Antibodies that bind to human fibrin or fibrinogen gamma C domain and methods of use - Patents.com

Abstract

Described herein are novel and improved antibodies that bind to human fibrin or fibrinogen γC domain, and methods of using the same. In certain aspects, described herein are methods of inhibiting microglial activation. In certain aspects, described herein are pharmaceutical compositions that include antibodies that bind to fibrin or fibrinogen γC domain. In certain aspects, the antibodies and methods described herein are used for the treatment of degenerative neuropathies involving inflammatory demyelination. TIFF2024522237000012.tif81128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 212,409, filed June 18, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing Not applicable. [Background technology]

[0003] background Degenerative neurological disorders such as multiple sclerosis (MS) can involve inflammatory demyelination and autoimmune responses. Microglia, particularly perivascular microglia, are thought to be necessary not only for the maintenance but also for the development of inflammatory demyelination in central nervous system (CNS) autoimmune diseases. Microglial activation contributes to the death of both neurons and oligodendrocytes through the release of cytokines and nitric oxide. In MS, the inflammatory process is associated with the destruction of the myelin sheath and may be accompanied by axonal damage that can lead to permanent functional impairments such as paralysis and vision loss. Resident microglia are involved in demyelination through their ability to phagocytose myelin and secrete pro-inflammatory cytokines.

[0004] In MS lesions, perivascular activation of microglia colocalizes with areas of blood-brain barrier (BBB) ​​disruption, and in vivo imaging studies have shown that BBB disruption causes immediate and localized activation of microglia. One of the earliest events associated with BBB disruption in MS is leakage of the blood protein fibrinogen in the nervous system, which leads to perivascular fibrin deposition. Fibrinogen is absent in the healthy CNS and only leaks into the brain after BBB disruption, thus acting as an environmental "danger" signal. Once fibrinogen is converted to fibrin, the CD11b / CD18 integrin receptor (also known as Mac-1, aMfl 2, and complement receptor 3) binds to fibrin and induces microglial activation leading to inflammatory demyelination. CD11b is the alpha chain of the receptor that regulates the phagocytosis of myelin during inflammatory demyelination. Immobilized fibrinogen and insoluble fibrin, but not soluble fibrinogen, have been identified as the physiological, high-affinity ligands of Mac-1.

[0005] The γ377-395 epitope of the fibrin or fibrinogen γC domain is the binding epitope for CDIIb of fibrin. 377~395 The peptide functions as an inhibitor of microglial activation by blocking the binding of fibrin to Mac-1. Fibrin binds to platelet integrin α IIb Because CD11b mediates blood clotting by binding to the β3 receptor via a distinct epitope, therapeutic agents (including antibodies) that block the CD11b binding epitope to fibrin can reduce the damaging effects of fibrin in the nervous system without affecting its beneficial effects in blood clotting. Therefore, safe and effective antibodies that inhibit fibrin-induced microglial activation without affecting its beneficial effects in blood clotting are needed as therapeutic agents for degenerative neuropathies involving inflammatory demyelination. Summary of the Invention

[0006] overview In one aspect, described herein is an isolated antibody that binds to a human fibrin or fibrinogen γC domain, comprising a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein (i) CDR-H1 comprises the sequence set forth in SEQ ID NO: 1; (ii) CDR-H2 comprises the sequence set forth in SEQ ID NO: 2; (iii) CDR-H3 comprises the sequence set forth in SEQ ID NO: 3, where X is glycine (G), valine (V), threonine (T), serine (S), alanine (A), or leucine (L); (iv) CDR-L1 comprises the sequence set forth in SEQ ID NO: 4; (v) CDR-L2 comprises the sequence set forth in SEQ ID NO: 5, and (vi) CDR-L3 comprises the sequence set forth in SEQ ID NO: 6.

[0007] In one embodiment, the isolated antibody comprises a VH sequence selected from the sequence set forth in one of SEQ ID NOs: 7-20. In one embodiment, the isolated antibody comprises a VL sequence selected from the sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence selected from the sequence set forth in one of SEQ ID NOs: 7-20, and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 7 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 8 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 9 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 10 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 11 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 12 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 13 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 14 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 15 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises the VH sequence set forth in SEQ ID NO: 16 and the VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 21.In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 18 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 19 and a VL sequence set forth in SEQ ID NO: 21. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 20 and a VL sequence set forth in SEQ ID NO: 21.

[0008] In one embodiment, the isolated antibody is a humanized antibody, a human antibody or a chimeric antibody. In one embodiment, the isolated antibody is a humanized antibody. In one embodiment, the isolated antibody comprises a heavy chain human constant region of a class selected from IgG, IgA, IgD, IgE and IgM. In one embodiment, the isolated human Fc region comprises a human heavy chain constant region of a class IgG and a subclass selected from IgG1, IgG2, IgG3 and IgG4. In one embodiment, the human Fc region comprises a wild type human IgG1 Fc. In one embodiment, the human Fc domain comprises a sequence set forth in SEQ ID NO: 22. In one embodiment, the heavy chain comprises a constant heavy chain sequence set forth by SEQ ID NO: 22. In one embodiment, the light chain comprises a constant light chain sequence set forth by SEQ ID NO: 23. In one embodiment, the antibody comprises a VH sequence set forth in SEQ ID NO:7, and a VL sequence set forth in SEQ ID NO:21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the antibody comprises a VH sequence set forth in SEQ ID NO:8, and a VL sequence set forth in SEQ ID NO:21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO:9, and a VL sequence set forth in SEQ ID NO:21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO:10, and a VL sequence set forth in SEQ ID NO:21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO:11, and a VL sequence set forth in SEQ ID NO:21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO:12, and a VL sequence set forth in SEQ ID NO:21; and the human Fc region comprises wild-type human IgG1 Fc.In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 13, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 14, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 15, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 16, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 17, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 18, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 19, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc. In one embodiment, the isolated antibody comprises a VH sequence set forth in SEQ ID NO: 20, and a VL sequence set forth in SEQ ID NO: 21; and the human Fc region comprises wild-type human IgG1 Fc.

[0009] In one embodiment, the Fc region comprises one or more amino acid substitutions, wherein the one or more substitutions result in increased antibody half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more substitutions.

[0010] In one embodiment, the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one embodiment, the isolated antibody is a monoclonal antibody. In one embodiment, the antibody binds to the γ377-395 epitope (SEQ ID NO: X) of the fibrin or fibrinogen γC domain.

[0011] In one embodiment, the isolated antibody has a titer of about 1, 2, 3, 4, 5, 6, 7, or 8 x 10 as measured by a surface plasmon resonance (SPR) single cycle kinetics (SCK) assay. -5 K below M D In one embodiment, the isolated antibody binds to a peptide comprising the sequence of the γ377-395 epitope of the human fibrin or fibrinogen γC domain at about 8×10 as measured by a surface plasmon resonance (SPR) single cycle kinetics (SCK) assay. -5 K below M D It binds to a peptide containing the γ377-395 epitope sequence of the γC domain of human fibrin or fibrinogen.

[0012] In one embodiment, the isolated antibody inhibits Mac-1 binding to fibrin or fibrinogen γC domain.In one embodiment, the isolated antibody exhibits inhibition of microglial adhesion to fibrin or fibrinogen γC domain.

[0013] In one embodiment, the isolated antibody is used in the treatment of a degenerative disorder of the nervous system.

[0014] In certain aspects, described herein is an isolated polynucleotide or set of polynucleotides, optionally cDNA, encoding an antibody, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof, of any of the preceding claims.

[0015] In certain aspects, described herein is a vector or set of vectors that comprises a polynucleotide or set of polynucleotides described herein.

[0016] In certain aspects, described herein is a host cell comprising a polynucleotide or set of polynucleotides encoding an isolated antibody, or a vector or set of vectors comprising a polynucleotide encoding an isolated antibody.

[0017] In certain aspects, described herein are methods of producing an antibody, the methods comprising expressing the antibody in a host cell comprising an isolated antibody-encoding polynucleotide or set of polynucleotides, or a vector or set of vectors comprising an isolated antibody-encoding polynucleotide, and isolating the expressed antibody.

[0018] In certain aspects, described herein is a pharmaceutical composition comprising an isolated antibody and a pharma- ceutically acceptable excipient.

[0019] In certain aspects, described herein are kits comprising an isolated antibody or a pharmaceutical composition comprising an isolated antibody and instructions for use.

[0020] In certain aspects, described herein are methods for treating a degenerative disorder of the nervous system, comprising administering to a mammalian subject a therapeutically effective amount of an isolated antibody or a pharmaceutical composition comprising the isolated antibody. In certain embodiments, the degenerative disorder of the nervous system is selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0021] In certain aspects, described herein are methods for treating a pathology associated with Mac-1 binding to fibrin or fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount of an isolated antibody or a pharmaceutical composition comprising an isolated antibody described herein.

[0022] In certain aspects, described herein are methods of inhibiting microglial activation, the methods comprising administering to a mammalian subject a therapeutically effective amount of an isolated antibody or a pharmaceutical composition comprising an isolated antibody described herein.

[0023] In certain aspects, described herein are methods of preventing degenerative disorders of the nervous system, the methods comprising administering to a mammalian subject a therapeutically effective amount of an isolated antibody or a pharmaceutical composition comprising an isolated antibody described herein.

[0024] In certain aspects, described herein are methods of treating colitis, the methods comprising administering to a mammalian subject a therapeutically effective amount of an isolated antibody or a pharmaceutical composition comprising the isolated antibody described herein. In certain aspects, described herein are methods of preventing colitis, the methods comprising administering to a mammalian subject a therapeutically effective amount of an isolated antibody or a pharmaceutical composition comprising the isolated antibody described herein.

[0025] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief description of the drawings]

[0026] [Figure 1] Schematic diagram showing humanized variant IgG1 antibodies designed with six variant heavy chains and five variant light chains. VH0 and Vk0 correspond to the variable heavy and variable light chain original chimeric antibodies carrying the original anti-fibulin 5B8 monoclonal antibody CDRs. [Diagram 2]Schematic diagram showing sequence alignment of chimeric antibody (VH0 and Vk0) and humanized variant sequences. [Diagram 3] Schematic diagram illustrating the amino acid residue differences between VH3 and VH4, with the positions that differ between VH3 and VH4 highlighted. [Diagram 5] 1 is an image of the results of non-reducing SDS-PAGE analysis performed on purified antibody protein to verify the concentration of the antibody for affinity analysis. [Figure 6] 1 is a graph showing the results of a thermal stability analysis showing the first thermal transition (Tm1) and second thermal transition (Tm2) of six humanized antibody variants. [Figure 7] FIG. 7A is a graph showing the results of an ELISA performed with mouse monoclonal 5B8 antibody that binds to human fibrinogen and human fibrin. FIG. 7B is a graph showing the results of an ELISA performed with mouse monoclonal 5B8 antibody that binds to human P2 peptide. [Figure 8] Figure 8A is a graph showing the results of an ELISA performed with a mouse IgG2b isotype control antibody that binds to human fibrinogen and human fibrin, and Figure 8B is a graph showing the results of an ELISA performed with a mouse IgG2b isotype control antibody that binds to human P2 peptide. [Figure 9] Figure 9A is a graph showing the results of an ELISA performed with a mouse IgG1 isotype control antibody that binds to human fibrinogen and human fibrin, and Figure 9B is a graph showing the results of an ELISA performed with a mouse IgG1 isotype control antibody that binds to human P2 peptide. [Figure 10] Figure 10A is a graph showing the results of an ELISA performed with a mouse chimeric VH0Vk0 antibody (chimeric mouse monoclonal 5B8 antibody with human Fc) that binds to human fibrinogen and human fibrin. Figure 10B is a graph showing the results of an ELISA performed with a mouse chimeric VH0Vk0 antibody (chimeric mouse monoclonal 5B8 antibody with human Fc) that binds to human P2 peptide. [Figure 11] Figure 11A is a graph showing the results of an ELISA performed with a humanized VH3Vk1 antibody that binds to human fibrinogen and human fibrin. Figure 11B is a graph showing the results of an ELISA performed with a humanized VH3Vk1 antibody that binds to human P2 peptide. [Figure 12] Figure 12A is a graph showing the results of an ELISA performed with a humanized VH3Vk2 antibody that binds to human fibrinogen and human fibrin, and Figure 12B is a graph showing the results of an ELISA performed with a humanized VH3Vk2 antibody that binds to human P2 peptide. [Figure 13] Figure 13A is a graph showing the results of an ELISA performed with a humanized VH4Vk1 antibody that binds to human fibrinogen and human fibrin, and Figure 13B is a graph showing the results of an ELISA performed with a humanized VH4Vk1 antibody that binds to human P2 peptide. [Figure 14] Figure 14A is a graph showing the results of an ELISA performed with a humanized VH4Vk2 antibody that binds to human fibrinogen and human fibrin, and Figure 14B is a graph showing the results of an ELISA performed with a humanized VH4Vk2 antibody that binds to human P2 peptide. [Figure 15] Figure 15A is a graph showing the results of an ELISA performed with a humanized VH5Vk2 antibody that binds to human fibrinogen and human fibrin, and Figure 15B is a graph showing the results of an ELISA performed with a humanized VH5Vk2 antibody that binds to human P2 peptide. [Figure 16] Figure 16A is a graph showing the results of an ELISA performed with a humanized VH6Vk2 antibody that binds to human fibrinogen and human fibrin, and Figure 16B is a graph showing the results of an ELISA performed with a humanized VH6Vk2 antibody that binds to human P2 peptide. [Figure 17] Figure 17A is a graph showing the results of a clotting assay control. Figure 17B is a graph showing the results of a clotting assay using 20 ug of selected humanized antibodies. [Figure 18] Schematic illustrating the procedure for affinity determination by steady-state analysis using single-cycle kinetics. [Figure 19] 1 is a graph showing the results of SEC-HPLC analysis of selected humanized variants. [Figure 20] Graph showing raw and fitted data from multi-cycle kinetics analysis of select variants. [Figure 21] 1 is a diagram illustrating potential amino acid residue liabilities within VH CDR sequences in humanized variant antibodies. [Figure 22] FIG. 13 is a graph illustrating capture levels obtained by manually loading HEK cell supernatants for Biacore steady-state analysis for a chimeric antibody, the original humanized variant antibody (VH4 / Vk2) and (VH5Vk2) with cysteine ​​102, and 12 humanized variant antibodies with cysteine ​​substitutions. [Figure 23] Steady-state analysis of 12 humanized variant antibodies with cysteine ​​substitutions using single-cycle kinetics is shown. [Figure 24] Figure 24: Effect of ICV injection of humanized anti-fibrin antibody variant VH5 C102G / VK2 on microglial activation, oxidative stress, and macrophage recruitment was evaluated. 10 ug of antibody was administered prophylactically by icv injection to FIE mice (Figure 24). Each circle represents an individual animal. Data are mean ± sem. One-way ANOVA with Tukey's multiple comparisons. [Diagram 25] 13 is an image of a tissue section from a mouse with chronic EAE bearing fibrinogen accumulation in the spinal cord lesion stained with 10 mg / ml VH5 C102G / VK2-biotin and CY3-streptavidin antibodies. [Figure 26]1 is a graph showing the pharmacokinetic profile of humanized anti-fibrin antibody variant VH5 C102G / VK2. Antibodies were detected by ELISA in plasma from EAE mice administered either 10 mg / Kg or 30 mg / Kg of VH5 C102G / VK2 antibody. [Figure 27] 1 is a graph showing the pharmacokinetic profile of humanized anti-fibrin antibody variant VH5 C102G / VK2. Antibodies were detected by ELISA in plasma and blood from wild-type Balb / c mice administered the VH5 C102G / VK2 antibody. [Figure 28] FIG. 28A is a graph showing the reduction of microglia in mice with fibrinogen-induced encephalomyelitis (FIE) administered either 10 mg / kg or 30 mg / kg of VH5 C102G / VK2 humanized antibody. Tissues were stained with Iba-1 (microglia marker, dilution 1:750). Iba-1 immunoreactivity (Iba-1+ area) was then calculated. FIG. 28B is a graph showing the reduction of macrophage infiltration in mice with fibrinogen-induced encephalomyelitis (FIE) administered either 10 mg / kg or 30 mg / kg of VH5 C102G / VK2 humanized antibody. Tissues were stained with Mac-2 (macrophage infiltration marker, dilution 1:750). Mac-2 immunoreactivity (Mac-2+ area) was then calculated. [Figure 29] FIG. 13 is a graph showing clinical scores of PLP EAE in N=10 mice prophylactically injected with humanized anti-fibrin antibody (5 mg / kg ip every 3 days). [Diagram 30] Figure 30A is a graph showing time to disease onset in PLP EAE in N=10 mice injected prophylactically with humanized anti-fibrin antibody (5 mg / kg ip every 3 days). Figure 30B is a graph showing paralysis rate in PLP EAE in N=10 mice injected prophylactically with humanized anti-fibrin antibody (5 mg / kg ip every 3 days). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description definition Unless otherwise defined, all technical terms, notations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and conditions, unless otherwise noted.

[0028] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0029] It is understood that the aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0030] For all compositions described herein and all methods of using the compositions described herein, the composition can include the recited components or steps or can "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" recited components, the composition can include the recited components and other components that do not substantially affect the condition being treated, but do not include any other components that do not substantially affect the condition being treated, other than the components explicitly recited, or when the composition includes additional components other than the recited components that substantially affect the condition being treated, the composition does not include the additional components in a concentration or amount sufficient to substantially affect the condition being treated. When a method is described as "consisting essentially of" recited steps, the method can include the recited steps and other steps that do not substantially affect the condition being treated, but the method does not include any other steps that substantially affect the condition being treated, other than the steps explicitly recited. As a non-limiting example, when a composition is described as "consisting essentially of" components, the composition can further include any amount of a pharma- ceutically acceptable carrier, vehicle, or diluent, and such other components that do not substantially affect the condition being treated.

[0031] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0032] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include "transformants" (or "transformed cells") and "transfectants" (or "transfected cells"), which include the primary transformed or transfected cells and their derived progeny, respectively. Such antibodies may not be completely identical in nucleic acid content to the parent cell and may contain mutations. "Recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells.

[0033] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocotyledons, dicotyledons, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0034] As used herein, the term "prokaryote" refers to prokaryotic organisms. For example, non-eukaryotes may be from the Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or Archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, e.g., Haloferax volcanii, etc.) phylogenetic domain. volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

[0035] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic compound, such as an anti-fibrin (FIBRIN) antibody, administered to an individual, either as a single dose or as part of a series of doses, that is effective to produce or contribute to a desired therapeutic effect, either alone or in combination with another therapy. Examples of desired therapeutic effects are enhanced immune response, slowing or delaying tumor development; stabilization of disease; amelioration of one or more symptoms. An effective amount can be given in one or multiple doses.

[0036] The term "treat" (and variations thereof, such as "treat" or "treatment") refers to a clinical intervention that seeks to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed during the course of clinical pathology. Desirable effects of treatment include inhibiting recurrence of disease, alleviating symptoms, reducing direct or indirect pathological consequences of disease, inhibiting metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and remission or improving prognosis.

[0037] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate an immune response in a subject.

[0038] As used herein, the term "subject" or "individual" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with the antibodies provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0039] The term "in vitro" refers to processes carried out within living cells that are grown apart from an organism, for example, in tissue culture.

[0040] The term "in vivo" refers to a process that takes place within an organism.

[0041] The term "package insert" is used to refer to instructions customarily included in a commercial package (e.g., a kit) of a therapeutic or diagnostic agent that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic or diagnostic agent.

[0042] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective in treating a subject, and that does not contain additional ingredients that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0043] The terms "co-administration," "co-administering," and "in combination with" include administration of two or more therapeutic agents simultaneously, concurrently, or sequentially without specific time limitations. In one embodiment, the agents are present in a cell or in a subject's body at the same time or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to administration of a second therapeutic agent.

[0044] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0045] The terms "increase" and "activate" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater increase in the recited variable.

[0046] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater decrease in the recited variable.

[0047] The term "about" connotes and encompasses the indicated value and a range above and below that value. In certain embodiments, the term "about" refers to the indicated value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "about" refers to the indicated value ± one standard deviation of that value.

[0048] The term "agonize" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to a receptor and agonizes the receptor.

[0049] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0050] For any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0051] The term "optionally," when used consecutively, means the inclusion of one to all of the listed combinations, and contemplates all subcombinations.

[0052] The term "amino acid" refers to the 20 common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0053] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope).

[0054] As used herein, "k d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is k off Also called value.

[0055] As used herein, "k a " " -1 ×sec -1 The term k ) refers to the binding rate constant for a particular antibody-antigen interaction. This value is k on Also called value.

[0056] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. D = k d / k a In some embodiments, the affinity of an antibody is determined by the K D For clarity, as known in the art, K D A smaller value indicates a higher affinity interaction, and K D Higher values ​​indicate lower affinity interactions.

[0057] As used herein, "K A " " -1 The term K ) refers to the binding equilibrium constant of a particular antibody-antigen interaction. A = k a / k d .

[0058] The term "antibody" is used herein in its broadest sense and includes certain immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0059] A "fibrin antibody", "anti-fibrin antibody" or "fibrin-specific antibody" as provided herein is an antibody that specifically binds to the antigen fibrin. In some embodiments, the antibody binds to the extracellular domain of fibrin. In certain embodiments, the fibrin antibody provided herein binds to an epitope of fibrin that is conserved between or within fibrin proteins from different species.

[0060] The term "epitope" refers to a portion of an antigen that specifically binds to an antibody.

[0061] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops").

[0062] The term "antigen-binding domain" means a part of an antibody capable of specifically binding to an antigen or epitope.

[0063] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0064] The term "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0065] The term "humanized antibody" refers to a protein having a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions such that the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response when administered to a human subject, as compared to the non-human species antibody.

[0066] The term "multispecific antibody" refers to an antibody that comprises two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes.

[0067] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is a natural IgG molecule that is bivalent (i.e., has two antigen-binding domains) but recognizes the same epitope at each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.

[0068] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope, except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present in small amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0069] The term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in a single chain Fab molecule. As described in more detail herein, an scFv has a variable domain of the light chain (VL) connected from its C-terminus to the N-terminus of the variable domain of the heavy chain (VH) by a polypeptide chain. Alternatively, an scFv comprises a polypeptide chain in which the C-terminus of the VH is connected to the N-terminus of the VL by a polypeptide chain.

[0070] The "Fab fragment" (also called fragment antigen-binding) contains the variable domains VL and VH of the light and heavy chains, respectively, as well as the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The variable domains contain the complementarity determining loops (CDRs, also called hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.

[0071] An "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0072] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0073] "Single-chain Fv" or "sFv" or "scFv" comprises the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0074] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. The Fc domain can follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0075] The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. sdabs are fairly stable and easy to express as fusion partners with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Appl. Microbiol Biotechnol. 77(1): 13-22).

[0076] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain including an Fc region. For example, when used to refer to an IgG molecule, a "full length antibody" is an antibody that contains two heavy chains and two light chains.

[0077] The term "antibody fragment" refers to an antibody that contains a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0078] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions.

[0079] The term "substantially purified" refers to constructs described herein, or variants thereof, which may be substantially or essentially free from components normally associated with or interacting with the protein as found in its naturally occurring environment, i.e., in a native cell, or, in the case of recombinantly produced heteromultimers, in a host cell, and in certain embodiments are substantially free of cellular material, including preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein.

[0080] The term percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., using publicly available computer software such as BLAST, BLASTP, BLASTN, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software or other algorithms available to those of skill in the art) or by visual inspection. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or can exist over the entire length of the two sequences being compared.

[0081] In sequence comparison, one sequence usually serves as reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinate is designated if necessary, and sequence algorithm program parameter is designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on designated program parameter.

[0082] Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0083] It is understood that the ranges recited herein are shorthand for all values ​​within the range, including the recited end points.For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0084] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0085] Antifibrin antibodies antibody structure The present application provides antibodies and compositions comprising the antibodies that bind to fibrin proteins.

[0086] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, several of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

[0087] An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50-70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The IgG1 heavy chain is composed of the VH, CH1, CH2 and CH3 domains, respectively, in the N-terminal to C-terminal direction. The light chain is composed of the VL and CL domains in the N-terminal to C-terminal direction. The IgG1 heavy chain includes a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin construct includes at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to a therapeutic polypeptide. In some embodiments, the immunoglobulin domain found in the antibody provided herein is from or derived from an immunoglobulin-based construct, such as a diabody or nanobody.In certain embodiments, the immunoglobulin construct described herein comprises at least one immunoglobulin domain from a heavy chain antibody, such as a camelid antibody.In certain embodiments, the immunoglobulin construct provided herein comprises at least one immunoglobulin domain from a mammalian antibody, such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody, or any chimeric antibody.

[0088] In some embodiments, the antibody provided herein comprises a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0089] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0090] Typically, a native four-chain antibody contains six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and / or complementarity determining regions (CDRs), the latter of which are most highly sequence variable and / or involved in antigen recognition. Except for CDR1 in VH, CDRs typically contain amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable regions that form the antigen-binding region. This particular region has been described by Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983), and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is intended that the application of either definition to refer to the CDR of an antibody or its variants is within the scope of the term as defined and used herein.The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues constitute a particular CDR given the variable region amino acid sequence of an antibody.

[0091] The amino acid sequence boundaries of the CDRs may be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme); each of which is incorporated by reference in its entirety.

[0092] Table A shows the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0093] CDRs can be assigned using antibody numbering software such as Abnum, available at www.bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, which is incorporated by reference in its entirety.

[0094] (Table A) Residues in the CDRs according to the Kabat and Chothia numbering scheme. TIFF2024522237000001.tif46145 * The C-terminus of CDR-H1, numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the CDR.

[0095] The "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise indicated, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0096] An example of an antigen-binding domain is the antigen-binding domain formed by the VH-VL dimer of an antibody.Another example of an antigen-binding domain is the antigen-binding domain formed by diversifying a specific loop from the tenth fibronectin type III domain of an adnectin.An antigen-binding domain can include CDRs 1, 2 and 3, in that order, from the heavy chain; and CDRs 1, 2 and 3, in that order, from the light chain.

[0097] Epitopes often consist of surface accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former may be lost in the presence of denaturing solvents, but the binding to the latter is not lost. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination, such as, for example, testing the binding of the antibody to fibrin variants with different point mutations or chimeric fibrin variants.

[0098] To screen for antibodies that bind to an epitope on a target antigen (e.g., fibrin) to which an antibody of interest binds, a routine cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, or in addition, epitope mapping can be performed by methods known in the art.

[0099] A chimeric antibody has a portion of the heavy and / or light chain derived from a particular source or species, and the remainder of the heavy and / or light chain derived from a different source or species.

[0100] A human antibody is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0101] A humanized antibody has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions such that the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response when administered to a human subject, compared to the non-human species antibody. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the immunogenic potential of the non-human antibody when administered to a human subject, either the altered amino acid residues are not important for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of methods for making humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0102] The two or more different epitopes may be epitopes on the same antigen (e.g., a single fibrin molecule expressed by a cell) or may be epitopes on different antigens (e.g., different fibrin molecules, or a fibrin molecule and a non-fibrin molecule, expressed by the same cell). In some aspects, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some aspects, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody").

[0103] Anti-fibrin antibodies can include those described herein, such as the clones listed in the figures and / or tables. In some embodiments, the antibody comprises an alternative scaffold. In some embodiments, the antibody consists of an alternative scaffold. In some embodiments, the antibody consists essentially of an alternative scaffold. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the antibody consists of an antibody fragment. In some embodiments, the antibody consists essentially of an antibody fragment.

[0104] In some embodiments, the antibody is a monoclonal antibody.

[0105] In some embodiments, the antibody is a polyclonal antibody.

[0106] In some embodiments, the antibody is produced by a hybridoma, hi other embodiments, the antibody is produced by a recombinant cell that has been engineered to express the desired variable and constant domains.

[0107] In some embodiments, the antibody may be a single chain antibody or other antibody derivative or variant thereof that retains the antigen specificity and lower hinge region.

[0108] In some embodiments, the antibody may be a polyfunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment or variant thereof. In certain embodiments, the antibody fragment or derivative thereof is selected from a Fab fragment, a Fab'2 fragment, a CDR and a ScFv.

[0109] In some embodiments, the antibody can form an immune complex, for example, the immune complex can be a tumor cell coated with the antibody.

[0110] In sequence comparison, one sequence usually serves as reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinate is designated if necessary, and sequence algorithm program parameter is designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on designated program parameter.

[0111] Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0112] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0113] Fibrin antibody sequence V H domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 7. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 8. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 11. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 13. H In some embodiments, the antibodies provided herein comprise the V HIn some embodiments, the antibodies provided herein comprise the V H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 16. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 17. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 18. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 19. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 20. H Contains arrays.

[0114] In some embodiments, the antibodies provided herein are selected from the exemplary VV ... H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H In some embodiments, the antibodies provided herein comprise a V or VL sequence as provided in SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 2 ... HThe antibody may be a nucleotide sequence of the antibody provided herein. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibody described in this paragraph is referred to herein as a "variant". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0115] V L domain In some embodiments, the antibody provided herein comprises a VL sequence selected from SEQ ID NO:21.

[0116] In some embodiments, the antibodies provided herein comprise a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity with the exemplary VL sequence provided in SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise a VL sequence provided in SEQ ID NO: 21 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0117] VH-VL combinations In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. H and V selected from SEQ ID NO: 21. L Contains arrays.

[0118] In some embodiments, the antibody provided herein comprises the V H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 8. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 11. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 13. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the V H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 16. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 17. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 18. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 19. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 20. H Sequence and V of SEQ ID NO: 21 L Contains arrays.

[0119] In certain aspects, any of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 can be combined with any of SEQ ID NOs: 21.

[0120] In some embodiments, the antibodies provided herein are selected from the exemplary VV ... H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence Hand V sequences having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary V sequences provided in SEQ ID NO: 21. L In some embodiments, the antibodies provided herein comprise a VH sequence as provided in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions, and a VL sequence as provided in SEQ ID NO: 21, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0121] CDR In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. H In some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. HIn some embodiments, the antibodies provided herein comprise 2-3 CDRs of a VH domain selected from SEQ ID NOs: 37, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some aspects, the CDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0122] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, the CDR-H1 is the CDR-H1 of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is a CDR-H2 of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 is a CDR-H3 of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., by the methods provided herein for obtaining antibodies.

[0123] In some embodiments, the antibodies provided herein comprise one to three CDRs of the VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise two to three CDRs of the VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise three CDRs of the VL domain of SEQ ID NO: 21. In some aspects, the CDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0124] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NO: 21. In some embodiments, CDR-L1 is CDR-L1 of the VL domain of SEQ ID NO: 21 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, CDR-L2 is CDR-L2 of the VL domain of SEQ ID NO: 21 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR-L3 is CDR-L3 of the VL domain of SEQ ID NO: 21 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0125] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and one to three CDRs of a VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and two to three CDRs of a VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and three CDRs of a VL domain of SEQ ID NO: 21. In some aspects, the CDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0126] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 24, 25, 26, 27, 28, 29, and 30. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 24, 25, 26, 27, 28, 29, and 30 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., by the methods provided herein for obtaining antibodies.

[0127] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 24. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 24. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0128] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 25. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 25. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0129] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 26. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0130] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 27. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 27. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 27 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0131] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 28. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 28. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0132] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 29. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 29. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 29 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0133] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 30. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 30. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 30 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0134] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 3. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 3. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 3 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0135] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1. In some aspects, the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H1 is a CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0136] In some embodiments, the antibodies provided herein comprise a selected CDR-H2 of SEQ ID NO: 2. In some aspects, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H2 of SEQ ID NO: 2. In some embodiments, the CDR-H2 is a selected CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0137] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 24 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 24, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0138] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 25, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0139] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 26 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 26, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 26, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0140] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 27 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 27, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 27, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 27 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0141] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 28 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 28, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 28, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0142] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H3 of SEQ ID NO: 29, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 29 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0143] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 30, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 30 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0144] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 6. In some aspects, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-L3 of SEQ ID NO: 6. In some embodiments, the CDR-L3 is a CDR-L3 of SEQ ID NO: 6 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0145] In some embodiments, the antibodies provided herein comprise a CDR-L2 of SEQ ID NO: 5. In some aspects, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-L2 of SEQ ID NO: 5. In some embodiments, the CDR-L2 is a CDR-L2 of SEQ ID NO: 5 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0146] In some embodiments, the antibodies provided herein comprise a CDR-L1 of SEQ ID NO: 4. In some aspects, the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-L1 is a CDR-L1 of SEQ ID NO: 4 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0147] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 6 and a CDR-L2 of SEQ ID NO: 5. In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 6, a CDR-L2 of SEQ ID NO: 5, and a CDR-L1 of SEQ ID NO: 4. In some embodiments, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO:6, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L2 of SEQ ID NO:5, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO:4. In some embodiments, the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0148] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:24, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 24, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 24 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0149] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:25, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 25, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6, and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 25 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0150] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:26, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 26, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 26 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0151] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:27, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 27, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 27 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0152] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:28, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 28, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 28 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0153] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:29, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 29, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 29 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0154] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30, a CDR-H2 of SEQ ID NO: 2, a CDR-H1 of SEQ ID NO: 1, a CDR-L3 of SEQ ID NO: 6, a CDR-L2 of SEQ ID NO: 5, and a CDR-L1 of SEQ ID NO: 4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 30, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 30 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0155] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 24, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0156] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 25, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0157] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 26, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0158] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 27, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0159] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 28, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0160] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 29, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0161] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 30, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0162] Fc area The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or a modified Fc region as described in the art or elsewhere in this disclosure.

[0163] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, the Fc polypeptide of a dimeric IgG Fc includes IgG CH2 and IgG CH3 constant domain sequences. Fc may be of IgA, IgD, IgE, IgG, and IgM classes, some of which may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0164] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, an FcR can be a native sequence human FcR. Generally, FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibitory receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes can also be bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0165] Modification of CH2 domain can affect FcR binding to Fc. Some amino acid modifications in Fc region are known in the art to selectively modify the affinity of Fc to different Fc gamma receptors. In some aspects, Fc comprises one or more modifications to promote selective binding to Fc-gamma receptors.

[0166] Exemplary mutations that alter binding of FcR to Fc are listed below: S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu Y, Vernes JM, Chiang N, et al. J Immunol Methods. 2011 Feb 28;365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen JB, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep 15;67(18):8882-90; Nordstrom JL, Gorlatov S, Zhang W, et al. Breast Cancer Res. 2011 Nov 30;13(6):R123); F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Sel. 2011 Sep;24(9):671-8.), S298A / E333A / K334A (Shields RL, Namenuk AK, Hong K, et al. J Biol Chem. 2001 Mar 2;276(9):6591-604); S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S, et al. Proc Natl Acad Sci US A. 2006 Mar 14;103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S, et al. Mol Immunol. 2008 Sep;45(15):3926-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other mutations listed in WO2011 / 120134 and WO2011 / 120135, which are incorporated herein by reference. Therapeutic Antibody Engineering (by William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012) lists the mutations on page 283.

[0167] In some embodiments, the antibody described herein comprises modifications to improve its ability to mediate effector function.Such modifications are known in the art and include defucosylation or engineering the affinity of Fc to activating receptors, mainly FCGR3a for ADCC and C1q for CDC.The following Table B summarizes the various designs reported in the literature for engineering effector function.

[0168] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn 297 EU numbering) without modifying the amino acid sequence are well known in the art. GlymaX® technology (ProBioGen AG) is based on the introduction of a gene for an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the addition of the sugar "fucose" to the N-linked antibody carbohydrate moiety by the antibody producing cells. (von Horsten et al. (2010) Glycobiology. 2010 Dec; 20 (12):1607-18. Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Pat. No. 8,409,572, which teaches that cell lines for producing antibodies are selected for their ability to produce lower levels of antibody fucosylation; the antibodies can be fully defucosylated (meaning that they contain no detectable fucose) or they can be partially defucosylated, meaning that the isolated antibody contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose normally found for a similar antibody produced by a mammalian expression system.

[0169] Thus, in one embodiment, the antibodies described herein can comprise a dimeric Fc that contains one or more amino acid modifications as described in Table B that confer improved effector function. In another embodiment, the antibodies can be defucosylated to improve effector function.

[0170] (Table B) CH2 domain and effector function engineering TIFF2024522237000002.tif86148

[0171] Fc modifications that reduce FcgR and / or complement binding and / or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curr Opin Biotech 20:685-691, and Strohl, WR and Strohl LM, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp 225-249). These strategies include reducing effector function by modifying glycosylation, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 regions of Fc. For example, U.S. Patent Application Publication No. 2011 / 0212087 (Strohl), International Patent Application Publication No. WO 2006 / 105338 (Xencor), U.S. Patent Application Publication No. 2012 / 0225058 (Xencor), U.S. Patent Application Publication No. 2012 / 0251531 (Genentech), and Strop et al ((2012) J. Mol. Biol. 420: 204-219) describe specific modifications that reduce binding of FcgR or complement to Fc.

[0172] Specific non-limiting examples of amino acid modifications known to reduce binding of FcgR or complement to Fc include those identified in Table C below.

[0173] (Table C) Modifications that reduce FcgR or complement binding to Fc TIFF2024522237000003.tif114128

[0174] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn 297 EU numbering) without modifying the amino acid sequence are well known in the art. GlymaxX® technology (ProBioGen AG) is based on the introduction of a gene for an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the addition of the sugar "fucose" to the N-linked antibody carbohydrate moiety by the antibody producing cells. (von Horsten et al. (2010) Glycobiology. 2010 Dec; 20 (12):1607-18.) Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD) (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells, which are deficient in protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Application Publication No. 2003 / 0157108; WO 2004 / 056312; These include knockout cell lines, such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (see Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107; each of which is incorporated by reference in its entirety). Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Patent No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to produce lower levels of antibody fucosylation.

[0175] Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD) (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells that are deficient in protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Application Publication No. 2003 / 0157108; WO 2004 / 056312; each of which is incorporated by reference in its entirety), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107; each of which is incorporated by reference in its entirety.

[0176] The antibodies can be completely defucosylated (meaning that they contain no detectable fucose) or they can be partially defucosylated, meaning that the isolated antibody contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15% or less than 5% of the amount of fucose typically detected for a similar antibody produced in a mammalian expression system.

[0177] In some aspects, the antibodies provided herein comprise an IgG1 domain with reduced fucose content at Asn 297 compared to naturally occurring IgG1 domains. Such Fc domains are known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, which is incorporated by reference in its entirety. In some aspects, such antibodies do not comprise fucose at Asn 297. The amount of fucose can be determined using any suitable method, for example, as described in WO 2008 / 077546, which is incorporated by reference in its entirety.

[0178] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of Fc region positions 298, 333, and 334. In some embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions at positions 239, 332, and 330, as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006,103:4005-4010, which is incorporated by reference in its entirety.

[0179] Other exemplary glycosylation variants that can be incorporated into the antibodies provided herein are described, e.g., in U.S. Patent Application Publication Nos. 2003 / 0157108, 2004 / 0093621, 2003 / 0157108, 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0132140, 2004 / 0110704, 2004 / 0110282, 2004 / 0109865; Nos. 2000 / 61739, 2001 / 29246, 2003 / 085119, 2003 / 084570, 2005 / 035586, 2005 / 035778; 2005 / 053742, 2002 / 031140; Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249; and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of which is incorporated by reference in its entirety.

[0180] In some embodiments, the antibody provided herein comprises an Fc region with at least one galactose residue in the oligosaccharide attached to the Fc region.Such antibody variants can have improved CDC function.Examples of such antibody variants are described in, for example, WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764; each of which is incorporated by reference in its entirety.

[0181] In some embodiments, the antibodies provided herein contain one or more modifications that improve or attenuate C1q binding and / or CDC. See U.S. Patent No. 6,194,551; WO 99 / 51642; and Idusogie et al., J. Immunol., 2000, 164:4178-4184; each of which is incorporated by reference in its entirety.

[0182] join The affinity of a molecule X for its partner Y is expressed as the dissociation equilibrium constant (K D The affinity can be expressed by the following equation: . The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0183] With respect to antibody binding to a target molecule, the terms "bind", "specifically binding", "specifically binds", "specific to", "selectively binds" and "selective to" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from non-specific or non-selective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics an epitope recognized on the target molecule. In that case, specific binding is indicated if binding of the antibody to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 50% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 40% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 30% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 20% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 10% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 1% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 0.1% of its affinity to fibrin.

[0184] When used herein in the context of two or more antibodies, the term "competes with" or "cross-competes with" indicates that two or more antibodies compete for binding to an antigen (e.g., fibrin). In one exemplary assay, fibrin is coated on a surface and contacted with a first fibrin antibody, followed by the addition of a second fibrin antibody. In another exemplary assay, a first fibrin antibody is coated on a surface and contacted with fibrin, followed by the addition of a second fibrin antibody. In either assay, antibodies compete with each other if the presence of the first fibrin antibody reduces the binding of the second fibrin antibody. The term "competes with" also includes antibody combinations in which one antibody reduces the binding of another antibody, but no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit each other's binding regardless of the order in which they are added. In some embodiments, an antibody reduces the binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% when measured in a competitive binding assay. A person skilled in the art can select the concentration of antibody used in a competitive assay based on the affinity of the antibody for fibrin and the valency of the antibody. The assays described in this definition are exemplary, and a person skilled in the art can use any suitable assay to determine whether antibodies compete with each other.Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated by reference in its entirety.

[0185] A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits or blocks the binding of the reference antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when measured in a competitive binding assay. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody for steric hindrance to occur. For example, a second competing antibody can be identified that competes with the first antibody described herein for binding to fibrin. In some cases, the second antibody can block or inhibit the binding of the first antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when measured in a competitive binding assay. In some cases, the second antibody can replace the first antibody by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0186] In some embodiments, the anti-fibrin antibody does not substantially bind to myeloid cells present outside the cancer tissue. In some embodiments, the anti-fibrin antibody does not substantially bind to stimulatory myeloid cells present within the cancer tissue.

[0187] In some embodiments, the anti-fibrin antibody binds to residues γ377-395 of the fibrin or fibrinogen γC domain of human fibrin (SEQ ID NO: 31). The binding epitope includes residues within a numerical range (e.g., residues 377-395 of fibrin), the beginning residue of each range (e.g., residues 377-394 of human fibrin) and the ending residue of each range (e.g., residues 378-395 of human fibrin), or any combination thereof.

[0188] In some embodiments, the antibodies provided herein have a molecular mass of about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 x 10 as measured by a Biacore assay. -6 K below M D In some embodiments, the K of the antibodies provided herein binds to human fibrin. D is approximately 0.001-0.01, 0.01-0.1, 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-1, 0.25-0.75, 0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-2, 1-5, 2-7, 3-8, 3-5, 4-6, 5-7, 6-8, 7-9, 7-10, or 5-10 x 10 as measured by Biacore assay. -6 In some embodiments, the antibodies provided herein have a titer of about 1×10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, or 1×10 -9 K below M D It binds to human fibrin.

[0189] In some embodiments, the antibodies provided herein have a molecular mass of about 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001 x 10 as measured by a Biacore assay. -5 K less than or equal to M D In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 5-3, 4-2, 3-1, 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, 1.9-1.5, 1.5-1, 1-0.8, 1-0.5, 0.9-0.6, 0.7-0.4, 0.6-0.2, 0.5-0.3, 0.3-0.2, 0.2-0.1, 0.1-0.01, 0.01-0.001, or 0.001-0.0001 x 10 as measured by a Biacore assay. -5 K of M D In some embodiments, the antibodies provided herein bind to human fibrin at about 10, 9.56, 9.5, 9.0, 8.88, 8.84, 8.5, 8, 7.5, 7.32, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 x 10 as measured by a Biacore assay. -4 (1 / s) or less, or K smaller d In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 7-10, 7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8.-8.5, 8.5-9, 9-9.5, or 9.5-10 x 10 as measured by a Biacore assay. -4 (1 / s)K dIn some embodiments, the antibodies provided herein bind to human fibrin at about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 45, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 7, 8, 9, or 10×10 as measured by a Biacore assay. 5 K greater than or equal to (1 / Ms) a In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 4-7, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10 x 10 as measured by a Biacore assay. 5 (1 / Ms)K a It binds to human fibrin (FIBRIN).

[0190] function "Effector function" refers to the biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include receptor ligand blocking, receptor agonism, or antagonism, C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the effector function of the fibrin antibodies described herein is antagonism, blocking Mac-1 receptor binding to fibrin.

[0191] Pharmaceutical Compositions The present application provides compositions comprising antibodies, including pharmaceutical compositions comprising any one or more of the antibodies described herein together with one or more pharma- ceutically acceptable excipients.In some embodiments, the compositions are sterile.The pharmaceutical compositions typically comprise an effective amount of antibodies.

[0192] These compositions may contain, in addition to one or more of the antibodies disclosed herein, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other materials may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal.

[0193] The pharmaceutical composition for oral administration can be in the form of tablet, capsule, powder or liquid. Tablets can contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions usually contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0194] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity and stability.Those skilled in the art can fully prepare appropriate solutions using isotonic vehicles such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary.

[0195] The anti-fibrin antibody given for administration to an individual is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (although in some cases prevention can also be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. The determination of treatment prescription, such as dosage, is within the responsibility of general practitioners and other physicians, and will usually take into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0196] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated.

[0197] method Preparation method The antibodies described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an antibody described herein. Such a nucleic acid can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of an antibody (e.g., the light and / or heavy chain of an antibody), or an amino acid sequence comprising the VHH of a single domain antibody. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In one embodiment, the nucleic acid is provided in a multicistronic vector. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antigen-binding polypeptide construct. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody is provided, the method comprising culturing a host cell comprising an antibody-encoding nucleic acid as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0198] For recombinant production of an antibody, for example, nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0199] When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein is present in certain embodiments at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1% of the dry weight of the cells or less. When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein is present in the medium in certain embodiments at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L of the dry weight of the cells or less. In certain embodiments, the "substantially purified" heteromultimers produced by the methods described herein have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically at least about 75%, 80%, 85%, and more specifically at least about 90%, at least about 95%, at least about 99% or more, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0200] Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0201] A recombinant host cell, or host cell, is a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for creating a recombinant host cell. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or may be integrated into the host genome. The host cell may include CHO, a derivative of CHO, NS0, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0202] For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, antibodies may be isolated in a soluble fraction from bacterial cell paste and can be further purified.

[0203] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0204] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. A number of baculovirus strains have been identified that can be used for transfection of insect cells, particularly Spodoptera frugiperda cells.

[0205] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0206] Vertebrate cells may be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); see, e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0207] In one embodiment, the antibodies described herein are produced in stable mammalian cells by a method comprising transfecting at least one stable mammalian cell with nucleic acids encoding the antibodies in a predetermined ratio; and expressing the nucleic acids in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that results in the highest percentage of antibody in the expression product.

[0208] In some embodiments, there are methods of producing antibodies in stable mammalian cells as described herein, wherein the expression product of at least one stable mammalian cell comprises a greater proportion of the desired glycosylated antibody as compared to monomeric heavy or light chain polypeptides, or other antibodies.

[0209] In some embodiments, there is a method of producing a glycosylated antibody in a stable mammalian cell as described herein, the method comprising identifying and purifying a desired glycosylated antibody, in some embodiments, the identification is by one or both of liquid chromatography and mass spectrometry.

[0210] If necessary, the antibody can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins can find use in the present invention for purification of antibodies. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification is often made possible by the specific fusion partner. For example, glutathione resin is used when GST fusions are utilized, Ni when His tags are utilized, etc. +2 The antibody can be purified using affinity chromatography or, if a flag tag is used, using immobilized anti-flag antibodies. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994, which is incorporated by reference in its entirety. The degree of purification required varies depending on the use of the antibody. In some cases, no purification is necessary.

[0211] In certain embodiments, antibodies are purified using anion exchange chromatography, including but not limited to, chromatography on Q-Sepharose, DEAE Sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0212] In certain embodiments, the proteins described herein are purified using cation exchange chromatography, including, but not limited to, SP Sepharose, CM Sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and equivalents.

[0213] Moreover, the antibodies described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids generally include, but are not limited to, D-isomers of the common amino acids, 2,4 diaminobutyric acid, alpha-aminoisobutyric acid, 4 aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6 aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoro-amino acids, designer amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs. Additionally, amino acids can be D (dextrorotatory) or L (levorotatory).

[0214] How to use In one aspect, the application provides a method of contacting fibrin with an anti-fibrin antibody, such as a human or humanized antibody, that results in inhibition of microglial adhesion to fibrin or fibrinogen γC domain.

[0215] In one aspect, the present application provides a method of using the isolated anti-fibrin antibody described herein for the treatment of degenerative disorders of the nervous system.In a particular aspect, the present application provides a method for treating degenerative disorders of the nervous system, comprising administering to a mammalian subject a therapeutically effective amount of the anti-fibrin antibody or a pharmaceutical composition comprising the anti-fibrin antibody described herein.In a particular embodiment, the present application provides a method for treating degenerative disorders of the nervous system selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0216] In certain aspects, described herein are methods for treating a pathology associated with Mac-1 binding to fibrin or Mac-1 binding to fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising an isolated anti-fibrin antibody described herein.

[0217] In certain aspects, described herein are methods of inhibiting microglial activation, the methods comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising the isolated antibody described herein.

[0218] In certain aspects, described herein are methods for preventing degenerative disorders of the nervous system, comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising an isolated anti-fibrin antibody described herein. In certain embodiments, the present application provides a method for preventing a degenerative disorder of the nervous system selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0219] In certain aspects, described herein are methods of treating or preventing colitis.

[0220] Method of administration In some embodiments, the methods provided herein are useful for treating a degenerative nervous system disorder in an individual. In one embodiment, the individual is a human and the antibody is a fibrin antibody described herein.

[0221] In some embodiments, the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally.An effective amount of anti-fibrin antibody can be administered for the treatment of cancer.The appropriate dosage of anti-fibrin antibody can be determined based on the type of cancer to be treated, the type of anti-fibrin antibody, the severity and course of cancer, the clinical condition of the individual, the clinical history of the individual and response to treatment, and the discretion of the attending physician.

[0222] In some embodiments, the antibody provided herein is administered with at least one additional therapeutic agent.Any suitable additional therapeutic or immunotherapeutic agent can be administered with the antibody provided herein.The additional therapeutic agent includes agents used to treat or prevent degenerative disorders of the nervous system selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0223] The additional therapeutic agent can be administered by any suitable means. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in the same pharmaceutical composition. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in different pharmaceutical compositions.

[0224] In embodiments in which the antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can be performed prior to administration of the additional therapeutic agent, simultaneously with administration of the additional therapeutic agent, and / or after administration of the additional therapeutic agent. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one month of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one week of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one day of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about 12 hours of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one hour of each other.

[0225] Kits and Articles of Manufacture The present application provides a kit comprising any one or more of the antibody compositions described herein.In some embodiments, the kit further comprises a component selected from any of the following: secondary antibody, immunohistochemistry analysis reagent, pharmaceutically acceptable excipient and instruction manual, and any combination thereof.In one particular embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein together with one or more pharmaceutically acceptable excipients.

[0226] The present application also provides an article of manufacture comprising any one of the antibody compositions or kits described herein. An example of an article of manufacture is a vial (including a sealed vial). EXAMPLES

[0227] Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should of course be allowed for.

[0228] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature, see, for example, TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (East, Pennsylvania: Mack Publishing Company, 1992); and, 1997, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2020, 2030, 2030, 2040, 2040, 2050, 2060, 2070, 2070, 2080, 2090, 2091, 2092, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, rd Ed. (Plenum Press) Vols A and B (1992).

[0229] Example 1: Humanization of anti-fibrin antibodies Composite Human Antibody™ VH and VL sequences were designed based on the murine monoclonal anti-fibrin antibody (5B8) sequence (Figures 1-2). A structural model of the murine 5B8 antibody V region was generated using the Swiss PDB and analyzed to identify key "constrained" amino acids in the V region that are likely essential for the binding properties of the antibody. Most residues contained within the CDRs (using both Kabat and Chothia definitions) along with some framework residues were deemed important. The murine 5B8 VH and Vκ sequences contain typical framework residues and the CDR 1, 2, and 3 motifs are comparable to many mouse antibodies.

[0230] From the above analysis, it was envisaged that a Composite Human sequence of 5B8 could be generated with a wide range of alternative residues outside the CDRs, but only a narrow menu of possible residues inside the CDR sequences. Preliminary analysis showed that corresponding sequence segments from several human antibodies could be combined to generate CDRs similar or identical to the CDRs in the murine sequences. Outside the CDRs and adjacent regions to the CDRs, a wide selection of human sequence segments was identified as potential building blocks for the novel humanized V-regions. During the analysis, it was noted that there was a potential unpaired cysteine ​​residue located within the VH CDR3 (Kabat residue 102). Although it is not possible to remove this residue during humanization, removal of the cysteine ​​should be considered, as unpaired cysteines may lead to potential instability, such as increased aggregation risk.

[0231] Based on the structural analysis, a large preliminary set of sequence segments that can be used to create 5B8 humanized variants was identified. These segments were selected and analyzed using iTope™ technology for in silico analysis of peptides binding to human MHC class II alleles, and TCED™ for known antibody sequence-associated T cell epitopes. Sequence segments identified as significant non-human germline binders to human MHC class II or that scored significant hits to TCED™ were discarded. This reduced the set of segments, and these combinations were analyzed again as above to ensure that the junctions between the segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V region sequences with reduced critical T cell epitopes. Initially, five heavy chain (VH1-VH6; no VH2) and four light chain (Vκ1-Vκ4) sequences were selected for gene synthesis and expression in mammalian cells. A total of 23 variant combinations were expressed as IgG1 antibodies. Figure 2 shows an alignment of the heavy and light chain variable region sequences of the chimeric antibody (VH0 and Vk0) and the humanized variants, and Figure 3 is a schematic diagram illustrating the differences in amino acid residues between VH3 and VH4.

[0232] Example 2: Production and characterization of anti-fibrin antibodies The number of variant combinations was narrowed down to a panel of six purified humanized variant antibodies. Transient transfection of the antibody constructs was performed in HEK293 EBNA cells and expression levels were assessed by Octet. The six lead humanized variants (VH3 / Vκ1, VH3 / Vκ2, VH4 / Vκ1, VH4 / Vκ2, VH5 / Vκ2 and VH6 / Vκ2) were transiently transfected in triple flasks into HEK EBNA adherent cells (LGC Standards, Teddington, UK) using the PEI transfection method and incubated for 10 days post-transfection. Due to low expression of the 5B8 chimera (VH0 / Vκ0), two batches were expressed. The titers of the IgG supernatants were assessed on day 10 by IgG titer ELISA.

[0233] Antibodies were purified and SDS-PAGE analysis of purified proteins was performed to confirm antibody concentration for affinity analysis (Figure 4). 5B8 chimeric and lead humanized variant antibodies were purified from cell culture supernatants using 1 mL and 5 mL Mab Select Sure columns (GE Healthcare, Little Chalfont, UK), respectively. The columns were washed with 1x TBS and proteins were eluted using Gentle elution Ag / Ab Elution buffer pH 6.6 (ThermoFisher, Loughborough, UK). Collected fractions were then further purified using a HiLoad™ 26 / 60 Superdex™ 200 pg preparative SEC column (GE Healthcare, Little Chalfont, UK) with PBS pH 7.4 as the mobile phase. Peak fractions containing monomeric protein were pooled, concentrated, filter sterilized and then quantified by A280 nm using the extinction coefficient (Ec (0.1%)) based on the predicted amino acid sequence. 2 μg of each purified protein was loaded per lane on reducing and non-reducing SDS-PAGE. All samples were loaded equally and the quantified antibody concentrations were shown to be accurate for affinity analysis. SEC-HPLC analysis was performed on selected antibody variants (Figure 5). Approximately 10 μg of antibody was analyzed by SEC-HPLC (post-preparative SEC) showing >99% monomer purity and <0.6% endotoxin (EU / mg) in the purified protein samples.

[0234] The six humanized variants were evaluated for thermal stability (Figure 6). To assess the thermal stability of the 5B8 chimeric antibody and the six lead Composite Human Antibody™ variants, a fluorescence-based thermal shift assay was used to determine the melting temperature (temperature at which 50% of the protein domains are misfolded). The six lead humanized antibodies, together with an irrelevant IgG1 standard (positive control) antibody, the 5B8 chimeric (VH0 / Vκ0) antibody and the murine 5B8 antibody, were diluted at 1 in 1000 dilutions to a final concentration of 0.1 mg / ml in 1xDPBS containing SYPRO® Orange (ThermoFisher, Loughborough, UK) and subjected to a temperature gradient from 25°C to 99°C over 56 minutes on a StepOnePlus™ Real-Time PCR System (ThermoFisher, Loughborough, UK). 1xDPBS was used as a negative control. Melting curves were analyzed using Protein Thermal Stability Software (version 1.2) (Figure 6). The negative control (PBS only) showed constant low fluorescence, therefore the derivative data is close to zero and is not shown in the melting curves in Figure 6. The murine 5B8 antibody showed a lower first thermal transition (Tm1) than the 5B8 chimera and the irrelevant IgG1 (Figure 6). Additionally, while murine 5B8 shows two thermal transition peaks, the second misfolding event was not discrete enough for the software to assign a value. In contrast, the chimera showed two clearly distinguishable thermal transitions occurring at 69.6 °C (CH2 and Fab) and 81.7 °C (CH3). The overall Tm values ​​suggest that the 5B8 chimera is more stable than the murine 5B8 and the irrelevant IgG1 standards.

[0235] Typically, the thermal denaturation of an antibody consists of three apparent transitions: one is a reversible transition of the CH2 domain, and the other two are consecutive irreversible transitions reflecting the denaturation of the CH3 domain and the Fab domain, respectively. The order of these transitions is usually CH2 < Fab < CH3. Chimeric antibodies show two transitions, while humanized variants show three transitions. In chimeric antibodies, the transition temperatures of the CH2 domain and the Fab domain are similar, suggesting overlap. Considering the increase in Tm2 in the humanized variant (most likely correlated with the Fab domain), These results indicate that the first thermal transition (Tm1) occurs at a similar temperature for chimeric and lead humanized variants. These results show the following order of thermal stability for six selected humanized variants: (lowest) VH0 / VK0 < VH3 / VK1 < VH4 / VK1 < VH3 / VK2 < VH4 / VK2 < VH5 / VK2 < VH6 / VK2 (highest).

[0236] The affinity of the humanized antibodies for fibrinogen, fibrin, and the P2 peptide was assessed by enzyme-linked immunosorbent assay (ELISA) (Figures 7-16). ELISA for fibrinogen binding was performed using plasminogen-depleted human fibrinogen (EMD / Calbiochem). 100 mL of 10 mg / mL IgG1-depleted fibrinogen (FGN) in 20 mM HEPES / saline buffer was added to each well of one half of a 96-well Nunc Maxisorp™ ELISA plate. ELISA for fibrin binding was prepared by adding 50 mL of 20 mM HEPES / saline buffer containing 14 mM CaCl2 and 2 units of thrombin to each well of the other half of the Nunc Maxisorp™ ELISA plate. Then, 50 mL of 20 mg of fibrinogen in 20 mM HEPES / saline buffer was added up to a total volume of 100 mL. The plates were placed in a 37°C incubator for 1 hour to allow fibrinogen to solidify into fibrin, followed by overnight placement in a 37°C plate dryer. ELISAs for P2 peptide were prepared by adding 100 mL of 30 mM P2 peptide in 20 mM HEPES / saline buffer to each well of a 96-well Nunc Maxisorp™ ELISA plate. The plates were transferred to a plate dryer and dried overnight at 37°C. All ELISAs were performed using Nunc MaxiSorp 96-well ELISA plates. Blocking was performed with 100 mL of 5% BSA in DPBS, incubated at RT for 1 hour. The plates were washed 5 times in DPBS containing 0.05% Tween-20 (EL406 was used in the wash buffer addition only). 50 mL of primary antibody was diluted in 0.5% BSA in DPBS, and the primary antibody was added to the plate and incubated at 37°C for 2 hours. The plate was washed 5 times in DPBS / Tween. 100 mL of secondary antibody was diluted in 0.5% BSA in DPBS. Secondary antibody was then added to the plate and incubated for 1 hour at RT. Afterwards, the plate was washed 5 times in DPBS / Tween. 100 mL of TMB / E substrate solution was added and the reaction was stopped after 5 minutes with 1N HCl.The results of the ELISA assay (Table D) show that all humanized clones tested bind to the P2 peptide and fibrin with similar increased affinity compared to their affinity for fibrinogen.

[0237] (Table D) Approximate EC50 of antibodies binding to human fibrinogen, fibrin and P2 peptide. TIFF2024522237000004.tif68164 * The curve did not reach saturation.

[0238] Clotting assays (in vitro fibrin polymerization assays) were also performed with the humanized variants (Figure 17). 20 mL of HEPES buffer was warmed in a 50 mL Falcon tube in a laboratory bath (or water bath) containing metal beads. This buffer was kept at 37°C throughout the fibrin polymerization assay. Frozen fibrinogen vials were thawed in the laboratory bath. Fibrinogen was diluted to 0.656 mg / mL in HEPES buffer (1 / 38 dilution; 100 μL of fibrinogen stock was mixed with 3.7 mL of HEPES buffer). Fibrinogen was then mixed with the humanized variant antibodies (total volume 160 μl) with or without preincubation at 37°C for 3 hours. Frozen thrombin was thawed in the laboratory bath and thrombin was diluted in HEPES buffer (8 μL of thrombin stock was mixed with 152 μL of HEPES buffer). A mixture of CaCl2 and thrombin in HEPES buffer was then prepared (3.6 mL total: 72 mL CaCl2 + 72 μL thrombin diluent and 3456 μL HEPES buffer). 40 μL of the CaCl2 and thrombin mixture was added to each well. 160 μL of the fibrinogen solution mixed with antibody was added to each well of the 96-well plate containing CaCl2 and thrombin. This resulted in the following final concentrations in each well: 10 mM CaCl2, 0.3 U / mL thrombin, 150 μg / mL fibrinogen, 50 μg / mL humanized antibody. The final buffer concentrations were 20 mM HEPES, 150 mM NaCl, 5 mM eACA, pH 7.4. A control for clot formation (no added fibrinogen), a control with known fibrinolysis inhibitors (fibrinogen + GPRP), and an antibody control (fibrinogen + IgG control) were included. Duplicate wells were analyzed for each condition. Clot formation controls were loaded first and last in each experiment to allow for "apparent" differences in lag time due to the time difference between loading the wells and starting the absorbance reading. Background absorbance of soluble fibrinogen in the absence of Ca / thrombin was very low (similar to buffer control). Absorbance was measured at A350 nm every 30 seconds for 40 minutes at 37°C using a SpectraMax M5 Microplate Reader.Upon addition of CaCl2 and thrombin, fibrinogen is converted to polymerized fibrin resulting in an increase in turbidity, as measured by an increase in absorbance at 350 nm and the formation of a gel-like structure in the well. Once maximum fibrin polymerization is reached, A350 nm remains stable (plateau).

[0239] Antibody binding affinity analysis was performed using HEK293 EBNA cell supernatants and affinity was evaluated by Biacore single cycle kinetics (Figures 18 and 19) and multi-cycle kinetics analysis (Figure 20). Figure 18 illustrates the procedure used for single cycle kinetics analysis. A Biacore T200 (serial number 1909913) instrument was used for single cycle kinetics analysis, using control software v2.0.1 and evaluation software V3.0. Anti-human capture chips were used with a running buffer consisting of HBS-P+ buffer (pH 7.4) containing 0.1% BSA at 25°C and a flow rate of 30 μl / min. Intact HEK supernatant was used as the ligand and loaded at 10 μl / min to approximately 1674 RU. P2 peptide containing SEQ ID NO: 31 (CPC Scientific 920712, Lot # CS-02-00350) was the analyte for analysis. Five-point 3-fold dilutions were performed ranging from 300,000 nM to 3703 nM with an injection time of 20 s and dissociation time of 25 s. Regeneration was performed with MgCl2. Antibodies were diluted to a final concentration of 20 μg / ml in running buffer based on the concentration assessed by Octet titration. Antibodies were loaded onto Fc2, Fc3 and Fc4 of an anti-human capture chip (GE Healthcare, Little Chalfont, UK) at the start of each cycle. IgG was captured at a flow rate of 10 μl / min to obtain a theoretical immobilization level (RL) of approximately 1674 RU and an RMax of approximately 50 RU. The surface was then allowed to stabilize. Single cycle kinetic data were obtained using P2 peptide as analyte at a flow rate of 30 μL / min to minimize potential mass transfer limitations. Multiple repeats with the reference 5B8 chimeric antibody were performed to confirm the stability of the surface and analyte to the kinetic cycles. The signal from reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A five-point, 3-fold dilution range of P2 peptide from 300,000 nM to 3703 nM was used without regeneration between each concentration.The association phase for five injections of increasing concentrations of P2 peptide was monitored for 20 seconds each time, and one dissociation phase was measured for 25 seconds after the last injected analyte. Regeneration of the anti-human capture surface was performed with one injection of 3.8 M magnesium chloride. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. The data were then analyzed using a steady-state fitting model due to the low affinity interactions.

[0240] Figure 8 shows the raw sensogram and fitted data of single cycle kinetics binding to the P2 peptide, and Table E summarizes the kinetic parameters, expression levels and estimated KDs of the humanized variants obtained from the single cycle kinetics analysis. All variants, including VK1 and VK2, are within 2-fold of the chimera (VH0 / VK0). From these results, six potential lead variants were identified as VH3 / VK1, VH3 / VK2, VH4 / VK1, VH4 / VK2, VH5 / VK2, and VH6 / VK2.

[0241] Multi-cycle kinetic analysis of the variants was also performed to determine the affinity of the antibodies. To establish the exact affinity for the P2 peptide, multi-cycle kinetic analysis was performed on the purified 5B8 chimeric antibody and the six lead humanized variant antibodies using a Biacore T200 (serial number 1909913) instrument running the Biacore T200 Evaluation Software V3.0.1 (Uppsala, Sweden). At the start of each cycle, the antibody was loaded onto Fc2, Fc3, and Fc4 of an anti-human capture chip (GE Healthcare, Little Chalfont, UK). IgG was captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 1674 RU, with a theoretical value to obtain an RMax of approximately 50 RU. The surface was then allowed to settle. Kinetic data was obtained using the P2 peptide as the analyte at a flow rate of 30 μL / min to minimize potential mass transfer effects. To confirm the stability of both the surface and analyte to kinetic cycling, multiple blank replicates and replicates of three concentrations of analyte were programmed into the kinetic run. For the kinetic analysis, a 2-fold dilution range of 58, from 300,000 nM to 2343.75 nM of P2 peptide, was selected and used. The association phase of the peptide was monitored for 20 seconds and the dissociation phase was monitored for 25 seconds. Regeneration of the anti-human capture surface was performed using a single injection of 3.8 M magnesium chloride. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface, and the data was then analyzed using steady-state analysis due to the low affinity interaction for the P2 peptide.

[0242] Figure 20 shows the raw sensograms and fitted data from the multi-cycle kinetic analysis of selected variants for binding to the P2 peptide, and Tables F and G summarize the kinetic data from both single-cycle and multi-cycle kinetic analyses. Table H summarizes the expression levels, binding affinity analysis, and thermostability analysis of the six humanized variant antibodies. These results show that all of the humanized variants have KDs within approximately two-fold of the chimeric (VH0 / VK0) antibody.

[0243] Table E. Steady-state data summary of humanized variants TIFF2024522237000005.tif247127

[0244] Table F. Kinetic analysis of lead variant antibodies TIFF2024522237000006.tif24746

[0245] Table G. Summary of humanized lead data TIFF2024522237000007.tif24766

[0246] Table H. Steady-state data and expression summary TIFF2024522237000008.tif24781

[0247] Example 3: Substitution of cysteine ​​102 improves antifibrin binding affinity to the fibrin γ377-395 epitope Three potential instabilities were identified within the VH CDRs of the parent antibody sequence that could not be addressed by humanization (Figure 21). A free cysteine ​​was identified at cysteine ​​102 position in CDR-H3 (SEQ ID NO: 3), and acid-labile sites were Asp 52 in CDR-H2 and Asp 96 in CDR-H3.

[0248] Sequence instability of cysteine ​​102 in CDR-H3 (SEQ ID NO: 3) was originally identified during humanization of the 5B8 murine monoclonal anti-fibrin antibody. Site-directed mutagenesis was performed to address the instability of cysteine ​​102 in the two humanized lead variants VH4 / Vκ2 and VH5 / Vκ2 (IgG1). A total of six substitutions (C102A, C102G, C102L, C102S, C102T, and C102V) were made in either VH4 (SEQ ID NO: 7) or VH5 (SEQ ID NO: 14) to generate a total of 12 new VH sequences. The 12 VH sequences and the Vκ2 light chain sequence (SEQ ID NO: 21) were used to generate 12 new antibodies. These 12 new antibodies, together with the parental humanized variants VH4 / Vκ2 and VH5 / Vκ2, were transiently transfected into HEK EBNA cells using the PEI method at small scale (6 wells). Supernatants were collected 7 days post-transfection and Octet titers and binding of fibrin peptide γ377-395 (SEQ ID NO: 31) were assessed by Biacore single cycle kinetics using steady state affinity.

[0249] To calculate the contact time for Biacore antibody loading, HEK supernatants were injected for 90 seconds at 10 ml / min (Figure 22). The signal after injection is an indication of the antibody expression level. Chimeric antibodies transfected at the same time as other variants had very low signals. As a result, sufficient capture levels could not be achieved (low expression was confirmed by Octet, indicating that the chimeras were below the detection level). For Biacore analysis, antibody variants carrying cysteine ​​substitutions were compared to the corresponding (unmodified) humanized variants (either VH4 / Vκ2 or VH5 / Vκ2). Figure 23 shows the results of steady-state analysis using single cycle kinetics of 12 humanized variant antibodies with cysteine ​​substitutions, and Table G summarizes the expression levels and binding affinities of 12 humanized variant antibodies with cysteine ​​substitutions. These results indicate that antibodies containing all six C102 substitutions were able to bind fibrin peptide γ377-395 (SEQ ID NO: 31) within approximately two-fold of the corresponding parent antibody. KD values ​​were consistent with those previously observed using supernatants, 8.5×10-5 (VH4 / Vκ2) and 1.3×10-4 (VH5 / Vκ2). C102G was identified as the best substitution for both VH4 / Vκ2 and VH5 / Vκ2 (C102G < C102V < C102T < C102S < C102A < C102L). Unexpectedly, humanization improved antibody expression, which was further improved by removal of the free cysteine ​​at cysteine ​​102 in CDR-H3.

[0250] Example 4: Humanized antibody variants have similar activity to 5B8 in fibrinogen-induced encephalomyelitis (FIE) The effect of ICV injection of humanized anti-fibrin antibody variant VH5 C102G / Vκ2 on microglial activation, oxidative stress, and macrophage recruitment was evaluated. Plasminogen-free fibrinogen was dissolved in endotoxin-free distilled water and diluted to 5 mg / ml in artificial cerebrospinal fluid (ACSF). Fibrinogen (1 μl of 5 mg / ml) was injected at a rate of 0.3 μl / min using a 10 μl Hamilton syringe attached to a 33-gauge needle at the following brain coordinates according to Paxinos and Watson: anteroposterior, -1.0 mm; mediolateral, -0.7 mm; dorsoventral, -1.325 mm from bregma. For prophylactic intracerebroventricular (icv) injections, 10 ug of antibody was delivered intracerebroventricularly (anteroposterior, -2.0 mm; mediolateral, 0 mm, dorsoventral, -2.0 mm) at a rate of 0.3 μl / min using a 10 μl syringe attached to a 33-gauge needle 30 min prior to fibrinogen injection. For prophylactic intravenous (iv) injections, antibody was injected retroorbitally using a 0.3 mL 29 g insulin syringe 1 h prior to fibrinogen injection. Mice were sacrificed 3 days after fibrinogen injection for histopathological evaluation of the brain.

[0251] 10 ug of antibody was administered prophylactically by icv injection to FIE mice (Figure 24). Stereotaxic fibrinogen was injected into the corpus callosum to induce encephalomyelitis. Each circle represents an individual animal. Data are means ± sem. One-way ANOVA with Tukey's multiple comparisons.

[0252] This data confirms that the humanized antibody variants described herein inhibit microglial activation, macrophage recruitment, and oxidative stress in FIE in vivo.

[0253] Example 5: Humanized antibody variants co-localize with fibrinogen in experimental autoimmune encephalomyelitis (EAE) Tissue sections from chronic EAE mice with fibrinogen accumulation in spinal cord lesions were stained with 10 mg / ml VH5 C102G / Vk2-biotin and CY3-streptavidin antibodies (FIG. 25).

[0254] Chronic EAE was induced in 8-9 week old female SJL / J mice by subcutaneous immunization with 15 ug of PLP139-151 in complete Freund's adjuvant supplemented with 400 ug of heat-inactivated Mycobacterium tuberculosis H37Ra, an epitope of amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG) ("MOG35-55 EAE") (day 0). Two days after immunization, mice were injected with 5 ng of pertussis toxin via IP administration. Antibodies were administered prophylactically IP at 0.2, 1, or 5 mg / kg twice weekly starting on day 0. Dexamethasone (0.5 mg / kg) was administered IP daily as a positive control. EAE lesion scores were monitored daily until the end of the study. The study was terminated around study days 14-16, 3 days after peak EAE, and spinal cords were harvested for histopathological analysis.

[0255] This data confirms that the humanized antibody variants described herein co-localize with fibrin(ogen).

[0256] Example 6: Pharmacokinetic analysis of humanized antibody variants Pharmacokinetic analysis was performed with the humanized anti-fibrin antibody variant VH5 C102G / Vk2. Antibodies were detected by ELISA in plasma from EAE mice administered either 10 mg / Kg or 30 mg / Kg of VH5 C102G / Vk2 antibody (Figure 26). Antibodies were detected by ELISA in plasma and blood from wild-type Balb / c mice administered VH5 C102G / Vk2 antibody (Figure 27). Similar pharmacokinetic trends were observed between BioAgilytix and Invicro despite different baselines, disease models, and analytical methods.

[0257] These results demonstrate efficient metabolism and / or clearance of the humanized antibody variants at the doses shown.

[0258] Example 7: Therapeutic Treatment of Fibrinogen-Induced Encephalomyelitis (FIE) We next evaluated the ability of humanized anti-fibrin antibodies to therapeutically inhibit microglial activation (Figure 28A) and macrophage infiltration (Figure 28B) in a fibrinogen-induced encephalomyelitis (FIE) mouse model. To induce FIE, mice were anesthetized with avertin and placed in a stereotaxic apparatus. Plasminogen-free fibrinogen was dissolved in endotoxin-free distilled water and diluted to 5 mg / ml in artificial cerebrospinal fluid (ACSF). Fibrinogen (1 μl of 5 mg / ml) was injected at a rate of 0.3 μl / min using a 10 μl Hamilton syringe attached to a 33-gauge needle at the following intracerebral coordinates according to Paxinos and Watson: anteroposterior, -1.0 mm; mediolateral, -0.7 mm; dorsoventral, -1.325 mm from bregma.

[0259] For prophylactic intracerebroventricular (icv) injections, 10 ug of antibody was delivered intracerebroventricularly (anteroposterior, -2.0 mm; mediolateral, 0 mm, dorsoventral, -2.0 mm) at a rate of 0.3 ul / min using a 10 ul syringe attached to a 33 gauge needle 30 min prior to fibrinogen injection. For prophylactic intravenous (iv) injections, antibody was injected retroorbitally using a 0.3 ml 29 g insulin syringe 1 h prior to fibrinogen injection.

[0260] Stereotaxic fibrinogen injections were performed into the corpus callosum to induce encephalomyelitis. A total of 78 mice were divided into 13 groups, and then n = 6 mice from each group were iv injected with VH5 C102G / VK2 humanized antibody at either 10 mg / kg or 30 mg / kg. Brain tissue collection and preparation were performed 3 days after injection. Sample exclusions: 5 mice; found dead on post-op day 1 (C 10 mg / kg, n =1) and post-op day 2 (B 10 mg / kg, n =1; D 10 mg / kg, n =1); wrong site injection (B 10 mg / kg, n =1; D 10 mg / kg, n =1). Blinding and quantification: All FIE experiments, image collection and quantification were performed in a blinded manner. Immunohistochemistry (IHC) and quantification were performed as follows: Samples from 73 mice were included for IHC and quantification. Coronal sections (30 um) were prepared on a cryostat. Tissues were stained with Iba-1 (microglia marker, 1:750 dilution) and Mac-2 (macrophage infiltration marker, 1:750 dilution). Iba-1 (Iba-1+ areas) and Mac-2 (Mac-2+ areas) immunoreactivity was then calculated. A reduction of both microglia and macrophages was detected in tissues from mice treated with the humanized anti-fibrin antibody variant.

[0261] These results demonstrate that the humanized antibody variants described herein are capable of therapeutically reducing microglial and macrophage infiltration in mice with FIE.

[0262] Example 8: Prophylactic treatment of relapsing-remitting EAE The ability of a humanized anti-fibrin antibody to prophylactically treat relapsing-remitting EAE ("PLP139-151 EAE") induced by an epitope at amino acids 139-151 of the proteolipid protein (PLP) was evaluated. EAE was induced in 8-9 week old female SJL / J mice by subcutaneous immunization with 15 ug of PLP139-151 in complete Freund's adjuvant supplemented with 400 ug of heat-inactivated Mycobacterium tuberculosis H37Ra (day 0). Two days after immunization, mice are injected with 5 ng of pertussis toxin via IP administration. Antibodies were administered prophylactically IP at 0.2, 1, or 5 mg / kg twice weekly starting on day 0. Dexamethasone (0.5 mg / kg) was administered IP daily as a positive control. Experimental design: 6 groups: n = 10 mice per group, total of 60 mice. Dose regimen: dexamethasone (5 mg / kg daily), humanized anti-fibrin antibodies (A, B, C, D_5 mg / kg every 3 days). EAE disability scores were monitored daily until study termination. The study was terminated 3 days after peak EAE around study days 14-16, and spinal cords were harvested for histopathological analysis.

[0263] Sample Exclusions: 3 mice; found dead on days 12 (Antibody B, n = 1), 15 (Antibody C, n = 1), or 16 (Antibody A, n = 1). Blinding and Quantification: All EAE experiments (antibody treatments and clinical scores) were performed in a blinded manner. 57 spinal cord samples were prepared for tissue processing.

[0264] Clinical scores of PLP EAE were evaluated in mice prophylactically injected with antibody (5 mg / kg ip every 3 days) (Figure 29). Clinical scores of mice injected with anti-fibrin humanized antibody were reduced compared to control mice injected with PBS or IgG1 only. Time to disease onset was also evaluated (Figure 30A). No mice injected with anti-fibrin humanized antibody had paralysis compared to control mice injected with PBS, IgG1, or dexamethasone only, in which 25%-50% or more of the mice had paralysis (Figure 30B).

[0265] Taken together, these results indicate that anti-fibrin humanized antibodies are effective for the prophylactic treatment of encephalomyelitis.

[0266] Example 9: Treatment of Neurodegenerative Diseases The purified humanized antibody variants described herein are formulated into pharmaceutical compositions that are administered to patients for the treatment of neurodegenerative diseases (e.g., multiple sclerosis or Alzheimer's disease). The pharmaceutical compositions comprising the humanized antibody variants described herein are administered in a dose sufficient to effectively reduce symptoms of the neurodegenerative disease. The pharmaceutical compositions are well tolerated and do not induce significant adverse side effects in patients.

[0267] Example 10: Humanized antibody variants for the treatment of colitis To initiate the dextran sulfate sodium (DSS)-induced colitis treatment, 8-10 week-old female C57BL / 6 mice were acclimated to the animal facility for at least 4 days, weighed, and randomly assigned to treatment groups based on body weight. Two types of studies were performed: acute (7 days) and chronic (28 days).

[0268] Acute DSS testing is performed by adding 2.5% DSS to drinking water for 7 days. Antibodies are administered IP at 10 and 30 mg / kg every 2 days (Q2D). Mice are euthanized on day 7 by isoflurane anesthesia, exsanguination, followed by cervical dislocation. Colons were removed and analyzed for histopathology.

[0269] Chronic DSS testing is performed by adding 2.0% DSS to drinking water for 1 week, then replacing with regular drinking water for 1 week, adding 2% DSS for another week, and then finishing with regular drinking water for another week. Humanized antibody variants described herein are administered IV prophylactically at 30 and 5 mg / kg twice weekly starting on day 0. Mice are euthanized after 28 days by isoflurane anesthesia, exsanguination, followed by cervical dislocation. Colons are removed and analyzed for histopathology.

[0270] This study confirms that the humanized antibody variants described herein are effective in treating colitis.

[0271] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

[0272] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0273] Unofficial sequence listing TIFF2024522237000009.tif227164TIFF2024522237000010.tif239164TIFF2024522237000011.tif111164

Claims

**Claim 1** A heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences CDR-H1, CDR-H2, and CDR-H3, and A light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences CDR-L1, CDR-L2, and CDR-L3 An isolated antibody that binds to the human fibrin γC or fibrinogen γC domain, comprising: a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 1, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 2, c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 3, where X is glycine (G), valine (V), threonine (T), serine (S), alanine (A), or leucine (L), d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 4, e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 5, and f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 6, The isolated antibody. **Claim 2** The isolated antibody according to claim 1, wherein the antibody comprises a VH sequence selected from the sequences set forth in any one of SEQ ID NOs: 7-20. **Claim 3** The isolated antibody according to claim 1, wherein the antibody comprises the VL sequence set forth in SEQ ID NO:

21. **Claim 4** The isolated antibody according to claim 1, wherein the antibody comprises: (a) The VH sequence set forth in SEQ ID NO: 7 and the VL sequence set forth in SEQ ID NO: 21; (b) The VH sequence set forth in SEQ ID NO: 8 and the VL sequence set forth in SEQ ID NO: 21; (c) The VH sequence set forth in SEQ ID NO: 9 and the VL sequence set forth in SEQ ID NO: 21; (d) The VH sequence set forth in SEQ ID NO: 10 and the VL sequence set forth in SEQ ID NO: 21; (e) The VH sequence set forth in SEQ ID NO: 11 and the VL sequence set forth in SEQ ID NO: 21; (f) The VH sequence set forth in SEQ ID NO: 12 and the VL sequence set forth in SEQ ID NO: 21; (g) The VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 21; (h) The VH sequence set forth in SEQ ID NO: 14 and the VL sequence set forth in SEQ ID NO: 21; (i) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 21; (j) the VH sequence set forth in SEQ ID NO: 16 and the VL sequence set forth in SEQ ID NO: 21; (k) the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 21; (l) the VH sequence set forth in SEQ ID NO: 18 and the VL sequence set forth in SEQ ID NO: 21; (m) the VH sequence set forth in SEQ ID NO: 19 and the VL sequence set forth in SEQ ID NO: 21; or (n) the VH sequence set forth in SEQ ID NO: 20 and the VL sequence set forth in SEQ ID NO:

21. **Claim 5** The antibody is (a) a humanized antibody, a human antibody, or a chimeric antibody; (b) comprising a heavy chain human constant region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM; (c) comprising a human Fc region, optionally wherein the human Fc region comprises the sequence set forth in SEQ ID NO: 22 and the human Fc region comprises the human heavy chain constant region of class IgG and subclasses selected from IgG1, IgG2, IgG3, and IgG4; (d) comprising wild-type human IgG1 Fc; and / or (e) a monoclonal antibody, The isolated antibody according to claim 1. **Claim 6** (a) The heavy chain comprises a constant heavy chain sequence described by SEQ ID NO: 22; and / or (b) The light chain comprises a constant light chain sequence described by SEQ ID NO: 23, The isolated antibody according to claim 1. **Claim 7** (a) Binds to the γ377-395 epitope of the fibrin γC or fibrinogen γC domain; (b) Inhibits Mac-1 binding to the fibrin γC or fibrinogen γC domain; and / or (c) Exhibits inhibition of microglia adhesion to the fibrin γC or fibrinogen γC domain, The isolated antibody according to claim 1. **Claim 8** A polynucleotide encoding the antibody according to claim 1. **Claim 9** A vector comprising the polynucleotide according to claim 8. **Claim 10** A host cell comprising the polynucleotide according to claim 8 or the vector according to claim 9. **Claim 11** The step of expressing an antibody by the host cell according to claim 10, and The step of isolating the expressed antibody A method for producing an antibody, comprising.

12. A pharmaceutical composition comprising the antibody according to any one of Claims 1 to 7 and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition according to Claim 12, for treating a neurodegenerative disorder in a subject in need thereof.

14. The pharmaceutical composition according to Claim 13, wherein the neurodegenerative disorder is selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

15. The pharmaceutical composition according to Claim 12, for treating a condition associated with Mac-1 binding to fibrin or Mac-1 binding to fibrinogen in a mammalian subject.

16. The pharmaceutical composition according to Claim 12, for inhibiting microglial activation in a mammalian subject.

17. The pharmaceutical composition according to Claim 12, for treating colitis in a subject in need thereof.

18. An isolated antibody that binds to the human fibrin γC or fibrinogen γC domain, comprising: (a) a VH sequence having at least 90% sequence identity to a sequence selected from the sequences set forth in any one of SEQ ID NOs: 7 to 20, and / or (b) a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO 21.

19. An isolated antibody that binds to the human fibrin γC or fibrinogen γC domain, comprising: (a) a VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (b) a VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (c) a VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (d) a VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 10 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (e) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 11 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (f) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 12 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (g) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 13 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (h) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 14 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (i) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 15 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (j) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 16 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (k) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 17 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (l) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 18 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; (m) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21; or (n) A VH sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20 and a VL sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

21. [

20. ] A composition comprising an antibody according to any one of claims 1 to 7, 18, and 19 for treating a neurodegenerative disorder or colitis.

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