WO2019006153A1 - Proteolytic cascade enhancement for the treatment of disease - Google Patents

Abstract

In some embodiments, the present invention provides methods and compositions including the use of complement-stabilizing reagents to treat disease. In some embodiments, the stabilization reagent of the present invention is useful in a method of enhancing complement activity, e.g., a method including a step of administering at least one complement-stabilizing reagent to subject in need thereof.

Description

PROTEOLYTIC CASCADE ENHANCEMENT FOR THE TREATMENT OF DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No.

62/527,073, filed on June 30, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND

[2] Proteolytic cascades involve proteases acting in a highly orchestrated sequence in living organisms. Proteases and proteolytic cascades are involved in the regulation of a wide variety of essential physiological and pathophysiologic processes and, therefore, are targets for therapeutic intervention. Examples of such proteolytic cascades include caspase-mediated apoptosis, blood coagulation, the matrix metalloproteinase cascade and the complement cascade.

[3] Teleologically, proteolytic cascades enable organisms to respond to injury, stress, infection and other stimuli, to amplify the response, and to regulate the response to avoid an excessive or harmful response. The initiation of the responses to these various stimuli involves activation of proteases and initiation of a series of proteolytic events (i.e. the proteolytic cascade) that produces effector molecules with biologic activity such as promotion of clotting, inflammation, etc. The regulation of the proteolytic cascade occurs by controlling the activity of individual proteases in the cascade. This regulation is achieved by several mechanisms including inherent instability of protein complexes, other proteases that degrade the protease, and regulatory proteins that bind to and inactivate the protease, etc. Several mechanisms can in turn interfere with these regulatory mechanisms. For example, an agent that can bind to and stabilize a protease can upregulate or enhance the proteolytic cascade. Any agent that can bind to and inactivate a regulatory protein can upregulate or enhance the cascade. Upregulation or enhancement of a proteolytic cascade can be used to promote a therapeutic effect such as promotion of clotting in the setting of bleeding or promotion of complement-mediated inflammation to kill tumor cells, etc.

[4] The complement system is an ancient branch of the innate immune system. It serves as a first line of defense against invading pathogens, particularly bacteria accessing the intravascular space. In addition to its key roles in clearance of foreign invaders and cell debris, the complement system also serves as a bridge to the adaptive immune system. The interplay between the innate and adaptive branches of the immune system contributes to the specificity of the response as well as modulation of the strength and duration of the immune response. The complement system was first recognized for its ability to "complement" the antibody response. Since its initial description however, other activities have been elucidated. These include opsonization of a target that can result in phagocytosis, complement dependent cellular cytotoxicity, B cell stimulation and antibody response, among others (Barrington et al. 2001, Immunological Reviews, 180:5-15).

[5] Three recognition pathways have been described through which complement activation can be triggered: classical pathway (CP), lectin pathway (LP) and alternative pathway (AP). While each is initiated through a distinct mechanism, common to all three pathways is a series of proteolytic activation and amplification steps that converge to cleave Complement protein 3 (C3). C3 is the central component of the complement system and its most abundant protein. It is a 190 kDa protein, synthesized predominantly in the liver that is encoded by 41 exons located on chromosome 19. It is translated as a single polypeptide chain, which is cleaved post-translationally to yield an alpha and a beta chain held together by disulfide bonds.

[6] C3 is activated via C3 convertases, enzyme complexes that proteolytically cleave

C3 to C3a and C3b. There are two C3 convertases: one is shared by the classical and lectin pathways and a second is used in the alternative pathway. The classical/lectin pathway C3 convertase is composed of two subunits: C4b, which covalently binds the enzyme to a target, and C2a, which is the catalytic subunit. The alternative pathway C3 convertase is composed of C3b and Bb, the larger subunit of the proenzyme Factor B. The subunits of both convertases are non- covalently linked to one another. These enzyme complexes are normally short-lived (a few minutes) due to spontaneous decay as well as to accelerated disassociation induced by complement regulatory proteins.

[7] Regardless of the cause, total deficiency of C3 predisposes to recurrent pyogenic infections. Point mutations or deletions in the C3 gene account for most cases of primary C3 deficiency. In secondary C3 deficiency, C3 is synthesized normally but excessive consumption leads to a marked reduction or complete absence of serum C3 protein. For example, a total deficiency of the regulatory protein factor H (FH) or factor I (FI) produces C3 depletion because of inadequate inhibition of the AP C3 convertase. Another cause of secondary C3 deficiency is C3 nephritic factor (C3-Nef), an autoantibody that stabilizes the AP C3 convertase leading to excessive C3 turnover. Subjects with secondary C3 deficiency may present with pyogenic infections as in primary deficiency but, in addition, also commonly develop glomerular disease. A few individuals with a C4 nephritic factor (C4-Nef) have also been described in the setting of lupus or glomerulonephritis. C4-Nef stabilizes the classical pathway C3 convertase (C4b2a) and leads to C3 consumption in a fashion analogous to C3-Nef.

SUMMARY OF THE INVENTION

[8] The present disclosure encompasses the surprising recognition that enhancing a proteolytic cascade such as complement may be used to treat certain diseases such as infections, cancer or any disease for which the killing and/or removal of unwanted cells provides a therapeutic benefit. In fact, the idea of enhancing complement as a therapeutic strategy is counterintuitive. Excessive or unregulated complement activation is almost uniformly associated with pathology, and it is directly involved in the pathophysiology of certain autoimmune inflammatory diseases. In some diseases, complement plays a key role in their pathogenesis. For that reason, the idea of complement depletion has evolved as a therapeutic concept for treatment of these conditions (Vogel, 2014, Molecular Immunology, 61(2): 191-203). Most therapeutic strategies that target complement proteins involve a mechanism of action that inhibits critical complement pathways or up-regulates complement regulatory proteins with the result that complement activation, or the production of complement effector proteins is reduced.

Therapeutic strategies often involve inhibitors of proteases and down-regulation of proteolytic cascades. Examples include anticoagulants that inhibit proteases such as factor Xa or thrombin in the blood clotting cascade, or anti-inflammatory compounds that inhibit complement proteins in the complement cascade. In particular, depletion has been proposed to be useful for ischemia reperfusion injuries and autoimmune diseases such as arthritis and myasthenia gravis. The idea that enhancing complement activation or stabilizing complement proteins could be used to treat disease is contrary to common understanding.

[9] Cobra venom factor, (CVF) is a well-studied complement-depleting agent but is not a suitable drug candidate at least in part because it is highly immunogenic. Production of humanized cobra venom factor (hCVF) has failed to circumvent the immunogenicity issue. Another concern with hCVF is that the natural convertases (C3bBb and C4b2a) act largely on target surfaces by covalently attaching to the surface via a thioester bond. In contrast, CVF does not deposit on surfaces, but rather acts in the fluid phase. Fluid phase activation of complement leads to generation of higher amounts of activated C3 fragments in circulation which will tend to locate in the kidney and thereby increase the risk of kidney disease. In contrast to agents like hCVF, according to various embodiments, provided compositions and methods comprising complement enhancing agents may be able to enhance complement activation on the cell surface of a specifically targeted cell or microbe.

[10] A further concern with hCVF is that immunogenicity has not been fully evaluated, and, as such, risk of an adverse immune response persists. Additionally, hCVF has only been evaluated for up to one month in animal models, so the long-term effects are not known. In contrast, at least because there are many humanized antibody drugs on the market, effects of humanized antibodies on the immune system and characteristics are better understood. Moreover, comprising complement-enhancing agents that are derived from human sources, including certain compositions disclosed herein, may be suited for therapeutic use without further humanization. Thus, in some embodiments, at least certain concerns associated with xeno-immune response may be alleviated by use of particular compositions disclosed herein.

[11] An additional concern with hCVF is that complement depletion could lead to adverse side effects such as severe, recurrent infections, e.g., by gram-positive bacteria. In contrast, in accordance with several embodiments, provided methods and compositions comprising a complement enhancing agents avoid (e.g., a CSR) this issue.

[12] The present invention is based, in part, on the surprising discovery that enhancing or stabilizing complement proteins may be used therapeutically to treat various diseases. In some embodiments, the present invention provides methods of generating agents that substantially stabilize or activate one or more protein complexes, enzyme, or set of enzymes (e.g., a complement or complement-associated complex, enzyme, and/or set of enzymes). In some embodiments, such agents as disclosed herein have the same or substantially similar binding and/or functional specificity to a naturally occurring compound found in a donor. In some embodiments, such agents (e.g., complement enhancing agents) then be used to modulate (e.g., down-regulate or up-regulate) the activity of one or more biochemical pathways, e.g., of the complement system. In accordance with various embodiments, an effect of this modulation may lead to, e.g., complement stabilization, for example, stabilization of the activity of one or more complement proteins.

[13] In some embodiments, methods provided herein allow for a new paradigm in the treatment of many diseases including but not limited to cancer and infection. As such, provided methods and compositions may increase the quality of life for sufferers of these indications.

[14] At least one aspect of the present disclosure includes a method of complement stabilization that includes administering to a subject in need thereof at least one complement- stabilizing reagent, such that the complement-stabilizing reagent increases the rate or duration of the activity of at least one protein or protein complex in a complement cascade. In certain embodiments, the complement-stabilizing reagent stabilizes a classical pathway C3 convertase. In certain embodiments, the complement-stabilizing reagent stabilizes an alternative pathway C3 convertase. In certain embodiments, the complement-stabilizing reagent stabilizes a classical pathway C5 convertase. In certain embodiments, the complement-stabilizing reagent stabilizes an alternative pathway C5 convertase. In certain embodiments, the complement-stabilizing reagent stabilizes a terminal pathway membrane attack complex (MAC). In certain

embodiments, the complement-stabilizing reagent stabilizes a classical pathway CI complex. In certain embodiments, the complement-stabilizing reagent stabilizes a lectin pathway mannose binding lectin complex.

[15] In certain embodiments, a complement-stabilizing reagent is or includes an aptamer, a peptide, a small molecule, a protein, an antibody, an antibody fragment, or a combination thereof.

[16] In certain embodiments, a complement-stabilizing reagent exhibits at least one of the following activities: promoting or increasing complement-dependent cytotoxicity (CDC) activation, activating complement-dependent cellular cytotoxicity (CDCC), enhancing antibody- dependent cellular cytotoxicity (ADCC), enhancing antibody-mediated phagocytosis, enhancing chemoattractant activity, upregulating activating Fc gamma receptor expression, enhancing opsonization by phagocytes, increasing production of complement adjuvant, stabilization of classical pathway C3 convertase, stabilization of alternative pathway C3 convertase, stabilization of classical pathway C5 convertase, stabilization of alternative pathway C5 convertase, stabilization of terminal pathway membrane attack complex, stabilization of lectin pathway mannose binding lectin complex, stabilization of classical pathway CI complex, enhanced generation of anaphylatoxins, enhanced immune cell activation, or any combination thereof.

[17] At least one aspect of the present disclosure includes a method of enhancing an immune system response including administering to a subject in need thereof a therapeutically effective amount of: (a) an antibody agent including a Fab portion; and (b) a complement- stabilizing reagent with at least one of the following activities: stabilization of classical pathway C3 convertase, stabilization of alternative pathway C3 convertase, stabilization of classical pathway C5 convertase, stabilization of alternative pathway C5 convertase, stabilization of terminal pathway membrane attack complex, stabilization of classical pathway CI complex, stabilization of lectin pathway mannose binding lectin complex, and combinations thereof. In certain embodiments, the antibody agent is an antibody that binds a tumor antigen. In certain embodiments, an antibody is or includes: an anti-CD-20 antibody, an anti-CD22 antibody, an anti-CD32b antibody, an anti-CD-33 antibody, an anti-CD40 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-VEGF antibody, an anti-HER2 receptor antibody, an anti- 17- IA antibody, an anti-CCR4 antibody, an anti-IGF-IR antibody, an anti-CTLA-4 antibody, or a combination thereof. In certain embodiments, an antibody is an antibody that binds a pathogen antigen. In certain embodiments, the antibody is or includes a monoclonal antibody. In certain embodiments, a complement-stabilizing reagent leads to complement-dependent cytotoxicity (CDC) activation, complement-dependent cellular cytotoxicity (CDCC) activation, antibody- dependent cellular cytotoxicity (ADCC) enhancement, enhanced antibody-mediated

phagocytosis, enhanced chemoattractant activity, upregulation of activating Fc gamma receptor expression, enhanced opsonization, increased production of complement adjuvant, stabilization of the classical pathway C3 convertase, stabilization of the alternative pathway C3 convertase, stabilization of the classical pathway C5 convertase, stabilization of the alternative pathway C5 convertase, stabilization of the terminal pathway membrane attack complex, stabilization of the lectin pathway mannose binding lectin complex, stabilization of the classical pathway CI complex, stabilization of complement protein complexes, enhanced generation of

anaphylatoxins, enhanced immune cell activation, or any combination thereof.

[18] In certain embodiments, an antibody is a bispecific antibody. In certain embodiments, a bispecific antibody binds: (a) a tumor antigen; and (b) a complement pathway component; such that binding of the bispecific antibody to the complement pathway component is complement-stabilizing. In certain embodiments, a bispecific antibody binds: (a) a pathogen antigen; and (b) a complement pathway component; such that binding of the bispecific antibody to the complement pathway component is complement-stabilizing.

[19] In certain embodiments, an anti-infective antibody includes a therapeutic antibody. In certain embodiments, a complement-stabilizing reagent is or includes: an aptamer, a peptide, a small molecule, a protein, an antibody, an antibody fragment, or any combination thereof.

[20] In certain embodiments, a subject is suffering from a disease, disorder, or condition that is selected from the group consisting of lymphoma, a leukemia, an X- linked lymphoproliferative disorder, an Epstein Barr Virus (EBV)-associated lymphoproliferative disorder, a breast cancer, a colorectal cancer, a lung cancer, a head and neck cancer, a prostate cancer, ovarian cancer, multiple myeloma, a melanoma, a bacterial infection, viral infection, fungal infection, parasitic infection, and any combination thereof.

[21] At least one aspect of the present invention includes a method of increasing the rate or duration of complement activity in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of a complement-stabilizing reagent to the subject in need thereof. In certain embodiments, the complement stabilizing reagent stabilizes a C3 convertase complex, optionally wherethe C3 convertase complex is a classical pathway C3 convertase complex. In certain embodiments, complement-stabilizing reagent is a classical pathway complement-stabilizing reagent. In certain embodiments, the classical pathway complement-stabilizing reagent is a classical pathway complement-stabilizing antibody.

[22] In certain embodiments, the complement-stabilizing antibody is an antibody that binds a C3 convertase, optionally where the C3 convertase complex is a classical pathway C3 convertase complex. In certain embodiments, the complement-stabilizing reagent is a donor autoantibody. In certain embodiments, the subject in need thereof is a subject at risk of or diagnosed as having cancer or an infectious disease.

[23] At least one aspect of the present invention includes a pharmaceutical

composition including a complement-stabilizing reagent and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient. In certain embodiments, the complement stabilizing reagent stabilizes a C3 convertase complex, optionally where the C3 convertase complex is a classical pathway C3 convertase complex. In certain embodiments, complement- stabilizing reagent is a classical pathway complement-stabilizing reagent. In certain

embodiments, the classical pathway complement-stabilizing reagent is a classical pathway complement-stabilizing antibody. In certain embodiments, the complement-stabilizing antibody is an antibody that binds a C3 convertase, optionally where the C3 convertase complex is a classical pathway C3 convertase complex. In certain embodiments, the complement-stabilizing reagent is a donor autoantibody.

[24] At least one aspect of the present invention includes a method of treating cancer or infectious disease, the method including administering to a subject in need thereof a pharmaceutical composition disclosed herein.

BRIEF DESCRIPTION OF THE DRAWING

[25] FIG. 1 is a schematic overview of the complement system. There are at least three known pathways for complement activation: Classical, Alternative and Lectin pathways. All three complement activation pathways involve the formation of short-lived enzyme complexes (convertases), which convertases contribute to the activation and amplification of complement. The three complement activation pathways converge on activation of intact C3. Proteolytic activation of C3 produces the C3 split products C3a and C3b. C3b is further proteolytically modified to form iC3b, a biomarker for C3 activation. Intact C3 and iC3b are circled in the schematic.

[26] FIG. 2 is a pair of schematics showing aspects of the complement system. Panel

A shows classical pathway C3 convertase (C4b2a), a key serine protease of the complement classical pathway (CP). C3 convertase (C4b2a) activates C3 to C3b. Typically, regulatory proteins, such as CR1, interact with the convertase and displace C2a, causing decay of the C3 convertase (C4b2a) complex, thus limiting C3 convertase (C4b2a) activity and, consequently, the amount of C3 activation that occurs. In a subject with an autoantibody to C3 convertase

(C4b2a), IgG autoantibody stabilizes C4b2a complex leading to increased C3 convertase (C4b2a) activity and, consequently, increased C3b generation, e.g., as compared to a subject without autoantibody to C3 convertase (C4b2a). Binding of antibody to C3 convertase (C4b2a) renders C3 convertase (C4b2a) complex refractory to CR1 decay activity, and for at least this reason stabilizes C3 convertase (C4b2a) complex and prolongs the active life of stabilized C3 convertase (C4b2a) complex.

[27] FIG. 3 is a graph showing decay of C3 convertase (C4b2a) complexes over time, as measured by a hemolysis assay. In the shown classical pathway hemolysis assay, purified IgG from a subject with a Classical Pathway Complement Stabilizing Antibody (CPCSA), here a C3 convertase (C4b2a) autoantibody, demonstrates stabilization of C3 convertase (C4b2a) as compared to natural decay. As shown, control IgG purified from a normal donor without a CPCSA had no effect on decay of convertase as compared to natural decay.

[28] FIG. 4 is a graph showing decay of C3 convertase (C4b2a) complexes over time, as measured by a hemolysis assay. In the shown classical pathway hemolysis assay, purified IgG from a patent with a Classical Pathway Complement Stabilizing Antibody (CPCSA), here a C3 convertase (C4b2a) autoantibody, demonstrates stabilization of C3 convertase (C4b2a) as compared to natural decay. In this figure, complement regulatory protein CR1 was added to both control and CPCSA samples. The graph demonstrates that convertase was still stabilized by subject CPCSA IgG in the presence of CR1. In contrast, CR1 accelerated decay of control not treated with CPCSA.

[29] FIG. 5 is a pair of plots showing C3 deposition on Raji cells. Raji cells were first treated with an anti-CD20 antibody, which was followed by build-up of the classical pathway C3 convertase (C4b2a) on the cell surface. CPCSA or a negative control was added to the Raji cell samples. C6-depleted human serum was subsequently added to the Raji cell samples either immediately after addition of CPCSA or 40 minutes of incubation after addition of CPCSA. The C6-depleted human serum provides a source of C3. Following incubation with serum, the cells were stained with an anti-C3 FITC antibody and the level of C3 deposited on the cell surface was assessed by flow cytometry.

[30] FIG. 6 is a bar graph showing Raji cell cytotoxicity. Raji cells were treated with anti-CD20 antibody, followed by addition of normal human serum, with or without CPCSA. Following an hour incubation, supernatants were collected and levels of cytotoxicity were measured using an LDH cytotoxicity kit. Enhanced cytotoxicity was observed in the presence of the CPCSA.

[31] FIG. 7 is a series of four schematics (panels A, B, C, and D) relating to the isolation of human monoclonal antibodies (MAbs) with C3 convertase (C4b2a)-stabilizing activity. Panel A shows production of human hybridomas from a donor with C3 convertase (C4b2a) Complement Stabilizing Antibody (C3CSA) in donor's serum. Human hybridomas can be used for producing human MAbs. Panel B shows use of screening of hybrid hybridomas to identify hybrid hybridoma encoding a C3CSA and subsequently isolate C3CSA monoclonal antibody (step B6a) and/or to identify the antibody sequence, sequence the antibody sequence, and clone the antibody sequence into an antibody (Ab) expression vector (step B6b). Panel C shows another means of identifying, sequencing, and cloning a C3CSA sequence into an antibody expression vector. In particular, the means shown in Panel C includes isolating white blood cells of a donor with C3CSA, isolating B cells or plasma cells by FACS, activating complement to identify cells with C3CSA, isolating cells with C3b by FACS, and then sequencing the antibody gene before cloning into an antibody expression vector. Panel D shows steps for molecular cloning and expression of MAbs with C3CSA from sequence data obtained from step B6b or from step C6. Panel D includes use of the antibody expression vector to transfect CHO cells, isolate transfectants, and purify monoclonal C3CSA antibody therefrom. A number of different antibody expression vectors can be used to confer on the C3CSA antibody the desired pharmacologic characteristics.

DEFINITIONS

[32] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject-matter belongs.

[33] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject-matter.

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

[35] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system, for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition.

[36] Agent or Reagent: In general, the interchangeable terms "agent" and "reagent," as used herein, refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system, or

phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). As used herein, the term "agent" or "reagent" encompasses a molecule including one or more structures functionally coupled to an antibody, or domain thereof, to create a therapeutic, and variations thereof.

[37] Aptamer: As used herein, the term "aptamer" encompasses a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and thus can also be referred to as "chemical antibodies." In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of agents for certain novel clinical applications.

[38] Alternative Pathway: "Alternative pathway" encompasses one of three arms of the complement system. The alternative pathway (AP) is one of the mechanisms by which C3 can become activated. The alternative pathway is typically activated by targets such as microbial surfaces and various complex polysaccharides and other materials. This alternative pathway can also be initiated spontaneously by the cleavage of the thioester bond in C3 by a water molecule to form C3(H20). C3(H20) binds factor B, which allows factor D to cleave factor B to Ba and Bb. Bb remains associated with C3(H20) to form C3(H20)Bb complex, which acts as a C3 convertase and cleaves C3, resulting in C3a and C3b.

[39] Amelioration: as used herein, refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes but does not require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury). [40] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kDa tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kDa each) and two identical light chain polypeptides (about 25 kDa each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino- terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy -terminal CH3 (located at the base of the Y's stem). A short region, known as the "switch", connects the heavy chain variable and constant regions. The "hinge" connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy -terminal constant (CL) domain, separated from one another by another "switch". Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., 3-, 4-, or 5- stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complement determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. In certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences, e.g., as found in natural antibodies, can be referred to and/or used as an "antibody", whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term "antibody" as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in certain embodiments, an antibody utilized in accordance with the present invention is an intact and/or full-length immunoglobulin of type IgA, IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgE, IgD, IgM, or IgY. In certain embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE, IgD, IgM, or IgY antibodies; antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; antigen-binding fragments or single chains of complete immunoglobulins (including, e.g., without limitation, single chain antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, scFv (single-chain variable), and dAb fragments); and other proteins that include at least one antigen-binding immunoglobulin variable region, e.g., a protein that comprises an immunoglobulin variable region, e.g., a heavy (H) chain variable region (VH) and a light (L) chain variable region (VL), bi- or multi- specific antibodies (e.g., Zybodies®, etc); single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); Small Modular

ImmunoPharmaceuticals ("SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;

Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody can lack a covalent modification (e.g., association with a glycan) otherwise characteristic of antibodies produced naturally. In some embodiments, an antibody can contain a covalent modification.

[41] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent can include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent can include one or more sequence elements are humanized, primatized, chimeric, etc, as is known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and

KALBITOR®s. In some embodiments, an antibody can lack a covalent modification (e.g., association with a glycan) that it would have if produced naturally. In some embodiments, an antibody can contain a covalent modification (e.g., association with a glycan, or other pendant group (e.g., poly-ethylene glycol, etc.)). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or 100%) sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%), or 100%) sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[42] Light chains of an antibody may be of type kappa or lambda. An antibody may be polyclonal or monoclonal. Polyclonal antibodies contain immunoglobulin molecules that differ in sequence of their complementarity determining regions (CDRs) and, therefore, typically recognize different epitopes of an antigen. Often a polyclonal antibody is derived from multiple different B cell lines each producing an antibody with a different specificity. A polyclonal antibody may be composed largely of several subpopulations of antibodies, each of which is derived from an individual B cell line. A monoclonal antibody is composed of individual immunoglobulin molecules that comprise CDRs with the same sequence, and, therefore, recognize the same epitope (i.e., the antibody is monospecific). Often a monoclonal antibody is derived from a single B cell line or hybridoma. An antibody may be a "humanized" antibody in which for example, a variable domain of rodent origin is fused to a constant domain of human origin or in which some or all of the complementarity-determining region amino acids often along with one or more framework amino acids are "grafted" from a rodent, e.g., murine, antibody to a human antibody, thus retaining the specificity of the rodent antibody.

[43] Antibody fragment: As used herein, an "antibody fragment" refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment

(particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids. "Antibody fragment" encompasses the Fc fragment and the antigen-binding region. The Fc fragment is the non-antigen binding part of an antibody molecule, the constant domain Fc mediates several immunological functions, such as binding to receptors on target cells and complement fixation (triggering effector functions that eliminate the antigen). The Fc domain is not essential for most biotechnical applications relying on antigen binding. The Fc fragment, which is glycosylated, can have different effector functions in the different classes of immunoglobulins. The second part of the antibody includes the antigen-binding region. The unique antigen-binding site of an antibody consists of the heavy and light chain variable domains (V_H and V_L). Each domain contains four conserved framework regions (FR) and three regions called CDRs (complementarity determining regions) or hypervariable regions. The CDRs strongly vary in sequence and determine the specificity of the antibody V_L and V_H domains together form a binding site, which binds a specific antigen. [44] Antigen: As used herein, the term "Antigen" refers to a molecule which is recognized by the adaptive immune system and can stimulate the generation of antibodies.

[45] Antigen-binding moiety: As used herein, the term, "Antigen-binding moiety," refers to the region of a complement enhancing agent or autoantibody capable of binding to antigen.

[46] Associated with: Two events or entities are "associated" with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[47] Autoantibody: "Autoantibody" encompasses an antibody produced by an organism, such as a human donor, which antibody recognizes an antigen of that organism's own tissue. Several mechanisms, without limitation, may trigger the production of autoantibodies: to provide just a few examples from human donors, an antigen, formed during fetal development and then sequestered, may be released as a result of infection, chemical exposure, or trauma, as occurs in autoimmune thyroiditis, sympathetic uveitis, and aspermia; there may be disorders of immune regulatory or surveillance function; antibodies produced against certain streptococcal antigens during infection may cross-react with myocardial tissue (Gulizia et al. 1991, The American Journal of Pathology, 138(2):285-301), causing rheumatic heart disease, or with glomerular basement membrane, causing glomerulonephritis; and normal body proteins may be converted to autoantigens by chemicals, infectious organisms, or therapeutic drugs. Some examples of autoantibodies are those found against gastric parietal cells in pernicious anemia (De Aizpurua et al. 1983, New England Journal of Medicine, 309(11):625-629), against platelets in autoimmune thrombocytopenia , and against antigens on the surface of erythrocytes in autoimmune hemolytic anemia (Fagiolo 1976, Acta Haematologica, 56(2):97-106). There is growing evidence that genetic factors increase the incidence and severity of autoimmune diseases.

[48] B cell: "B cell" encompasses a type of white blood cell and, specifically, a type of lymphocyte. Many B cells mature into what are called plasma cells that produce antibodies (proteins) necessary to fight off infections while other B cells mature into memory B cells. All of the plasma cells descended from a single B cell typically produce the same antibody, which antibody is typically directed against at least the antigen that stimulated the single B cell to mature. The same principle holds with memory B cells. Thus, all of the plasma cells and memory cells "remember" the stimulus that led to their formation.

[49] Binding: It will be understood that the term "binding", as used herein, typically refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

[50] Biological Sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample can be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell- containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces; other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term "sample" refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semipermeable membrane. Such a "processed sample" can comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

[51] Bispecific antibody. "Bispecific antibody" encompasses an engineered protein composed of fragments of two distinct antibodies and thus binds to two separate antigens.

[52] Cancer: As used herein, the term "cancer" refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they display an abnormally elevated proliferation rate and/or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer can be characterized by one or more tumors. In some embodiments, a cancer can be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. In some embodiments, a relevant cancer can be characterized by a solid tumor. In some embodiments, a relevant cancer can be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, Breast cancer (HER2/Neu positive), Colorectal cancer, non-squamous non-small cell lung cancer, glioblastoma, renal cell carcinoma, Squamous cell carcinoma of the head and neck, Metastatic colorectal carcinoma, Metastatic melanoma, Non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Chronic lymphocytic leukemia, Acute myeloid leukemia, Hodgkin's lymphoma, and systemic anaplastic lymphoma.

[53] Chemotherapeutic. The term "chemotherapeutic", has used herein has its art- understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic moieties are useful in the treatment of cancer. In some embodiments, a chemotherapeutic moiety can be or include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g. microtubule targeting moieties such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhihitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum- based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic moiety can be or include one or more of

Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan,

Maytansine and/or analogs thereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic moiety can be utilized in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic moiety is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLLl- Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG- 7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, FMGN-853, FMGN-529,

vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, a

chemotherapeutic moiety can be or comprise one or more of farnesyl-thiosalicylic acid (FTS), 4- (4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2),

tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin, or curcumin. [54] Classical pathway. "Classical pathway" is one of three arms of the complement system and is primarily activated by immune complexes, specifically IgG/IgM antibodies bound to antigen. Other activators include lipopolysaccharide, myelin, polyanionic compounds, C reactive protein (CRP), and microbial DNA and RNA.

[55] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic moieties). In some embodiments, the two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy can involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, can be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[56] Comparable. As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions can reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied. [57] Composition: Those skilled in the art will appreciate that the term

"composition", as used herein, can be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition can be of any form - e.g., gas, gel, liquid, solid, etc.

[58] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential to a particular aspect or embodiment, but other elements or steps can be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method

"consisting essentially of (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and can also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of (or "consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step can be substituted for that element or step.

[59] Complement activation: "Complement activation" used herein, refers to any process that involves initiation, propagation, and prolongation of the complement cascade.

[60] Complement activity: As used herein, the term "complement activity" can refer to any of one or more of (a) cleavage of C3 by C3 convertase; (b) deposition of C3 on complement-targeted cells; (c) lysis of complement-targeted cells; (d) complement-dependent cytotoxicity (CDC), complement-dependent cellular cytotoxicity (CDCC); (e) antibody- dependent cellular cytotoxicity (ADCC); (f) antibody-mediated phagocytosis; (g)

chemoattractant activity; (h) upregulating Fc gamma receptor expression; (i) opsonization by phagocytes; (j) production of complement adjuvant; (k) generation of anaphylatoxins; (1) enhanced immune cell activation; (m) any other measure of complement activity known in the art; or (n) any combination thereof. Complement activity may be said to increase or decrease if there is a change in the rate or duration of any complement activity disclosed herein or otherwise known in the art. In the clinic, levels of complement activity are routinely determined by assays including, without limitation, CH50 (complete complement titer), APH50 (alternative pathway complement titer), C3 and C4 nepholometry (to determine C3 and C4 concentrations). For more in depth assessment of the complement system, assays to determine the concentration and/or function of one or more of CI, C2, C3, C4, C5, C6, C7, C8 and C9, among other things, are known in the art(as described in Kirschfink and Mollnes, Clinical and Diagnostic Laboratory Immunology, 2003, 10(6): 982-989). Methods of assessment of complement activity according to one or more of the measures disclosed herein are known in the art and disclosed herein.

[61] Complement-enhancing or complement-stabilizing. The terms "complement- enhancing" and "complement-stabilizing," used interchangeably herein, refer to reagents and/or processes that increase the rate, duration, and/or propagation of complement activity, or the activity of a complement pathway component, as compared to a reference.

[62] Complement protein complex. "Complement protein complex" as used herein, refers to any group of two or more complement proteins that transiently bind with one another as part of the complement cascade.

[63] Epitope. "Epitope" refers to the minimum portion of a molecule that is recognized by an antibody, which antibody may be, e.g., a particular antibody, a particular group of antibodies, or a hypothetical, desired, or conceptualized antibody or group of antibodies. The term "epitope" is also used herein to refer to the minimum portion of a molecule that is recognized by a non-antibody specific binding agent. Unless otherwise indicated, it is assumed herein that a specific binding agent that binds to a complement protein binds to an epitope present and accessible for binding under one or more conditions.

[64] Excipient: "Excipient" encompasses any inert substance used to bulk up or dilute a drug, or as a vehicle for a drug.

[65] Fc region: "Fc region" encompasses a non-antigen binding part of an antibody molecule. Constant domain Fc mediates several immunological functions, such as binding to receptors on target cells, antibody-dependent cell-mediated cytotoxicity (ADCC) and

complement fixation (triggering effector functions that eliminate the antigen).

[66] Immune cell subset: "Immune cell subset" refers to a group of immune cells that express the same surface markers. [67] "Improved " "increased" or "reduced": As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a baseline value or reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be "improved" relative to that obtained or expected in the absence of treatment or with a comparable reference agent or control. Alternatively, or additionally, in some embodiments, an assessed value achieved with an agent of interest may be "improved" relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.

[68] Infection or Infectious disease. The terms "infection" and "infectious disease," as used interchangeably herein, refers to conditions caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi; the diseases can be spread, directly or indirectly, from one person to another. Zoonotic diseases are infectious diseases of animals that can cause disease when transmitted to humans. Examples of pathogenic microorganisms include but are not limited to bacteria such as Neisserial meningococcus, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, viruses such as influenza virus, hepatitis C virus, protozoa such as Pneumocystis, helminths such as strongyloides, ectoparasites, and fungi such as Candida, Histoplasma, and Cryptococcus.

[69] Neoepitope. "Neoepitope" refers to an epitope that is generated or becomes detectable as a result of proteolytic cleavage of a complement component, other self-protein or cleavage product.

[70] Nephritic factor. "Nephritic factor" encompasses an autoantibody that stabilizes one of the C3 or C5 convertases of the complement system.

[71] Non-antigen-binding moiety, as used herein, "non-antigen-binding moiety," can refer to any moiety of an antibody, such as an autoantibody, that is not a variable domain.

[72] Peptide. "Peptide" encompasses a chain of amino acid monomers linked by peptide (amide) bonds. The covalent chemical bonds are formed when the carboxyl group of one amino acid reacts with the amine group of another. A peptide is typically a short chain of amino acid monomers.

[73] Pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving predetermined therapeutic effects when administered to a relevant population. In some embodiments, a pharmaceutical composition can be specially formulated for administration in a particular form (e.g., in a solid form or a liquid form), and/or can be specifically adapted for, for example: oral administration (for example, as a drenche (aqueous or non-aqueous solutions or suspensions), tablet, capsule, bolus, powder, granule, paste, etc, which can be formulated specifically for example for buccal, sublingual, or systemic absorption); parenteral administration (for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained- release formulation, etc); topical application (for example, as a cream, ointment, patch or spray applied for example to skin, lungs, or oral cavity); intravaginal or intrarectal administration (for example, as a pessary, suppository, cream, or foam); ocular administration; nasal or pulmonary administration, etc.

[74] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[75] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. In certain instances, "pharmaceutically acceptable carrier" refers to substances that improve the effectiveness and/or delivery of a therapeutic agent, and can include, e.g., sustained release systems, agents that reduce toxicity and/or alter drug metabolism. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose;

starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt;

gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[76] Pharmaceutically acceptable excipient. As used herein, "pharmaceutically acceptable excipient" refers to substances formulated with the active ingredient of the medicine. They are added to stabilize, bulk up or otherwise enhance the effect of the active ingredient.

[77] Prevent or prevention , as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention can be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[78] Protein: As used herein, the term "protein" refers to a polypeptide {i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins can include moieties other than amino acids {e.g., can be glycoproteins, proteoglycans, etc.) and/or can be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a

"protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D- amino acids, or both and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[79] Stabilization of a protein or complex. As used herein, "stabilization" of a protein or protein complex refers to any process which prolongs the life and/or activity of one or more proteins or protein complexes of the complement cascade.

[80] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[81] Small molecule. "Small molecules" encompass a low molecular weight (<900

Daltons) organic compound that may help regulate a biological process, with a size on the order of 10^"9 m.

[82] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.

[83] Donor: A donor is a subject from which a material, agent, or sequence (including without limitation any portion of serum, an antibody, or a protein or nucleic acid sequence of an antibody) is derived [84] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount that produces the desired effect for which it is

administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount can be that amount that provides a particular desired pharmacological response in a significant number of subjects when

administered to subjects in need of such treatment. In some embodiments, reference to a therapeutically effective amount can be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy can be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent can be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

[85] Treatment. As used herein, the term "treatment" (also "treat" or "treating") refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively, or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. In various examples, treatment is of a cancer. As used herein, the term "treatment" also encompasses any diagnostic testing. In certain embodiments, a therapeutic agent is selected from the group consisting of antibiotics, anti-inflammatory agents, and inhibitors of complement. In other embodiments, treatment encompasses modifying a treatment the individual has already received or is receiving. For example, in one embodiment treating an individual on a ventilator encompasses optimizing the ventilator.

[86] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while exemplifying certain embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

DETAILED DESCRIPTION

Overview of the Complement System

[87] The complement system comprises more than 30 serum and cellular proteins and plays important roles in innate and adaptive immunity (Trouw and Daha 2011). There are three major pathways of complement activation. The classical pathway is primarily activated by immune complexes, specifically IgG/IgM antibodies bound to antigen. Other activators include lipopolysaccharide, myelin, polyanionic compounds, C-reactive protein (CRP), and microbial DNA and RNA. The lectin pathway is activated by polysaccharides with free-mannose groups and other sugars common to fungi and bacteria. The alternative pathway is mediated by direct C3 activation by "foreign" substances that often include microbial cell wall components. All three major pathways of complement activation converge on the central protein complement component 3 (C3). C3 is a central mediator of inflammation. See FIG. 1 for a schematic overview of the complement system.

[88] The classical pathway is typically triggered by immune complexes, which are complexes of antigen bound with antibodies, generally belonging to the IgM or IgG isotypes. Immune complexes in turn bind to complement component CI, which is comprised of Clq, Clr, and Cls. The binding of Clq to an antibody-antigen complex triggers activation of Clr and Cls. Activated Cls then cleaves component C4 to produce C4a and C4b. C4b is capable of covalent attachment to cell surfaces, although only about five percent does so. The remaining 95 percent reacts with water to form a soluble, activated C4b. Component 2 can then associate with C4b, which after which it is activated by Cls to C2a and C2b. C4b and C2a combine to form

C4bC2a, the classical pathway (CP) C3 convertase.

[89] The CP convertase cleaves C3 to form C3a and C3b. Like activated C4b, C3b can covalently bind to cell surfaces or react with H20 and stay in solution. Activated C3b has multiple roles. By itself, it can serve as an opsonin to make the decorated cell or particle more easily ingested by phagocytes. In addition, C3b can associate with C4bC2a (the CP C3 convertase) to form a C5 convertase. The complex, termed C4bC2aC3b is termed the CP C5 convertase. Alternatively, C3b can form the core of another C3 convertase called the alternative pathway (AP) C3 convertase.

[90] The alternative pathway (AP) is another mechanism by which C3 can become activated. It is typically activated by targets such as microbial surfaces and various complex polysaccharides and other materials. This alternative pathway can also be initiated

spontaneously by the cleavage of the thioester bond in C3 by a water molecule to form C3(H20). C3(H20) binds factor B, which allows factor D to cleave factor B to Ba and Bb. Bb remains associated with C3(H20) to form C3(H20)Bb complex, which acts as a C3 convertase and cleaves C3, resulting in C3a and C3b.

[91] C3b formed either via this process or via the classical or lectin pathways binds to targets (e.g., on cell surfaces) and forms a complex with factor B, which is subsequently cleaved by factor D and form Bb, resulting in C3bBb, which is termed the alternative pathway (AP) C3 convertase. Binding of another molecule of C3b to the AP C3 convertase produces C3bBbC3b, which is the AP C5 convertase.

[92] The lectin complement pathway is initiated by binding of mannose-binding lectin

(MBL) and MBL-associated serine protease (MASP) to carbohydrates. The MB11 gene (known as LMANl in humans) encodes a type 1 integral membrane protein localized in the intermediate region between the endoplasmic reticulum and the Golgi. The MBL2 gene encodes the soluble mannose-binding protein found in serum. In the human lectin pathway, MASP1 and MASP2 are involved in proteolysis of C4 and C2, leading to C3 convertase, which lead to production of a C5 convertase as described above for the CP.

[93] C5 convertase generated via any of the three pathways cleave C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8, which catalyzes polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The assembling MAC inserts itself into the target cell membrane, forming a pore delineated by a ring of C9 molecules. MAC formation causes cell lysis of invading microbes, MAC formation on host cells can also cause lysis, but not necessarily. Sublytic amounts of MAC on the membrane of cells may affect cell function in a variety of ways. The small cleavage products C3a, C4a, and C5a are anaphylatoxins and mediate multiple reactions in the acute inflammatory response. C3a and C5a are also potent chemotactic factors that attract immune system cells such as neutrophils and macrophages into the area of crisis.

Complement-Stabilizing Reagent (CSR)

[94] The present disclosure provides, among other things, complement-stabilizing reagents (CSRs). As disclosed herein, a CSR is a reagent that increases the rate and/or duration of complement activity when contacted with one or more complement system components as compared to the rate and/or duration of complement activity of a reference control. In various embodiments, a CSR is said to "stabilize" complement when application of the CSR to a complement system increases the rate and/or duration of complement activity as compared to a reference control. The complement activity may be activity of any one or more of the Classical, Alternative and Lectin pathways.

Classical Pathway Complement-Stabilizing Reagent (CPCSR)

[95] The present specification discloses, among other things, the identification and use of reagents that enhance classical complement pathway activity. A Classical Pathway

Complement-Stabilizing Reagent (CPCSR) can be, e.g., an antibody. In various instances, a CPCSR physically interacts with a classical complement pathway component in a manner that directly or indirectly increases the rate and/or duration of the activity of the classical complement pathway component, and/or directly or indirectly increases the rate and/or duration of classical complement pathway activity.

[96] Certain exemplary CPCSRs stabilize a complement pathway component that is or includes a complex that determines or contributes to the rate and/or duration of classical complement pathway activity, such as the C3 convertase (C4b2a) complex shown in FIG. 2. [97] In various embodiments, a CPCSR comprises, or is derived at least in part from, a

"C4 nephritic factor" autoantibody of a producing donor.

[98] In some embodiments, a complement-enhancing agent derived from a C4 nephritic factor stabilizes a classical pathway C3 convertase and/or increase complement activation.

[99] In some embodiments, a CPCSR may inhibit complement regulatory proteins

(CRP). Many complement regulatory proteins such as the C4b-binding protein, complement receptor type 1, membrane cofactor protein, decay accelerating factor (DAF) act to down regulate complement activity by either preventing association of complement proteins, cleaving complement proteins, or causing the dissociation of complement protein complexes. In some embodiments, antibodies that bind to CRPs and inhibit their activity can enhance complement activation and complement-mediated events.

[100] In some embodiments, a CSR will stabilize the C5b-9 membrane attack complex

(MAC). The MAC is generated by terminal pathway activation which first activates C5 to C5b and then sequentially recruits C6, C7, C8 and C9 which forms a pore in membrane of the target cell. In some embodiments, antibodies and other molecules that bind to epitopes of the MAC complex of C5b, C6, C7, C8, and C9 could stabilize the complex and increase the lysis of cells. Those of skill in the art will appreciate that stabiliziation of the C5b-9 membrane attack complex (MAC), e.g., by a molecule that binds an eptiope of the MAC complex of C5b, C6, C7, C8, and C9, will not necessarily be specific to either the classical or the alternative pathway.

Accordingly, such embodiments could be utilized in various instances, including without limitation as a CPCSR or APCSR.

[101] In some embodiments, a CPCSR described herein can demonstrate, in a same or comparable assay, activity equal to, comparable to, or greater than a CPCSR found in or derived from a subject who presented as C3 deficient. For example, in Miller et al. (2012 Clin. Immunol. 145(3): 241-250), which is herein incorporated by reference in its entirety, a reported subject produced a Classical Pathway Complement Stabilizing Antibody capable of stabilizing the classical pathway (CP) C3 convertase (C4b2a). The stabilized C3 convertase (C4b2a) depleted the subject's C3 reserves through consumption.

[102] In some embodiments, a CPCSR may lead to complement dependent cytotoxicity

(CDC) initiated by Clq recognition of IgG or IgM on a target. In some embodiments, C3b is then generated and deposited on the target, initiating the terminal pathway and membrane attack complex formation. In some embodiments, a CPCSR will stabilize one or more complement protein complexes generated in the CDC pathway.

[103] In some embodiments, a CPCSR will lead to antibody dependent cellular cytotoxicity (ADCC), for example, as initiated by IgG through the Fc receptor on the following effectors: macrophages, monocytes, polymorphonuclear cells and natural killer cells.

[104] In some embodiments, a CPCSR will lead to complement dependent cellular cytotoxicity (CDCC), for example, as initiated by Clq, C3b, C4b or iC3b binding to one or more of the following receptors: C lqR, CR2, CR3 and CR4 which are present effector cells such as macrophages, monocytes, polymorphonuclear cells and natural killer cells.

[105] In some embodiments, a CPCSR stimulates phagocytosis by CR3-iC3b engagement.

[106] In some embodiments, a CPCSR will enhance opsonization by either C3b or C4b.

In some embodiments, downstream proteolytic events will lead to production of C3d and C4d which can further engage other effector cells.

[107] In some embodiments, a CPCSR will promote anaphylatoxin activity by generating chemoattractants, which recruit inflammatory cells and modulate antigen presenting cell responses to toll-like receptor and T cells.

[108] In some embodiments, a CPCSR will stabilize one or more proteins in the classical pathway (CP) C3 convertase. Complement activation (CA) via antibody-antigen complexes is referred to as the classical pathway of complement activation. The CI complex (Clq, r, s) binds to the Fc portion of antibody bound to antigen and cleave C4 and C2 into C4b and C2a, which combine to form the CP C3 convertase (C4b2a). The C4b2a complex is a serine protease which can cleave C3 into C3a and C3b (i.e. C3 convertase activity) (see FIG. 2). The C4b2a complex has a very short half-life to avoid excessive complement activation. In addition, complement regulatory proteins can promote C4b2a inactivation to down-regulate CA. Certain antibodies to the C4b2a convertase can stabilize the enzyme and prolong its half-life, thus enhancing CA even in the presence of regulatory proteins. In some embodiments, such an antibody, or analogous C4b2a-binding molecules may be used to promote and enhance CA on the surface of a tumor cell and increase the tumor killing of anti-CD monoclonal antibodies such as anti-CD20 mAbs (e.g. rituximab). [109] In some embodiments, a CPCSR will stabilize the C5 convertase of the classical pathway. When complement activation (CA) is initiated via the classical pathway the complement cascade continues when the C4b2a complex binds to a C3b molecule to form the C4b2a3b complex which is a serine protease that can cleave C5 into C5a and C5b (i.e. C5 convertase activity). The C4b2a3b complex has a very short half-life to avoid excessive complement activity. In addition, complement regulatory proteins can promote C4b2a3b inactivation to down regulate the activity of the terminal complement pathway. Certain antibodies to the C5 convertase can stabilize the enzyme and prolong its half-life and thus enhance complement activity even in the presence of regulatory proteins. In some embodiments, such an antibody, or analogous C4b2a3b-binding molecule, could be used to promote and enhance CA on the surface of a tumor cell and increase the tumor killing of anti-CD monoclonal antibodies such as anti-CD20 mAbs (e.g. rituximab).

[110] In some embodiments, a CPCSR may be in the form of bispecific antibodies and localize the convertase stabilization to the tumor cell membrane by binding to a tumor cell surface marker and to the convertase.

[Ill] In some embodiments, CPCSR molecules may act in the fluid phase or on cell membranes. In accordance with various embodiments, by directing the convertase stabilizing activity to the tumor cell membrane the off-target effects of the convertase-stabilizing activity will be reduced.

[112] In some embodiments, a CPCSR will activate complement-dependent cytotoxicity thus enhancing target cell killing.

[113] In some embodiments, a CPCSR will activate complement-dependent cellular cytotoxicity thus enhancing target cell killing.

[114] In some embodiments, a CPCSR will amplify the antibody-dependent cellular cytotoxicity thus enhancing target cell killing.

[115] In some embodiments, a CPCSR will magnify antibody-mediated phagocytosis.

[116] In some embodiments, a CPCSR will promote chemoattractant activity.

[117] In some embodiments, a CPCSR can lead to upregulating expression of the activating Fc gamma receptor.

[118] The present invention includes a nucleic acid encoding CPCSR as disclosed herein. Nucleic acid sequences encoding a CPCSR are disclosed herein, any of which may be present or included in a composition of the present invention. A nucleic acid molecule of the present invention can be a nucleic acid molecule encoding, alone or among other encoded elements, a CPCSR. A nucleic acid molecule of the present invention can be a linear nucleic acid, plasmid, or vector. In certain instances, a CPCSR composition is a nucleic acid in solution, such as a linear nucleic acid, plasmid, or vector in solution. Solutions for storage of nucleic acid reagents are known in the art. Numerous plasmids and vectors are known in the art. For example, a vector including a nucleic acid sequence encoding a CPCSR can be an expression vector, e.g., a vector for expression of an antibody or antigen-binding polypeptide.

Classical Pathway Complement Stabilizing Antibody (CPCSA)

[119] In some embodiments, a CPCSR is or comprises an antibody that stabilizes a classical complement pathway component in a manner that directly or indirectly increases the rate and/or duration of the activity of the classical complement pathway component, and/or directly or indirectly increases the rate and/or duration of classical complement pathway activity.

[120] Certain exemplary CPCSAs stabilize a complement pathway component that is or includes a complex that determines or contributes to the rate and/or duration of classical complement pathway activity, such as C3 convertase (C4b2a) complex.

[121] A CPCSA epitope can be any epitope associated with a classical complement pathway component. A CPCSA epitope can include an active site of a classical complement pathway component. In certain instances, binding of a CPCSA to its epitope can occlude an active site of a classical complement pathway component. A CPCSA epitope can include a site of a classical complement pathway component that is a site involved in interaction of the classical complement pathway component with another molecule, such as another classical complement pathway component. In certain instances, binding of a CPCSA to its epitope can occlude a site of a classical complement pathway component that is a site involved in interaction of the classical complement pathway component with another molecule, such as another classical complement pathway component. In some embodiments, a CPCSA epitope can be or comprise an epitope present when a classical complement pathway component is in a first state of that molecule but which is not present when that component is in a second state. For example, in some instances, a CPCSA epitope can be or comprise an epitope that is presented when a classical complement pathway component is present in a complex, but which is not presented when the classical complement pathway component is not in complex.

[122] CPCSAs can be derived from various sources. In certain instances, a CPCSA is designed and/or synthesized in vitro. In certain instances, a CPCSA is derived from an organism (e.g., a naturally occurring organism). In certain instances, the organism is an organism immunized with one or more classical complement pathway component molecules, or complexes thereof, or with one or more epitopes present in such molecules and/or complexes. In certain instances, a CPCSA is or comprises an antibody produced by a human subject, or an antibody including a sequence (e.g., at least one CDR or variable region) thereof.

[123] In some embodiments, a CPCSA can be found and/or derived from serum of a mammalian (e.g., a human) subject having a condition characterized by classical complement dysfunction. Human conditions characterized by classical complement dysfunction are known in the art (see, e.g., Miller et al. 2012 Clin. Immunol. 145(3): 241-250, which is herein incorporated by reference in its entirety).

[124] In certain some embodiments, a CPCSA is or comprises an antibody that binds to and stabilizes C3 convertase complex (C4b2a). Such a CPCSA can be referred to as a C3 convertase Complement Stabilizing Antibody (C3CSA, more specifically a CP-C3CSA). To provide just one example, an IgG autoantibody that stabilized the classical pathway C3 and C5 convertases, preventing decay of convertase complexes was identified from subject serum in Miller et al. (2012 Clin. Immunol. 145(3): 241-250, which is herein incorporated by reference in its entirety).

[125] In some embodiments, a CPCSA can be an antibody found in or derived from a donor, or produced by manipulation of such an antibody, or produced by manipulation of a fragment thereof (see, e.g., FIG. 7).

[126] In some instances, an exemplary CPCSA is or comprises a C4 nephritic factor, e.g., a C4 nephritic factor found in or derived from a donor.

[127] In some embodiments, a CPCSA can be found and/or derived from serum of a mammalian (e.g., a human) subject having a condition characterized by classical complement dysfunction and/or producing C4 nephritic factor.

[128] In some embodiments, a CPCSA described herein can demonstrate, in a same or comparable assay, activity equal to, comparable to, or greater than a reference CPCSA found in or derived from a donor, such as a C4 nephritic factor. In certain embodiments, a CPCSA can have properties that differ from those of a CPCSA found in or derived from a donor.

[129] In some embodiments, a CPCSA described herein can demonstrate, in a same or comparable assay, activity equal to, comparable to, or greater than a reference CPCSA found in or derived from a subject who presented as C3 deficient. In certain embodiments, a CPCSA can have properties that differ from those of a CPCSA found in or derived from a donor. For example, in Miller et al. (2012 Clin. Immunol. 145(3): 241-250), which is herein incorporated by reference in its entirety, a reported subject produced a CPCSA capable of stabilizing the classical pathway (CP) C3 convertase (C4b2a), and a CPCSA of the present invention can modulate the rate or duration of complement activity, e.g., in a given assay, in a manner equal to, comparable to, greater than, or with otherwise different properties than does a donor-derived CPCSA such as that described in Miller et al. (2012 Clin. Immunol. 145(3): 241-250), which is herein incorporated by reference in its entirety. In some embodiments, a portion (e.g., an antigen- binding fragment or portion thereof) of a CPCSA, e.g., a CPCSA found in or derived from a donor, can be manipulated to generate a CPCSA that differs from an initial CPCSA, e.g., a CPCSA as found in or derived from the donor. Such a product can, in some instances, be used as a therapeutic, e.g., in the subject the original CPCSA was found in or derived from, or in another subject.

[130] As will be appreciated by those of skill in the art, an antibody (e.g., a CPCSA, e.g., a CPCSA found in or derived from a donor or produced by manipulation of such an antibody or produced by manipulation of a fragment thereof) as described herein can be or comprise an immunoglobulin molecule that recognizes and specifically binds to a target through at least one antigen recognition site within a variable, optimized, or selected region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (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 the like, or any combination thereof), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified

immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as glucans, toxins, radioisotopes, and the like. As used herein, an antibody can be, e.g., an "intact antibody" or an "antibody fragment." As used herein, "antibody" additionally encompasses various alternative formats as may be known in the art, e.g., camelid antibodies. As used herein, an antibody or intact antibody can be an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable (V_H) region and a heavy chain constant region (C_H). The heavy chain constant region comprises three domains, C_HI , C_H2, and C_H3. Each light chain comprises a light chain variable (V_L) region and a light chain constant region (C_L). The V_H and V_L regions can be further subdivided into regions of hypervariability, termed

complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Other intact antibodies, e.g., intact camelid antibodies, are known in the art.

[131] In some embodiments, changes in the non-antigen binding domain may cause or support a therapeutic response in the individual receiving treatment with the CPCSR.

[132] In some embodiments, provided compositions may include one or more changes in the effector functions of a CPCSA as compared to a form in which the CPCSA was initially synthesized, or as compared to a form in which the CPCSA was initially found in or derived from a donor.

[133] Many antibodies and other moieties contribute to disease pathology. Often, it is not just the antigen specificity of the antibody that determines pathology; the Fc region can contribute to it as well. The Fc region contributes to many of the physiologic properties of the antibody. In some embodiments, the provided complement enhancing agents may comprise one or more such property, including, but not limited to: extended circulatory half-life, tissue distribution (homing), the ability to enhance ADCC, the ability to activate CDC and the ability to enhance antibody mediated phagocytosis. [134] It is recognized that the Fc region may contribute to one or more other properties as well. In some embodiments, one could tailor the physiological properties of an agent's specificity. In some embodiments, this may comprise changing the native Fc region for other Fc regions from other Ab isotypes. It should also be noted that some modifications of the CPCSA will not be simple swaps of the Fc region. In some embodiments, modifications can be made at the N-terminus of the CPCSA. In some embodiments, adding an IgM Fc region may make a monoclonal multivalent CPCSA with strong complement activation/stabilization potential. In some embodiments, the half-life of a CPCSA may be between 1-90 days. Other embodiments may include using an IgA Fc to create a CPCSA capable of being transported across mucosal surfaces as well as conferring complement activation via the alternative pathway (e.g., IgAl). In still other embodiments, the inclusion of one or more IgGl and IgG3 Fc regions may confer strong complement activation potential and placental transfer and may also promote antibody- dependent cell cytotoxicity (ADCC), another important mechanism of tumor cell killing. In addition, inclusion of one or more IgGl and IgG3 Fc regions may activate CDC and provide the ability to enhance antibody-mediated phagocytosis. In embodiments where IgG4 is added to the Fc region of a CPCSA, it may serve as a way to minimize immune effector functions, such as CDC and ADCC. Fc regions are not the only substitutions or additions that could be used to customize molecules.

[135] In some embodiments, antibody engineering may be used to activate, enhance and/or stabilize a CPCSA. In some embodiments, this may be accomplished by one of the following: modifying the hinge region flexibility, modifying the Fc region, for example generating the IgGl/IgG3 chimeric region, modifying glycosylation sites, increasing the affinity of IgG for Clq by mutagenesis and increasing Fc-Fc interactions by mutagenesis (to enable hexamerization). In some embodiments one can use antibodies to activate complement to varying degrees. There are several natural isotypes of human IgG- IgGl, IgG2, IgG3, and IgG4. Additionally, IgM, IgA, IgE and IgD are other types of antibody expressed. IgM is the most efficient at activating complement due to the multi-valent structure of the molecule. In some embodiments one could use several isotypes of IgG to activate complement such as IgGl and IgG3.

[136] Broadly, in various embodiments, therapeutics of a new class can be produced by functionally associating two or more antigen-binding moieties and/or non-antigen-binding moieties to each other, providing a new platform of therapeutics that are complement-enhancing agents. In accordance with various embodiments, exemplary methods of associating include, but are not limited to: mixing, blending or combining under pressure substantially equal to atmospheric pressure, mixing, blending or combining under pressures elevated above

atmospheric pressure, mixing, blending or combining under pressure less than atmospheric pressure (e.g. vacuum). In some embodiments, associating may be or include covalent interactions. In some embodiments, associating may be a direct binding or other association between a CPCSR and a complement protein complex. In some embodiments, associating may be or comprise indirect binding or other association, for example, through use of a linker or other intermediate moiety.

[137] In some embodiments, provided methods and compositions may include use of one or more antigen-binding moieties from one or more complement stabilizing moieties to create a non-natural complement enhancing agent. In some embodiments, provided

compositions may include a single chain antibody.

[138] In some embodiments, provided methods include making a complement enhancing agent comprising isolating an antigen-binding moiety and functionally coupling the antigen-binding moiety to a non-antigen-binding moiety to create a non-natural complement enhancing agent. In some embodiments, a moiety is or comprises proteins, peptides, small molecules, nucleotide sequences, antibody fragments, nanoparticles, microparticles,

carbohydrates, aptamers, polyethylene glycol, lipids or combinations of the above.

[139] In some embodiments, the antigen-binding moiety and/or non-antigen-binding moiety exhibits at least one of the following activities: activation of complement-dependent cytotoxicity (CDC), activation of complement dependent cellular cytotoxicity (CDCC), enhances antibody-dependent cellular cytotoxicity (ADCC), enhances antibody-mediated phagocytosis, enhances chemoattractant activity, upregulates activating Fc gamma receptor expression, generates opsonization signals, generates complement adjuvant, enhances C3 classical pathway convertase activity, enhances C5 classical pathway convertase activity, enhances C3 alternative pathway convertase activity, enhances C5 alternative pathway convertase activity, promotes anaphylatoxin generation, enhances the activity of other complement protein complexes or any combination thereof. [140] In some embodiments, provided compositions comprise one or more agents capable of stabilizing complement protein complexes from one of the following: wherein the second agent may include but is not limited to the activation of complement-dependent cytotoxicity (CDC) such as a convertase stabilizer, a MAC stabilizer, a complement-dependent cellular cytotoxicity (CDCC) activation event by C3b, C4b or iC3b, an antibody-dependent cellular cytotoxicity (ADCC) enhancement event by IgG, an enhanced antibody mediated phagocytosis event by iC3b binding to CR2, an enhanced chemoattractant activity by enhancing C5a generation, an upregulated activating event by Fc gamma receptor expression through signaling the C5a receptor, an opsonization event which includes deposition of C3b, C4b or iC3b, generation of complement adjuvant such as C3d, stabilization of C3 classical pathway convertase activity, stabilization of C5 classical pathway convertase activity, promotion of anaphylatoxin signaling or any combination thereof.

[141] In some embodiments, provided methods may include functionally coupling at least one antigen-binding moiety of an autoantibody to a distinct antigen-binding moiety to create a bispecific autoantibody agent wherein the distinct antigen-binding moiety binds to a pathogen antigen or tumor antigen subset.

[142] In some embodiments, provided compositions are capable of being used for treatment of one or more of cancer or infectious diseases, which may include but are not limited to viral and bacterial infections. In some embodiments, in the case of a viral or bacterial infection, the pathogen may expresses complement regulatory proteins, and comprises one or more pharmaceutically acceptable carriers or excipients.

[143] In some embodiments, provided compositions are capable of causing removal of unwanted, non-cancerous tissue from the body such as pathologic neovasculature such as that found in solid tumors, choroidal neovasculature, or the pannus of diseased tissue such as the joint in rheumatoid arthritis or osteoarthritis. The targeted vasculature might also be that of undesired tissue such as excess adipose tissue. There are several vascular surface markers that might be targeted. Examples include but are not limited to: integrins alpha-v beta-3 (ανβ3) and alpha-v beta-5 (ανβ5), Endoglin (CD 105), VEGF Receptors (1-3), Receptors for Insulin-like Growth Factors (1-6), Somatostatin Receptors (1-5), Fibroblast Growth Factor (FGF) Receptors, Platelet- derived Growth Factor (PDGF) Receptors, CD44, extradomain B (ED-B) of fibronectin, Tenascin C, the human target of the L19 antibody, Tissue Factor Receptor, and Heme Oxygenase 1 (HO-1). Other receptors might include E-Selectin, P-Selectin, ICAM-1, CD133, complement component 3 (C3) split products such as iC3b and C3d, and von Willebrand Factor (vWF)

[144] In some embodiments, provided methods include treating or reducing the severity of a human disease including the step of administering one or more complement enhancing agent(s) to a subject in need thereof. In some embodiments, one or more complement enhancing agents are administered at least twice. In some embodiments, one or more complement enhancing agents are administered at most once per day. In some embodiments, one or more complement enhancing agents are administered at least once per week. In some embodiments, one or more complement enhancing agents are administered at most once per week. In some embodiments, one or more complement enhancing agents are administered at least once per month. In some embodiments, one or more complement enhancing agents are administered at most once per month.

[145] In some embodiments, provided methods and compositions may be used alone, or in combination with other therapies including, but not limited to, at least one of the following: immunotherapy, anti-infective therapies, anti-neovascularization therapy, and anti-tumor therapy, and vaccinations.

[146] These and other objects, features, embodiments and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims.

Alternative Pathway Complement-Stabilizing Reagent (APCSR)

[147] The present specification discloses, among other things, the identification and use of reagents that enhance alternative complement pathway activity. An Alternative Pathway Complement-Stabilizing Reagent (APCSR) can be, e.g., an antibody. In various instances, an APCSR physically interacts with an alternative complement pathway component in a manner that directly or indirectly increases the rate and/or duration of the activity of the alternative complement pathway component, and/or directly or indirectly increases the rate and/or duration of alternative complement pathway activity.

[148] Certain exemplary APCSRs stabilize a complement pathway component that is or comprises a complex that determines or contributes to the rate and/or duration of alternative complement pathway activity. [149] In some embodiments, an APCSR described herein can demonstrate, in a same or comparable assay, activity equal to, comparable to, or greater than an autoantibody of a subject that produces nephritic factor, which autoantibody stabilizes the C3bBb convertase of the alternative pathway.

[150] In some embodiments, a complement-enhancing agent will stabilize the alternative pathway (AP) C3 convertase. Complement activation (CA) via the AP occurs when C3b binds to factor B which is then cleaved by factor D to form the C3bBb complex. The C3bBb complex is a serine protease which can cleave C3 into C3a and C3b (i.e. C3 convertase activity). The C3bBb complex has a very short half-life to avoid excessive complement activation. In addition, complement regulatory proteins can promote C3bBb inactivation to down regulate CA. A third protein, properdin (P), is a positive regulator of the AP C3 convertase, and stabilizes the complex, but, the half-life is still only minutes. Certain antibodies to the C3bBb or C3bBbP convertase can stabilize the enzyme and prolong its half-life and thus enhance CA even in the presence of regulatory proteins. In some embodiments, such an antibody, or analogous C3bBb(P) -binding molecules, could be used to promote and enhance CA on the surface of a tumor cell and increase the tumor killing of anti-CD monoclonal antibodies such as anti-CD20 mAbs (e.g. rituximab).

[151] In some embodiments, a complement-enhancing agent will stabilize the C5 convertase of the alternative pathway. When complement is initiated via the alternative pathway the complement cascade continues when the C3bBb complex binds to additional C3b molecule to form the C3bBb3b complex which is a serine protease that can cleave C5 into C5a and C5b (i.e. C5 convertase activity). The C3bBbC3b complex has a very short half-life to avoid excessive complement activity. In addition, complement regulator proteins can promote C3bBbC3b inactivation to down regulate the activity of the terminal complement components. Certain antibodies to the C5 convertase can stabilize the enzyme and prolong its half-life and thus enhance complement activity even in the presence of regulatory proteins. In some embodiments, such an antibody, or analogous C3bBbC3b -binding molecules, could be used to promote and enhance CA on the surface of a tumor cell and increase the tumor killing of anti-CD monoclonal antibodies such as anti-CD20 mAbs (e.g. rituximab).

[152] In various embodiments, an APCSR comprises, or is derived at least in part from, a "nephritic factor" autoantibody of a producing donor, which nephritic factor can in some instances stabilize C3bBb or C3bBbP convertase of the alternative pathway. Such an APCSR may have antibacterial properties due, at least in part, to enhancement of complement-mediated bacterial lysis and phagocytosis.

[153] In some embodiments, a complement-enhancing agent derived from nephritic factor stabilizes a alternative pathway C3 convertase and/or increases complement activation.

[154] In some embodiments, an APCSR may be in the form of bispecific antibodies and localize the convertase stabilization to the tumor cell membrane by binding to a tumor cell surface marker and to the convertase.

[155] In some embodiments, APCSR molecules may act in the fluid phase or on cell membranes. In accordance with various embodiments, by directing the convertase stabilizing activity to the tumor cell membrane the off-target effects of the convertase-stabilizing activity will be reduced.

[156] In some embodiments, an APCSR will activate complement-dependent cytotoxicity thus enhancing target cell killing.

[157] In some embodiments, an APCSR will activate complement-dependent cellular cytotoxicity thus enhancing target cell killing.

[158] In some embodiments, an APCSR will amplify the antibody-dependent cellular cytotoxicity thus enhancing target cell killing.

[159] In some embodiments, an APCSR will magnify antibody-mediated phagocytosis.

[160] In some embodiments, an APCSR will promote chemoattractant activity.

[161] In some embodiments, an APCSR can lead to upregulating expression of the activating Fc gamma receptor.

[162] The present invention includes a nucleic acid encoding APCSR as disclosed herein. Nucleic acid sequences encoding an APCSR are disclosed herein, any of which may be present or included in a composition of the present invention. A nucleic acid molecule of the present invention can be a nucleic acid molecule encoding, alone or among other encoded elements, an APCSR. A nucleic acid molecule of the present invention can be a linear nucleic acid, plasmid, or vector. In certain instances, an APCSR composition is or comprises a nucleic acid in solution, such as a linear nucleic acid, plasmid, or vector in solution. Solutions for storage of nucleic acid reagents are known in the art. Numerous plasmids and vectors are known in the art. For example, a vector including a nucleic acid sequence encoding an APCSR can be an expression vector, e.g., a vector for expression of an antibody or antigen-binding polypeptide.

Alternative Pathway Complement Stabilizing Antibody (APCSA)

[163] In some embodiments, an APCSR is or comprises an antibody that stabilizes an alternative complement pathway component in a manner that directly or indirectly increases the rate and/or duration of the activity of the alternative complement pathway component, and/or directly or indirectly increases the rate and/or duration of alternative complement pathway activity.

[164] Certain exemplary APCSAs stabilize a complement pathway component that is or comprises a complex that determines or contributes to the rate and/or duration of alternative complement pathway activity, such as C3bBb convertase complex.

[165] In some embodiments, an APCSA can be an antibody found in or derived from a donor, or produced by manipulation of such an antibody, or produced by manipulation of a fragment thereof (see, e.g., methods provided in FIG. 7).

[166] In some instances, an exemplary APCSA is or comprises a nephritic factor, e.g., a nephritic factor found in or derived from a donor.

[167] An APCSA epitope can be or comprise any epitope associated with an alternative complement pathway component. An APCSA epitope can include an active site of an alternative complement pathway component. In certain instances, binding of an APCSA to its epitope can occlude an active site of an alternative complement pathway component. An APCSA epitope can include a site of an alternative complement pathway component that is a site involved in interaction of the alternative complement pathway component with another molecule, such as another alternative complement pathway component. In certain instances, binding of an APCSA to its epitope can occlude a site of an alternative complement pathway component that is a site involved in interaction of the alternative complement pathway component with another molecule, such as another alternative complement pathway component. In some embodiments, an APCSA epitope can be an epitope present when an alternative complement pathway component is in a first state of that molecule, but which is not present when that component is in a second state. For example, in some instances, APCSA epitope can be an epitope that is presented when an alternative complement pathway component is present in a complex, but which is not presented when the alternative complement pathway component is not in complex.

[168] APCSAs can be derived from various sources. In certain instances, an APCSA is designed and/or synthesized in vitro. In certain instances, an APCSA is derived from an organism. In certain instances, the organism is an organism immunized with one or more alternative complement pathway component molecules, or complexes thereof, or with one or more epitopes present in such molecules and/or complexes. In certain instances, an APCSA is or includes an antibody produced by a human subject, or an antibody including a sequence (e.g., at least one CDR or variable region) thereof.

[169] In some embodiments, an APCSA can be found and/or derived from serum of a mammalian (e.g., a human) subject having a condition characterized by alternative complement dysfunction and/or producing nephritic factor.

[170] In certain some embodiments, an APCSA is or comprises an antibody that binds to and stabilizes C3 convertase complex (C3bBb). Such an APCSA can be referred to as a C3 convertase Complement Stabilizing Antibody (C3CSA, more specifically an AP-C3CSA).

[171] In some embodiments, an APCSA described herein can demonstrate, in a same or comparable assay, activity equal to, comparable to, or greater than a reference APCSA found in or derived from a donor, such as a nephritic factor. In certain embodiments, an APCSA can have properties that differ from those of an APCSA found in or derived from a donor.

[172] In some embodiments, a portion (e.g., an antigen-binding fragment or portion thereof) of an APCSA, e.g., an APCSA found in or derived from a donor, can be manipulated to generate an APCSA that differs from an initial APCSA, e.g., an APCSA as found in or derived from the donor. Such a product can, in some instances, be used as a therapeutic, e.g., in the subject the original APCSA was found in or derived from, or in another.

[173] As will be appreciated by those of skill in the art, an antibody (e.g., an APCSA, e.g., an APCSA found in or derived from a donor or produced by manipulation of such an antibody or produced by manipulation of a fragment thereof) as described herein can be or include an immunoglobulin molecule that recognizes and specifically binds to a target through at least one antigen recognition site within a variable, optimized, or selected region of an immunoglobulin molecule. [174] In some embodiments, changes in the non-antigen binding domain may cause or support a therapeutic response in the individual receiving treatment with the APCSR.

[175] In some embodiments, provided compositions may include one or more changes in the effector functions of an APCSA as compared to a form in which the APCSA was initially synthesized, or as compared to a form in which the APCSA was initially found in or derived from a donor.

[176] In some embodiments, a provided APCSA may comprise one or more FC- mediated properties.

[177] In some embodiments, one could tailor the physiological properties of APCSA' s specificity. In some embodiments, this may comprise changing the native Fc region for other Fc regions from other Ab isotypes. It should also be noted that some modifications of the APCSA will not be simple swaps of the Fc region. In some embodiments, modifications can be made at the N-terminus of the APCSA. In some embodiments, adding an IgM Fc region may make a monoclonal multivalent APCSA with strong complement activation/stabilization potential. In some embodiments, the half-life of an APCSA may be between 1-90 days. Other embodiments may include using an IgA Fc to create an APCSA capable of being transported across mucosal surfaces as well as conferring complement activation via the alternative pathway (e.g., IgAl). In still other embodiments, the inclusion of one or more IgGl and IgG3 Fc regions may confer strong complement activation potential and placental transfer and may also promote antibody- dependent cell cytotoxicity (ADCC), another important mechanism of tumor cell killing. In addition, inclusion of one or more IgGl and IgG3 Fc regions may activate CDC and provide the ability to enhance antibody-mediated phagocytosis. In embodiments where IgG4 is added to the Fc region of an APCSA, it may serve as a way to minimize immune effector functions, such as CDC and ADCC. Fc regions are not the only substitutions or additions that could be used to customize molecules.

[178] In some embodiments, antibody engineering may be used to activate, enhance and/or stabilize an APCSA. In some embodiments, this may be accomplished by one of the following: modifying the hinge region flexibility, modifying the Fc region, for example generating the IgGl/IgG3 chimeric region, modifying glycosylation sites, increasing the affinity of IgG for Clq by mutagenesis and increasing Fc-Fc interactions by mutagenesis (to enable hexamerization). In some embodiments one can use antibodies to activate complement to varying degrees. There are several natural isotypes of human IgG- IgGl, IgG2, IgG3, and IgG4. Additionally, IgM, IgA, IgE and IgD are other types of antibody expressed. IgM is the most efficient at activating complement due to the multi-valent structure of the molecule. In some embodiments one could use several isotypes of IgG to activate complement such as IgGl and IgG3.

Treatment of Cancer and Infection

[179] Administration of a CSR as disclosed herein can be used to treat a subject at risk of, having, or diagnosed as having, a cancer or infection, of the present disclosure includes administration of CSR to a subject in order to harness subject's complement protein system against infectious diseases and cancers. Such diseases include, without limitation, infectious diseases in which complement enhancement will promote the killing and/or removal of a microbial pathogen such as a virus, bacteria, parasite, or fungus. Such diseases also include various types of cancer including solid tumors such as lymphomas, melanoma, adenocarcinomas, squamous cell carcinomas, and circulating cancers such as leukemias, including but not limited to, acute and chronic lymphocytic leukemia (ALL and CLL), acute and chronic myleocytic leukemia (AML and CML). In some embodiments, the present invention provides new methods and compositions of making non-natural complement enhancing agents.

[180] In various instances, a CSR as disclosed herein is administered in combination with at least a second agent or therapy, which second agent or therapy can be selected to further treatment of cancer or infection. For instance, anti-tumor monoclonal antibody -based

immunotherapy has been developed into a highly successful strategy to treat many solid cancers, lymphoma, and leukemia. In some instances, in which a CSR is administered to a subject for treatment of cancer, a second agent or therapy is also administered to the subject, which second agent or therapy can be, for example, a monoclonal antibody specific to a tumor antigen associated with the cancer. In various instances, a CSR as disclosed herein is administered in combination with at least a second agent or therapy, which second agent or therapy can be, include, or complement-enhancing agent and may be or include an antibody. An antibody administered in combination with a CSR can be, for example, a complement-enhancing agent that recognizes a cancer neoantigen. A cancer neoantigen may be or include a protein normally only expressed by a fetus. In other cases, a neoantigen may be or include a mutated or modified version of a normal human protein. One example neoantigen, HDM-2, is found on the surface of several tumor types but not on healthy cells. In other cases, the protein will be expressed in atypical locations, such as cell surface expression of nucleolin. Other tumor antigens recognized by antibodies are described in the literature (Scott and Renner, 2001).

[181] In certain embodiments, a method of treating cancer can include administration of a CSR as described herein and administration of an anti-cancer agent or therapy that is not a CSR. Various agents and therapies are known in the art or described herein for the treatment of cancer. In particular embodiments, agents and therapies used in the treatment of subjects having cancer include, for example, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel,

temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomysin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, amifostine, and a combination thereof, and others described herein or known in the art. Anti-cancer agents for use in the present invention include biologies and/or antibody therapies including, without limitation, Arzerra (Ofatumumab), Avastin

(Bevacizumab), Bexxar (Tositumomab), Campath (Alemtuzumab), Erbitux (Cetuximab), Herceptin (Trastuzumab), Mylotarg (Gemtuzumab ozogamicin), Rituxan (Rituximab), Vectibix (Panitumumab), and Zevalin (Ibritumomab tiuxetan). Anti-cancer agents for use in the present invention further include cancer immunotherapy agents. Treatment regimens can include chemotherapy, surgery, and/or radiation therapy.

[182] In various instances, a CSR as disclosed herein is administered in combination with at least a second agent or therapy, which second agent or therapy is a checkpoint blockade or checkpoint blockade therapy that provides a second anti-tumor signal. The immune system contains checkpoints, at least in part, to prevent the immune system from attacking a host's own cells. These negative regulators include cell surface molecules such as PD-1 and CTLA-4. PD-1 and CTLA-4 are present on T cell surfaces and, when recognized by cell surface molecules on other cells (such as B7-1 and B7-2), send negative signals to the T cell to prevent activation. Cancer cells can also upregulate the binding partners of these checkpoint molecules to protect themselves from the immune system attack. [183] In certain embodiments, one or more CSRs may be used to improve the efficacy of a second agent or therapy that is or includes a cancer vaccine. One such vaccine is primarily an autologous vaccine utilizing tumor cells from the same subject in which the vaccine will later be used. In some embodiments, in this type of vaccine, tumor cells are collected from a subject, altered and killed in the laboratory to make them more immunogenic and then returned to the subject. The subject's immune system will then attack these cells while also attacking the tumor cells present in the body. Co-treatment of the CSR with the tumor cell vaccine would further stimulate the immune system to respond to the cancer.

[184] In some embodiments, a cancer vaccine may be or comprise an antigen vaccine.

In some embodiments, such vaccines may boost the immune system by exposing it to one or more tumor antigens that are peptides or proteins. Accordingly, the immune system will typically recognize tumors that are expressing the same antigens. In some embodiments, CSRs may boost the response to the antigen vaccine by providing a complement activation adjuvant.

[185] In some embodiments, comprising one or more cancer vaccine embodiments, it is possible a CSR may be used in conjunction with a dendritic cell vaccine. In some embodiments, such vaccines utilize this specialized cell type of the immune system to "present" tumor antigens for T cells to recognize and initiate an immune response.

[186] Without wishing to be held to a particular theory, it is contemplated that, in at least some instances, complement system may play a role in subject response to vaccine.

Through the CR2 receptor on B cells, antibody responses may be enhanced when CR2 binds to C3d on an antigen. Additionally, in some embodiments, other complement receptors may be present on dendritic cells, T cells and B cells which can further stimulate these cells to mount an immune response.

[187] Combination therapy of a CSR and a second agent or therapy can be more effective than the second agent or therapy alone. For instance, combination therapy of a CSR and a cancer immunotherapeutic second agent can be more effective than cancer

immunotherapeutic alone. Monoclonal antibody -based immunotherapy is associated with certain inherent limitations, and cancers can develop mechanisms that circumvent anti-cancer effects. For example, many tumor cells demonstrate increased levels of complement regulatory proteins on their cell surface, which regulatory proteins are expressed on the cell surface of healthy host tissue to abort complement activation before it can cause damage. On tumor cells however, increased levels of these regulators prevent amplification of complement activation that may otherwise have been triggered by sensing of damage or non-self-characteristics (Carter and Lieber 2014, FEBS Letters 588:334-340).

[188] It is further appreciated that, in some embodiments, there is interplay (e.g., synergistic and/or antagonistic effects) between complement and cell-mediated effectors. In addition to cell killing, enhanced complement activation may also attract and/or activate effector cells leading to additional activity(ies). Moreover, various cancer immunotherapeutics will differ in complement activation. For example, in some embodiments, even between two drugs that target the same tumor antigen (e.g., CD20), differential complement response may be observed. In one particular example, ofatumab-treated cells bind Clq with more avidity than rituximab-treated cells, due at least in part to difference in Fc regions of the antibodies.

[189] In certain embodiments, a CSR of the present can be administered separately from an additional agent or therapy. In certain embodiments, administration of a CSR can be to a subject having previously received, scheduled to receive, or in the course of a treatment regimen including an additional anti-cancer therapy.

[190] In some embodiments, an agent or therapy used in combination with CSR can be administered in a single therapeutic composition or dose together with CSR, at the same time as CSR in the form of a separate composition, or in a manner temporally distinct from the administration of CSR. When a CSR is to be used in combination with an additional agent, the CSR can be co-formulated with the additional agent or the CSR can be formulated separately from the additional agent formulation. For example, the respective CSR and additional agent compositions can be, e.g., mixed prior to administration and administered together, or can be administered separately, e.g., at the same or at different times. In various embodiments, an additional agent or therapy administered in combination with a CSR as described herein can be administered at the same time as CSR, on the same day as CSR, or in the same week as CSR. In various embodiments, an additional agent or therapy administered in combination with a CSR as described herein can be administered such that administration of the CSR and the additional agent or therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of CSR. In various embodiments, the administration frequency of one or more additional agents can be the same as, similar to, or different from the administration frequency of a CSR. In some embodiments, a second agent is administered periodically rather than being continually present in the body.

[191] In some embodiments, an administration regimen, e.g. timing and dosage, of a

CSR and that of any of one or more additional agents or therapies can be determined

independently and administered independently, while in certain circumstances dosages can be co-modulated, interdependent, co-administered, or have any other relationship known to those of skill in the art. It is contemplated that CSR combination therapies can demonstrate synergy between CSR and one or more additional agents or therapies and can in some embodiments demonstrate greater-than-additive effects. A CSR can be administered in any effective amount as determined independently or as determined by the joint action of CSR and any of one or more additional agents or therapies administered. Administration of the CSR may, in some

embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional agent or therapy relative to a reference regimen for administration of additional agent or therapy or therapy absent the CSR. In certain embodiment, a composition described herein can replace or augment other previously or currently administered therapy. For example, upon treating with CSR, administration of one or more additional agents or therapies can cease or diminish, e.g., be administered at lower levels.

[192] Encompassed within combination therapy is a treatment regimen that includes administration of two distinct CSR as described herein and/or a treatment regimen that includes administration of a CSR as described herein by a plurality of formulations and/or routes of administration.

[193] In various instances, a CSR composition can be formulated to include an acceptable carrier or excipient, e.g., a carrier or excipient suitable for laboratory use, a carrier or excipient suitable for in vitro use, or a pharmaceutically acceptable carrier or excipient.

Examples of carriers include, without limitation, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Compositions of the present invention can include a salt, e.g., a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt.

[194] Selection or use of any particular form may depend, in part, on the intended mode of application. In various embodiments, a CSR can be formulated as, or in a manner appropriate to use as, a laboratory reagent or reagent for in vitro use. In various embodiments, a CSR will be formulated in a liquid form.

[195] In various embodiments, a composition including an antibody as described herein, e.g., a sterile formulation, can be formulated in accordance with conventional practices, e.g., using distilled water as a carrier. Suitable carriers can include, without limitation, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D- mannose, D-mannitol, and sodium chloride, optionally in combination with a suitable

solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™, HCO-50 and the like.

[196] As disclosed herein, a CSR composition may be in any form known in the art.

Such forms include, e.g., liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[197] In various embodiments, a composition of the present invention can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, optionally followed by filter sterilization. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of powder forms, e.g., sterile powders, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (e.g., as described herein). In some embodiments, proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. A composition described herein can include monostearate salts or gelatin.

[198] A CSR composition can be formulated by suitably combining the CSR with one or more vehicles or media, such as sterile water, physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, optionally in a concentration suitable to an intended use. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a stabilizer such as benzyl alcohol or phenol, and an antioxidant.

[199] A CSR composition can be packaged in a suitable ampule or other suitable packaging, e.g., for laboratory use.

[200] In some embodiments, a CSR composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 11/2 years, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[201] In some embodiments, a CSR composition can be formulated as a solution. In some embodiments, a composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage at 2-8°C (e.g., 4°C).

In vitro diagnostics

[202] In certain embodiments, ability of a candidate CSR, such as a C3CSR, to increase complement activity is measured according to a method disclosed herein. Exemplary assay methods disclosed herein include a hemolysis assay, a C3 deposition assay, or a biochemical assay. In certain instances, assessment of a CSR disclosed herein provides a reference value. In certain instances, a CSR disclosed herein is provided as a competitor to a candidate CSR in an assay. In certain embodiments, a candidate CSR is present in or isolated from serum removed from a donor.

Clinical assays

[203] In various embodiments, a CSR as disclosed herein can be used to sort cells based on FACS, which cells may then be used for clinical testing. For example, to determine whether a population of patient cells will be susceptible to complement mediated cytotoxicity when treated with a certain therapeutic, cells may be treated with the therapeutic in the presence of a CSR and a source of complement, i.e. human serum. Following this treatment, cells can be stained with an anti-C3 antibody to assess the level of C3 deposited on their membranes. This could similarly be performed to look for deposition of other complement proteins such as C4b or C5b. EXAMPLES

[204] Examples described herein demonstrate that a CPCSA can stabilize C3 convertase

(C4b2a) complex. Examples described herein demonstrate that stabilization of C3 convertase (C4b2a) by CPCSA enhances rate and/or duration of complement activity. Accordingly, the present Examples demonstrate, among other things, diagnostic and clinical utility of

compositions and methods disclosed in the present specification.

[205] Reagents and cell lines utilized in the present Examples include Raji and Ramos

B cell lines obtained from American Type Tissue Collection (ATCC). Anti-CD20 antibodies were purchased from InvivoGen, FITC labeled anti-C3 came from AssayPro (St Louis, MO). CpG DNA for B cell stimulation was purchased from InvivoGen and cytokines (IL-2, IL-15, IL- 21) came from Stem Cell Technologies. Purified human complement proteins (CI (l .Omg/mL), C2 (0.5mg/mL), C3 (l .Omg/mL), C4 (l .Omg/mL)), antibody sensitized sheep erythrocytes (EA cells) and C6 depleted serum were purchased from Complement Technologies (Tyler, TX). Protein G agarose was purchased from Thermo Scientific (Rockford, IL). DGVB⁺⁺ buffer consisted of veronal buffered saline: 0.015% sodium 5',5"-diethylbarbiturate (pH 7.35) and 71 mM NaCl supplemented with 2.5% dextrose, 0.1% gelatin, 1 mM MgCl₂ and 0.15 mM CaCl₂. EDTA-GVB buffer consisted of 0.1% gelatin, 0.015% sodium 5', 5'-diethylbarbiturate (pH 7.35), 71 mM NaCl and 40 mM EDTA.

Example 1 : CPCSA stabilizes C3 convertase (C4b2a) complex

[206] The present example demonstrates stabilization of C3 convertase (C4b2a), as measured by a hemolysis assay, in samples treated with IgG CPCSA purified from a subject. The classical pathway C3 convertase (C4b2a) was assembled on antibody sensitized sheep erythrocytes (EA). Immediately following convertase assembly, the cells were subjected to a decay period of various times (0-90 min) in the presence of the CPCSA, negative control molecule or no additional molecule. At the end of the decay period, NHS in EDTA-GVB was added to allow for terminal pathway activation and lysis of the EA cells. The level of lysis of the EA cells was used as a read out for convertase activity.

[207] To purify IgG CPCSA, primary B cells were isolated and transformed. Whole blood was harvested from the donor with the autoantibody of interest under informed consent. PBMCs were isolated from the whole blood sample by Ficoll density gradient centrifugation. The buffy coat was isolated by careful pipetting and either aliquoted and frozen in liquid nitrogen or put into culture for Epstein-barr virus (EBV) transformation.

[208] For the hemolysis assays, sheep erythrocyte (EA) cell intermediates were prepared. To prepare EA cell intermediates, classical pathway C3 convertase was assembled on antibody-sensitized sheep erythrocytes (EA) as previously described, e.g., in Medof (J Exp Med. 1984 Nov l; 160(5): 1558-78), Ohi (Clin. Exp. Immunol. 1994; 95:316-321), and Kuttner-Kondo (J. Biol. Chem. 2007;282: 18552-18562). Briefly, EA cells (5 ^χ 10⁸ cells/ml) were washed three times in DGVB⁺⁺. Purified human CI was added and the cells incubated at 30° C for 15 min, followed by washing in DGVB⁺⁺. Human C4 was added and the mixture incubated for 15 min at 30° C. Following washing in DGVB⁺⁺, purified human C2 was added in a limiting fashion (0.3 μg C2 to 5 x 10⁸ cells). The cells were then incubated at RT for 4 min and washed. EAC14b2a cells were resuspended in DGVB⁺⁺ and utilized immediately.

[209] To measure C3 convertase (C4b2a) stability, EAC14b2a cells (50 μΐ) were mixed with 50 μΐ of DGVB⁺⁺, CPCSA or a negative control (IgG purified from a reference donor that has not developed a CPCSA) and incubated at 30° C to allow decay of the convertase. At specified time points, normal human serum, diluted 1 :20 in 40 mM EDTA-GVB buffer, was added as a source of complement components and the samples were incubated for 1 h at 37° C. After centrifugation, the absorbance at 414 nm was determined in the supernatant as a readout of complement activation.

[210] As shown in FIG. 3, hemolysis data demonstrate that CPCSA, but not a control sample, maintains higher levels of convertase activity, observed as hemolysis, over time as compared to natural decay of hemolysis of antibody sensitized sheep red blood cells. The results demonstrate that in the presence of CPCSA the convertase was still active after the decay period, out to 90 min (FIG. 3). In contrast, in the presence of a negative control molecule or buffer alone decay of the convertase can be seen across the time course, with essentially all of the convertase activity gone by 60 min. These data confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo.

Example 2: CPCSA stabilizes C3 convertase (C4b2a) complex in the presence of

physiologically relevant convertase inhibitor [211] Natural decay of convertase is accelerated in the presence of certain complement regulatory proteins. The present Example examines whether the CPCSA stabilized convertase is resistant to that activity. The present example utilizes a hemolysis assay to determine the effect of CR1 convertase inhibitor on hemolysis decay in the presence or absence CPCSA. When the hemolysis assay described in Example 1 was repeated in the presence of the complement regulatory protein CD35 (Complement regulatory protein 1; CR1), the CPCSA stabilized convertase remained active, whereas the samples that were incubated with the negative control molecule demonstrated accelerated decay (FIG. 4).

[212] IgG CPCSA were purified as described in example 1. A hemolysis assay was carried out as described in Example 1, as applied to both CPCSA-treated sample and a control sample treated with IgG purified from a normal donor without a CPCSA. All samples were further treated with CR1, a physiologically relevant C3 convertase (C4b2a) inhibitor.

[213] As shown in FIG. 4, data demonstrate that CPCSA inhibits decay of hemolysis as compared to control samples lacking CPCSA, and further that potentiation of complement activity in the presence of CPCSA is resistant to inhibition by CR1. These data confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo, and that CPCSA activity is resistant to regulatory proteins.

Example 3 : CPCSA stabilizes C3 convertase (C4b2a) complex and enhances C3 deposition on B cells treated with an anti-CD20 antibody

[214] This Example utilizes a C3 deposition assay on Raji cells to demonstrate that

CPCSA improves C3 deposition. Raji cells are a human B cell line derived from a Burkitt's lymphoma subject that are often used as a model cell line for therapeutic development. They express CD20 on their surface and are somewhat susceptible to Rituximab (anti-CD20) mediated killing. One of the mechanisms that contributes to the killing of tumor cells is complement dependent cytotoxicity (CDC). However, it has been well established that some B cell lymphomas are not susceptible to rituximab, which could be overcome if CDC can be enhanced, by, for example, the CPCSA. To demonstrate enhanced complement activation on Raji cells in the presence of the CPCSA, a complement deposition assay was developed for B cells.

[215] The Raji cell complement activation assay of the present example was modeled, at least in part, after the hemolysis assay. Complement was activated in a step-wise fashion. First cells were treated with anti-CD20 humanized antibody to trigger the classical pathway of complement. Next, complement proteins CI, C4 and C2 were added. Because B cells express a number of complement regulatory proteins on their surface, these steps had to be optimized to identify the ideal concentration, temperature and incubation time to allow for convertase assembly. Once the convertase was assembled, NHS was added in the presence of EDTA as a source of C3. Following an incubation period, the amount of C3b deposited on the cell membrane was assessed by staining and FACS.

[216] For the above-mentioned optimization of the present B cell complement deposition assay, Raji and Ramos B cell lines were sensitized with an anti-CD20 monoclonal antibody (InvivoGen) by incubation on ice for 1 hour in RPMI media. Following sensitization, cells were washed and resuspended at a concentration of 10⁷ cells/ml. To assess the level of complement activation that occurs using complete serum, normal human serum (NHS), which lacks CPCSA, was then added at various concentrations (7.5-30%). Following an incubation for 30 minutes at 37°C, cells were centrifuged, washed and resuspended in FACS buffer (2% fetal bovine serum, FBS, in PBS). Cells were stained with a FITC labeled anti-C3 antibody and samples were analyzed on a BD Accuri flow cytometer. Results demonstrated that cells were competent for complement activation (data not shown).

[217] For further optimization of the present B cell complement deposition assay, a step-wise complement activation assay was performed on the Raji cells to assess classical pathway activation, using varied parameters. In a method similar to the step-wise hemolysis assay, CI, C4 and C2 were added step-wise to anti-CD20 sensitized Raji cells. Following addition of the C2, cells were washed and resuspended in RPMI. It was necessary to find optimal conditions for this stepwise complement activation assay. Two isotypes of anti-CD20 (IgGl or IgG3) were used alone or in combination at various concentrations (4 μg/mL - 16 μg/mL). Following an incubation on ice from 30 min to 60 min, CI was tested in concentrations ranging between 10 μg/mL- 200 μg/mL. The cells and CI were incubated for 30 min to 60 min and at temperatures ranging from 30°C to 37°C. The C4 complement protein was then added in various concentrations (20 μg/ml-200 μg/ml) followed by an incubation for 15 min ranging in temperature from 23°C to 37°C. The C2 complement protein was then added at dilutions of (0.5 μg/ml-5 μg/ml) followed by various incubation times (5 min - 30 min) and temperatures ranging between 30°C to 37°C. The CPSCA was next added or not added to samples. The samples were then incubated at temperatures ranging from 30°C - 37°C to allow decay of the C3 convertase (C4b2a), followed by addition of NHS or C6 depleted serum with concentrations ranging from 1.7% to 30% in GVB-EDTA buffer to allow C3 activation and C3b deposition on the B cell surface. At the conclusion of the serum incubation, the cells were washed, resuspended in FACS buffer and stained with an anti-C3 FITC antibody. Samples were analyzed by flow cytometry. The final conditions identified were 4 μg/ml anti-CD20 IgG3 with a 30 min incubation on ice; CI was used at 40 μg/ml and incubated at 30°C for one hour; C4 was used at 20 μg/ml and incubated at 30°C for 15 min. C2 was used at 5 μg/ml and incubated for 5 min at room temperature. The final concentration of NHS or C6-dpl serum was 7.5% and samples were incubated for 30 min at 37°C.

[218] As shown in FIG. 5, data demonstrated that, prior to decay, CPCSA and control- treated samples were comparable in C3 deposition. However, following a 40 minute decay period, data showed that the CPCSA-treated sample demonstrated greater C3 deposition than control. Control, by contrast, resembled no complement samples following the 40 minute decay period. The results demonstrated that in the presence of the CPCSA more C3b is deposited on the Raji cell surface (FIG. 5). The CPCSA also allowed the convertase to be activated for a longer time than unstabilized convertase (FIG. 5). Results further confirmed that CPCSA potentiates complement activity. These data confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo.

Example 4: CPCSA enhances complement-mediated cytotoxicity

[219] To determine whether the increased complement deposition seen in the presence of the CPCSA translates to an increase in CDC, cytotoxicity assays were performed. This Example utilizes a B cell cytotoxicity assay to demonstrate that CPCSA potentiates complement activity. In these assays, Raji cells were treated with anti-CD20 and complement as described earlier to assemble the convertase. Then, NHS was added as a source of the terminal pathway in the presence or absence of CPCSA. Following an incubation period, supernatants from the cells were collected and the levels of LDH release were assessed.

[220] In the B cell cytotoxicity assay of the present Example, Raji cells were plated in

96 well plates and sensitized with anti-CD20 antibody to initiate the classical pathway of complement. NHS was added to wells and the samples were incubated for 2 hours. Supernatants were collected from the wells and analyzed using the Lactate dehydrogenase release assay from Promega.

[221] As shown in FIG. 6, results demonstrated that treatment of Raji cells with

CPCSA in conjunction with anti-CD20 leads to increased complement mediated cell death. The results demonstrated that there was an increase in LDH from the cells treated with CPCSA (FIG. 6), indicating higher levels of cytotoxicity in those samples. These data confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo.

Example 5: Biochemical assay demonstrates that CPCSA stabilizes C3 convertase (C4b2a) against decay

[222] A biochemical assay is used to measure enzymatic activity of stabilized C3 convertase (C4b2a). The assay utilizes a fluorescently labeled substrate. In this assay, when convertase is assembled in vitro in wells not treated with CPCSA, fluorescent substrate is initially cleaved by C3 convertase (C4b2a) activity, but after a decay period cleavage of the substrate is no longer observed. In wells treated with CPCSA, fluorescent substrate will continue to be cleaved by C3 convertase (C4b2a) following the C3 convertase (C4b2a) decay period. These data would indicate that, in the presence of CPCSA, C3 convertase (C4b2a) activity is extended over a significantly longer period of time as compared to C3 convertase (C4b2a) not treated with CPCSA.

Example 6: CPCSA stabilizes C3 convertase (C4b2a) complex in the presence of

physiologically relevant convertase inhibitor

[223] The present Example examines whether the CPCSA-stabilized convertase is resistant to decay in the presence of decay accelerating factor (DAF). The present example utilizes a hemolysis assay (see Examples 1 and 2) to determine the effect of DAF convertase inhibitor on hemolysis decay in the presence or absence CPCSA. When the hemolysis assay is repeated in the presence of DAF, the CPCSA-stabilized convertase remains active, as compared to samples that are incubated with a negative control molecule, which control samples demonstrate decay.

[224] IgG CPCSA is purified as described in example 1. A hemolysis assay is carried out as described in Example 1, applied to both CPCSA-treated sample and a control sample that is treated with IgG purified from a normal donor without a CPCSA. All samples are further treated with DAF, a physiologically relevant C3 convertase (C4b2a) inhibitor.

[225] Data will demonstrate that CPCSA inhibits decay of hemolysis as compared to control samples lacking CPCSA, and further that potentiation of complement activity in the presence of CPCSA is resistant to inhibition by DAF. Data will confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo, and that CPCSA activity is resistant to regulatory proteins.

Example 7: CPCSA stabilizes C3 convertase (C4b2a) complex in the presence of

physiologically relevant convertase inhibitor

[226] The present Example examines whether the CPCSA-stabilized convertase is resistant to decay in the presence of Factor H. The present example utilizes a hemolysis assay (see Examples 1 and 2) to determine the effect of Factor H convertase inhibitor on hemolysis decay in the presence or absence CPCSA. When the hemolysis assay is repeated in the presence of Factor H, the CPCSA-stabilized convertase remains active, as compared to samples that are incubated with a negative control molecule, which control samples demonstrate decay.

[227] IgG CPCSA is purified as described in example 1. A hemolysis assay is carried out as described in Example 1, applied to both CPCSA-treated sample and a control sample that is treated with IgG purified from a normal donor without a CPCSA. All samples are further treated with Factor H, a physiologically relevant C3 convertase (C4b2a) inhibitor.

[228] Data will demonstrate that CPCSA inhibits decay of hemolysis as compared to control samples lacking CPCSA, and further that potentiation of complement activity in the presence of CPCSA is resistant to inhibition by Factor H. Data will confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo, and that CPCSA activity is resistant to regulatory proteins.

Example 8: CPCSA stabilizes C3 convertase (C4b2a) complex in the presence of

physiologically relevant convertase inhibitor

[229] The present Example examines whether the CPCSA-stabilized convertase is resistant to decay in the presence of Factor I. The present example utilizes a hemolysis assay (see Examples 1 and 2) to determine the effect of Factor I convertase inhibitor on hemolysis decay in the presence or absence CPCSA. When the hemolysis assay is repeated in the presence of Factor I, the CPCSA-stabilized convertase remains active, as compared to samples that are incubated with a negative control molecule, which control samples demonstrate decay.

[230] IgG CPCSA is purified as described in example 1. A hemolysis assay is carried out as described in Example 1, applied to both CPCSA-treated sample and a control sample that is treated with IgG purified from a normal donor without a CPCSA. All samples are further treated with Factor I, a physiologically relevant C3 convertase (C4b2a) inhibitor.

[231] Data will demonstrate that CPCSA inhibits decay of hemolysis as compared to control samples lacking CPCSA, and further that potentiation of complement activity in the presence of CPCSA is resistant to inhibition by Factor I. Data will confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo, and that CPCSA activity is resistant to regulatory proteins.

Example 9: CPCSA stabilizes C3 convertase (C4b2a) complex in the presence of

physiologically relevant convertase inhibitor

[232] The present Example examines whether the CPCSA-stabilized convertase is resistant to decay in the presence of membrane cofactor protein (MCP or CD46). The present example utilizes a hemolysis assay (see Examples 1 and 2) to determine the effect of membrane cofactor protein (MCP or CD46) convertase inhibitor on hemolysis decay in the presence or absence CPCSA. When the hemolysis assay is repeated in the presence of membrane cofactor protein (MCP or CD46), the CPCSA-stabilized convertase remains active, as compared to samples that are incubated with a negative control molecule, which control samples demonstrate decay.

[233] IgG CPCSA is purified as described in example 1. A hemolysis assay is carried out as described in Example 1, applied to both CPCSA-treated sample and a control sample that is treated with IgG purified from a normal donor without a CPCSA. All samples are further treated with membrane cofactor protein (MCP or CD46), a physiologically relevant C3 convertase (C4b2a) inhibitor.

[234] Data will demonstrate that CPCSA inhibits decay of hemolysis as compared to control samples lacking CPCSA, and further that potentiation of complement activity in the presence of CPCSA is resistant to inhibition by membrane cofactor protein (MCP or CD46). Data will confirm the ability of CPCSA to potentiate complement activity in vitro or in vivo, and that CPCSA activity is resistant to regulatory proteins.

OTHER EMBODIMENTS

[235] While we have described a number of embodiments, it is apparent that our basic disclosure and examples may be altered to provide other embodiments that utilize the compounds and methods described herein. Therefore, it will be appreciated that the scope of is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

[236] All references cited herein are hereby incorporated by reference.

Claims

CLAIMS We claim:

1. A method of complement stabilization comprising administering to a subject in need thereof at least one complement-stabilizing reagent, wherein the complement-stabilizing reagent which increases the rate or duration of an activity of at least one protein or protein complex in a complement cascade.

2. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the classical pathway C3 convertase.

3. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the alternative pathway C3 convertase.

4. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the classical pathway C5 convertase.

5. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the alternative pathway C5 convertase.

6. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the terminal pathway membrane attack complex (MAC).

7. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the classical pathway CI complex.

8. The method of claim 1, wherein the complement-stabilizing reagent stabilizes the lectin pathway mannose binding lectin complex.

9. The method of any one of the above claims, wherein the complement-stabilizing reagent is or comprises an aptamer, a peptide, a small molecule, a protein, an antibody, an antibody fragment, or a combination thereof.

10. The method of any one of the above claims, wherein the complement-stabilizing reagent exhibits at least one of the following activities: promoting or increasing complement-dependent cytotoxicity (CDC) activation, activating complement-dependent cellular cytotoxicity (CDCC), enhancing antibody-dependent cellular cytotoxicity (ADCC), enhancing antibody-mediated phagocytosis, enhancing chemoattractant activity, upregulating activating Fc gamma receptor expression, enhancing opsonization by phagocytes, increasing production of complement adjuvant, stabilization of classical pathway C3 convertase, stabilization of alternative pathway C3 convertase, stabilization of classical pathway C5 convertase, stabilization of alternative pathway C5 convertase, stabilization of terminal pathway membrane attack complex, stabilization of lectin pathway mannose binding lectin complex, stabilization of classical pathway CI complex, , enhanced generation of anaphylatoxins, enhanced immune cell activation, or any combination thereof.

11. A method of enhancing an immune system response comprising administering a

therapeutically effective amount of:

(a) an antibody agent comprising a Fab portion; and

(b) a complement-stabilizing reagent with at least one of the following activities:

stabilization of classical pathway C3 convertase, stabilization of alternative pathway C3 convertase, stabilization of classical pathway C5 convertase, stabilization of alternative pathway C5 convertase, stabilization of terminal pathway membrane attack complex, stabilization of classical pathway CI complex, stabilization of lectin pathway mannose binding lectin complex, and a combination thereof;

to a subject in need thereof.

12. The method of claim 11, wherein the antibody agent is an antibody that binds a tumor antigen.

13. The method of claim 11, wherein the antibody is or comprises: an anti-CD-20 antibody, an anti-CD22 antibody, an anti-CD32b antibody, an anti-CD-33 antibody, an anti-CD40 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-VEGF antibody, an anti-HER2 receptor antibody, an anti-17-IA antibody, an anti-CCR4 antibody, an anti-IGF-IR antibody, an anti- CTLA-4 antibody, or a combination thereof.

14. The method of claim 11, wherein the antibody is an antibody that binds a pathogen antigen.

15. The method of claim 14, wherein the antibody is or comprises a monoclonal antibody.

16. The method of claim 11, wherein the complement-stabilizing reagent leads to complement- dependent cytotoxicity (CDC) activation, complement-dependent cellular cytotoxicity (CDCC) activation, antibody-dependent cellular cytotoxicity (ADCC) enhancement, enhanced antibody- mediated phagocytosis, enhanced chemoattractant activity, upregulation of activating Fc gamma receptor expression, enhanced opsonization, increased production of complement adjuvant, stabilization of the classical pathway C3 convertase, stabilization of the alternative pathway C3 convertase, stabilization of the classical pathway C5 convertase, stabilization of the alternative pathway C5 convertase, stabilization of the terminal pathway membrane attack complex, stabilization of the lectin pathway mannose binding lectin complex, stabilization of the classical pathway CI complex, stabilization of complement protein complexes, enhanced generation of anaphylatoxins, enhanced immune cell activation, or any combination thereof.

17. The method of claim 11, wherein the antibody is a bispecific antibody.

18. The method of claim 17, wherein the bispecific antibody binds:

(a) a tumor antigen; and

(b) a complement pathway component;

wherein binding of the bispecific antibody to the complement pathway component is complement-stabilizing.

19. The method of claim 17, wherein the bispecific antibody binds: (a) a pathogen antigen; and

(b) a complement pathway component;

wherein binding of the bispecific antibody to the complement pathway component is

complement-stabilizing.

20. The method of claim 19, wherein the anti-infective antibody comprises a therapeutic antibody.

21. The method of any of the above claims, wherein the complement-stabilizing reagent is or comprises: an aptamer, a peptide, a small molecule, a protein, an antibody, an antibody fragment, or a combination thereof.

22. The method of any one of the above claims, wherein the subject is suffering from a disease, disorder, or condition that is selected from the group consisting of lymphoma, a leukemia, an X- linked lymphoproliferative disorder, an Epstein Barr Virus (EBV)-associated

lymphoproliferative disorder, a breast cancer, a colorectal cancer, a lung cancer, a head and neck cancer, a prostate cancer, ovarian cancer, multiple myeloma, a melanoma, a bacterial infection, viral infection, fungal infection, parasitic infection, or any combination thereof.

23. A method of increasing the rate or duration of complement activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a complement-stabilizing reagent to the subject in need thereof.

24. The method of claim 23, wherein the complement stabilizing reagent stabilizes a C3 convertase complex, optionally wherein the C3 convertase complex is a classical pathway C3 convertase complex.

25. The method of claim 23, wherein complement-stabilizing reagent is a classical pathway complement-stabilizing reagent.

26. The method of claim 25, wherein the classical pathway complement-stabilizing reagent is a classical pathway complement-stabilizing antibody.

27. The method of claim 26, wherein the complement-stabilizing antibody is an antibody that binds a C3 convertase, optionally wherein the C3 convertase complex is a classical pathway C3 convertase complex.

28. The method of any one of claims 23 to 27, wherein the complement-stabilizing reagent is a donor autoantibody.

29. The method of any one of claims 23 to 28, wherein the subject in need thereof is a subject at risk of or diagnosed as having cancer or an infectious disease.

30. A pharmaceutical composition comprising a complement-stabilizing reagent and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient

31. The pharmaceutical composition of claim 30, wherein the complement stabilizing reagent stabilizes a C3 convertase complex, optionally wherein the C3 convertase complex is a classical pathway C3 convertase complex.

32. The method of claim 30, wherein complement-stabilizing reagent is a classical pathway complement-stabilizing reagent.

33. The method of claim 32, wherein the classical pathway complement-stabilizing reagent is a classical pathway complement-stabilizing antibody.

34. The method of claim 33, wherein the complement-stabilizing antibody is an antibody that binds a C3 convertase, optionally wherein the C3 convertase complex is a classical pathway C3 convertase complex.

35. The method of any one of claims 30 to 34, wherein the complement-stabilizing reagent is a donor autoantibody.

36. A method of treating cancer or infectious disease, the method comprising administering to a subject in need thereof a pharmaceutical composition according to any one of claims 30-35.