Antibodies targeting complement factor D and their use

Abstract

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Antibodies that specifically bind complement factor D in a pH-sensitive manner, as well as methods for making and using such antibodies, are described.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 173,092, filed on 9 April 2021, the entire contents of which are incorporated herein by reference.

[0002] Inclusion by referencing the sequence list This application is filed together with an electronic sequence listing. The sequence listing is provided as an ASCII text file named SHR-2005WO_SL.txt, with a size of 41,684 bytes, created on April 7, 2022. The electronic information of the sequence listing is incorporated in its entirety by reference. [Background technology]

[0003] The complement system contains several proteins that can interact with pathogens and promote their elimination. This system plays a crucial role in various biological processes, such as inflammation, and complement dysfunction can lead to or contribute to disease. Various medical conditions resulting from abnormal complement activity can be treated by administering antibodies or other binding molecules that can bind to complement factors. [Overview of the project]

[0004] In the art, there is a need to develop therapeutic molecules for treating patients with complement-related conditions. The present invention addresses this need by providing a therapeutic agent that targets complement factor D (CFD). In one embodiment, the present invention provides an isolated antibody that specifically binds to complement factor D (CFD). In some embodiments, the isolated antibody that specifically binds to complement factor D (CFD) comprises a heavy chain variable region amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs. 5, 27, 29, 34, or 36, and / or a light chain variable region amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs. 6, 8, 26, 28, 35, or 37.

[0005] In some embodiments, isolated antibodies that specifically bind to complement factor D (CFD) contain the same heavy chain variable region amino acid sequence as SEQ ID NOs. 5, 27, 29, 34, or 36, and / or the same light chain variable region amino acid sequence as SEQ ID NOs. 6, 8, 26, 28, 35, or 37.

[0006] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0007] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLES (SEQ ID NO: 20), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0008] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0009] In some embodiments, the isolated antibody contains a light chain variable region amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs. 6, 8, 26, 28, 35, or 37.

[0010] In some embodiments, the isolated antibody contains the same light chain variable region amino acid sequence as SEQ ID NOs. 6, 8, 26, 28, 35, or 37.

[0011] In some embodiments, the isolated antibody comprises a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSASSNDDAV (SEQ ID NO: 18).

[0012] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSASSNDDAV (SEQ ID NO: 18).

[0013] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 5 and the same light chain variable region amino acid sequence as SEQ ID NO: 6.

[0014] In some embodiments, the isolated antibody comprises a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSADLNDDAV (SEQ ID NO: 19).

[0015] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSADLNDDAV (SEQ ID NO: 19).

[0016] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 5 and the same light chain variable region amino acid sequence as SEQ ID NO: 8.

[0017] In some embodiments, the isolated antibody comprises a light chain CDR1 containing the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), a light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0018] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLES (SEQ ID NO: 20), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), a light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0019] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 29 and the same light chain variable region amino acid sequence as SEQ ID NO: 28.

[0020] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), a light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0021] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 27 and the same light chain variable region amino acid sequence as SEQ ID NO: 26.

[0022] In one embodiment, the present invention provides an isolated antibody that specifically binds to complement factor D (CFD), wherein the heavy chain CDR1 comprises the amino acid sequence YYAWS (SEQ ID NO: 12), the heavy chain CDR2 comprises the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), the heavy chain CDR3 comprises the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), the light chain CDR1 comprises the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), the light chain CDR2 comprises the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or the light chain CDR3 comprises the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0023] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0024] In some embodiments, the isolated antibody comprises a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0025] In some embodiments, the isolated antibody comprises a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0026] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 34 and the same light chain variable region amino acid sequence as SEQ ID NO: 35.

[0027] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 36 and the same light chain variable region amino acid sequence as SEQ ID NO: 37.

[0028] In some embodiments, the isolated antibody comprises a light chain variable domain having at least 90% identity to the amino acid sequence described in SEQ ID NO: 35 or SEQ ID NO: 37, a heavy chain variable domain having at least 90% identity to the amino acid sequence described in SEQ ID NO: 34 or SEQ ID NO: 36, and / or the light chain variable domain of (a) and the heavy chain variable domain of (b).

[0029] In some embodiments, the isolated antibody contains a light chain variable domain having at least 95% identity with the amino acid sequence described in SEQ ID NO: 35 or SEQ ID NO: 37.

[0030] In some embodiments, the isolated antibody contains a heavy chain variable domain having at least 95% identity with the amino acid sequence described in SEQ ID NO: 34 or SEQ ID NO: 36.

[0031] In some embodiments, the isolated antibody is a monoclonal antibody or a fragment thereof.

[0032] In some embodiments, the isolated antibody further comprises an IgG constant region.

[0033] In some embodiments, the isolated antibody contains a light chain amino acid sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 3, 30, or 32.

[0034] In some embodiments, the isolated antibody contains a heavy chain amino acid sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, 31, or 33.

[0035] In some embodiments, the isolated antibody inhibits the complement accessory pathway. In some embodiments, the isolated antibody inhibits the cleavage of complement factor B.

[0036] In some embodiments, the isolated antibody binds to the CFD at pH 7.4 with an affinity dissociation constant (KD) between approximately 1 pM and 50 pM.

[0037] In some embodiments, the isolated antibody binds to the CFD at pH 7.4 with affinity dissociation constants (KD) of less than approximately 50 pM, less than approximately 45 pM, less than approximately 40 pM, less than approximately 35 pM, less than approximately 30 pM, less than approximately 25 pM, less than approximately 20 pM, less than approximately 15 pM, less than approximately 10 pM, less than approximately 9 pM, less than approximately 8 pM, less than approximately 7 pM, less than approximately 6 pM, or less than approximately 5 pM.

[0038] In some embodiments, the isolated antibody binds to the CFD at pH 5.5 with an affinity dissociation constant (KD) between approximately 15 nM and 150 nM.

[0039] In some embodiments, the isolated antibody binds to CFD at pH 5.5 with affinity dissociation constants (KD) greater than approximately 15 nM, greater than approximately 20 nM, greater than approximately 25 nM, greater than approximately 30 nM, greater than approximately 35 nM, greater than approximately 40 nM, greater than approximately 45 nM, greater than approximately 50 nM, greater than approximately 100 nM, or greater than approximately 150 nM.

[0040] In some embodiments, the isolated antibody has an off-rate from the antibody in CFD at pH 5.5 of 0.010s.-1 Super, 0.015s -1 Super, 0.02s -1 Super, 0.025s -1 Super, 0.03s -1 Ultra, 0.035s -1 Ultra-long, or 0.04s -1 It's incredible.

[0041] In some embodiments, the isolated antibody has a serum half-life of more than about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 60 days, about 70 days, about 80 days, about 90 days, about 95 days, about 100 days, or about 125 days or more.

[0042] In one embodiment, the present invention provides a nucleic acid sequence encoding an isolated antibody described herein.

[0043] In one embodiment, the present invention provides a vector comprising a nucleic acid sequence encoding an isolated antibody described herein.

[0044] In one embodiment, the present invention provides a host cell comprising a nucleic acid sequence encoding an isolated antibody described herein.

[0045] In one embodiment, the present invention provides a method for producing an antibody, comprising culturing host cells under conditions suitable for antibody expression.

[0046] In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof described herein for pharmaceutical use.

[0047] In one embodiment, the present invention provides a method for treating a complement-mediated disorder, comprising administering an effective amount of an antibody described herein to a subject in need thereof.

[0048] In some embodiments, the complement-mediated disorder or condition is atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH).

[0049] In some embodiments, administration of the antibody inhibits intravascular hemolysis and extravascular hemolysis.

[0050] Definitions A or An: The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0051] Affinity: As used herein, the term “affinity” refers to the characteristics of the binding interaction between a binding moiety (e.g., an antigen-binding moiety (e.g., a variable domain described herein) and / or an Fc receptor-binding moiety (e.g., an FcRn-binding moiety)) and a target (e.g., an antigen (e.g., CFD) and / or an FcR (e.g., FcRn)), and indicates the strength of the binding interaction. In some embodiments, the measure of affinity is represented as the dissociation constant (K D ). In some embodiments, the binding moiety has a high affinity for the target (e.g., less than about 10 -7 M, less than about 10 -8 M, or less than about 10 -9 M of K D ). In some embodiments, the binding moiety has a low affinity for the target (e.g., higher than about 10 -7 M, higher than about 10 -6 M, higher than about 10 -5 M, or higher than about 10 -4 M of K D ). In some embodiments, the binding moiety has a high affinity for the target at a first pH, a low affinity for the target at a second pH, and an intermediate affinity for the target at a pH level between the first pH and the second pH.

[0052] Approximately or about: As used herein, the terms “approximately” or “about” refer to a value that is similar to the described reference value when applied to one or more values ​​for a given purpose. In certain embodiments, unless otherwise specified or the context makes clear, the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the described reference value in either direction (greater than or less than) (except where such a number exceeds 100% of the possible value).

[0053] Antibody: As used herein, the term “antibody” means a polypeptide comprising at least one immunoglobulin variable region, e.g., an immunoglobulin variable domain or an amino acid sequence providing an immunoglobulin variable domain sequence. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody may include two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments) and complete antibodies, e.g., intact immunoglobulins of type IgA, IgG, IgE, IgD, and IgM (and their subtypes). The light chain of an immunoglobulin may be of kappa or lambda type.

[0054] Binding moiety: As used herein, “binding moiety” means any molecule or part of a molecule that can specifically bind to a target, for example, a target of interest (e.g., an antigen (e.g., CFD) and / or an FcR (e.g., FcRn)). Examples of binding moieties include antibodies, their antigen-binding fragments, Fc regions or their Fc fragments, antibody mimetic compounds, peptides, and aptamers.

[0055] Constant Region: As used herein, the term “constant region” refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. A constant region may include any or all of the following immunoglobulin domains: CH1 domain, hinge region, CH2 domain, CH3 domain (derived from IgA, IgD, IgG, IgE, or IgM), and CH4 domain (derived from IgE or IgM).

[0056] Fc region: As used herein, the term “Fc region” refers to a dimer of two “Fc polypeptides,” each “Fc polypeptide” containing the constant region of an antibody, excluding the first constant region immunoglobulin domain. In some embodiments, the “Fc region” contains two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. “Fc polypeptide” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, as well as the last three constant region immunoglobulin domains of IgE and IgM, and may also include some or all of the mobile hinges on the N-terminal side of these domains. In the case of IgG, the “Fc polypeptide” includes the immunoglobulin domains C-gamma 2 (Cγ2) and C-gamma 3 (Cγ3), as well as the lower part of the hinge between C-gamma 1 (Cγ1) and Cγ2. While the boundaries of Fc polypeptides can vary, human IgG heavy chain Fc polypeptides are typically defined as containing a residue beginning at T223, C226, or P230 at their carboxyl terminus, where numbering follows the EU index similar to that of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). In the case of IgA, the Fc polypeptide includes the immunoglobulin domains C alpha 2 (Cα2) and C alpha 3 (Cα3), as well as the lower part of the hinge between C alpha 1 (Cα1) and Cα2. The Fc region may be synthetic, recombinant, or generated from natural sources such as IVIG.

[0057] K a :When used in this specification, "K a "Association rate" refers to the rate at which a specific binding site and its target site form a binding site / target complex.

[0058] K d :When used in this specification, "K d " refers to the dissociation rate of a specific binding site / target complex.

[0059] K D :When used in this specification, "K D " is K d K a The ratio to (i.e., K d / K a This refers to the dissociation constant, which is obtained from ) and expressed as molar concentration (M). D The value can be determined using methods well established in the art, for example, by using surface plasmon resonance or by using a biosensor system such as the Biacore® system.

[0060] Reference: The "reference" entity, "reference" system, "reference" quantity, and set of "reference" conditions are the objects on which the test entity, test system, test quantity, and set of test conditions are compared as described herein. For example, in some embodiments, the "reference" antibody is a control antibody that has not been manipulated as described herein.

[0061] Selective binding: As used herein, “selective binding,” “selectively binding,” “specific binding,” or “specifically binding” means, in relation to the binding portion and the target, the preferential association of the binding portion with the target rather than with non-target entities. Some degree of non-specific binding may occur between the binding portion and the non-target. In some embodiments, the binding portion selectively binds to the target if the binding between the binding portion and the target is more than 2 times, more than 5 times, more than 10 times, or more than 100 times compared to the binding between the binding portion and the non-target. In some embodiments, the binding portion has a binding affinity of about 10 -5 Less than M, approximately 10-6 Less than M, approximately 10 -7 Less than M, approximately 10 -8 Less than M, or about 10 -9 If the value is less than M, it selectively binds to the target.

[0062] Subject: The term “subject” as used herein means any subject for which diagnosis, prognosis, or therapy is desired. For example, a subject may be a mammal, e.g., human or non-human primate (e.g., ape, monkey, orangutan, or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

[0063] Target: As used herein, “target” is any molecule to which the binding portion of a multispecific binding molecule specifically binds. In some embodiments, the target is an antigen described herein (e.g., CFD). In some embodiments, the target is FcR (e.g., FcRn). The terms “first target” and “second target” are used herein to refer to two distinct molecular species, rather than two molecules of the same molecular species. For example, in some embodiments, the first target is a serum protein and the second target is FcRn.

[0064] Therapeutic Dose: As used herein, the term “therapeutic dose” means the amount of a therapeutic molecule (e.g., an engineered antibody as described herein) that imparts a therapeutic effect to the subject being treated at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows signs of or feels an effect). In particular, “therapeutic dose” means the amount of a therapeutic molecule or composition effective in treating, restoring, or preventing a particular disease or condition, or in exhibiting a detectable therapeutic or preventive effect, such as restoring symptoms associated with a disease, preventing or delaying the onset of a disease, and / or even reducing the severity or frequency of symptoms of a disease. Therapeutic doses may be administered in a dosing regimen that may contain multiple unit doses. For any particular therapeutic molecule, the therapeutic dose (and / or appropriate unit dose in an effective dosing regimen) may vary depending, for example, the route of administration, and combinations with other pharmaceuticals. Furthermore, the specific therapeutically effective dose (and / or unit dose) for any particular subject may depend on various factors, including the disorder being treated and its severity; the activity of the particular drug used; the particular composition used; the subject's age, weight, overall health, sex, and diet; the administration time, route of administration, and / or excretion or metabolic rate of the particular therapeutic molecule used; the duration of treatment; and similar factors well known in the medical field.

[0065] Treatment: As used herein, the term “treatment” (and also “to treat” or “to treat”) means any administration of a therapeutic molecule (e.g., an engineered antibody as described herein) that partially or completely reduces, restores, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Such treatment may be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition in question, and / or to subjects who exhibit only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be administered to subjects who exhibit one or more established signs of the disease, disorder, and / or condition in question. This disclosure further provides the following aspects. [Item 1] A heavy chain variable region amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 5, 27, 29, 34, or 36, and / or Light chain variable region amino acid sequences having at least 90%, 95%, 98%, or 99% sequence identity with sequence numbers 6, 8, 26, 28, 35, or 37. An isolated antibody that specifically binds to complement factor D (CFD), including [specific antibody name]. [Item 2] An isolated antibody as described in item 1, containing the same heavy chain variable region amino acid sequence as SEQ ID NOs. 5, 27, 29, 34, or 36. [Item 3] An isolated antibody as described in any one of the items, containing the same light chain variable region amino acid sequence as SEQ ID NOs. 6, 8, 26, 28, 35, or 37. [Item 4] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), and / or The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), An isolated antibody as described in any one of items 1 to 3. [Item 5] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANEGSTYYSPSLES (SEQ ID NO: 20), and / or The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), An isolated antibody as described in any one of items 1 to 3. [Item 6] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), and / or The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), An isolated antibody as described in any one of items 1 to 3. [Item 7] The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSASSNDDAV (SEQ ID NO: 18). An isolated antibody as described in any one of items 1 through 6. [Item 8] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSASSNDDAV (SEQ ID NO: 18). The isolated antibody described in item 7. [Item 9] The same heavy chain variable region amino acid sequence as SEQ ID NO. 5, and The same light chain variable region amino acid sequence as SEQ ID NO: 6 Isolated antibodies, including those listed in item 8. [Item 10] The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSADLNDDAV (SEQ ID NO: 19). An isolated antibody as described in any one of items 1 through 6. [Item 11] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSADLNDDAV (SEQ ID NO: 19). The isolated antibody described in item 10. [Item 12] The same heavy chain variable region amino acid sequence as SEQ ID NO. 5, and The same light chain variable region amino acid sequence as SEQ ID NO: 8 Isolated antibodies, including those described in item 11. [Item 13] The light chain CDR1 contains the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), The light chain CDR2 contains the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or The light chain CDR3 contains the amino acid sequence QSADSNDDAV (SEQ ID NO: 11), An isolated antibody as described in any one of items 1 through 6. [Item 14] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANEGSTYYSPSLES (SEQ ID NO: 20), The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), The light chain CDR1 contains the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), The light chain CDR2 contains the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or The light chain CDR3 contains the amino acid sequence QSADSNDDAV (SEQ ID NO: 11), The isolated antibody described in item 13. [Item 15] The same heavy chain variable region amino acid sequence as SEQ ID NO: 29, and Same light chain variable region amino acid sequence as SEQ ID NO: 28 Isolated antibodies, including those listed in item 14. [Item 16] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), The light chain CDR1 contains the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), The light chain CDR2 contains the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or The light chain CDR3 contains the amino acid sequence QSADSNDDAV (SEQ ID NO: 11), The isolated antibody described in item 13. [Item 17] The same heavy chain variable region amino acid sequence as SEQ ID NO: 27, and The same light chain variable region amino acid sequence as SEQ ID NO: 26 Isolated antibodies, including those described in item 16. [Item 18] The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSADSNDDAV (SEQ ID NO: 11), The isolated antibody described in item 6. [Item 19] The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSADSNDDAV (SEQ ID NO: 11), The isolated antibody described in item 18. [Item 20] The same heavy chain variable region amino acid sequence as SEQ ID NO: 34, and The same light chain variable region amino acid sequence as SEQ ID NO: 35 Isolated antibodies, including those described in item 19. [Item 21] The same heavy chain variable region amino acid sequence as SEQ ID NO: 36, and Same light chain variable region amino acid sequence as SEQ ID NO: 37 Isolated antibodies, including those described in item 19. [Item 22] An isolated antibody that specifically binds to complement factor D (CFD), The heavy chain CDR1 contains the amino acid sequence YYAWS (SEQ ID NO: 12), The heavy chain CDR2 contains the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), The heavy chain CDR3 contains the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), The light chain CDR1 contains the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), The light chain CDR2 contains the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or The light chain CDR3 contains the amino acid sequence QSADSNDDAV (SEQ ID NO: 11), The isolated antibody mentioned above. [Item 23] A heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34, and Light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35 The isolated antibodies described in item 22, including [the specified antibody]. [Item 24] The same heavy chain variable region amino acid sequence as SEQ ID NO: 34, and The same light chain variable region amino acid sequence as SEQ ID NO: 35 Isolated antibodies, including those described in item 23. [Item 25] A heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36, and Light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 37. The isolated antibodies described in item 22, including [the specified antibody]. [Item 26] The same heavy chain variable region amino acid sequence as SEQ ID NO: 36, and Same light chain variable region amino acid sequence as SEQ ID NO: 37 Isolated antibodies, including those described in item 25. [Item 27] An isolated antibody, described in any one of the preceding items, which is a monoclonal antibody or a fragment thereof. [Item 28] An isolated antibody as described in any one of the preceding items, further comprising an IgG constant region. [Item 29] An isolated antibody according to any one of the preceding items, comprising a light chain amino acid sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 3, 30, or 32. [Item 30] An isolated antibody according to any one of the preceding items, comprising a heavy chain amino acid sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, 31, or 33. [Item 31] An isolated antibody described in any one of the preceding items that inhibits the complement accessory pathway. [Item 32] The isolated antibody described in item 31 inhibits the cleavage of complement factor B. [Item 33] At pH 7.4, the affinity dissociation constant (K) is between approximately 1 pM and approximately 50 pM. D An isolated antibody described in any one of the preceding items that binds to CFD. [Item 34] At pH 7.4, affinity dissociation constants (K) less than approximately 50 pM, less than approximately 45 pM, less than approximately 40 pM, less than approximately 35 pM, less than approximately 30 pM, less than approximately 25 pM, less than approximately 20 pM, less than approximately 15 pM, less than approximately 10 pM, less than approximately 9 pM, less than approximately 8 pM, less than approximately 7 pM, less than approximately 6 pM, or less than approximately 5 pM. D An isolated antibody described in any one of the preceding items that binds to CFD. [Item 35] At pH 5.5, the affinity dissociation constant (K) is between approximately 15 nM and approximately 150 nM. D An isolated antibody described in any one of the preceding items that binds to CFD. [Item 36] At pH 5.5, affinity dissociation constants (K) greater than approximately 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 100 nM, or 150 nM are considered to be the optimal balance. D An isolated antibody described in any one of the preceding items that binds to CFD. [Item 37] The off-rate of CFD from the antibody at pH 5.5 is 0.010 s. -1 Super, 0.015s -1 Super, 0.02s -1 Super, 0.025s -1 Super, 0.03s -1 Ultra, 0.035s -1 Ultra-long, or 0.04s -1 An isolated antibody described in any one of the preceding items, which is superseded by the description in any one of the preceding items. [Item 38] An isolated antibody as described in any one of the preceding items, having a serum half-life of more than approximately 5 days, approximately 10 days, approximately 15 days, approximately 20 days, approximately 25 days, approximately 30 days, approximately 35 days, approximately 40 days, approximately 45 days, approximately 50 days, approximately 60 days, approximately 70 days, approximately 80 days, approximately 90 days, approximately 95 days, approximately 100 days, or approximately 125 days or more. [Item 39] A nucleic acid sequence encoding an isolated antibody as described in any one of items 1 through 38. [Item 40] A vector containing the nucleic acid sequence described in item 39. [Item 41] A host cell containing the nucleic acid sequence described in item 39 or the vector described in item 40. [Item 42] A method for producing an antibody, comprising culturing a host cell described in item 41 under conditions suitable for the expression of the antibody. [Item 43] Antibodies or antigen-binding fragments described in items 1 to 38, for use as pharmaceuticals. [Item 44] A method for treating a complement-mediated disorder, comprising administering an effective amount of an antibody described in any one of items 1 to 38 to a subject in need thereof. [Item 45] The method according to item 44, wherein the complement-mediated disorder or condition is atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH). [Item 46] The method according to item 44 or 45, wherein the administration of the antibody inhibits intravascular and extravascular hemolysis.

[0066] The drawings are for illustrative purposes only and are not intended to be restrictive. [Brief explanation of the drawing]

[0067] [Figure 1] This is a schematic diagram illustrating the activation of secondary pathways and anti-CFD strategies in PNH patients who experience hemolysis. [Figure 2A] This is a schematic diagram showing an in vitro assay for evaluating the efficacy of anti-CFD mAb inhibition. Hemolysis levels can be measured by optical density (OD) from released hemoglobin. MAC levels can be measured using complement activation in a terminal complement complex (TCC) kit. [Figure 2B] Exemplary results from a terminal complement complex (TCC) assay measuring membrane invasion complex (MAC) formation in the presence of two anti-CFD antibodies are shown. CFD-depleted serum shows no secondary pathway activity. [Figure 3] This shows the exemplary maximum geometric mean percentage of C3 deposition in RBCs using a hemolysis assay. Anti-CFD antibody 2 (169C8) inhibits C3b deposition in rabbit RBCs as well as anti-CFD antibody 1 (benchmark anti-CFD antibody) compared to a control without the antibody (left). [Figure 4] This shows exemplary Biacore sensorgrams of 2-binding affinity of anti-CFD antibodies against human CFD and cyno-CFD. [Figure 5] This shows exemplary concentrations (μg / ml) of anti-CFD antibody 2 (clone 169C8) over time in knock-in human FcRn mice. [Figure 6] This shows exemplary levels (μg / ml) of total hIgG over time in non-human primates (NHPs) injected with anti-CFD mAbs. [Figure 7] This shows the time course and exemplary TCC formation percentage in anti-CFD antibodies administered intravenously (IV) or subcutaneously (SC) to NHP. [Figure 8] The top panel shows exemplary levels of total hIgG (μg / ml) over time in animals subcutaneously injected with anti-CFD mAbs, and the concentration of free CFD and TCC formation percentage over time in non-human primates. The middle panel shows free target levels after anti-CFD injection. The bottom panel shows the duration of CFD inhibition after anti-CFD antibody injection. [Figure 9] The arrows indicate exemplary total hIgG (μg / ml) levels over time in NHP patients with multiple injections. [Figure 10] The arrows indicate the percentage of TCC formation over time in NHP with multiple injections. [Modes for carrying out the invention]

[0068] Inhibition of complement factor D may prevent activation of secondary pathways. This disclosure is partly based on the discovery of engineered antibodies that remarkably exhibit pH-dependent binding to CFD (e.g., human CFD) and / or altered (e.g., increased, e.g., pH-dependent) binding to Fc receptors (e.g., FcRn), resulting in CFD depletion. CFD inhibition using the antibodies described herein prevents intravascular and extravascular hemolysis.

[0069] Complement factor D Complement factor D (CFD), also known as adipsin, is a serine protease essential for initiating a secondary pathway of the complement system. Human complement factor D is synthesized as a 253-amino acid precursor containing a signal peptide (aa1-20), a 5-residue activation / propeptide (aa21-25), and a mature chain (aa26-253). Mature human factor D shares 98% aa sequence identity with chimpanzee factor D, 96% with rhesus monkey factor D, 84% with porcine factor D, and 66% with mouse factor D. Factor D is expressed in multiple tissues, including monocytes / macrophages, muscle, sciatic nerve, endometrium, kidney, and intestine, and is expressed at particularly high levels in adipocytes.

[0070] Abnormal activation of the complement accessory pathway can lead to complement-mediated destruction of red blood cells (RBCs). For example, CD55 / 59 deficiency on the surface of RBCs can cause terminal complement-mediated deposition and destruction of sensitive RBCs. Captured oxyhemoglobin can cause hemolysis, releasing free hemoglobin and leading to nitric oxide depletion. Nephrotoxicity is due to the deposition of free hemoglobin iron and hemosiderin from hemolytic red blood cells. Repeated exposure to toxic substances, thrombosis, and necrosis can lead to renal failure. Hemolysis can also lead to complications including renal failure, pulmonary hypertension, and cholelithiasis.

[0071] antibody The anti-CFD antibodies described herein are designed to inhibit complement factor D (CFD) activity by inhibiting factor B cleavage in a secondary pathway. This inhibitory activity prevents intravascular and extravascular hemolysis. The anti-CFD antibodies according to the present invention demonstrate species cross-reactivity to humans and cynomolgus monkeys (cyno) for both acid-switched half-life extension (ASHE) and CFD inhibition. In some embodiments, the anti-CFD antibodies are engineered to bind to CFD with higher affinity at pH 7.4 than at pH 5.5. In some embodiments, antibody binding at pH 7.4 is less than or equal to 20 pM. In some embodiments, CFD is acidic at pH 5.5: kd > 20 nM, off-rate > 10 E -2 s -1It is released in the cyno. In some embodiments, the anti-CFD antibody has a half-life of approximately 20 days in cyno or more than 45 days in knock-in huFcRn mice.

[0072] The manipulated antibodies described herein may be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds having antibody-like properties, and other immunological binding sites known in the art, such as Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibody, diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, TandAb, or any combination thereof. The subunit structures and three-dimensional arrangements of various classes of antibodies are known in the art.

[0073] An antibody may be an immunoglobulin molecule consisting of four polypeptide chains, for example, two heavy (H) chains and two light (L) chains. The heavy chain may include a heavy chain variable domain and a heavy chain constant domain. The heavy chain constant domain may include CH1, hinge, CH2, CH3, and in some cases, CH4 regions. A preferred heavy chain constant region may be derived from any immunoglobulin (e.g., IgA, IgG, or IgE). In some embodiments, a preferred heavy chain constant region may be derived from IgG1, IgG2, or IgG4. In certain embodiments, a preferred heavy chain constant region is derived from IgG1. The light chain may include a light chain variable domain and a light chain constant domain. The light chain constant domain may include either a kappa light chain or a lambda light chain. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain can typically be further subdivided into variable regions called complementarity-determining regions (CDRs), which are inserted between more conserved regions called framework regions (FRs). Such heavy and light chain variable domains may each include three CDRs and four framework regions, namely FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, arranged in the following order from the amino terminus to the carboxyl terminus, one or more of which may be manipulated as described herein.

[0074] Manipulated heavy chain In some embodiments, the manipulated antibodies described herein may include or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. EVQVQESGPGLVKPSQTLSLTCTVSGGSITTSYYAWSWIRQPPGKGLEWMGDIANDGSTYYSPSLESRTSISRDTSKNQFSLQLSSVTPEDTAVYYCARLRSLYTDYDPHYYDYW GQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK(Sequence ID 2)

[0075] In some embodiments, the manipulated antibodies described herein may include a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 27. EVQVQESGPGLVKPSQTLSLTCTVSGGSITTSYYAWSWIRQPPGKGLEWMGDIANDGSTYYSPSLESRTSISRDTSKNQFSLQLSSVTPEDTAVYYCARLRSLYTDYDPHYYDYWGQGTQVTVSS(Sequence ID 27)

[0076] In some embodiments, the manipulated antibodies described herein may include or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 4. QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWIGDIANEGSTYYSPSLESRVTISRDTSKNQFSLQLSSVTAADTAVYYCARLRSLYTDYDPHYYDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPG(Sequence ID 4)

[0077] In some embodiments, the manipulated antibodies described herein may include or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 31. QVQVQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWMGDIANDGSTYYSPSLESRVTISRDTSKNQFSLQLSSVTAQDTAVYYCARLRSLYTDYDPHYYDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPG (Sequence ID 31)

[0078] In some embodiments, the manipulated antibodies described herein may include or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. QVQVQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWMGDIANDGSTYYSPSLESRVTISRDTSKNQFSLQLSSVTAQDTAVYYCARLRSLYTDYDPHYYDYW GQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPG (Sequence ID 33)

[0079] In some embodiments, the engineered antibody contains a heavy chain amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 2, 4, 31, or 33. In some embodiments, the engineered antibody contains the same heavy chain amino acid sequence as SEQ ID NOs: 2, 4, 31, or 33. In some embodiments, the engineered antibody contains 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or two or fewer amino acid substitutions compared to SEQ ID NOs: 2, 4, 31, or 33. In some embodiments, the manipulated antibody contains a heavy chain amino acid sequence having one or more amino acid substitutions at one or more of the positions 1, 4, 30, 50, 56, 69, 70, 89, 90, 120, 222, 364, or 366, compared to SEQ ID NOs: 2, 4, 31, or 33.

[0080] In some embodiments, the manipulated antibodies described herein may include a heavy chain variable region comprising or relating to the amino acid sequence of SEQ ID NO: 29. QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWIGDIANEGSTYYSPSLESRVTISRDTSKNQFSLQLSSVTAADTAVYYCARLRSLYTDYDPHYYDYWGQGTLVTVSS (Sequence ID 29)

[0081] In some embodiments, the manipulated antibodies described herein may include a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 5. QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWIGDIANEGSTYYSPSLKSRVTISRDTSKNQFSLQLSSVTAADTAVYYCARLRSLYTDYDPHYYDYWGQGTLVTVSS (Sequence ID 5)

[0082] In some embodiments, the manipulated antibodies described herein may include or comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34. QVQVQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWMGDIANDGSTYYSPSLESRVTISRDTSKNQFSLQLSSVTAQDTAVYYCARLRSLYTDYDPHYYDYWGQGTLVTVSS (Sequence ID 34)

[0083] In some embodiments, the manipulated antibodies described herein may include a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 36. QVQVQESGPGLVKPSQTLSLTCTVSGGSISTSYYAWSWIRQPPGKGLEWMGDIANDGSTYYSPSLESRVTISRDTSKNQFSLQLSSVTAQDTAVYYCARLRSLYTDYDPHYYDYWGQGTQVTVSS (Sequence ID 36)

[0084] In some embodiments, the manipulated antibody contains a heavy chain amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 5, 27, 29, 34, or 36. In some embodiments, the manipulated antibody contains the same heavy chain amino acid sequence as SEQ ID NOs. 5, 27, 29, 34, or 36.

[0085] In some embodiments, the engineered antibody contains a heavy chain amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 5, 27, 29, 34, or 36. In some embodiments, the engineered antibody contains the same heavy chain amino acid sequence as SEQ ID NOs. 5, 27, 29, 34, or 36. In some embodiments, the engineered antibody contains 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or two or fewer amino acid substitutions compared to SEQ ID NOs. 5, 27, 29, 34, or 36. In some embodiments, the manipulated antibody contains a heavy chain amino acid sequence having one or more amino acid substitutions at one or more of the positions 1, 4, 30, 50, 56, 69, 70, 89, 90, 120, or 222, compared to SEQ ID NOs. 5, 27, 29, 34, or 36.

[0086] In some embodiments, the manipulated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0087] In some embodiments, the manipulated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLES (SEQ ID NO: 20), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0088] In some embodiments, the manipulated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), and / or a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14).

[0089] As can be understood by those skilled in the art, any such heavy chain CDR sequence can be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or a portion thereof, which may be present in any format of antibody or binding molecule disclosed herein or otherwise known in the art, for example by molecular biological techniques.

[0090] Manipulated heavy chain constant domain In the various engineered antibodies described herein, the heavy chain constant domain may be of any class (or subclass). In the various engineered antibodies described herein, the heavy chain constant domain may contain one or more amino acid sequences of IgG, IgM, IgA, IgD, or IgE, including subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In various cases, the constant domain of the engineered antibody described herein may contain a mixture of two or more classes (or subclasses) of immunoglobulin heavy chain constant domains. For example, an engineered antibody may contain a first portion of a constant domain having a sequence of an immunoglobulin constant domain selected from the IgG, IgM, IgA, IgD, or IgE class, and a second portion of a constant domain having a sequence of an immunoglobulin constant domain selected from the IgG, IgM, IgA, IgD, or IgE class, different from the first portion. In some cases, the constant domain of the manipulated antibody described herein may comprise a mixture of two or more subclasses of a particular class of constant domains, for example, a first portion of a constant domain having a sequence of an immunoglobulin constant domain selected from the constant domains of the IgG1, IgG2, IgG3, or IgG4 subclasses, and a second portion of a constant domain having a sequence of an immunoglobulin constant domain selected from the constant domains of the IgG1, IgG2, IgG3, or IgG4 subclasses, different from the first portion. In some specific embodiments, the constant domain comprises all or part of an IgG2 constant domain and all or part of an IgG4 constant domain.

[0091] In some cases, the manipulated antibody includes an antibody constant region, Fc region, or Fc fragment that exhibits modified binding (compared to a reference constant region) to one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptors). In some embodiments, the constant region, Fc region, or Fc fragment is manipulated to bind to a target (e.g., FcRn receptor) in a modified manner (e.g., in a pH-sensitive manner (e.g., in a manner that is more or less pH-sensitive) and / or in a reduced or increased binding) compared to a reference constant region, Fc region, or Fc fragment. In some embodiments, the engineered antibody includes an antibody constant region, Fc region, or Fc fragment that exhibits reduced binding (compared to a reference constant region) to one or more Fcγ receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, or FcγRIV). In some embodiments, the engineered antibody includes an antibody constant region, Fc region, or Fc fragment that exhibits increased binding (compared to a reference constant region) to FcRn receptors at serum pH and / or intracellular pH.

[0092] For example, the engineered antibody may include a constant region, Fc region, or Fc fragment of an IgG antibody engineered to include the addition, deletion, or substitution of one or more amino acids from amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)). While we do not wish to be bound by theory, one or more of these constant region, Fc region, or Fc fragment amino acids are thought to mediate interactions with Fc receptors, such as FcRn. In some embodiments, one or more of these disclosed amino acids are substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. In some embodiments, non-histidine residues are substituted with histidine residues. In some embodiments, histidine residues are substituted with non-histidine residues.

[0093] In some embodiments, the manipulated antibody comprises a constant region, Fc region, or Fc fragment of an IgG antibody having amino acid modifications at one or more of the 308, 309, 311, 312, and 314 positions, more specifically, having substitutions of threonine, proline, serine, aspartic acid, and leucine at one or more of the 308, 309, 311, 312, and 314 positions, respectively. In some embodiments, one or more residues at the 308, 309, and 311 positions are substituted with isoleucine, proline, and glutamic acid, respectively. In yet another embodiment, one or more residues at the 308, 309, 311, 312, and 314 positions are substituted with threonine, proline, serine, aspartic acid, and leucine, respectively.

[0094] In some embodiments, the manipulated antibody comprises a constant region, Fc region, or Fc fragment of an IgG antibody having amino acid modifications at one or more of the 251, 252, 254, 255, and 256 positions, more specifically, substitutions at one or more of these positions. In some embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with leucine, tyrosine, phenylalanine, serine, tryptophan, or threonine, residue 254 is substituted with threonine or serine, residue 255 is substituted with leucine, glycine, isoleucine, or arginine, and / or residue 256 is substituted with serine, phenylalanine, arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagine, or threonine. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine or leucine, residue 254 is substituted with threonine or serine, and / or residue 255 is substituted with arginine. In yet other embodiments, residue 252 is substituted with phenylalanine, and / or residue 256 is substituted with aspartic acid. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, and / or residue 255 is substituted with arginine.

[0095] In some embodiments, the manipulated antibody comprises a constant region, Fc region, or Fc fragment of an IgG antibody having amino acid modifications at one or more of the positions 428, 433, 434, 435, and 436, more specifically, having substitutions at one or more of these positions. In some embodiments, residue 428 is substituted with methionine, threonine, leucine, phenylalanine, or serine; residue 433 is substituted with lysine, arginine, serine, isoleucine, proline, glutamine, or histidine; residue 434 is substituted with phenylalanine, tyrosine, or histidine; residue 435 is substituted with tyrosine; and / or residue 436 is substituted with histidine, asparagine, arginine, threonine, lysine, methionine, or threonine. In some embodiments, one or more residues at positions 433, 434, 435, and 436 are substituted with lysine, phenylalanine, tyrosine, and histidine, respectively. In some embodiments, residue 428 is substituted with methionine and / or residue 434 is substituted with tyrosine.

[0096] In some embodiments, the manipulated antibody comprises a constant region, Fc region, or Fc fragment of an IgG antibody having amino acid modifications at one or more of the 385, 386, 387, and 389 positions, more specifically, substitutions at one or more of these positions. In some embodiments, residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, or alanine; residue 386 is substituted with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine; residue 387 is substituted with arginine, histidine, serine, threonine, alanine, or proline; and / or residue 389 is substituted with proline or serine. In some embodiments, one or more residues at the 385, 386, 387, and 389 positions are substituted with arginine, threonine, arginine, and proline, respectively. In some embodiments, one or more residues at positions 385, 386, and 389 are substituted with aspartic acid, proline, and serine, respectively.

[0097] In some embodiments, the manipulated antibody comprises a constant region, Fc region, or Fc fragment of an IgG antibody having one or more of the following substitutions: leucine at residue 251, tyrosine or leucine at residue 252, threonine or serine at residue 254, arginine at residue 255, threonine at residue 308, proline at residue 309, serine at residue 311, aspartic acid at residue 312, leucine at residue 314, arginine at residue 385, threonine at residue 386, arginine at residue 387, proline at residue 389, methionine at residue 428, lysine at residue 433, phenylalanine or tyrosine at residue 434, tyrosine at position 435, and / or tyrosine at position 436. Examples of additional amino acid substitutions that may be included in the constant region, Fc region, or Fc fragment are those described in U.S. Patent Nos. 6,277,375, 8,012,476, and 8,163,881.

[0098] In some embodiments, the engineered antibodies described herein include a heavy chain constant domain containing, for example, the Ala-Ala mutation described in PCT publication numbers WO94 / 28027 and WO98 / 47531, and Xu et al. (2000) Cell Immunol 200:16-26. Thus, in some embodiments, an engineered antibody having one or more mutations containing the Ala-Ala mutation within the heavy chain constant region has reduced effector function or no effector function at all. According to these embodiments, the constant region of the engineered antibodies described herein may include a substitution to alanine at position 234 and / or a mutation to alanine at position 235 (EU numbering).

[0099] As can be understood by those skilled in the art, any such heavy chain constant domain sequence can be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or a portion thereof, which may be present in any format of antibody or binding molecule disclosed herein or otherwise known in the art, for example by molecular biological techniques.

[0100] Manipulated light chain In some embodiments, the manipulated antibodies described herein may include or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1. SSALTQPSALSVTKGQTAKITCQGDLLPRHYAHWYQQKTGQAPKLIVYDDDIRPSGIPERFSGSNSGGVATLTIAGAQAEDEADYHCQSADSNDDAVFGGGTHLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(Sequence ID 1)

[0101] In some embodiments, the manipulated antibodies described herein may include or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 3. SYELTQPPALSVSPGQTARITCQGDLLPRHYAHWYQQKTGQAPKLVIYDDDIRPSGIPERFSGSSSGTMATLTISGAQAEDEADYHCQSADSNDDAVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(Sequence ID 3)

[0102] In some embodiments, the manipulated antibodies described herein may include or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 30. SSALTQPPALSVSKGQTARITCQGNLLPRHYAHWYQQKTGQAPKLIVYDDNIRPSGIPERFSGSNSGGVATLTISGAQAEDEADYHCQSADSNDDAVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(Sequence ID 30)

[0103] In some embodiments, the manipulated antibodies described herein may include or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 32. SSALTQPSALSVSKGQTARITCQGNLLPRHYAHWYQQKTGQAPKLIVYDDNIRPSGIPERFSGSNSGGVATLTISGAQAEDEADYHCQSADSNDDAVFGGGTHLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(Sequence ID 32)

[0104] In some embodiments, the engineered antibody comprises a light chain amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1, 3, 30, or 32. In some embodiments, the engineered antibody comprises a light chain amino acid sequence identical to SEQ ID NO: 1, 3, 30, or 32. In some embodiments, the engineered antibody comprises 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or two or fewer amino acid substitutions compared to SEQ ID NO: 1, 3, 30, or 32. In some embodiments, the engineered antibody comprises a light chain amino acid sequence having one or more amino acid substitutions at one or more of positions 1, 2, 3, 4, 8, 13, 14, 19, 46, 47, 65, 68, 69, 75, 103, 108 compared to SEQ ID NO: 1, 3, 30, or 32.

[0105] In some embodiments, the engineered antibody described herein may comprise a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 26. SSALTQPSALSVTKGQTAKITCQGDLLPRHYAHWYQQKTGQAPKLIVYDDDIRPSGIPERFSGSNSGGVATLTIAGAQAEDEADYHCQSADSNDDAVFGGGTHLTVLSQPKAAPSVTLFPPSS (SEQ ID NO: 26)

[0106] In some embodiments, the engineered antibody described herein may comprise a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 28. SYELTQPPALSVSPGQTARITCQGDLLPRHYAHWYQQKTGQAPKLVIYDDDIRPSGIPERFSGSSSGTMATLTISGAQAEDEADYHCQSADSNDDAVFGGGTQLTVLGQPKAAPSVTL (SEQ ID NO: 28)

[0107] In some embodiments, the manipulated antibodies described herein may include a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 6. SYELTQPPALSVSPGQTARITCQGNLLPRHYAHWYQQKTGQAPKLVIYDDNIRPSGIPERFSGSSSGTTATLTISGAQAEDEADYHCQSASSNDDAVFGGGTQLTVL(Sequence ID 6)

[0108] In some embodiments, the manipulated antibodies described herein may include a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8. SYELTQPPALSVSPGQTARITCQGNLLPRHYAHWYQQKTGQAPKLVIYDDNIRPSGIPERFSGSSSGTTATLTISGAQAEDEADYHCQSADLNDDAVFGGGTQLTVL(Sequence ID 8)

[0109] In some embodiments, the manipulated antibodies described herein may include a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 35. SSALTQPPALSVSKGQTARITCQGNLLPRHYAHWYQQKTGQAPKLIVYDDNIRPSGIPERFSGSNSGGVATLTISGAQAEDEADYHCQSADSNDDAVFGGGTQLTVL(Sequence ID 35)

[0110] In some embodiments, the manipulated antibodies described herein may include a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 37. SSALTQPSALSVSKGQTARITCQGNLLPRHYAHWYQQKTGQAPKLIVYDDNIRPSGIPERFSGSNSGGVATLTISGAQAEDEADYHCQSADSNDDAVFGGGTHLTVL(Sequence ID 37)

[0111] In some embodiments, the engineered antibody contains a light chain variable region amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 6, 8, 26, 28, 35, or 37. In some embodiments, the engineered antibody contains the same light chain variable region amino acid sequence as SEQ ID NOs. 6, 8, 26, 28, 35, or 37. In some embodiments, the engineered antibody contains 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or two or fewer amino acid substitutions compared to SEQ ID NOs. 6, 8, 26, 28, 35, or 37. In some embodiments, the manipulated antibody contains a light chain variable region amino acid sequence having one or more amino acid substitutions at one or more of the positions 1, 2, 3, 4, 8, 13, 14, 19, 46, 47, 65, 68, 69, 75, 103, or 108, compared to SEQ ID NOs: 6, 8, 26, 28, 35, or 37.

[0112] In some embodiments, the manipulated antibodies described herein include a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSASSNDDAV (SEQ ID NO: 18).

[0113] In some embodiments, the manipulated antibodies described herein include a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSADLNDDAV (SEQ ID NO: 19).

[0114] In some embodiments, the manipulated antibodies described herein include a light chain CDR1 containing the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), a light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0115] In some embodiments, the manipulated antibodies described herein include a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and / or a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0116] As can be understood by those skilled in the art, any such light chain CDR sequence can be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or a portion thereof, which may be present in any format of antibody or binding molecule disclosed herein or otherwise known in the art, for example by molecular biological techniques.

[0117] Manipulated light chain constant domain In some embodiments, the manipulated antibodies described herein include a light chain containing any light chain constant domain sequence, for example, a light chain constant sequence known to those skilled in the art. As those skilled in the art will recognize, the light chain constant domain may be a kappa light chain constant domain or a lambda light chain constant domain. In certain embodiments, the light chain constant domain disclosed herein is a kappa light chain constant domain.

[0118] In various cases, the manipulated antibodies described herein include a light chain constant domain comprising or consisting of the amino acid sequence of SEQ ID NO: 434. As can be understood by those skilled in the art, any such light chain constant domain sequence can be readily combined with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or a portion thereof, which may be present in any format of antibody or conjugate molecule disclosed herein or otherwise known in the art, for example, by molecular biological techniques.

[0119] Exemplary manipulated antibodies The engineered antibody may contain various heavy and light chains as described herein. In some embodiments, the engineered antibody may contain two heavy and light chains. In various cases, this disclosure includes an antibody comprising at least one heavy and / or light chain as disclosed herein, at least one heavy and / or light chain framework domain as disclosed herein, at least one heavy and / or light chain CDR domain as disclosed herein, and / or any heavy and / or light chain constant domain as disclosed herein.

[0120] In various cases, the engineered antibodies disclosed herein are homodimer monoclonal antibodies. In various cases, the engineered antibodies disclosed herein are heterodimer antibodies. In various cases, the engineered antibodies are, for example, typical antibodies or diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandabs, DVDs, BiTe, scFv, TandAb, scFv, Fab, Fab2, Fab3, F(ab')2, etc., or any combination thereof.

[0121] In some embodiments, the engineered isolated antibodies described herein include or consist of at least one light chain variable domain having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to any one of the amino acid sequences of SEQ ID NO: 6, 8, 26, 28, 35, or 37.

[0122] In some embodiments, the engineered isolated antibodies described herein include or consist of at least one heavy chain variable domain having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to any one of the amino acid sequences of SEQ ID NO: 5, 27, 29, 34, or 36.

[0123] In some embodiments, the engineered isolated antibodies described herein include a heavy chain CDR1 that includes the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 that includes the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), a heavy chain CDR3 that includes the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 that includes the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 that includes the amino acid sequence DDNIRPS (SEQ ID NO: 17), and a light chain CDR3 that includes the amino acid sequence QSASSNDDAV (SEQ ID NO: 18).

[0124] In some embodiments, the isolated antibody includes a heavy chain variable region amino acid sequence identical to SEQ ID NO: 5 and a light chain variable region amino acid sequence identical to SEQ ID NO: 6.

[0125] In some embodiments, the manipulated isolated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and a light chain CDR3 containing the amino acid sequence QSADLNDDAV (SEQ ID NO: 19).

[0126] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 5 and the same light chain variable region amino acid sequence as SEQ ID NO: 8.

[0127] In some embodiments, the manipulated isolated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and a light chain CDR3 containing the amino acid sequence QSADLNDDAV (SEQ ID NO: 19).

[0128] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 29 and the same light chain variable region amino acid sequence as SEQ ID NO: 28.

[0129] In some embodiments, the manipulated isolated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGDLLPRHYAH (SEQ ID NO: 9), a light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10), and a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0130] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 27 and the same light chain variable region amino acid sequence as SEQ ID NO: 26.

[0131] In some embodiments, the manipulated isolated antibodies described herein include a heavy chain CDR1 containing the amino acid sequence YYAWS (SEQ ID NO: 12), a heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13), a heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYDY (SEQ ID NO: 14), a light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16), a light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17), and a light chain CDR3 containing the amino acid sequence QSADSNDDAV (SEQ ID NO: 11).

[0132] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 34 and the same light chain variable region amino acid sequence as SEQ ID NO: 35.

[0133] In some embodiments, the isolated antibody contains the same heavy chain variable region amino acid sequence as SEQ ID NO: 36 and the same light chain variable region amino acid sequence as SEQ ID NO: 37.

[0134] In some embodiments, the light chain and heavy chain comprise a signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 21), MAWTPLLLPLLTFCTVSEA (SEQ ID NO: 38), or MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 39).

[0135] Manipulated fusion protein In some embodiments, the Disclosure provides a fusion protein comprising (i) one or more antigen-binding regions described herein (e.g., antigen-binding regions of immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds having antibody-like properties, and other antigen-binding regions known in the Art, such as Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibody, diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, TandAb, etc.), for example, one or more variable domains described herein, or a portion thereof (e.g., one or more CDRs described herein), and (ii) one or more additional polypeptides. For example, albumin is an abundant serum protein protected from degradation by pH-dependent recycling mediated by interaction with FcRn. In some embodiments, one or more variable domains or engineered antibodies described herein, or a portion thereof (e.g., one or more CDRs described herein), fuse with albumin, a portion thereof (e.g., a portion of albumin that binds to FcRn), and / or an engineered variant of albumin that binds to FcRn with improved affinity. In other embodiments, one or more variable domains or engineered antibodies described herein, or a portion thereof (e.g., one or more CDRs described herein), fuse with a polypeptide, which binds to albumin to form a fusion protein-albumin complex, which can bind to FcRn. In some embodiments, the polypeptide that binds to albumin is a single-stranded variable fragment (scFv). Albumin or a portion thereof may contain mutations in one or more amino acids that can alter the binding of albumin or a portion thereof to FcRn. Such mutations are known in the art (see, for example, Andersen et al., Nature Communications 3:610 doi:10.1038 / nocmms1607 (2012)).

[0136] In other cases, one or more variable domains or manipulated antibodies, or parts thereof (e.g., one or more CDRs described herein), fused with transferrin. Transferrin is recycled by binding to the transferrin receptor (see, e.g., Widera et al., Adv. Drug Deliv. Rev. 55:1439-66 (2003)).

[0137] In some embodiments, the present disclosure provides a fusion protein comprising one or more variable domains or manipulated antibodies, or a portion thereof, described herein, and one or more additional polypeptides and / or scFvs that bind to FcRn.

[0138] Nucleotide sequence This disclosure includes nucleotide sequences that encode one or more heavy chains, heavy chain variable domains, heavy chain framework regions, heavy chain CDRs, heavy chain constant domains, light chains, light chain variable domains, light chain framework regions, light chain CDRs, light chain constant domains, or other immunoglobulin-like sequences, antibodies, or binding molecules, as disclosed herein. In various cases, such nucleotide sequences may be present in a vector. In various cases, such nucleotides may be present in the genome of a cell, for example, a target cell requiring treatment, or a cell for antibody production, for example, a mammalian cell for antibody production.

[0139] PEGylation In certain embodiments, the manipulated antibodies described herein can be PEGylated to include mono or poly(e.g., 2-4) PEG moieties. Such PEGylated antibodies may exhibit an increased half-life compared to non-PEGylated reference antibodies, e.g., those having the same amino acid sequence but with different PEGylation, different amounts of PEGylation, or an unPEGylated antibody.

[0140] PEGylation can be carried out by any suitable reaction known in the art. Methods for preparing PEGylated proteins may generally include (a) reacting a polypeptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions that cause the polypeptide to bind to one or more PEG groups, and (b) obtaining a reaction product(s). Generally, the conditions for the reaction can be determined individually based on known parameters and the desired results.

[0141] Several PEG bonding methods are available to those skilled in the art. For example, the step of PEGylating the multispecific binding molecule described herein can be carried out via an acylation or alkylation reaction with a reactive polyethylene glycol molecule.

[0142] pH-dependent binding of manipulated antibodies to CFD and / or Fc receptors The manipulated antibodies described herein exhibit pH-dependent or enhanced pH-dependent affinity for CFD (e.g., mediated by one or more variable domains described herein) and / or modified (e.g., increased, e.g., pH-dependent) affinity for FcRn (e.g., mediated by one or more constant domains described herein). For example, in some embodiments, an antibody capable of binding to CFD, or a variable domain capable of binding to CFD, binds to CFD with higher affinity at serum pH (e.g., neutral pH or pH higher than 7.4) than at compartment (e.g., endosome) pH (e.g., acidic pH or pH equal to or lower than pH 6.0). In various cases where CFD is bound by an antibody having pH-dependent CFD binding, the transition of pH from serum pH to compartment pH (e.g., from serum to endosomes) facilitates the separation (i.e., "debinding") of CFD and the antibody at compartment pH and / or in a specific compartment, e.g., in endosomes. In various cases, such pH-dependent binding may mediate antibody recycling and / or CFD degradation. In certain cases, the transition from serum pH to compartmental pH (e.g., from serum to endosomes) facilitates the separation (i.e., "debinding") of CFDs and antibodies at compartmental pH and / or in specific compartments, e.g., endosomes, resulting in the recycling of antibodies by FcRn and the degradation of antigens in lysosomes. In some such cases, the pH dependence of CFD binding improves the antibody's "processing capacity," at least in that, if recycled, the antibody can return to the serum and freely bind to target circulating CFDs. In some cases, the recycling of antibodies exhibiting pH-dependent CFD binding can continue until the antibody is finally degraded or degraded, and a single antibody molecule may have bound to multiple CFD molecules, not just one, and mediated their inactivation up to this point.

[0143] In certain embodiments, the engineered antibodies disclosed herein include a constant domain (e.g., an Fc domain) that exhibits increased affinity to an Fc receptor such as FcRn compared to a control (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2). In some embodiments, such increased affinity compared to a control is at serum pH values ​​(e.g., pH greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2 or higher). In some embodiments, such increased affinity compared to the control is at a specific pH (e.g., pH <7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5.0 or less). In certain embodiments, the engineered antibody disclosed herein includes a constant domain (e.g., an Fc domain) that exhibits pH-dependent (or enhanced pH-dependent compared to a control, e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2) affinity for an Fc receptor such as FcRn. Neonatal Fc receptors (FcRn) are MHC class I-like molecules that function to protect IgG and albumin from catabolism, mediate the transepithelial transport of IgG and participate in antigen presentation by professional antigen-presenting cells. IgG antibody subtypes exhibit long serum half-lives, primarily due to the removal of antibodies from endosomes by FcRn, which recycles IgG outside the cell.

[0144] In some specific cases, the manipulated antibodies described herein exhibit greater pH dependence than endogenous IgG in binding to FcRn, in that the antibodies described herein show a greater absolute and / or relative differential change in affinity between serum pH and compartment (e.g., endosome) pH (or between serum and endosomes) than endogenous IgG. In some cases, antibodies with pH-dependent binding to FcRn show greater binding to FcRn than endogenous IgG at compartment (e.g., endosome) pH (e.g., acidic pH or pH equal to or below pH 6.0). In some cases, antibodies with pH-dependent binding to FcRn show greater binding to FcRn than endogenous IgG at serum pH (e.g., neutral pH or serum with a pH higher than pH 7.4).

[0145] In certain cases, the engineered antibodies described herein exhibit pH-dependent or enhanced pH-dependent binding to FcRn and compete with endogenous IgG for interaction with FcRn. Therefore, in some cases, the engineered antibodies described herein bind to FcRn at a higher level than endogenous IgG molecules (i.e., outperform endogenous IgG molecules for binding to FcRn as a result of greater affinity for FcRn), and / or result in a net increase in the rate of recycling of the FcRn affinity-enhanced antibody compared to endogenous IgG molecules. In some cases, such preferential interaction between the engineered antibodies described herein and FcRn compared to endogenous IgG molecules results in an increased antibody half-life compared to the reference antibody.

[0146] In some embodiments, the manipulated antibodies described herein exhibit a) increased affinity for FcRn at acidic or compartmental pH compared to serum pH, and / or b) decreased affinity for CFD at acidic or compartmental pH compared to serum pH. In some cases, such a combination of features results in a synergistic increase in the antibody's ability to process the antigen, reducing the formation of antigen-antibody complexes, and / or significantly increasing the antibody's half-life if the antigen-antibody complexes are eliminated faster than the antibody itself. This technique enables highly effective targeting and elimination of CFD.

[0147] In some embodiments, the engineered antibody further comprises an FcRn-binding moiety having a higher affinity for CFD at serum pH than at compartment pH (e.g., endosomal pH) and a lower affinity for FcRn at serum pH than at compartment pH (e.g., endosomal pH), in which such antibody binds to serum CFD with high affinity to form an antibody / CFD complex. In the internalization of the antibody / CFD complex into an internal compartment (e.g., endosome) having a pH lower than serum pH by cells (e.g., by pinocytosis), the lower affinity of the antibody for CFD facilitates the separation of the antibody from the CFD, and the subsequently released CFD can be degraded by cellular mechanisms (e.g., by lysosomes). Furthermore, the increased affinity for FcRn at compartment pH facilitates the formation of an antibody / FcRn complex, which can be recycled into serum via an FcRn recycling pathway (in which the complex is exposed to serum or serum-like pH conditions, this pH facilitating the release of the antibody from the FcRn complex into, for example, serum). One possible end result of such a process is a decrease in the serum half-life of CFD. Another possible end result of such a process is an increase in the serum half-life of the antibody.

[0148] In various cases disclosed herein, serum pH may be, for example, a typical or characteristic pH or pH range of serum, an individual or average serum pH or pH range of one or more subjects, a standard pH value of serum, a measured pH value of serum, an estimated pH value of serum, or a selected pH value of serum. In various cases disclosed herein, serum pH may be greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2 or greater. In various cases, serum pH is in the range of 6.8–8.2, 7.0–8.0, 7.0–7.8, 7.0–7.6, 7.0–7.4, or 7.0–7.2. In some embodiments, serum pH is pH 7.4 or approximately pH 7.4.

[0149] In various cases disclosed herein, compartment pH may be, for example, a typical or characteristic pH or pH range of an endosomal compartment (e.g., within an endosome), an individual or average endosomal compartment pH or pH range of one or more subjects, a standard pH value of an endosomal compartment, a measured pH value of an endosomal compartment, an estimated pH value of an endosomal compartment, or a selected pH value of an endosomal compartment. In various cases disclosed herein, compartment pH may be less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5.0 or less. In various cases, the pH of the compartment is in the range of 5.0-7.2, 5.0-7.0, 5.0-6.8, 5.0-6.6, 5.0-6.4, 5.0-6.2, 5.0-6.0, 5.0-5.8, 5.0-5.6, 5.0-5.4, or 5.0-5.2.

[0150] In some embodiments, the serum half-life of serum CFD protein is reduced. For example, binding of a manipulated antibody to serum CFD reduces the serum half-life of CFD to approximately 7, 6, 5, 4, 3, 2, or less than 1 day, or approximately 24, 18, 12, or less than 6 hours.

[0151] In some embodiments, the serum half-life of the manipulated antibody is increased. For example, binding of the manipulated antibody to FcRn increases the serum half-life of the antibody to about 4 to about 45 days, for example, about 5 to about 30 days, about 10 to about 30 days, or about 20 to about 30 days. In certain embodiments, the manipulated antibodies described herein have a serum half-life of about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days or more.

[0152] In certain embodiments, the manipulated antibodies described herein exhibit a pH-dependent change in affinity for CFD. Affinity is determined by the K of the antibody and antigen. D It can be measured as the (equilibrium dissociation constant), K D The affinity is inversely related. In various embodiments, the K of the manipulated antibody described herein against CFD under serum pH (e.g., pH greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2 or higher) or serum conditions. D It is about 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , or 10 -15 It is less than M. In certain cases, the K of the antibody described herein against CFD at serum pH. DK is between 0.001 and 1 nM, for example, 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM. In some embodiments, K to CFD under compartmental pH (e.g., pH less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or pH less than or equal to 5.0) or compartmental conditions. D This refers to the K of the same antibody against CFD under serum pH or serum conditions. D For example, at least twice as high, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 times higher (and / or the affinity of an antibody for CFD under compartmental pH or compartmental conditions may decrease by the same proportion compared to its affinity under serum pH or serum conditions). In some embodiments, the pH of the compartment (e.g., less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or pH 5.0 or less) or K to CFD under compartment conditions D For example, 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , or 10 -3 M may be greater than M. In certain cases, the K of the manipulated antibodies described herein against CFD under compartmental pH or compartmental conditions. Dis, for example, equal to or greater than 1 nM, for example, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 mM or more.

[0153] In some embodiments, the engineered antibodies described herein exhibit a pH-dependent change in affinity for a receptor, such as an Fc receptor, such as FcRn. In various embodiments, the K of the engineered antibodies described herein for FcRn at a compartment pH (e.g., a pH less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5.0 or less) or compartment conditions D is 10 -4 is 10 -5 is 10 -6 is 10 -7 is 10 -8 is 10 -9 is 10 -10 is 10 -11 is 10 -12 is 10 -13 is 10 -14 or 10 -15 M or less. In some embodiments, the K of FcRn at a serum pH (e.g., a pH greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2 or more) or serum conditions D is the K of the same antibody for FcRn at a compartment pH or compartment conditions DHigher than, for example, at least 2-fold, for example, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7000-fold, 8,000-fold, 9,000-fold, or 10,000-fold or more (and / or the affinity of the antibody for FcRn under serum pH or serum conditions can be reduced by the same proportion compared to the affinity under compartment pH or compartment conditions). In some embodiments, the K D is, for example, 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , or 10 -3 M or more. In certain cases, the K D of the engineered antibody described herein for FcRn under serum pH or serum conditions is, for example, equal to or greater than 1 nM, for example, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 mM or more.

[0154] In some embodiments, the manipulated antibodies described herein exhibit both a pH-dependent change in affinity for CFD and a pH-dependent change in affinity for Fc receptors, such as FcRn. Therefore, in some embodiments, the manipulated antibodies described herein exhibit affinity for CFD under serum conditions or at a compartmentalized pH or above, and affinity for FcRn under compartmentalized conditions or at a compartmentalized pH or above, serum conditions or above, respectively. In various embodiments, the K of the manipulated antibody against CFD under serum pH (e.g., pH greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2 or higher) or serum conditions. D is 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , or 10 -15 It may be less than M. In certain cases, the K of the manipulated antibody against CFD at serum pH. D This is between 0.001 and 1 nM, for example, 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM. In certain cases, the K of the manipulated antibody described herein against CFD at compartment pH. D K is between 0.001 and 1 nM, for example, 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM. In some embodiments, K to CFD under compartmental pH (e.g., pH less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or pH less than or equal to 5.0) or compartmental conditions. D This refers to the K of the same antibody against CFD under serum pH or serum conditions.D For example, at least twice as high, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 times higher (and / or the affinity of the antibody for CFD under compartmental pH or compartmental conditions decreases by the same proportion compared to its affinity under serum pH or serum conditions). In some embodiments, the pH of the compartment (e.g., less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or pH 5.0 or less) or K to CFD under compartment conditions D For example, 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , or 10 -3 It is greater than M. In certain cases, the K of the manipulated antibody against CFD under compartmental pH or compartmental conditions. D This is, for example, equal to or greater than 1 nM, such as 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 mM or greater. In some embodiments, the K of the manipulated antibody against FcRn under serum pH (e.g., pH greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or pH greater than 8.2) or serum conditions. DThis refers to the K of the same antibody against FcRn under compartmentalized pH or compartmentalized conditions. D For example, at least twice as high, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 times higher (and / or the affinity of the antibody for FcRn under serum pH or serum conditions decreases by the same proportion compared to its affinity under compartment pH or compartment conditions). In some embodiments, the K of the manipulated antibody against FcRn under serum pH conditions (e.g., pH greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2 or higher) or serum conditions. D For example, 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , or 10 -3 It is greater than M. In certain cases, the K of the manipulated antibody against FcRn under serum pH or serum conditions. DThis is, for example, equal to or greater than 1 nM, such as 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 mM or greater. In some embodiments, the K of the manipulated antibody against FcRn under compartmental pH (e.g., less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or pH 5.0 or less) or compartmental pH conditions. D is 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , or 10 -15 It is less than M.

[0155] In some embodiments, the engineered antibodies described herein exhibit a longer half-life than a reference antibody (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2) when administered to a subject, for example, in the subject's serum. In various cases, the serum half-life of a reference antibody (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2) may be, for example, 250 to 300 hours. In various cases, the serum half-life of the engineered antibodies described herein may be, for example, at least 250 hours, for example, at least 260, 270, 280, 290, or 300 hours. In certain embodiments, the serum half-life of the engineered antibodies described herein may be at least 300 hours, for example, at least 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 hours. In certain embodiments, the serum half-life of the manipulated antibody described herein may be at least 1,000 hours, for example, at least 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, or 15,000 hours or more. In various embodiments, the serum half-life of the manipulated antibody described herein may be at least 12 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 2 months, 3 months, 4 months, 5 months, or 6 months or more. In various cases, the serum half-life of the manipulated antibodies described herein may be increased by at least, for example, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 75x, or 100x or more compared to a reference antibody (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2).

[0156] In certain embodiments, the manipulated antibodies described herein exhibit an increased plasma half-life, an increased mean retention time in plasma, and / or an increased CFD clearance level (compared to, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2). These parameters can be determined by methods known to those skilled in the art (e.g., described in Nestorov et al., J. Clin. Pharmacol. 48:406-417 (2008), Leveque et al., Anticancer Research 25:2327-2344 (2005), and Igawa et al., PLoS One 8:e63236.doi:10.1371 / journal.pone.0063236 (2013)). For example, the engineered antibodies described herein (e.g., a single dose of such an engineered antibody) reduce the plasma level of CFD to at least 1 / 10, 1 / 50, 1 / 100, 1 / 250, 1 / 500, 1 / 750, 1 / 1000, or 1 / 1500 of a reference antibody, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0157] In some embodiments, the engineered antibodies described herein inhibit CFD cleavage (and / or serum levels of CFD). In various embodiments, the engineered antibodies disclosed herein inhibit CFD cleavage (and / or serum levels of CFD) compared to prior measurements or reference values ​​from the same patient. In certain embodiments, administration of the engineered antibodies disclosed herein reduces the level or amount of CFD cleavage (and / or the level or amount of CFDa and / or CFDb) compared to an equivalent administration of a reference antibody, e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2 (e.g., reducing the level by more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200%).

[0158] Manipulated multiple specificity molecules In the context of engineered anti-CFD antibodies, the methods and compositions described herein can be applied to further proteins to produce multispecific binding molecules. The multispecific binding molecules according to this disclosure are engineered to include one or more binding moieties that specifically bind to one or more targets of interest in a pH-dependent manner. Multispecific binding molecules include nucleic acids (e.g., RNA and DNA), proteins (e.g., antibodies), and combinations thereof. The pH-dependent binding moieties may be, or include, nucleic acids (e.g., RNA and DNA) and aptamers, polypeptides (e.g., antibodies or fragments thereof, albumin, receptors, ligands, signal peptides, avidin, and protein A), polysaccharides, biotin, hydrophobic groups, hydrophilic groups, drugs, and any organic molecules that bind to receptors.

[0159] Antibodies or fragments of antibodies as binding sites. In some embodiments, the multispecific binding molecules described herein are engineered antibodies. In some cases, one or more binding sites described herein are or include an antibody, its antigen-binding fragment, and / or its Fc region (or Fc fragment). The basic structure of an IgG antibody consists of two identical polypeptide light chains and two identical polypeptide heavy chains linked together by a disulfide bond. The first domain located at the amino terminus of each chain has a variable amino acid sequence and provides the antibody-binding specificity found in each individual antibody. These are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain have relatively invariant amino acid sequences and are known as constant heavy (CH) and constant light (CL) regions. In the case of an IgG antibody, the light chain contains one variable region (VL) and one constant region (CL). The IgG heavy chain contains a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain contains an additional constant region (CH4).

[0160] Antibodies described herein include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fv (scFv), disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies, as well as any of the antigen-binding fragments described above. Antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0161] The term "Fc fragment," as used herein, refers to one or more fragments of an Fc region that retain the Fc function and / or activity described herein, such as binding to an Fc receptor. The term "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments (Ward et al., (1989) Nature 341:544-546), and isolated complementarity-determining regions (CDRs). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same way as intact antibodies.

[0162] Antibodies or fragments can be prepared by any method for synthesizing antibodies known in the art (see, for example, Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Brinkman et al., 1995, J.Immunol.Methods 182:41-50, WO92 / 22324, WO98 / 46645). Chimeric antibodies can be prepared, for example, using the method described in Morrison, 1985, Science 229:1202, and humanized antibodies can be prepared, for example, by the method described in U.S. Patent No. 6,180,370.

[0163] Additional antibodies for the compositions and methods described herein include, for example, bispecific antibodies and polyvalent antibodies as described in Segal et al., J.Immunol.Methods 248:1-6 (2001) and Tutt et al., J.Immunol.147:60 (1991).

[0164] In some embodiments, the multispecific molecules described herein are engineered antibodies (for example, engineered to have pH-sensitive binding to an antigen and FcRn).

[0165] Manipulated antigen-binding domain of a manipulated multispecific molecule In some embodiments, the binding site is an antibody (e.g., an IgG antibody, e.g., IgG1, IgG2, or IgG3 antibody) or antigen-binding fragment that has been manipulated to bind to a target (i.e., an antigen) in a modified manner compared to a reference antibody or antigen-binding fragment (e.g., in a pH-sensitive manner, e.g., in a manner that is more or less pH-sensitive). For example, an antibody can be manipulated by modifying (e.g., by addition, deletion, or substitution) amino acids within and / or at positions involved in the antibody CDR structure of one or more antibody CDRs. Exemplary and non-limiting antibody sites that may be modified include the following (amino acid positions are indicated based on Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)):

[0166] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102

[0167] Light chains: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94.

[0168] In some embodiments, one or more of these disclosed amino acids may be substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. While we do not wish to be bound by theory, it is believed that substituting one or more amino acids at these positions with histidine may yield antibodies with pH-dependent antigen-binding properties. In some embodiments, non-histidine residues are substituted with histidine residues. In some embodiments, histidine residues are substituted with non-histidine residues. Further manipulated antigen-binding regions include, for example, those described in U.S. Publication No. 20110229489.

[0169] Manipulated constant region of manipulated multispecific molecules In some cases, the binding region is or includes an antibody constant region, Fc region, or Fc fragment that binds to one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptors). In some embodiments, the constant region, Fc region, or Fc fragment is engineered to bind to a target (e.g., an Fc receptor) in a modified manner compared to a reference constant region, Fc region, or Fc fragment (e.g., in a pH-sensitive manner, e.g., in a manner that is more or less pH-sensitive).

[0170] In some cases, the binding site may be or include a constant region, Fc region, or Fc fragment of an IgG antibody that has been manipulated to include the addition, deletion, or substitution of one or more amino acids from the amino acid residues described herein (e.g., 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH))).

[0171] Fabrication of multi-specificity binding molecules In some embodiments, the multispecific binding molecules described herein are manipulated by mutagenesis using known techniques to include one or more binding sites exhibiting pH-sensitive binding to one or more targets. For example, the sequence of a reference polypeptide (e.g., a therapeutic antibody or therapeutic fusion protein) can be obtained, and one or more amino acid residues can be added, deleted, or substituted. In some embodiments, one or more amino acid residues are substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. In some embodiments, one or more amino acids are substituted with histidine. While we do not wish to be bound by theory, it is thought that substitution of amino acid residues with histidine results in the insertion of a protonation site that can enhance the pH sensitivity of the binding site. Polypeptides can be prepared using standard methods and assayed for binding to the target of interest described herein. Further methods for increasing the pH sensitivity of the binding site are described, for example, in Sarkar et al., Nature Biotechnology 20:908-913 (2002), Murtaugh et al., Protein Science 20:1619-1631 (2011), and U.S. Publication No. 20110229489.

[0172] In some embodiments, a first target of interest is selected, and an antibody that selectively binds to the target is provided, obtained, and / or produced (e.g., using known methods described herein). One or more amino acids in the antigen-binding region and / or Fc region are substituted (e.g., with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine) to determine the pH sensitivity of binding to the target (and additionally or alternatively to FcRn). An antibody demonstrating a desired binding affinity is selected as a multispecific binding molecule.

[0173] In some embodiments, polypeptides that naturally bind to a target of interest are provided, obtained, and / or produced. The polypeptide is conjugated to an Fc region or Fc fragment described herein (e.g., binding to an FcRn with a desired binding affinity) using known methods. For example, the polypeptide and the Fc region or Fc fragment can be conjugated as a fusion protein by chemical means or by recombinant expression. Additionally or alternatively, one or more amino acids of the polypeptide may be substituted (e.g., histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine) to determine the pH sensitivity of the polypeptide-target binding.

[0174] In some embodiments, the multispecific binding molecules described herein are manipulated to include one or more binding moieties identified and / or selected by screening. For example, an antigen-binding moiety that binds to an antigen in a pH-sensitive manner can be identified using a library expressing the antigen-binding moiety, such as a phage library. Such a library can be screened for antigen-binding moieties that have a first affinity for the antigen at a first pH (e.g., pH 7.4) and a second affinity for the antigen at a second pH (e.g., pH 5.5). The multispecific binding molecules described herein can be manipulated to include such identified pH-sensitive antigen-binding moieties. Additionally and / or alternatively, an FcRn-binding moiety that binds to FcRn in a pH-sensitive manner can be identified using a library. Methods for screening recombinant antibody libraries are publicly known (see, for example, Hoogenboom, Nature Biotech. 23:1105-1116 (2005), U.S. Patent No. 5,837,500, U.S. Patent No. 5,571,698, and WO2012 / 044831).

[0175] Measurement of the interaction between the binding site and the target The binding properties of the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) to their targets (e.g., CFD and / or FcRn) can be measured by methods known in the art, such as: BIACORE analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequence analysis, and systematic mutagenesis. The binding interaction between the antibody and CFD and / or FcRn can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling any of the interacting substances. A change in mass at the binding surface of the BIA chip (indicating a binding event) results in a change in the refractive index of light near the surface. The change in refractive index generates a detectable signal that can be measured as an indicator of real-time reactions between biomolecules. Methods using SPR are described, for example, in U.S. Patent No. 5,641,640, Raether (1988) Surface Plasmons, Springer Verlag, Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345, Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705, and online resources provided by BIAcore International AB (Uppsala, Sweden). In addition, the KinExA® (bound equilibrium exclusion) assay, available from Sapidyne Instruments (Boise, Id.), can also be used.

[0176] Information from SPR provides the equilibrium dissociation constant (K) for binding the binding site to its target (e.g., to the CFD and / or FcRn of the manipulated antibody). D ) and K on and K offIt can be used to obtain accurate and quantitative measures of dynamic parameters, including K.S. Such data can be used to compare different molecules. Information from SPR can also be used to elucidate structure-activity relationships (SAR). For example, dynamic and equilibrium binding parameters of a particular binding site to a target can be evaluated at various pH levels. Specific binding parameters at specific pH levels, such as high affinity, low affinity, and slow K.S. off It is possible to identify variant amino acids at a given position that correlate with [the given expression].

[0177] Treatment method In some embodiments, the multispecific conjugating molecules described herein (e.g., the engineered antibodies described herein) are used in methods for treating one or more complement-related conditions. In some embodiments, the multispecific conjugating molecules described herein (e.g., the engineered antibodies described herein) are for pharmaceutical use. Complement-related conditions include, but are not limited to, conditions caused by symptoms that are all or partly caused by elevated or decreased complement activity, or conditions that are known to occur in conjunction with elevated or decreased complement activity. CFD is important in pathology and is particularly susceptible to CFD blockers in hematological, neurological, and renal disorders. Examples of complement-related conditions include, but are not limited to, peripheral neuropathy, cryoglobulinemia, cryoglobulinemia-related neuropathy, neurosarcoidosis, age-related macular degeneration (AMD), Alzheimer's disease, amyotrophic lateral sclerosis (ALS), antiphospholipid syndrome (or antiphospholipid antibody syndrome or Hughes' syndrome), vascular neuropathy, reflex sympathetic dystrophy, complex regional pain syndrome, chronic inflammatory demyelinating polyneuropathy, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, brain injury, C3 nephropathy, capillary leak syndrome, cardiopulmonary bypass and hemodialysis, cardiovascular disorders, fulminant antiphospholipid syndrome, cerebrovascular disorders, chronic inflammatory demyelinating neuropathy, cold agglutinin disease (CAD), and degossinus Diseases, dense deposit disease (DDD), dermatitis, inflammatory myopathy, dermatomyositis, myositis, antibody-induced myositis, diabetic vasculopathy, diabetic retinopathy, dilated cardiomyopathy, disseminated intravascular coagulation (DIC), elevated liver enzymes, epidermolysis bullosa, lupus-associated vasculitis, glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, HELLP syndrome, hemolysis, sickle cell angioplasty Anemia, Henoch-Schönlein purpura nephritis, idiopathic thrombocytopenic purpura (ITP), injury resulting from myocardial infarction, ischemia-reperfusion injury, Kawasaki disease, lupus nephritis, immune complex vasculitis, macular degeneration (e.g., age-related macular degeneration (AMD)), mesenteric / intestinal vascular disorders, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myocarditis, neonatal alloimmune thrombocytopenia (NAITP), neuromyelitis optica,Organ or tissue transplantation, paroxysmal cold hemoglobinuria, paroxysmal nocturnal hemoglobinuria (PNH), microimmune vasculitis, pemphigus, percutaneous coronary intervention, peripheral vascular disease, post-transfusion purpura, psoriasis, recurrent miscarriage, renal vascular disease, restenosis after stent placement, revascularization of grafts and / or re-grafts, rotational atherectomy, rheumatoid arthritis, psoriatic arthritis, scleroderma, sepsis, septic shock, Shiga toxin E. coli-associated hemolytic uremic syndrome (STEC-HUS), spinal cord injury Examples include injury, spontaneous abortion, systemic inflammatory response, glomerulonephritis, systemic lupus, systemic lupus erythematosus (SLE), systemic lupus erythematosus-associated vasculitis, Takayasu's arteriovenous arthritis, thoracoabdominal aortic aneurysm, thrombotic thrombocytopenic purpura (TTP), graft rejection, traumatic brain injury, type 1 diabetes mellitus, typical or infectious hemolytic uremic syndrome, vasculitis, vasculitis associated with rheumatoid arthritis, and venous gas embolism, or any complement-related inflammatory response.

[0178] Complement-related disorders include, but are not limited to, complement-related lung disorders such as asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, alpha-1 antitrypsin deficiency, emphysema, bronchiectasis, bronchiolitis obstructive, alveolitis, sarcoidosis, pulmonary fibrosis, and collagen vascular disorders.

[0179] In some embodiments, the multispecific conjugating molecules described herein (e.g., the engineered antibodies described herein) can be used to treat graft rejection / graft-versus-host disease (GVHD), reperfusion injury (e.g., after cardiopulmonary bypass or tissue transplantation), and tissue injury following other forms of traumatic injury such as burns (e.g., severe burns), blunt trauma, spinal injury, or frostbite. In some embodiments, the multispecific conjugating molecules described herein (e.g., the engineered antibodies described herein), either alone or in combination with a second anti-inflammatory agent, can be used to treat inflammatory disorders, such as, but not limited to, rheumatoid arthritis (RA) (above), inflammatory bowel disease, sepsis (above), septic shock, acute lung injury, disseminated intravascular coagulation (DIC), or Crohn's disease. In some embodiments, the second anti-inflammatory agent may be selected from the group consisting of NSAIDs, corticosteroids, anti-TNF agents such as methotrexate, hydroxychloroquine, etanercept and infliximab, B-cell depletion agents such as rituximab, interleukin-1 antagonists, or T-cell costimulators such as abatacept. In some embodiments, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) are useful for treating allergic asthma, allergic rhinitis, hyper-IgE syndrome / Job syndrome, food allergies, paroxysmal nocturnal hemoglobinuria (PNH), inflammatory bowel disease, and / or other macroorgan cytokine-mediated inflammatory conditions.

[0180] In some embodiments, the manipulated antibodies described herein are used in methods for treating PNH or aHUS. Paroxysmal nocturnal hemoglobinuria (PNH) (formerly known as Marquia-Fava-Micheli syndrome) is a rare, acquired, life-threatening blood disorder characterized by complement system-mediated destruction of red blood cells.

[0181] In patients with PNH, uncontrolled AP activation of red blood cells (RBCs) causes hemolytic anemia. Approximately 15–30% of patients with PNH have extravascular hemolysis that manifests with C5 inhibition and continue to require habitual transfusions despite therapy. Approximately 1–5% of patients do not respond to some C5 treatments due to polymorphisms in C5 and CR1. Despite vaccination, Neisseria meningitidis infection may occur in a small number of patients treated with some C5 treatments. Despite the availability of C5 inhibitor therapy, there are unmet medical needs in complement disorders (e.g., PNH and aHUS).

[0182] If left untreated, PNH erythrocytes can undergo intravascular hemolysis. In the presence of anti-C5 therapy, C3 fragment deposition can lead to breakthrough and extravascular hemolysis. C3 opsonization via RES macrophages in the liver and spleen can also occur. Treatment with factor D inhibitors can protect PNH erythrocytes from both intravascular and extravascular hemolysis.

[0183] In various cases, the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) treat, alleviate, reduce the prevalence, decrease the frequency, or reduce the level or amount of one or more symptoms or biomarkers of PNH. Symptoms and biomarkers of PNH include, but are not limited to, hemolysis, abdominal pain (e.g., severe abdominal or stomach pain), leg pain, leg swelling, headache (e.g., severe headache), back pain, weakness, fatigue (e.g., tiredness, difficulty performing daily activities, difficulty concentrating, dizziness, weakness), shortness of breath, dysphagia, jaundice of the skin, jaundice of the eyes, erectile dysfunction, anemia, pulmonary hypertension, recurrent infections, susceptibility to infection, colored urine (e.g., dark urine), Budd-Chiari syndrome, palpitations, myelodysplasia, acute leukemia, menorrhagia, confusion, and Symptoms include irritability, red blood in the urine, thrombosis (e.g., venous thrombosis, e.g., hepatic vein thrombosis or sagittal vein thrombosis), smooth muscle dystonia, abdominal contractions, esophageal spasms, chronic kidney disease, hamate hemolysis test results, sucrose hemolysis test results, binding of monoclonal antibodies to cell-binding complement regulators (CD59, CD24, CD66b, CD16, fluorescently labeled erolidine (FLAER)) to peripheral blood samples, elevated serum lactate dehydrogenase, serum creatinine levels, fibrinolysis, plasmin-mediated blood clot breakdown, D-dimer levels, and others known in the art. Symptoms may increase after infection, alcohol, exercise, or stress. Specific symptoms and their progression vary among subjects.

[0184] Therefore, in some embodiments, the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) are administered to subjects in need, such as subjects with paroxysmal nocturnal hemoglobinuria (PNH) or subjects with atypical hemolytic uremic syndrome (aHUS).

[0185] In various cases, administration of a multispecific binding molecule as described herein (e.g., an engineered antibody as described herein) results in a reduction in the prevalence, frequency, level, and / or amount of one or more symptoms or biomarkers of PNH as described herein or otherwise known in the art, e.g., a reduction of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms or biomarkers compared to prior measurements or reference values ​​in the subject.

[0186] In some embodiments, administration of the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) to subjects having PNH results in a greater reduction or improvement of one or more symptoms or biomarkers of PNH than a reference antibody under equivalent conditions, e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. Therefore, treatment of subjects having PNH may result in a reduction in the frequency or likelihood of symptom exacerbation following infection, alcohol, exercise, or stress.

[0187] Atypical hemolytic uremic syndrome (aHUS) is a disorder that primarily affects kidney function. This condition can occur at any age and can lead to the formation of abnormal blood clots (thrombi) in the small blood vessels of the kidneys. These clots can cause serious medical problems if they restrict or block blood flow. Atypical hemolytic uremic syndrome is characterized by three main features related to abnormal clotting: hemolytic anemia, thrombocytopenia, and renal failure. Gene mutations associated with atypical hemolytic uremic syndrome cause uncontrolled activation of the complement system. This overactive system attacks the cells lining the blood vessels of the kidneys, leading to inflammation and the formation of abnormal blood clots. These abnormalities lead to kidney damage, and often to renal failure and ESRD.

[0188] In various cases, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) treat, alleviate, reduce the prevalence, decrease the frequency, or reduce the level or amount of one or more symptoms or biomarkers of aHUS. Symptoms of aHUS include, but are not limited to, nausea, vomiting, confusion, shortness of breath (dyspnea), fatigue, anemia, thrombocytopenia, renal injury, renal failure, end-stage renal disease, stroke, gastrointestinal problems (e.g., severe stomach pain), colitis, vascular injury, heart attack, neurological problems (e.g., seizures), anemia, hemolysis, pallor, jaundice, edema, tachycardia, jaundice, thrombotic microangiopathy (TMA), transplant-associated thrombotic microangiopathy (TA-TMA), stroke, heart attack, malaise, microangiogenic anemia, bloody diarrhea, pulmonary complications, pancreatitis, fragmented red blood cells, encephalopathy, coma, malignant hypertension, proteinuria, thrombocytopenia, decreased hemoglobin, decreased heptaglobin, increased lactate dehydrogenase (LDH), increased creatine, and / or increased blood urea nitrogen.

[0189] In various cases, administration of a multispecific binding molecule described herein (e.g., an antibody described herein) to a subject having aHUS results in a reduction in the prevalence, frequency, level, or amount of one or more symptoms or biomarkers of aHUS described herein or otherwise known in the art, for example, a reduction of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms compared to prior measurements or reference values ​​in the subject.

[0190] In some embodiments, administration of the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) to subjects having aHUS results in a greater reduction or improvement of one or more symptoms of aHUS than a reference antibody under equivalent conditions, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0191] In some embodiments, the manipulated antibodies described herein are used in methods for treating acquired microthrombotic disorders. Acquired microthrombotic disorders, characterized by thrombocytopenia, hemolytic anemia, and renal failure, affect 4 per million people worldwide and 20% of hematopoietic stem cell recipients in the United States (8,000 per year in the US). Thrombotic microangiopathy (TMA) is a condition that results in thrombosis of capillaries and arterioles due to endothelial damage. These disorders may result from genetic complement mutations such as aHUS. They may also be secondary to certain drug treatments, stem cell transplantation, infection-related events, pregnancy, surgical procedures, malignancies, or STEC-related events. This may be confirmed in association with thrombocytopenia, anemia, purpura, and renal failure. Classical TMAs are hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Other conditions associated with TMA include atypical hemolytic uremic syndrome, disseminated intravascular coagulation, scleroderma renal crisis, malignant hypertension, antiphospholipid antibody syndrome, and drug toxicity, such as calcineurin inhibitor toxicity. TMA often results in decreased endothelial thrombotic resistance, leukocyte adhesion to damaged endothelium, complement consumption, increased vascular shear stress, and abnormal fragmentation of von Willebrand factor (vWF).

[0192] Transplant-associated thrombotic microangiopathy (TA-TMA) can present with complications of transplantation itself, including infections, graft-versus-host disease, and disseminated intravascular coagulation, as well as side effects of immunosuppressants, potentially mimicking TMA. Because the pathophysiology of TA-TMA is not fully understood, current treatment options are suboptimal, and this condition results in a very high mortality rate. In various cases, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) treat, alleviate, reduce the prevalence, decrease the frequency, or reduce the level or amount of one or more symptoms or biomarkers of TMA or TA-TMA. Symptoms of TA-TMA include, but are not limited to, fever, microangiogenic hemolytic anemia (confirmation of fragmented red blood cells in blood smears), renal failure, thrombocytopenia, and neurological findings, and may also include multi-organ failure or injury affecting the brain, kidneys, heart, liver, and other major organs.

[0193] The majority of TA-TMA patients lack suppression of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity to less than 5% to 10% of normal levels and do not have a complete response to plasmapheresis. In addition, TA-TMA expression is highly heterogeneous, ranging from asymptomatic and low levels of red blood cell fragmentation to fulminant disease. The diagnosis of TA-TMA is most reliably made by examination of peripheral hemocardium for red blood cell fragments.

[0194] In various cases, administration of a multispecific binding molecule described herein (e.g., an antibody described herein) to a subject having TA-TMA results in a reduction in the prevalence, frequency, level, or amount of one or more symptoms or biomarkers of TA-TMA described herein or otherwise known in the art, for example, a reduction of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms compared to prior measurements or reference values ​​in the subject.

[0195] In some embodiments, administration of the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) to subjects having TA-TMA results in a greater reduction or improvement of one or more symptoms of TA-TMA than a reference antibody under equivalent conditions, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0196] In some embodiments, the manipulated antibodies described herein are used in methods for treating C3 glomerulosis (C3G). C3 glomerulosis (C3G) is a rare group of kidney diseases characterized by C3 deposition without immunoglobulin deposits. These kidney diseases can be subdivided histologically into dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). C3G patients commonly have mutations in complement accessory pathway genes, the presence of C3 glomerulonephritis factors, and substantial risk of both ESRD and disease relapse after kidney transplantation. C3 convertase autoantibodies stabilize the C3 convertase complex and increase local production of accessory pathway complement. Renal failure occurs within 10 years in 50% of patients, and kidney transplantation in C3 disease is limited by a 50% relapse and graft loss rate after transplantation.

[0197] Dense deposit disease (DDD) is a very rare kidney disease characterized by abundant C3 in the glomeruli on kidney biopsy and named for the extremely high density of deposits found in the glomerular basement membrane (GBM) using electron microscopy. In both DDD and C3GN, the deposits of C3 and other proteins in the GBM impair kidney function. Progressive damage to the glomeruli occurs, eventually leading to kidney failure. When kidney failure occurs, dialysis must be initiated or a transplant must be performed. The rate of progression to end-stage renal failure and the need for dialysis are thought to be similar in both DDD and C3GN. In addition to high density deposits in the kidneys, people with DDD may develop deposits in the eyes. The signs and symptoms of DDD and C3GN are similar and include, but are not limited to, hematuria, proteinuria, leukocytes in the urine; edema, hypertension, decreased urine output; and decreased alertness.

[0198] If C3G (either DDD or C3GN) is suspected, immunofluorescence analysis should show abundant C3 in the glomerular capillaries. In addition to C3, the glomeruli contain other complement system proteins, and levels of complement proteins in the bloodstream may be reduced. Causes of complement dysregulation include gene variants and autoantibodies against convertases in complement regulatory proteins [5, 9, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29]; mutations in complement factor H (CFH), complement factor I (CFI), MCP (also known as membrane cofactor protein or CD46), complement factor B (CFB), complement factor C3, and CFHR5. Complement dysregulation can also be due to acquired factors.

[0199] In various cases, administration of a multispecific binding molecule described herein (e.g., an antibody described herein) to a subject having DDD or C3GN results in a reduction in the prevalence, frequency, level, or amount of one or more symptoms or biomarkers of DDD or C3GN described herein or otherwise known in the art, for example, a reduction of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms compared to prior measurements or reference values ​​in the subject.

[0200] In some embodiments, administration of the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) to subjects having DDD or C3GN results in a greater reduction or improvement of one or more symptoms of DDD or C3GN than a reference antibody under equivalent conditions, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0201] In some embodiments, the manipulated antibodies described herein are used in methods for treating polyneuropathy. Complement activation in peripheral neurons during injury leads to subtypes of polyneuropathy, such as hereditary / familial amyloid neuropathy; Guillain-Barré syndrome (GBS), a demyelinating neuropathy that can damage motor, sensory, and autonomic nerve fibers; and diabetic neuropathy. Peripheral neuropathy (PN) can be caused by inflammation or damage to nerves. This can result in stinging, numbness, and burning pain in any part of the body, but is commonly felt in the hands, feet, and lower legs. Some patients may experience increased sensitivity to pain, decreased sensitivity to temperature, and sensorimotor dysfunction due to nerve damage.

[0202] Systemic amyloidosis is a diverse group of disorders that can lead to multi-organ failure due to the deposition of abnormal amyloid fibrils. Hereditary amyloid peripheral neuropathy can be further classified according to the type of amyloid protein that causes the disease course. These include transthyretin, apolipoprotein A1, gelzolin, and Aβ2-microglobulin. Mutations in the TTR gene cause the most common form of hereditary amyloidosis, while amyloid light chain (AL) amyloidosis is the most common acquired form. Peripheral nervous system involvement is common and can manifest as length-dependent sensorimotor polyneuropathy, focal neuropathy, multifocal neuropathy, or autonomic neuropathy. Familial amyloid polyneuropathy (FAP) refers to a group of hereditary amyloidosis that typically have prominent clinical findings involving the peripheral sensorimotor and / or autonomic nervous system.

[0203] Guillain-Barré syndrome (GBS) is a rapidly developing muscle weakness caused by the immune system damaging the peripheral nervous system. Initial symptoms are typically sensory or painful changes, often accompanied by muscle weakness, starting in the feet and hands and spreading to the arms and upper body, with bilateral involvement. Symptoms of Guillain-Barré syndrome often begin with stabbing pain and weakness, difficulty with eye or facial movements, including speech, chewing, or swallowing, severe pain, difficulty with bladder control or bowel function, tachycardia, hypotension or hypertension, and difficulty breathing, often starting in the feet and legs and spreading to the upper body and arms. As GBS progresses, muscle weakness may develop into paralysis. Guillain-Barré syndrome is now known to occur in several forms. The main types are acute inflammatory demyelinating polyradiculoneuropathy (AIDP), Miller-Fischer syndrome (MFS), acute motor axonal neuropathy (AMAN), and acute motor sensory axonal neuropathy (AMSAN).

[0204] Diabetic neuropathy is a system of nerve damage caused by diabetes. People with diabetes may develop nerve damage throughout their body over time. Some people with nerve damage are asymptomatic. Others may experience symptoms such as pain, tingling, or numbness, i.e., loss of sensation in the hands, arms, feet, and legs. Nerve problems can occur in any organ system, including the digestive tract, heart, and reproductive organs. Symptoms of nerve damage may include numbness, tingling, or pain in the toes, feet, legs, hands, arms, and fingers; muscle wasting; indigestion; nausea or vomiting; diarrhea or constipation; dizziness or syncope due to a drop in blood pressure after standing or sitting; difficulty urinating; erectile dysfunction in men; or vaginal dryness in women.

[0205] In various cases, administration of a multispecific binding molecule described herein (e.g., an antibody described herein) to a subject having peripheral neuropathy results in a reduction in the prevalence, frequency, level, or amount of one or more symptoms or biomarkers of peripheral neuropathy described herein or otherwise known in the art, for example, a reduction of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms compared to prior measurements or reference values ​​in the subject.

[0206] In some embodiments, administration of the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) to subjects with peripheral neuropathy results in a greater reduction or improvement of one or more symptoms of peripheral neuropathy than a reference antibody under equivalent conditions, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0207] In some embodiments, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) exhibit a reduced effective dose compared to a reference protein (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2). For example, the effective dose of the engineered antibodies described herein may be, for example, less than 1,000 mg / dose, e.g., less than 900 mg / dose, 800 mg / dose, 700 mg / dose, 600 mg / dose, 500 mg / dose, 550 mg / dose, 400 mg / dose, 350 mg / dose, 300 mg / dose, 200 mg / dose, 100 mg / dose, 50 mg / dose, or 25 mg / dose or less. In certain cases, the effective dose of the engineered antibody disclosed herein may be lower than the effective, recommended, or approved dose of a reference antibody, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, where the dose of the reference antibody may be, for example, 900 mg / dose or 600 mg / dose. Alternatively, or in combination with the doses disclosed herein, the engineered antibody described herein may be effectively or usefully administered at a frequency of less than once per week, for example, less than once per week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year. In certain specific cases, the effective or useful administration frequency of the manipulated antibody disclosed herein is lower than the effective, recommended, or approved administration frequency of a reference antibody, e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, and the administration frequency of the reference antibody may be weekly (e.g., a dose of 300-600 mg depending on the subject's body weight) or every two weeks (e.g., a dose of 300-1200 mg depending on the subject's body weight).

[0208] In some embodiments, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) can be administered at reduced doses compared to a reference antibody comprising a reference protein, e.g., a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. However, if the engineered antibody is administered in the same, equivalent, or substantially equivalent formulation and / or via the same, equivalent, or substantially equivalent route of administration as the reference (e.g., the reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2), it will achieve the same, equally effective, relatively effective, or substantially effective outcomes. In some embodiments, the engineered antibodies described herein may be administered at increased intervals compared to a reference antibody (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2), but when the engineered antibodies are administered in the same, equivalent, or substantially equivalent formulation and / or via the same, equivalent, or substantially equivalent route of administration as the reference (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2), they will achieve the same, equally effective, relatively effective, or substantially effective outcomes. In some embodiments, the engineered antibodies described herein may be administered in a reduced number of unit doses and / or a shortened treatment period compared to a reference antibody (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2), but when the engineered antibodies are administered in the same, equivalent, or substantially equivalent formulation and / or via the same, equivalent, or substantially equivalent route of administration as the reference (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2), they will achieve the same, equally effective, relatively effective, or substantially effective outcomes.

[0209] In some such embodiments, the administered dose of the engineered antibody described herein is considered to have a lower likelihood of inducing an adverse response, such as an adverse immune response, when administered to a subject, than an effective dose of a reference antibody, such as a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. Therefore, in various embodiments, the engineered antibody disclosed herein is considered to have a lower likelihood of inducing an adverse reaction or side effect per administered active unit than a reference antibody, such as a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. In various embodiments, the engineered antibody disclosed herein is considered to have a lower likelihood of inducing an adverse reaction or side effect of a certain degree of severity per administered active unit than a reference antibody, such as a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. In various embodiments, the manipulated antibodies disclosed herein may induce one or more adverse reactions or side effects in patients to a smaller degree or fewer per administered active unit than a reference antibody, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. Examples of adverse reactions or side effects that may be associated with the administration of conventional antibodies, such as antibodies that can bind to CFD, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, include headache, nasopharyngitis, back pain, nausea, diarrhea, hypertension, upper respiratory tract infection, abdominal pain, vomiting, anemia, cough, peripheral edema, and / or urinary tract infection.

[0210] In some embodiments, administration of the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) reduces the serum CFD titer. The typical human plasma concentration of CFD is approximately 0.37 μM. When one or more doses of the engineered antibodies disclosed herein are administered, the plasma concentration of human CFD in the subject may be reduced compared to a previously measured concentration in the same subject or to a standard value, for example, compared to approximately 0.37 μM. In various cases, the serum concentration of CFD after administration of a manipulated antibody, including any number of doses (e.g., one dose, three doses, or several doses prescribed over a period of several months or years) administered to one or more subjects or populations of subjects over any period of time (e.g., 1 to 4 weeks, 1 to 12 months, or 1 to 3 years or more), may be equal to or less than, for example, 0.35 μM, 0.325 μM, 0.30 μM, 0.275 μM, 0.25 μM, 0.225 μM, 0.20 μM, 0.175 μM, 0.15 μM, 0.125 μM, 0.10 μM, 0.075 μM, 0.05 μM, or 0.025 μM. In some embodiments, when administered to a subject, the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) result in a greater reduction in serum CFD titers than a reference antibody under equivalent conditions, for example, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0211] In some embodiments, when administered to a subject (e.g., in a single dose), the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) are measured at elevated levels in plasma at a defined time after administration (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more) compared to the levels of a control (e.g., a reference protein, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2) at the same defined time. For example, at a defined time after a single dose, the level of the engineered antibody described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% higher than the corresponding level of the reference antibody, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0212] In some embodiments, the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) are measured at elevated levels in plasma at a defined time after administration (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more) compared to the levels of a control (e.g., a reference protein, a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2) at the same defined time. For example, at a defined time after administration, the level of the manipulated antibody described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% higher than the corresponding level of the reference antibody (e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2).

[0213] In some embodiments, the manipulated antibodies described herein have an increased half-life (compared to, for example, a control, e.g., a reference antibody, e.g., a reference antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2), and therefore the manipulated antibodies can be administered to subjects at increased dosing intervals. For example, the manipulated antibodies can be administered once every week, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, or at longer intervals.

[0214] In some embodiments, the therapeutically effective dose of the multispecific binding molecule described herein (e.g., the engineered antibody described herein) is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the effective dose of a reference therapeutic protein, e.g., a reference antibody containing a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2. In some embodiments, a single dose of the engineered antibody described herein achieves the same therapeutic effect as two or more doses of a reference antibody, e.g., a reference antibody containing a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2.

[0215] In some embodiments, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) are administered at doses of approximately 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the concentration of the target antigen (e.g., CFD) in the serum of interest.

[0216] In some embodiments, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) may be administered via routes other than intravenous administration, such as subcutaneous administration. Thus, in various embodiments, the antibodies disclosed herein can be administered subcutaneously. In some embodiments, the engineered antibodies described herein may be administered via intravenous and subcutaneous routes, for example, as components of a single-treatment strategy. Intravenous and subcutaneous administration may be simultaneous or not.

[0217] In some embodiments, the multispecific binding molecules described herein (e.g., the engineered antibodies described herein) can be used in several diagnostic and therapeutic applications. For example, the engineered antibodies described herein in a detectably labeled form can be used in assays to detect the presence or amount of CFD in a sample (e.g., a biological sample). The engineered antibodies described herein can be used in in vitro assays to study the inhibition of CFD activity and / or cleavage. In some embodiments, the engineered antibodies described herein can be used as a positive control in assays designed to identify further novel compounds that inhibit complement activity or otherwise are useful for treating complement-related disorders. For example, the engineered antibodies described herein can be used as a positive control in assays to identify further compounds (e.g., small molecules, aptamers, or antibodies) that reduce or suppress CFD production or MAC formation.

[0218] The multispecific binding molecules described herein (e.g., the engineered antibodies described herein) may be used in monitoring subjects, e.g., subjects having, suspected of having, at risk of developing, or being treated with one or more complement-related conditions. Monitoring may include determining the amount or activity of CFD in the subject, e.g., in the subject's serum. In some embodiments, the evaluation is performed at least one (1) hour after administration of the engineered antibody described herein, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1, 2, 4, 10, 13, or 20 days or more, or at least 1, 2, 4, 10, 13, or 20 weeks or more. Subjects may be evaluated during one or more of the following periods: before the start of treatment, during treatment, or after the administration of one or more elements of treatment. Evaluation may include assessing the need for further treatment, e.g., whether the dosage, frequency of administration, or duration of treatment should be changed. The evaluation may also include assessing the need to add or remove selected therapeutic modalities, for example, adding or removing any of the treatments for complement-related disorders described herein.

[0219] Formulation and administration In various embodiments, the multispecific binding molecules described herein (e.g., the manipulated antibodies described herein) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions may be useful, for example, for the prevention and / or treatment of diseases, such as PNH and / or aHUS, or other complement-related disorders. Pharmaceutical compositions can be formulated by methods known to those skilled in the art (e.g., as described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985)).

[0220] A suitable means of administration may be selected based on the age and condition of the subject. A single dose of a pharmaceutical composition containing a multispecific binding molecule as described herein (e.g., an engineered antibody as described herein) may be selected from the range of 0.001 to 1000 mg / kg body weight. On the other hand, the dose may be selected from the range of 0.001 to 100,000 mg / kg body weight, but this disclosure is not limited to such a range. The dose and method of administration will vary depending on the patient's weight, age, condition, etc., and can be suitably selected as needed by those skilled in the art.

[0221] In various cases, pharmaceutical compositions can be formulated to include pharmaceutically acceptable carriers or excipients. Examples of pharmaceutically acceptable carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic and absorption retardant agents. The compositions of the present invention may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0222] In various embodiments, compositions containing the antibodies described herein, such as sterile formulations for injection, can be formulated according to conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection in combination with optionally suitable solubilizers, such as alcohols such as ethanol, polyhydric alcohols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80™ and HCO-50.

[0223] As disclosed herein, the pharmaceutical composition may be in any form known in the art. Such forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injections and infusions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[0224] The selection or use of any particular form may, in part, depend on the intended mode of administration and therapeutic use. For example, compositions containing a composition intended for systemic or local delivery may be in the form of an injection or infusion. Thus, compositions may be formulated for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to, but is not limited to, modes of administration other than enteral and local administration, usually by injection, and includes intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions.

[0225] The route of administration may be parenteral, for example, by injection, nasal administration, pulmonary administration, or transdermal administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0226] In various embodiments, the pharmaceutical compositions of the present invention can be formulated as solutions, microemulsions, dispersants, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the compositions described herein into a suitable solvent having one or a combination of the components listed above, and subsequently sterilizing by filtration, if necessary. Generally, dispersants are prepared by incorporating the compositions described herein into a sterile vehicle containing a base dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying to obtain the powder of the compositions described herein and any additional desired components (see below) from a pre-sterilized filtered solution of those components. Adequate fluidity of the solution can be maintained, for example, by the use of a coating agent such as lecithin, maintaining the required particle size in the case of dispersants, and by the use of a surfactant. Sustained absorption of the injectable composition can be achieved by including absorption-delaying reagents, such as monostearate and gelatin, in the composition.

[0227] Pharmaceutical compositions can be administered parenterally in the form of injectable formulations, including sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, a pharmaceutical composition can be formulated by suitably combining a therapeutic molecule with a pharmaceutically acceptable vehicle or culture medium, such as sterile water and saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, and binders, and then mixing them in unit dose forms required by generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dose within a specified range is provided. Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffer or sodium acetate buffer, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injectable formulations can be packaged in suitable ampoules.

[0228] In some embodiments, the composition may be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition may be formulated for storage at 2–8°C (e.g., 4°C) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1 1 / 2 years, or 2 years). Thus, in some embodiments, the compositions described herein are stable for storage at 2–8°C (e.g., 4°C) for at least 1 year.

[0229] In certain cases, pharmaceutical compositions can be formulated as solutions. In some embodiments, compositions can be formulated, for example, as buffer solutions of a suitable concentration that are suitable for storage at 2–8°C (e.g., 4°C).

[0230] Compositions comprising one or more manipulated antibodies described herein can be formulated into immunoliposome compositions. Such formulations can be prepared by methods known in the art. Liposomes with increased circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0231] In certain embodiments, the composition may be formulated with a carrier capable of protecting the compound from rapid release, such as a release-controlled formulation comprising an embedding agent and a microencapsulation delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978), "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0232] In some embodiments, the composition can be formulated into a composition suitable for intrapulmonary administration to mammals such as humans (e.g., administration via an inhaler or nebulizer). Methods for formulating such compositions are well known in the art. Dry powder inhalation formulations and systems suitable for administering the formulations are also well known in the art. Lung administration may be oral and / or nasal. Examples of pharmaceutical devices for lung delivery include metered-dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, the compositions described herein may be administered to the target lung by a dry powder inhaler. These inhalers are spray-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well-known in the medical field, but are not limited to, TURBOHALER® (AstraZeneca; London, England), AIR® inhalers (ALKERMES®; Cambridge, Mass.), ROTAHALER® (GlaxoSmithKline; London, England), and ECLIPSE® (Sanofi-Aventis; Paris, France). See also, for example, PCT publication numbers WO04 / 026380, WO04 / 024156, and WO01 / 78693. DPI devices are used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, the compositions described herein can be administered intrapulmonaryly by metered-dose inhalers. These inhalers rely on a spray to deliver different doses of the compound to the lungs. Further devices and intrapulmonary administration methods are described, for example, in U.S. Patent Application Publications 20050271660 and 20090110679, the respective disclosures of which are incorporated herein by reference in their entirety.

[0233] In some embodiments, the compositions may be formulated for delivery to the eye, for example, in the form of a pharmaceutically acceptable solution, suspension, or ointment. Preparations for use in ocular treatment may be in the form of sterile aqueous solutions containing, for example, additional components, for example, but not limited to, preservatives, buffers, tonics, antioxidants, and stabilizers, nonionic wetting or clarifying agents, and thickeners. Preparations described herein may be administered topically to the eye of a subject requiring treatment (e.g., a subject suffering from AMD) by conventional methods, for example, in the form of eye drops, or by immersing the eye in a therapeutic solution containing one or more compositions.

[0234] In certain embodiments, various devices for introducing a drug into the vitreous cavity of the eye may be suitable for administering the compositions described herein. For example, U.S. Publication No. 2002 / 0026176 describes a drug-containing plug that can be inserted through the sclera to protrude into the vitreous cavity and deliver the drug into the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantable device for introducing a drug into the suprachoroidal cavity or an avascular region for sustained release of the drug into the eye. U.S. Patents No. 5,773,019 and No. 6,001,386 disclose implantable drug delivery devices that can be attached to the scleral surface of the eye, respectively. Further methods and devices for the delivery of therapeutic agents to the eye (e.g., transscleral patches and delivery via contact lenses) are described, for example, in Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151, Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181, Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10), Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347, Kim et al. (2007) Ophthalmic Res 39:244-254, and PCT publication number WO04 / 073551, and these disclosures are incorporated herein by reference in their entirety.

[0235] In certain embodiments, the administration of the antibodies described herein is achieved by targeting the nucleic acid encoding the antibody. The nucleic acids encoding the therapeutic antibodies described herein can be incorporated into gene constructs used as part of a gene therapy protocol for delivering nucleic acids that can be used to express and produce antibodies in cells. Such expression constructs of components can be administered in any therapeutically effective carrier, e.g., any formulation or composition that can effectively deliver the component gene to cells in vivo. Techniques include viral vectors, including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1 (HSV-1), or insertion of the target gene into a recombinant bacterial or eukaryotic plasmid. Viral vectors can be directly transfected into cells, and plasmid DNA can be delivered using, for example, cationic liposomes (lipofection) or derivatized polylysine conjugates, gramicidin S, artificial viral envelopes, or other such intracellular carriers, and direct injection or CaPO4 precipitation of the gene construct (see, e.g., WO04 / 060407).Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are known to those skilled in the art (e.g., Eglitis et al. (1985) Science 230:1395-1398, Danos and Mulligan (1988) Proc Natl Acad Sci USA 85:6460-6464, Wilson et al. (1988) Proc Natl Acad Sci USA 85:3014-3018, Armentano et al. (1990) Proc Natl Acad Sci USA 87:6141-6145, Huber et al. (1991) Proc Natl Acad Sci USA 88:8039-8043, Ferry et al. (1991) Proc Natl Acad Sci USA 88:8377-8381, Chowdhury et al. See also al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644, Kay et al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc Natl Acad Sci USA 89:10892-10895, Hwu et al. (1993) J Immunol 150:4104-4115, U.S. Patent Nos. 4,868,116 and 4,980,286, and PCT Publication Nos. WO89 / 07136, WO89 / 02468, WO89 / 05345, and WO92 / 07573). Another viral gene delivery system utilizes adenovirus-derived vectors (see, for example, Berkner et al. (1988) BioTechniques 6:616, Rosenfeld et al. (1991) Science 252:431-434, and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenovirus vectors derived from adenovirus strain Ad5 dl324 or other adenovirus strains (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art.Another viral vector system useful for delivering target genes is adeno-associated virus (AAV). See, for example, Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349-356, Samulski et al. (1989) J Virol 63:3822-3828, and McLaughlin et al. (1989) J Virol 62:1963-1973.

[0236] In various embodiments, subcutaneous administration can be performed by devices such as syringes, pre-filled syringes, autoinjectors (e.g., disposable or reusable), pen-type injectors, patch-type injectors, wearable injectors, portable syringe infusion pumps with subcutaneous infusion sets, or other devices for subcutaneous injection in combination with antibody drugs.

[0237] The injection system of this disclosure may utilize a delivery pen as described in U.S. Patent No. 5,308,341. Pen-type devices are most commonly used for the self-delivery of insulin to patients with diabetes and are well known in the art. Such a device may include at least one injection needle (e.g., a 31-gauge needle about 5–8 mm long) and is typically pre-filled with one or more therapeutic unit doses of therapeutic solution, which is useful for rapidly delivering the solution to the target with as little pain as possible. A single dosing pen comprises a vial holder in which a vial of therapeutic drug or other drug can be stored. The pen may be a fully mechanical device or may be combined with electronic circuitry to precisely set and / or display the dosage of drug to be injected to the user. See, for example, U.S. Patent No. 6,192,891. In some embodiments, the needle of the pen-type device is disposable, and the kit includes one or more disposable replacement needles. Pen-type devices suitable for delivering any one of the compositions having the features of the present invention are described, for example, in U.S. Patent Nos. 6,277,099, 6,200,296, and 6,146,361, the disclosures of which are incorporated herein by reference in their entirety. Microneedle-based pen-type devices are described, for example, in U.S. Patent No. 7,556,615, the disclosures of which are incorporated herein by reference in their entirety. See also the high-precision pen-type injector (PPI) device MOLLY® manufactured by Scandinavian Health Ltd.

[0238] In some embodiments, the compositions described herein can be therapeutically delivered to a target by topical administration. As used herein, “topical administration” or “topical delivery” may mean delivery of a composition or drug to a target tissue or site that does not rely on vascular transport. For example, a composition may be delivered by injection or implantation of the composition or drug, or by injection or implantation of a device containing the composition or drug. In certain embodiments, after topical administration near the target tissue or site, the composition or drug, or one or more of its components, may diffuse to a target tissue or site other than the administration site.

[0239] In some embodiments, the compositions described herein can be administered topically to joints (e.g., joint junctions). For example, in some embodiments where the disorder is arthritis, therapeutically appropriate compositions can be administered directly to or near the joint (e.g., the joint space). Examples of intraarticular joints to which the compositions described herein can be administered topically include, for example, the hip, knee, elbow, wrist, sternoclavicular, temporalis-mandible, carpal, tarsal, ankle, and any other joints prone to arthritis. The compositions described herein can also be administered to bursae, such as the acromion, biceps-radial, cubitoradial, deltoid, infrapatellar, ischial bursa, and any other bursae known in the medical field.

[0240] In some embodiments, the compositions provided herein are available in unit dosage forms that may be suitable for self-administration. Such unit dosage forms may be provided in containers, typically, for example, vials, cartridges, pre-filled syringes, or disposable pens. Administration devices, such as the administration device described in U.S. Patent No. 6,302,855, may also be used in conjunction with the injection systems described herein, for example.

[0241] The preferred dose of a composition described herein that can treat or prevent a disorder in a subject may depend on various factors, including, for example, the age, sex, and weight of the subject being treated, and the specific inhibitory compound used. For example, a different dose of a composition containing an antibody described herein may be required to treat a subject with RA compared to doses of different formulations of that antibody. Other factors that may influence the dose administered to a subject include, for example, the type or severity of the disorder. For example, a subject with RA may require a different dose than a subject with PNH. Other factors include, for example, other medical disorders affecting the subject at the time or in the past, the subject's overall health, the subject's genetic predisposition, diet, administration time, excretion rate, drug combinations, and any other additional therapeutic agents administered to the subject. It should also be understood that specific doses and treatment regimens for any particular subject may also be adjusted at the discretion of the treating physician.

[0242] The compositions described herein may be administered in fixed doses or in milligrams / kilogram (mg / kg) doses. In some embodiments, the dose may also be selected to reduce or avoid antibody production or other host immune responses to one or more antigen-binding molecules in the composition. Exemplary doses of antibodies, such as those in the compositions described herein, are, for example, 1-1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg. Exemplary doses of the compositions described herein, but not limited to, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg.

[0243] A pharmaceutical solution may contain a therapeutically effective amount of the composition described herein. Such an effective amount can be readily determined by those skilled in the art, in part, based on the effect of the composition administered, or, if two or more agents are used, the combined effect of the composition and one or more additional activators. The therapeutically effective amount of the composition described herein may also vary depending on the individual's medical condition, age, sex, and weight, as well as factors such as the ability of the composition (and one or more additional activators) to induce a desired response in the individual, e.g., the recovery of at least one state parameter, e.g., the recovery of at least one symptom of complement-mediated disorder. For example, the therapeutically effective amount of the composition described herein may inhibit (reduce the severity of or eliminate the occurrence of) and / or prevent a particular disorder and / or any one of the symptoms of that particular disorder that is known in the art or described herein. The therapeutically effective amount is also the amount in which the therapeutically beneficial effect outweighs any toxic or harmful effect of the composition.

[0244] A suitable human dose of any of the compositions described herein can be further evaluated, for example, in a Phase I dose-escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718, Hanouska et al. (2007) Clin Cancer Res 13(2,part 1):523-531, and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0245] The toxicity and therapeutic efficacy of the composition can be determined by known pharmaceutical procedures in cell culture or experimental animals (e.g., animal models of any of the complement-mediated disorders described herein). These procedures may include, for example, LD 50 (A lethal dose in 50% of the population) and ED 50It can be used to determine the dose that is therapeutically effective in 50% of the population. The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which is the LD (Low Life Quantitative). 50 / ED 50 It can be expressed as a ratio. Compositions described herein that exhibit a high therapeutic index are preferred. Compositions that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue and to minimize potential damage to normal cells, thereby reducing side effects.

[0246] Those skilled in the art will understand that data obtained from cell culture assays and animal studies can be used to determine the range of dosages for human use. Appropriate dosages of the compositions described herein are generally those with little to no toxicity. 50 The circulating concentration of the composition containing the substance is within this range. The dose may vary within this range depending on the dosage form used and the route of administration utilized. For the compositions described herein, the therapeutically effective dose can be initially estimated from a cell culture assay. The dose can be determined in an animal model that achieves the circulating plasma concentration range including ICFD0 (i.e., the concentration of the antibody that achieves median inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine a useful dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography. In some embodiments where local administration (e.g., to the eye or joint) is desired, cell culture or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

[0247] Combination therapy In various embodiments, the engineered antibodies described herein may be included in a course of treatment further comprising the administration of at least one additional agent to the subject. In various cases, the additional agent administered in combination with the engineered antibodies described herein may be a complement inhibitor, for example, a terminal complement inhibitor. In various cases, the additional agent administered in combination with the antibodies described herein may be an anti-inflammatory agent. In various cases, the additional agent administered in combination with the antibodies described herein may be an agent that treats the symptoms of PNH. In various cases, the additional agent administered in combination with the antibodies described herein may be an agent that treats the symptoms of aHUS.

[0248] In some embodiments, the method can be carried out in conjunction with other therapies for complement-related disorders. For example, the composition can be administered to the subject concurrently with, before, or after plasmapheresis, IVIG therapy, or plasma exchange. See, for example, Appel et al. (2005) J Am Soc Nephrol 16:1392-1404. In some embodiments, the composition can be administered to the subject concurrently with, before, or after kidney transplantation.

[0249] In various cases, additional agents administered in combination with the manipulated antibody described herein may be administered simultaneously with the manipulated antibody, on the same day as the manipulated antibody, or in the same week as the manipulated antibody. In various cases, additional agents administered in combination with the manipulated antibody described herein may be administered together with the manipulated antibody in a single formulation. In certain embodiments, additional agents may be administered at a time interval from the administration of the manipulated antibody described herein, for example, more than one hour before or after, more than one day before or after, more than one week before or after, or more than one month before or after the administration of the manipulated antibody. In various embodiments, the frequency of administration of one or more additional agents may be the same as, similar to, or different from the frequency of administration of the manipulated antibody described herein.

[0250] Treatment regimens including the administration of two distinct antibodies described herein and / or treatment regimens including the administration of antibodies described herein by multiple formulations and / or routes of administration are included in combination therapy.

[0251] In some embodiments, the composition may be formulated with one or more additional therapeutic agents, for example, additional therapies to treat or prevent complement-related disorders in the subject (e.g., AP-related disorders or CP-related disorders). Additional agents for treating complement-related disorders in the subject may vary depending on the specific disorder being treated and may include, but are not limited to, antihypertensive agents (e.g., angiotensin-converting enzyme inhibitors) [e.g., for use in the treatment of HELLP syndrome], anticoagulants, corticosteroids (e.g., prednisone), or immunosuppressants (e.g., vincristine or cyclosporine A). Examples of anticoagulants include, for example, warfarin (coumadin), aspirin, heparin, phenindione, fondaparinux, hydraparinux, and thrombin inhibitors (e.g., argatroban, repiridine, bivalirudine, or dabigatran). The compositions described herein may also be formulated with fibrinolytic agents (e.g., anclod, ε-aminocaproic acid, antiplasmin-α1, prostacyclin, and defibrotide) for the treatment of complement-related disorders. In some embodiments, the compositions may be formulated with lipid-lowering agents such as hydroxymethylglutaryl-CoA reductase inhibitors. In some embodiments, the compositions may be formulated with, or for use with, anti-CD20 agents such as rituximab (RITUXAN®; Biogen Idec, Cambridge, Mass.). In some embodiments, for example, for the treatment of rheumatoid arthritis (RA), the compositions may be formulated with one or both of infliximab (REMICADE®; Centocor, Inc.) and methotrexate (RHEUMATREX®, TREXALL®). In some embodiments, the compositions described herein may be formulated with nonsteroidal anti-inflammatory drugs (NSAIDs).Many different NSAIDs are available, some of which are over-the-counter (OTC) and include ibuprofen (ADVIL®, MOTRIN®, NUPRIN®) and naproxen (ALLEVE®), as well as meloxicam (MOBIC®), etodolac (LODINE®), nabumetone (RELAFEN®), sulindac (CLINORIL®), trementine (TOLECTIN®), and magnesium choline salicylate. Many other NSAIDs, including (TRILASATE®), diclofenac (CATAFLAM®, VOLTAREN®, ARTHROTEC®), diflunisal (DOLOBID®), indomethacin (INDOCIN®), ketoprofen (ORUDIS®, ORUVAIL®), oxaprozin (DAYPRO®), and piroxicam (FELDENE®), are available by prescription. In some embodiments, the compositions can be formulated for use with antihypertensive agents, antiseizure agents (e.g., magnesium sulfate), or antithrombotic agents. Examples of antihypertensive agents include labetalol, hydralazine, nifedipine, calcium channel antagonists, nitroglycerin, or sodium nitroprusside. (See, for example, Mihu et al. (2007) J Gastrointestin Liver Dis 16(4):419-424). Examples of antithrombotic agents include heparin, antithrombin, prostacyclin, or low-dose aspirin.

[0252] In some embodiments, compositions comprising the manipulated antibodies described herein can be formulated for administration with one or more additional therapeutic agents for use in the treatment of ocular complement-related disorders. Such additional therapeutic agents may be, for example, bevacizumab or bevacizumab Fab fragment or ranibizumab, all marketed by Roche Pharmaceuticals, Inc., and pegaptanib sodium (MUCOGEN®; Pfizer, Inc.). Such kits may also optionally include instructions for administering the compositions.

[0253] In some cases, combination therapy may involve administering one or more additional agents (e.g., anti-IgE antibodies, anti-IL-4 antibodies, anti-IL-5 antibodies, or antihistamines) that provide therapeutic benefit to subjects who have, are at risk of developing, or are suspected of having complement-related lung disorders such as COPD or asthma.

[0254] In some embodiments, a composition formulated for intrapulmonary administration may include at least one additional activator for treating lung injury. At least one activator may be, for example, an anti-IgE antibody (e.g., omalizumab), an anti-IL-4 antibody or anti-IL-5 antibody, an anti-IgE inhibitor (e.g., montelukast sodium), a sympathomimetic (e.g., albuterol), an antibiotic (e.g., tobramycin), a deoxyribonuclease (e.g., PULMOZYME®), an anticholinergic (e.g., ipratropium bromide), a corticosteroid (e.g., dexamethasone), a β-adrenergic receptor agonist, a leukotriene inhibitor (e.g., Ziloton), a 5-lipoxygenase inhibitor, a PDE inhibitor, a CD23 antagonist, an IL-13 antagonist, a cytokine release inhibitor, a histamine H1 receptor antagonist, an antihistamine, an anti-inflammatory agent (e.g., cromolyn sodium), or a histamine release inhibitor.

[0255] In some embodiments, the composition can be formulated for administration to subjects undergoing intravenous gamma globulin therapy (IVIG), plasmapheresis, plasma replacement, or plasma exchange. In some embodiments, the composition can be formulated for use before, during, or after kidney transplantation.

[0256] When the composition is used in combination with a second activator, the composition may be a combination with the second drug, or the composition may be formulated separately from the formulation of the second drug. For example, each pharmaceutical composition may be mixed, for example, immediately before administration, administered together, or administered separately, for example, at the same or different times.

[0257] The compositions described herein may replace or enhance previously or currently administered therapies. For example, when treatment is performed using the compositions described herein, the administration of one or more additional activators may be discontinued or reduced, for example, by being administered at a lower level. For example, after administration of the engineered antibody described herein, a lower level of reference antibody containing a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2 may be administered. In some embodiments, the administration of previous therapies may be maintained. In some embodiments, previous therapies may be maintained until the level of the composition reaches a level sufficient to produce a therapeutic effect. The two therapies may be administered in combination.

[0258] Recombinant gene technology According to this disclosure, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the art can be used. Such techniques are described in the literature (e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Usubel et al. (eds.), Current See Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0259] Recombinant expression of genes, such as nucleic acids encoding polypeptides, such as manipulated antibodies, as described herein, may involve the construction of an expression vector containing the nucleic acid encoding the polypeptide. Once the polynucleotide is obtained, a vector for polypeptide production can be prepared by recombinant DNA techniques using techniques known in the art. Known methods can be used to construct an expression vector containing a polypeptide coding sequence and appropriate transcriptional and translational regulatory signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0260] The expression vector can be transferred into host cells using conventional techniques, and the transfected cells can then be cultured using conventional techniques to produce polypeptides.

[0261] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In addition, materials, methods, and examples are illustrative and not intended to limit the scope. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Similar or equivalent methods and materials may be used in the practice or testing of the invention, but preferred methods and materials are described herein. [Examples]

[0262] The following examples describe several preferred embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0263] Example 1. In vitro assay for evaluating the efficacy of anti-CFD mAb inhibition. This embodiment demonstrates an in vitro assay for evaluating the efficacy of anti-CFD mAb inhibition. As shown in Figure 2A, the hemolysis level can be measured by optical density (OD) from released hemoglobin, and the MAC level can be measured using complement activation in a terminal complement complex (TCC) kit assay. Pooled human or cyno serum was combined with anti-CFD mAb1 or anti-CFD mAb2 (light chain SEQ ID NO: 1, heavy chain SEQ ID NO: 2). As shown in Figure 2B, CFD-depleted serum showed no side-pathway activity.

[0264] Figure 3 shows the maximum geometric mean percentage of C3 deposition in RBCs using a hemolysis assay format. C5-depleted serum in contact with rabbit RBCs was thought to demonstrate C3b deposition in rabbit erythrocytes visualized by FACS. Anti-CFD antibody 2 inhibits C3b deposition in rabbit RBCs, similar to the benchmark antibody (anti-CFD antibody 1).

[0265] Example 2. Acid switching and half-life extension of anti-CFD antibodies This example demonstrates that the anti-CFD antibodies described herein exhibit acid-switching properties for human and cyno CFD. As shown in Figure 4 and Table 1, anti-CFD antibody 2 demonstrates picomolar binding affinity to human CFD and cyno CFD at pH 7.4 and nM affinity at pH 5.5. Half-life experiments in knock-in human FcRn mice showed that anti-CFD antibody 2 reached a half-life of more than 45 days (Figure 5).

[0266] [Table 1]

[0267] Example 3. Pharmacokinetic and pharmacodynamic (PK / PD) evaluation of anti-CFD mAbs in non-human primates (NHPs). This example demonstrates the (PK / PD) evaluation of anti-CFD mAbs in non-human primates (NHPs). In one study, animals received either a single intravenous injection of 30 mg / kg of anti-CFD antibody 1 or a single intravenous or subcutaneous injection of 30 mg / kg of anti-CFD antibody 2. Each treatment group included n=3 animals. Antibody 2 demonstrated prolonged PK in cynomolgus monkeys compared to animals injected with antibody 1. As shown in Figure 6, a 2-log difference in total hIgG levels was observed in animals injected with anti-CFD mAb 2. The ASHE properties demonstrated by anti-CFD antibody 2 translate to a significantly improved PD duration. As shown in Figure 7, the duration of PD was approximately 10-fold with the ASHE mAb (antibody 2) compared to a control CFD antibody without ASHE properties. The duration of PD was associated with the suppression of free target CFD (Figure 8).

[0268] In the second study, animals were treated with multiple intravenous injections of anti-CFD antibody 2 at doses of 50 mg / kg, 5 mg / kg, 1 mg / kg, or subcutaneously at 25 mg / kg. Injections were administered on day 1 (hour 0), day 22 (528 hours), day 29 (696 hours), and day 36 (864 hours). Two animals were included in each dose group. As shown in Figure 9, the cumulative effect of multiple injections on PK in total hIgG was observed after IV and SC injections. Improved PD duration was also observed in animals treated with multiple injections. Four subcutaneous injections of antibody 2 administered at 0 hours, 528 hours (day 22), 696 hours (day 29), and 864 hours (day 36) maintained complete inhibition beyond 504 hours (day 21) (Figure 11). Subcutaneously administered anti-CFD ASHE mAb showed a longer PD duration of more than two weeks. An intravenously administered anti-CFD antibody (Antibody 1) that did not exhibit ASHE characteristics had a 2-day disease progression (PD) period.

[0269] Other Embodiments While several embodiments of the present invention are described herein, this disclosure and examples may be modified to provide other methods and compositions of the present invention. It will be understood that the scope of the present invention should be defined by the appended claims, in addition to the specific embodiments shown as examples. All references cited herein are incorporated herein by reference.

Claims

1. An isolated antibody that specifically binds to complement factor D (CFD), (a) Heavy chain CDR1 containing amino acid sequence YYAWS (SEQ ID NO: 12); Heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15); Heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYYDY (SEQ ID NO: 14); Light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16); Light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17); and, Light chain CDR3 containing the amino acid sequence QSASSNDDAV (SEQ ID NO: 18), or (b) Heavy chain CDR1 containing amino acid sequence YYAWS (SEQ ID NO: 12); Heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLKS (SEQ ID NO: 15); Heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYYDY (SEQ ID NO: 14); Light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16); Light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17); and Light chain CDR3 containing the amino acid sequence QSADLNDDAV (SEQ ID NO: 19), or (c) Heavy chain CDR1 containing amino acid sequence YYAWS (SEQ ID NO: 12); Heavy chain CDR2 containing the amino acid sequence DIANEGSTYYSPSLES (SEQ ID NO: 20); Heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYYDY (SEQ ID NO: 14); Light chain CDR1 containing the amino acid sequence QGDLPRHYAH (SEQ ID NO: 9); Light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10); and, Light chain CDR3 containing amino acid sequence QSADSNDDAV (SEQ ID NO: 11), (d) Heavy chain CDR1 containing amino acid sequence YYAWS (SEQ ID NO: 12); Heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13); Heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYYDY (SEQ ID NO: 14); Light chain CDR1 containing the amino acid sequence QGDLPRHYAH (SEQ ID NO: 9); Light chain CDR2 containing the amino acid sequence DDDIRPS (SEQ ID NO: 10); and, Light chain CDR3 containing the amino acid sequence QSADSNDAV (SEQ ID NO: 11), or (e) Heavy chain CDR1 containing amino acid sequence YYAWS (SEQ ID NO: 12); Heavy chain CDR2 containing the amino acid sequence DIANDGSTYYSPSLES (SEQ ID NO: 13); Heavy chain CDR3 containing the amino acid sequence LRSLYTDYDPHYYYDY (SEQ ID NO: 14); Light chain CDR1 containing the amino acid sequence QGNLLPRHYAH (SEQ ID NO: 16); Light chain CDR2 containing the amino acid sequence DDNIRPS (SEQ ID NO: 17); and, Light chain CDR3 containing amino acid sequence QSADSNDDAV (SEQ ID NO: 11), Isolated antibodies.

2. (a) A heavy chain variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and The light chain variable region amino acid sequence is at least 90% identical to that of SEQ ID NO: 6, or (b) A heavy chain variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and The light chain variable region amino acid sequence is at least 90% identical to that of Sequence ID No. 8, or (c) A heavy chain variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 29, and The light chain variable region amino acid sequence is at least 90% identical to sequence number 28, or (d) A heavy chain variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 27, and The light chain variable region amino acid sequence is at least 90% identical to sequence number 26, or (e) A heavy chain variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 34, and The light chain variable region amino acid sequence is at least 90% identical to that of Sequence ID No. 35, or (f) A heavy chain variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 36, and The light chain variable region amino acid sequence is at least 90% identical to that of Sequence ID No.

37. The isolated antibody according to claim 1, comprising:

3. An isolated antibody according to claim 1, comprising a light chain amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 1, 3, 30, or 32.

4. An isolated antibody according to claim 1, comprising a heavy chain amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 2, 4, 31, or 33.

5. An isolated antibody according to claim 1, which inhibits the complement accessory pathway.

6. An isolated antibody according to claim 1, which inhibits the cleavage of complement factor B.

7. At pH 7.4, the affinity dissociation constant (K) between 1 pM and 50 pM is Ｄ The isolated antibody according to claim 1, which binds to CFD.

8. At pH 5.5, the affinity dissociation constant (K) is between 15 nM and 150 nM. Ｄ The isolated antibody according to claim 1, which binds to CFD.

9. At pH 5.5, affinity dissociation constant (K) greater than 15 nM Ｄ The isolated antibody according to claim 1, which binds to CFD.

10. The off-rate of CFD from the antibody at pH 5.5 is 0.010 s. －１ from 0.04s －１ The isolated antibody according to claim 1.

11. The isolated antibody according to claim 1, wherein the isolated antibody has a serum half-life of 5 to 125 days.

12. The isolated antibody according to claim 1, having a serum half-life of more than 45 days.

13. A composition for use as a pharmaceutical, comprising the antibody or antigen-binding fragment described in claim 1.

14. A composition for treating complement-mediated diseases or disorders, comprising the antibody or antigen-binding fragment described in claim 1.

15. The composition according to claim 14, wherein the complement-mediated disorder or condition is atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH).

16. The composition according to claim 15, wherein administration of the composition inhibits intravascular hemolysis and extravascular hemolysis.

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