Humanized mAb107

Abstract

Described herein are humanized antibodies that bind with high affinity to the leukocyte integrin CD11b / CD18 (CD11b, αMβ2, CR3), and methods of their use.

Description

[Technical field]

[0001] Claiming priority This application claims priority to U.S. Provisional Patent Application No. 63 / 166,711, filed March 26, 2021. The entire contents of the foregoing are incorporated herein by reference.

[0002] Federally Sponsored Research or Development This invention was made with Government support under Grant No. DK48549 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Description of sequence listing This specification contains a sequence listing that has been submitted electronically as an ASCII text file entitled "sequencelisting." The ASCII text file was created on Mar. 24, 2022 and is 21.4 kilobytes in size. The material in the ASCII text file is incorporated herein by reference in its entirety.

[0004] Described herein are humanized antibodies that bind with high affinity to the leukocyte integrin CD11b / CD18 (CD11b, αMβ2, CR3), and methods of their use. [Background technology]

[0005] Leukocyte integrin CD11b / CD18 (also known as CD11b, αMβ2, CD3, herein referred to as CD11b) is a typical innate immune receptor expressed on innate immune cells. CD11b binds more than 40 ligands, thus mediating leukocyte adhesion-dependent functions, including homing, phagocytosis, adhesion-dependent superoxide generation, protease and cytokine production, antibody-dependent cytolytic activity, and promoting adaptive immunity (Arnaout, F1000Res. 2016 Oct 4;5:F1000 Faculty Rev-2433; van den Elsen et al., Science (2011) 332:608-1; Bajic et al., Proc Natl Acad Sci USA. (2013) 110:16426-31). Binding of physiological ligands such as ICAM-1, complement iC3b and fibrinogen to CD11b induces activation of tertiary and quaternary changes of CD11b leading to proadhesive cell signaling. Here, we establish that current anti-integrin agents that stabilize this activated proinflammatory state can cause significant adverse events in humans that were not predicted in rodent studies (Raab-Westphal et al., Cancers (Basel). 2017 Aug 23;9(9):110). Summary of the Invention

[0006] Provided herein are antibodies or antigen-binding fragments thereof that bind to CD11b, and methods of use thereof. In some embodiments, the antibodies or antigen-binding fragments thereof have the following complementarity determining regions (CDRs): 1) CDR1 of the VH of SEQ ID NO: 1, e.g., GFNIKD (SEQ ID NO: 5); 2) CDR1 of the VL of SEQ ID NO:2, e.g., SQNLLYSSNQKNY (SEQ ID NO:8); 3) CDR2 of the VH of SEQ ID NO: 1, for example, PADDKTK (SEQ ID NO: 6); 4) CDR2 of the VL of SEQ ID NO:2, e.g., WASTRESGVPDR (SEQ ID NO:9); 5) CDR3 of the VH of SEQ ID NO: 1, for example, GHYGYDGYA (SEQ ID NO: 7); and 6) CDR3 of the VL of SEQ ID NO: 2, for example, YYSYPL (SEQ ID NO: 10) The amino acid sequence comprises:

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising VH CDR1, 2, 3, and a light chain variable region comprising VL CDR1, 2, 3, wherein VH CDR1, 2, 3 are identical to the complementarity determining regions of SEQ ID NO:1, and VL CDR1, 2, 3 are identical to the complementarity determining regions of SEQ ID NO:2.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0009] [ka] (SEQ ID NO:3)

[0010] [ka] (Heavy chain, SEQ ID NO: 17)

[0011] [ka] (heavy chain, SEQ ID NO: 13), and

[0012] [ka] (Light chain, SEQ ID NO:4) and wherein the complementarity determining regions of said sequence are unaltered.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a single chain variable fragment (scFv). In some embodiments, the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 15.

[0014] Further provided herein is a composition comprising an antibody or antigen-binding fragment thereof described herein and a pharma- ceutically acceptable carrier.

[0015] Also provided herein is a method for ameliorating a condition associated with ischemia-reperfusion injury in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0016] In some embodiments, the condition is post-ischemic renal fibrosis.

[0017] In some embodiments, the condition is a renal fibroinflammatory disease.

[0018] In some embodiments, the condition is pulmonary fibrosis.

[0019] In some embodiments, the condition is post-myocardial infarction left ventricular adverse remodeling.

[0020] In some embodiments, the composition is administered to the subject within about 5 hours after ischemia-reperfusion injury.

[0021] In some embodiments, the composition is administered to the subject within about 2 hours after ischemia-reperfusion injury.

[0022] Also provided herein is a method for treating a subject having or at risk of developing a disorder associated with ischemia-reperfusion injury in an organ, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0023] In some embodiments, the organ is a kidney, hi some embodiments, the injury is acute kidney injury.

[0024] In some embodiments, the organ is the heart. In some embodiments, the disorder is an acute coronary syndrome. In some embodiments, the disorder is an acute myocardial infarction (MI).

[0025] In some embodiments, the organ is a lung.

[0026] In some embodiments, the composition is administered to the subject within about 5 hours after ischemia-reperfusion injury.

[0027] In some embodiments, the composition is administered to the subject within about 2 hours after ischemia-reperfusion injury.

[0028] Further provided herein is a method for providing an organ for transplantation, comprising administering to an organ donor a composition comprising an antibody or antigen-binding fragment thereof described herein; and removing the organ from the organ donor.

[0029] In some embodiments, the organ is a kidney, heart, or lung.

[0030] Also provided herein is a method for reducing delayed organ graft function following organ transplantation, comprising administering to an organ recipient a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein prior to transplantation of the organ into the recipient or within one day of transplantation, thereby reducing delayed organ graft function following organ transplantation.

[0031] In some embodiments, the organ is a kidney, heart, or lung.

[0032] Further provided herein is a method for treating an organ prior to transplantation into a recipient, the method comprising contacting the organ with a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0033] In some embodiments, the organ is a kidney, heart, or lung.

[0034] In some embodiments, the contacting step comprises perfusing the organ with a composition comprising a polypeptide or an antibody that immunospecifically binds to an epitope recognized by mab107.

[0035] Further provided herein is a method for treating a subject having an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0036] In some embodiments, the autoimmune disease is cytoplasmic antineutrophil cytoplasmic antibody (cANCA)-associated vasculitis.

[0037] Further provided herein is a method for treating a subject having diabetic nephropathy, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0038] Also provided herein is a method for ameliorating a condition associated with chemotherapy in a subject, 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 condition is adriamycin nephropathy.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in this invention; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are for illustration 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, controls.

[0040] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0041] [Figure 1] FIG. 13 shows Coomassie staining of SDS-PAGE showing the migration of murine mAb107 and humanized IgG4κ mAb107 mutated to carry a new Asn257 / Asp substitution in the CDR2 heavy chain under non-reducing (left panel) and reducing (right panel) conditions. [Diagram 2] Coomassie stain of SDS-PAGE showing migration of humanized IgG4κ mAb107 containing wild type Ser228(S) or Pro228(P) in the IgG4 core hinge region under reducing (left panel) or non-reducing (right panel) conditions. This substitution reduced the band corresponding to the known Fab arm exchange, which resulted in a functionally monovalent and bispecific antibody with unknown specificity and therefore potentially reduced therapeutic efficiency (Silva et al., J Biol Chem. 2015 Feb 27;290(9):5462-9). Molecular weight standards (in kDa) are shown in one lane of the stained gel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The following paragraphs provide certain definitions to provide a clearer and more consistent understanding of the specification and claims.

[0043] About: The term "about" when used herein with reference to a value refers to the same value in the context of the referenced value. In general, a person skilled in the art familiar with the context will recognize the appropriate degree of difference encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% of the referenced value.

[0044] Administered / Administered: As used herein, the term "administration" typically refers to administration of a composition to a subject or a system for achieving delivery of an agent that is or is contained in a composition. Those skilled in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (e.g., may include one or more of topical to the dermis, intradermal, subcutaneous, transdermal, etc.), intestinal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a particular organ (e.g., in the liver), mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), intravaginal, vitreous, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion) for at least a selected period of time.

[0045] Affinity: As known in the art, "affinity" is a measure of how strongly a particular ligand binds to its partner. Affinity can be measured in a variety of ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the binding partner concentration can be fixed higher than the ligand concentration to mimic physiological conditions. Alternatively, or in addition, in some embodiments, the binding partner concentration and / or the ligand concentration can be different. In some such embodiments, affinity can be compared to a reference under comparable conditions (e.g., concentrations).

[0046] Animal: As used herein, refers to each species of the animal kingdom. In some embodiments, "animal" refers to humans of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0047] Binding: The term "binding", as used herein, will be understood to refer to a non-covalent bond between or among two or more entities. "Direct" binding involves physical contact between the entities or moieties; direct binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including all those in which the interacting entities or moieties are studied in isolation or in the context of a more complex system (e.g., while covalently or otherwise bound to a carrier entity and / or in a biological system or cell).

[0048] CDR: as used herein, refers to a complementarity determining region in an antibody variable region. There are three CDRs in each of the heavy and light chain variable regions, and they are called CDR1, CDR2 and CDR3 for each variable region. A "set of CDRs" or "CDR set" refers to a group of three or six CDRs occurring in either a single variable region capable of binding to an antigen or the CDRs of cognate heavy and light chain variable regions capable of binding to an antigen. Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.); one skilled in the art can recognize the differences between these systems and understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0049] Epitope: as used herein includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a suitable three-dimensional conformation. In some embodiments, such chemical atoms or groups are spatially and physically close to each other when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative (e.g., linearized) conformation.

[0050] Humanized: As known in the art, the term "humanized" generally refers to a method for modifying a V antibody whose amino acid sequence is derived from a reference antibody that originated in a non-human species (e.g., mouse). H and V LThe term "humanized" refers to an antibody (or antibody component) that contains a variable region sequence, but also includes modifications intended to make the sequence associated with a reference antibody more "human-like", i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is one that immunospecifically binds to an antigen of interest and has framework regions (FR) that have substantially the same amino acid sequence as a human antibody, and complementarity determining regions (CDRs) that have substantially the same amino acid sequence as a non-human antibody. A humanized antibody contains substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions and all or substantially all of the framework regions that correspond to those of a non-human immunoglobulin (i.e., donor immunoglobulin) are of human immunoglobulin consensus sequences. In some embodiments, a humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains both a light chain and at least the variable domains of a heavy chain. The antibody also comprises the heavy chain constant region, C H 1, hinge, C H 2. C H 3, and possibly C. H It may contain four regions.

[0051] Polypeptide: The term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three amino acids. Those skilled in the art will appreciate that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the entire sequences cited herein, but also polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Moreover, those skilled in the art will appreciate that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that preserves activity, shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and more usually includes at least one region of greater identity, often greater than 90%, or greater than 95%, 96%, 97%, 98%, or 99%, with one or more highly conserved regions, and usually includes at least 3-4, and often up to 20 or more amino acids with another polypeptide of the same class, is encompassed by the related term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0052] Prevent or prophylaxis: As used herein, when used in relation to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing a disease, disorder, and / or condition and / or delaying the onset and / or severity of one or more characteristics or symptoms of a disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population suspected of the disease, disorder, or condition.

[0053] Recombinant: as used herein, is intended to refer to a polypeptide designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that has been transgenic or otherwise engineered to express one or more genes or genetic components that encode and / or direct the expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or a polypeptide prepared, expressed, created, or isolated by any other means that involves splicing or ligating a selected nucleic acid sequence element to another, chemically synthesizing a selected sequence element, and / or otherwise generating a nucleic acid that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements arise from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources, such as, for example, in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0054] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity, stabilizes one or more characteristics, and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will recognize that the term "therapeutically effective amount" does not in fact require achieving successful treatment in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a particular desired pharmacological response in a significant number of subjects when administered to a patient in need of such treatment. Those skilled in the art will recognize that in some embodiments, a therapeutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and / or administered in multiple doses, for example as part of a dosing regimen.

[0055] Vector: As used herein, refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors with bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell, thereby being replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electrophoresis, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above may generally be carried out according to conventional methods well known in the art, and as described in various general and more detailed references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0056] Antibodies of the present disclosure Antibodies of the present disclosure include humanized forms of mAb107, as described in the Examples. In some embodiments, an antibody of the present disclosure is an anti-CD11b antibody, or a fragment thereof. In some embodiments, an antibody of the present disclosure is a recombinant antibody. In some embodiments, an anti-CD11b antibody of the present disclosure comprises one, two, or three heavy chain CDR sequences that are or include SEQ ID NOs: 5, 6, and / or 7. In some embodiments, an anti-CD11b antibody of the present disclosure comprises one, two, or three light chain CDR sequences that are or include SEQ ID NOs: 8, 9, and / or 10. In some embodiments, a humanized form of mAb107, or an antigen-binding antibody fragment thereof, comprises one or more sequences including SEQ ID NOs: 1-18. In some embodiments, the humanized form of mAb107 or an antigen-binding antibody fragment thereof comprises a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NOs:1-18.

[0057] The amino acid sequence of the humanized form of the mAb107 antibody or its antigen-binding fragment of the present disclosure can be substituted through conservative substitution.As used herein, the term "conservative substitution" refers to a modification of a polypeptide in which one or more amino acids are replaced with amino acids having similar biochemical properties, without causing the loss of biological or biochemical function of the corresponding polypeptide.As used herein, the term "conservative sequence variant" or "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain.Amino acid residues having a similar side chain are defined in the art. These residues include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Thus, it is expected that the antibodies of the present invention may have conservative amino acid substitutions and still retain activity.

[0058] In some embodiments, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding humanized form of mAb107 and fragments thereof.Humanized form of mAb107 antibody and fragments thereof can be generated from nucleic acid molecules using molecular biology methods known in the art.Nucleic acids of the present disclosure include, for example, DNA and / or RNA.

[0059] The nucleic acid construct of the present disclosure can be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecule can be operably linked to an expression control sequence. A vector comprising any of the above-mentioned nucleic acid molecules or fragments thereof is further provided by the present disclosure. Any of the above-mentioned nucleic acid molecules or fragments thereof can be cloned into any suitable vector and used to transform or transfect any suitable host. The selection of vectors and methods for constructing them are generally known to those skilled in the art and are described in general technical references (see generally "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).

[0060] In some embodiments, exogenous nucleic acid (DNA or RNA) can be introduced into prokaryotic or eukaryotic host cells using conventional techniques such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc. Desirably, and depending on the need and whether the vector is DNA or RNA, the vector can contain regulatory sequences, such as transcriptional and translational initiation and termination codons, specific to the type of host into which the vector is introduced (e.g., bacteria, fungi, plants, or animals). In some embodiments, the vector contains regulatory sequences specific to the genus of the host. Preferably, the vector contains regulatory sequences specific to the species of the host.

[0061] Suitable viral vectors include, for example, retroviral vectors, parvovirus-based vectors, such as adeno-associated virus (AAV)-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors, and lentiviral vectors such as herpes simplex (HSV)-based vectors.These viral vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0062] In some embodiments, the nucleic acid molecule is inserted into a vector capable of expressing the humanized mAb107 antibody or a fragment thereof when introduced into a suitable host cell. Suitable host cells include, but are not limited to, bacteria, yeast, insect, and mammalian cells. Exemplary host cells include prokaryotes (e.g., E. coli) and eukaryotes (e.g., COS or CHO cells). Mammalian host cells that may be used include human Hela293, Expi293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7 and CV1, quail QCl-3 cells, mouse L cells, and Chinese Hamster Ovary (CHO) cells (e.g., DG44 cells). In some embodiments, mammalian host cells suitable for expressing the antibody may be Chinese Hamster Ovary (CHO) cells (e.g., including DHFR-CHO cells used with a DHFR selectable marker), NSO myeloma cells, COS cells, or SP2 cells.

[0063] In some embodiments, the nucleic acid and vector of the present disclosure can be isolated and / or purified.The present disclosure also provides a composition comprising the above-mentioned isolated or purified nucleic acid molecule, optionally in the form of a vector.The isolated nucleic acid and vector can be prepared using standard techniques known in the art, including, for example, alkaline / SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis and other techniques well known in the art.The composition can include other components as further described herein.

[0064] Mouse IgG1 mAb107 (mAb107) blocks multiligand binding to CD11b and CD11b-mediated transendothelial neutrophil / monocyte migration and phagocytosis (Li et al., J Immunol. 2002 Feb 1;168(3):1219-25). Crystal structure determination of mouse mAb107 in complex with CD11b revealed that the mAb is a ligand mimic and occupies the ligand-binding site of CD11b. An unexpected structural feature coincident with cellular integrins was that binding of mAb107 stabilized the integrin in its inactive, non-signaling conformation, the first example of an integrin inhibitor (Mahalingam et al., J Immunol. 2011 Dec 15;187(12):6393-401). Most recently, mAb107 has been shown to protect native kidneys from otherwise irreversible renal failure caused by severe ischemia-reperfusion injury (IRI) in non-human primates (NHPs) (Dehnadi et al., Nat Commun. 2017 Jan 10;8:13899). mAb107 blocked homing of leukocytes to the reperfused organ, phagocytosis, and production of IL-18, RANTES, C3, IL-6, and IFN-γ proteins in ischemia-reperfused NHP kidneys.

[0065] IRI is a common acute inflammatory response mediated by activated innate immune cells that infiltrate ischemic organs after reperfusion. It is the basis for post-ischemic failure of multiple organs, including the heart, brain, and kidneys. It is also the cause of delayed organ transplant function and early allograft failure, which is widely recognized to have detrimental effects on graft survival, initial hospital stay, medical costs, and mortality (Siedlecki et al., Kidney Int. 2011 Aug;80(3):263-71; Menke et al., Curr Opin Organ Transplant. 2014 Aug;19(4):395-400; Cooper et al., Curr Opin Nephrol Hypertens. 2013 Nov;22(6):698-703; Renders and Heeman, Curr Opin Organ Transplant. 2012 Dec;17(6):634-9). Previous efforts to limit IRI, primarily performed in rodents, have targeted individual proinflammatory mediators and shown promise in rodents, but this approach has been less effective in larger animals and has lacked human trials (Cerda et al., Clin J Am Soc Nephrol. 2015 Oct 7;10(10):1859-67; Molitoris et al., Clin. J. Am. Soc. Nephrol. 7, 842-843 (2012); Anderson et al., Cold Spring Harb Perspect Med. 2013 Sep 1;3(9):a015503; Gallagher et al., Expert Opin Investig Drugs. 2017 Feb;26(2):141-154; Benoit et al., Pediatr Nephrol. 2018 May;33(5):779-787).Contributing factors likely include significant interspecies differences in immune responses between rodents and primates (Seok et al., Proc Natl Acad Sci US A. 2013 Feb 26;110(9):3507-12), heterogeneity of immune cell populations in primates, and the dual role of innate immune cells in tissue damage as well as tissue repair, complicating target selection and timing of intervention. The impressive success of mAb107 in preventing postischemic renal failure in non-human primates is a breakthrough and forms a compelling rationale for testing its utility in humans. Described herein are antibodies or antigen-binding fragments thereof, including humanized and mutagenized antibodies or antigen-binding fragments thereof, that bind to CD11b.

[0066] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity determining regions (CDRs) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and can specifically bind 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 contain an Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0067] As used herein, the term "antibody" includes not only complete polyclonal or monoclonal antibodies, but also any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof, fusion proteins comprising an antibody, and any other modified form of an immunoglobulin molecule that contains an antigen recognition site. Antibodies include any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of a particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, an immunoglobulin may be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional shapes of the different classes of immunoglobulins are well known. Exemplary antibodies and antibody fragments include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope-binding fragments (e.g., bispecific antibodies), camelid antibodies, chimeric antibodies, single-chain variable fragments (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments (e.g., antigen-binding portions) exhibiting a desired biological activity, disulfide-stabilized Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to an antibody of the invention), intrabodies, and epitope-binding fragments of any of the above.

[0068] Fv fragments are antibody fragments that contain a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain that is tightly bound, and this binding can be essentially covalent, for example in scFv. It is in this configuration that 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 that contains only three CDRs specific for an antigen) can have the ability to recognize and bind an antigen, although usually with a lower affinity than the entire binding site.

[0069] Single chain Fv or (scFv) antibody fragments comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding. One exemplary scFv is shown in SEQ ID NO: 15. This scFv comprises a humanized VH segment, a humanized VL segment, and a linker between these two segments.

[0070] The Fab fragment contains 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 generally contain a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0071] Diabodies are small antibody fragments with two antigen-binding sites, which comprise a VH linked to a VL (VH and VL) in the same polypeptide chain. 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, resulting in two antigen-binding sites.

[0072] A linear antibody comprises a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific. The antibodies and antibody fragments of the present disclosure may be modified in the Fc region to provide desired effector functions or serum half-life.

[0073] In some embodiments, the Fc region may be conjugated to PEG or albumin to increase serum half-life, or some other conjugation that produces a desired effect. Alternatively, certain other Fc regions may be used to minimize side effects or therapeutic complications when it is desirable to eliminate or reduce effector functions.

[0074] How to use The present disclosure provides methods of treatment comprising administering to a subject a composition disclosed herein.

[0075] The term "subject" as used herein refers to any animal. In some embodiments, the subject is a mammal. In some embodiments, the term "subject" as used herein refers to a human (e.g., a male, female, or child). Samples for use in the methods may include, for example, serum samples obtained from a selected subject.

[0076] In some embodiments, subject selection may include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample to indicate that the subject is suitable for selection. In some embodiments, the subject may have or be confirmed or identified as having a condition or disease, for example, by a medical professional. In some embodiments, indicating a positive response to a condition or disease may be a patient record, a family history, and / or detection of a positive response symptom. In some embodiments, multiple parties may be involved in subject selection. For example, a first party may obtain a sample from a candidate subject and a second party may test the sample. In some embodiments, the subject may be selected and / or referred by a medical practitioner (e.g., a general practitioner). In some embodiments, subject selection may include obtaining a sample from a selected subject and storing the sample and / or using the sample in a method disclosed herein. The sample may include, for example, a cell or a population of cells.

[0077] The antibodies described herein may be administered to patients who have suffered from acute myocardial infarction in hopes of preventing damage from ischemia-reperfusion injury. In some embodiments, the myocardial infarction condition is adverse left ventricular remodeling after myocardial infarction. In some embodiments, the myocardial infarction condition includes myocardial stunning. In some embodiments, the myocardial infarction condition includes microvascular dysfunction. In some embodiments, the disclosed anti-CD11b antibodies are administered to a subject with ischemia-reperfusion injury within about 5 hours of injury. In some embodiments, the disclosed anti-CD11b antibodies are administered to a subject with ischemia-reperfusion injury within about 2 hours of injury. In some embodiments, the disclosed anti-CD11b antibodies are administered before ischemia-reperfusion injury. Currently, it is estimated that more than 30% of the 860,000 myocardial infarction patients in the United States each year suffer from complications from reperfusion injury, which is the leading cause of adverse events.

[0078] The antibodies described herein may also be used to prevent acute kidney injury (AKI) caused by ischemia-reperfusion injury, which complicates recovery from cardiac surgery in up to 30-40% of patients, totaling an estimated 1.8 million per year in the United States, and costing the U.S. healthcare system approximately $10 billion annually. Renal replacement therapy (dialysis) occurs in 2-5% of such patients, is associated with a 50% case fatality rate, an increased risk of long-term mortality (HR, 1.31; 95% CI, 1.16-1.47; p<0.00001), and there is no effective preventative therapy. This clinical setting is similar to and supported by results in non-human primate models of renal ischemia / reperfusion injury in that it is voluntary, common, causes relatively standardized injury, and observations are available (Dehnadi et al., Nat Commun. 2017 Jan 10;8:13899). In some embodiments, the renal injury includes renal fibroinflammatory disease. In some embodiments, the renal injury comprises post-ischemic renal fibrosis. In some embodiments, the disclosed anti-CD11b antibody is administered to a subject with ischemia-reperfusion injury within about 5 hours of injury. In some embodiments, the disclosed anti-CD11b antibody is administered to a subject with ischemia-reperfusion injury within about 2 hours of injury. In some embodiments, the disclosed anti-CD11b antibody is administered before ischemia-reperfusion injury.

[0079] The antibodies described herein may also be used to prevent (reduce the risk of) delayed graft function (DGF, orphan drug name approved by the FDA) resulting from ischemia-reperfusion injury of the kidney graft. This is a common and major complication in kidney transplantation, rising to 31% of patients receiving allografts from deceased donors in 2015. Of the 19,060 kidney transplants performed in the United States alone in 2016, 70.5% (approximately 13,437 cases per year) were from deceased donors, compared with approximately 14,000 cases per year in the EU. There is no approved therapy. Moreover, the clinical setting herein is similar to the experimental setup in non-human primate models (Dehnadi et al., Nat Commun. 2017 Jan 10;8:13899). DGF increases post-operative costs by more than $4,000, increases post-transplant hospitalization by up to 75% (usually 5-10 days), and over the course of 5 years after transplant, DGF patients have significantly greater time on dialysis, graft rejection, and mortality. mAb107 to DGF could reach annual sales of $300M+, with less than one-third of transplants entering inpatient care and one-third of these transitioning to home care.

[0080] The antibodies of the present disclosure may also be used to improve xenograft acceptance and induce immune tolerance to allografts when delivered in combination with anti-CD40 antibodies. Treatment with anti-CD11b antibodies attenuates early xenograft rejection in animal models, and anti-CD40 / CD11b therapy is statistically similar to anti-CD154 antibody treatment for preventing xenograft rejection (Liu and Ford, Am J Transplant. 2020; 20:2216-2225; DA Faber et al., "Combined CD11b / CD40 Blockade is Superior to CD40 Blockade Alone in Prolonging Survival in Pig-to-Nonhuman Primate Renal Xenotransplantation," Abstract 2 at 2021 American Transplant Congress).

[0081] The antibody of the present disclosure can also be administered to a subject with pulmonary reperfusion injury.For example, the antibody of the present disclosure can be administered to a subject with pulmonary fibrosis.In some embodiments, the disclosed anti-CD11b antibody is administered to a subject with reperfusion injury within about 5 hours of injury.In some embodiments, the disclosed anti-CD11b antibody is administered to a subject with reperfusion injury within about 2 hours of injury.In some embodiments, the disclosed anti-CD11b antibody is administered before reperfusion injury.

[0082] The antibodies of the present disclosure may also be administered to subjects with autoimmune diseases, such as cytoplasmic antineutrophil cytoplasmic antibody (cANCA)-associated vasculitis or diabetic nephropathy. Additionally, the antibodies of the present disclosure may also be administered to subjects undergoing or having undergone chemotherapy.

[0083] The antibody of the present disclosure may also be administered to a subject who has or has been diagnosed with dry eye disease. In some embodiments, the antibody of the present disclosure may be administered topically. In some embodiments, the antibody of the present disclosure may be administered to the cornea. In some embodiments, the antibody of the present disclosure may be administered to the conjunctival epithelium. Topical application of risuteganib, which partially blocks leukocyte homing through CD11b, reduces inflammation and improves signs and symptoms of dry eye disease (Donnenfeld, et al., "Prospective, Randomized, Double-Masked, Vehicle-Controlled, Safety and Efficacy Study of Topical Risuteganib in Treating Dry Eye Disease," ASCRS 2021 meeting, paper 77664).

[0084] The antibody of the present disclosure may also be administered to subjects who have or have been diagnosed with Alzheimer's disease. In some embodiments, the antibody of the present disclosure may be administered with or without other components to reduce microglial activation. Microglial activation mediates early neuronal synapse loss in Alzheimer's mouse models (Hong et al., Science. 2016 May 6. 352:6286; Merlini et al., Neuron. 2019 March 20. 101:1099-1108). Thus, blocking or attenuating microglial activation through administration of the anti-CD11b antibody of the present disclosure may be used to treat subjects who have or have been diagnosed with Alzheimer's disease.

[0085] The antibodies described herein include humanized forms of mAb107, which may contain one or more amino acid substitutions based on our crystal structure, improving the affinity of humanized mAb107 for CD11b relative to the murine form.

[0086] See also U.S. Patent Application Publication No. 2018 / 0244782, U.S. Patent No. 7,323,552, U.S. Patent No. 7,998,738, and U.S. Patent No. 10,738,121, the disclosures of which are incorporated by reference in their entireties herein.

[0087] Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of pharmaceutical compositions comprising, as an active ingredient, an antibody or antigen-binding fragment thereof described herein.

[0088] Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the phrase "pharma- ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, consistent with pharmaceutical regulations.

[0089] A pharmaceutical composition is typically formulated to correspond to its intended route of administration, which includes parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0090] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0091] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols, sodium chloride, such as mannitol, sorbitol, etc. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0092] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of above-listed components, and optionally followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other required components from above-listed components.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which produces powder of active ingredient plus any additional desired components from the solution that has been previously sterilized by filtration.

[0093] Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primojel, or corn starch; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0094] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, such as those described in U.S. Patent No. 6,468,798.

[0095] Systemic administration of the therapeutic compounds described herein may also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0096] Pharmaceutical compositions can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0097] In one embodiment, the therapeutic compound can be prepared with a carrier that protects the therapeutic compound from rapid elimination from the body, such as sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques or can be obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (containing liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0098] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES

[0099] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0100] [Example 1] Humanized mAb107 Simple method Design: The sequences flanking each of the three CDRs of the variable and heavy and light chains of mouse mAb107 were changed to human immunoglobulin sequences. We also introduced a change in CDR2 of the variable heavy chain that replaces asparagine (N) with aspartic acid (D) to improve affinity. The cDNA was produced commercially.

[0101] Cloning: The variable regions of our designed cDNAs encoding the variable heavy and light chains of humanized mAb107 were reverse transcribed using an online program and synthesized by IDT. A secretion signal sequence was added to the 5' end of each chain. N / D mutations were introduced into the heavy chain using QuikChange II XL site-directed mutagenesis kit (Agilent). The variable region of the heavy chain was joined to the human IgG4 kappa constant region by overlap PCR and cloned into pcDNA3.4 using XbaI and XhoI restriction sites. Similarly, the light chain variable region was fused to the human kappa light chain constant region.

[0102] Production, purification, and evaluation: Recombinant antibodies were produced using Expi293 cells (Thermo Fisher). Cells were grown in Expi293 expression medium (Thermo Fisher) at 37°C in 5% CO2 on an orbital shaker (125 rpm). Expi293 cells were transiently transfected with 1:1 heavy and light chain plasmids using the manufacturer's protocol. Culture supernatants were collected after one week, and IgG was purified using protein G agarose beads (Thermo Scientific). We routinely obtained 1-3 mg of purified IgG per 100 ml of cell culture. Heavy and light chain protein sizes as well as IgG purity were confirmed by SDS-PAGE. Affinity of humanized mAb107 versus mouse mAb107 was measured using the A-domain of CD11b as a ligand by biolayer interferometry (BLI) using the Octet assay system.

[0103] result The optimized amino acid sequences of the heavy and light chains of humanized N55D substituted mAb107 are as follows:

[0104] A) Humanized heavy chain (CDR1, 2 and 3 are in bold; N57 substitution in CDR2 is shown in lower case).

[0105] [ka]

[0106] B) Humanized light chain (CDRs 1, 2 and 3 are in bold)

[0107] [ka]

[0108] Sequence of humanized mAb107 IgG4κ Heavy (H) chain (CDRs in bold; N57D substitution in CDR2 in lower case; constant region in italics). Lower case "d" in CDR3 is the D residue that binds metal ions in MIDAS.

[0109] [ka]

[0110] mAb107 IgG1 is

[0111] [ka]

[0112] Light (L) chain (CDRs are in bold; constant regions are in italics)

[0113] [ka]

[0114] The protein sizes of the heavy and light chains and the purity of the IgG were confirmed by SDS-PAGE and showed the expected sizes (Figure 1).

[0115] Affinities for the CD11b A domain were determined by BLI. Affinities for the parental mouse mAb107 were 1-5 nM. Affinities for humanized humAb107 were 5-9 nM.

[0116] [Example 2] Humanized Mutagenized Anti-CD11b As shown in FIG. 2, an S to P mutation at position

[0228] in the IgG4 hinge region (VESKYGPPCPPCPAPEFLGG) (italics in SEQ ID NO: 12) reduced Fab arm exchange.

[0117] DNA sequence of humanized mutagenized mAb107 IgG4k heavy chain V5 - affinity 5-9nM

[0118] [ka]

[0119] Protein Translation:

[0120] [ka]

[0121] Leader sequence: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 13) N to D substitution in CDR2H: bold (to enhance affinity) [N57 in the crystal structure of the 107Fab / CD11b A domain complex]. S to P substitution: bold (to prevent Fab arm conversion [JBC, VOL. 290, NO. 9, pp. 5462-5469, February 27, 2015]).

[0122] DNA sequence of humanized mAb107 IgG4k light chain

[0123] [ka]

[0124] Protein Translation:

[0125] [ka]

[0126] Molecular weight=24,569 Leader sequence: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 13)

[0127] Humanized mutant mAb107 in scFv format

[0128] [ka]

[0129] Features: Two foreign amino acids: EF GS linker: GGGGSGGGGSGGGGS (SEQ ID NO: 16) Heavy and light chain CDRs 1-3 are in bold

[0130] Other embodiments Although the present invention has been described in conjunction with the detailed description thereof, it is to be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. The following complementarity determining regions (CDRs): 1) CDR1 of the VH of SEQ ID NO:1, comprising the sequence of SEQ ID NO:5; 2) CDR1 of the VL of SEQ ID NO:2, comprising the sequence of SEQ ID NO:8; 3) CDR2 of the VH of SEQ ID NO:1 comprising the sequence of SEQ ID NO:6; 4) CDR2 of the VL of SEQ ID NO:2, comprising the sequence of SEQ ID NO:9; 5) the CDR3 of the VH of SEQ ID NO:1, comprising the sequence of SEQ ID NO:7; and 6) CDR3 of the VL of SEQ ID NO: 2, comprising the sequence of SEQ ID NO: 10 An antibody or antigen-binding fragment thereof comprising an amino acid sequence comprising:

2. a heavy chain variable region comprising VH CDR1, 2, 3, and a light chain variable region comprising VL CDR1, 2, 3; the VH CDR1, 2, 3 are identical to the complementarity determining regions of SEQ ID NO: 1; the VL CDR1, 2, 3 are identical to the complementarity determining regions of SEQ ID NO:2; An antibody or an antigen-binding fragment thereof. 【Request 3】 【Chemical 1】 (SEQ ID NO: 3) 【Chemistry 2】 (Heavy chain, SEQ ID NO: 17) 【Chemistry 3】 (heavy chain, SEQ ID NO: 13), and 【Chemistry 4】 (Light chain, SEQ ID NO:4) and wherein the amino acid sequence comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of: The antibody or antigen-binding fragment thereof described in claim 2.

4. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

5. The antibody or antigen-binding fragment thereof of claim 4, comprising SEQ ID NO:

15.

6. A pharmaceutical composition for ameliorating a condition associated with ischemia-reperfusion injury in a subject, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in claims 1 to 5.

7. The condition is post-ischemic renal fibrosis. , renal fibroinflammatory diseases, Pulmonary fibrosis, or adverse left ventricular remodeling after myocardial infarction.

8. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is administered to the subject within about 2 hours or within about 5 hours after the ischemia-reperfusion injury.

9. 1. A pharmaceutical composition for treating a subject having or at risk of developing a disorder associated with ischemia-reperfusion injury in an organ, comprising: The pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to claims 1 to 5.

10. The pharmaceutical composition of claim 9, wherein the organ is a kidney, a heart, or a lung.

11. 10. The pharmaceutical composition of claim 9, wherein the disorder is acute kidney injury, acute coronary syndrome, or acute myocardial infarction (MI).

12. 10. The pharmaceutical composition of claim 9, wherein the composition is administered to the subject within about 2 hours or within about 5 hours after the ischemia-reperfusion injury.

13. A pharmaceutical composition for reducing delayed graft function after organ transplantation, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, administering to an organ transplant recipient a therapeutically effective amount of said pharmaceutical composition prior to or within one day of transplantation of an organ into said recipient; The pharmaceutical composition thereby reducing delayed graft function following organ transplantation.

14. The pharmaceutical composition of claim 13, wherein the organ is a kidney, a heart, or a lung.

15. A method for treating an organ prior to transplantation into a recipient, comprising contacting the organ with a composition comprising the antibody or antigen-binding fragment thereof according to claims 1 to 5.

16. 16. The method of claim 15, wherein the organ is a kidney, a heart, or a lung.

17. 16. The method of claim 15, wherein the contacting step comprises perfusing the organ with a composition comprising a polypeptide or an antibody that immunospecifically binds to the epitope recognized by mAb107.

18. A pharmaceutical composition for treating a subject having an autoimmune disease, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to claims 1 to 5.

19. The pharmaceutical composition of claim 18, wherein the autoimmune disease is cytoplasmic antineutrophil cytoplasmic antibody (cANCA)-associated vasculitis.

20. A pharmaceutical composition for treating a subject with diabetic nephropathy, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to claims 1 to 5.

21. A pharmaceutical composition for ameliorating a disease condition associated with chemotherapy in a subject, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to claims 1 to 5.

22. 22. The pharmaceutical composition of claim 21, wherein the condition is adriamycin nephropathy.

23. A composition comprising the antibody or antigen-binding fragment thereof according to claims 1 to 5 and a pharma- ceutically acceptable carrier.

24. A nucleic acid molecule encoding the humanized mAb107 antibody or antigen-binding fragment of claims 1 to 5.

25. A recombinant vector comprising the nucleic acid molecule of claim 24.

26. 26. A host cell comprising a recombinant vector according to claim 25 and / or a nucleic acid molecule according to claim 24.

27. 27. The host cell of claim 26, selected from the group consisting of E. coli, P. pastoris, Sf9, COS, HEK293, and CHO.

28. (a) one or more of the nucleic acid molecule of claim 24, the recombinant vector of claim 25, or the host cell of claim 26 or 27; and (b) a pharma- ceutically acceptable carrier.

13. A pharmaceutical composition comprising:

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