting group (-N-, which may be selected from allyloxycarbonyl (Alloc), 9-fluorenylmethylcarbonyl (Fmoc), t-butylcarbonyl (BOC), and benzylcarbonyl (Cbz).

[0171] (24) The conjugated antibody of any of the preceding embodiments, wherein the linker comprises a spacer arm having a distance of about 10-50 angstroms (alternatively 10-40 angstroms, or 10-30 angstroms, or 10-20 angstroms).

[0172] (25) The linker comprises a spacer arm, [ka] and [ka] 5. The conjugated antibody of any of the preceding embodiments, having a formula selected from:

[0173] (26) The linker is [ka] and [ka] (wherein n is an integer selected from 3 to 24). 26. The conjugated antibody of embodiment 24 or 25, having a formula selected from:

[0174] (27) The spacer arm is a peptide sequence comprising an amino acid selected from glycine, serine, and alanine, such as (G4S) m [wherein m is an integer selected from 1 to 5].

[0175] (28) The linker is a polyethylene glycol (Peg) moiety (i.e., (-O-CH2-CH2) 1-24 5. The conjugated antibody of any preceding embodiment, comprising:

[0176] (29) The linker is -((Peg) 1-24 )-(AA) 1-2 -((Peg) 1-24 )-, wherein AA is a glutamic acid residue attached through its side chain gamma carboxyl group, or a lysine residue attached through its side chain epsilon amino group.

[0177] (30) The peptide-linker is frm-M(CF3)-Ile-Phe-Leu-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Leu-Phe-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-Nle-γE-Peg12-NH-(CH2) 2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg -2Nal-αMeF-Nle-γE-Peg6-γE-γE-Peg6-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF- 5. The conjugated antibody of any of the preceding embodiments, having a formula selected from Nle-γE-Peg6-εK-εK-Peg6-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-D-Nle-γE-Peg12-NH-(CH2)2-Y, frm-M(CF3)-Dpg-2Nal-αMeF-γE-Peg12-NH-(CH2)2-Y, and frm-M(CF3)-Dpg-4Pal-αMeF-Nle-γE-Peg12-NH-(CH2)2-Y, wherein Y comprises an amino group or a cysteine ​​reactive moiety for conjugating the peptide-linker to the antibody.

[0178] (31) The conjugated antibody of embodiment 30, wherein the cysteine ​​reactive moiety is selected from maleimide, maleimide-diaminopropione, iodoacetamide, or vinylsulfone.

[0179] (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 by a thioether bond formed between the maleimide moiety and a cysteine ​​residue of the antibody.

[0180] (33) The antibody comprises an IgG heavy chain constant region and a light chain region, and the cysteine ​​residue is H Residue 124 of domain 1, H Residue 378 of the C3 domain, orH Residue 124 of domain C1 and the C H 33. The conjugated antibody of embodiment 32, wherein both of residues 378 and 379 of the three domains are selected.

[0181] (34) The conjugated antibody of any of the preceding embodiments, wherein the antibody is a human antibody, a chimeric or hybrid antibody, or a humanized antibody.

[0182] (35) The conjugated antibody of any of the preceding embodiments, wherein the antibody comprises an IgG heavy chain constant region selected from the human IgG1 or human IgG4 isotype.

[0183] (36) The conjugated antibody of any of the preceding embodiments, wherein the antibody comprises an IgG heavy chain constant region comprising an isoleucine substituted at residue 247, a glutamic acid substituted at residue 332, or both an isoleucine substituted at residue 247 and a glutamic acid substituted at residue 332, numbered according to EU index numbering.

[0184] (37) The conjugated antibody of any of the preceding embodiments, wherein 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.

[0185] (38) The conjugated antibody of any of the preceding embodiments, wherein the antibody is a monoclonal antibody.

[0186] (39) The conjugated antibody of any of the preceding embodiments, wherein the antibody is a bispecific antibody.

[0187] (40) The conjugated antibody of any of the preceding embodiments, wherein the antibody binds to an antigen selected from HER2, PSMA, TROP2, MUC-1, nectin 4, LIV-1, mesothelin, MUC-16, folate receptor-R1, CEACAM5, GPNMB, CD56, STEAP1, ENPP3, guanylate cyclase C, SLC44A4, 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 CD98.

[0188] (41) The antibody is selected from the group consisting of T-DM1, ARX788, SYD985, MLN2704, PSMA-ADC, TACSTD2, sacituzumab govitecan, (IMMU-132)), mucin 1, sialoglycotope CA6; SAR-566658, enfortumab vedotin (ASG-22M6E), ASC-22CE), ZIP6, SGN-LIV1A, DMOT4039A, anetumab ravtansine (BAY-94-9343), BMS-986148, sofituzumab vedotin, mirvetuximab soravtansine (IMGN-853), vintafolide, labetuzumab SN-38, and glenbatumumab. Vedotin, lorvotuzumab mertansine (IMGN-901), bundletuzumab vedotin (RG-7450), AGS-16M8F, indusatumab vedotin (MLN-0264), ASG-5ME, rifastuzumab vedotin, TNFSF7, DNIB0600A, AMG-172, MDX-1243, borsetuzumab mafodotin (SGN-75), BAY79-4620, TPBG, PF06263507, SLTRK6 (ASG-15ME), anti-Fyn3, SC16LD6.5, HuMax-TF-ADC (TF-011-MMAE), PCA062, human mAb L19 and F8, RG-7636, CD309; anti-VEGFR-2ScFv-As2O3-stealth nanoparticles, anti-TnC-A1 antibody SIP(F16), anti-periostin antibody, rovalpituzumab soravtansine, ABT-414, IMGN289 AMG-595, brentuximab vedotin, iratumumab MDX-060, inotuzumab ozogamicin(CMC-544), pinatuzumab vedotin, epratuzumab SN38, polatuzumab vedotin, coltuximab ravtansine, SAR-3419, SGN-CD19A, indatuximab ravtansine, milatuzumab doxorubicin, IMGN-529, gemtuzumab ozogamicin, IMGN779, SGN 5. The conjugated antibody of any of the preceding embodiments, comprising one or more of the following: CD33A, and IGN523.

[0189] (42) A pharmaceutical composition comprising a conjugated antibody according to any of the preceding embodiments and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0190] (43) A method for treating a solid or liquid tumor, comprising administering to a patient in need of treatment an effective amount of a conjugated antibody according to any of embodiments 1 to 41 or a pharmaceutical composition according to embodiment 42.

[0191] (44) The method according to embodiment 43 for treating breast cancer, lung cancer, prostate cancer, skin cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, uterine cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibroid tumors, leukemia, or lymphoma.

[0192] (45) A conjugated antibody according to any one of embodiments 1 to 41 for use in therapy.

[0193] (46) A conjugated antibody according to any one of embodiments 1 to 41 for use in the treatment of a solid tumor or a liquid tumor.

[0194] (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, uterine cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibroid tumors, leukemia, or lymphoma.

[0195] (48) Use of an antibody according to any one of embodiments 1 to 47 for the manufacture of a therapeutic agent for a solid cancer or a liquid tumor.

[0196] (49) The use of embodiment 48, wherein the solid or liquid tumor is selected from breast cancer, lung cancer, prostate cancer, skin cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, uterine cancer, muscle cancer, bone cancer, mesothelial cancer, vascular cancer, fibroid tumor, leukemia, or lymphoma.

[0197] (50) A method for activating reactive oxygen species (ROS) production in neutrophils, comprising contacting neutrophils with a conjugated antibody according to any one of embodiments 1 to 41 under conditions in which the conjugated antibody activates ROS production in neutrophils.

[0198] (51) The method of embodiment 48, wherein the linker of the conjugated antibody comprises a polyethylene glycol spacer comprising at least 12 monomers.

[0199] (52) Formula: RP 1 -P 2 -P 3 -NH-(CH2CH2O) n -CH2CH2-Y [wherein R is HC(=O)-, P 1 is Met(C(halogen) m ) [wherein m is 1 to 3] (e.g., Met(CF3), Met(CHF2), or Met(CH2F)); P 2 , P 1 P is 1 to 6 proteinogenic or non-proteinogenic amino acids linked to each other by peptide bonds; 3 is an amino acid containing a side chain containing a -COOH moiety (e.g., glutamic acid or aspartic acid) or a -NH2 moiety (e.g., lysine), optionally containing a glutamic acid residue linked through its side chain gamma carboxyl group or a lysine residue linked through its side chain epsilon amino group; P 3 is a peptide bond. 2 wherein n is an integer selected from 3 to 24; and Y comprises an amino or cysteine ​​reactive moiety, where appropriate Y is selected from maleimide, maleimido-diaminopropione, iodoacetamide, or vinylsulfone, or a salt thereof.

[0200] (53) Formula: RP 1 -P 2 -P 3-NH-(CH2CH2O) n -CH2CH2-Y [wherein R is HC(=O)-, and P 1 is Met or Met(C(halogen) m ) [wherein m is 1 to 3] (e.g., Met(CF3), Met(CHF2), or Met(CH2F)); P 2 , P 1 P is 1 to 6 proteinogenic or non-proteinogenic amino acids linked to each other by peptide bonds; 3 is an amino acid containing a side chain containing a -COOH moiety (e.g., glutamic acid or aspartic acid) or a -NH2 moiety (e.g., lysine), optionally containing a glutamic acid residue linked through its side chain gamma carboxyl group or a lysine residue linked through its side chain epsilon amino group; P 3 is a peptide bond. 2 wherein n is an integer selected from 3 to 24; and Y comprises an amino or cysteine ​​reactive moiety, where appropriate Y is selected from maleimide, maleimido-diaminopropione, iodoacetamide, or vinylsulfone, or a salt thereof.

Claims

1. A conjugated antibody or an antigen-binding fragment thereof, comprising an antibody or an antigen-binding fragment thereof conjugated to a peptide containing an N-formyl-halogenated methionine residue at the N-terminus.

2. The conjugated antibody according to claim 1, wherein the N-formyl-halogenated methionine residue is N-formyl-trifluoromethionine.

3. The conjugated antibody according to claim 1, wherein the antibody is conjugated to the peptide via a linker.

4. The conjugated antibody is 【Chemical Formula 1】 The conjugated antibody according to claim 3, having a formula represented as

5. The conjugated antibody according to claim 3, wherein the peptide contains a C-terminal glutamic acid residue, and the peptide is conjugated to the linker by an amide bond formed between the gamma-carboxyl group of the glutamic acid and the amino group of the linker.

6. The conjugated antibody according to claim 3, wherein the peptide contains a C-terminal lysine residue, and the peptide is conjugated to the linker by an amide bond formed between the epsilon-amino group of the lysine and the carboxyl group of the linker.

7. The conjugated antibody is 【Chemical Formula 2】 The conjugated antibody according to claim 3, having a formula represented as

8. The conjugated antibody according to claim 1, wherein the peptide contains 2 to 10 amino acids.

9. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from D - amino acids.

10. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from homologs of amino acids lacking one or more methylene groups between the α - carbon and the side chain, or homologs of amino acids having an additional methylene group between the α - carbon and the side chain.

11. The conjugated antibody according to claim 10, wherein the peptide is selected from homologs of amino acids lacking one or more methylene groups between the α - carbon and the side chain.

12. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from homo - amino acids.

13. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from bis - homo - amino acids.

14. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from alkylated amino acids having an alkyl substituent on the α - carbon.

15. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from dialkylated amino acids having a dialkyl substituent on the α - carbon.

16. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from alkylated amino acids having an alkyl substituent on the amino group.

17. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from phenylalanine, tyrosine, tryptophan, histidine, proline, naphthylalanine.

18. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from nor - amino acids and linear core amino acids.

19. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from amino acids containing substituents selected from alkynyl, azide, thiophenyl, thienyl, pyridyl, anthrenyl, cycloalkyl, diphenyl, furyl, and naphthyl.

20. The peptide is of the formula: NH ２ -CH ２ -CH ２ -(O-CH ２ -CH ２ ) ｎ -COOH (wherein n is selected from 1 to 24), and the conjugated antibody according to claim 1, which comprises 2 to 10 amino acids selected from the amino acids so represented.

21. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from β - amino acids, γ - amino acids, δ - amino acids, ε - amino acids, and ζ - amino acids.

22. The conjugated antibody according to claim 1, wherein the peptide comprises 2 to 10 amino acids selected from cyclo - amino acids.

23. The conjugated antibody according to claim 1, wherein the peptide comprises an amino - protecting group.

24. The conjugated antibody according to claim 3, wherein the linker comprises a spacer arm having a distance of about 10 to 50 angstroms.

25. The linker comprises a spacer arm, and the linker is 【Chemical formula 3】 and 【Chemical formula 4】 The conjugated antibody according to claim 3, having a formula selected from

26. wherein the linker is 【Chemical Formula 5】 and 【Chemical Formula 6】 (wherein n is an integer selected from 3 to 24) The conjugated antibody according to claim 25, having a formula selected from

27. The conjugated antibody according to claim 24, wherein the spacer arm is a peptide sequence containing an amino acid selected from glycine, serine, and alanine.

28. wherein the linker contains a polyethylene glycol (Peg) moiety (i.e., (-O-CH ２ -CH ２ ) １－２４ ) of the conjugated antibody according to claim 3.

29. wherein the linker is -((Peg) １－２４ )-(AA) １－２ -((Peg) １－２４ ) - [wherein AA is a glutamic acid residue bonded by its side-chain gamma carboxyl group or a lysine residue bonded by its side-chain epsilon amino group] and contains a segmented polyethylene glycol moiety represented as the conjugated antibody according to claim 3.

30. wherein the peptide-linker is 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 contains an amino group or a cysteine reactive moiety for conjugating the peptide-linker to the antibody] The conjugated antibody according to claim 3, having a formula selected from

31. The conjugated antibody according to claim 30, wherein the cysteine reactive moiety is selected from maleimide, maleimide-diaminopropion, iodoacetamide, or vinyl sulfone.

32. The conjugated antibody according to claim 3, wherein the linker contains a maleimide moiety, and the linker is conjugated to the antibody by a thioether bond formed between the maleimide moiety and a cysteine residue of the antibody.

33. The antibody includes an IgG heavy chain constant region and a light chain region, and the cysteine residue is the C Ｈ residue 124 of domain 1, the C Ｈ residue 378 of domain 3, or both the residue 124 of domain 1 and the residue 378 of domain 3 selected from the C Ｈ residue 124 of domain 1 and the C Ｈ The conjugated antibody according to claim 32, which is selected from both the residue 378 of domain 3.

34. The conjugated antibody according to claim 1, wherein the antibody is a human antibody, a chimeric or hybrid antibody, or a humanized antibody.

35. The conjugated antibody according to claim 1, wherein the antibody includes an IgG heavy chain constant region selected from human IgG1 isotype or human IgG4 isotype.

36. When the antibody is numbered based on the EU index numbering, the IgG heavy chain constant region includes 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. The conjugated antibody according to claim 35.

37. The conjugated antibody according to claim 36, wherein the antibody includes a heavy chain constant region having an amino acid sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53, 54, 55, 56, or 57.

38. The conjugated antibody according to claim 1, wherein the antibody is a monoclonal antibody.

39. The conjugated antibody according to claim 1, wherein the antibody is a bispecific antibody.

40. The conjugated antibody according to claim 1, wherein the antibody binds to an antigen selected from HER2, PSMA, TROP2, MUC-1, nectin 4, LIV-1, mesothelin, MUC-16, folate receptor-R1, CEA CAM5, GP NMB, CD56, STEAP1, ENPP3, guanylate cyclase C, SLC44A4, 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 CD98.

41. The conjugated antibody according to claim 1, comprising one or more of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody selected from T-DM1, ARX788, SYD985, MLN2704, PSMA-ADC, TACSTD2, sacituzumab govitecan, (IMMU-132), SAR-566658, enfortumab vedotin (ASG-22M6E), ASC-22CE, ZIP6, SGN-LIV1A, DMO4039A, anetumab ravtansine (BAY-94-9343), BMS-986148, sophituzumab vedotin, mirvetuximab soravtansine (IMGN-853), vintafolide, labetuzumab SN-38, glenvantamab vedotin, lorvotuzumab mertansine (IMGN-901), bunderatuzumab vedotin (RG-7450), AGS-16M8F, indusatumab vedotin (MLN-0264), ASG-5ME, rifatuzumab vedotin, TNF-SF7, DNI-B0600A, AMG-172, MDX-1243, borsetuzumab mafodotin (SGN-75), BAY79-4620, TPBG, PF06263507, SLTRK6 (ASG-15ME), anti-Fyn3, SC16LD6.5, HuMax-TF-ADC (TF-011-MMAE), PCA062, human mAb L19, human mAb F8, RG-7636, anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles, anti-TnC-A1 antibody SIP (F16), anti-periostin antibody, robatupizumab soravtansine, ABT-414, IMGN289 AMG-595, brentuximab vedotin, alemtuzumab MDX-060, inotuzumab ozogamicin (CMC-544), pinatuzumab vedotin, epratuzumab SN38, polatuzumab vedotin, cortsuximab ravtansine, SAR-3419, SGN-CD19A, indatuximab ravtansine, miratuzumab doxorubicin, MGN-529, gemtuzumab ozogamicin, IMGN779, SGN CD33A, and IGN523.

42. A pharmaceutical composition comprising the conjugated antibody according to claim 1 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

43. The pharmaceutical composition according to claim 42 for treating solid cancer or liquid tumors.

44. The pharmaceutical composition according to 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, mesothelioma, angiosarcoma, fibrosarcoma, leukemia, or lymphoma.

45. A medicament comprising the conjugated antibody according to claim 1 for use in therapy.

46. A medicament comprising the conjugated antibody according to claim 1 for use in the treatment of solid cancer or liquid tumors.

47. The medicament according to 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, mesothelioma, angiosarcoma, fibrosarcoma, leukemia, or lymphoma.

48. Use of the antibody according to claim 1 for the manufacture of a therapeutic agent for solid cancer or liquid tumors.

49. The use according to claim 48, wherein the solid cancer or liquid tumor is 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, mesothelioma, angiosarcoma, fibrosarcoma, leukemia, or lymphoma.

50. An agent for activating the production of reactive oxygen species (ROS) in neutrophils, the agent comprising the conjugated antibody according to claim 1.

51. The pharmaceutical according to claim 45, wherein the linker of the conjugated antibody comprises a polyethylene glycol spacer comprising at least 12 monomers.

52. Formula: R-P １ -P ２ -P ３ -NH-(CH ２ CH ２ O) ｎ -CH ２ CH ２ -Y [wherein, R is HC(=O)-; P １ is Met(C(halogen)) ｍ [wherein, m is an integer from 1 to 3]; P ２ is 1 to 6 proteinaceous or non-proteinaceous amino acids bonded to P １ by peptide bonds; P ３ is an amino acid containing a side chain containing a -COOH moiety or a -NH ２ moiety, and P ３ is bonded to P ２ by a peptide bond; n is an integer selected from 3 to 24; and Y contains an amino or cysteine-reactive moiety] A compound represented by the formula or a salt thereof.

53. Formula: R-P １ -P ２ -P ３ -NH-(CH ２ CH ２ O) ｎ -CH ２ CH ２ -Y [wherein, R is HC(=O)-; P １ is Met or Met(C(halogen)) ｍ [wherein, m is an integer from 1 to 3]; P ２ is P １1 to 6 proteinaceous or non-proteinaceous amino acids mutually bonded by peptide bonds; P ３ is an amino acid containing a side chain including a -COOH moiety, and P ３ is bonded to P by a peptide bond; ２ n is an integer selected from 3 to 24; and Y is a compound represented by [including an amino or cysteine-reactive moiety] or a salt thereof.

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