Anti-CitH3 antibodies and uses thereof

Abstract

The present disclosure relates to anti-CitH3 (citrullinated histone H3) antibodies, antigen-binding fragments, and uses thereof.

Description

[Technical Field]

[0001] The present disclosure relates to anti-CitH3 (citrullinated histone H3) antibodies and uses thereof.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 438,778, filed January 12, 2023, which is incorporated herein by reference in its entirety.

[0003] (Incorporation of electronic filing documents) This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on December 27, 2023, has the file name "HNC0002PCT.xml" and is 21,128 bytes in size. [Background technology]

[0004] Neutrophil extracellular traps (NETs) are networks of extracellular fibers composed primarily of DNA from neutrophils that bind to pathogens. Neutrophils are traditionally thought to be the immune system's first line of defense against infection, killing invading pathogens.

[0005] NETs can have detrimental effects on the host, as extracellular exposure of histone complexes may play a role during the development of autoimmune diseases such as systemic lupus erythematosus. NETs can also play a role in inflammatory diseases, as they can be identified in preeclampsia, a pregnancy-associated inflammatory disorder in which neutrophils are known to be activated.

[0006] Excessive NETs have been detected in sepsis and are associated with significant organ damage. Histone citrullination / desorption induced by peptidylarginine deiminase (PAD) is a key post-translational modification that promotes chromatin decondensation during NET formation. Furthermore, citrullinated histones are found in the extracellular space of neutrophils along with DNA as components of NETs.

[0007] Citrullinated histone H3 (CitH3) has recently been shown to be significantly involved in the process of NETosis. In clinical trials, CitH3 levels were found to be significantly correlated with the Respiratory Sequential Organ Failure Assessment score. Importantly, released CitH3 induces the formation of more NETs through a positive feedback mechanism in neutrophils. Furthermore, CitH3 also activates macrophage pyrogenesis, causing further tissue damage and exacerbating immune dysfunction. This phenomenon, called a "vicious cycle," generates more circulating CitH3 and induces a "cytokine storm."

[0008] Elevated levels of CitH3 have also been detected in patients with various cancers, causing changes in the tumor microenvironment and thus affecting tumor progression and the effectiveness of cancer treatment. Furthermore, CitH3 / NET-induced immune dysfunction may also affect tissue repair and regeneration processes, such as in chronic wounds and multiple organ failure.

[0009] The recent clinical and commercial success of antibodies has generated considerable interest in antibody-based therapeutics. Summary of the Invention [Problem to be solved by the invention]

[0010] There is a need to develop antibodies against CitH3 to treat immune dysfunction caused by NET activation and release. [Means for solving the problem]

[0011] The present disclosure relates to anti-CitH3 antibodies, antigen-binding fragments thereof, and uses thereof.

[0012] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to CitH3 (citrullinated histone H3), comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence. and a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to the CDR3 amino acid sequence of the selected VH CDR1, 2, and 3 and the selected VL CDR1, 2, and 3. In some embodiments, the selected VH CDR1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, and 6, respectively, and (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, and 21, respectively.

[0013] In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0014] In some embodiments, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.

[0015] In some embodiments, the antibody or antigen-binding fragment specifically binds to human CitH3.

[0016] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0017] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (eg, a bispecific antibody).

[0018] In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide, the nucleic acid comprising: (1) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, wherein the immunoglobulin heavy chain or fragment thereof binds to CitH3 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 11, 12, 13, or 15; or (2) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, wherein the immunoglobulin light chain or fragment thereof binds to CitH3 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, or 14.

[0019] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.

[0020] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0021] In some embodiments, the VH when paired with a VL specifically binds human CitH3, or the VL when paired with a VH specifically binds human CitH3.

[0022] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a humanized immunoglobulin heavy chain or fragment thereof and the immunoglobulin light chain or fragment thereof is a humanized immunoglobulin light chain or fragment thereof.

[0023] In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv) or a multispecific antibody (eg, a bispecific antibody).

[0024] In some embodiments, the nucleic acid is cDNA.

[0025] In one aspect, the present disclosure provides a vector comprising one or more of the nucleic acids described in this disclosure. In one aspect, the present disclosure provides a vector comprising two of the nucleic acids described in this disclosure. In some embodiments, the vector encodes a VL region and a VH region that together bind to CitH3.

[0026] In one aspect, the present disclosure provides a pair of vectors, each vector comprising one of the nucleic acids described in the present disclosure, hi some embodiments, the pair of vectors encode a VL region and a VH region that together bind to CitH3.

[0027] In one aspect, the present disclosure provides a cell comprising a vector described in this disclosure, or a pair of vectors described in this disclosure.

[0028] In some embodiments, the cells are CHO cells.

[0029] In one aspect, the present disclosure provides a cell comprising one or more of the nucleic acids described in this disclosure.

[0030] In one aspect, the present disclosure provides a cell comprising two of the nucleic acids described in this disclosure.

[0031] In some embodiments, the two nucleic acids together encode a VL region and a VH region that together bind to CitH3.

[0032] In one aspect, the disclosure provides a manufacturing method for producing an antibody or antigen-binding fragment thereof, the manufacturing method comprising: (a) culturing a cell described in this disclosure under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; (b) harvesting the antibody or antigen-binding fragment produced by the cell.

[0033] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to CitH3, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, or 100% identical to SEQ ID NO: 7, 8, 9, 10, or 14, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, or 100% identical to SEQ ID NO: 11, 12, 13, or 15.

[0034] In some embodiments, the VH comprises the sequence of SEQ ID NO:7 and the VL comprises the sequence of SEQ ID NO:11.

[0035] In some embodiments, the VH comprises the sequence of SEQ ID NO:7 and the VL comprises the sequence of SEQ ID NO:12.

[0036] In some embodiments, the VH comprises the sequence of SEQ ID NO:7 and the VL comprises the sequence of SEQ ID NO:13.

[0037] In some embodiments, the VH comprises the sequence of SEQ ID NO:8 and the VL comprises the sequence of SEQ ID NO:11.

[0038] In some embodiments, the VH comprises the sequence of SEQ ID NO:8 and the VL comprises the sequence of SEQ ID NO:12.

[0039] In some embodiments, the VH comprises the sequence of SEQ ID NO:8 and the VL comprises the sequence of SEQ ID NO:13.

[0040] In some embodiments, the VH comprises the sequence of SEQ ID NO:9 and the VL comprises the sequence of SEQ ID NO:11.

[0041] In some embodiments, the VH comprises the sequence of SEQ ID NO:9 and the VL comprises the sequence of SEQ ID NO:12.

[0042] In some embodiments, the VH comprises the sequence of SEQ ID NO:9 and the VL comprises the sequence of SEQ ID NO:13.

[0043] In some embodiments, the VH comprises the sequence of SEQ ID NO:10 and the VL comprises the sequence of SEQ ID NO:11.

[0044] In some embodiments, the VH comprises the sequence of SEQ ID NO:10 and the VL comprises the sequence of SEQ ID NO:12.

[0045] In some embodiments, the VH comprises the sequence of SEQ ID NO:10 and the VL comprises the sequence of SEQ ID NO:13.

[0046] In some embodiments, the VH comprises the sequence of SEQ ID NO:14 and the VL comprises the sequence of SEQ ID NO:15.

[0047] In some embodiments, the antibody or antigen-binding fragment specifically binds to human CitH3.

[0048] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0049] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (eg, a bispecific antibody).

[0050] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to CitH3, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 7, 8, 9, 10, or 14; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 identical to VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 11, 12, 13, or 15.

[0051] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CitH3, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected antibody or antigen-binding fragment; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of the selected antibody or antigen-binding fragment.

[0052] In some embodiments, the selected antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof described in the present disclosure.

[0053] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof described in this disclosure.

[0054] In one aspect, the present disclosure provides a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in this disclosure.

[0055] In some embodiments, the subject has a solid tumor or a hematological malignancy.

[0056] In some embodiments, the cancer is associated with CitH3-induced nephrosis.

[0057] In one aspect, the present disclosure provides a method of treating a subject having an immune disorder (e.g., an autoimmune disease), the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein. In some embodiments, the immune disorder is associated with nephrosis.

[0058] In one aspect, the present disclosure provides a method of treating a subject having an infectious disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in this disclosure.

[0059] In some embodiments, the infection is caused by bacteria and / or viruses, eg, sepsis.

[0060] In one embodiment, the infectious disease is influenza.

[0061] In one aspect, the present disclosure provides a method for inhibiting CitH3-nephrosis in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in the present disclosure.

[0062] In some embodiments, CitH3-nephrosis is caused by infection, sepsis, cancer, a chronic wound, or an autoimmune disorder.

[0063] In one aspect, the present disclosure provides a method for treating a subject having organ damage, such as cardiac, hepatic, or cerebral myocardial infarction, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in the present disclosure.

[0064] In one aspect, the present disclosure provides a method of treating a subject having a skin ulcer, e.g., a diabetic foot ulcer, tissue injury, or a chronic wound disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in this disclosure.

[0065] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in this disclosure and a pharmaceutically acceptable carrier.

[0066] In one aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently linked to a therapeutic agent, hi some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0067] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate described in this disclosure and a pharmaceutically acceptable carrier.

[0068] As used in this disclosure, the term "antibody" refers to any antigen-binding molecule that comprises at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody may comprise the Fc region of a human antibody. The term antibody also includes derivatives formed from antibody fragments, e.g., bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies.

[0069] As used in this disclosure, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion of an antibody can specifically bind to an antigen. In some embodiments, an antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0070] As used in this disclosure, the term "human antibody" refers to an antibody encoded by endogenous nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in a human cell culture (e.g., a human hybridoma cell). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or a yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a cow) that includes an unrearranged or rearranged human immunoglobulin locus (e.g., a heavy or light chain human immunoglobulin locus).

[0071] As used in this disclosure, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and murine antibodies). A non-limiting example of a chimeric antibody is an antibody that contains variable domain sequences (e.g., all or part of the light chain variable domain sequence and / or heavy chain variable domain sequence) of a non-human (e.g., murine) antibody and the constant domain of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

[0072] As used in this disclosure, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (e.g., murine) immunoglobulin and includes sequence derived from human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., donor antibody), e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of a human immunoglobulin are replaced by corresponding non-human (e.g., murine) immunoglobulin residues. In some embodiments, a humanized antibody may include residues that are not present in the recipient or donor antibody. These modifications may be made to further refine antibody performance. In some embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., murine) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described in this disclosure.

[0073] As used in this disclosure, the terms "subject" and "patient" are used interchangeably throughout and refer to an animal, human, or non-human to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. A human patient can be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, dwarf pigs), horses, dogs, cats, cows, and other pets, livestock, and zoo animals.

[0074] As used in this disclosure, the phrases "specifically binding" and "specifically bind," when referring to an antibody, mean that the antibody interacts preferentially with its target molecule (CitH3) over other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule. In other words, the reagent does not generally recognize and bind to all molecules, but rather recognizes and binds to molecules containing a particular structure. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a CitH3 molecule may be referred to as a CitH3-specific antibody or an anti-CitH3 antibody.

[0075] As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

[0076] As used in this disclosure, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications.

[0077] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0078] Other features and advantages of the present invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0079] [Figure 1] FIG. 1 shows the results of an initial ELISA screen of humanized antibodies that bind to CitH3. [Figure 2A] Figure 2A shows the ELISA binding results of humanized antibodies untreated or heat-treated. Chimeric antibody VH / VL was used as a control. [Figure 2B] Figure 2B shows the ELISA binding results of the humanized antibodies untreated or heat-treated. A chimeric antibody VH / VL was used as a control. [Figure 2C] Figure 2C shows the ELISA binding results of the humanized antibody under untreated or heat treatment. The chimeric antibody VH / VL was used as a control. [Figure 2D] Figure 2D shows the ELISA binding results of the humanized antibodies untreated or heat-treated. The chimeric antibody VH / VL was used as a control. [Figure 2E] Figure 2E shows the ELISA binding results of the humanized antibodies under untreated or heat treatment. The chimeric antibody VH / VL was used as a control. [Figure 3]Figure 3 shows the results of non-specific baculovirus binding of humanized antibodies by ELISA. 7A4E11 and Rituxan were used as negative controls. [Figure 4] Figure 4 shows the validated ELISA binding results of humanized antibodies that bind to CitH3. 7A4E11 was used as a positive control. [Figure 5] Figure 5 shows a non-reducing SDS-PAGE gel of the chimeric antibody VH / VL (lane 1) and three humanized antibodies VH2 / VL2 (lane 2), VH2 / VL3 (lane 3), and VH3 / VL3 (lane 4). MW is a protein marker. [Figure 6A] FIG. 6A shows the reduction-CE-SDS results of standard IgG. [Figure 6B] FIG. 6B shows the reduction-CE-SDS results for humanized antibody VH3 / VL3. [Figure 6C] FIG. 6C shows the results of non-reducing CE-SDS of standard IgG. [Figure 6D] FIG. 6D shows the non-reducing CE-SDS results for humanized antibody VH3 / VL3. [Figure 7A]Figures 7A-7B show a comparative analysis of Cayman CitH3-mAb and hCitH3-mAb binding affinities. Four distinct peptides, histone H3 (H3), citrullinated histone H3 (CitH3; 4 Cit), acetylated histone H3 (AceH3), and methylated histone H3 (MetH3) (New England Peptide™, Inc., Gardner, MA), were electrophoresed in duplicate at 0.5 micrograms each by SDS-polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were split into two sections for parallel overnight probing with equal amounts of Cayman CitH3-mAb (Cayman Chemicals, Ann Arbor, MN) or hCitH3-mAb (2 μg / ml). For convenience, hCitH3-mAb is the same as VH3 / VL3. After three extensive washes, the sections were incubated with HRP-conjugated secondary antibodies specific for mouse IgG and human IgG, respectively, for 2 hours (7A). For simultaneous signal generation, both membrane sections were exposed simultaneously for the same period. Quantitative analysis of signal intensity was performed using Image Studio Lite, and the results are shown as the mean ± SEM of three independent experiments (n = 3 / group). **: P < 0.01 (7B). [Figure 7B]Figures 7A-7B show a comparative analysis of Cayman CitH3-mAb and hCitH3-mAb binding affinities. Four distinct peptides, histone H3 (H3), citrullinated histone H3 (CitH3; 4 Cit), acetylated histone H3 (AceH3), and methylated histone H3 (MetH3) (New England Peptide™, Inc., Gardner, MA), were electrophoresed in duplicate at 0.5 micrograms each by SDS-polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were split into two sections for parallel overnight probing with equal amounts of Cayman CitH3-mAb (Cayman Chemicals, Ann Arbor, MN) or hCitH3-mAb (2 μg / ml). For convenience, hCitH3-mAb is the same as VH3 / VL3. After three extensive washes, the sections were incubated with HRP-conjugated secondary antibodies specific for mouse IgG and human IgG, respectively, for 2 hours (7A). For simultaneous signal generation, both membrane sections were exposed simultaneously for the same period. Quantitative analysis of signal intensity was performed using Image Studio Lite, and the results are shown as the mean ± SEM of three independent experiments (n = 3 / group). **: P < 0.01 (7B). [Figure 8A] Figures 8A-8B show a schematic of the assay (8A) and results (8B), demonstrating that hCitH3-mAb improves survival in mice undergoing LPS-induced septic shock. C57BL6 / J mice were exposed to a lethal LPS dose (25 mg / kg, intraperitoneally), resulting in complete death within 24 hours (IgG was used as a control). Tail vein administration of hCitH3-mAb (20 mg / kg, tail vein) significantly improved survival. [Figure 8B]Figures 8A-8B show a schematic of the assay (8A) and results (8B), demonstrating that hCitH3-mAb improves survival in mice undergoing LPS-induced septic shock. C57BL6 / J mice were exposed to a lethal LPS dose (25 mg / kg, intraperitoneally), resulting in complete death within 24 hours (IgG was used as a control). Tail vein administration of hCitH3-mAb (20 mg / kg, tail vein) significantly improved survival. [Figure 9A] Figures 9A-9C show the developed multiplex platform and results (9C) using "pre-equilibration digital ELISA (PEd-ELISA)" technology (9A and 9B), demonstrating early detection of elevated CitH3 levels after endotoxin shock. [Figure 9B] Figures 9A-9C show the developed multiplex platform and results (9C) using "pre-equilibration digital ELISA (PEd-ELISA)" technology (9A and 9B), demonstrating early detection of elevated CitH3 levels after endotoxin shock. [Figure 9C] Figures 9A-9C show the developed multiplex platform and results (9C) using "pre-equilibration digital ELISA (PEd-ELISA)" technology (9A and 9B), demonstrating early detection of elevated CitH3 levels after endotoxin shock. [Figure 10A] Figures 10A-10B show a schematic of the assay (10A) and results (10B), demonstrating that hCitH3-mAb (H3L3) improves survival in mice subjected to Pseudomonas aeruginosa (PA)-induced sepsis. C57BL6 / J mice (10-12 weeks, male) were subjected to PA-induced septic shock (2.5 x 10 CFU, intranasal), and hCitH3-mAb (20 mg / kg) was delivered to the mice via tail vein injection 30 minutes after PA administration. As a control, mice received a similar treatment with human IgG (purchased from Sigma). All mice receiving human IgG died within 3 days (n = 6), whereas approximately 80% of mice receiving hCitH3-mAb survived (n = 5). [Figure 10B]Figures 10A-10B show a schematic of the assay (10A) and results (10B), demonstrating that hCitH3-mAb (H3L3) improves survival in mice subjected to Pseudomonas aeruginosa (PA)-induced sepsis. C57BL6 / J mice (10-12 weeks, male) were subjected to PA-induced septic shock (2.5 x 10 CFU, intranasal), and hCitH3-mAb (20 mg / kg) was delivered to the mice via tail vein injection 30 minutes after PA administration. As a control, mice received a similar treatment with human IgG (purchased from Sigma). All mice receiving human IgG died within 3 days (n = 6), whereas approximately 80% of mice receiving hCitH3-mAb survived (n = 5). [Figure 11A] Figures 11A-11B show the results of histological evaluation of PA-induced ALI (11A). Separate experiments were performed on mice subjected to PA-induced septic shock and sacrificed 24 h after hCitH3-mAb administration (n = 5 for hCitH3-mAb, n = 5 for IgG). Acute lung injury (ALI) scores were assessed using mouse lung sections by a board-certified pathologist blinded to animal treatment (11B). [Figure 11B] Figures 11A-11B show the results of histological evaluation of PA-induced ALI (11A). Separate experiments were performed on mice subjected to PA-induced septic shock and sacrificed 24 h after hCitH3-mAb administration (n = 5 for hCitH3-mAb, n = 5 for IgG). Acute lung injury (ALI) scores were assessed using mouse lung sections by a board-certified pathologist blinded to animal treatment (11B). [Figure 12A]Figures 12A-12B show a schematic (12A) and survival curve results (12B) of C57BL / 6 mice (12 weeks old) treated with or without hCitH3-mAb in a mouse model of lethal colonic puncture (CLP)-induced sepsis. C57BL / 6 mice were administered a single dose of hCitH3-mAb (20 mg / kg body weight) or human IgG (20 mg / kg body weight) 0.5 hours before CLP. Survival was monitored for 10 days (n = 7 per group). The results show that hCitH3-mAb significantly improved mouse survival compared with the IgG group. [Figure 12B] Figures 12A-12B show a schematic (12A) and survival curve results (12B) of C57BL / 6 mice (12 weeks old) treated with or without hCitH3-mAb in a mouse model of lethal colonic puncture (CLP)-induced sepsis. C57BL / 6 mice were administered a single dose of hCitH3-mAb (20 mg / kg body weight) or human IgG (20 mg / kg body weight) 0.5 hours before CLP. Survival was monitored for 10 days (n = 7 per group). The results show that hCitH3-mAb significantly improved mouse survival compared with the IgG group. [Figure 13A] Figures 13A-13C show the results of evaluating the role of hCitH3-mAb in bacterial clearance during septic shock induced by Pseudomonas aeruginosa. Mice were inoculated intranasally with 2.5 x 10 P. aeruginosa cells and then administered a tail vein injection of hCitH3-mAb (20 mg / kg) or human IgG (20 mg / kg). After infection, lung and spleen tissues were harvested and homogenized for bacterial burden assessment by culturing on LB agar plates (13B - lung, 13C - spleen). Images of agar plates (13A) are shown. [Figure 13B]Figures 13A-13C show the results of evaluating the role of hCitH3-mAb in bacterial clearance during septic shock induced by Pseudomonas aeruginosa. Mice were inoculated intranasally with 2.5 x 10 P. aeruginosa cells and then administered a tail vein injection of hCitH3-mAb (20 mg / kg) or human IgG (20 mg / kg). After infection, lung and spleen tissues were harvested and homogenized for bacterial burden assessment by culturing on LB agar plates (13B - lung, 13C - spleen). Images of agar plates (13A) are shown. [Figure 13C] Figures 13A-13C show the results of evaluating the role of hCitH3-mAb in bacterial clearance during septic shock induced by Pseudomonas aeruginosa. Mice were inoculated intranasally with 2.5 x 10 P. aeruginosa cells and then administered a tail vein injection of hCitH3-mAb (20 mg / kg) or human IgG (20 mg / kg). After infection, lung and spleen tissues were harvested and homogenized for bacterial burden assessment by culturing on LB agar plates (13B - lung, 13C - spleen). Images of agar plates (13A) are shown. [Figure 14] Figure 14 shows the synergistic effect of hCitH3-mAb and rhMG53 in protecting against CLP-sepsis in aged mice. Aged C57BL / 6J mice (male, 24-26 months) were obtained from the National Institute of Aging (NIA). Mice were anesthetized with isoflurane according to a protocol approved by the University of Virginia IACUC. Mouse sepsis was performed using a colonic puncture (CLP) model. The cecum of each mouse was isolated from the abdominal cavity with sterile forceps, ligated with 4-0 silk sutures, and punctured using a 20G needle at the midpoint between the tail end and the ligation set. Mice were divided into four groups: CLP, CLP treated with rhMG53, CLP treated with hCitH3-mAb, and CLP treated with rhMG53 + hCitH3-mAb. hCitH3-mAb was administered via tail vein injection at a dose of 20 mg / kg, and recombinant human MG53 (rhMG53) protein was administered via intraperitoneal injection at a dose of 5 mg / kg after CLP. [Figure 15]Figure 15 shows that CLP induced an increase in IL-6 in aged mice compared with sham controls. Administration of rhMG53 (5 mg / kg, intraperitoneal injection) or hCitH3-mAb (20 mg / kg, tail vein injection) attenuated the increase in IL-6. Coadministration of rhMG53 and hCitH3-mAb almost completely abolished the increase in IL-6. [Figure 16A] Figures 16A and 16B show the change in body weight (% of initial body weight) of plotted animals infected with influenza virus and treated with rhMG53 (16A) or hCitH3-mAb (16B) compared to animals treated with saline as a control. [Figure 16B] Figures 16A and 16B show the change in body weight (% of initial body weight) of plotted animals infected with influenza virus and treated with rhMG53 (16A) or hCitH3-mAb (16B) compared to animals treated with saline as a control. [Figure 17A] Figures 17A-17D show representative microscopic images of H&E-stained lung tissue sections from mice infected with influenza virus and treated with saline (17A), rhMG53 (17B), or hCitH3-mAb (17C). Quantitative analysis of cellularity in tissue sections (17D) was performed using ImageJ software. We observed a decrease in cellular infiltration in lung tissues harvested from animals 9 days after influenza virus infection and treated with rhMG53 (or hCitH3-mAb) compared to animals treated with saline (n = 3 per group, p values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 17B]Figures 17A-17D show representative microscopic images of H&E-stained lung tissue sections from mice infected with influenza virus and treated with saline (17A), rhMG53 (17B), or hCitH3-mAb (17C). Quantitative analysis of cellularity in tissue sections (17D) was performed using ImageJ software. We observed a decrease in cellular infiltration in lung tissues harvested from animals 9 days after influenza virus infection and treated with rhMG53 (or hCitH3-mAb) compared to animals treated with saline (n = 3 per group, p values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 17C] Figures 17A-17D show representative microscopic images of H&E-stained lung tissue sections from mice infected with influenza virus and treated with saline (17A), rhMG53 (17B), or hCitH3-mAb (17C). Quantitative analysis of cellularity in tissue sections (17D) was performed using ImageJ software. We observed a decrease in cellular infiltration in lung tissues harvested from animals 9 days after influenza virus infection and treated with rhMG53 (or hCitH3-mAb) compared to animals treated with saline (n = 3 per group, p values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 17D] Figures 17A-17D show representative microscopic images of H&E-stained lung tissue sections from mice infected with influenza virus and treated with saline (17A), rhMG53 (17B), or hCitH3-mAb (17C). Quantitative analysis of cellularity in tissue sections (17D) was performed using ImageJ software. We observed a decrease in cellular infiltration in lung tissues harvested from animals 9 days after influenza virus infection and treated with rhMG53 (or hCitH3-mAb) compared to animals treated with saline (n = 3 per group, p values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 18A]Figures 18A-18B show the results of ELISA quantification of IFNβ (18A) and IL-6 (18B) in lung tissue collected from mice without influenza virus infection or mice infected with influenza virus for 9 days and treated with saline, rhMG53, or hhCitH3-mAb (n=3 per group, p-values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 18B] Figures 18A-18B show the results of ELISA quantification of IFNβ (18A) and IL-6 (18B) in lung tissue collected from mice without influenza virus infection or mice infected with influenza virus for 9 days and treated with saline, rhMG53, or hhCitH3-mAb (n=3 per group, p-values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 19A] Figures 19A-19C present the results of a multiplex assay for mouse cytokines showing increased plasma levels of inflammatory markers, including GM-CSF (19B), IFNγ (19C), and LIX (19A), in mice with influenza virus infection compared to uninfected control animals, which were significantly reduced in animals treated with rhMG53 or hCitH3-mAb (n=3 per group, p-values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 19B] Figures 19A-19C present the results of a multiplex assay for mouse cytokines showing increased plasma levels of inflammatory markers, including GM-CSF (19B), IFNγ (19C), and LIX (19A), in mice with influenza virus infection compared to uninfected control animals, which were significantly reduced in animals treated with rhMG53 or hCitH3-mAb (n=3 per group, p-values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 19C]Figures 19A-19C present the results of a multiplex assay for mouse cytokines showing increased plasma levels of inflammatory markers, including GM-CSF (19B), IFNγ (19C), and LIX (19A), in mice with influenza virus infection compared to uninfected control animals, which were significantly reduced in animals treated with rhMG53 or hCitH3-mAb (n=3 per group, p-values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). [Figure 20] FIG. 20 shows Western blots demonstrating that treatment with rhMG53 or hCitH3-mAb suppressed inflammation as indicated by reduced levels of NLRP3, MDA-5, IFITM3 after 9 days of influenza virus. [Figure 21] FIG. 21 shows Western blots demonstrating that treatment with rhMG53 or hCitH3-mAb suppressed apoptosis as indicated by reduced levels of cleaved caspase 2 / 3 / 11 and PARP after 9 days of influenza virus infection. [Figure 22] Figure 22 shows Western blots demonstrating that treatment with rhMG53 or hCitH3-mAb suppressed the expression of full-length Gasdermin D and reduced the levels of cleaved Gasdermin D after 9 days of influenza virus infection. [Figure 23] FIG. 23 shows Western blot analysis demonstrating that expression of MPO, a neutrophil marker, was observed in ischemia-reperfusion-induced liver injury, with upregulation detected from 6 to 72 hours after reperfusion. [Figure 24] FIG. 24 shows Western blot analysis demonstrating increased expression of CitH3 in ischemia-reperfusion-induced liver injury in mice 24 hours after reperfusion, which continued to increase up to 72 hours. [Figure 25]Figures 25A and 25B show that hCitH3-mAb treatment eliminated serum CitH3 levels by Western blot analysis. (Figure 25A) Mice subjected to hepatic ischemia and reperfusion were intravenously injected with human IgG antibody or hCitH3-mAb immediately after reperfusion. CitH3 levels were measured in serum samples collected before surgery and on days 1, 3, and 7 after surgery. (Figure 25B) Ponceau S staining of the loading control is shown. [Figure 26] FIG. 26 shows the results of a neutralization assay of CitH3 using hCitH3-mAb, and the results of cardiac function recovery after hepatic ischemia and reperfusion. [Figure 27A] Figures 27A and 27B show images of whole brain tissue from mice treated with CitH3-mAb versus control. (Figure 27A) Coronal sections of normal versus failing brain tissue samples taken from treated and control mice 24 hours after reperfusion are shown. (Figure 27B) The extent of ischemic myocardial infarction was tracked, and the integrated volume was calculated and shown in a bar graph. [Figure 27B] Figures 27A and 27B show images of whole brain tissue from mice treated with CitH3-mAb versus control. (Figure 27A) Coronal sections of normal versus failing brain tissue samples taken from treated and control mice 24 hours after reperfusion are shown. (Figure 27B) The extent of ischemic myocardial infarction was tracked, and the integrated volume was calculated and shown in a bar graph. [Figure 28]Figure 28 shows images of the wound area of ​​hCit-mAb-treated and untreated wounds in 14-week-old B6.BKS(D)-Leprdb / J (db / db) mice. 14-week-old B6.BKS(D)-Leprdb / J (db / db) mice were purchased from Jackson Laboratory. After anesthesia with 1.5% isoflurane, the mice's dorsal hair was removed using depilatory cream (Nair), and a 5 mm round, full-thickness skin excision wound was created at the midline, 2 cm caudal to the skull. hCitH3-mAb (20 mg / kg) and human IgG (Sigma-Aldrich, I4506; 20 mg / kg) were injected subcutaneously on days 0 and 3 after injury. The wound was covered with a hydrocolloid dressing (Tegaderm; 3M Health Care) to maintain a moist environment and was changed daily. Wound area was measured on days 2, 4, 7, 10, and 12 after injury. The data demonstrated the benefit of hCitH3-mAb in improving cutaneous wound healing in db / db mice (n=3 each). [Figure 29A] Figures 29A-29C show that co-treatment of hydrogel formulations of hCitH3-mAb and recombinant human Mitsugumin 53 (rhMG53) protein synergistically controls inflammation and improves diabetic ulcer healing. rhMG53 is a TRIM (trisite motif-containing) family protein. [Figure 29B] Figures 29A-29C show that co-treatment of hydrogel formulations of hCitH3-mAb and recombinant human Mitsugumin 53 (rhMG53) protein synergistically controls inflammation and improves diabetic ulcer healing. rhMG53 is a TRIM (trisite motif-containing) family protein. [Figure 29C] Figures 29A-29C show that co-treatment of hydrogel formulations of hCitH3-mAb and recombinant human Mitsugumin 53 (rhMG53) protein synergistically controls inflammation and improves diabetic ulcer healing. rhMG53 is a TRIM (trisite motif-containing) family protein. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to CitH3 (citrullinated histone H3). In some embodiments, the antibodies or antigen-binding fragments described herein can be used to treat immune deficiencies caused by multi-organ damage and / or tumor growth accompanied by nephrosis. In some embodiments, such conditions include infections and sepsis caused by bacteria and / or viruses. In other embodiments, the conditions include cancer progression and / or autoimmune disorders, as well as chronic wounds and organ failure.

[0081] Histones bind to nuclear DNA, packaging it into nucleosomes, and can be released into the bloodstream upon cellular activation or injury. Therefore, several clinical studies have shown that circulating cell-free DNA (cfDNA) and nucleosomes function as potential blood markers in various malignancies, but investigations of the diagnostic or prognostic relevance of circulating histones have been scarce and primarily focused on sepsis and trauma patients. Once released extracellularly, histones can mediate harmful effects on the host, suggesting their potential as both prognostic markers and therapeutic targets.

[0082] Post-translational histone modifications can have profound effects on histone structure and function and are associated with various diseases. Citrullinated histone H3 (CitH3) is the product of the post-translational conversion of peptidylarginine on the N-terminus of histone H3 to citrulline. The subsequent reduction of the positive charge of histone residues results in weak binding to negatively charged DNA, leading to chromatin decondensation. Histone citrullination is catalyzed by the enzymes peptidylarginine deiminase 4 (PAD4) and PAD2, which are primarily located in the cytoplasm of immune cells; only the PAD isozyme can translocate to the nucleus upon cell activation. This important role of CitH3 in immune cell chromatin decondensation makes it a central marker for the recently described neutrophil release of decondensed, web-like nuclear chromatin, termed neutrophil extracellular traps (NETs). CitH3 can be detected in the nucleus of neutrophils upon stimulation but can also be released into the bloodstream during NETosis. Importantly, the released CitH3 can induce the formation of more NETs through a positive feedback mechanism. Furthermore, PAD2 and PAD4 have been shown to be overexpressed in various types of tumors, suggesting that CitH3 can be released into the bloodstream upon cancer cell death. Thus, tumor cells may be an additional and unrecognized source of CitH3 in cancer.

[0083] The present disclosure provides several anti-CitH3 antibodies and humanized anti-CitH3 antibodies that can effectively inhibit NET activation and release and can be used to treat various diseases.

[0084] (histone citrullination) Citrullination is the Ca-mediated conversion of arginine residues to citrulline, catalyzed by the PAD family. 2+Citrullination, or deamination, is a catalytically driven enzyme transformation. Citrullination, or deamination, refers to the modification of a primary ketimine group (=NH) to a ketone group (=O), resulting in ammonia as a by-product. Thus, it hydrolyzes the strongly alkaline and positively charged arginine side chain to form neutral urea. Charge transfer can affect protein-protein interactions, hydrogen bond formation, and protein structure, potentially leading to denaturation. Various proteins, including cytoplasmic, nuclear, membrane, and mitochondrial proteins, can be citrullinated. Citrullinated proteins can be detected using antibody-based detection systems, such as mass spectrometry.

[0085] Citrullinated histones account for approximately 10% of all histone molecules in HL-60 granulocytes, highlighting the importance of PTMs in many nuclear-related processes. Various tumors are associated with overexpression of PAD and increased citrullination. The challenge is to identify the specific citrullinated proteins involved in various autoimmune diseases. Furthermore, protein identification is fundamental for further mechanistic studies of the citrullination machinery. Specifically, histone citrullination influences tumorigenesis in various ways, including regulating gene transcription, cell differentiation, and apoptosis. The discovery of histone citrullination, one of the reactions in which histones are substrates for PAD, could be a breakthrough in tumor research.

[0086] With regard to tumor immunity, novel epitopes induced by PTMs may provide new targets for tumor-specific immunotherapy. Nutrient deprivation, hypoxia, redox stress, DNA damage, and other tumor microenvironment conditions can increase PAD activity and the expression of citrullinated proteins. Recently, the mean serum CitH3 concentration in patients with indolent malignant tumors was found to be three-fold increased compared with that in healthy individuals, indicating that citrullinated proteins may be a cancer biomarker. In a group of cancer patients, the mean serum CitH3 levels in invasive tumors were higher than those in localized tumors, consistent with previous reports of PAD-mediated citrullination and metastasis. Notably, CitH3 levels in the plasma of cancer patients were associated with higher levels of cell-free DNA and neutrophil activation. Notably, researchers found that high plasma levels of CitH3 (>29.8 ng / mL, >75%) were strongly associated with the risk of short-term mortality. Increased CitH3 expression is considered a novel prognostic blood marker in patients with advanced cancer. The proportion of CitH3-positive neutrophils increases in patients with more severe disease. Serum CitH3 levels are closely correlated with neutrophil activation markers such as elastase, myeloperoxidase, IL-6, and IL-8. Therefore, CitH3 is considered a useful biomarker for assessing the inflammatory response and prognosis of patients with advanced cancer.

[0087] Histone citrullination has also been reported to potentially play a role in cancer therapy. Venous thromboembolism (VTE) frequently occurs during cancer treatment and can be life-threatening. Studies have shown that CitH3 is independently associated with VTE in cancer patients and plays an important role in predicting the occurrence of VTE during cancer treatment. Furthermore, inhibition of PAD2-mediated H3Cit26 reduces IL-6 expression in BMMSCs and mediates malignant plasma cell resistance to chemotherapeutic agents. One group found that effective inhibitors of PAD2 can enhance antitumor activity by inhibiting CitH3 in an HCT-116 xenograft mouse model. Furthermore, PAD2-H3Cit26 is considered a novel therapeutic target in castration-resistant prostate cancer. Furthermore, crosstalk between histones undergoing deacetylation and histones undergoing citrullination is associated with cancer cell growth, suggesting the combination of PAD and histone deacetylase inhibitors as a strategy for cancer treatment. In MCF-7 cells, Cl-amidine regulates the expression of the tumor suppressor protein OKL38 by reducing histone citrullination at the OKL38 promoter. Finally, a ubiquitous compound used in traditional Chinese medicine inhibits hematopoietic metastasis of certain tumors by targeting CitH3 and NET.

[0088] The present disclosure provides several anti-CitH3 antibodies, antigen-binding fragments thereof, and methods of using these anti-CitH3 antibodies and antigen-binding fragments to treat various diseases.

[0089] (Anti-CitH3 antibodies and antigen-binding fragments) The present disclosure provides an antibody and its antigen-binding fragment that specifically binds to CitH3.The antibody and antigen-binding fragment described in the present disclosure are highly purified and thermostable, and can specifically bind to CitH3.The present disclosure provides, for example, mouse anti-CitH3 antibody Y1128, and chimeric antibody and humanized antibody thereof.

[0090] The CDR sequences of Y1128 and Y1128-derived antibodies (e.g., humanized antibodies) include the CDRs of the heavy chain variable domain defined by Kabat numbering, SEQ ID NOS: 1-3, and the CDRs of the light chain variable domain defined by SEQ ID NOS: 4-6. CDRs may also be defined by the Chothia system. Based on Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOS: 16-18, and the CDR sequences of the light chain variable domain are set forth in SEQ ID NOS: 19-21.

[0091] The amino acid sequences of the heavy and light chain variable regions of the humanized antibodies are also provided. Because there are different methods for humanizing mouse antibodies (e.g., sequences can be replaced with different amino acids), the heavy and light chains of the antibody may be configured with two or more versions of the humanized sequence. The amino acid sequence of the heavy chain variable region of the humanized Y1128 antibody is set forth in SEQ ID NOS: 7-10. The amino acid sequence of the light chain variable region of the humanized 9H3 antibody is set forth in SEQ ID NOS: 11-13. Any of these heavy chain variable region sequences (SEQ ID NOS: 7-10) can be paired with any of these light chain variable region sequences (SEQ ID NOS: 11-13).

[0092] As shown in Table 1, the humanization percentage refers to the percentage identity of the heavy or light chain variable region sequence compared to human antibody sequences in the International Immunogenetics Database (IMGT). A top hit means that the heavy or light chain variable region sequence is closer to a particular species than to other species. For example, a top hit for human means that the sequence is closer to human than to other species. A top hit for mouse means that the sequence is closer to mouse than to other species, and these percentage identities are the highest compared to the sequences of other species. In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of methods for determining the humanization percentage and determining the top hit are known in the art. A high humanization percentage often has various advantages, such as being safer and more effective in humans, more likely to be tolerated by human subjects, and / or less likely to have side effects.

[0093] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described in the present disclosure may comprise one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1 to 3 and SEQ ID NOs: 16 to 18, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4 to 6 and SEQ ID NOs: 19 to 21.

[0094] [Table 1]

[0095] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence and the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence. The light chain variable region (VL) may comprise or consist of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to the CDR2 amino acid sequence, and the CDR3 region may comprise or consist of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, and 3 amino acid sequences and selected VL CDR1, 2, and 3 amino acid sequences are shown in Table 2 (Kabat CDRs) and Table 3 (Chothia CDRs).

[0096] [Table 2]

[0097] [Table 3]

[0098] In some embodiments, an antibody or antigen-binding fragment described in the present disclosure may comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0099] In some embodiments, an antibody or antigen-binding fragment described in the present disclosure may comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0100] In some embodiments, an antibody or antigen-binding fragment described in this disclosure may comprise a light chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 6 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0101] In some embodiments, an antibody or antigen-binding fragment described in this disclosure may comprise a light chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0102] Insertions, deletions and substitutions may be within the CDR sequences or at one or both termini of the CDR sequences.

[0103] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to CitH3. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 7, 8, 9, 10, or 14, and the selected VL sequence is SEQ ID NO: 11, 12, 13, or 15.

[0104] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. For purposes of this disclosure, comparison of sequences and determination of the percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0105] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding an immunoglobulin heavy chain or a polypeptide comprising an immunoglobulin heavy chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises the CDRs set forth in Table 2 or Table 3, or has the sequence set forth in Table 1 or SEQ ID NOs: 14, 15, and 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CitH3 (e.g., human CitH3).

[0106] Anti-CitH3 antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided in the present disclosure include polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intracellular), and antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0107] Antibody fragments are suitable for use in the provided methods as long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to CitH3 retain the ability to bind to CitH3. Fv fragments are antibody fragments that contain the complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight association, which may be covalent in nature, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs, or a subset thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, although usually with lower affinity than the entire binding site.

[0108] Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0109] Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments contain a pair of Fab fragments generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are known in the art.

[0110] Diabodies are small antibody fragments with two antigen-binding sites, which comprise a VH connected to a VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0111] Linear antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0112] The antibodies and antibody fragments of the present disclosure may be modified within the Fc region to provide desired effector functions or serum half-lives.

[0113] Antibody multimerization can be achieved through spontaneous antibody aggregation or through chemical or recombinant conjugation techniques known in the art. For example, a proportion of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0114] Alternatively, antibody homodimers can be formed through chemical bonding techniques known in the art.For example, heterobifunctional crosslinkers such as, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acylthioacetate) can be used to form antibody multimers.Antibody homodimers can be converted into Fab'2 homodimers through digestion with pepsin.Another method of forming antibody homodimers is by using the self-T15 peptide.

[0115] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be generated by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may comprise at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of a second antibody molecule by replacing the large amino acid side chain(s) with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers. Methods for preparing bispecific antibodies are known in the art.

[0116] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin, while the other can be bound to biotin. Heteroconjugate antibodies can also be produced using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art.

[0117] Methods for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage, starting with proteolytic cleavage of intact antibodies to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenate to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then reconverted to the Fab' thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'TNB derivative to form the bispecific antibody.

[0118] Any of the antibodies or antigen-binding fragments described in this disclosure can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumins, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0119] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. Antibody-drug conjugates comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytocidal or cytostatic agent (e.g., cytochalasin B, gramcidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

[0120] (Antibodies and antigen-binding fragments) The present disclosure provides anti-CitH3 antibodies and antigen-binding fragments thereof. Generally, antibodies (also called immunoglobulins) are composed of two classes of polypeptide chains: light chains and heavy chains. A non-limiting example of an antibody of the present disclosure may be an intact four-immunoglobulin chain antibody, comprising two heavy chains and two light chains. The heavy chain of the antibody may be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a kappa light chain or a lambda light chain. The antibody may be configured to contain two identical copies of the light chain and two identical copies of the heavy chain. The heavy chains each contain one variable domain (or variable region, V H ) and multiple constant domains (or constant regions), which are linked together via disulfide bonds within the constant domains to form the "stem" of the antibody. Each light chain contains one variable domain (or variable region, V L ) and one constant domain (or constant region), each of which is bound to one heavy chain via a disulfide bond. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0121] These hypervariable regions, known as complementarity-determining regions (CDRs), form the loops that comprise the primary antigen-binding surface of an antibody. The four framework regions adopt a primarily beta-sheet structure, with the CDRs forming loops that connect, and in some cases form part of, the beta-sheet structure. The CDRs of each chain are held in close proximity by the framework regions, and the CDRs from the other chain contribute to forming the antigen-binding region.

[0122] The method of identifying the CDR region of an antibody by analyzing the amino acid sequence of the antibody is well known, and several definitions of CDR are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the position of the structural loop region. Unless otherwise specified in this disclosure, the Kabat numbering is used as the default in this disclosure.

[0123] CDR is important for recognizing the epitope of antigen.As used in this disclosure, " epitope " is the smallest part of target molecule that can be specifically bound by the antigen binding domain of antibody.The smallest size of epitope can be about 3, 4, 5, 6 or 7 amino acids, but these amino acids do not need to be in the continuous linear sequence of the primary structure of antigen, because it can depend on the three-dimensional structure of antigen based on the secondary structure and tertiary structure of antigen.

[0124] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art.

[0125] An antibody may also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed in the present disclosure also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to a portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that can specifically bind to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof may be, for example, an scFv, Fv, Fd, dAb, diabody, bispecific antibody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is or is homologous to an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs from either the heavy or light chain of an intact antibody.

[0126] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) described herein fused to a CD3-zeta transmembrane domain and endodomain. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-CitH3, to increase efficacy. Thus, in one aspect, the present disclosure further provides cells (e.g., T cells) expressing the chimeric antigen receptors described herein.

[0127] In some embodiments, the scFv has one heavy chain variable domain and one light chain variable domain.

[0128] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described in this disclosure. Cross-competition assays are known in the art. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described in this disclosure. Epitope binding assays are known in the art.

[0129] (Characteristics of antibodies) In some embodiments, the antibodies or antigen-binding fragments thereof described in this disclosure are CitH3 agonists. In some embodiments, the antibodies or antigen-binding fragments thereof are CitH3 antagonists.

[0130] In some implementations, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than, or 0.0001s -1 Specifically binds to CitH3 (e.g., human CitH3, monkey CitH3, mouse CitH3, and / or chimeric CitH3) with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.0001s -1 Greater than, or 0.00001s -1 It's super.

[0131] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1 × 10 6In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 × 10 7 / Ms is less than.

[0132] Affinity can be estimated from the quotient of the rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 30 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1 × 10 -12 It's over M.

[0133] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human CitH3 (SEQ ID NO: 22), monkey CitH3, and / or mouse CitH3. In some embodiments, the antibody does not bind to human CitH3, monkey CitH3, and / or mouse CitH3.

[0134] In some embodiments, an antibody or antigen-binding fragment thereof described herein is resistant to post-incubation heat treatment at about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes. In some embodiments, heat resistance is determined by measuring binding between the antibody or antigen-binding fragment and an antigen (e.g., CitH3). For example, loss of CitH3 binding compared to an untreated sample indicates heat vulnerability of the antibody or antigen-binding fragment.

[0135] In some embodiments, an antibody or antigen-binding fragment thereof described in the present disclosure has equivalent (e.g., less than about 150%, about 140%, about 130%, about 120%, about 110%, about 100%, about 90%, about 80%, about 70%, about 60%, or about 50%) binding ability to baculovirus compared to a reference antibody (e.g., 7A4E11 or Rituxan).

[0136] In some embodiments, an antibody or antigen-binding fragment thereof (e.g., a humanized anti-CitH3 antibody) described in the present disclosure has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% of the binding ability to CitH3 compared to a chimeric anti-CitH3 antibody described in the present disclosure or a reference antibody (e.g., 7A4E11).

[0137] In some embodiments, an antibody or antigen-binding fragment thereof described in this disclosure can be purified to a purity of at least 90, 99.8%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, or 99.9%.

[0138] In some embodiments, the thermal stability of an antibody or antigen-binding fragment thereof is determined, for example, by differential scanning fluorimetry (DSF). The antibodies or antigen-binding fragments described herein may have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. Because IgG can be described as a multidomain protein, the melting curve may show multiple transitions, with a first denaturation temperature, Tm1, a second denaturation temperature, Tm2, ​​and a third denaturation temperature, Tm3. The presence of these three peaks often indicates the denaturation of the CH2 domain (Tm1), CH3 domain (Tm2), and Fab domain (Tm3), respectively. Thus, in some embodiments, an antibody or antigen-binding fragment described in this disclosure has a Tm1 of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, an antibody or antigen-binding fragment described in this disclosure has a Tm2 of greater than 60, 94, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 95°C. In some embodiments, Tm1 and Tm2 are less than 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 60°C, 94°C, or 95°C.

[0139] In some embodiments, the risk of thermal aggregation of an antibody or antigen-binding fragment thereof is determined, for example, by static light scattering (SLS). Tag is the temperature at which SLS begins to detect aggregation. In some embodiments, tag 266 (for detecting smaller aggregate particles) is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80°C. In some embodiments, tag 473 (for detecting larger aggregate particles) is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80°C. In some embodiments, tag 266 and / or tag 473 is greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80°C.

[0140] In some embodiments, antibody or antigen-binding fragment aggregation is determined, for example, by dynamic light scattering (DLS). In some embodiments, the polydispersity index (PDI) of the antibody or antigen-binding fragment thereof is 0.3 or less, 0.29 or less, 0.28 or less, 0.27 or less, 0.26 or less, 0.25 or less, 0.24 or less, 0.23 or less, 0.22 or less, 0.21 or less, 0.2 or less, or 0.19 or less. In some embodiments, the mode diameter of the antibody or antigen-binding fragment thereof is less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, or less than 50 nm.

[0141] In some embodiments, the antibody has a tumor growth inhibition (TGI%) of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be determined, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after initiation of treatment, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after initiation of treatment. As used in this disclosure, tumor growth inhibition (TGI%) is calculated using the following formula:

[0142] [Number 1] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0143] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day zero. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day zero.

[0144] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, and Fv fragment.

[0145] (Method for producing anti-CitH3 antibodies) Isolated fragments of human CitH3 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected with at least one adjuvant. In some embodiments, the antigenic peptides or proteins may be conjugated to an agent that is immunogenic in the species being immunized. Animals may be injected with the antigenic peptides or proteins multiple times (e.g., two, three, or four times).

[0146] The full-length polypeptide or protein can be used, or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of CitH3 and encompasses an epitope of the protein such that antibodies raised against the peptide form specific immune complexes with the protein. As mentioned above, the N-terminal sequence of human CitH3 is known in the art (SEQ ID NO: 22, CitH3 amino acid N-terminal sequence: ACTKQTACKSTGGKAPCKQLATKAACKSAP).

[0147] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation may contain, for example, a recombinantly expressed or chemically synthesized polypeptide (e.g., a fragment of human CitH3). The preparation may further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0148] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a CitH3 polypeptide or its antigenic peptide (e.g., a portion of CitH3) as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized CitH3 polypeptides or peptides. If necessary, antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A in protein G chromatography, to obtain an IgG fraction. At an appropriate time after immunization, for example, when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as hybridoma or trioma technology. Techniques for producing hybridomas are well known in the art. Monoclonal antibody-producing hybridoma cells can be detected by screening hybridoma culture supernatants for antibodies that bind to the target polypeptide or epitope, for example, using a standard ELISA assay.

[0149] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding the human, humanized, or chimeric antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence that constitutes the antigen-binding site of the antibody or antigen-binding domain. Within a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for a target protein, e.g., CitH3. Any combination of deletion, insertion, and / or combination can be made to arrive at an antibody or antigen-binding fragment with increased binding affinity for the target. Amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications to the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in cells), or introducing new glycosylation sites.

[0150] The antibodies disclosed in this disclosure may be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0151] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences as) human germline immunoglobulin sequences. Human antibodies may be constructed, for example, in the CDRs, to include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0152] Humanized antibodies typically have a human framework (FR) into which nonhuman CDRs have been grafted. Thus, a humanized antibody has one or more amino acid sequences introduced into it from a nonhuman source. These nonhuman amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization can be essentially performed, for example, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, a "humanized" antibody is a chimeric antibody in which substantially less than an intact human V domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically murine antibodies in which some CDR and some FR residues are substituted by residues from analogous sites in human antibodies.

[0153] The selection of human VH and VL domains used to create humanized antibodies is very important to reduce immunogenicity.According to the so-called "best fit" method, the V domain sequence of mouse antibody is screened against the entire library of known human domain sequences.Then, the human sequence that is closest to the mouse sequence is adopted as the human FR of humanized antibody.

[0154] It is further important that antibodies be humanized while retaining high specificity and affinity for the antigen, as well as other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve desired antibody properties, such as increased affinity for one or more target antigens.

[0155] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-CitH3 antibody comprises an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with the sequence present in the light or heavy chain of the original antibody.

[0156] Identity or homology with respect to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to sequences present in a human, humanized, or chimeric anti-CitH3 antibody or fragment, after aligning the sequences and introducing gaps if necessary, and not considering conservative substitutions as part of the sequence identity.

[0157] Additional modifications can be made to the anti-CitH3 antibody or antigen-binding fragment. For example, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus produced can optionally have increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional crosslinkers. Alternatively, antibodies with dual Fc regions can be engineered.

[0158] In some embodiments, anti-CitH3 antibody or its antigen-binding fragment can be covalently modified.These covalent modifications can be produced by chemical synthesis or enzymatic synthesis, or by enzymatic or chemical cleavage.Other types of covalent modifications of antibody or antibody fragment can be introduced into the molecule by reacting the target amino acid residue of antibody or fragment with an organic derivatizing agent that can react with selected side chain or N-terminal or C-terminal residue.

[0159] In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, for example, by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (position 314 in the EU numbering or Kabat numbering of Fc region residues). However, due to minor antibody sequence variations, Asn297 may be located upstream or downstream of position 297, i.e., approximately ±3 amino acids between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody can be further engineered to replace asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0160] (recombinant vector) The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed in this disclosure (e.g., polynucleotides encoding polypeptides disclosed in this disclosure), host cells into which the recombinant vectors are introduced (i.e., such that the host cell contains the polynucleotides and / or vectors containing the polynucleotides), and production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0161] As used in this disclosure, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An expression vector can deliver and express one or more polynucleotides of interest as encoded polypeptides in a host cell into which the "expression vector" is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to regulatory elements, such as a promoter, enhancer, and / or polyA tail, either within the vector or in the genome of the host cell at or near or adjacent to the integration site of the polynucleotide of interest, such that the polynucleotide of interest is translated in a host cell into which it is introduced with the expression vector.

[0162] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors associated with cationic condensing agents (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors.

[0163] In some implementations, a polynucleotide disclosed in the present disclosure (e.g., a polynucleotide encoding a polypeptide disclosed in the present disclosure) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-deficient virus. In the latter case, viral propagation generally occurs only in the complementing virus-packaging cells. Suitable systems are known in the art. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may also be "naked," and uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0164] For expression, a DNA insert containing a polynucleotide encoding an antibody or a polynucleotide encoding a polypeptide disclosed in this disclosure can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to those skilled in the art. The expression construct may further comprise sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct may comprise translation beginning at the translation initiation and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0165] As indicated, expression vectors may also contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK293 cells, and plant cells. Appropriate culture media and conditions for the host cells described in this disclosure are known in the art.

[0166] Non-limiting examples of vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Pharagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting examples of eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0167] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs, such as those of Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0168] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PHH may be used.

[0169] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods described in many standard laboratory manuals and known in the art.

[0170] Transcription of DNA encoding the antibodies of the present disclosure in higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act to increase transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0171] Appropriate secretion signals may be incorporated into the expressed polypeptide for secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. The signals may be endogenous to the polypeptide or may be heterologous signals.

[0172] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and may contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in host cells, during purification, or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before final preparation of the polypeptide. Adding peptide moieties to polypeptides to facilitate secretion or excretion, to improve stability and facilitate purification, is, inter alia, well-known and routine in the art.

[0173] (Treatment method) The antibody(ies) or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes. In one aspect, the present disclosure provides a method of treating an immune dysfunction in a subject, a method of reducing symptoms of an immune dysfunction in a subject, or a method of reducing the risk of developing an immune dysfunction. In some embodiments, the treatment can halt, slow, hinder, or inhibit the progression of the immune dysfunction. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of the immune dysfunction in the subject. In some embodiments, the immune dysfunction is caused by NET activation and release (NETosis). In some embodiments, the methods described in this disclosure can inhibit NETosis.

[0174] In one aspect, the present disclosure provides methods for treating, preventing, or reducing the risk of developing a disorder associated with an aberrant or unwanted immune response, e.g., an immune response caused by an infection or an autoimmune disorder, whereby the use of anti-CitH3 antibodies can improve immune function or reduce nephrosis / pyroptosis-associated tissue damage.

[0175] In some embodiments, these autoimmune disorders include, but are not limited to, alopecia areata, lupus, ankylosing spondylitis, Menneil's disease antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis autoimmune hepatitis, pemphigus vulgaris, Behcet's disease persistent anemia, bullous pemphigoid, nodular polyarthritis, cardiomyopathy, polychondritis, celiac-spur dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary non-gamma globulinemia, and rheumatoid arthritis. These include rheumatic fever, Schallg-Strauss syndrome, primary biliary cirrhosis, buccal pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, gammopathy sarcoidosis, fibromyalgia, scleroderma, Grave's disease, Sjögren's syndrome, Guillain-Barré syndrome, stiff-man syndrome, Hashimoto's thyroiditis / arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type 1), vasculitis, lichen planus, and vitiligo.An anti-CitH3 antibody or antigen-binding fragment thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing a disorder associated with an abnormal or unwanted immune response associated with nephropathy. In some embodiments, the subject has Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the subject has autoimmune thyroid disease, Graves' disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjogren's syndrome, autoimmune nephritis, or systemic lupus erythematosus.

[0176] In some embodiments, the infection is caused by bacteria and / or a virus (e.g., COVID-19). In one aspect, the present disclosure provides methods for treating, preventing, or reducing the risk of developing an infection, or reducing or ameliorating symptoms associated with an infection, e.g., sepsis or septic shock, caused by bacteria and / or a virus.

[0177] The antibody(ies) or antigen-binding fragments thereof of the present disclosure can be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides a method of treating cancer in a subject, reducing the rate of volumetric growth of a tumor in a subject over time, reducing the risk of developing metastases, or reducing the risk of developing further metastases in a subject. In some embodiments, treatment can stop, slow, hinder, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0178] As used in this disclosure, the term "cancer" refers to cells capable of autonomous growth. Examples of such cells include cells with an abnormal state characterized by rapidly proliferating cell proliferation. The term is intended to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems, as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small intestine cancer. "Spontaneous" cancers include any cancer not experimentally induced by the implantation of cancer cells into a subject, including, for example, naturally occurring cancers, cancers caused by patient exposure to carcinogens, cancers caused by the insertion of transgenes or the knockout of tumor suppressor genes, and cancers caused by infection, e.g., viral infection. The term "cancer" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal derivation. The term "hematopoietic neoplastic disorder" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders can arise from the myeloid, lymphoid, or erythroid lineages, or their precursor cells.

[0179] In one aspect, the present disclosure provides a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed in the present disclosure to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer such as breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a malignant hematological disease). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer.

[0180] In one aspect, the present disclosure provides a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed in the present disclosure to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a bacterial or viral infection), or reducing inflammation resulting from a bacterial or viral infection. In some aspects, the compositions and methods disclosed in the present disclosure can be used to treat patients at risk for viral or bacterial infection. Patients with viral or bacterial infections can be identified using various methods known in the art. In one aspect, the present disclosure relates to a method for reducing adverse effects of viral or bacterial infection, such as lung damage, weight loss, and reduced levels of inflammatory markers such as cytokines. The method of treatment comprises administering an effective amount of an antibody or antigen-binding fragment thereof disclosed in the present disclosure, alone or in combination with one or more therapeutic agents or compounds effective for such treatment, such as rhMG53, and others known in the art.

[0181] In some embodiments, the compositions and methods disclosed in this disclosure can be used to treat patients at risk for cancer. Patients with cancer can be identified by various methods known in the art.

[0182] In one aspect, the present disclosure relates to a method of reducing the rate of tumor growth comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or antibody-drug conjugate described in this disclosure. In one aspect, the present disclosure relates to a method of killing tumor cells comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or antibody-drug conjugate described in this disclosure.

[0183] As used in this disclosure, "effective amount" means an amount or dosage sufficient to bring about beneficial or desired results, including halting, slowing, hindering, or inhibiting the progression of a disease, e.g., immunodeficiency and chronic wounds. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of symptoms, and the route of administration, and therefore, dosing may be determined on an individual basis.

[0184] An effective amount can be administered in one or more doses. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to improve, stop, stabilize, reverse, inhibit, delay, and / or delay the progression of a patient's disease. As understood in the art, the effective amount of an antibody or antigen-binding fragment can vary depending on other factors, such as the patient's medical history and the type (and / or dosage) of antibody used, among others.

[0185] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed in the present disclosure may be determined empirically, and making such determinations is within the skill of the art. One of ordinary skill in the art will understand that the dosage that must be administered will vary depending, for example, on the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed in the present disclosure, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed in the present disclosure used, and other agents administered to the mammal. Guidance on selecting appropriate doses for antibodies or antigen-binding fragments can be found in the literature on therapeutic uses of antibodies and antigen-binding fragments.

[0186] A typical daily dose of an effective amount of antibody is between 0.01 mg / kg and 100 mg / kg. In some embodiments, the dosage may be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0187] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition comprising at least one antibody or antigen-binding fragment and a solid oral composition comprising at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0188] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administering at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is a period of overlap in the period of physiological activity of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0189] In some embodiments, the subject may be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described in the disclosure) for an extended period of time (e.g., for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). One of skill in the art may determine the length of the treatment period using any of the methods described in the present disclosure for diagnosing the effectiveness of the treatment or based on the effectiveness of the treatment (e.g., observation of at least one symptom of the disease, e.g., immunodeficiency). As described herein, a competent medical practitioner can also vary the type or number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one of the antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject based on assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0190] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two or more, three or more, or four) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated by any method known in the art.

[0191] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial or antifungal agents, such as benzyl alcohol or methylparabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride); or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. The compositions may be formulated and packaged in ampoules, disposable syringes, or multiple-dose vials. Where necessary (e.g., as injectable formulations), proper fluidity can be maintained by the use of a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which may be comprised of biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza® Corporation and Nova Pharmaceutical, Inc.).

[0192] Compositions comprising one or more of any of the antibodies or antigen-binding fragments described in this disclosure may be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0193] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population) can be measured. The therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0194] Data obtained from cell culture assays and animal studies can be used to formulate an appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described in this disclosure) is an amount that treats a disease in a subject, the subject identified as being at risk for developing the disease, and reduces / shortens the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described in this disclosure can be determined by a medical professional or veterinarian using methods known in the art and by observation of one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and the presence of other diseases).

[0195] Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, or about 1 μg / kg to about 50 μg / kg). While these doses vary widely, those of skill in the art will appreciate that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in potency and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered initially, and the attending medical or veterinary professional (for therapeutic applications) or researcher (if still working in the development phase) can then gradually increase the dose until an appropriate response is obtained. It will further be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the particular compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life of the antibody or antibody fragment in vivo.

[0196] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods of producing the antibodies or antigen-binding fragments thereof for the various uses described in this disclosure. [Example]

[0197] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0198] Example 1: Generation of mouse anti-CitH3 antibody To generate mouse antibodies against human CitH3 (SEQ ID NO: 22), BALB / c mice were immunized with human CitH3 protein. Spleen tissue from the immunized mice was isolated, and the spleen cells were fused with myeloma cells to generate hybridomas. Hybridomas were screened, and those producing high-titer mouse antibodies were subjected to sequencing. Specifically, the lead mouse anti-CitH3 antibody Y1128 was selected for humanization.

[0199] Example 2: Humanization of mouse antibodies The starting point for humanization was the murine antibody Y1128. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of these murine antibodies were determined as SEQ ID NO: 14 (EVQVQQSGAELVRPGALVKLSCKASGFNIKDYYMHWMKQRPEQGLEWIGWIDPENGNTIYDPKFQGKATITSDTSSNTAYLQLNSLTSEDTAVYYCAPFGNYVWFAYWGQGTLVTVSA) and SEQ ID NO: 15 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGITYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFILKISRVEAEDLGLYYCFQGSHVPFTFGSGTKLEIKR), respectively. A chimeric antibody was constructed with the same VH and VL of Y1128, which contains a human heavy chain constant region (eg, CH1, CH2, and CH3 domains) and light chain constant region (eg, CL domain).

[0200] During the humanization process, the murine CDRs were grafted into a human framework acceptor, and residues in the human framework that differed from those in the murine framework were examined. Back mutations from human to murine residues were designed.

[0201] Four humanized heavy chain variable region variants (SEQ ID NOs: 7-10) and three humanized light chain variable region variants (SEQ ID NOs: 11-13) for Y1128 were constructed containing different modifications or substitutions.

[0202] These humanized heavy chain variable region variants can be combined with any of the light chain variable region variants derived from the same mouse antibody. For example, Y1128-VH1 (SEQ ID NO: 7) can be combined with any of the humanized light chain variable region variants based on the same mouse antibody Y1128 (e.g., SEQ ID NOs: 11-13), and the antibody will be labeled accordingly. For example, when Y1128-VH1 is combined with Y1128-VL3 (SEQ ID NO: 13), the antibody will be labeled as Y1128-VH1 / VL3 (or VH1 / VL3).

[0203] Example 3: Initial binding screening of humanized antibody candidates by ELISA The binding affinity of humanized anti-CitH3 antibodies to CitH3 was screened by ELISA. Specifically, a high-binding ELISA plate was coated with 100 μL of test antibody at 1 μg / mL in phosphate-buffered saline (PBS). The plate was covered and incubated overnight at 4°C. The next day, the plate was washed three times with 1×PBS / 0.05% Tween® buffer (0.05% PBST), spun, and the washing cycle was repeated a total of six times. Each subsequent wash was performed in the same manner. After the final wash, the plate was placed on a towel to dry the wells. The ELISA plate was then blocked with 150 μL / well of blocking buffer (1% bovine serum albumin (BSA) in 0.05% PBST) on a plate shaker (covered) for 1 hour at room temperature (RT). After blocking, the plate was washed six times, and 100 μL of each peptide (serial diluted in blocking buffer) was added. The plate was incubated at room temperature for 1 hour on a plate shaker. After incubation, the plate was washed six times, and 100 μL / well of rabbit anti-histone H3 (citrulline R2+R8+R17) secondary antibody (Abcam®, Cambridge, MA, catalog number: ab5103) in blocking buffer was added at a dilution of 1:3000. The plate was then incubated at room temperature for 1 hour on a plate shaker. After incubation, the plate was washed six times, and 100 μL / well of goat anti-rabbit IgG (H&L) HRP secondary antibody (Jackson ImmunoResearch, West Grove, PA, catalog number: 111-035-144) in blocking buffer was added at a dilution of 1:10000. The plate was incubated at room temperature for 1 hour on a plate shaker. After incubation, the plate was washed six times and 100 μL / well of Amplex™ red-developing substrate was added. The plate was kept in a dark drawer for 15 minutes and then analyzed on a plate reader using the following parameters: excitation (Ex) 530, emission (Em) 590, and cutoff 570 nm. The results are shown in the table below and in Figure 1.

[0204] [Table 4]

[0205] As shown in Figure 1, initial screening ELISA results showed strong signals from multiple variants when CitH3 was added at 20,000 pg / ml. Humanized antibodies VH1 / VL3 (VH: SEQ ID NO: 7, VL: SEQ ID NO: 13), VH2 / VL3 (VH: SEQ ID NO: 8, VL: SEQ ID NO: 13), VH2 / VL2 (VH: SEQ ID NO: 8, VL: SEQ ID NO: 12), VH3 / VL3 (VH: SEQ ID NO: 9, VL: SEQ ID NO: 13), and VH3 / VL2 (VH: SEQ ID NO: 9, VL: SEQ ID NO: 12) showed the strongest responses and were selected for subsequent experiments.

[0206] Example 4: Heat treatment and binding of humanized antibody candidates by ELISA The effect of heat treatment of selected anti-CitH3 antibodies was determined by measuring their binding affinity to CitH3. ELISA assays were performed using chimeric (VH: SEQ ID NO: 14, VL: SEQ ID NO: 15) or humanized antibodies to assess their thermal stability.

[0207] Specifically, a high-binding ELISA plate was coated with 100 μL of goat anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog number: 109-005-008) at a concentration of 5 μg / mL in PBS. The plate was covered and incubated overnight at 4°C. The next day, the plate was washed three times with 1×PBS / 0.05% Tween® buffer (0.05% PBST), spun, and the washing cycle was repeated a total of six times. Each subsequent wash was performed in the same manner. After the final wash, the plate was placed on a towel to dry the wells. The ELISA plate was then blocked with 150 μL / well of blocking buffer (1% BSA in 0.05% PBST) on a plate shaker (covered) for 1 hour at room temperature. After blocking, the plate was washed six times, and 100 μL of each diluted antibody was added. Before loading, 50 μL of diluted antibody was heated to 70°C for 5 minutes in a PCR machine. The plate was incubated for 1 hour at room temperature on a plate shaker. After incubation, the plate was washed six times, and 100 μL / well of each peptide (serial dilution) was added. The plate was then incubated for 1 hour at room temperature on a plate shaker. After incubation, the plate was washed six times, and 100 μL / well of rabbit anti-histone H3 (citrulline R2+R8+R17) secondary antibody (Abcam®, catalog number: ab5103) in blocking buffer was added at a dilution ratio of 1:3000. The plate was then incubated for 1 hour at room temperature on a plate shaker. After incubation, the plate was washed six times, and 100 μL / well of goat anti-rabbit IgG (H&L) HRP secondary antibody (Jackson ImmunoResearch, catalog number: 111-035-144) in blocking buffer was added at a dilution ratio of 1:10000. The plate was incubated for 1 hour at room temperature on a plate shaker. After incubation, the plate was washed six times and 100 μL / well of Amplex™ red-developing substrate was added. The plate was kept in a dark drawer for 15 minutes and then analyzed on a plate reader using the following parameters: Ex 530, Em 590, and cutoff 570 nm. The results are shown in the table below and in Figures 2A-2E.

[0208] [Table 5]

[0209] The humanized antibodies VH2 / VL3, VH2 / VL2, and VH3 / VL3 in Figures 2A-2E did not show any loss of CitH3 binding after heat treatment, demonstrating robust binding and thermal stability under heat stress. In contrast, VH1 / VL3 and VH3 / VL2 showed decreased CitH3 binding after heat treatment. As a result, these antibodies were not selected for further experiments.

[0210] Example 5: Nonspecific baculovirus binding of humanized lead candidates by ELISA The nonspecific binding of the selected anti-CitH3 antibodies to baculovirus was determined by ELISA. Specifically, a high-binding ELISA plate was coated with 100 μL of baculovirus diluted 1:500 in PBS. The plate was covered and incubated overnight at 4°C. The next day, the plate was washed three times with 1x PBS / 0.05% Tween® buffer (0.05% PBST), spun, and the washing cycle was repeated a total of six times. Each subsequent wash was performed in the same manner. After the final wash, the plate was placed on a towel to dry the wells. The ELISA plate was then blocked with 150 μL / well of blocking buffer (1% BSA in 0.05% PBST) on a plate shaker (covered) for 1 hour at room temperature. After blocking, the plate was washed six times, and 100 μL of each diluted antibody was added. The plate was then incubated on a plate shaker at room temperature for 1 hour. After incubation, the plate was washed six times, and 100 μL / well of goat anti-human IgG Fc HRP secondary antibody (Jackson ImmunoResearch, Catalog No. 109-035-098) or goat anti-mouse IgG Fc HRP (Jackson ImmunoResearch, Catalog No. 115-035-008) in blocking buffer was added at a 1:5000 dilution ratio. The plate was then incubated for 1 hour at room temperature on a plate shaker. After incubation, the plate was washed six times, and 100 μL / well of Amplex™ red-developing substrate was added. The plate was kept in a dark drawer for 15 minutes and then analyzed on a plate reader using the following parameters: Ex 530, Em 590, and cutoff 570 nm. The results are shown in the table below and in Figure 3.

[0211] [Table 6]

[0212] As shown in Figure 3, the humanized antibodies VH2 / VL3 and VH3 / VL3 showed similar low binding capabilities to baculovirus compared to the negative controls (7A4E11 and Rituxan (clinically approved anti-CD20 monoclonal antibody drug)). This indicated that humanization did not increase their nonspecific binding. However, both the humanized antibody VH2 / VL2 and the chimeric antibody (VH / VL) showed higher binding signals compared to the negative controls, indicating increased nonspecific binding capabilities.

[0213] Example 6: Evaluation of final binding properties of humanized lead candidates by ELISA The binding affinity of the humanized lead anti-CitH3 antibodies to CitH3 was screened by ELISA. The same method as described in Example 3 was used. The results are shown in the table below and in Figure 4.

[0214] [Table 7]

[0215] As shown in Figure 4, the humanized antibody lead VH3 / VL3 showed a higher binding signal compared to 7A4E11, while VH2 / VL3 and VH2 / VL2 showed similar binding signals compared to 7A4E11. Furthermore, the chimeric antibody (VH / VL) showed the lowest binding signal among the antibodies tested. The strong signal from the humanized lead antibody indicates that humanization did not significantly affect binding to CitH3.

[0216] Example 7: Determination of antibody purity by SDS-PAGE The purified antibodies, including the chimeric antibody (VH / VL) and three humanized antibodies (VH2 / VL2, VH2 / VL3, and VH3 / VL3), were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 1 μg of each protein was loaded. As shown in Figure 5, all antibodies showed a single major band, indicating high purity.

[0217] Example 8: Determination of thermal stability by DSF / SLS Immunoglobulin G (IgG) has a multidomain structure, with each domain having its own melting temperature (Tm). For example, the CH2 domain typically has a Tm of approximately 70°C in PBS, while the CH3 domain is more stable at a Tm of approximately 80°C. The fragment antigen-binding region (Fab region) can have a wide Tm range, e.g., approximately 50-85°C, due to sequence variation. Therefore, the Tm values ​​measured by various analytical techniques are typically "apparent" transition temperatures, rather than the actual Tm of each domain. This DSF assay can generate more than one Tm value, but only Tm1 is important when evaluating thermal stability for antibody therapeutics. The Tm value is the temperature at which SLS begins to detect aggregation. Tm 266 measures SLS at 266 nm, which is more sensitive and suitable for detecting smaller aggregate particles. Tm 473 measures SLS at 473 nm, which is better for detecting larger aggregate particles.

[0218] The chimeric antibody (VH / VL) and three humanized antibodies (VH2 / VL2, VH2 / VL3, and VH3 / VL3) were subjected to differential scanning fluorimetry (DSF) and static light scattering (SLS) analysis to determine their thermal stability. Specifically, each sample was submitted to the UNcle system (Unchained Labs) for analysis. For DSF and SLS, a 1.3°C / min temperature ramp was performed while monitoring from 25°C to 95°C. Thermal aggregation was measured at 266 nm and 473 nm by SLS using UNcle. Melting temperatures (TM) and aggregation temperatures (TAG) were calculated and analyzed using UNcle analysis software.

[0219] [Table 8]

[0220] As shown in the summary table above, DSF and SLS results represent the mean and standard deviation of triplicate sample measurements. The DSF / SLS results indicate that all three humanized antibody candidates have similar melting temperatures (Tm1) and thermal aggregation risk compared to the chimeric antibody.

[0221] Example 9: Measurement of aggregation by DLS Dynamic light scattering (DLS) was used to detect aggregation in antibody samples. Specifically, DLS was performed on an UNcle (Unchained Labs) at 25°C immediately prior to DSF / SLS. UNcle analysis software was used for data calculation and analysis.

[0222] [Table 9]

[0223] Typically in DLS, "mode diameter" refers to the protein particle diameter, and "mass fraction" refers to the percentage amount of each particle group. "PDI" refers to the polydispersity index. Typically, the higher this index, the more polydisperse the sample. If the PDI is 0.25 or less, the sample can be considered monodisperse (Unchained Lab technical note). DLS results show the mean and standard deviation (if significant) of triplicate measurements of these samples. Each sample measurement itself is the average of eight consecutive acquisitions.

[0224] As shown in the summary table above, all five samples contained antibodies of typical diameter with slight to undetectable levels of aggregation at room temperature. The humanized antibodies VH3 / VL3 and VH2 / VL3 were monodisperse, while VH2 / VL2 and the chimeric antibodies showed limited polydispersity.

[0225] Example 10: Analysis of heterogeneity by capillary electrophoresis (CE) Purified samples of VH3 / VL3 (with the highest expression level), along with standard IgG, were prepared in reducing and non-reducing labeling buffer before being submitted to CE analysis. "IS" is a 10 kD protein that served as an internal standard. The results of reduced-CE-SDS and non-reduced-CE-SDS are shown in the table below.

[0226] [Table 10]

[0227] [Table 11]

[0228] In the electropherogram of the reduced VH3 / VL3 sample (Figure 6B), the peak at 1.256 relative migration time (RMT) corresponds to the peak at 1.218 RMT in the reduced IgG standard sample (Figure 6A), i.e., derived from the IgG light chain. In the electropherogram of the reduced VH3 / VL3 sample (Figure 6B), the peak at 1.634 RMT corresponds to the peak at 1.580 RMT in the reduced IgG standard sample (Figure 6A), i.e., derived from the IgG heavy chain. No other species were observed in the VH3 / VL3 electropherogram, indicating the high purity of the sample, free of molecular weight (MW) variants.

[0229] In the electropherogram of the non-reduced VH3 / VL3 sample (Figure 6D), the peak observed at 2.396 RMT in the non-reduced VH3 / VL3 electropherogram, which accounts for 98.37% of the total mass and corresponds to the peak at 2.306 in the non-reduced IgG standard sample (Figure 6A), is derived from intact IgG, indicating the high purity of the intact VH3 / VL3 antibody sample. The very small peak in the non-reduced VH3 / VL3 (Figure 6D) at 2.264 RMT (total area 1.6%) corresponds to the peak at 2.208 RMT observed in the non-reduced IgG sample (Figure 6C), i.e., should be derived from an IgG lacking one light chain.

[0230] In conclusion, VH3 / VL3, hereafter hCitH3-mAb, was selected as the final lead antibody based on its desirable characterization and developability analysis results.

[0231] Example 11: Comparative analysis of binding affinity of CAYMAN CitH3-mAb and hCitH3-mAb Four distinct peptides, histone H3 (H3), citrullinated histone H3 (CitH3; 4Cit), acetylated histone H3 (AceH3), and methylated histone H3 (MetH3), at concentrations of 0.5 micrograms each, were run in duplicate through SDS-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Figure 7A). The membrane was divided into two sections for parallel overnight probing with equal amounts of Cayman CitH3-mAb (Ann Arbor, MI) or hCitH3-mAb (2 μg / ml). After three extensive washes, the sections were incubated with HRP-conjugated secondary antibodies specific for mouse IgG and human IgG (Jackson ImmunoResearch Labs, Cat. No. 309-035-008) for 2 hours, respectively. For simultaneous signal development, both membrane sections were exposed simultaneously for the same period of time. Quantitative analysis of signal intensity was performed using Image Studio Lite, and the results are shown as the mean ± SEM of three independent experiments (n = 3 / group). **: P < 0.01 (Figure 7B).

[0232] As expected, hCitH3-mAb has superior binding affinity to the commercially available Cayman CitH3-mAb (Figure 7B). As shown in Figure 7A, hCitH3-mAb maintains high specificity in recognizing CitH3 as an antigen, but not unmodified H3, acetylated H3, or methylated H3. More importantly, hCitH3-mAb contains >100-fold higher binding capacity than the commercially available Cayman CitH3-mAb (Figure 7A-B).

[0233] Example 12: Use of hCitH3-mAb as a therapeutic agent to treat sepsis-induced lung injury and multiple organ failure (method) Mouse studies. Eight- to 12-month-old C57BL / 6J mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Under isoflurane anesthesia, mice were inoculated with 2.5 × 10 of P. aeruginosa for the sterile shock model in accordance with University of Michigan Animal Care and Use Committee protocols. 6 Mice were infected intranasally with colony-forming units (CFU) of the strain PA 19660. For the LPS-induced intraperitoneal toxic shock model, LPS was administered intraperitoneally at 25 mg / kg. For the CLP-induced septic shock model, 75% of the cecum was ligated from the apex and punctured with a 21-gauge needle. After model induction, mice were treated with hCitH3-mAb or IgG. Survival was monitored over 10 days.

[0234] Western Blot—For Western blot analysis, cell and tissue lysates were prepared using RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitors (Sigma-Aldrich®, St. Louis, MO) and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA). Lysates were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Membranes were washed with TBST, blocked with 5% milk in TBST for 1 hour, and probed with specific antibodies as detailed in the figures. Detection was performed using the Pierce™ ECL Plus kit.

[0235] PEdELISA—a pre-equilibrated digital ELISA (PEdELISA)—enables rapid quantification of protein biomarkers, requiring only 10 mL of serum with incubation times of 15–300 seconds across a four-log dynamic range. In our study, plasma samples were collected at 3-h intervals for CitH3 and cytokine level analysis using PEdELISA (Figure 9A–C). The PEd-ELISA microarray platform combines ultrasensitive single-molecule digital counting and transient biosensing for CitH3 quantification in whole blood. This technology provides rapid (<20 min) and sensitive detection of CitH3, enabling real-time monitoring. Its high multiplexing capability (up to 16 complexes) and machine learning-based data analysis make it a valuable tool for early diagnosis of microbial infections, timely administration of therapeutic interventions, and the ability to prevent sepsis-ALI progression.

[0236] Using this platform, elevated CitH3 levels could be detected as early as 3 hours after endotoxic shock. Most notably, administration of hCitH3-mAb effectively blocked CitH3 elevation and the development of cytokine storm. These findings suggest a potential threshold for elevated CitH3 levels that could serve as a diagnostic biomarker for the onset of sepsis, as well as a critical time window for therapeutic intervention.

[0237] Acute lung injury—Histologic analysis of lung tissue was performed according to our previous study. Briefly, lung tissue was embedded in paraffin, sliced ​​into 5 μm sections, and stained with hematoxylin and eosin (H&E). A pathologist blinded to the specimen group assignment evaluated the sections.

[0238] Bacterial load determination - PBS (1 mL) and the homogenized lung and spleen tissue samples were serially diluted 10-fold in sterile PBS. Ten microliters of each sample was plated onto a nutrient agar plate and incubated at 37°C for 15 hours. The number of bacterial colonies was counted from the plate.

[0239] During systemic microbial infection, activated neutrophils undergo an intrinsic cell death process, releasing DNA and cellular proteins, including elastase and histone H3, leading to the formation of "neutrophil extracellular traps (NETs)." Emerging evidence suggests that uncontrolled and excessive NET formation contributes to sepsis-induced acute lung injury (ALI).

[0240] Citrullination of histone H3 (CitH3), catalyzed by peptidylarginine deiminases (PAD2 and PAD4), plays a critical role in initiating NET-induced immune cell death and tissue damage during severe infection. Sepsis-induced acute lung injury (ALI) and multiple organ failure pose a significant threat to public health, often requiring extended hospitalization in intensive care units and resulting in a significant burden on healthcare systems. The complex nature of ALI / ARDS, along with the potential complication of multiple organ failure, makes the management and treatment of ALI patients extremely challenging.

[0241] As shown in Figure 8B, hCitH3-mAb improves survival in mice undergoing LPS-induced septic shock. C57BL6 / J mice were exposed to a lethal LPS dose (25 mg / kg, intraperitoneally) that caused complete death within 24 hours (IgG was used as a control). Tail vein administration of hCitH3-mAb (20 mg / kg, tail vein) significantly improved survival.

[0242] Using the PEd-ELISA microarray platform (Figure 9A-9B), elevated CitH3 levels can be detected as early as 3 hours after endotoxic shock (Figure 9C). Most notably, administration of hCitH3-mAb effectively blocked the elevation of CitH3 and the development of cytokine storm. These findings suggest a potential threshold for elevated CitH3 levels that can serve as a diagnostic biomarker for the onset of sepsis, as well as a critical time window for therapeutic intervention.

[0243] Example 13: hCitH3-mAb (H3L3) improves survival in mice subjected to Pseudomonas aeruginosa (PA)-induced sepsis PA-induced septic shock (2.5 × 10 6 A study was conducted to evaluate the effects of hCitH3-mAb in C57BL6 / J mice (10–12 weeks, male) receiving PA (CFU, intranasal). hCitH3-mAb (20 mg / kg) was delivered to mice via tail vein injection 30 min after PA administration. As a control, mice received a similar treatment with human IgG (Sigma). As shown in Figure 10B, all mice administered human IgG died within 3 days (n = 6), while approximately 80% of mice administered hCitH3-mAb survived (n = 5). For histological evaluation of PA-induced ALI, separate experiments were performed in mice subjected to PA septic shock and sacrificed 24 h after hCitH3-mAb administration (n = 5 for hCitH3-mAb and n = 5 for IgG). Acute lung injury (ALI) scores were assessed using mouse lung sections by a certified pathologist blinded to the animal experiments (Figure 11A). As shown in Figures 11A and 11B, mice administered hCitH3-mAb showed a significant reduction in lung injury.

[0244] Example 14: Humanized CitH3 monoclonal antibody (hCitH3-mAb) improves survival in young mice subjected to CLP sepsis Figure 12B shows the survival curves of C57BL / 6 mice (12 weeks old) treated with or without hCitH3-mAb in a mouse model of lethal colonic puncture (CLP)-induced sepsis. C57BL / 6 mice received a single dose of hCitH3-mAb (20 mg / kg body weight) or human IgG (20 mg / kg body weight) 0.5 hours before CLP. Survival rates were monitored for 10 days (n = 7 per group). The results showed that hCitH3-mAb significantly improved mouse survival compared with the IgG group.

[0245] Example 15: Evaluation of the role of hCitH3-mAb in bacterial clearance during Pseudomonas aeruginosa-induced septic shock Figure 13 shows the 2.5x10 6Evaluation of bacterial burden in the lungs and spleen of mice inoculated intranasally with P. aeruginosa cells and then administered a tail vein injection of hCitH3-mAb (20 mg / kg) or human IgG (20 mg / kg) is shown. After infection, lung and spleen tissues were harvested and homogenized for bacterial burden assessment by culturing on LB agar plates.

[0246] Example 16: hCitH3-mAb can be used to treat septic multi-organ injury in aging (method) Aged C57BL / 6J mice (male, 24-26 months) were obtained from the National Institute on Aging. Mice were anesthetized with isoflurane according to a protocol approved by the University of Virginia IACUC. Mouse sepsis was performed using a colonic puncture (CLP) model. The cecum of the mouse was isolated from the abdominal cavity with sterile forceps, ligated with 4-0 silk sutures, and punctured using a 20G needle at the midpoint between the tail end and the ligated set.

[0247] Mice were divided into four groups: CLP, CLP treated with rhMG53, CLP treated with hCitH3-mAb, and CLP treated with rhMG53 + hCitH3-mAb. hCitH3-mAb was administered via tail vein injection at a dose of 20 mg / kg, and recombinant human MG53 (rhMG53) protein was administered via intraperitoneal injection at a dose of 5 mg / kg after CLP.

[0248] The results showed that hCitH3-mAb treatment improved the survival of aged mice subjected to CLP-sepsis (Figure 14). Furthermore, cotreatment with hCitH3-mAb and rhMG53 synergistically improved the survival of aged mice after CLP.

[0249] Twenty-four hours after CLP, lung tissues were collected from aged C57BL6 mice (25 months old). WB was performed using an anti-IL-6 antibody (Cat. No. 12912T, Cell Signaling Technology®, Danvers, MA). Referring to Figure 15, CLP induced an increase in IL-6 in aged mice compared with sham controls. Administration of rhMG53 (5 mg / kg, intraperitoneal injection) or hCitH3-mAb (20 mg / kg, tail vein injection) attenuated the increase in IL-6. Co-administration of rhMG53 and hCitH3-mAb almost completely abolished the increase in IL-6.

[0250] Example 17: hCitH3-mAb can be used to treat virus-induced multi-organ damage (method) Virus Stock—Influenza virus strain A / Puerto Rico / 8 / 34 (H1N1) (PR8, kindly provided by Dr. Jacob Yount, Ohio State University) was propagated in 10-day-old embryonated chicken eggs (Charles River Laboratories, Wilmington, MA) and titered as previously described.

[0251] Mouse Study - Aged male C57BL / 6J mice (22-24 months old) were obtained from the National Institute on Aging. Mice were infected intranasally under anesthesia with isoflurane according to a protocol approved by the University of Virginia Animal Care and Use Committee. Mice were infected with PR8 at a dose of 150 TCID50. Two doses of humanized CitH3 antibody (hCitH3-mAb) were administered via tail vein injection at a dose of 20 mg / kg 24 and 72 hours after influenza virus infection. rhMG53 protein was administered via tail vein injection at a dose of 2 mg / kg daily for 7 days. The first dose of rhMG53 was administered 24 hours after influenza infection. Mouse weights were monitored and recorded daily. Weight loss of more than 20% was the primary criterion used for human endpoint euthanasia in mouse experiments. To determine lung cytokine levels, tissues were harvested, homogenized in 1 ml of PBS, snap-frozen, and stored at -80°C prior to analysis via ELISA. Mouse IFNβ and IL-6 ELISAs were performed on supernatants from lung homogenate supernatants using the respective R&D Systems® Duoset® ELISA kits (Minneapolis, MN, catalog numbers DY8234-05 and DY406, respectively) according to the manufacturer's instructions. Lung tissue samples were fixed in 10% neutral buffered formalin for 24 hours at 4°C and then transferred to 70% ethanol. Lungs were embedded in paraffin, sectioned, stained with H&E, and imaged via a Leica DMi8 widefield microscope. Plasma samples were collected at the time of animal euthanasia and subjected to multiplex assays of mouse cytokines (GM-CSF, IFNγ, IL13, and LIX) at the core facility for flow cytometry at the University of Virginia Medical Center.

[0252] Western Blot—For Western blot analysis, snap-frozen lung tissue was processed in radioimmunoprecipitation assay (RIPA) lysis buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% SDS, and 0.5% deoxycholate) supplemented with a cocktail of protease inhibitors (Sigma-Aldrich®) and phosphatase inhibitors (Thermo Fisher Scientific). Mouse lung lysates were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were washed with Tris-buffered saline with Tween 20 (TBST), blocked with 5% milk in TBST for 1 hour, and incubated with different antibodies as indicated. Immunoblots were visualized using the Pierce™ ECL Plus kit (Pierce).

[0253] The change in body weight (% of initial body weight) was plotted for animals infected with influenza virus and treated with rhMG53 (Figure 16A) or hCitH3-mAb (Figure 16B) compared with saline-treated animals as a control.

[0254] Representative microscopic images of H&E-stained lung tissue sections from mice infected with influenza virus and treated with saline, rhMG53, or hCitH3-mAb are shown in Figures 17A-17D. Quantitative analysis of cellulose in the tissue sections was performed using ImageJ software. Reduced cellular infiltration was observed in lung tissues collected from animals 9 days after influenza virus infection and treated with rhMG53 or hCitH3-mAb compared with animals treated with saline (n = 3 per group, p-value calculated using one-way ANOVA with Tukey's multiple comparison test).

[0255] Figures 18A and 18B show the results of ELISA quantification of IFNβ and IL-6 in lung tissues collected from mice without influenza virus infection, or mice infected with influenza virus for 9 days and treated with saline, rhMG53, or hhCitH3-mAb (n = 3 per group, p values ​​calculated using one-way ANOVA with Tukey's multiple comparison test). The results demonstrated that administration of hCitH3-mAb, like rhMG53, significantly reduced the cytokine storm in lung tissues after virus infection.

[0256] Multiplex mouse cytokine assays showed increased plasma levels of inflammatory markers, including GM-CSF, IFNγ, and LIX, in mice with influenza virus infection compared with uninfected control animals, which were significantly reduced in animals treated with rhMG53 or hCitH3-mAb (n = 3 per group, p values ​​calculated using one-way ANOVA with Tukey's multiple comparison test) (Figures 19A-C).

[0257] Western blot analysis showed that treatment with rhMG53 or hCitH3-mAb suppressed inflammation, as indicated by reduced levels of NLRP3, MDA-5, and IFITM3 after 9 days of influenza virus infection (Figure 20). Furthermore, treatment with rhMG53 or hCitH3-mAb suppressed apoptosis, as indicated by reduced levels of cleaved caspase 2 / 3 / 11 and PARP (poly(ADP-ribose) polymerase) after 9 days of influenza virus infection (Figure 21). Suppression of full-length GSDMD expression and reduced levels of cleaved GSDMD were also observed (Figure 22). Because GSDMD cleavage is a known trigger of pyroptotic tissue damage, the attenuation of GSDMD cleavage by hCitH3-mAb supports the beneficial effect of hCitH3-mAb in protecting against virus-induced lung injury.

[0258] Example 18: hCitH3-mAb can be used to treat sterile-inflammation-induced multi-organ injury. (method) Animals - Male wild-type mice (C57BL / 6, 10-12 weeks old) were purchased from Jackson Laboratory. Animal handling and surgical procedures were performed according to protocols approved by the Institutional Animal Care and Use Community (IACUC) at the University of Virginia, in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

[0259] Hepatic ischemia - Twelve-week-old male mice were used for partial hepatic warm ischemia surgery. All surgical tools were sterilized by autoclaving before surgery. Mice were anesthetized in an induction chamber containing 5% isoflurane in 100% oxygen at a flow rate of 1 L / min. Mice were then placed supine on a warming pad on a nose cone connected to continuous oxygen with 5% isoflurane. Body temperature was monitored with a rectal temperature probe and maintained at 37°C with a heating pad. Partial hepatic (70%) warm ischemia was used. Ischemia was initiated by depilation of the abdomen and three washes with betadine. Under a dissecting microscope, the hepatic hilum was removed free of surrounding tissue. All structures of the portal triad (hepatic artery, portal vein, and bile duct) to the left and middle liver lobes were occluded with microvascular clamps for 90 minutes, followed by removal of the clamps to initiate reperfusion. Ischemia was confirmed by visualization of a pale slope in the ischemic lobe. Evidence of reperfusion was based on an immediate color change in the ischemic lobe. The abdominal incision was then closed using 5-0 polypropylene sutures. Animals were sacrificed at 0.5, 6, 24, and 72 hours after reperfusion for collection of serum, liver, heart, lungs, kidneys, and spleen.

[0260] Antibody treatment—One group of mice received hCitH3-mAb, and another group of mice received human IgG antibody (Sigma-Aldrich®, I4506) via IV injection at 20 mg antibody per kg mouse immediately after reperfusion and on day 3 after surgery.

[0261] Echocardiography - For echocardiography, mice underwent baseline echocardiography followed by hepatic ischemia-reperfusion surgery. All surviving mice were then subjected to follow-up echocardiography on days 7 and 14 after surgery.

[0262] Western blot analysis: Total protein was extracted from flash-frozen liver tissue using protein lysis buffer containing protease inhibitors. Denatured proteins were resolved on a 4-12% gradient gel (Invitrogen™, NP0336BOX), and the separated proteins were transferred to a PVDF membrane. The membrane was stained with Ponceau S solution to demonstrate equal protein loading for each sample. The membranes were further probed with primary antibodies and incubated overnight at 4°C. They were then washed with TBST and probed with secondary antibodies. Protein expression was detected by an Invitrogen Imaging system using a chemiluminescent substrate (Thermo Scientific™, PI34580). The antibodies used were: MPO (R&D Systems™, AF3667), GAPDH (Cell Signaling Technology™, 2118S). For serum samples, 1 μl of serum was loaded onto SDS-PAGE for Western blot analysis.

[0263] In addition to in vivo studies with hCitH3-mAb using mouse models of sepsis and virus-induced multiple organ failure, a surprising and intriguing finding is that hCitH3-mAb is effective in treating sterile inflammation-induced tissue damage.

[0264] Experiments using ischemia-reperfusion-induced liver injury were conducted, and the results showed that hCitH3-mAb has significant benefits in treating sterile inflammation-induced liver injury. More surprisingly, ischemia-reperfusion-induced liver injury can propagate to myocardial infarction, as reflected by a decrease in the ejection fraction (EF) of the mouse heart. Interestingly, administration of hCitH3-mAb can attenuate the decrease in EF in mice subjected to ischemia-reperfusion-induced liver injury.

[0265] Western blot analysis showed that expression of MPO, a neutrophil marker, was observed in ischemia-reperfusion-induced liver injury with upregulation detected from 6 hours to 72 hours after reperfusion (Figure 23), and expression of CitH3 was increased in ischemia-reperfusion-induced liver injury at 24 hours after reperfusion and continued to increase up to 72 hours (Figure 24).

[0266] The results showed that hCitH3-mAb treatment eliminated serum CitH3 levels (Figure 25A). Mice that underwent hepatic ischemia and reperfusion were intravenously injected with human IgG antibody or hCitH3-mAb immediately after reperfusion. CitH3 levels were measured in serum samples collected before surgery and on days 1, 3, and 7 after surgery. (Figure 25B) shows Ponceau S staining of the loading control.

[0267] FIG. 26 shows that neutralization of CitH3 with hCitH3-mAb restores cardiac function after liver I / R, while the group treated with human IgG antibody shows impaired cardiac function.

[0268] These findings support the broader value of hCitH3-mAb for treating multi-organ injury under conditions of sterile inflammation.

[0269] Example 19: hCitH3-mAb protects against ischemic stroke in mice (method) C57BL / 6J mice (male, 16-20 weeks old, weighing 25-30 g) were obtained from the Jackson Lab. Mice were anesthetized using isoflurane according to a protocol approved by the University of Virginia IACUC. A commercially available silicone-coated monofilament (Doccol Corporation, Sharon, MA) was introduced into the medial carotid artery (ICA) via transection of the lateral carotid artery (ECA) until the monofilament occluded the base of the MCA. Cerebral ischemia-reperfusion was established for 60 min using transient middle cerebral artery occlusion (tMCAO) according to our previous study.

[0270] Mice were divided into two groups: (i) a tMCAO group treated with IgG as a control, and (ii) a tMCAO group treated with hCitH3-mAb. IgG and hCitH3-mAb were administered via tail vein injection at a dose of 20 mg / kg immediately after reperfusion. At 24 h after reperfusion, whole brain tissue was collected and frozen at -20°C for 10 min upon animal euthanasia. Then, the tissue was sliced ​​into five serial 2 mm coronal sections, incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich®) at 37°C for 20 min, gently rotated every 5 min, and fixed in 4% paraformaldehyde in phosphate buffer at 4°C. White tissue indicated inflamed areas, while red tissue indicated normal areas. The extent of ischemic myocardial infarction was tracked, and the integrated volume was calculated using Image J software. The results are shown in Figures 27A-B.

[0271] Example 20: hCitH3-mAb can be used as a therapeutic agent to treat diabetes and diabetic wounds. (method) 14-week-old B6.BKS(D)-Lepr db db / db mice were purchased from the Jackson Laboratory (Bar Harbor, ME). After anesthesia with 1.5% isoflurane, the mice's dorsal hair was removed using depilatory cream (Nair), and a 5 mm round, full-thickness excision wound was created at the midline of each mouse, 2 cm caudal to the skull. hCitH3-mAb (20 mg / kg) and human IgG (Sigma-Aldrich, I4506; 20 mg / kg) were injected subcutaneously on days 0 and 3 after injury. The wound was covered with a hydrocolloid dressing (Tegaderm™; 3M Health Care, St. Paul, MN) to maintain a moist environment and was changed daily. Wound area was measured on days 2, 4, 7, 10, and 12 after injury. Data demonstrated the benefit of hCitH3-mAb in improving skin wound healing in db / db mice (n = 3 for each).

[0272] Wound healing is impaired in diabetes, and diabetic foot ulcers (DFUs) present a significant risk of morbidity and mortality. A combination of neuropathy and vasculopathy contributes to DFUs, but the underlying cellular and molecular mechanisms that impair tissue healing in diabetes remain poorly understood. This lack of understanding limits therapeutic strategies beyond glucose control, revascularization, and conventional wound care.

[0273] NETs are activated by pro-inflammatory cytokines and reactive oxygen species, particularly in diabetes, a condition known for its chronic inflammation and oxidative stress. Citrullination of histone H3 (CitH3), catalyzed by peptidylarginine deiminases (PAD2 and PAD4), plays a key role in initiating NET-induced immune cell death and tissue damage. Elevated CitH3 has been identified as a risk factor for impaired wound healing and amputation in patients with DFUs.

[0274] FIG. 28 shows that hCitH3-mAb enhances wound healing in diabetic mice, and the combination of hCitH3-mAb and rhMG53 adds synergistic effects for treating diabetic ulcers.

[0275] Previously, a hydrogel formulation of recombinant human MG53 (rhMG53) protein was shown to be effective in treating skin wounds in db / db mice. We demonstrate that cotreatment with hCitH3-mAb and rhMG53 may have a synergistic effect to control inflammation (via hCitH3-mAb) and improve healing (via rhMG53), thus effectively treating diabetic ulcers (Figures 29A-29C). The composition of ROSS-A6 hydrogels for encapsulation of rhMG53 protein for wound healing applications was previously described. Sustained delivery of rhMG53 was previously shown to promote diabetic wound healing and hair follicle development. Surprisingly, our results indicate that the combination of hCitH3-mAb and rhMG53 may provide a synergistic effect for enhancing the treatment of diabetic ulcers, possibly by complementing the functions of rhMG53 in promoting wound healing and hCitH3-mAb in attenuating diabetes-associated nephropathy dysfunction.

[0276] <Other embodiments> While the present invention has been described in conjunction with its detailed description, it will be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CitH3 (citrullinated histone H3), a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence; The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, and 6, respectively; and (2) An antibody or antigen-binding fragment thereof, wherein the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, and 21, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which specifically binds to human CitH3.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a humanized antibody or antigen-binding fragment thereof.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

7. A nucleic acid comprising a polynucleotide encoding a polypeptide, (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3, respectively, wherein the VH binds to CitH3 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NOs: 11, 12, 13, or 15; or (2) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, wherein the VL binds to CitH3 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 7, 8, 9, 10, or 14. A nucleic acid comprising an immunoglobulin light chain or a fragment thereof.

8. The nucleic acid of claim 7, comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.

9. The nucleic acid of claim 7, comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

10. 10. The nucleic acid of any one of claims 7 to 9, wherein the VH when paired with the VL specifically binds to human CitH3, or the VL when paired with the VH specifically binds to human CitH3.

11. The nucleic acid of any one of claims 7 to 10, wherein the immunoglobulin heavy chain or fragment thereof is a humanized immunoglobulin heavy chain or fragment thereof and the immunoglobulin light chain or fragment thereof is a humanized immunoglobulin light chain or fragment thereof.

12. 12. The nucleic acid of any one of claims 7 to 11, encoding a single chain variable fragment (scFv) or a multispecific antibody (e.g. a bispecific antibody).

13. The nucleic acid according to any one of claims 7 to 12, which is a cDNA.

14. A vector comprising one or more of the nucleic acids according to any one of claims 7 to 13.

15. A vector comprising two of the nucleic acids according to any one of claims 7 to 13, encoding the VL and VH regions which together bind to CitH3.

16. A pair of vectors, each vector comprising one of the nucleic acids of any one of claims 7 to 13, and the pair of vectors together encoding the VL and VH regions that together bind to CitH3.

17. A cell comprising a vector according to claim 14 or 15, or a pair of vectors according to claim 16.

18. 18. The cell of claim 17, which is a CHO cell.

19. A cell comprising one or more of the nucleic acids according to any one of claims 7 to 13.

20. A cell comprising two of the nucleic acids according to any one of claims 7 to 13.

21. The cell of claim 20, wherein the two nucleic acids together encode the VL region and the VH region that together bind to CitH3.

22. 1. A manufacturing method for producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing the cell of any one of claims 17 to 21 under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; (b) harvesting the antibody or antigen-binding fragment produced by the cell.

23. An antibody or antigen-binding fragment thereof that binds to CitH3, An antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, or 100% identical to SEQ ID NO: 7, 8, 9, 10, or 14, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, or 100% identical to SEQ ID NO: 11, 12, 13, or 15.

24. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 7 and the VL comprises the sequence of SEQ ID NO:

11.

25. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 7 and the VL comprises the sequence of SEQ ID NO:

12.

26. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 7 and the VL comprises the sequence of SEQ ID NO:

13.

27. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 8 and the VL comprises the sequence of SEQ ID NO:

11.

28. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 8 and the VL comprises the sequence of SEQ ID NO:

12.

29. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 8 and the VL comprises the sequence of SEQ ID NO:

13.

30. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 9 and the VL comprises the sequence of SEQ ID NO:

11.

31. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 9 and the VL comprises the sequence of SEQ ID NO:

12.

32. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 9 and the VL comprises the sequence of SEQ ID NO:

13.

33. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 10 and the VL comprises the sequence of SEQ ID NO:

11.

34. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 10 and the VL comprises the sequence of SEQ ID NO:

12.

35. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 10 and the VL comprises the sequence of SEQ ID NO:

13.

36. The antibody or antigen-binding fragment thereof of claim 23, wherein the VH comprises the sequence of SEQ ID NO: 14 and the VL comprises the sequence of SEQ ID NO:

15.

37. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 36, which specifically binds to human CitH3.

38. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 37, which is a humanized antibody or antigen-binding fragment thereof.

39. 39. The antibody or antigen-binding fragment thereof of any one of claims 23 to 38, which is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

40. An antibody or antigen-binding fragment thereof that binds to CitH3, a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 7, 8, 9, 10, or 14; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 11, 12, 13, or 15.

41. An antibody or antigen-binding fragment thereof that binds to CitH3, a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected antibody or antigen-binding fragment thereof; a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3 that are identical to the VL CDR1, a VL CDR2, and a VL CDR3 of the selected antibody or antigen-binding fragment thereof; The selected antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof according to any one of claims 23 to 40.

42. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 23 to 41.

43. 43. A method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and 23 to 42.

44. 44. The method of claim 43, wherein the subject has a solid tumor or a hematological malignancy.

45. 44. The method of claim 43, wherein the cancer is associated with CitH3-induced nephrosis.

46. 43. A method of treating a subject having an immune disorder (e.g., an autoimmune disease), comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6 and 23-42.

47. 47. The method of claim 46, wherein the immune disorder is associated with nephrosis.

48. 43. A method of treating a subject having an infectious disease, comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and 23 to 42.

49. 49. The method of claim 48, wherein the infection is caused by bacteria and / or viruses, such as sepsis.

50. A method for inhibiting CitH3-nephrosis in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 6 and 23 to 42.

51. 51. The method of claim 50, wherein the CitH3-nephrosis is caused by infection, sepsis, cancer, a chronic wound, ischemia, or an autoimmune disorder.

52. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and 23 to 42, and a pharmaceutically acceptable carrier.

53. 52. The method of claim 51, wherein the chronic wound is a skin wound.

54. 54. The method of claim 53, wherein the skin wound is a diabetic foot ulcer.

55. 52. The method of claim 51, wherein the sepsis results in lung injury.

56. 52. The method of claim 51, wherein the ischemia is hepatic ischemia, cerebral ischemia, or cardiac ischemia.

57. 52. The method of claim 51, further comprising the co-administration of another therapeutic compound, said compound being rhMG53.

Images