Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2839—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
  • C07K16/2845—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the ASCII text file created on March 24, 2022, is 21.4 kilobytes in size.
• the material in the ASCII text file is hereby incorporated by reference in its entirety.
• TECHNICAL FIELD 15 Described herein are humanized antibodies that bind to Leukocyte integrin CD11b/CD18 (CD11b, ⁇ M ⁇ 2, CR3) with enhanced affinity, and methods of use thereof.
• BACKGROUND Leukocyte integrin CD11b/CD18 also known as CD11b, ⁇ M ⁇ 2, CR3, referred to herein as CD11b
• CD11b is the archetypal innate immune receptor expressed on innate immune 20 cells.
• CD11b binds more than 40 ligands, thus mediating leukocyte adhesion-dependent functions including homing, phagocytosis, adhesion-dependent superoxide generation, proteolytic enzyme and cytokine production, antibody-dependent cytolytic activity, and enhancing 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 25 USA. (2013) 110:16426–31).
• Binding of physiologic ligands such as ICAM-1, 1 Attorney Docket No.29539-0415WO1/MGH 25105 complement iC3b and fibrinogen to CD11b induces activating tertiary and quaternary changes in CD11b leading to proadhesive cell signaling. It is now established that current anti-integrin agents that stabilize this activated proinflammatory state can cause serious adverse outcomes in humans that were not predicted from the rodent studies (Raab- 5 Westphal et al., Cancers (Basel). 2017 Aug 23;9(9):110). SUMMARY Provided herein are antibodies or antigen-binding fragments thereof that bind to CD11b, and methods of use thereof.
• the antibodies or antigen- binding fragments thereof comprise an amino acid sequence that comprises the following 10 complementarity determining regions (CDR): 1) CDR 1 of the VH of SEQ ID NO:1, e.g., GFNIKD (SEQ ID NO:5); 2) CDR 1 of the VL of SEQ ID NO:2, e.g., SQNLLYSSNQKNY (SEQ ID NO:8); 3) CDR 2 of the VH of SEQ ID NO:1, e.g., PADDKTK (SEQ ID NO:6); 4) CDR 2 of the VL of SEQ ID NO:2, e.g., WASTRESGVPDR (SEQ ID NO:9); 15 5) CDR3 of the VH of SEQ ID NO:1, e.g., GHYGYDGYA (SEQ ID NO:7); and 6) CDR 3 of the VL of SEQ ID NO:2, e.g., YYSYPL (SEQ ID NO:10).
• CDR complementarity
• the antibodies or antigen-binding fragments thereof comprise a heavy chain variable region comprising VH CDRs 1, 2, 3, and a light chain variable region comprising VL CDRs 1, 2, 3, wherein the VH CDRs 1, 2, 3 are identical 20 to complementarity determining regions in SEQ ID NO: 1, and the VL CDRs 1, 2, 3 are identical to complementary determining regions in SEQ ID NO: 2.
• the antibodies or antigen-binding fragments thereof comprise an amino acid sequence that comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of: 25 QVQLVQSGAEVKKPGASVKVSCKPSGFNIKDIYMQWVRQAPGQRLEWIGRIDPA dDKTKYDPKFQGRATITADTSASTAYLELSSLRSEDTAVYYCASEGHYGYdGYAM DYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTPSSSLGTKTYTCNVDHKPSNTKVDKR (SEQ ID NO:3); 30 QVQLVQSGAEVKKPGASVKVSCKPSGFNIKDIYMQWVRQAPGQRLEWIGRIDPA 2 Attorney Docket No.29539-0415WO1/MGH 25105 dDKTKYDPKFQGRATITADTSASTAY
• the antibodies or antigen-binding fragments thereof comprise a single chain variable fragment (scFv). In some embodiments, the antibodies or antigen-binding fragments thereof comprise SEQ ID NO:15.
• compositions comprising an antibody or antigen- binding fragment thereof as described herein, and a pharmaceutically acceptable carrier 25
• methods for ameliorating a pathology associated with ischemia reperfusion injury in a subject include administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described herein.
• the pathology is post-ischemic renal fibrosis. 30 In some embodiments, the pathology is a kidney fibroinflammatory disease.
• the pathology is pulmonary fibrosis. 3 Attorney Docket No.29539-0415WO1/MGH 25105 In some embodiments, the pathology is post-myocardial infarction left ventricular adverse remodeling. In some embodiments, the composition is administered to the subject within about 5 hours after the ischemia reperfusion injury. 5 In some embodiments, the composition is administered to the subject within about 2 hours after the ischemia reperfusion injury. Also provided herein are methods for treating a subject having or at risk of developing a disorder associated with ischemia reperfusion injury in an organ. The methods include administering to the subject a composition comprising a therapeutically 10 effective amount of an antibody or antigen-binding fragment thereof as described herein. In some embodiments, the organ is a kidney.
• the disorder is acute kidney injury.
• the organ is a heart.
• the disorder is acute coronary syndrome.
• the disorder is acute myocardial 15 infarction (MI).
• the organ is a lung.
• the composition is administered to the subject within about 5 hours after the ischemia reperfusion injury. In some embodiments, the composition is administered to the subject within about 20 2 hours after the ischemia reperfusion injury. Additionally, provided herein are methods for providing an organ for transplantation. The methods include administering to an organ donor a composition comprising an antibody or antigen-binding fragment thereof as described herein; and harvesting the organ from the organ donor.
• the organ is a kidney, a heart, or a lung.
• methods for reducing delayed graft function following organ transplantation include administering to an organ recipient a therapeutically effective amount of composition comprising an antibody or antigen- binding fragment thereof as described herein prior to transplantation of the organ to the 30 recipient or within a day of the transplantation, thereby reducing delayed graft function following organ transplantation. 4 Attorney Docket No.29539-0415WO1/MGH 25105
• the organ is a kidney, a heart, or a lung. Further, provided herein are methods for treating an organ prior to transplantation into a recipient.
• the methods include contacting the organ with a composition comprising an antibody or antigen-binding fragment thereof as described herein. 5
• the organ is a kidney, a heart, or a lung.
• the contacting step comprises perfusing the organ with the composition comprising a polypeptide or antibody that immunospecifically binds the epitope recognized by mab107.
• methods for treating a subject having an 10 autoimmune disease comprising administering to the subject a therapeutically effective amount of composition comprising an antibody or antigen-binding fragment thereof as described herein.
• the autoimmune disease is cytoplasmic antineutrophil cytoplasmic antibodies (cANCA)-associated vasculitis.
• compositions comprising an antibody or antigen-binding fragment thereof as described herein.
• methods for ameliorating a pathology associated with 20 chemotherapy in a subject comprising administering to the subject a therapeutically effective amount of composition comprising an antibody or antigen-binding fragment as described herein.
• the pathology is Adriamycin nephropathy.
• FIG.2. Coomassie stain of an SDS-PAGE showing migration of humanized IgG4 ⁇ mAb107 containing wild-type Ser 228 (S) or Pro 228 (P) in the core hinge region of IgG4 under reducing (left) panel of unreduced (right panel) conditions.
• the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.
• Administration/Administered typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition.
• administration may be ocular, oral, parenteral, topical, etc.
• administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, 5 intravenous, intraventricular, within a specific organ (e. g.
• administration may involve only a single dose.
• administration may involve application of a fixed number of doses.
• administration may involve dosing that is intermittent 10 (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
• administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
• affinity is a measure of the tightness with a particular ligand binds to its partner. Affinities can be measured in different ways. In 15 some embodiments, affinity is measured by a quantitative assay. In some such embodiments, binding partner concentration may be fixed to be in excess of ligand concentration so as to mimic physiological conditions. Alternatively or additionally, in some embodiments, binding partner concentration and/or ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under 20 comparable conditions (e.g., concentrations).
• Animal as used herein refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, of either sex and at any stage of development.
• animal refers to non-human animals, at any stage of development.
• the non-human animal is a mammal (e.g., a 25 rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
• animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
• an animal may be a transgenic animal, genetically engineered animal, and/or a clone.
• Binding typically 30 refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves 7 Attorney Docket No.29539-0415WO1/MGH 25105 physical interaction by way of 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 where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with 5 a carrier entity and/or in a biological system or cell). CDR: as used herein, refers to a complementarity determining region within an antibody variable region.
• CDR1, CDR2 and CDR3 There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
• a “set of CDRs” or “CDR set” refers to a group of three or six 10 CDRs that occur in either a single variable region capable of binding the antigen or the CDRs of cognate heavy and light chain variable regions capable of binding the antigen.
• Epitope includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
• an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
• such chemical atoms or groups are surface-exposed 20 when the antigen adopts a relevant three-dimensional conformation.
• such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation.
• at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).
• Humanized is commonly used to refer to antibodies (or antibody components) whose amino acid sequence includes V H and VL region sequences from a reference antibody raised in a non-human species (e.g., a mouse), but also includes modifications in those sequences relative to the reference antibody intended to render them more “human-like”, i.e., more similar to human 30 germline variable sequences.
• a “humanized” antibody is one that immunospecifically binds to an antigen of interest and 8 Attorney Docket No.29539-0415WO1/MGH 25105 that has a framework (FR) region having substantially the amino acid sequence as that of a human antibody, and a complementary determining region (CDR) having substantially the amino acid sequence as that of a non-human antibody.
• FR framework
• CDR complementary determining region
• a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', 5 F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor immunoglobulin) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
• a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant 10 region.
• a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain.
• the antibody also may include a CH1, hinge, CH2, CH3, and, optionally, a CH4 region of a heavy chain constant region.
• Polypeptide generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in 15 the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without 20 destroying activity.
• 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.
• proteins may comprise natural amino acids, non-natural amino acids, 30 synthetic amino acids, and combinations thereof.
• the term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 9 Attorney Docket No.29539-0415WO1/MGH 25105 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
• proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
• Prevent or prevention refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset and/or severity of one or more characteristics or symptoms of the 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 10 significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition.
• Recombinant is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by 15 recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of 20 the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the poly
• one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for 30 example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).
• a therapeutically effective amount means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
• a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
• therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual.
• a therapeutically 10 effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
• a therapeutically effective amount may be formulated and/or administered in a single dose.
• a therapeutically effective amount may be 15 formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
• Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• vector refers to a circular double stranded DNA loop into which additional DNA 20 segments may be ligated.
• a viral vector Another type of vector, wherein additional DNA segments may be ligated into the viral genome.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a 25 host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors are capable of directing the expression of genes to which they are operatively linked.
• Such vectors are referred to herein as “expression vectors.”
• Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
• 30 Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described 11 Attorney Docket No.29539-0415WO1/MGH 25105 herein.
• the foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.
• Antibodies of the Disclosure include humanized forms of mAb107, as described in 10 the examples.
• the antibodies of the disclosure are anti-CD11b antibodies, or fragments thereof.
• the antibodies of the disclosure are recombinant antibodies.
• the anti-CD11b antibodies of the disclosure includes 1, 2, or 3 heavy chain CDR sequences that are or include a sequence of SEQ ID NOs: 5, 6, and/or 7.
• the anti-CD11b antibodies of the 15 disclosure includes 1, 2, or 3 light chain CDR sequences that are or include a sequence of SEQ ID NOs: 8, 9, and/or 10.
• the humanized forms of mAb107 or an antigen-binding antibody fragment thereof includes one or more sequences including SEQ ID NOs: 1-18.
• the humanized forms of mAb107 or an antigen-binding antibody fragment includes a sequence that is at least 95%, 96%, 20 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.
• Amino acid sequences of a humanized form of mAb107 antibody or antigen- binding fragment binds of the present disclosure may be substituted through conservative substitution.
• the term “conservative substitution” used herein refers to modification of a 25 polypeptide in which one or more amino acids are substituted with an amino acid having a similar biochemical property so as not to cause the loss of a biological or biochemical function of the corresponding polypeptide.
• the term “conservative sequence variant” or “conservative amino acid substitution” used herein is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Amino acid residues 30 having a similar side chain are defined in the art.
• amino acids with a basic side chain e.g., lysine, arginine, and histidine
• amino acids with an acidic 12 Attorney Docket No.29539-0415WO1/MGH 25105 side chain e.g., aspartic acid and glutamate
• amino acids with a non-charged polar side chain e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine
• amino acids with a non-polar side chain e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan
• amino acids with a betabranched side chain 5 e.g., threonine, valine, and isoleucine
• amino acids with an aromatic side chain e.g., tyrosine, phenylalanine, tryptophan, and histidine
• the disclosure provides polynucleotides comprising a 10 nucleotide sequence encoding humanized forms of mAb107 and fragments thereof. Humanized forms of mAb107 antibodies and fragments therefore may be produced from nucleic acid molecules using molecular biological methods known to the art.
• Nucleic acids of the present disclosure include, for example DNA and/or RNA. Nucleic acid constructs of the present disclosure may be inserted into an 15 expression vector or viral vector by methods known to the art, and nucleic acid molecules may be operably linked to an expression control sequence.
• a vector comprising any of the above described nucleic acid molecules, or fragments thereof, is further provided by the present disclosure.
• Any of the above nucleic acid molecules, or fragments thereof, can be cloned into any suitable vector and can be used to transform or transfect any 20 suitable host.
• the selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, "Recombinant DNA Part D,” Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).
• conventionally used techniques such as, for example, 25 electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc.
• a vector may include regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be 30 introduced, as appropriate and taking into consideration whether the vector is DNA or RNA
• a vector comprises regulatory sequences that are specific to 13 Attorney Docket No.29539-0415WO1/MGH 25105 the genus of the host.
• a vector comprises regulatory sequences that are specific to the species of the host.
• Suitable viral vectors include, for example, retroviral vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, AA V-adenoviral chimeric 5 vectors, and adenovirus-based vectors, and lentiviral vectors, such as Herpes simplex (HSV)-based vectors.
• retroviral vectors e.g., adeno-associated virus (AAV)-based vectors, AA V-adenoviral chimeric 5 vectors, and adenovirus-based vectors
• lentiviral vectors such as Herpes simplex (HSV)-based vectors.
• HSV Herpes simplex
• nucleic acid molecules are inserted into a vector that is able to express humanized mAb107 antibody or fragment thereof when introduced into an appropriate host cell.
• Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells.
• Exemplary host cells include prokaryotes (e.g., E. 15 coli) and eukaryotes (e.g., a COS or a CHO cell).
• Mammalian host cells that could be used include human Hela 293, Expi293, H9 and Jurkat cells, mouse NIH3T3 and C 127 cells, Cos 1, Cos 7 and CV 1, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells (e.g., DG44 cells).
• a mammalian host cell suitable for the expression of the antibody may be a Chinese Hamster Ovary (CHO) cell (for 20 example, including DHFR-CHO cells used along with a DHFR-selectable marker), an NSO myeloma cell, a COS cell or an SP2 cell.
• CHO Chinese Hamster Ovary
• nucleic acids and vectors of the present disclosure may be isolated and/or purified.
• the present disclosure also provides a composition comprising an above-described isolated or purified nucleic acid molecule, optionally in the form of a 25 vector.
• Isolated nucleic acids and vectors may be prepared using standard techniques known in the art including, for example, alkali/SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis and other techniques well known in the art.
• the composition can comprise other components as described further herein.
• the mouse IgG1 mAb107 blocks multiligand binding to CD11b, and 30 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 14 Attorney Docket No.29539-0415WO1/MGH 25105 mAb107 in complex with CD11b revealed that the mAb is a ligand-mimic, occupying the ligand-binding site in CD11b.
• IRI is common acute inflammatory response mediated by the activated innate immune cells that infiltrate the ischemic organ following reperfusion. It underlies post- ischemic failure of multiple organs including the heart, brain and kidney. It is also the cause of delayed graft function and primary allograft non-function, which have been 15 widely acknowledged to have detrimental effects on graft survival, initial length of stay, medical costs, and mortality (Siedlecki et al., Kidney Int.
• antibodies or antigen-binding fragments thereof comprising humanized and mutagenized antibodies or antigen-binding fragments thereof that bind CD11b.
• antibody refers to any antigen-binding molecule that 5 contains at least one (e.g., one, two, three, four, five, or six) complementary 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.
• CDR complementary determining region
• Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), 10 single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
• an antibody can contain an Fc region of a human antibody.
• the term antibody also includes derivatives, e.g., bi-specific antibodies, single- chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
• antibody encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding fragment (i.e., “antigen-binding portion”) or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
• An antibody includes an antibody of any class, such as IgG, IgA, or IgM 20 (or sub-class thereof), and the antibody need not be of any particular class.
• immunoglobulins can be assigned to different classes.
• immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
• the 25 heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
• the subunit structures and three-dimensional configurations of 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 30 antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), camelid antibodies, chimeric antibodies, single 16 Attorney Docket No.29539-0415WO1/MGH 25105 chain variable fragments (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g.
• an Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to 10 define an antigen binding site on the surface of the VH-VL dimer.
• Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
• the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
• scFV is shown in SEQ ID NO:15.
• This scFV includes a humanized VH 20 segment, a humanized VL segment, and a linker between these two segments.
• the Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain.
• F(ab')2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of 25 antibody fragments are also known in the art.
• Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL).
• Linear antibodies comprise 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 can be bispecific or monospecific. Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide 5 desired effector functions or serum half-life.
• the Fc region can be conjugated to PEG or albumin to increase the serum half-life, or some other conjugation that results in the desired effect.
• certain other Fc regions may be used 10
• Methods of Use The disclosure provides methods of treatment that include administering to a subject a composition disclosed herein.
• 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 15 to a human (e.g., a man, a woman, or a child).
• Samples for use in the methods can include serum samples, e.g., obtained from the selected subject.
• subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection.
• the subject can be confirmed or identified, 20 e.g. by a health care professional, as having had or having a condition or disease.
• exhibition of a positive response towards a condition or disease can be made from patient records, family history, and/or detecting an indication of a positive response.
• multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party 25 can test the sample.
• subjects can be selected and/or referred by a medical practitioner (e.g., a general practitioner).
• subject selection can include obtaining a sample from a selected subject and storing the sample and/or using the in the methods disclosed herein.
• Samples can include, for example, cells or populations of cells.
• the antibodies described herein can be administered to patients that have suffered an acute myocardial infarction in the hopes of preventing damage due to ischemia 18 Attorney Docket No.29539-0415WO1/MGH 25105 reperfusion injury.
• the myocardial infarction condition is post myocardial infarction left ventricular adverse remodeling.
• the myocardial infarction condition includes myocardial stunning.
• the myocardial infarction condition includes microvascular dysfunction.
• the disclosed anti-CD11b antibodies are administered to a subject with an ischemia reperfusion injury within about 5 hours of the injury. In some embodiments, the disclosed anti-CD11b antibodies are administered to a subject with an ischemia reperfusion injury within about 2 hours of the injury. In some embodiments, the disclose anti-CD11b antibodies are administered prior to the ischemic reperfusion injury. 10 Currently it is estimated that more than 30% of the 860,000 myocardial infarction patients in the U.S. each year suffer complications due to reperfusion injury and it is a chief cause of adverse outcomes.
• the antibodies described herein can also be used in preventing acute kidney injury (AKI) caused by ischemia reperfusion injury that complicates recovery from 15 cardiac surgery in up to 30-40 % of patients, totaling an estimated 1.8 million patients in the U.S each year and at an annual cost to the U.S. health system of approximately $10 billion.
• Renal replacement therapy occurs in 2–5 % of such patients and is associated with 50 % mortality, and with increased risk of long-term mortality (HR, 1.31; 95% CI, 1.16-1.47; p ⁇ 0.00001), with no effective preventive therapy.
• the kidney injury includes kidney fibroinflammatory disease.
• the kidney injury includes post-ischemic renal fibrosis.
• the disclosed anti-CD11b antibodies are administered to a subject with an ischemia reperfusion injury within about 5 hours of the injury. In some embodiments, the disclosed anti-CD11b antibodies are administered to a subject with an ischemia reperfusion injury within about 2 hours of the injury.
• the disclose anti-CD11b antibodies are administered prior to the ischemic reperfusion injury.
• the antibodies described herein can also be used to prevent (reduce risk of) delayed graft function (DGF, orphan drug designation approved by FDA) resulting from 19 Attorney Docket No.29539-0415WO1/MGH 25105 ischemia reperfusion injury of the kidney graft. This is a common and major complication in kidney transplants, rising in 2015 to 31% of patients receiving allografts from deceased donors. In 2016, of the 19,060 kidney transplants performed in the US alone, 70.5% ( ⁇ 13,437 per year) came from deceased donors, and ⁇ 14,000 per year in the 5 EU. There is no approved therapy.
• DGF delayed graft function
• DGF increases post-surgical costs by more than $4,000, increases posttransplant hospital stay (usually 5-10 days) by up to 75%, and over five years post- transplant, DGF patients had significantly higher time on dialysis, transplant rejection, 10 and mortality.
• mAb107 for DGF could reach $300M+ annual revenues, with less than one-third transplant penetration for hospital treatment, and one-third of those moving on to home treatment.
• the antibodies of the disclosure can also be used to improve the acceptance of xenografts and in tolerance induction to allografts when delivered in combination with 15 anti-CD40 antibodies.
• anti-CD11b antibodies mitigates early xenograft rejection in animal models, and anti-CD40/CD11b therapy is statistically similar to anti- CD154 antibody treatment for preventing xenotransplantation rejection (Liu and Ford, Am J Transplant.2020; 20:2216-2225; D.A. Faber et al., “Combined CD11b/CD40 Blockade is Superior to CD40 Blockade Alone in Prolonging Survival in Pig-to- 20 Nonhuman Primate Renal Xenotransplantation,” Abstract 2 at 2021 American Transplant Congress).
• the antibodies of the disclosure can also be administered to a subject with a reperfusion injury of the lungs.
• the antibodies of the disclosure can be administered to a subject having pulmonary fibrosis.
• the 25 disclosed anti-CD11b antibodies are administered to a subject with a reperfusion injury within about 5 hours of the injury. In some embodiments, the disclosed anti-CD11b antibodies are administered to a subject with a reperfusion injury within about 2 hours of the injury. In some embodiments, the disclose anti-CD11b antibodies are administered prior to the reperfusion injury. 30
• the antibodies of the disclosure can also be administered to subjects with an autoimmune disease, for example cytoplasmic antineutrophil cytoplasmic antibodies 20 Attorney Docket No.29539-0415WO1/MGH 25105 (cANCA)-associated vasculitis or diabetic nephropathy. Additionally the antibodies of the disclosure can also be administered to subjects undergoing or having undergone chemotherapy.
• the antibodies of the disclosure can also be administered to a subject having or 5 diagnosed with dry eye disease.
• the antibodies of the disclosure can be administered topically.
• the antibodies of the disclosure can be administered to the cornea.
• the antibodies of the disclosure can be administered to the conjunctival epithelium.
• Topical application of risuteganib which blocks leukocyte homing in part through CD11b, reduces inflammation and improves the 10 signs and symptoms of dry eye disease.
• the antibodies of the disclosure can also be administered to a subject having or diagnosed with Alzheimer’s disease.
• the antibodies of the 15 disclosure can be administered with or without other components to decrease microglia activation.
• Microglia activation mediates early neuronal synapse loss in Alzheimer mouse models. (Hong et al., Science.2016 May 6. 352:6286; Merlini et al., Neuron. 2019 March 20.101:1099-1108). Therefore, blocking or attenuating microglia activation through administration of the anti-CD11b antibodies of the disclosure can be used to treat 20 a subject having or diagnosed with Alzheimer’s disease.
• the antibodies described herein include humanized versions of mAb107, that can include one or more amino-acid substitution, based on our crystal structure, include those that improve affinity of humanized mAb107 to CD11b vs. the mouse version. See also, US 2018/0244782, US 7,323,552, US 7,998,738, and 10,738,121, the 25 entire disclosures of which are incorporated herein by reference.
• Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof described herein as an active ingredient. 30 Pharmaceutical compositions typically include a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier includes 21 Attorney Docket No.29539-0415WO1/MGH 25105 saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
• Pharmaceutical compositions are typically formulated to be compatible with its 5 intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
• solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as 15 benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
• antibacterial agents such as 15 benzyl alcohol or methyl parabens
• compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, 25 Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
• the carrier can be a solvent or dispersion medium containing, for 30 example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can 22 Attorney Docket No.29539-0415WO1/MGH 25105 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 dispersion 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, 5 thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
• 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.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated 15 above.
• a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated 15 above.
• the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Oral compositions generally include an inert diluent or an edible carrier.
• the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
• Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
• Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain 25 any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, 30 methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid
• the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Such methods include those described in U.S. Patent No.6,468,798.
• Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
• the pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other 15 glycerides) or retention enemas for rectal delivery.
• the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl 20 acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
• Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.
• Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be 25 prepared according to methods known to those skilled in the art, for example, as described in U.S.
• Patent No.4,522,811 The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
• EXAMPLES 30 The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. 24 Attorney Docket No.29539-0415WO1/MGH 25105
• variable regions of our designed cDNAs encoding the variable heavy and light chain of humanized mAb107 were reverse translated using online program, and 10 synthesized by IDT. Secretion signal sequence was added at 5’-end of each chain.
• the N/D mutation was introduced to the heavy chain using QuikChange II XL site-directed mutagenesis kit (Agilent).
• the variable region of the heavy chain was joined to human IgG4 ⁇ constant region by overlapping PCR, and cloned into pcDNA3.4 using XbaI and XhoI restriction sites. Similarly, light chain variable region was fused to the human kappa 15 light constant region.
• Expi293 cells (Thermo Fisher) were used to produce recombinant antibodies. Cells were grown on orbital shaker (125 rpm) in Expi293 Expression Medium (Thermo Fisher) at 37°C in 5% CO2. Expi293 cells were transiently transfected with 1:1 heavy chain and light chain plasmids, using 20 manufacturer’s protocol. Culture supernatant was harvested 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. The protein sizes of the heavy and light chains and purity of the IgG were confirmed on SDSPAGE.
• Affinity of the humanized mAb107 vs. mouse mAb107 was measured using the A-domain of CD11b as ligand by 25 Bio-layer interferometry (BLI) using the Octet Assay system.
• Results The optimized amino acid sequences of heavy and light chains of humanized N55D-substituted mAb107 are as follows: A) Humanized Heavy Chain (CDR1, 2 and 3 are in bold; N57D substitution in 30 CDR2 indicated in lower case).
• the lower case “d” in CDR3 is the D residue that binds the metal ion at MIDAS.
• mAB107 IgG1 is QVQLVQSGAEVKKPGASVKVSCKPSGFNIKDIYMQWVRQAPGQRLEWIGRIDPAdDKTKYDPKFQ GRATITADTSASTAYLELSSLRSEDTAVYYCASEGHYGYdGYAMDYWGQGTTVTVSS

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