Targeted complement activating molecules and methods of use thereof

Abstract

In one aspect, the present disclosure provides targeted complement activating molecules comprising a target binding domain and a complement activating serine protease effector domain. In some embodiments, the target binding domain is derived from an antibody or an antigen-binding fragment thereof. Also provided are compositions and methods for treating a microbial infection using targeted complement activating molecules.

Description

[0001] TARGETED COMPLEMENT ACTIVATING MOLECULES AND METHODS OF USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to targeted complement activating molecules comprising a targeting domain and a serine protease domain for use in targeting complement activation, and related compositions and methods.

[0004] STATEMENT REGARDING SEQUENCE LISTING

[0005] The sequence listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is IT l_0342_US2_PCT_Sequence Listing_ST26, the file is 103,222 bytes, was created on January’ 29, 2026; and is being submitted via the Patent Center with the filing of the specification.

[0006] BACKGROUND

[0007] The complement system supports innate host defense against pathogens and other acute insults (M. K. Liszewski and J. P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W. E. Paul, Raven Press, Ltd., New York), and also has a role in immune surveillance against cancer (P, Macor, et al., Front. Immunol., 9:2203, 2018). More than 30 fluid-phase and membrane-bound glycoproteins, cofactors, receptors, and regulatory proteins are involved in tlie complement system (S. Meyer, et al., mAbs, 6: 1133, 2014). Many of them are serine proteases, which form a highly regulated cascade of activation events. The complement system responds rapidly to molecular stress signals through a cascade of sequential proteolytic reactions initiated by the binding of pattern recognition receptors (PRRs) to distinct structures on damaged cells, biomaterial surfaces, or microbial intruders (Reis et al,, Nat. Rev. Immunol., 18:5, 2018). Activation of the complement cascade induces diverse immune effector functions, such as cell lysis, phagocytosis, chemotaxis, and immune activation (S. Meyer, et al., 2014). Furthermore, the complement system also acts as a bridge between the innate immune response and the subsequent activation of adaptive immunity. In addition to its anti-infectious properties, the complement system is also involved in the clearance of immune complexes and apoptotic cells, tissue regeneration, mobilization of hematopoietic progenitor cells, and angiogenesis (T. M. Pierpont et al., Front. Oncol., 8: 163, 2018).The complement system can be activated through three distinct pathways: the classical pathway, the alternative pathway, and the lectin pathway. See FIGURE 1.

[0008] Activation of the classical pathway is triggered by a conformational change of the classical pathway initiation complex Cl, composed of Clq, a hexamer of trimeric chains, and a heterotetramer of the Clq-associated serine proteases Clr and C Is, as detailed below. The binding of Clq to complexes composed of host antibodies bound to a foreign particle (i.e., an antigen) initiates the activation of Cl complex. Since activation of the classical pathway largely depends on a prior adaptive immune response by the host, the classical pathway is an effector mechanism of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

[0009] The classical pathway (CP) is primarily initiated by antibody-antigen complexes. Antibodies of subclasses IgM and IgG bind to an antigen on the surface of a pathogen or a target cell and recruit the Cl complex, which is composed of the multimolecular recognition subcomponent Clq (composed of six heterotrimers of the Clq A-chain, B-chain, and C-chain) and the Clq-associated serine proteases Clr and Cis. Upon binding of Clq to the Fc-region of either an IgM bound to an antigen or to at least two IgG antibodies bound to their antigens, the serine protease Clr is converted from its zymogen form into its enzymatically active form and subsequently cleaves and activates its substrate Cis. Once activated, Cis cleaves C4 into its fragments C4a and C4b. C4b binds to complement component C2 and this complex, C4bC2, is cleaved by Cis in a second cleavage step to release C2b, forming tire complement C3 converting enzyme complex C4bC2a, a so-called C3 convertase, which cleaves the abundant plasma complement component C3 into C3a and C3b.

[0010] The lectin pathway is triggered by the binding of pattern recognition molecules, such as mannose-binding lectin (MBL), ficolins or collectin- 11 and collectin- 10, to pathogen-associated molecular patterns (PAMPs) or apoptotic or distressed host cells. The recognition molecules form a complex with the MBL-associated serine proteases, MASP-1 and MASP-2, and activate them upon binding, which results in the cleavage of C2 and C4 and the formation of the C3 convertase (C4bC2a).

[0011] The alternative pathway (AP) is initiated by spontaneous hydrolysis of C3 (“tickover”) to C3(H2O), which binds to factor B (fB). The conversion of the resulting C3(H2O)fB complexes requires the enzymatic activity of another highly specific serine protease called factor D. The availability of enzymatically active factor D is thought to be a limiting factor for the alternative pathway amplification loop and tire availability of factor Drequires the action of another enzyme, MASP-3, which is required for conversion of pro¬ Factor D (proCFD) into its active form, mature factor D (matCFD) (Dobo et al., 2016).

[0012] MatCFD, another serine protease, cleaves the C3(H2O)-bound ffl into Ba and Bb. Bb is also a serine protease and participates in the formation of alternative C3 convertase C3(H2O)Bb, which cleaves C3 into C3a and C3b. By this mechanism, the alternative pathway is constitutively active at low levels. The AP amplification loop is formed when freshly generated C3b, formed either by C3(H2O)Bb or by the classical and lectin pathway C3 convertase C4bC2a, binds to the target surfaces and sequesters fB to form C3bfB complexes that, upon cleavage by matCFD, create another C3 convertase complex C3bBb. This convertase can be further stabilized by properdin, which prevents decay of the complex and conversion of C3b by factor H and factor I. C3bBb is the functional convertase of the alternative pathway.

[0013] The three pathways converge after formation of the C3 convertases C4bC2a and C3bBb. The C3 cleavage fragment C3a is an anaphylatoxin which promotes inflammation. C3b functions as an opsonin by binding covalently through a thioester bond on the surface of target cells, marking them for circulating complement receptor (CR)-displaying effector cells, such as NK cells and macrophages, which contribute to complement-dependent cellular cytotoxicity (CDCC) and complement-dependent cellular phagocytosis (CDCP), respectively. C3b also binds to the C3 convertase (either C4bC2a or C3bBb) to form a C5 convertase (C4bC2a(C3b)n or C3bBb(C3b)n, respectively), which leads to MAC formation and subsequent CDC. Additionally, C3b’s cell-bound degradation fragments, iC3b and C3dg, can promote complement-receptor-mediated cytotoxicity (CDCC and CDCP) as well as adaptive immune response through B cell activation (M. C. Carroll, Nat. Immunol., 5:981, 2004).

[0014] Formation of C5 convertase leads to the cleavage of C5 into C5a and C5b. C5a is another anaphylatoxin. C5b recruits C6-9 to form the membrane attack complex (MAC, or C5b-9 complex). The MAC causes pore formation resulting in membrane destruction of the target cell and cell lysis (so called complement-dependent cytotoxicity, CDC). Direct cell lysis through the MAC formation has been traditionally recognized as a terminal effector mechanism of the complement system, however, C3b mediated opsonization and pro-inflammatory signaling as well as the anaphylatoxin function of C3a are thought to play a significant role in the mediation of complement dependent inflammatory pathology.

[0015] Complement regulatory proteins (CRPs) prevent unwanted complement activation and consumption of complement components. These proteins are present in most cells andvia tight control they play an important role in protecting the host cells from complement-mediated damage. CRPs can be soluble proteins (sCRPs) or membrane-bound complement regulatory proteins (mCRPs) (P. F. Zipfel and C. Skerka, Nat. Rev. Immunol. 9:729, 2009). One of the most abundant protease inhibitors in circulating blood is the Cl inhibitor (Clinh), with an average plasma concentration of 0.25 g / L (H. Gregorek, Comp, and Inflamm. 8:310, 1991). Clinh binds to and inactivates Cl r, Cis, and two of the MBL-associated serine proteases, MASP-1 and MASP-2; hence it is the primary inhibitor for the classical and lectin pathway. Other sCRPs include C4 binding protein (C4BP), and factors H and I (P. F. Zipfel and C. Skerka, 2009).

[0016] In contrast to sCRPs, the mCRPs regulate the complement pathways by targeting both C3 and C4 (P. F. Zipfel and C. Skerka, 2009). For example, CD46 (membrane cofactor protein; MCP) is a co-factor for factor I, which mediates cleavage of C3b and C4b into their inactive degradation products, iC3b and iC4b, respectively, and thereby leads to inhibition of all three complement pathways. CD55 (decay acceleration factor; DAF) accelerates the decay of C3 and C5 convertases, which inhibits all three complement pathways. CD59 (protectin) prevents assembly of the MAC by inhibiting the polymerization of C9 and its subsequent binding to C5b-8, thus inhibiting all three pathways.

[0017] For most microbial organisms, the first line of defense provided by complement is sufficient to prevent infection and preserve the integrity of the host organism. Pathogens are micro-organisms that have acquired ways to undermine the host’s immune system, break through the barriers that protect the host against microbial invasion, and establish an infection. Pathogens have developed various ways to undermine the host’s immune defense. For example, the bacterium Neisseria meningitidis has a surface protein called Factor H-binding protein that sequesters and binds the host’s negative complement regulatory' component factor H (fH) to the bacterial surface. This, in turn, protects tire bacteria from complement activation since surface bound factor H decays and inactivates complement C3 and C5 convertases that have formed on the pathogen surface and thereby prevents the host complement system from neutralizing, killing, or opsonizing the pathogen. Other strategies that pathogens have developed to evade complement attack include the sequestration of host C4-binding protein by bacterial surface proteins like PspA and PspC of Streptococcus pneumoniae to prevent the formation of classical and lectin pathway C3 and C5 convertases C4bC2a and C4bC2a(C3b)n respectively on the bacterial surface (Haleem KS, et al. Infect immun. 2018 Dec 19;87(l):e00742-18.), and the release of complement activating toxins that consume complement away from the vulnerable pathogen surface.Complement’s central role in multiple physiological processes requires that complement activation be tightly regulated. Pathogens (P. F. Zipfel and C Skerka, 2009), however, have been shown to use evasion strategies to block complement activity, including expression of negative complement regulatory proteins. Thus, there is a need for therapies that enhance complement activity against pathogens, such as by targeting complement activation to the pathogens, or to tissues where the pathogens are present.

[0018] As previously described in the patent application published as WO 2023 / 060167, a new technology has been recently developed for directing complement activation to the surface of pathogens. This technology, Targeted Complement Activation Therapy, or T-CATTM, combines a targeting domain with a complement activating serine protease domain, Tire previous application described the development of tire technology and results relating to a variety of pathogens. The present application builds on this previous work. Provided herein are new antibodies and new T-CAT molecules that permit the targeting of additional pathogens.

[0019] SUMMARY

[0020] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0021] In one aspect, the present disclosure provides Targeted Complement Activation Therapy (T-CAT™) molecules that target Klebsiella bacteria. In some embodiments, the T-CAT molecules comprise a targeting domain that targets an antigen present on a Klebsiella bacterium. In some embodiments, the targeting domain is a target binding domain that binds to an antigen present on the surface of a Klebsiella bacterium. In some embodiments, the target binding domain binds to an antigen present on the surface of a Klebsiella pneumoniae bacterium. In some embodiments, the antigen is MrkA, In some embodiments, the target binding domain is derived from an antibody. In some embodiments, the target binding domain comprises an antigen-binding fragment of an antibody. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-MrkA antibody Clone 3. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-MrkA antibody Clone 37. In some embodiments, the T-CAT molecules comprise (or further comprise) a complement activating serine protease effector domain. In some embodiments, the complement activating serine protease effectordomain in catalytically active, while in other embodiments tlie complement activating serine protease effector domain is in a zymogen form.

[0022] In another aspect, the present disclosure provides T-CAT molecules that target Pseudomonas bacteria. In some embodiments, the T-CAT molecules comprise a targeting domain that targets an antigen present on a Pseudomonas bacterium. In some embodiments, the targeting domain is a target binding domain that binds to an antigen present on the surface of a Pseudomonas bacterium. In some embodiments, the target binding domain binds to an antigen present on the surface of a Pseudomonas aeruginosa bacterium. In some embodiments, the antigen is Fla-B. In some embodiments, tire target binding domain is derived from an antibody. In some embodiments, the target binding domain comprises an antigen-binding fragment of an antibody. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-Fla-B Clone 14. In some embodiments, the T-CAT molecules comprise (or further comprise) a complement activating serine protease effector domain. In some embodiments, the complement activating serine protease effector domain in catalytically active, while in other embodiments the complement activating serine protease effector domain is in a zymogen form.

[0023] In another aspect, the present disclosure provides T-CAT molecules that target Neisseria bacteria. In some embodiments, the T-CAT molecules comprise a targeting domain that targets an antigen present on a Neisseria bacterium. In some embodiments, the targeting domain is a target binding domain that binds to an antigen present on the surface of a Neisseria bacterium. In some embodiments, the target binding domain binds to an antigen present on the surface of a. Neisseria meningitidis bacterium. In some embodiments, the target binding domain binds to an antigen present on the surface of a Neisseria gonorrhoeae bacterium. In some embodiments, the antigen is NHBA. In some embodiments, the target binding domain is derived from an antibody. In some embodiments, the target binding domain comprises an antigen-binding fragment of an antibody. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-NHBA Clone 4. In some embodiments, the T-CAT molecules comprise (or further comprise) a complement activating serine protease effector domain. In some embodiments, the complement activating serine protease effector domain in catalytically active, while in other embodiments the complement activating serine protease effector domain is in a zymogen form.

[0024] In another aspect, the present disclosure provides T-CAT molecules that target Streptococcus bacteria. In some embodiments, the T-CAT molecules comprise a targetingdomain that targets an antigen present on a Streptococcus bacterium. In some embodiments, the targeting domain is a target binding domain that binds to an antigen present on the surface of a Streptococcus bacterium. In some embodiments, the target binding domain binds to an antigen present on the surface of a Streptococcus pneumoniae bacterium. In some embodiments, the antigen is PspA. In some embodiments, the target binding domain is derived from an antibody. In some embodiments, the target binding domain comprises an antigen-binding fragment of an antibody. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-PspA Clone 148. In some embodiments, the T-CAT molecules comprise (or further comprise) a complement activating serine protease effector domain. In some embodiments, the complement activating serine protease effector domain in catalytically active, while in other embodiments the complement activating serine protease effector domain is in a zymogen form.

[0025] In another aspect, the present disclosure provides T-CAT molecules that target Staphylococcus bacteria. In some embodiments, the T-CAT molecules comprise a targeting domain that targets an antigen present on a Staphylococcus bacterium, in some embodiments, the targeting domain is a target binding domain that binds to an antigen present on the surface of a Staphylococcus bacterium. In some embodiments, the target binding domain binds to an antigen present on the surface of a Staphylococcus aureus bacterium. In some embodiments, the antigen is Protein A. In some embodiments, the target binding domain is derived from an antibody. In some embodiments, the target binding domain comprises an antigen -binding fragment of an antibody. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-Protein A Clone 32. In some embodiments, the T-CAT molecules comprise (or further comprise) a complement activating serine protease effector domain. In some embodiments, the complement activating serine protease effector domain in catalytically active, while in other embodiments the complement activating serine protease effector domain is in a zymogen form.

[0026] In any of the above embodiments, the T-CAT molecules may comprise a fusion protein comprising the N-terminus of the serine protease effector domain fused to the C- terminus of the antibody heavy chain or fragment thereof or to the C-terminus of the antibody light chain or fragment thereof. Alternatively, the fusion protein may comprise the C-terminus of the serine protease effector domain fused to the N-terminus of the antibody heavy chain or fragment thereof or to the N-terminus of the antibody light chain or fragment thereof. In some embodiments, the targeted complement activating molecules comprise such a fusion protein and a second antibody chain, which is a light chain or fragment thereof if thefusion protein comprises a heavy chain or fragment thereof and which is a heavy chain or fragment thereof if the fusion protein comprises a light chain or fragment thereof. In some embodiments, the targeted complement activating molecules comprise a fusion protein comprising the N-tenninus of tire serine protease effector domain fused to the C-terminus of a single-chain antibody or fragment thereof, or a fusion protein comprising the C-terminus of the serine protease effector domain fused to the N-tenninus of a single-chain antibody or fragment thereof.

[0027] In some embodiments, the serine protease effector domain comprises complement factor D or a fragment thereof, Clr or a fragment thereof, Cis or a fragment thereof, MASP-2 or a fragment thereof, MASP-3 or a fragment thereof, MASP-1 or a fragment thereof, C2a or a fragment thereof, or Bb or a fragment thereof.

[0028] In another aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to an antigen on the surface of a Klebsiella bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to an antigen on the surface of a Klebsiella pneumoniae bacterium. In some embodiments, the antibodies and antigen- binding fragments thereof bind to the Klebsiella pneumoniae antigen MrkA. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti -MrkA Clone 3. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-MrkA antibody Clone 37.

[0029] In a further aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to an antigen on the surface of a Pseudomonas bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to an antigen on the surface of a Pseudomonas aeruginosa bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to the Pseudomonas aeruginosa antigen flagellin B (Fla-B). In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-Fla-B Clone 14.

[0030] In a further aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to an antigen on the surface of a Neisseria bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to an antigen on the surface of a Neisseria meningitidis bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to the Neisseria meningitidis antigen NHBA. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti -NHBA Clone 4.In a further aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to an antigen on the surface of a Streptococcus bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to an antigen on the surface of a Streptococcus pneumoniae bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to the Streptococcus pneumoniae antigen PspA. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti-PspA Clone 148.

[0031] In a further aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to an antigen on the surface of a Staphylococcus bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to an antigen on the surface of a Staphylococcus aureus bacterium. In some embodiments, the antibodies and antigen-binding fragments thereof bind to the Staphylococcus aureus antigen Protein A. In some embodiments, the antibodies and antigen-binding fragments thereof bind to more than one isolate of Staphylococcus aureus. In some embodiments, the antibodies and antigen-binding fragments thereof bind to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more different isolates of Staphylococcus aureus. In some embodiments, the antibody is, or the antigen-binding fragment of an antibody is derived from, anti -Protein A Clone 32.

[0032] Also provided herein are polynucleotides encoding T-CAT molecules or portions thereof and antibodies or antigen -binding fragments thereof, and cloning vectors or expression cassettes comprising such polynucleotides.

[0033] Further provided herein are host cells expressing T-CAT molecules and antibodies or antigen- binding fragments thereof, and methods of producing T-CAT molecules and antibodies or antigen-binding fragments thereof, comprising culturing the host cells under conditions allowing for expression of the molecules and isolating the molecules.

[0034] Also provided herein are methods of activating at least one complement pathway in a mammalian subject using the T-CAT molecules. In some embodiments, the T-CAT molecules may be used to induce complement dependent cytotoxicity (CDC), complement¬ dependent cellular cytotoxicity (CDCC), or complement-dependent cellular phagocytosis (CDCP) in a target cell. In some embodiments, the T-CAT molecules may be used to treat a microbial infection, such as a bacterial infection. In some embodiments, the bacterial infection is a Klebsiella, Pseudomonas, Neisseria, Streptococcus, or Staphylococcus infection.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conj unction with the accompanying drawings, wherein:

[0036] FIGURE 1 is a diagram illustrating the classical, lectin, and alternative complement pathways.

[0037] FIGURE 2 is a diagram illustrating certain formats for the targeted complement activating (T-CAT) molecules described herein. Such molecules may comprise a targeting domain derived from an antibody and a serine protease effector domain fused to either the heavy chain or light chain of the antibody. Shown are an unmodified antibody (far left) and targeted complement activating molecules comprising a serine protease effector domain fused to: the C-terminus of the heavy chain (second from left), the N-terminus of the heavy chain (center), tire C-terminus of the light chain (second from right), or the N-terminus of the light chain (far right).

[0038] FIGU RE 3 is an image of SDS-PAGE analysis of a monoclonal antibody that binds Klebsiella pneumoniae antigen MrkA and the T-CAT molecule comprising a targeting domain of the same antibody. The left two columns show the separation of the monoclonal antibody heavy chain (HC) and light chain (LC), next to a size marker ladder. Hie center two columns show the shift in size when the antibody HC is fused to the CCP1-CCPII-SP domains of Cis. The right three columns show that the Cis serine protease is active in the T-CAT molecule, as C4 cleavage was observed when purified C4 was incubated with the T- CAT molecule but not observed when purified C4 was incubated with the parental antibody.

[0039] FIGURE 4 is a graphic representation of the results of experiments testing for the presence of monoclonal antibody or T-CAT molecule after administration to mice by intraperitoneal (i.p.) injection. The levels of antibody or T-CAT molecule were measured in blood samples, bronchoalveolar lavage (BAL) samples, and lung homogenate samples. Measurements were taken up to 48 hours post-administration. High levels of both antibody and T-CAT molecule were detected at all post-administration timepoints measured.

[0040] FIGURE 5 is a graphic representation of the results of binding experiments using an antibody or a T-CAT molecule targeted to a Klebsiella pneumoniae antigen, ELISA plates were coated with Klebsiella pneumoniae. Serial dilutions of ei ther antibody or T-CAT targeted to Klebsiella pneumoniae antigen MrkA were added to the ELISA plates and incubated for 1 hour. Serial dilutions of control isotype antibody or control isotype T-CATmolecule were also tested. Binding was detected using anti-human antibodies. The antibody and T-CAT molecules targeted to Klebsiella pneumoniae antigen MrkA bound to the bacteria to a similar extent, while tire isotype controls did not bind.

[0041] FIGURE 6 is a graphic representation of the results of C3b deposition assays using an antibody or a T-CAT molecule targeted to a Klebsiella pneumoniae antigen. Bacteria were opsonized with 2.5% NHS with antibody or T-CAT molecule targeted to Klebsiella pneumoniae antigen MrkA, or with control isotype antibody or control isotype T-CAT molecule. Levels of C3b deposition were measured by ELISA (left panel) or FACS analysis (right panel). High levels of complement C3b deposition were observed on the surface of bacteria opsonized with the targeted T-CAT molecule. Low levels of C3b deposition -were observed with the targeted antibody or the isotype controls.

[0042] FIGURE 7 is a graphic representation of the results of binding experiments using an antibody or a T-CAT molecule targeted to a Pseudomonas antigen. ELISA plates were coated with Pseudomonas aeruginosa. Serial dilutions of targeted antibody or T-CAT targeted to Pseudomonas aeruginosa antigen Fla-B, or control isotype antibody or control isotype T-CAT molecule, were added to the ELISA plates and incubated for 1 hour. Binding was detected using anti-human antibodies. The antibody and T-CAT molecules targeted to Pseudomonas aeruginosa antigen Fla-B bound to the bacteria to a similar extent, -while the isotype controls did not bind.

[0043] FIGURE 8 is a graphic representation of the results of C3b deposition assays using an antibody or a T-CAT molecule targeted to a Pseudomonas aeruginosa antigen. Bacteria were opsonized with 2.5% NHS with antibody or T-CAT molecule targeted to Pseudomonas aeruginosa antigen Fla-B, or with control isotype antibody or control isotype T-CAT molecule. Levels of C3b deposition were measured by ELISA (left panel) or FACS analysis (right panel). High levels of complement C3b deposition -were observed on the surface of bacteria opsonized with the targeted T-CAT molecule. Low levels of C3b deposition were observed with the targeted antibody or the isotype controls,

[0044] FIGURE 9 is a graphic representation of the results of experiments testing the use of an antibody or T-CAT molecule targeted to Klebsiella pneumoniae in a mouse model of Klebsiella pneumoniae infection. Mice infected intranasally with 5x108cfu of Klebsiella pneumoniae were treated 12 and 36 hours post-infection with antibody or T-CAT molecules targeted to Klebsiella pneumoniae antigen MrkA, or with a control isotype antibody or a control isotype T-CAT molecule. Treatment with the targeted T-CAT molecule significantlyincreased the survival time of mice after infection compared to either the isotype controls or the targeted antibody.

[0045] FIGURE 10 is a graphic representation of the results of experiments testing the use of an antibody or T-CAT molecule targeted to Klebsiella pneumoniae in a mouse model of Klebsiella pneumoniae infection. Mice were treated as described above. The bacterial load in blood samples (left panel) and lung tissue (right panel) collected at 24 and 48 hours postinfection was significantly lower in mice treated with the targeted T-CAT molecule as compared to either the targeted antibody or the isotype controls.

[0046] FIGURE 11 shows lung sections from mice treated with an antibody or T-CAT molecule targeted to Klebsiella pneumoniae in a mouse model of Klebsiella pneumoniae infection. Mice were treated as described above. Lung sections from infected animals treated with targeted T-CAT showed a marked reduction in the severity' of lung pathology, with a significant reduction in leukocyte infiltration into the lung parenchyma compared to the targeted antibody or isotype controls. Lung injury score was evaluated by three independent evaluators, and is shown in the graph at bottom right.

[0047] FIGURE 12 is a graphic representation of the results of experiments testing the use of an antibody or T-CAT molecule targeted to Pseudomonas aeruginosa in a mouse model of Pseudomonas aeruginosa infection. Mice infected intranasally with 2.5x106cfu of Pseudomonas aeruginosa were treated 24, 48, and 72 hours post-infection with antibody or T-CAT molecules targeted to Pseudomonas aeruginosa antigen Fla-B or with a control isotype antibody or control isotype T-CAT molecule. Treatment with the targeted T-CAT molecule significantly increased the survival time after infection compared to either the isotype controls or the targeted antibody.

[0048] FIGURE 13 is a graphic representation of the results of experiments testing the use of an antibody or T-CAT molecule targeted to Pseudomonas aeruginosa in a mouse model of Pseudomonas aeruginosa infection. Mice were treated as described above. The bacterial load in blood samples (left panel) and lung tissue (right panel) collected at 24, 48, and 72 hours post-infection was significantly lower in mice treated with the targeted T-CAT molecule as compared to either the targeted antibody or the isotype controls.

[0049] FIGURE 14 shows the lung pathology of animals from the mouse model of Pseudomonas aeruginosa infection described above. Lung sections from infected mice treated with the targeted antibody, targeted T-CAT molecule, control isotype antibody, or control isotype T-CAT molecule were stained with hematoxylin and eosin. Lung sections from targeted T-CAT-treated mice showed an improvement of lung pathology, with a markedreduction in leukocyte infiltration into the lung parenchyma, as compared to mice treated with targeted antibody or isotype controls. Lung sections from targeted T-CAT-treated mice also showed improvement over time, as shown in the sections from 3, 4, and 5 days post¬ infection. Lung sections from infected mice treated with targeted antibody, control isotype antibody, or control isotype T-CAT molecule taken two days post-infection are shown for comparison, along with a section of normal lung from an uninfected mouse,

[0050] FIGURE 15 shows a lung injury score for the lung sections shown in FIG. 14. Lung injury was evaluated by three independent evaluators.

[0051] FIGURE 16 is a graphic representation of the results of binding experiments using an antibody targeted to a Staphylococcus aureus antigen. ELISA plates were coated with one of twenty isolates of multidrug-resistant Staphylococcus aureus. An ELISA plate was also coated with Enterococcus faecalis, as a negative control. Antibodies targeted to Staphylococcus aureus antigen Protein A were added to the ELISA plates and incubated for 1 hour. Binding was detected using rabbit anti-mouse HRP-conjugated antibodies. The antibody targeted to Staphylococcus aureus antigen Protein A showed binding to all the tested Staphylococcus aureus isolates and no binding to the Escherichia faecalis sample.

[0052] FIGURE 17 is a graphic representation of the results of C4b deposition assays using a T-CAT molecule targeted to a Staphylococcus aureus antigen. Bacteria were opsonized with 2.5% NHS with antibody or T-CAT molecule targeted to Staphylococcus aureus antigen Protein A, or with control isotype T-CAT molecule. Levels of C4b deposition were measured by ELISA. High levels of complement C4b deposition were observed on the surface of bacteria opsonized with the targeted T-CAT molecule. Lower levels of C4b deposition were observed with the targeted antibody or the isotype control.

[0053] FIGURE 18 is a graphic representation of the results of binding experiments using an antibody targeted to a Neisseria meningitidis antigen. ELISA plates were coated with Neisseria meningitidis or Neisseria gonorrhoeae. Serial dilutions of one of two targeted antibodies. Clone 4 and Clone 9 were added to the ELISA plates and incubated for 1 hour. Binding was detected using anti-human antibodies. The antibody Clone 4 targeted to Neisseria meningitidis antigen NHBA bound to both Neisseria meningitidis and Neisseria gonorrhoeae to a similar extent. The antibody Clone 9 bound to Neisseria meningitidis but not to Neisseria gonorrhoeae.

[0054] FIGURE 19 is a graphic representation of the results of C4b deposition assays using a T-CAT molecule targeted to a Neisseria meningitidis antigen. Bacteria were opsonized with 2.5% NHS with antibody Clone 4 or the corresponding T-CAT molecule targeted toNeisseria meningitidis antigen NHBA, or with control isotype antibody or control isotype T-CAT molecule. Levels of C4b deposition were measured by ELISA. High levels of complement C4b deposition were observed on the surface of bacteria opsonized with the targeted T-CAT molecule. Lower levels of C4b deposition were observed with the targeted antibody or the isotype controls.

[0055] FIGURE 20 is a graphic representation of the results of binding experiments using an alternative antibody or a T-CAT molecule targeted to a Klebsiella pneumoniae antigen. ELISA plates were coated with Klebsiella pneumoniae. Serial dilutions of targeted antibody Clone 37 or the corresponding T-CAT targeted to Klebsiella pneumoniae antigen MrkA, or control isotype antibody or control isotype T-CAT molecule, were added to the ELISA plates and incubated for 1 hour. Binding was detected using anti-human antibodies. The Clone 37 antibody and corresponding T-CAT molecules targeted to Klebsiella pneumoniae antigen MrkA bound to the bacteria to a similar extent, while the isotype controls did not bind.

[0056] FIGURE 21 is a graphic representation of the results of C4b deposition assays using a T-CAT molecule targeted to a Klebsiella pneumoniae antigen. Bacteria were opsonized with 2.5% NHS with antibody Clone 37 or the corresponding T-CAT molecule targeted to Klebsiella pneumoniae antigen MrkA, or with control isotype antibody or control isotype T-CAT molecule. Levels of C4b deposition w'ere measured by ELISA. High levels of complement C4b deposition were observed on the surface of bacteria opsonized with the targeted T-CAT molecule. Lower levels of C4b deposition were observed with the targeted antibody or tire isotype controls.

[0057] DETAILED DESCRIPTION

[0058] I. Definitions

[0059] Unless specifically defined herein, all terms used herein have the same meaning as would be understood by those of ordinary skill in the art of the present invention, Tire following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention. Additional definitions are set forth throughout this disclosure.

[0060] In the present descriptions, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated or evident from the context. Any number range recited hereinrelating to any physical feature, such as polymer subunits, size, or thickness, is to be understood to include any integer within the recited range and, when appropriate, fractions thereof, unless otherwise indicated or evident from the context. As used herein, the term “about” is meant to specify that the range or value provided may vary by ±10% of the indicated range or value, unless otherwise indicated.

[0061] It should be understood that the terms “a”, “an”, and “the” as used herein refer to one or more of the referenced components. Tire use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms “include”, “have”, and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0062] “Optional” or “optionally” means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element component, event, or circumstance occurs and instances in which it does not.

[0063] It should be understood that the individual constructs or groups of constructs derived from the various combinations of the structures and subunits described herein are disclosed by the present application to the same extent as if each construct or group of constructs was set forth individually. Thus, selection of particular structures or particular subunits is within the scope of the present disclosure.

[0064] The term "consisting essentially of is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain) or a protein "consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy-terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

[0065] As used herein, the terms “treat”, “treatment”, or “ameliorate” refer to medical management of a disease, disorder, or condition of a subject. In general, an appropriate dose or treatment regimen comprising a targeted complement activating molecule or compositionof the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof.

[0066] A "therapeutically effective amount" or "effective amount" of a targeted complement activating molecule, polynucleotide, vector, host cell, or composition of this disclosure refers to an amount of the composition or molecule sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner, Wheri referring to an individual active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient or a cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously.

[0067] As used herein, "‘a subject” includes all mammals, including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents. A subject may be male or female, and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

[0068] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemicalcompounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0069] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).

[0070] In the broadest sense, the naturally occurring amino acids can be divided into groups based on the chemical characteristic of the side chain of the respective amino acids. By "hydrophobic" amino acid is meant either He, Leu, Met, Phe, Trp, Tyr, Vai, Ala, Cys or Pro. By "hydrophilic" amino acid is meant either Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg or His.

[0071] A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp orW). Additionally or alternati vely, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g,, acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp.

[0072] Additional information can be found in Creighton (1984) Proteins, W. H, Freeman and Company.

[0073] As used herein, "protein" or ‘"peptide” or "polypeptide" refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to aminoacid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated, in certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.

[0074] " Nucleic acid molecule" or “oligonucleotide” or "polynucleotide" or "polynucleic acid" refers to an oligomeric or polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), which includes, for example, mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes, for example, cDNA, genomic DNA, and synthetic DNA. Both RNA and DNA may be single or double stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as tire result of the redundancy or degeneracy of the genetic code, or by splicing.

[0075] Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule.

[0076] " Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, tire sequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX), or Megalign (DNASTAR) software. The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res.

[0077] 25:3389-3402, 1997. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0078] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such a nucleic acid could be part of a vector and / or such a nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. " Isolated" can, in some embodiments, also describe an antibody, antigen¬ binding fragment, polynucleotide, vector, host cell, or composition that is outsi de of a human body.

[0079] The term "gene" means the segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes regions preceding and following the coding region (e.g., 5’ untranslated region (UTR) and 3‘ UTR) as well as intervening sequences (introns) between individual coding segments (exons).

[0080] A "functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one or more base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100% level of activity of the parent polypeptide, or a level of activity greater than that of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding," "similar affinity" or "similaractivity" when the functional variant displays an improvement in performance, or no more than a 50% reduction in performance, in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring enzymatic activity or binding affinity.

[0081] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100% level of activity of the parent polypeptide, or a level of activity greater than that of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of this disclosure has "similar binding" or "similar activity " when the functional portion or fragment displays an improvement in performance, or no more than a 50% reduction in performance, in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10% reduction, or no more than a log difference as compared to the parent or reference with regard to affinity).

[0082] As used herein, the term "engineered," "recombinant," or "non-natural" refers to an organism, microorganism, cell, protein, polypeptide, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory' regions in which the modifications alter expression of a polynucleotide, gene, or operon.

[0083] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules (e.g.,receptors, ligands, etc.) may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra- chromosomal genetic material (e.g., as a plasmid or other self-replicating vector), The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity7, may be from the same species, a different species, or a combination thereof.

[0084] In certain embodiments, a nucleic acid molecule or portion thereof native to a host cell will be considered heterologous to the host cell if it has been altered or mutated, or a nucleic acid molecule native to a host cell may be considered heterologous if it has been altered with a heterologous expression control sequence or has been altered with an endogenous expression control sequence not normally associated with the nucleic acid molecule native to a host cell. In addition, the term "heterologous" can refer to a biological activity that is different, altered, or not endogenous to a host cell. As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding an antibody or antigen¬ binding fragment (or other polypeptide), or any combination thereof.

[0085] As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.

[0086] The term "expression", as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, posttranslational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0087] The term "operably linked" refers to the association of tw o or more nucleic acid molecules on a single nucleic acid fragment so that tire function of one is affected by tireother. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). " Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

[0088] As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., a heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of different encoding nucleic acid molecules or the number of different protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

[0089] Tire term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, when the context clearly indicates, a fusion protein of the present disclosure). A (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al.. Mol. Ther. 8: 108, 2003: Mátés et al., Nat. Genet. 41:753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0090] As used herein, "expression vector" or "vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences typically include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be

[0091] T1a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence. In the present specification, "plasmid," "expression plasmid," "viral vector," “expression vector”, and "vector" are used interchangeably.

[0092] The term "introduced" in the context of inserting a nucleic acid molecule into a cell, means "transfection", "transformation," or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0093] In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to affect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest may also be considered operatively linked.

[0094] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a lentiviral vector or a γ-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double -stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g,, vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.,Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing transgenes are known in the art and have been previous described, for example, in: U. S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno- associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HS V and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0095] Other vectors that can be used with the compositions and methods of this disclosure include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. Hie Development of Human Gene Therapy. New7York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0096] When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allow'ing for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

[0097] Plasmid vectors, including DNA -based plasmid vectors for expression of one or more proteins in vitro or for direct administration to a subject, are also known in the art. Such vectors may comprise a bacterial origin of replication, a viral origin of replication, genes encoding components required for plasmid replication, and / or one or more selection markers, and may also contain additional sequences allowing for bicistronic or multicistronic expression.

[0098] As used herein, the term "host" refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure).

[0099] A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. Hie term also encompasses progenyof the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al.. Molecular Cloning: A Laboratory Manual 2d ed, (Cold Spring Harbor Laboratory, 1989).

[0100] As used herein, the term "‘complement activating” refers to a molecule that is capable of participating in one or more complement pathways in such a manner as to lead to deposition of complement components on a target cell surface and, optionally, to target cell death. The precise sequence of events that results from complement activation depends on the complement pathway activated (i.e., classical, lectin, or alternative) and the role of the specific complement activating molecule within that pathway. As described above, each of the complement pathways entails the sequential activation of a series of serine proteases. Thus, the serine proteases of the complement pathway, such as mannan-binding lectin- associated serine proteases (MASP) MASP-1, MASP-2, and MASP-3, C1r, C1s, C2a, complement factor D (CFD), and complement factor Bb, are examples of complement activating molecules.

[0101] " Antigen", as used herein, refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, activation of complement, antibody dependent cytotoxicity, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary' biological samples that can contain one or more antigens include tissue samples, stool samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen. Antigens can also be present in or on an infectious agent, such as present in a virion, or expressed or presented on the surface of a cell infected by infectious agent.

[0102] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules,such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Where an antigen is or comprises a peptide or protein, the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.

[0103] The term "antibody" refers to an immunoglobulin molecule consisting of one or more polypeptides that specifically binds an antigen through at least one epitope recognition site. For example, the term “antibody” encompasses an intact antibody comprising at least two heavy chains and two light chains connected by disulfide bonds, as well as any antigenbinding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab’2 fragment. The term also encompasses full-length or fragments of antibodies of any class or sub-class, including IgG and sub-classes thereof (such as IgGl, IgG2, IgG3, and IgG4), IgM, IgE, IgA, and IgD.

[0104] The term "antibody" is used herein in the broadest sense, encompassing antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), or from a hybridoma, phage selection, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments). It is not intended that the term “antibody” be limited as regards to the source of the antibody or manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (e.g,, bispecific antibodies); humanized antibodies; fully human antibodies, murine antibodies; chimeric, mouse-human, mouse -primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact molecule or fragment thereof. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab')?., Fv), single-chain (such as ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity. The term encompasses genetically engineered and-or otherwise modified formsof immunoglobulins such as intrabodies, peptibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv, and the like, including antigen-binding fragments thereof The terms “VII” and “VL” refer to the variable binding regions from an antibody heavy chain and an antibody light chain, respectively. A VL may be a kappa class chain or a lambda class chain. The variable binding regions comprise discrete, well-defined sub-regions known as complementarity' determining regions (CDRs) and framework regions (FRs). Tire CDRs are located within a hypervariable region (HVR) of the antibody and refer to sequences of amino acids within antibody variable regions which, in general, together confer the antigen specificity and / or binding affinity of the antibody. Consecutive CDRs (i.e., CDR1 and CDR2, and CDR2 and CDR3) are separated from one another in primary structure by a framework region.

[0105] As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from an antibody of one animal species (e.g., a rodent), while the remainder of the antibody molecule is derived from an antibody of a different animal species (e.g., a human). In some embodiments, a chimeric antibody is comprised of an antigen-binding domain of one antibody operably linked or otherwise fused to heterologous constant regions of a different antibody. For example, a mouse-human chimeric antibody comprises an antigen-binding domain of a mouse antibody fused to a constant region derived from a human antibody. In some embodiments, the heterologous constant region may be from a different Ig class from the parent antibody, including IgA (including subclasses IgA 1 and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3 and IgG4) and IgM.

[0106] As used herein, a “humanized antibody” is a molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and / or sequence of a human immunoglobulin. A humanized antibody differs from a chimeric antibody in that typically only the CDRs from the non-human species are used, grafted onto appropriate framework regions in a human variable domain. Antigen binding sites may be wild-type or may be modified by one or more amino acid substitutions. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, generally including the antigen-binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0107] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and / or light chains, and that specifically binds to the antigen to which the antibody was raised. An antigen-binding fragment may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence from an antibody.

[0108] A " Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Both the Fab and F(ab’)2 are examples of "antigen-binding fragments," Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the con stant domains bear a free thiol group. F(ab')2 antibody fragments are often produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0109] Fab fragments may be joined, e.g., by a peptide linker, to form a single-chain Fab, also referred to herein as "scFab." In these embodiments, an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain. A heavy-chain derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VH + CHI, or " Fd") and a light chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VL + CL) may be linked in any arrangement to form a scFab. For example, a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment -linker - light chain Fab fragment) or (light chain Fab fragment - linker - heavy chain Fab fragment)." Fv" is a small antibody fragment that contains a complete antigen-recognition and antigen- binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.

[0110] " Single-chain Fv" also abbreviated as "sFv" or "scFv", are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The scFv polypeptide may comprise a polypeptide linker disposed between and linking tire VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding, although a linker is not always required. Such a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art. Additionally, or alternatively, Fv can have a disulfide bond formed between and stabilizing the VH and the VL. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). In certain embodiments, the antibody or antigen-binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL orientation or in a VL linker- VH orientation. Any scFv of the present disclosure may be engineered so that the C-terminal end of the VI, domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C)). Alternatively, in some embodiments, a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.

[0111] Peptide linker sequences for use in scFv or in other fusion proteins, such as the targeted complement activating molecules described herein, may be chosen, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides and / or on a target molecule; and / or (3) the lack or relative lack of hydrophobic or charged residues that might react with the polypeptides and / or target molecule. Other considerations regarding linker design (e.g., length) can include the conformation or range of conformations in which the VH and VL can form a functional antigen-binding site. In certain embodiments, peptide linker sequences contain, for example, Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, may also beincluded in a linker sequence. Other amino acid sequences which may be usefully employed as linker include those disclosed in Maratea et al., Gene 40:3946(1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U. S. Pat. No.4,935,233, and U. S. Pat. No.

[0112] 4,751,180. Other illustrative and non-limiting examples of linkers may include, for example, the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO:99) when present in a single iteration or repeated one to five times or more, and may begin or end in a partial iteration; see, e.g,, SEQ ID NO: 100. Any suitable linker may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformational movement betw een tw o regions, domains, motifs, fragments, or modules connected by the linker), and w ill preferably be biologically inert and / or have a low risk of immunogenicity in a human.

[0113] Antibodies may be monospecific (e.g., binding to a single epitope) or multispecific (e.g., binding to multiple epitopes and / or target molecules). A bispecific or multispecific antibody or antigen-binding fragment may, in some embodiments, comprise one, tw7o, or more antigen-binding domains (e.g., a VH and a VL). Two or more binding domains may be present that bind to the same or different epitopes, and a bispecific or multispecific antibody or antigen-binding fragment as provided herein can, in some embodiments, two or more binding domains, that bind to different antigens or pathogens altogether.

[0114] Antibodies and antigen-binding fragments may be constructed in various formats. Exemplary antibody formats disclosed in Spiess et al., Mol. Immunol.

[0115]

[0116] (2015), and in Brinkmann and Kontermann, mAbs 9(2): 182-212 (2017), which formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH Common Light-Chain antibodies, TandAbs, Triple Bodies, TriBi Minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, κλ-bodies, orthogonal Fabs, DVD-Igs (e.g., US Patent No. 8,258,268, which formats are incorporated herein by reference in their entirety), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L, H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one), as well as so-called FIT-Ig (e.g., PCT 5 Publication No. WO 2015 / 103072, which formats are incorporated hereinby reference in their entirety), so-called WuxiBody formats (e.g., PCT Publication No. WO 2019 / 057122, which formats are incorporated herein by reference in their entirety), and so- called In-Elbow -Insert Ig formats (lEI-Ig; e.g., PCT Publication Nos. WO 2019 / 024979 and WO 2019 / 025391, which formats are incorporated herein by reference in their entirety).

[0117] An antibody or antigen-binding fragment may comprise two or more VH domains, two or more VL domains, or both (i.e., two or more VH domains and two or more VL domains). In particular embodiments, an antigen-binding fragment comprises the format (N- terminal to C -terminal direction) VH-linker-VL-linker-VH-linker-VL, wherein the two VH sequences can be the same or different and the two VL sequences can be the same or different. Such linked scFvs can include any combination of VH and VL domains arranged to bind to a given target, and in formats comprising two or more VH and / or two or more VL, one, two, or more different epitopes or antigens may be bound. It will be appreciated that formats incorporating multiple antigen -binding domains may include VH and / or VL sequences in any combination or orientation. For example, the antigen -binding fragment can comprise the format VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker-VH-linker-VH-linker-VL.

[0118] As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single¬ chain variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as the hybridoma method described by Kohler, G., et al., Nature 256:495, 1975, or they may be made by recombinant DNA methods (see, e.g., U. S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al.. Nature 352:624-628, 1991, and Marks, J. D., et al., J. Mol. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.The recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains, or equivalents in other species. Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids) similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[0119] The basic four-chain antibody unit is a heterotetrameric gly coprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody differs from this plan in that it consists of five of the basic heterotetramer units along with an additional polypeptide called the J chain, and therefore contains 10 antigen- binding sites. Secreted IgA antibodies also differ from the basic structure in that they can polymerize to form polyvalent assemblages comprising two to five of the basic four-chain units along with a J chain. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. The pairing of a VH and VL together forms a single antigen-binding site.

[0120] Each H chain has, at the N-terminus, a variable domain (VH) followed by three constant domains (CHI, CH2, CH3), in the case of alpha, gamma, and delta chains, or four CH domains (CHI, CH2, CH3, CH4), in the case of mu and epsilon chains.

[0121] Each L chain has, at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. When an L chain and an H chain are paired, the VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CHI). Tire L chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains (CL).

[0122] Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated alpha (a), delta (5), epsilon (s), gamma (y) and mu ( u). respectively. The y and a classes are further divided into subclasses on the basis of minor di fferences in CH sequence and function, for example, humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds); Appleton and Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

[0123] The term "variable" refers to that fact that certain segments of the V domains differ extensively in sequence among antibodies, The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable domains. Rather, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the n-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions.

[0124] As used herein, “effector functions” refer to those biological activities attributable to the Fc region of an antibody. Examples of antibody effector functions include participation in antibody-dependent cellular cytotoxicity (ADCC), Clq binding and complement¬ dependent cytotoxicity, Fc receptor binding, phagocytosis, down-regulation of cell surface receptors, and B cell activation. Modifications such as amino acid substitutions may be made to an Fc domain in order to modify (e.g., enhance or reduce) one or more functions of an Fc-containing polypeptide. Such functions include, for example, Fc receptor binding, antibody¬ half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding. Amino acid modifications that modify Fc functions include, for example, T250Q / M428L, M252Y / S254T / T256E, H433K / N434F, M428L / N434S, E233P / L234V / L235A / G236A / A327G / A330S / P331S, E333A, S239D / A330L / I332E, P257I / Q311, K326W / E333S, S239D / I332E / G236A, N297Q, K322A, S228P, L235E / E318 A / K320A / K322A, L234A / L235A, and L234A / L235A / P329G mutations. Other Fc modifications and their effect on Fc function are known in the art.

[0125] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding, The hypervariable region containsseveral “complementarity determining regions” (CDRs). The heavy chain comprises three CDR sequences (CDRH1, CDRH2, and CDRH3) and the light chain comprises three CDR sequences (CDRL1, CDRL2, and CDRL3). A variety of systems exist for identifying and numbering the amino acids that make up the CDRs. For example, the hypervariable region generally comprises CDRs at around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and at around about 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy' chain variable domain when numbering in accordance with the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991); and / or at about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and 26-32 (Hl), 52-56 (H2) and 95-102 (H3) in tire heavy chain variable domain when numbered in accordance with the Chothia numbering system, as described in Chothia and Lesk, J Mol. Biol. 796:901-917 (1987); and / or at about residues 27-38 (LI), 56-65 (L2) and 105-117 (L3) in the VL, and 27-38 (Hl), 56-65 (H2), and 105-117 (H3) in the VH when numbered in accordance with the IMGT numbering system as described in Lefranc, J. P., et al., Nucleic Acids Res 27:209-212; Ruiz, M., et al., Nucleic Acids Res 25:219-221 (2000). Equivalent residue positions can be annotated and compared for different molecules using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). Accordingly, identification of CDRs of an exemplary variable domain (VH or VL) sequence as provided herein according to one numbering scheme is not exclusive of an antibody comprising CDRs of the same variable domain as determined using a different numbering scheme.

[0126] As used herein, “specifically binds” refers to an antibody or antigen-binding fragment that binds to an antigen with a particular affinity, while not significantly associating or uniting with any other molecules or components in a sample. Affinity may be defined as an equilibrium association constant (Ka), calculated as the ratio of kon / koff, with units of 1 / M or as an equilibrium dissociation constant Ka), calculated as the ratio of kofrikon with units of M.

[0127] In some contexts, antibody and antigen-binding fragments may be described with reference to affinity and / or to avidity for antigen. Unless otherwise indicated, avidity refers to the total binding strength of an antibody or antigen-binding fragment thereof to antigen, and reflects binding affinity, valency of the antibody or antigen-binding fragment (e.g,, whether the antibody or antigen -binding fragment comprises one, two, three, four, five, six, seven, eight, nine, ten, or more binding sites), and, for example, whether another agent is presentthat can affect the binding (e.g., a non-competitive inhibitor of the antibody or antigen¬ binding fragment).

[0128] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. it is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0129] II. Overview

[0130] Tire present disclosure provides compositions and methods for the targeted activation of the complement pathway. Antibodies, also known as immunoglobulins (Ig), are the key¬ effector molecules of the humoral immune response. These glycoproteins are synthesized by B lymphocytes when exposed to specific antigens. Antigens may be found on pathogens, such as bacteria, viruses, or fungi, or on non-biological particles such as dust or toxins. Once antibodies are secreted, they bind with high specificity to their corresponding antigens, neutralising the biological activity of tire antigen-bearing body. Antibodies also facilitate the activation of the classical pathway of the complement system. This interaction enhances microbial destruction through complement-mediated lysis and / or opsonization, promoting efficient phagocytosis by innate immune cells such as macrophages and neutrophils. Because antibodies direct a robust and effector function of the immune system against the target cells, antibodies they are considered useful in fighting against infectious microbes in clinical settings.

[0131] The use of monoclonal antibody (mAb) therapy is becoming increasingly promising in the field of infectious diseases. Neutralising monoclonal antibodies have been used to protect against the damaging effect of the toxins produced by some pathogens. For example, mAbs can neutralise diphtheria toxin by binding to the toxin's active sites or blocking its ability to bind to host cells. (Kakita et al. Isolation of a human monoclonal antibody- with strong neutralizing activity against diphtheria toxin. Infect Immun (2006) 74:3682.) Recently, the FDA approved bezlotoxumab, a mAb targeting Clostridium difficile toxin TcdB, to prevent the recurrence of C. difficile infection, but bezlotoxumab has not been shown to cure active infection. (Gerding et al. Bezlotoxumab for prevention of recurrent Clostridium difficile infection in patients at increased risk for recurrence. Clin Infect Dis (2018) 67:649.) Similarly, using mAb against Staphylococcus aureus alpha-hemolysin effectively neutralised the toxin and prevented tissue invasion by the bacterium. However, the use of this toxin-specific mAb alone was not enough to clear the infection. (Ruzin et al. Characterisation of anti-alpha toxin antibody levels and colonisation status after administration of an investigational human monoclonal antibody, MEDI4893, against Staphylococcus aureus alpha toxin. Clin Transl Immunol (2018) 7:el009.)

[0132] The present disclosure relates to the mAb platform called ‘targeted complement activation therapy’ (T-CAT) that exploits the full potential of complement to maximize the activity of therapeutic mAbs. This platform is able to target complement activity to any cell expressing an antigen to which antibody can be generated. Thus, the T-CAT platform may be used for a wide variety of pathogenic infections, including bacterial, viral, fungal, and parasitic infections. Targeted complement activating molecules, which comprise fusion proteins having both a targeting domain derived from an antibody and a serine protease effector domain capable of activating one or more complement pathways, deliver targeted complement activation activity to the location of the antigen targeted by the antibody. The cells targeted are determined by the antigen-binding domain selected for use in the fusion protein.

[0133] The T-CAT platform supports the host’s natural immune defense by combining the specificity of host immunoglobulins against microbial surface components with the ability to initiate complement activation directly on a microbial target surface without relying on the tightly controlled and complex pattern recognition-dependent activation pathways that can be undermined by pathogens’ escape mechanisms. For antibody / antigen complexes (i.e., immune complexes) to initiate activation of the classical pathway in response to a pathogen infection or the presence of any non-self antigen, recognition component Clq must bind at least two immune complexes in close sterical proximity in order to undergo a conformational change and subsequently activated its associated serine proteases Clr and Cis. Since pathogens are naturally selected through their ability to avoid surface activation of complement that could lead to their elimination, a selective advantage is given to those bacteria with an antigen distribution that prevents activation of the C l complex, an evasion strategy that explains why antimicrobial antibody therapies so far have failed to meet expectations. T-CAT molecules, however, overcome the limitations of conventional antimicrobial mAbs. Unlike conventional antimicrobial mAbs, T-CAT molecules require only a single binding event to initiate cleavage of complement C3 and achieve an effective complement response, thereby bypassing the temporo-spatial limitations of the classical pathway initiation complex C 1 posed by pathogen evasion mechanisms, such as low antigen density.III, Targeted Antibodies

[0134] Provided herein are antibodies and antigen -binding fragments thereof that bind to antigens present on the surface of a microbial pathogen. In some embodiments, the pathogen is a bacterial pathogen. In some embodiments, the bacterial pathogen is Klebsiella pneumoniae. In some embodiments, the bacterial pathogen is Pseudomonas aeruginosa. In some embodiments, the bacterial pathogen is Neisseria meningitidis. In some embodiments, the bacterial pathogen is Streptococcus pneumoniae. In some embodiments, the bacterial pathogen is Staphylococcus aureus. In some embodiments, the antigen is expressed on the surface of a microbial pathogen or on the surface of a cell infected by a microbial pathogen. For example, the antigen may be K. pneumoniae MrkA, S', pneumoniae pneumococcal surface protein A (PspA), P. aeruginosa Fla-B, N. meningitidis neisserial heparin binding antigen (NHBA), or S. aureus Protein A.

[0135] The antibody may be a naturally occurring type of antibody, of any class or sub-class, or any type of engineered antibody. For example, the antibody or antigen-binding fragment thereof may comprise an antibody Fab fragment, F(ab’)2 fragment, Fab’ fragment, Fv fragment, a single-chain antibody fragment, a single-chain variable fragment (scFv), a single¬ domain antibody (e.g., sdAb, sdFv, or nanobody) or a fragment thereof, or an intrabody, peptibody, chimeric antibody, humanized antibody, multispecific antibody, or a fragment thereof.

[0136] In some embodiments, the antibody or antigen -binding fragment thereof comprises an Fc region, or fragment thereof. In some embodiments, the Fc region comprises one or more mutations that modify (e.g., enhance or reduce) one or more functions of an Fc-containing polypeptide. Such functions include, for example, Fc receptor binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding.

[0137] In some embodiments, the antibody is an anti-MrkA antibody or antigen-binding fragment thereof, or an anti-PspA antibody or antigen-binding fragment thereof, or an anti-Fla-B antibody or antigen-binding fragment thereof, an anti-NHBA antibody or antigen¬ binding fragment thereof, or a Protein A antibody or antigen-binding fragment thereof, in some embodiments, the antibody is the anti-MrkA antibody Clone 3 or an antigen-binding fragment thereof, or the anti-MrkA antibody Clone 37 or an antigen-binding fragment thereof, or the anti-PspA antibody Clone 148 or an antigen-binding fragment thereof, or the anti-Fla-B antibody Clone 14 or antigen-binding fragment thereof, or the anti-NHBAantibody Clone 4 or antigen-binding fragment thereof, or the anti-Protein A antibody Clone 32. In some embodiments, the antibody or antigen-binding fragment thereof comprises an anti-NHBA Clone 4 heavy chain (SEQ ID NO:39) and / or an anti-NHBA Clone 4 light chain (SEQ ID NO:44); an anti-Fla-B Clone 14 heavy chain (SEQ ID NO:48) and / or an anti-Fla-B Clone 14 light chain (SEQ ID NO:53); an anti-MrkA Clone 3 heavy chain (SEQ ID NO:57) and / or an anti-MrkA Clone 3 light chain (SEQ ID NO:62); and anti-MrkA Clone 37 heavy chain (SEQ ID NO:84) and / or an anti-MrkA Clone 37 light chain (SEQ ID NO:89); an anti-PspA Clone 148 heavy chain (SEQ ID NO:66) and / or an anti-PspA Clone 148 light chain (SEQ ID NO:71); or an anti-Protein A Clone 32 heavy chain (SEQ ID NO:75 and / or an anti¬ Protein A Clone 32 light chain (SEQ ID NO: 80).

[0138] In some embodiments, the antibody is an anti-NHBA antibody or antigen-binding fragment thereof comprising an HCDR1 as set forth in SEQ ID NO: 40, an HCDR2 as set forth in SEQ ID NO:41, an HCDR2 as set forth in SEQ ID NO:43, an LCDRl as set forth in SEQ ID NO:45, an LCDR2 as set forth in SEQ ID NO:46, and an LCDR3 as set forth in SEQ ID NO: 47.

[0139] In some embodiments, the antibody is an anti-Fla-B antibody or antigen- binding fragment thereof comprising an HCDR1 as set forth in SEQ ID NO:49, an HCDR2 as set forth in SEQ ID NO:50, an HCDR2 as set forth in SEQ ID NO:51, an LCDRl as set forth in SEQ ID NO:54, an LCDR2 as set forth in SEQ ID NO:55, and an LCDR3 as set forth in SEQ ID NO:56.

[0140] In some embodiments, the antibody is an anti-MrkA antibody or antigen-binding fragment thereof comprising an HCDR1 as set forth in SEQ ID NO:58, an HCDR2 as set forth in SEQ ID NO: 59, an HCDR2 as set forth in SEQ ID NO: 60, an LCDRl as set forth in SEQ ID NO:63, an LCDR2 as set forth in SEQ ID NO:64, and an LCDR3 as set forth in SEQ ID NO:65.

[0141] In some embodiments, the antibody is an anti-MrkA antibody or antigen-binding fragment thereof comprising an HCDR1 as set forth in SEQ ID NO: 85, an HCDR2 as set forth in SEQ ID NO:86, an HCDR2 as set forth in SEQ ID NO:87, an LCDRl as set forth in SEQ ID NO: 90, an LCDR2 as set forth in SEQ ID NO:91, and an LCDR3 as set forth in SEQ ID NO: 92.

[0142] In some embodiments, the antibody is an anti-PspA antibody or antigen-binding fragment thereof comprising an HCDR1 as set forth in SEQ ID NO:67, an HCDR2 as set forth in SEQ ID NO:68, an HCDR2 as set forth in SEQ ID NO:69, an LCDRl as set forth inSEQ ID NO:72, an LCDR2 as set forth in SEQ ID NO:73, and an LCDR3 as set forth in SEQ ID NO: 74.

[0143] In some embodiments, the antibody is an anti-Protein A antibody or antigen -binding fragment thereof comprising an HCDR1 as set forth in SEQ ID NO: 76, an HCDR2 as set forth in SEQ ID NO:77, an HCDR2 as set forth in SEQ ID NO:78, an LCDR1 as set forth in SEQ ID NO:81, an LCDR2 as set forth in SEQ ID NO:82, and an LCDR3 as set forth in SEQ ID NO: 83.

[0144] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more mutations relative to the wild-type sequence of the corresponding antibody domain. For example, the antibody or antigen-binding fragment thereof may comprise mutations that inhibit protein degradation, inhibit glycosylation, enhance or reduce binding affinity or avidity, or increase in vivo half-life of the targeted complement activating molecule. Accordingly, in some embodiments, the antibody or antigen-binding fragment thereof comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91% at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% identity with the wild-type sequence of the corresponding antibody domain.

[0145] IV. Targeted Complement activating Molecules

[0146] Provided herein are targeted complement activating therapy (T-CATTM) molecules comprising a) a target binding domain and b) a complement activating serine protease effector domain. Such molecules have the ability to deliver targeted complement activation activity to a cell surface, thereby leading to complement-mediated lysis of the targeted cell. The complement activation activity may be delivered to individual cells expressing the target antigen, or to tissues within which the target antigen is expressed.

[0147] A. Complement activating serine protease effector domains

[0148] In certain embodiments, the complement activating serine protease effector domain of the targeted complement activating molecules are derived from components of the complement system. In some embodiments, the complement activating serine protease effector domain comprises MASP-1, MASP-2, MASP-3, Clr, Cis, complement factor D (CFD), C2a, or factor Bb. In some embodiments, the complement activating serine protease effector domain comprises a fragment of any of the aforementioned proteases having serine protease activity. For example, the serine protease domain may comprise the CCP1-CCP2-SP domains of MASP-1, MASP-2, MASP-3, Clr, or Cis. Any serine protease that activates any of the classical, lectin, or alternative complement pathways may be used, as may anyfragment of such a serine protease that retains such activity. In some embodiments, the complement activating serine protease effector domain comprises a serine protease effector domain ofMASP-1 (SEQ ID NO: 11), MASP-2 (SEQ ID NO:1), MASP-3 (SEQ ID NO: 10), Clr (SEQ ID NO: 13), Cis (SEQ ID NO:20), C2a (SEQ ID NO:32), Bb (SEQ ID NO:33), mature CFD (SEQ ID NO:34), or pro-CFD (SEQ ID NO:36).

[0149] In some embodiments, the complement activating serine protease effector domain is in an inactive, zymogen form that requires activation in order to form an active serine protease. Such activation may be provided by other molecules comprising the same serine protease effector domain, by molecules comprising a different serine protease effector domain, or by any other chemical or enzymatic means. In some embodiments, the complement activating serine protease effector domain is in a catalytically active form. One example of a complement activating serine protease effector domain in zymogen form is pro- CFD, which is converted to the active form, mature CFD, by removal of a 6 amino acid activation peptide. Many other complement activating serine proteases, including MASP-1, MASP-2, MASP-3, Clr, and Cis, also have both active and zymogen forms.

[0150] In some embodiments, the complement activating serine protease effector domain comprises one or more mutations relative to a wild-type serine protease. Any number of mutations may be present in the complement activating serine protease effector domain, provided that it retains some level of serine protease activity. Accordingly, in some embodiments, the complement activating serine protease effector domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the wild-type sequence of the corresponding serine protease effector domain. Such mutations may confer a beneficial effect on the targeted complement activating molecule, such as increased resistance to protein degradation or increased resistance to inhibition by endogenous serpins, such as Cl inhibitor, or other inhibitors of serine protease activity. In some embodiments, the complement activating serine protease effector domain comprises one or more mutations relative to a wild-type serine protease, such as MASP-2R444K(SEQ ID NO:2), MASP-2K317Q, R444K(SEQ ID NO:5), MASP-2K321Q’R444K(SEQ ID NO:6), MASP-2K342Q, R444K(SEQ ID NO:7), MASP-2K350Q’R444K(SEQ ID NO:8), MASP- 2K356Q= R444K(SEQID NO:9)5 MASP-lR504Q(SEQ ID NO: 12), C 1 rK3742’ (SEQ ID NO: 14), C1rR380Q(SEQ ID NO:15), C1rH484W(SEQ ID NO:16), ClrG485W(SEQ ID NO:17), C1rR486W(SEQ ID NO: 18), ClsK308Q(SEQ ID NO:22), ClsK310Q(SEQ ID NO:23), ClsR314Q(SEQ ID

[0151]

[0152] NO:24), C1sR331Q(SEQ ID NO:25), CIsK346Q(SEQ ID NO:26), ClsK351Q(SEQ ID NO:27),C1sK353Q(SEQ ID NO:28), C1sD456W(SEQ ID NO:29), C1sN457W(SEQ ID NO:30), and C

[0153]

[0154] C1sP458W(SEQ ID NO:31).

[0155] B. Target binding domains

[0156] In certain embodiments, the target binding domain of the T-CAT molecule is derived from an antibody. The antibody may be a naturally occurring type of antibody, of any class or sub-class, or any type of engineered antibody. For example, the target binding domain may be derived from an antibody Fab fragment, F(ab’)2 fragment, Fab’ fragment, Fv fragment, a single -chain antibody fragment, a single -chain variable fragment (scFv), a single - domain antibody (e.g., sdAb, sdFv, or nanobody) or a fragment thereof, or an intrabody, peptibody, chimeric antibody, humanized antibody, multispecific antibody, or a fragment thereof. In some embodiments, the target binding domain of the T-CAT molecule comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the target binding domain comprises an antibody VH and / or VL. In some embodiments, the target binding domain comprises from one to six CDRs of an antibody.

[0157] In some embodiments, the target binding domain comprises an Fc region, or fragment thereof. In some embodiments, the Fc region comprises one or more mutations that modify (e.g., enhance or reduce) one or more functions of an Fc-containing polypeptide. Such functions include, for example, Fc receptor binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding.

[0158] In some embodiments, the target binding domain binds to an antigen present on a microbial pathogen. Tire pathogen may be a bacterial pathogen. In some embodiments, the bacterial pathogen is Klebsiella pneumoniae. In some embodiments, the bacterial pathogen is Pseudomonas aeruginosa. In some embodiments, the bacterial pathogen is Neisseria meningitidis. In some embodiments, the bacterial pathogen is Streptococcus pneumoniae. In some embodiments, the bacterial pathogen is Staphylococcus aureus. In some embodiments, the antigen is expressed on the surface of a microbial pathogen or on the surface of a cell infected by a microbial pathogen. For example, the antigen may be K. pneumoniae MrkA, S. pneumoniae pneumococcal surface protein A (PspA), P. aeruginosa Fla-B, N. meningitidis NHBA, or 5. aureus Protein A.

[0159] In some embodiments, the target binding domain comprises an anti-MrkA antibody or antigen-binding fragment thereof, or an anti-PspA antibody or antigen-binding fragment thereof, or an anti-Fla-B antibody or antigen-binding fragment thereof, or an anti -NHBA antibody or antigen-binding fragment thereof, or a Protein A antibody or antigen-bindingfragment thereof, in some embodiments, the target binding domain comprises the anti-MrkA antibody Clone 3 or antigen- binding fragment thereof, or the anti-MrkA antibody Clone 37 or antigen-binding fragment thereof, or the anti-PspA antibody Clone 148 or an antigen-binding fragment thereof, or the anti-Fla-B antibody Clone 14 or antigen-binding fragment thereof, or the anti-NHBA antibody Clone 4 or antigen-binding fragment thereof, or the anti-Protein A antibody Clone 32 or antigen-binding fragment thereof. In some embodiments, the target binding domain comprises an anti-NHBA Clone 4 heavy chain (SEQ ID NO:39) and / or an anti-NHBA Clone 4 light chain (SEQ ID NO:44); an anti-Fla-B Clone 14 heavy chain (SEQ ID NO:48) and / or an anti-Fla-B Clone 14 light chain (SEQ ID NO:53); an anti-MrkA Clone 3 heavy chain (SEQ ID NO:57) and / or an anti-MrkA Clone 3 light chain (SEQ ID NO:62); an anti-MrkA Clone 37 heavy chain (SEQ ID NO: 84) and / or an anti-MrkA Clone 37 light chain (SEQ ID NO:89); an anti-PspA Clone 148 heavy chain (SEQ ID NO:66) and / or an anti-PspA Clone 148 light chain (SEQ ID NO:71); or an anti-Protein A Clone 32 heavy chain (SEQ ID NO:75) and / or an anti-Protein A Clone 32 light chain (SEQ ID NO:80).

[0160] In some embodiments, the target binding domain comprises one or more mutations relative to the wild-type sequence of the corresponding antibody domain. For example, the target binding domain may comprise mutations that inhibit protein degradation, inhibit glycosylation, enhance or reduce binding affinity or avidity, or increase in vivo half-life of the targeted complement activating molecule. Accordingly, in some embodiments, the target binding domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the wild-type sequence of the corresponding antibody domain.

[0161] C. Fusion proteins and multi-chain molecules

[0162] In certain embodiments, the T-CAT molecule comprises a fusion protein. The fusion protein comprises a complement activating serine protease effector domain fused to a target binding domain. The fusion protein may have any of several configurations: a) the N-terminus of tire complement activating serine protease effector domain fused to the C-tenninus of an antibody heavy chain or fragment thereof, b) the C-terminus of the complement activating serine protease effector domain fused to the N-terminus of an antibody heavy chain or fragment thereof, c) the N-terminus of the complement activating serine protease effector domain fused to the C-terminus of an antibody light chain or fragment thereof, d) the C-terminus of the complement activating serine protease effectordomain fused to the N-terminus of an antibody light chain or fragment thereof, e) the N-terminus of the complement activating serine protease effector domain fused to the C-terminus of a single-chain or single-domain antibody or fragment thereof, or f) the C- terminus of the complement activating serine protease effector domain fused to the N-terminus of a single-chain or single-domain antibody or fragment thereof.

[0163] In some embodiments the target binding domain and the serine protease effector domain within the fusion protein are connected by a linker. Any suitable linker may be used. An example of one such linker is the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO:37), which may be present in a single iteration or repeated one to five times or more, and may begin or end in a partial iteration; see, e.g., SEQ ID NO:38.

[0164] In some embodiments, the fusion protein comprises a target binding domain derived from anti-NHBA Clone 4 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-NHBA Clone 4 and a serine protease effector domain derived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 39 or 44 and a serine protease effector domain comprising any of SEQ ID NOs: 1-36. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO:39 or 44 and a serine protease effector domain comprising SEQ ID NO:20. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO:43,

[0165] In some embodiments, the fusion protein comprises a target binding domain derived from anti-Fla-B Clone 14 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-Fla-B Clone 14 and a serine protease effector domain derived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO:48 or 53 and a serine protease effector domain comprising any of SEQ ID NOs: 1-36. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO:48 or 53 and a serine protease effector domain comprising SEQ ID NO:20. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO:52.

[0166] In some embodiments, the fusion protein comprises a target binding domain derived from anti-MrkA Clone 3 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-MrkA Clone 3 and a serine protease effector domainderived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 57 or 62 and a serine protease effector domain comprising any of SEQ ID NO: 1-36. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO:57 or 62 and a serine protease effector domain comprising SEQ ID NO:20. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO:61,

[0167] In some embodiments, the fusion protein comprises a target binding domain derived from anti-MrkA Clone 37 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-MrkA Clone 37 and a serine protease effector domain derived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 84 or 89 and a serine protease effector domain comprising any of SEQ ID NO: 1-36, In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 84 or 89 and a serine protease effector domain comprising SEQ ID NO:20. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO: 88.

[0168] In some embodiments, the fusion protein comprises a target binding domain derived from anti-PspA Clone 148 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-PspA Clone 148 and a serine protease effector domain derived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 66 or 71 and a serine protease effector domain comprising any of SEQ ID NO: 1-36. In some embodiments, tire fusion protein comprises the sequence set forth as SEQ ID NO:70.

[0169] In some embodiments, the fusion protein comprises a target binding domain derived from anti-Protein A Clone 32 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-Protein A Clone 32 and a serine protease effector domain derived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO:75 or 80 and a serine protease effector domain comprising any of SEQ ID NO: 1 -36. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO:79.

[0170] In certain embodiments, the fusion protein comprises a target binding domain derived from an antibody heavy chain or an antibody light chain. In such cases, the T-CAT moleculemay comprise an additional polypeptide that enhances antigen binding, effector function, stability, etc. of the molecule. In some embodiments, the T-CAT molecule comprises: a) a fusion protein comprising a target binding domain derived from an antibody heavy chain and b) an antibody light chain or fragment thereof. In some embodiments, the T-CAT molecule comprises: a) a fusion protein comprising a target binding domain derived from an antibody light chain and b) and antibody heavy chain or fragment thereof. In some embodiments, the antibody heavy chain and the antibody light chain are derived from the same antibody. In some embodiments, the T-CAT molecule may comprise a) a fusion protein comprising a target binding domain derived from an antibody heavy chain and b) a fusion protein comprising a target binding domain derived from an antibody light chain.

[0171] In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-NHBA Clone 4 heavy chain and b) an anti- NHBA Clone 4 light chain or fragment thereof. In some embodiments, the T-CAT molecule comprises a fusion protein comprising the sequence set forth as SEQ ID NO:43 and an antibody light chain comprising the sequence set forth as SEQ ID NO:44. In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-NHBA Clone 4 light chain and b) an anti-NHBA Clone 4 heavy chain or fragment thereof.

[0172] In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-Fla-B Clone 14 heavy chain and b) an anti-Fla-B Clone 14 light chain or fragment thereof. In some embodiments, the T-CAT molecule comprises a fusion protein comprising the sequence set forth as SEQ ID NO:52 and an antibody light chain comprising the sequence set forth as SEQ ID NO: 53. In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-Fla-B Clone 14 light chain and b) an anti-Fla-B Clone 14 heavy chain or fragment thereof.

[0173] In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-MrkA Clone 3 heavy chain and b) an anti-MrkA Clone 3 light chain or fragment thereof. In some embodiments, the T-CAT molecule comprises a fusion protein comprising the sequence set forth as SEQ ID NO:61 and an antibody light chain comprising the sequence set forth as SEQ ID NO:62. In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-MrkA Clone 3 light chain and b) an anti-MrkA Clone 3 heavy chain or fragment thereof.In some embodiments, the fusion protein comprises a target binding domain derived from anti-MrkA Clone 37 and a serine protease effector domain derived from MASP-2, MASP-3, Clr, Cis, C2a, Bb, or CFD. In some embodiments, the fusion protein comprises a target binding domain derived from anti-MrkA Clone 37 and a serine protease effector domain derived from Cis. In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 84 or 89 and a serine protease effector domain comprising any of SEQ ID NO: 1-36, In some embodiments, the fusion protein comprises a target binding domain comprising SEQ ID NO: 84 or 89 and a serine protease effector domain comprising SEQ ID NO:20. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO:88.

[0174] In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-PspA Clone 148 heavy chain and b) an anti-PspA Clone 148 light chain or fragment thereof. In some embodiments, the T-CAT molecule comprises a fusion protein comprising the sequence set forth as SEQ ID NO:70 and an antibody light chain comprising the sequence set forth as SEQ ID NO:71. In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-PspA Clone 148 light chain and b) an anti-PspA Clone 148 heavy chain or fragment thereof.

[0175] In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-Protein A Clone 32 heavy chain and b) an antiProtein A Clone 32 light chain or fragment thereof. In some embodiments, the T-CAT molecule comprises a fusion protein comprising the sequence set forth as SEQ ID NO:79 and an antibody light chain comprising the sequence set forth as SEQ ID NO: 80. In some embodiments, the T-CAT molecule comprises a) a fusion protein comprising a target binding domain derived from an anti-Protain A Clone 32 light chain and b) an anti-Protein A Clone 32 heavy chain or fragment thereof.

[0176] V, Polynucleotides, Vectors, and Host Cells

[0177] Further provided herein are isolated polynucleotides that encode any of the presently disclosed targeted complement activating molecules or a portion thereof (e.g., fusion protein, antibody heavy chain or fragment thereof, and / or antibody light chain or fragment thereof). In certain embodiments, the polynucleotide is codon -optimized for expression in a host cell. Once a coding sequence is known or identified, codon optimization can be performed using known techniques and tools, such as the GenScript® OptimumGene™ tool or theThermoFisher Scientific® GeneArt GeneOptimizer™. Codon-optimized sequences include sequences that are partially codon optimized, having one or more codons optimized for expression in the host cell, and those that are fully codon-optimized. It will also be appreciated that polynucleotides encoding targeted complement activating molecules and portions thereof may possess different nucleotide sequences while still encoding the same protein due to the degeneracy of the genetic code, splicing, etc.

[0178] In certain embodiments, a polynucleotide encoding a targeted complement activating molecule or portion thereof may be comprised in a polynucleotide that includes other sequences and / or features. For example, a polynucleotide may include one or more sequences useful for control or expression of the encoding proteins, such as promoter sequence(s), polyadenylation sequence(s), sequence(s) encoding signal peptides, etc. The polynucleotide may comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0179] Also provided are vectors comprising or containing a polynucleotide that encodes any of the presently disclosed targeted complement activating molecules or a portion thereof. Any appropriate vector may be used, including viral vectors and plasmid vectors. In certain embodiments, a vector comprises a polynucleotide that encodes both a fusion protein and the corresponding antibody heavy chain or light chain that together make up a targeted complement activating molecule. The sequence encoding the fusion protein and the sequence encoding the antibody heavy chain or light chain may be contained within a single open reading frame, in which case they may optionally be separated by a polynucleotide encoding a protease cleavage site and / or a polynucleotide encoding a self-cleaving peptide.

[0180] Alternatively, the sequence encoding the fusion protein and the sequence encoding the antibody heavy chain or light chain may be contained within separate open reading frames on a single vector. In other embodiments, the sequence encoding the fusion protein and the sequence encoding the antibody heavy chain or light chain are present on two different vectors, such that a first vector encodes the fusion protein and a second vector encodes the antibody heavy chain or light chain.

[0181] In a further aspect, the present disclosure also provides a host cell comprising a polynucleotide or vector disclosed herein. Any appropriate cell into which such a polynucleotide or vector may be introduced may be used. Examples of such cells include eukaryotic cells, including yeast cells, animal cells, insect cells, mammalian cells, and plant cells, and prokary otic cells, including bacterial cells such as E. coli. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is an immortalizedmammalian cell line. Cells appropriate for use in producing and expressing polynucleotides and vectors are known in the art.

[0182] In some embodiments, the cell may be transfected with a polynucleotide or vector disclosed herein. The term “transfection” encompasses any method known to one of skill in the art for introducing nucleic acid molecules into cells. Such methods include, for example, electroporation, lipofection, nanoparticle-based transfection, virus-based transfection, etc. Host cells may be transfected stably or transiently.

[0183] In some embodiments, the host cell expresses the targeted complement activating molecule or portion thereof encoded by the polynucleotide or vector. Such expression may include post-translational modifications such as removal of signal sequence, glycosylation, and other such modifications. In a related aspect, the present disclosure provides methods for producing targeted complement activating molecules or portions thereof, which methods comprise culturing a host cell for a sufficient time under conditions allowing for expression of the molecules and isolating the molecules. Methods useful for isolating and purifying recombinantly produced proteins include, for example, obtaining supernatant from suitable host cells that secrete the proteins into culture medium, concentrating the medium, and purifying the protein by passing the concentrate through a suitable purification matrix or series of matrices. Methods for purification of proteins are well known in the art.

[0184] VI. Pharmaceutical Compositions

[0185] Also provided herein are compositions that comprise a therapeutic agent selected from any one or more of the presently disclosed targeted complement activating molecules, polynucleotides, vectors, or host cells, singly or in any combination, and may also include other selected therapeutic agents. Such compositions may further comprise one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0186] A pharmaceutically acceptable carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity' of the therapeutic agent (and any other therapeutic agents combined therewith). Examples of pharmaceutically acceptable carriers for peptides are described in U. S. Patent No. 5,211,657 to Yamada. The therapeutic agents described herein may be formulated into preparations in solid, semi solid, gel, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections allowing for oral, parenteral, or surgical administration. Local administration of the compositions by coating medical devices and the like is also contemplated.Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solutions, dextrose solution. Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any biocompatible oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable s. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0187] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay, or regulate) the delivery' of the agent(s) or to enhance the delivery?, uptake, stability, or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery systems include, for example, the PEO: PHB: PEO copolymers and copolymer / cyclodextrin complexes disclosed in WO 2004 / 009664 A2 and the PEO and PEO / cyclodextrin complexes disclosed in U. S. Patent Application Publication No.

[0188] 2002 / 0019369 Al. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.

[0189] Compositions of the present invention may be formul ated for delivery’ by any appropriate method including, without limitation, oral, topical, transdermal, sublingual, buccal, subcutaneously, intra-muscularly, intravenously, intra-arterially or as an inhalant.

[0190] The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

[0191] Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject may take the form of one or more dosage units, and a container of a herein described therapeutic agent may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain an effective amount of therapeutic agent or composition of tire presentdisclosure, for treatment of a disease or condition of interest in accordance with teachings herein.

[0192] A composition may be in the form of a solid or liquid. In some embodiments, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid, or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0193] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, or the like. Such a solid composition will typically contain one or more inert fillers or diluents such as sucrose, com starch, or cellulose. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in tire form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0194] The composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, preservative, dye / colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, di spersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included.

[0195] Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following excipients: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred excipient. An injectable pharmaceutical composition is preferably sterile.

[0196] A liquid composition intended for either parenteral or oral administration should contain an amount of a therapeutic agent as described herein such that a suitable dosage will be obtained. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intrastemal, or intra-arterial injection or infusion. Typically, the therapeutic agent is at least 0.01% of the composition. When intended for oral administration, this amount may be varied to be between about 0.1% and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% therapeutic agent.

[0197] The composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.

[0198] Thickening agents may be present in a composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0199] A composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. Tire composition in solid or liquid form may include an agent that binds to the therapeutic agent(s) of the disclosure and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include one or more proteins or a liposome.

[0200] Tire composition may consist essentially of dosage units that can be administered as an aerosol. Tlie term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri -phasic system in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, sub-containers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation, may determine preferred aerosols.

[0201] It will be understood that compositions of the present disclosure also encompass carrier molecules for polynucleotides, as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).

[0202] The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a composition that comprises therapeutic agent as described herein and optionally, one or more of salts, buffers and / or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the composition so as to facilitate dissolution or homogeneous suspension in the aqueous delivery system.

[0203] VII. Methods and Uses

[0204] Further provided herein are methods for use of a T-CAT molecule, polynucleotide, vector, host cell, or composition of the present disclosure in activating one or more complement pathways in a mammalian subject. In some embodiments, the complement classical pathway, complement lectin pathway, or complement alternative pathway are activated. In some embodiments, any two or all three of the complement pathways are activated. In some embodiments, the targeted complement activating molecule, polynucleotide, vector, host cell, or composition of the present disclosure may be used to induce complement-dependent cell death (CDC), complement-dependent cell-mediated cytotoxicity’ (CDCC), or complement-dependent cellular phagocytosis (CDCP) of a target cell. Such methods comprise contacting a target cell with the T-CAT molecule or a composition comprising the T-CAT molecule, wherein said contacting results in complement deposition on the target cell, thereby leading to complement-mediated cell death.

[0205] Also provided herein are methods of treating a microbial infection in a subject comprising administering a therapeutically effective amount of a T-CAT molecule or composition comprising the T-CAT molecule to the subject. In some embodiments, a microbial infection is treated using a T-CAT molecule comprising a targeting domain thatbinds an antigen present of the surface of a microbial pathogen or on the surface of a cell infected with a microbial pathogen. In some embodiments, the infection is a bacterial infection. In some embodiments, the bacterial pathogen is Neisseria meningitidis, Neisseria gonorrhea, Klebsiella pneumoniae. Streptococcus pneumoniae, Pseudomonas aeruginosa, or Staphylococcus aureus.

[0206] Provided herein is the use of a T-CAT molecule, polynucleotide, vector, host cell, or composition of the present disclosure for treatment of a microbial infection. In some embodiments, the targeted complement activating molecule for use in treatment of a microbial infection comprises a targeting domain that binds an antigen present of the surface of a microbial pathogen or on the surface of a cell infected with a microbial pathogen. In some embodiments, the infection is a bacterial infection. In some embodiments, the bacterial pathogen is Neisseria meningitidis, Neisseria gonorrhea, Klebsiella pneumoniae.

[0207] Streptococcus pneumoniae. Pseudomonas aeruginosa, or Staphylococcus aureus.

[0208] Provided herein is a T-CAT molecule, polynucleotide, vector, host cell, or composition of the present disclosure for use in the manufacture of a medicament for treating a microbial infection. In some embodiments, the medicament for treating a microbial infection comprises a T-CAT molecule comprising a targeting domain that binds an antigen present of the surface of a microbial pathogen or on the surface of a cell infected with a microbial pathogen. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the bacterial pathogen is Neisseria meningitidis, Neisseria gonorrhea, Klebsiella pneumoniae. Streptococcus pneumoniae, Pseudomonas aeruginosa, or Staphylococcus aureus.

[0209] Administration of the T-CAT molecules or compositions of the present disclosure may be by any appropriate route, including oral, topical, transdermal, sublingual, buccal, subcutaneously, intra-muscularly, intravenously, intra-arterially or as an inhalant. Tire T- CAT molecules or compositions of the present disclosure are administered in a therapeutically effective amount, which amount will vary depending upon a variety of factors including the specific molecules employed, the metabolic stability and length of action of the molecules, the age, sex, body weight, general health, and diet of the subject, the mode and time of administration, the rate of excretion, any additional therapeutic agents administered to the subject in the same time frame, the severity’ of the particular disorder or disease, and the genetic and epigenetic makeup of the subject. In certain embodiments, the T-CAT molecules or compositions may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, ormore. Successive administration may be carried out at any interval, including about 6, about 12, about 24, about 36, about 48, about 74, about 96, or about 108 hours apart, or more.

[0210] In some embodiments, the T-CAT molecules, polynucleotides, vectors, host cells, or compositions of the present disclosure are used in combination with other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation that contains T-CAT molecules or compositions of the present disclosure together w ith one or more additional therapeutic agents, or the T-CAT molecules or compositions of the present disclosure and the additional therapeutic agents may each be administered as a separate dosage formulation. Where separate dosage formulations are used, the T-CAT molecules or compositions of the present disclosure and the additional therapeutic agents may be administered at essentially the same time, i.e., concurrently, or at separate times, i.e., sequentially in any order. In some embodiments, a combination therapy may comprise administration of two or more different T-CAT molecules of the present disclosure, or two or more compositions each comprising a different T-CAT molecule of the present disclosure.

[0211] VIII. Sequences

[0212] Tire sequences referred to within the present specification are summarized in Table 1

[0213] TABLE 1:

[0214] SEQ Description Sequence

[0215] ID NO.

[0216] 1 MASP-2 CCP1 / 2SP QPCPYPMAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGH LPLKS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTY KAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVL I LGGT T AAGALL Y DNWVLT AAHAVY E QKHDAS ALD I RMGT L KRL S P H Y T Q AW S E AV F I H E GY T H DAG FDND I AL I KLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPJEVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T K V I NY I P W I E N 11 S D F

[0217]

[0218] MASP-2R444KQPCPYPMAPPNGHVSPVQAKY ILKDS FS I FCETGYELLQGH CCP1 / 2SP LPLKS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGKIYGGQKAKPGDFPWQVL ILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTL KRL, S P H Y TQ AW S E A V F I H E GY 4’ H DAG FDND I AL, I KLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T KV I N Y I P W I E N 11 S D F

[0219] MASP-2R444QQPCPYPMAPPNGHVSPVQAKY ILKDS FS I FCETGYELLQGH CCP1 / 2SP LPLKS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGQIYGGQKAKPGDFPWQVL I LGGT T AAGALL Y DNWVLT AAHAVY E QKHDAS ALD I RMGT L KRL S P H Y T Q AW S E AV F I H E G Y T H DAG FDND I AL I KLNNKV V I NS N I T P I CL PRKE AE S FMRT DD I GT AS GWGLT QRG FLARN LMY VD I P I VDHQKCTAAYE KP PY PRG S VTANMLCAGLE SGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T KV I N Y I P W I E N 11 S D F

[0220] MASP-2S633AQ PC PY PMAP PNGHVS PVQAKY ILKDS FS I FCETGYELLQGH CCP1 / 2SP LPLKS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVL ILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTL KRL S P H Y T Q AW S E AV F I H E G Y T H DAG FDND I AL I KLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY GVYTKVINY I PW I ENI I SD F MASP-2K317Q,R444KQPC PYPMAPPNGHVSPVQAQY ILKDS FS I FCETGYELLQGH CCP1 / 2SP LPLKS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGKIYGGQKAKPGDFPWQVL

[0221]

[0222] I LGGT T AAGALL Y DNWVLT AAHAVY E QKHDAS ALD I RMGT L KRL S P H Y T Q AW S E AV F I H E GY T H DAG FDND I AL I KLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T K V I NY I P W 1 E N 1 I S D F

[0223] MASP-2K321Q,R444KQPCPYPMAPPNGHVSPVQAKY ILQDS FS I FCETGYELLQGH CCP1 / 2SP LPLKS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGKIYGGQKAKPGDFPWQVL I LG GT T AAGALL Y DNWVLT AAHAVY E QKHDAS ALD I RMGT L KRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYY’DIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T KV I N Y I P W I E N 11 S D F

[0224] MASP-2K342Q,R444KQPCPYPMAPPNGHVSPVQAKY ILKDS FS I FCETGYELLQGH CCP1 / 2SP LPLQS FTAVCQKDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGKIYGGQKAKPGDFPWQVL I LGGT T AAGALL Y DNWVLT AAHAVY E QKHDAS ALD I RMGT L KRL S P H Y T Q AW S E AV F I H E G Y T H DAG FDND I AL I KLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T KV I N Y I P W I E N 11 S D F

[0225] MASP-2 K350Q. R444K QPCPYPMAPPNGHVSPVQAKY ILKDS FS I FCETGYELLQGH CCP1 / 2SP LPLKS FTAVCQQDGSWDRPMPACS IVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGKIYGGQKAKPGDFPWQVL ILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKVV INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG

[0226]

[0227] KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY G VY T K V I N Y I P W I E N 11 S D F

[0228] MASP-2R356Q,R444KQPCPYPMAPPNGHVSPVQAKY ILKDSFSIFCETGY ELLQGH CCP1 / 2SP LPLKSFTAVCQKDGSWDRPMPACSIVDCGPPDDLPSGRVEY ITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKGEKSLPVCEPVCGLSARTTGGKIYGGQKAKPGDFPWQVL I LGGT T AAGALL Y DNWVLT AAHAVY E QKHDAS ALD I RMGT L KRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKV V INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARN LMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGG KDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY GVYTKVINYIPWIENIISDF MASP-3 CCP1 / 2SP NECPELQPPVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDN VEMDTFQIECLKDGTWSNKIPTCKIVDCRAPGELEHGLITF STRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTCSAQGVWM NKVLGRSLPTCLPECGQPSRSLPSLVKRIIGGRNAEPGLFP WQAL I VVE DT S RVPNDKW EG S GALL SAS W I LT AAR VL RSQ R RDTTV1PVSKEHVTVYLGLHDVRDKSGAVNSSAARVVLHPD FNIQNYNHDIALVQLQEPVPLGPHVMPVCLPRLEPEGPAPH MLGLVAGWGISNPNVTVDE I I SSGTRTLSDVLQYVKLPVVP RAE CRT S Y E S RS GN Y S VT ENM FCAGY Y E GGKDT CLGD SGGA FVIFDDLSQRWWQGLVSWGGPEECGSKQVYGVYTKVSNYV DWVWEQMGLPQSVVEPQVER MASP-1 CCP1 / 2SP NECPELQPPVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDN VEMDTFQIECLKDGTWSNKIPTCKIVDCRAPGELEHGLITF STRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTCSAQGVWM NKVLGRSLPTCLPVCGLPKFSRKLMARI FNGRPAQKGTTPW IAMLSHLNGQPFCGGSLLGSSWIVTAAYRCLHQSLDPEDPTL RDSDLLSPSDFKIILGKHWRLRSDENEQHLGVKHTTLHPQY DPNT FENDVALVELLES PVLNAFVMP ICLPEGPQQEGAMVI VSGWGKQFLQRFPETLME1EIPIVDHSTCQKAYAPLKKKVT RDM I GAG E KE G G RD AC AG D S GG PM VT LN RE RG Q W Y L VGT V S WGDDCGKKDRYGVY SY I HHNKDWIQRVTGVRN

[0229]

[0230] MASP-1R504QNECPELQPPVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDN CCP1 / 2SP VEMDTFQIECLKDGTWSNKIPTCKIVDCRAPGELEHGLITF STRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTCSAQGVWM N KVLGRSL PTCL PVCGL PK FS RKLMARI FNGRPAQKGTT PW IAMLSHLNGQPFCGGSLLGSSWIVTAAYCLHQSLDPEDPTL QDSDLLSPSDFKIILGKHWRLRSDENEQHLGVKHTTLHPQY DPNT FENDVAL V ELLES PVLNAFVMP ICLPEGPQQEGAMVI VSGWGKQ FLQRFPETLME I E I PI VDHSTCQKAYAPLKKKVT RDMICAGEKEGGRDACAGDSGGPMVTLNRERGQWYLVGTVS WGDDCGKKDRYGVY SY I HHNKDWIQRVTGVRN

[0231] C1r CCP1 / 2SP IKCPQPKTLDEFTIIQNLQPQYQFRDYFIATCKQGYQLIEG NQVLHSFTAVCQDDGTWHRANPRCKIKDCGQPRNLPNGDFR Y T T TMG VNT Y KAR I Q Y Y C H E P Y Y KMQ T RAG S RE S E QG VY T C TAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRI IGGQKA KMGNFPWQVFTNIHGRGGGALLGDRWILTAAHTLYPKEHEA Q SNAS LD VFLGHTN VE E LMKLGNH P I RRVS VH P DY RQ DE S Y N FEGD I ALL ELE NS VTLGPNL LP I CL PDNDT FY DLGLMGY V S G F G VME E K I AH DL R F V RL P V AN P Q AC E N WL RG KN RM D V F S QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WG I GC S RGY G FY T KVLN Y VDW I KKEME E E D

[0232] ClrK374QCCP1 / 2SP I KC PQ PKTLDE FT I IQNLQ PQ YQ FRDY F I ATCKQGYQL I EG NQVLHSFTAVCQDDGTWHRAMPRCKIQDCGQPRNLPNGDFR YTTTMGVNTYKARIQYYCHEPYYKM QTRAGSRESEQGVYTC TAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRI IGGQKA KMGN FPWQVFTNIHGRGGGALLGDRWILTAAHTLY PKEHEA Q SNAS LD VFLGHTN VE E LMKLGNH P I RRVS VH P DY RQ DE S Y NFEGDIALLELENSVTLGPNLLPICLPDNDTFYDLGLMGYV S G F G VME E K I AH DL R F V RL P V AN P Q AC E N WL RG KN RM D V F S QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WGIGCSRGYGFYTKVLNYVDWIKKEMEEED

[0233] C1rR380QCCP1 / 2SP I KCPQPKTLDEFTI IQNLQPQYQFRDYFIATCKQGYQLIEG NQVLHSFTAVCQDDGTWHRAMPRCKIKDCGQPQNLPNGDFR Y T T TMG VNT Y KAR I Q Y Y C H E P Y Y KMQ T RAG S RE S E QG VY T C TAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRI IGGQKA

[0234]

[0235] KMGNFPWQVFTNIHGRGGGALLGDRWILTAAHTLYPKEHEA Q SNAS LD VFLGHTN VE E LMKLGNH P I RRVS VH P DY RQ DE S Y NFEGDIALLELENSVTLGPNLLPICLPDNDT FYDLGLMGYV SGFGVMEEKIAHDLRFVRLPVANPQACENWLRGKNRMDVFS QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WG I GC S RGY G FY T K VLN Y VDW I KKEME E E D

[0236] C1rH484WCCP1 / 2SP IKCPQPKTLDEFTIIQNLQPQYQFRDYFIATCKQGYQLIEG NQVLHSFTAVCQDDGTWHRAMPRCKIKDCGQPRNLPNGDFR YTTTMGVNTYKARIQYYCHEPYYKM QTRAGSRESEQGVYTC TAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRIIGGQKA KMGNFPWQVFTNIWGRGGGALLGDRWILTAAHTLYPKEHEA QSNASLDVFLGHTNVEELMKLGNHPIRRVSVHPDYRQDESY NFEGDIALLELENSVTLGPNLLPICLPDNDTFYDLGLMGYV S G F G VME E K I AH DL R F V RL P V AN P Q AC E N WL RG KN RM D V F S QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WG I GC S RGY G FY T KVLN Y VDW I KKEME E E D

[0237] C1rG485WCCP1 / 2SP I KC PQ PKTLDE FT I IQNLQ PQ YQ FRDY F I ATCKQGYQL I EG NQVLHSFTAVCQDDGTWHRAMPRCKI KDCGQPRNLPNGDFR Y T T TMG VNT Y KAR I Q Y Y C H E P Y Y KMQ T RAG S RE S E QG VY T C TAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRI IGGQKA KMGNFPWQVFTNIHWRGGGALLGDRWILTAAHTLYPKEHEA Q SNAS LD VFLGHTN VE E LMKLGNH P I RRVS VH P DY RQ DE S Y NFEGDIALLELENSVTLGPNLLPICLPDNDT FYDLGLMGYV SGFGVMEEKIAHDLRFVRLPVANPQACENWLRGKNRMDVFS QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WG I GC S RGY G FY T KVLN Y VDW I KKEME E E D

[0238] C1rR486WCCP1 / 2SP IKCPQPKTLDEFTIIQNLQPQYQFRDYFIATCKQGYQLIEG NQVLHSFTAVCQDDGTWHRAMPRCKI KDCGQPRNLPNGDFR Y T T TMG VNT Y KAR I Q Y Y C H E P Y Y KMQ T RAG S RE S E QG VY T C TAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRI IGGQKA KMGNFPWQVFTN I HGWGGGALLGDRW I LTAAHTLYPKEHEA Q SNAS LD VFLG HTN VE E LMKLGNH P I RRVS VH P DY RQ DE S Y NFEGDIALLELENSVTLGPNLLPICLPDNDT FYDLGLMGYV S G F G VME E K I AH DL R F V RL P VAN P Q AC E N WL RG KN RM D V F S

[0239]

[0240] QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WG I GC S RGY G FY T KVLN Y VDW I KKEME E E D

[0241] C1rS654ACCP1 / 2SP IKCPQPKTLDE FTI IQNLQPQYQFRDYFIATCKQGYQLIEG NQVLHSFTAVCQDDGTWHRAMPRCKIKDCGQPRNLPNGDFR YTTTMGVNTYKARIQYYCHEPYYKM QTRAGSRESEQGVYTC TAQGIWKNEQKGEKIPRCLP VCGKPVNPVEQRQRI IGGQKA KMGNFPWQVFTNIHGRGGGALLGDRWILTAAHTLYPKEHEA Q SNAS LD VFLGHTN VE E LMKLGNH P I RR VS VH P DY RQ DE S Y NFEGDIALLELENSVTLGPNLLPICLPDNDTFYDLGLMGYV S G FGVM E E K I AH DL R FVRL P VAN P Q AC E NWL R G KN RM D V F S QNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVS WGIGCSRGYGFYTKVLNYVDWIKKEMEEED

[0242] C1s CCP1 / 2SP MPC PKEDT PNS VWE PAKAKY VFRDVVQ I TCLDG FE VVEGRV GAT S FYSTCQSNGKWSNSKLKCQPVDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFI HPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL P VAPL RKC KE VKVE KPT AL)AE AY V FT PNMI CA GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0243] C1sS632ACCP1 / 2SP MPCPKEDTPNSVWEPAKAKYVFRDVVQITCLDGFEVVEGRV GATSFYSTCQSNGKWSNSKLKCQPVDCGIPESIENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDAGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0244] C1sK308QCCP1 / 2SP MPCPKEDTPNSVWEPAQAKY VFRDVVQITCLDGFEVVEGRV GAT S FYSTCQSNGKWSNSKLKCQPVDCGI PE S I ENGKVEDP

[0245]

[0246] ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGAL INE YWVLT AAHVVEGNRE PTMY VG ST S VQT S R LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD PVKMGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDR AVRLKAARLPVAPLRKCKEVKVEKPTADAEAYVFTPNMICA GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0247] C1sK310QCCP1 / 2SP MPC PKEDT PNS VWE PAKAQ Y VFRDVVQ I TCLDG FE VVEGRV GAT S FYSTCQSNGKWSNSKLKCQ P VDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGAL INE YWVLT AAHVVEGNRE PTMY VGST S VQT S R LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0248] C1sR314QCCP1 / 2SP MPCPKEDTPNSVWEPAKAKYVFQDVVQITCLDGFEVVEGRV GAT S FYSTCQSNGKWSNSKLKCQPVDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD PVKMGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0249] C1sR331QCCP1 / 2SP MPCPKEDTPNSVWEPAKAKYVFRDVVQITCLDGFEVVEGQV GAT S FYSTCQSNGKWSNSKLKCQPVDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL G PE L P KC VP VCG VP RE P FE E KQRI I GG S DAD I KN F PWQ VF F DNPWAGGAL INE YWVLT AA. HVVEGNRE PTMY VGST S VQT S R LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD

[0250]

[0251] P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARLPVAPLRKCKEVKVEKPTADAEAYVFTPNMICA GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG Y GL Y T R V KN Y V DW I MKTM Q E N S T P R E D

[0252] C1sK346QCCP1 / 2SP MPC PKEDT PNSVWEPAKAKYVFRDVVQITCLDGFEVVEGRV GAT S FYSTCQSNGQWSNSKLKCQPVDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL PVAPLRKCKEVKVEKPTADAEAYV FTPNMICA GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0253] C1sK351QCCP1 / 2SP MPC PKEDT PNS VWE PAKAKY VFRDVVQ I TCLDG FE VVEGRV GATSFYSTCQSNGKWSNSQLKCQPVDCGIPESIENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGAL INE YWVLT AAHVVEGNRE PTMY VG ST S VQT S R LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED CIS K353Q CCP1 / 2SP MPC PKEDT PNS VWE PAKAKY VFRDVVQ I TCLDG FE VVEGRV GATSFYSTCQSNGKWSNSKLQCQPVDCGIPESIENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DNPWAGGAL INE YWVLT AAHVVEGNRE PTMY VGST S VQT S R LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD PVKMGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0254]

[0255] C1sD456WCCP1 / 2SP MPC PKEDT PNS VWE PAKAKY VFRDVVQ I TCLDG FE VVEGRV GAT S FYSTCQSNGKWSNSKLKCQPVDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRIIGGSDADIKNFPWQVFF WNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFI HPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI CA GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0256] C1sN457WCCP1 / 2SP MPCPKEDTPNSVWEPAKAKYVFRDVVQITCLDGFEVVEGRV GATSFYSTCQSNGKWSNSKLKCQPVDCGIPESIENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL GPELPKCVPVCGVPREPFEEKQRI IGGSDADIKNFPWQVFF DWPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD PVKMGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0257] C1sP458WCCP1 / 2SP MPCPKEDTPNSVWEPAKAKYVFRDVVQITCLDGFEVVEGRV GAT S FYSTCQSNGKWSNSKLKCQPVDCGI PE S I ENGKVEDP ESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVL G PE L P KC VP VCG VP RE P FE E KQRI I GG S DAD I KN F PWQ VF F DNWWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSR LAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I S GWGRT E KRDR AVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V FT PNMI C A GGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED

[0258] C2a VWFA-SP KIQIQRSGHLNLYLLLDCSQSVSENDFLIFKESASLMVDRI FS FE INVSVAI IT EASE PKVLMSVLNDNSRDMTEVI S SLEN ANY KD H E NGT GT NT Y AALN S V Y LMMNNQMRL LGME TMAWQ E IRHAIILLTDGKSNMGGSPKTAVDHIREILNINQKRNDYLD

[0259]

[0260] IYAIGVGKLDVDWR. ELNELGSKKDGERHAFILQDTKALHQV FE HML DVS KLT DT I CGVGNMS ANAS DQE RT P WH VT I KPKS Q ETCRGALISDQWVLTAAHCFRDGNDHSLWRVNVGDPKSQWG KEFLIEKAVISPGFDVFAKKNQGILE FYGDDI ALLKLAQKV KMSTHARPICLPCTMEANLALRRPQGSTCRDHENELLNKQS VPAHFVALNGSKLNINLKMGVEWTSCAEWSQEKTMFPNLT DVREVVTDQFLCSGTQEDESPCKGESGGAVFLERRFRFFQV GLVSWGLYNPCLGSADKNSRKRAPRSKVPPPRDFHINLFRM QPWLRQHLGDVLNFLPL

[0261] Bb VWFA-SP KIVLDPSGSMNI YLVLDGSDSIGASNFTGAKKCL'YNLIEKV ASYGVKPRYGLVTYATYPILIWVKVSEADSSNADWVTKQLNE INYEDHKLKSGTNTKKALQAVYSMMSWPDDVPPEGWNRTRH VIILMTDGLHNMGGDPITVIDEIRDLLYIGKDRKNPREDYL DVYVFGVGPLVNQVNINALASKKDNEQHVFKVKDMENLEDV FYQMI DE SQ SLSLCGMVWE HRKGT DY HKQPWQAKI S VI RP S KGHESCMGAVVSEYFVLTAA. HCFTVDDKEHSIKVSVGGEKR DLEIEVVLFHPNYNINGKKEAGIPEFYDYDVALIKLKNKLK YGQTIRPICLPCTEGTTRALRLPPTTTCQQQKEELLPAQDI KALFVSEEEKKLTRKEVYIKNGDKKGSCERDAQYAPGYDKV KD I S E VVT P R FL CT GG V S P Y AD PNT C RG D S GG P L I VH KRS R FIQVGVISWGVVDVCKNQKRQKQVPAHAR. DFHINLFQVLPW LKEKLQDEDLGFL

[0262] MatCFD ILGGREAEAHARPYITASVQLNGAHLCGGVLVAEQWVLSAAH C L E DAADGKVQ VLL GAH S L S Q P E P S KRL Y D VL RAV P H P D S Q PDTIDHDLLLLQLSEKATLGPAVRPLPWQRVDRDVAPGTLC DVAGWGIVNHAGRRPDSLQHVLLPVLDRATCNRRTHHDGAI TERLMCAESNRRDSCKGDSGGPLVCGGVLEGVVTSGSRVCG NRKKPGI YT RVAS Y AAW I DS VLA

[0263] MatCFDS20SAILGGREAEAHARPYMASVQLNGAHLCGGVLVAEQWVLSAAH C L E DAADGKVQ VLL GAH S L S Q P E P S KRL Y D VL RAV P H P D S Q PDTIDHDLLLLQLSEKATLGPAVRPLPWQRVDRDVAPGTLC DVAGWGIVNHAGRRPDSLQHVLLPVLDRATCNRRTHHDGAI TERLMCAESNRRDSCKGDAGGPLVCGGVLEGVVTSGSRVCG NRKKPGI YT RVAS Y AAW I DS VLA

[0264]

[0265] ProCFD APPRGRILGGREAEAHARPYMASVQLNGAHLCGGVLVAEQW VLSAAHCLEDAADGKVQVLLGAHSLSQPEPSKRLYDVLRAV P H P DS Q P DT I DH DLLLLQL S E KAT LG PAVRPL P W Q RVDRD V APGTLCDVAGWGIVNHAGRRPDSLQHVLLPVLDRATCNRRT HHDGAITERLMCAESNRRDSCKGDSGGPLVCGGVLEGVVTS G S R VCGN RKKPG 1 Y 7’ RV AS Y AAW I DS VL A

[0266] Linker GGGGS

[0267] Linker GGGGSGGGGSGGGG

[0268] aN4_HC EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQNH GKSLEWIGDINPKNGDTFYNQKFKGRATLTVDKSSSTAYMQ FNSLTSEDSAVYYCTRSNW FDYWGQGTTLTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHT FPAVLQ S SGLY SLS SWT VPS S SLGTQT Y I CNVNHKP S NT KVD KKVE P KS C D KT H T 0 P P C P AP E LL GG P S V FL F P P KP K DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPALPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0269] aN4 HCDR1 DYYMK

[0270] aN4 HCDR2 DINPKNGDT FYNQKFKG

[0271] aN4_HCDR3 SNWFDY

[0272] aN4-Cls_HC EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQNH GKSLEWIGDINPKNGDTFYNQKFKGRATLTVDKSSSTAYMQ FNSLTSEDSAVYYCTRSNWFDYWGQGTTLTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHT FPA. VLQ S SGLY SLS SWT VPS S SLGTQT Y I CNVNHKP S NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT K P R E E Q Y N S T Y R V V S VLTVLHQDWLNG K E Y KC K V S N KAL PA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGG

[0273]

[0274] GGSGGGGMPCPKEDTPNSVWEPAKAKYVFRDVVQITCLDGF EVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCGIPESIE NGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNG SWVNEVLGPELPKCVPVCGVPREPFEEKQR1 IGGSDADIKN F PWQ V F FDN PWAGGAL I NE Y W VLT AAH VVEGNRE PTMY VG S TSVQTSRLAKSKMLTPEHVFI HPGWKLLEVPEGRTNFDND1 AL VRL KD P VKMG PT VS P I CL PGT S S D YNLMDGDLGL I SGWG RT E KRDRAVRLKAARL P VAPL RKC KE VKVE KPT ADAE AY V F TPNMICEAGGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLV SWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED

[0275] aN4 LC DIVMTQAAPSVPVTPGESVSISSCRSSKSLLHSNGNTYLYFW LQRPGQSPQLLIYRMSN LASGVPDRFSSSGSGAAFTLRISR VEAEDVGVY YCMQHLEY PVT FGAGTKLELKRTVAAPS VFI F P P S DE QL KS GT AS VVCLLNN F Y PRE AKVQWKVDNALQ SGN S QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG L S S PVTKS FNRGEC

[0276] aN4 LCDR1 RSSKSLLHSNGNTYLY

[0277] aN4_LCDR2 RMSNLAS

[0278] aN4_LCDR3 MQHLEYPVT

[0279] aPal4_HC D VQ LQ E S G P GT, V KP S Q S T, S L, I C S VT G Y S I ’I' S G Y Y WNW I RQ F PGNKLEWMG Y I S YDGRDNYNPSLKNRI S VTRDT SKNQ FFLK LNSVTTEDTATYYCSRD1RSYAMDQWGQGTSVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL T SGVHT FPAVLQ S SGLY SL S SWT VP S S SLGTQT Y ICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKAISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aPa!4 HCDR1 SGYYWN

[0280] aPa!4 HCDR2 Y I S YDGRDNYNPSLKN

[0281] aPa!4_HCDR3 DIRSYAMDQ

[0282]

[0283] aPa14-C1s_HC DVQLQESGPGLVKPSQSLSLICSVTGYSITSGYYWNWIRQF PGNKLEWMGYISYDGRDNYNPSLKNRISVTRDTSKNQFFLK LNSVTTEDTATYYCSRDIRSYAMDQWGQGTSVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAA’EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGG SGGGGSGGGGMPCPKEDTPNSVWEPAKAKYVFRDVVQITCL DGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCGIPE SIENGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEYHCA GNGSWVNEVLGPELPKCVPVCGVPREPFEEKQRIIGGSDAD IKNFPWQVFFDNPWAGGALINEYWVLTAAHVVEGNREPTMY VGSTSVQTSRLAKSKMLTPEHVFIHPGWKLLEVPEGRTNFD NDIALVRLKDPVKMGPTVSPICLPGTSSDYNLMDGDLGLIS GWGRTEKRDRAVRLKAARLPVAPLRKCKEVKVEKPTADAEAY YVFTPNMICAGGEKGMDSCKGDSGGAFAVQDPNDKTKFYAA GLVSWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED

[0284] aPa!4JLC DIQMTQSPASLYASVGETVTITCRASGDIHSYLAWYQQKQG RSPQLLVYNAETLADGVPSRFSGTGSVTQYSLR1SSLQPED FGSYYCQHFWSTPYTFGGGTKLEMKRTVAAPSVFI FPPSDE QLKSGTASVVCLLNNFYPREAEKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSI-GADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

[0285] aPa14_LCDR1 RASGDIHSYLA

[0286] aPa14_LCDR2 NAETLAD

[0287] aPa14_LCDR3 QHFWSTPYT

[0288]

[0289] aKp3_HC QVQLQESGPGLVKPSETLSLTCTVSGGSMNSNSNTYYWGWI RQPPGKGLEWIGTIHSSGRTYYNPSLKSRVTISVDMSKNQF SLNLT SATAADTAVYYCARDLSGASLAPRRP FNYY YYNMDV WGRGTLVT VS SAST KGP SV FPLAP S S KST SGGT AALGCL VK DY FPE PVT VSWNSGALT SGVHT FPAVLQ S SGLY SL S S VVT V PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ D WLNG KE Y KC KV SN KAL PAP I E KT I S KAKGQ P RE P Q VY T L P P S RDE LT KNQ VS LT CL VKG FY P S D I AVE WE SNGQ P ENN Y KT T P P VL D S DG S F F L Y S KL T V D K S R W Q Q GN V F S C S VN H E AL H N HYTQKSLSLSPGK

[0290] aKp3 HCDR1 SNSNTYYWG

[0291] aKp3 HCDR2 T I H S S G R T Y Y N P S L K S

[0292] aKp3 HCDR3 DL S GASL AP RRP FN Y Y Y YNMD V

[0293] aKp3-C1s_HC QVQLQESGPGLVKPSETLSLTCTVSGGSMNSNSNTYYWGWI RQPPGKGLEWIGTIHSSGRTYYNPSLKSRVTISVDMSKNQF SLNLT SATAADTAVYYCARDLSGASLAPRRP FNYYYYNMDV WGRGTLVT VS SAST KGP SVFPLAP S S KST SGGT AALGCLVK DY FPE PVT VSWNSGALT SGVHT FPAVLQ S SGLY SL S S VVT V PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ D WLNG KE Y KC KV SN KAL P AP I E KT I S KAKGQ P RE P Q V Y T L P P S RDE LT KNQ VS LT CL VKG FY PSD I AV E WE SNGQ PENNY KT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGGGSGGGGSGGGGMPCPKEDTPNSVWEP AKAKYVFRDVVQITCLDGFEVVEGRVGATSFYSTCQSNGKW SNSKLKCQPVDCGI PESIENGKVEDPESTLFGSVIRYTCEE PYYYMENGGGGEYHCAGNGSWVNEVLGPELPKCVPVCGVPR EPFEEKQRIIGGS DAD I KN F P WQ V F F DN P WAGG AL I N E Y W V LTAAHVVEGNREPTMYVGSTSVQTSRLAKSKMLTPEHVFIH PGWKLLEVPEGRTNFDNDIALVRLKDPVKMGPTVSPICLPG T S S DYNLMDGDLGL I SGWGRT EKRDRAVRLKAARL PVAPLR

[0294]

[0295] KCKEVKVEKPTADAEAYVFTPNMICAGGEKGMDSCKGDSGG AFAVQDPNDKTKFYAAGLVSWGPQCGTYGLYTRVKNYVDWI MKTMQENSTPRED

[0296] aKp3_LC DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWF QQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISR VE AE D V G VY Y CMQGT H W P P I T FGQ GT RL E I KRT V AAP S V F I FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN S Q E S VT E Q D S KD S T Y S L S S T L T L S RADY E KH K V Y AC E VT H Q GL S S P VT KS FNRGE C

[0297] aKp3 LCDR1 R S S Q S L V Y S DGNT Y LN

[0298] aKp3_LCDR2 KVSNRDS

[0299] aKp3_LCDR3 MQGTHWPPIT

[0300] aSp148_HC EVKLVESGGGLVKPGGSLKLSCAASGFTFSRYGMSWVRQTP EKRLEWVATISGGGSYTYYPDSVKGRFTMSRDNAKNKLYLQ MSSLRSEDTALYYCARHNDDGYFYFDYWGQGTTLTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALT SGVHT FPAVLQ S SGLY SL S SWT VP S S SLGTQT Y ICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0301] aSp148 HCDR1 RYGMS

[0302] aSp148 HCDR2 TISGGGSYTYYPDSVKG

[0303] aSp148 HCDR3 HNDDGYFYFDY

[0304] aSp148-C1s_HC EVKLVESGGGLVKPGGSLKLSCAASGFTFSRYGMSWVRQTP EKRLEWVATISGGGSYTYYPDSVKGRFTMSRDNAKNKLYLQ MSSLRSEDTALYYCARHNDDGYFYFDYWGQGTTLTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALT SGVHT FPAVLQ S SGLY SL S SWT VP S S SLGTQT Y ICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0305]

[0306] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL P AP I E KT I S KAKGQ P RE P Q VY T L P P S RD E L T KNQ VS L T CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY S KL T V D K S R WQ Q GN V F S C S VN H E AL H N H Y ’I' Q KS L S L S P GG G GGSGGGGSGGGGMPCPKEDTPNSVWEPAKAKYVFRDVVQIT CLDGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCGI PESIENGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEYH CAGNGSWVNEVLGPELPKCVPVCGVPREPFEEKQRIIGGSD ADI KN F P WQ V F F DN P WAGG AL I NE Y W VL T AAH V VE GN RE P T MYVGSTSVQTSRLAKSKMLTPEHVFIHPGWKLLEVPEGRTN FDNDIALVRLKDPVKMGPTVSPICLPGTSSDYNLMDGDLGL I S G WG RT E KRDRAVRL KAARL P VAPL RKC KE VKVE KP TAD A EAYVFTPNMICAGGEKGMDSCKGDSGGAFAVQDPNDKTKFY AAGLVSWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED

[0307] aSp148_LC DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQ QKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPV EEEDAATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTL S K AD Y E K H K V Y AC E VT H QGL SSPVTKSFNRGEC

[0308] aSp148 LCDR1 KASQSVDYDGDSMYN

[0309] aSp148 LCDR2 AASNLES

[0310] aSp148_LCDR3 QQSNEDPYT

[0311] aSa32_HC E VKL VE S GGGL VQ P RG S L RL S CAT S G FT FT E N FMS W VRQ S P G KAL E WL T F S RD KAHGH T T D Y S S S V I GR FT I ARDN S Q S I L Y LQMNALRTEDSATYYCVRGGGPHYFDYWGQGTIVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALT SGVHT FPAVLQ S SGLY SL S SWT VP S S SLGTQT Y ICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL P AP I E KT I S KAKGQ P RE P Q VY T L P P S RD E L T KNQ VS L T CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0312]

[0313] aSa32_HCDR1 ENFMS

[0314] aSa32_HCDR2 F S R D K AH G H T T D Y S S S VI G

[0315] aSa32_HCDR3 GGGPHYFDY

[0316] aSa32-Cls HC E VKL VE S GGGL VQ P RG S L RL S CAT S G FT FT E N FMS W VRQ S P G KAL E WL T F S RD KAHGH T T D Y S S S V I GR FT I ARDN S Q S I L Y LQMNALRTEDSATYYCVRGGGPHYFDYWGQGTIVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL P AP I E KT I S KAKGQ P RE P Q VY T L P P S RD E L T KNQ VS L T CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGMPCPKEDTPNSVWEPAKAKYV FRDVVQIT CLDGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCGI PESIENGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEYH CAGNGSWVNEVLGPELPKCVPVCGVPREPFEEKQRIIGGSD ADI KN F P WQ V F F DN P WAGG AL I NE Y W VL T AAH V VE GN RE P T MYVGSTSVQTSRLAKSKMLTPEHVFIHPGWKLLEVPEGRTN FDNDIALVRLKDPVKMGPTVSPICLPGTSSDYNLMDGDLGL ISGWGRTEKRDRAVRLKAARLPVAPLRKCKEVKVEKPTADA EAYVFTPNMICAGGEKGMDSCKGDSGGAFAVQDPNDKTKFY AAGLVSWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED

[0317] aSa32_LC D I VMT Q S H K FM S S S VG D R V Y I T C KA S Q D VG S AV AW Y Q Q K P G QSPKLLIYWTSTRHTGVPDRFTGSESGTDFTLTISNVQSED LADY FCQQYNSY PWT FGGGTKLE I KRTVAAPSVFI FPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

[0318] aSa32_LCDR1 KASQDVGSAVA

[0319] aSa32_LCDR2 WTSTRHT

[0320] aSa32_LCDR3 QQYNSYPWT

[0321]

[0322] aKp37_HC QVQLQQSGAELMRPGASVRISCKATGYTFSSYWIEWIKQRP GHGLEWIGETLPESGSTNYNEKFKGK. ATFTA. DTSSNTAYMQ ISSLTSEDSAVYYCARQGKYPYYYW1DYWGQGTSVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKAL PAP I E KT I S KAKGQ P RE PQ VYT L P P S RDE LT KNQ VS L T CL VKG F Y P S D I AVE WE SNGQ PENNY KT T P P VL DS DG S F FL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0323] aKp37_HCDR1 SYWIE

[0324] aKp37_HCDR2 ETLPESGSTNYNEKFKG

[0325] aKp37_HCDR3 QGKYPYYYVMDY

[0326] aKp37-Cls_HC QVQLQQSGAELMRPGASVRISCKATGYTFSSYWIEWIKQRP GHGLEWIGETLPESGSTNYNEKFKGKATFTADTSSNTAYMQ ISSLTSEDSAVYYCARQGKYPYYYVMDYWGQGTSVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CL VKG F Y P S D I AVE WE SNGQ PENNY KT T P P VL DS DG S F FL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG GGGSGGGGSGGGGMPCPKEDTPNSVWEPAKAKYVFRDVVQI TCLDGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCG IPESIENGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEY HCAGNGS WVNE VLGPEL PKCVPVCGVPRE P FEE KQRI IGG S DADEEKNFPWQVFFDNPWAGGALINEYWVLTAAHVVEGNREP TMY VGST S VQT S RLAKS KMLT PER VF I H PGWKLLE VPEGRT N FDND I ALVRLKDP VKMGPT VS P I CL PGT S S DYNLMDGDLG LIS GWGRT E KRDRAVRL KAARL P VAPLRKC KE VKVE KPT AD

[0327]

[0328] AEAYA’FTPNMICAGGEKGMDSCKGDSGGAFAVQDPNDKTKF YAAGLVSWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED 89 aKp37_LC DIQMNQSPSSLSASLGDTITITCHASQNIYVWLSWYQQQPG NIPNLLIYKSSNLHTGVPSRFSGSGSGTGFTLTISSLQPED I AT Y Y CQQ S Q S Y PWT FGGGT KL E I KRT VAAP S V F I F P P S DE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

[0329] 90 aKp37_LCDR1 HASQNIYVWLS

[0330] 91 aKp37_LCDR2 KSSNLHT

[0331] 92 aKp37_LCDR3 QQSQSYPWT

[0332]

[0333] IX. Examples

[0334] Example 1

[0335] Preparation of Monoclonal Antibodies and T-CAT Molecules

[0336] Monoclonal specific antibodies against A meningitidis NHB, K, pneumoniae MrkA, 5. pneumoniae PspA, P. aeruginosa flagellin B (Fla-B), and 5. aureus protein A were generated. Briefly, 7-week old BALB-C mice were immunized subcutaneously with 10 pg of recombinant antigen protein emulsified with alum. After 7 immunisation steps, mice were sacrificed and spleens were collected. Hybridoma cells were generated by fission of splenocytes and NS0 cell line from murine myeloma (Sigma Aldrich) according to standard protocol. After 2 weeks, positive clones were identified using binding ELISA, where ELISA plates were costed with the bacterial antigens. The isotype of the isolated antibodies was identified using IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit (Sigma Aldrich). Total RNA from positive clones was prepared using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Nucleotides encoding antibody V regions were amplified from the total RN with isotype-specitic reverse primers using the SMARTer RACE 573’ kit (Takara Bio). After verifying the sequences, the V regions were re-amplified with designed cloning primers and cloned into expression vectors carrying the human yl heavy chain and human K light chain constant regions using the In-Fusion HD cloning kit (Clontech). All plasmid constructs were verified by sequencing, propagated in Escherichia coli Mach1 (Invitrogen) and extracted using a Qiagen kit. The resulting constructs were transfectedtransiently into Expi293 or ExpiCHO cells (Life Technologies). After culture, secreted recombinant antibodies were purified from supernatants by protein A chromatography. Antibodies generated are listed in Table 2. Numbers indicate the SEQ ID NO reference for die indicated antibody region.

[0337] TABLE 2:

[0338] Antibody HC HCDR1 HCDR2 HCDR3 LC LCDR1 LCBR2 LCDR3 Neisseria 39 40 41 42 44 45 46 47

[0339] anti-NIIBA

[0340] Clone 4

[0341] Pseudomonas 48 49 50 50 53 54 55 aeruginosa

[0342] anti-FlaB

[0343] Clone 14

[0344] Klebsiella 57 58 59 60 62 63 64 65 pneumoniae

[0345] anti-MIrkA

[0346] Clone 3

[0347] Klebsiella 84 85 86 87 89 90 91 92 pneumoniae

[0348] anti-MrkA

[0349] Clone 37

[0350] Streptococcus 66 67 68 69 71 72 73 74 pneumoniae

[0351] anti-PspA

[0352] Clone 148

[0353] Staphylococcus 75 76 77 78 80 81 82 83 aureus

[0354] anti-Protein A

[0355] Clone 32

[0356]

[0357] Antibody sequences were used to generate T-CAT fusion proteins. For T-CAT fusion proteins, nucleotides encoding a catalytic fragment of Cis were fused to the heavy chain of tire antibodies with a G4S linker on the vectors. All plasmid constructs were verified by sequencing, propagated in Escherichia coli Mach1 (Invitrogen) and extracted using a Qiagen kit. The resulting constructs were transfected transiently into Exp-293 or ExpiCHO cells (Life Technologies). After culture, secreted recombinant antibodies and fusion proteins were purified from supernatants by protein A chromatography. The T-CAT fusion proteins generated are shown in Table 3.

[0358] TABLE 3:

[0359] Target NHBA Fla-B AirkA MrkA PspA Protein A binding Clone 4 Clone 14 Clone 3 Clone 37 Clone 148 Clone 32 domain

[0360] Serine Cis Cis Cis Cis Cis Cis protease

[0361] effector

[0362] domain

[0363] HC / Cls SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID fusion NO: 43 NO:52 NO:6I NO:88 NO:70 NO:79

[0364]

[0365] A size shift was observed in the T-CAT fusion proteins as compared to the corresponding antibody heavy chain or light chain, as expected. See FIGURE 3, left four columns. The serine protease function is active in the T-CAT fusion protein See FIGURE 3, right three columns.

[0366] T-CAT molecules were obtained by combining a heavy chain fusion protein with the corresponding antibody light chain or combining a light chain fusion protein with the corresponding antibody heavy chain.

[0367] Example 2

[0368] Binding of Monoclonal Antibodies and T-CAT Molecules to Target Bacteria Tire monoclonal antibodies and T-CAT molecules prepared as described in Example 1 were assayed for binding to the target bacteria. Maxisorp polystyrene microtiter ELISAplates were coated with formalin-fixed bacteria (OD600 = 0.6) in carbonate buffer (15mM Na2CCh, 35 mM NaHCCh, pH 9.6). After overnight incubation at 4 °C, ELISA plates were blocked using BSA in TBS buffer (lOmM Tris-HCl, 140mM NaCl, pH7.4) for 2 hours then washed with TBS buffer containing 0.05% (v / v) Tween 20 and 5 mM CaCh. Specific monoclonal antibodies, T-CAT molecules, control isotype antibodies, and control isotype T- CAT molecules were serially diluted in TBS and incubated with ELISA plates for 1 hour at room temperature (RT) followed by peroxidase-conjugated rabbit anti-human IgG (Dako). After 1 hour, wells were washed and 100 uL of 1-Step Ultra TMB Solution (Thermo Fisher Scientific) were then added to each well and incubated for 5 minutes at room temperature. Tire reaction w as stopped by the addition of 2M H2SO4 and the optical density at 450 nm was immediately measured.

[0369] Results for Klebsiella pneumoniae anti-MrkA Clone 3 and the corresponding T-CAT molecule are shown in FIGURE 5. Results for Klebsiella pneumoniae anti-MrkA Clone 37 and the corresponding T-CAT molecule are shown in FIGURE 20. Results for Pseudomonas aeruginosa anti-Fla-B Clone 14 and the corresponding T-CAT molecule are shown in FIGURE 7. In each case, the antibody and T-CAT molecule bound the bacteria to the same extent, while the isotype controls did not bind.

[0370] Results for Neisseria meningitidis anti-NHBA Clone 4 are shown in FIGURE 18. Clone 4 and another anti-NHBA antibody, Clone 9, were tested for binding to both Neisseria meningitidis and Neisseria gonorrhea. Anti-NHBA Clone 4 showed binding to both Neisseria species, while anti-NHBA Clone 9 showed binding only to Neisseria meningitidis.

[0371] For Staphylococcus aureus anti-protein A Clone 32, binding was assessed for twenty different isolates of multidrug-resistant A aureus. Maxisorp polystyrene microtiter ELISA plates were coated with the one of the A aureus strains or with Enterococcus faecalis, as a negative control, as described above. The Fc receptors of S. aureus were blocked with an Fc blocking agent. Monoclonal antibody Clone 32 was incubated with the ELISA plates as described above. Results are shown in FIGURE 16. Monoclonal antibody Clone 32 was observed to bind to all twenty N aureus isolates. No binding to negative control bacteria E. faecalis was observed.

[0372] Example 3

[0373] Pharmacokinetic Study of Monoclonal Antibodies and T-CAT molecules in Mice Tlie monoclonal antibodies and T-CAT molecules prepared as described in Example 1 were injected into mice to study their pharmacokinetic (PK) properties. 5mg / kg of antibodyor T-CAT molecule was injected interperitoneally (i.p.). Briefly, ELISA plates were coated with purified antigen and blocked as described above. Serum samples were collected at different time points post administration of antibodies. Bronchoalveolar lavage (BAL) and lung homogenates were also prepared. Samples were diluted 1 / 100 in PBS and incubated with the ELISA plates. Binding of antibodies or T-CAT molecules was detected using peroxidase-conjugated rabbit anti-human IgG as described above. Known concentrations of purified T-CAT molecules or antibodies were serially diluted and incubated with the ELISA plate to generate standard curves, which were used to calculate antibody and T-CAT concentrations in the samples. Results are shown in FIGURE 4 for a representative antibody and T-CAT molecule, in this case Klebsiella pneumoniae anti-MrkA Clone 3 and the corresponding T-CAT molecule. High levels of both antibody and T-CAT molecule were detected in all tissues tested at all timepoints tested, which were up to 48 hours post-administration.

[0374] Example 4

[0375] Complement Deposition Assays

[0376] Tire ability of the monoclonal antibodies and T-CAT molecules prepared as described above to trigger deposition of C3b on the surface of the target bacterial cells was assayed using two different methods.

[0377] For ELISA-based assays, Maxisorp polystyrene microtiter ELISA plates were coated with formalin-fixed bacteria (OD600 = 0.6) in carbonate buffer. The next day, wells were blocked and washed as mentioned above. 2.5% normal human serum (NHS) diluted in barbital buffer saline (BBS++) buffer containing Ca++and Mg++(4mM barbital, 145mM NaCl, 2mM CaCh, 1 mM MgCb, pH 7.4) and supplemented with 1,5 nM of either antibody, T-CAT molecule, or isotype control antibody was incubated at 4°C with ELISA plates for different time points and then washed. Deposition of C3b was detected using rabbit anti-C3c (Dako), followed by peroxidase-conjugated goat anti-rabbit IgG. After 1 hour, wells were washed and 100 L of 1-Step Ultra TMB Solution (Thermo Fisher Scientific) was then added to each well and incubated for 5 minutes at room temperature. The reaction was stopped by the addition of 2M H2SO4 and the optical density at 450nm was immediately measured.

[0378] For FACS analysis, bacterial cultures were washed twice with TBS buffer and resuspended in BBS4to a final concentration of 106CFU / mL. Bacterial suspension (100 |iL) was opsonised with 2.5% (vol / vol) NHS supplemented with either antibody, T-CAT molecule, isotype control antibody, or isotype control T-CAT molecule for 10 min at 4°C.Nonopsonised bacteria were used as a negative control. After opsonisation, the bacterial samples were washed twice with TBS buffer. C3b deposition on tire surface of bacteria was detected using rabbit anti-human C3c followed by Alexa Fluor 488 Donkey anti-rabbit IgG. Fluorescence intensity was measured with a FACS Calibur cell analyzer (BD Biosciences).

[0379] Results for Klebsiella pneumoniae anti-MrkA Clone 3 and the corresponding T-CAT molecule are shown in FIGURE 6. Results for Pseu domonas aeruginosa anti-Fla-B Clone 14 and the corresponding T-CAT molecule are shown in FIGURE 8. In each figure, the left panel shows the results of the ELISA assay and the right panel shows the results of the FACS analysis. In each case, the targeted T-CAT molecule induced high levels of C3b deposition, while the targeted monoclonal antibody performed no better in these assays than the controls.

[0380] The ability of the monoclonal antibodies and T-CAT molecules prepared as described above to trigger deposition of C4b on the surface of the target bacterial cells w as assayed. The ELISA-based assay for C4b deposition was similar to that for C3b deposition described above, except that detection was carried out using rabbit anti-human C4c that cross-reacts with C4b (Dako). Fluorescence intensity was measured as described above.

[0381] Results for Staphylococcus aureus protein A-targeted antibodies and T-CAT molecules are shown in FIGURE 17. The targeted T-CAT molecules showed significantly enhanced levels of C4b deposition on the surface of N. aureus as compared to either the targeted antibody or an isotype control T-CAT molecule. Results for Neisseria meningitidis anti-NHBA Clone 4 and the corresponding T-CAT molecule are shown in FIGURE 19. Results for 'Klebsiella pneumoniae anti-MrkA Clone 37 and the corresponding T-CAT molecule are shown in FIGURE 21. In each case, the targeted T-CAT molecule induced high levels of C4b deposition, while the targeted monoclonal antibody performed no better than the controls.

[0382] Example 5

[0383] Treatment of Klebsiella infection with T-CAT in a mouse model

[0384] Animal infection studies were performed using 12-14-wk-old female BALB / C mice from Charles River Laboratory were used for A. pneumoniae infection studies. For respiratory tract infection, mice were lightly anesthetised with 2.5% (vol / vol) fluothane (AstraZeneca) over oxygen (1.5-2 L / min), after which 50 pL of PBS containing 5 × 107cfu of K. pneumoniae was administered into the mouse nostrils. The infected mice were treated with 5mg / kg of Klebsiella pneumoniae anti-MrkA Clone 3, the anti-MrkA Clone 3 T-CAT molecule, or an isotype control antibody via i.p. route at 12, and 36 hours post infection.Mice were monitored for progression of clinical signs and euthanised when they became lethargic. Blood samples were obtained at predetermined time points, and viable counts were calculated after serial dilution in PBS and plating out on the corresponding agar / blood agar plates. Lungs were collected under aseptic conditions then homogenised in 10 mL of PBS. Counts of viable bacteria in lung homogenate were calculated. For histopathology, mice were euthanised at pre-determined time points and then lungs (n=3) were collected and fixed using paraformaldehyde 4% in PBS. 7pm lung sections were stained with haematoxylin and eosin and subsequently fixed with DPX mount (BDH). Lung injury and pathology scores were assessed by three independent researchers

[0385] Results are shown in FIGURE 9, FIGURE 10, and FIGURE 11. FIGURE 9 shows survival over time for the treated and control mice. Mice treated with the T-CAT molecule showed significantly improved survival time over mice treated with the targeted antibody or isotype control antibody. FIGURE 10 shows bacterial load in blood samples (left panel) and lung tissue (right panel) collected at 24 and 48 hours post-infection. The bacterial load in both tissues is significantly lower in mice treated with the T-CAT molecule as compared to either the targeted antibody or the isotype control. FIGURE 11 shows lung sections from mice treated with targeted antibody, targeted T-CAT molecule, or isotype control antibody. Lung sections from infected animals treated with the T-CAT molecule showed a marked reduction in the severity of lung pathology, with a significant reduction in leukocyte infiltration into the lung parenchyma compared to the targeted antibody or isoty pe control. Lung injury score is shown in the graph below the lung sections.

[0386] Example 6

[0387] Treatment of Pseudomonas infection with T-CAT in a mouse model Animal infection studies were performed using 12-14-wk-old female BALB / C mice from Charles River Laboratory were used for P. aeruginosa infection studies. For respiratory tract infection, mice were lightly anesthetised with 2.5% (vol / vol) fluothane (AstraZeneca) over oxygen (1.5-2 L / min), after which 50 pL of PBS containing 2.5 × 106of P. aeruginosa was administered into the mouse nostrils. The infected mice were treated with 5mg / kg of Pseudomonas aeruginosa anti-Fla-B Clone 4, the anti-Fla-B Clone 4 T-CAT molecule, or an isotype control antibody via i.p. route at 24, 48, and 72 hours post infection. Mice were monitored for progression of clinical signs and euthanised when they became lethargic. Blood samples were obtained at predetermined time points, and viable counts were calculated after serial dilution in PBS and plating out on the corresponding agar / blood agar

[0388] 19plates. Lungs were collected under aseptic conditions then homogenised in 10 mL of PBS. Counts of viable bacteria in lung homogenate were calculated. For histopathology, mice were euthanised at pre-determined time points and then lungs (n=3) were collected and fixed using paraformaldehyde 4% in PBS. 7pm lung sections were stained with haematoxylin and eosin and subsequently fixed with DPX mount (BDH). Lung injury and pathology scores were assessed by three independent researchers

[0389] Results are shown in FIGURE 12, FIGURE 13, and FIGURE 14. FIGURE 12 shows survival over time for the treated and control mice. Mice treated with the T-CAT molecule showed significantly improved survival time over mice treated with the targeted antibody or isotype control antibody. FIGURE 13 shows bacterial load in blood samples (left panel) and lung tissue (right panel) collected at 24 and 48 hours post-infection. The bacterial load in both tissues is significantly lower in mice treated with the T-CAT molecule as compared to either the targeted antibody or the isotype control. FIGURE 14 shows lung sections from mice treated with targeted antibody, targeted T-CAT molecule, or isotype control antibody. Lung sections from infected animals treated with the T-CAT molecule showed a marked reduction in tire severity of lung pathology, with a significant reduction in leukocyte infiltration into the lung parenchyma compared to the targeted antibody or isotype control. Lung sections from T-CAT-treated mice showed improvement overtime, as shown in the sections from 3, 4, and 5 days post-infection. Lung injury score is shown in the graph below the lung sections,

[0390] X. Other Embodiments

[0391] All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

[0392] While certain embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the specific embodiments described that are obvious to those skilled in the fields of medicine, immunology, pharmacology, or related fields are intended to be within the scope of the invention.

[0393] Accordingly, the following numbered paragraphs describing specific embodiments are provided for clarity, but should not be construed to limit the claims.1. A targeted complement activating molecule comprising:

[0394] (a) a target binding domain; and

[0395] (b) a complement activating serine protease effector domain;

[0396] wherein the target binding domain comprises an antibody or an antigen-binding fragment thereof and wherein the antibody or antigen-binding fragment thereof is, or is derived from:

[0397] i) an anti-Klebsiella pneumoniae MrkA antibody Clone 3;

[0398] ii) anti-Klebsiella pneumoniae MrkA antibody Clone 37;

[0399] iii) anti-Streptococcus pneumonia PspA antibody Clone 148;

[0400] iv) anti-Pseudomonas aeruginosa Fla-B Clone 14;

[0401] v) anti-Neisseria meningitidis NHBA Clone 4; or

[0402] vi) anti-Staphylococcus aureus Protein A Clone 32.

[0403] 2. The molecule of paragraph 1, wherein the complement activating serine protease effector domain comprises MASP-1 or a fragment thereof, MASP-2 or a fragment thereof, MASP-3 or a fragment thereof, Clr or a fragment thereof, Cis or a fragment thereof, factor D or a fragment thereof, C2a or a fragment thereof, or factor Bb or a fragment thereof.

[0404] 3. The molecule of paragraph 1 or paragraph 2, wherein the complement activating serine protease effector domain is catalytically active.

[0405] 4. The molecule of paragraph 1 or paragraph 2, wherein the complement activating serine protease effector domain is in a zymogen form.

[0406] 5. The molecule of any one of paragraphs 1 -4, wherein the target binding domain comprises an antibody heavy chain or fragment thereof and an antibody light chain or fragment thereof.

[0407] 6. The molecule of paragraph 5, wherein the molecule comprises:

[0408] a) a fusion protein comprising:

[0409] i) the N -terminus of the complement activating serine protease effector domain fused to the C-terminus of the antibody heavy chain or fragment thereof; or

[0410] ii) the C-terminus of the complement activating serine protease effector domain fused to the N-terminus of the antibody heavy chain or fragment thereof; and

[0411] an antibody light chain or fragment thereof; or

[0412] b) a fusion protein comprising:i) the N-terminus of the complement activating serine protease effector domain fused to the C -terminus of the antibody light chain or fragment thereof; or

[0413] ii) the C -terminus of the complement activating serine protease effector domain fused to the N-terminus of the antibody light chain or fragment thereof; and

[0414] an antibody heavy chain or fragment thereof.

[0415] 7. The molecule of any one of paragraphs 1-6, wherein the molecule comprises a fusion protein comprising:

[0416] a) the N-terminus of the complement activating serine protease effector domain fused to the C-terminus of a single-chain antibody or fragment thereof or single-domain antibody or fragment thereof; or

[0417] b) the C-terminus of the complement activating serine protease effector domain fused to the N-terminus of a single-chain antibody or fragment thereof or single-domain antibody or fragment thereof.

[0418] 8. The molecule of any one of paragraphs 1-7, wherein the target binding domain and the serine protease effector domain are connected by a linker.

[0419] 9. The molecule of any of paragraphs 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 3.

[0420] 10. The molecule of paragraph 9, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 3 and the serine protease effector domain comprises C1s or a fragment thereof.

[0421] 11. The molecule of paragraph 9 or 10, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:57.

[0422] 12. The molecule of paragraph 9 or 10, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:62.

[0423] 13. The molecule of paragraph 9 or 10, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:57 and a light chain as set forth in SEQ ID NO:62.

[0424] 14. The molecule of any of paragraphs 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 37.

[0425] 15. The molecule of paragraph 14, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 37 and the serine protease effector domain comprises C1s or a fragment thereof.16. The molecule of paragraph 14 or 15, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 84.

[0426] 17. The molecule of paragraph 14 or 15, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:89.

[0427] 18. The molecule of paragraph 14 or 15, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:84 and a light chain as set forth in SEQ ID NO:89.

[0428] 19. The molecule of any of paragraphs 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Streptococcus pneumonia PspA antibody Clone 148.

[0429] 20. The molecule of paragraph 19, wherein the target binding domain comprises anti- Streptococcus pneumonia PspA Clone 148 or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

[0430] 21. The molecule of paragraph 19 or 20, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:66.

[0431] 22. The molecule of paragraph 19 or 20, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:71.

[0432] 23. The molecule of paragraph 19 or 20, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:66 and a light chain as set forth in SEQ ID NO: 71.

[0433] 24. The molecule of any of paragraphs 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Pseudomonas aeruginosa Fla-B Clone 14,

[0434] 25. The molecule of paragraph 24, wherein the target binding domain comprises anti- Pseudomonas aeruginosa FlaB Clone 14 or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

[0435] 26. The molecule of paragraph 24 or 25, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:48.

[0436] 27. The molecule of paragraph 24 or 25, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:53.

[0437] 28. The molecule of paragraph 24 or 25, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:48 and a light chain as set forth in SEQ ID NO:53.

[0438] 29. The molecule of any of paragraphs 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Neisseria meningitidis NHBA Clone 4.

[0439] 30. The molecule of paragraph 29, wherein the target binding domain comprises anti- NHBA Clone 4 or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.31. The molecule of paragraph 29 or 30, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:39.

[0440] 32. The molecule of paragraph 29 or 30, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:44.

[0441] 33. The molecule of paragraph 29 or 30, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:39 and a light chain as set forth in SEQ ID NO:44.

[0442] 34. The molecule of any of paragraphs 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Staphylococcus aureus Protein A Clone 32.

[0443] 35. The molecule of paragraph 34, wherein the target binding domain comprises anti- Staphylococcus aureus Protein A Clone 32or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

[0444] 36. The molecule of paragraph 34 or 35, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:75,

[0445] 37. The molecule of paragraph 34 or 35, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO: 80.

[0446] 38. The molecule of paragraph 34 or 35, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:75 and a light chain as set forth in SEQ ID NO:80, 39. The molecule of paragraph 6 or 7, wherein the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs:43, 52, 61, 70, and 79.

[0447] 40. The molecule of paragraph 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:43.

[0448] 41. The molecule of paragraph 40, further comprising a light chain as set forth in SEQ ID NO: 44.

[0449] 42. The molecule of paragraph 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:52

[0450] 43. The molecule of paragraph 42, further comprising a light chain as set forth in SEQ ID NO:53.

[0451] 44. The molecule of paragraph 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:61.

[0452] 45. The molecule of paragraph 44, further comprising a light chain as set forth in SEQ ID NO:62.

[0453] 46. The molecule of paragraph 39, -wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 70.47. The molecule of paragraph 46, further comprising a light chain as set forth in SEQ ID NO: 71.

[0454] 48. The molecule of paragraph 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:79.

[0455] 49. The molecule of paragraph 48, further comprising a light chain as set forth in SEQ ID NO: 80.

[0456] 50. The molecule of any one of paragraphs 1-49, wherein the serine protease effector domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-36.

[0457] 51. The molecule of any one of paragraphs 1-49, wherein the serine protease effector domain comprises an amino acid sequence set forth in SEQ ID NO:20.

[0458] 52. The molecule of paragraph 50 or 51, wherein the complement activating serine protease effector domain comprises one or more mutations relative to a wild-type serine protease and / or the target binding domain comprises one or more mutations relative to a wildtype antibody.

[0459] 53. The molecule of paragraph 52, wherein the one or more mutations inhibit protein degradation.

[0460] 54. The molecule of paragraph 52, wherein the one or more mutations confer resistance to serine protease inhibition by Cl inhibitor or other serpins.

[0461] 55. The molecule of paragraph 52, wherein the one or more mutations inhibit glycosylation of the molecule at one or more amino acid residues.

[0462] 56. The molecule of any one of paragraphs 1-55, wherein the molecule binds to a target with an affinity between 1 pM and 1 pM.

[0463] 57. The molecule of any one of paragraphs 1-55, wherein the molecule binds to a target on a cell surface with an affinity between 1 pM and 1 pM.

[0464] 58. The molecule of any one of paragraphs 1-55, wherein the molecule has a serine protease activity that is at least 70% of the serine protease activity of the serine protease domain alone.

[0465] 59. The molecule of any one of paragraphs 1-55, wherein the molecule has a serine protease activity that is at least 80% of the serine protease activity of the serine protease domain alone.

[0466] 60. The molecule of any one of paragraphs 1-55, wherein the molecule has a serine protease activity that is at least 90% of the serine protease activity of the serine protease domain alone.61. The molecule of any one of paragraphs 1-55, wherein the molecule binds to a target on a cell surface and activates a complement pathway when administered to a mammalian subject.

[0467] 62. The molecule of any one of paragraphs 1-55, wherein the molecule induces complement dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity’ (CDCC), and / or complement-dependent cellular phagocytosis (CDCP).

[0468] 63. A polynucleotide encoding the molecule of any one of paragraphs 1-55.

[0469] 64. A cloning vector or expression cassette comprising the polynucleotide of paragraph 63.

[0470] 65. A host cell expressing the molecule of any one of paragraphs 1-55, or comprising the cloning vector or expression cassette of paragraph 64.

[0471] 66. A method of producing a molecule comprising:

[0472] (a) a target binding domain; and

[0473] (b) a complement activating serine protease effector domain; the method comprising culturing the host cell of paragraph 65 under conditions allowing for expression of the molecule and isolating the molecule.

[0474] 67. The use of the molecule of any one of paragraphs 1-55 to activate at least one complement pathway in a mammalian subject.

[0475] 68. The use of paragraph 67, wherein the activation of the at least one complement pathway comprises:

[0476] a) activation of the complement classical pathway;

[0477] b) activation of the complement lectin pathway;

[0478] c) activation of the complement alternative pathway; or

[0479] d) two or more of (a)-(c).

[0480] 69. The use of the molecule of any one of paragraphs 1-55 to induce complement dependent cell death (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), or complement-dependent cellular phagocytosis (CDCP) in a target cell.

[0481] 70. The use of the molecule of any one of paragraphs 1-55 to treat a microbial infection in a mammalian subject.

[0482] 71. The use of paragraph 70, wherein the infection is a bacterial infection.

[0483] 72. A composition comprising the molecule of any one of paragraphs 1-55 and one or more excipients.73. A method of activating at least one complement pathway in a mammalian subject by administering the molecule of any one of paragraphs 1-55 or the composition of paragraph 72.

[0484] 74. The method of paragraph 73, wherein the activation of the at least one complement pathway comprises:

[0485] a) activation of the complement classical pathway;

[0486] b) activation of the complement lectin pathway;

[0487] c) activation of the complement alternative pathway; or

[0488] d) two or more of (a)-(c).

[0489] 75. A method of inducing complement dependent cell death (CDC) in a target cell, comprising contacting the target cell with the molecule of any one of paragraphs 1-55 or the composition of paragraph 72, w herein said contacting results in complement deposition on the target cell, thereby leading to complement-mediated cell death,

[0490] 76. A method of inducing complement-dependent cell-mediated cytotoxicity (CDCC) or complement-dependent cellular phagocytosis (CDCP) toward a target cell, comprising contacting the target cell with the molecule of any one of paragraphs 1-55 or the composition of paragraph 72, wherein said contacting results in complement deposition on the target cell, thereby leading to complement-mediated cell death.

[0491] 77. A method of treating a microbial infection in a mammalian subject, comprising administering the molecule of any one of paragraphs 1-55 or the composition of paragraph 72 to the subject.

[0492] 78. The method of paragraph 77, wherein the infection is a bacterial infection.

[0493] 79. The method of paragraph 78, wherein the bacterial pathogen is Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus.

[0494] 80. The method of paragraph 79, wherein the bacterial pathogen is multidrugresistant Staphylococcus aureus.

[0495] 81. The targeted complement activating molecule of any one of paragraphs 1-55 or the composition of paragraph 72 for use in the manufacture of a medicament for treating a microbial infection.

[0496] 82. The composition of paragraph 72 for use in treating a microbial infection.

[0497] 83. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Neisseria meningitidis antigen NHBA, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO:40, a HCDR2 as set forth in SEQ IDNO:41, a HCDR3 as set forth in SEQ ID NO:42, a LCDR1 as set forth in SEQ ID NO:45, a LCDR2 as set forth in SEQ ID NO:46, and a LCDR3 as set forth in SEQ ID NO:47.

[0498] 84. Tire monoclonal antibody or antigen binding fragment thereof of paragraph 83, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:39 and a light chain as set forth in SEQ ID NO: 44.

[0499] 85. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Pseudomonas aeruginosa antigen Fla-B, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO:49, a HCDR2 as set forth in SEQ ID NO:50, a HCDR3 as set forth in SEQ ID NO:51, a LCDR1 as set forth in SEQ ID NO:54, a LCDR2 as set forth in SEQ ID NO:55, and a LCDR3 as set forth in SEQ ID NO:56.

[0500] 86. The monoclonal antibody or antigen binding fragment thereof of paragraph 85, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:48 and a light chain as set forth in SEQ ID NO: 53,

[0501] 87. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Streptococcus pneumoniae antigen PspA, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO: 67, a HCDR2 as set forth in SEQ ID NO:68, a HCDR3 as set forth in SEQ ID NO:69, a LCDR1 as set forth in SEQ ID NO:72, a LCDR2 as set forth in SEQ ID NO:73, and a LCDR3 as set forth in SEQ ID NO: 74,

[0502] 88. The monoclonal antibody or antigen binding fragment thereof of paragraph 87, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:66 and a light chain as set forth in SEQ ID NO:71.

[0503] 89. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Staphylococcus aureus antigen Protein A, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO: 76, a HCDR2 as set forth in SEQ ID NO:77, a HCDR3 as set forth in SEQ ID NO:78, a LCDR1 as set forth in SEQ ID NO:81, a LCDR2 as set forth in SEQ ID NO:82, and a LCDR3 as set forth in SEQ ID NO:83.

[0504] 90. The monoclonal antibody or antigen -binding fragment thereof of paragraph 89, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 75 and a light chain as set forth in SEQ ID NO: 80.

[0505] 91. lire antibody or antigen-binding fragment thereof of any one of paragraphs 83-90, wherein the molecule binds to a target with an affinity’ between 1 pM and 1 M,

[0506] 92. A polynucleotide encoding the antibody or antigen-binding fragment of any one of paragraphs 83-90.93. A cloning vector or expression cassette comprising the polynucleotide of paragraph 92.

[0507] 94. A host cell expressing the antibody or antigen-binding fragment thereof of any¬ one of paragraphs 83-90, or comprising the cloning vector or expression cassette of paragraph 93.

[0508] 95. A method of producing an antibody or antigen -binding fragment thereof, the method comprising culturing the host cell of paragraph 94 under conditions allowing for expression of the antibody or antigen-binding fragment thereof and isolating the antibody or antigen- binding fragment thereof.

[0509] 96. Tire use of the antibody or antigen-binding fragment thereof of any one of paragraphs 83-90 to treat a microbial infection in a mammalian subject.

[0510] 97. A composition comprising the antibody or antigen-binding fragment thereof of any one of paragraphs 83-90 and one or more excipients.

[0511] 98. The antibody or antigen-binding fragment thereof of any one of paragraphs 83-90 for use in the manufacture of a medicament for treating a microbial infection.

[0512] 99. The composition of paragraph 97 for use in treating a microbial infection.

Claims

1. CLAIMSWhat is claimed is:

1. A targeted complement activating molecule comprising:(a) a target binding domain; and(b) a complement activating serine protease effector domain;wherein the target binding domain comprises an antibody or an antigen-binding fragment thereof and wherein the antibody or antigen-binding fragment thereof is, or is derived from:i) atAanti-Klebsiella pneumoniae MrkA antibody Clone 3;ii) anti-Klebsiella pneumoniae MrkA antibody Clone 37;iii) anti-Streptococcus pneumonia PspA antibody Clone 148;iv) anti-Pseudomonas aeruginosa Fla-B Clone 14;v) anti-Neisseria meningitidis NHBA Clone 4; orvi) anti-Staphylococcus aureus Protein A Clone 32.

2. The molecule of claim 1, wherein the complement activating serine protease effector domain comprises MASP-1 or a fragment thereof, MASP-2 or a fragment thereof, MASP-3 or a fragment thereof, C1r or a fragment thereof, C1s or a fragment thereof, factor D or a fragment thereof, C2a or a fragment thereof, or factor Bb or a fragment thereof.

3. The molecule of claim 1 or claim 2, wherein the complement activating serine protease effector domain is catalytically active.

4. The molecule of claim 1 or claim 2, wherein the complement activating serine protease effector domain is in a zymogen form.

5. The molecule of any one of claims 1-4, wherein the target binding domain comprises an antibody heavy chain or fragment thereof and an antibody light chain or fragment thereof.

6. The molecule of claim 5, wherein the molecule comprises:a) a fusion protein comprising:i) the N-terminus of the complement activating serine protease effector domain fused to the C-terminus of the antibody heavy chain or fragment thereof; orii) the C-terminus of the complement activating serine protease effector domain fused to the N-terminus of the antibody heavy chain or fragment thereof; and an antibody light chain or fragment thereof; orb) a fusion protein comprising:i) the N-terminus of the complement activating serine protease effector domain fused to the C -terminus of the antibody light chain or fragment thereof; orii) the C -terminus of the complement activating serine protease effector domain fused to the N-terminus of the antibody light chain or fragment thereof; and an antibody heavy chain or fragment thereof.

7. The molecule of any one of claims 1-6, wherein the molecule comprises a fusion protein comprising:a) the N-terminus of the complement activating serine protease effector domain fused to the C -terminus of a single-chain antibody or fragment thereof or single-domain antibody or fragment thereof; orb) tire C-terminus of the complement activating serine protease effector domain fused to the N-terminus of a single-chain antibody or fragment thereof or single-domain antibody or fragment thereof.

8. The molecule of any one of claims 1-7, wherein the target binding domain and the serine protease effector domain are connected by a linker.

9. Tire molecule of any of claims 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 3.

10. The molecule of claim 9, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 3 and the serine protease effector domain comprises Cis or a fragment thereof.

11. The molecule of claim 9 or 10, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 57.

12. The molecule of claim 9 or 10, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:62.

13. The molecule of claim 9 or 10, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:57 and a light chain as set forth in SEQ ID NO:62.

14. The molecule of any of claims 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 37.

15. lire molecule of claim 14, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Klebsiella pneumoniae MrkA antibody Clone 37 and the serine protease effector domain comprises Cis or a fragment thereof.

16. The molecule of claim 14 or 15, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 84.

17. The molecule of claim 14 or 15, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO: 89.

18. The molecule of claim 14 or 15, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 84 and a light chain as set forth in SEQ ID NO: 89.

19. The molecule of any of claims 1-8, wherein the antibody or antigen-binding fragment thereof is, oris derived from, anti-Streptococcus pneumonia PspA antibody Clone 148.

20. The molecule of claim 19, wherein the target binding domain comprises anti-Streptococcus pneumonia PspA Clone 148 or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

21. Hie molecule of claim 19 or 20, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:66.

22. The molecule of claim 19 or 20, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO: 71.

23. The molecule of claim 19 or 20, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:66 and a light chain as set forth in SEQ ID NO:71.

24. The molecule of any of claims 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Pseudomonas aeruginosa Fla-B Clone 14.

25. The molecule of claim 24, wherein the target binding domain comprises anti-Pseudomonas aeruginosa FlaB Clone 14 or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

26. The molecule of claim 24 or 25, w herein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 48.

27. The molecule of claim 24 or 25, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:53.

28. The molecule of claim 24 or 25, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:48 and a light chain as set forth in SEQ ID NO:53.

29. The molecule of any of claims 1-8, wherein the antibody or antigen-binding fragment thereof is, or is derived from, anti-Neisseria meningitidis NHBA Clone 4.

30. lire molecule of claim 29, wherein the target binding domain comprises anti-NHBA Clone 4 or an antigen -binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

31. Hie molecule of claim 29 or 30, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 39.

32. The molecule of claim 29 or 30, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:44.

33. The molecule of claim 29 or 30, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO: 39 and a light chain as set forth in SEQ ID NO: 44.

34. The molecule of any of claim 1-8, wherein tire antibody or antigen-binding fragment thereof is, oris derived from, anti-Staphylococcus aureus Protein A Clone 32.

35. The molecule of claim 34, wherein the target binding domain comprises anti-Staphylococcus aureus Protein A Clone 32or an antigen-binding fragment thereof and the serine protease effector domain comprises Cis or a fragment thereof.

36. Tire molecule of claim 34 or 35, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:75.

37. The molecule of claim 34 or 35, wherein the target binding domain comprises a light chain as set forth in SEQ ID NO:80,38. The molecule of claim 34 or 35, wherein the target binding domain comprises a heavy chain as set forth in SEQ ID NO:75 and a light chain as set forth in SEQ ID NO:80.

39. lire molecule of claim 6 or 7, wherein the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs:43, 52, 61, 70, and 79.

40. Tire molecule of claim 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:43.

41. The molecule of claim 40, further comprising a light chain as set forth in SEQ ID NO:44.

42. The molecule of claim 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:52.

43. The molecule of claim 42, further comprising a light chain as set forth in SEQ ID NO:53.

44. The molecule of claim 39, w herein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:61.

45. The molecule of claim 44, further comprising a light chain as set forth in SEQ ID NO:62.

46. lire molecule of claim 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 70.

47. The molecule of claim 46, further comprising a light chain as set forth in SEQ ID NO: 71.

48. The molecule of claim 39, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 79.

49. The molecule of claim 48, further comprising a light chain as set forth in SEQ ID NO:80.

50. The molecule of any one of claims 1-49, wherein the serine protease effector domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-36.

51. The molecule of any one of claims 1-49, wherein the serine protease effector domain comprises an amino acid sequence set forth in SEQ ID NO:20.

52. The molecule of claim 50 or 51, wherein the complement activating serine protease effector domain comprises one or more mutations relative to a wild-type serine protease and / or the target binding domain comprises one or more mutations relative to a wild¬ type antibody.

53. The molecule of claim 52, wherein the one or more mutations inhibit protein degradation.

54. The molecule of claim 52, wherein the one or more mutations confer resistance to serine protease inhibition by CI inhibitor or other serpins.

55. The molecule of claim 52, wherein the one or more mutations inhibit glycosylation of the molecule at one or more amino acid residues.

56. The molecule of any one of claims 1-55, wherein the molecule binds to a target with an affinity between 1 pM and 1 pM,57. The molecule of any one of claims 1-55, wherein the molecule binds to a target on a cell surface with an affinity betw een 1 pM and I pM.

58. The molecule of any one of claims 1-55, wherein the molecule has a serine protease activity that is at least 70% of the serine protease activity of the serine protease domain alone.

59. The molecule of any one of claims 1-55, wherein the molecule has a serine protease activity that is at least 80% of the serine protease activity of the serine protease domain alone.

60. Tire molecule of any one of claims 1-55, wherein the molecule has a serine protease activity that is at least 90% of the serine protease activity of the serine protease domain alone,61. The molecule of any one of claims 1 -55, wherein the molecule binds to a target on a cell surface and activates a complement pathway when administered to a mammalian subject.

62. The molecule of any one of claims 1-55. wherein the molecule induces complement dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), and / or complement-dependent cellular phagocytosis (CDCP).

63. A polynucleotide encoding the molecule of any one of claims 1-55.

64. A cloning vector or expression cassette comprising the polynucleotide of claim 63.

65. A host cell expressing the molecule of any one of claims 1-55, or comprising the cloning vector or expression cassette of claim 64.

66. A method of producing a molecule comprising:(a) a target binding domain; and(b) a complement activating serine protease effector domain; the method comprising culturing the host cell of claim 65 under conditions allowing for expression of the molecule and isolating the molecule.

67. The use of the molecule of any one of claims 1-55 to activate at least one complement pathway in a mammalian subject.

68. lire use of claim 67, wherein the activation of the at least one complement pathway comprises:a) activation of the complement classical pathway;b) activation of the complement lectin pathway;c) activation of the complement alternative pathway; ord) two or more of (a)-(c).

69. The use of the molecule of any one of claims 1-55 to induce complement dependent cell death (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), or complement-dependent cellular phagocytosis (CDCP) in a target cell,70. The use of the molecule of any one of claims 1-55 to treat a microbial infection in a mammalian subject.

71. The use of claim 70, wherein the infection is a bacterial infection.

72. A composition comprising the molecule of any one of claims 1 -55 and one or more excipients.

73. A method of activating at least one complement pathway in a mammalian subject by administering the molecule of any one of claims 1-55 or the composition of claim 72.

74. The method of claim 73, -wherein the activation of the at least one complement pathway comprises:a) activation of tire complement classical pathway;b) activation of the complement lectin pathway;c) activation of the complement alternative pathway; ord) two or more of (a)-(c).

75. A method of inducing complement dependent cell death (CDC) in a target cell, comprising contacting the target cell with the molecule of any one of claims 1-55 or the composition of claim 72, wherein said contacting results in complement deposition on the target cell, thereby leading to complement-mediated cell death.

76. A method of inducing complement-dependent cell-mediated cytotoxicity (CDCC) or complement-dependent cellular phagocytosis (CDCP) toward a target cell, comprising contacting the target cell with the molecule of any one of claims 1-55 or the composition of claim 72, wherein said contacting results in complement deposition on the target cell, thereby leading to complement-mediated cell death.

77. A method of treating a microbial infection in a mammalian subject, comprising administering the molecule of any one of claims 1-55 or the composition of claim 72 to the subject.

78. lire method of claim 77, wherein the infection is a bacterial infection.

79. The method of claim 78, wherein the bacterial pathogen is Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus.

80. The method of claim 79, wherein the bacterial pathogen is multidrug-resistant Staphylococcus a ureus.

81. The targeted complement activating molecule of any one of claims 1-55 or the composition of claim 72 for use in the manufacture of a medicament for treating a microbial infection.

82. Tire composition of claim 72 for use in treating a microbial infection.

83. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Neisseria meningitidis antigen NHBA, wherein the antibody or antigen-binding fragment thereof comprises a IICDR1 as set forth in SEQ ID NO:40, a HCDR2 as set forth in SEQ ID NO:41, a HCDR3 as set forth in SEQ ID NO:42, a LCDR1 as set forth in SEQ ID NO:45, a LCDR2 as set forth in SEQ ID NO:46, and a LCDR3 as set forth in SEQ ID NO:47.

84. The monoclonal antibody or antigen binding fragment thereof of claim 83, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:39 and a light chain as set forth in SEQ ID NO: 44.

85. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Pseudomonas aeruginosa antigen Fla-B, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO:49, a HCDR2 as set forth in SEQ ID NO:50, a HCDR3 as set forth in SEQ ID NO:51, a LCDR1 as set forth in SEQ ID NO:54, a LCDR2 as set forth in SEQ ID NO:55, and a LCDR3 as set forth in SEQ ID NO:56.

86. The monoclonal antibody or antigen binding fragment thereof of claim 85, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:48 and a light chain as set forth in SEQ ID NO: 53.

87. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Streptococcus pneumoniae antigen PspA, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO:67, a HCDR2 as set forth in SEQ ID NO:68, a HCDR3 as set forth in SEQ ID NO:69, a LCDR1 as set forth in SEQ ID NO:72, a LCDR2 as set forth in SEQ ID NO:73, and a LCDR3 as set forth in SEQ ID NO:74.

88. The monoclonal antibody or antigen binding fragment thereof of claim 87, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 66 and a light chain as set forth in SEQ ID NO:71.

89. A monoclonal antibody or antigen-binding fragment thereof that selectively binds Staphylococcus aureus antigen Protein A, wherein the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO:76, a HCDR2 as set forth in SEQ ID NO:77, a HCDR3 as set forth in SEQ ID NO:78, a LCDR1 as set forth in SEQ ID NO:81, a LCDR2 as set forth in SEQ ID NO:82, and a LCDR3 as set forth in SEQ ID NO:83.

90. The monoclonal antibody or antigen-binding fragment thereof of claim 89, wherein tire antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:75 and a light chain as set forth in SEQ ID NO: 80.

91. The antibody or antigen-binding fragment thereof of any one of claims 83-90, wherein the molecule binds to a target with an affinity between 1 pM and 1 jiM.

92. A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 83-90.

93. A cloning vector or expression cassette comprising the polynucleotide of claim 92.

94. A host cell expressing the antibody or antigen-binding fragment thereof of any one of claims 83-90, or comprising the cloning vector or expression cassette of claim 93.

95. A method of producing an antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of claim 94 under conditions allowing forexpression of the antibody or antigen-binding fragment thereof and isolating the antibody or antigen- binding fragment thereof.

96. Tire use of the antibody or antigen-binding fragment thereof of any one of claims 83-90 to treat a microbial infection in a mammalian subject.

97. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 83-90 and one or more excipients.

98. The antibody or antigen-binding fragment thereof of any one of claims 83-90 for use in the manufacture of a medicament for treating a microbial infection.

99. The composition of claim 97 for use in treating a microbial infection.

Images