Fusion protein constructs for complement-associated diseases

Abstract

To provide a monomeric fusion protein construct that binds a complement-associated antigen, wherein the monomeric fusion protein construct can bind a complement-associated antigen and comprises a targeting moiety and a complement modulator protein, or a fragment thereof or a variant thereof.SOLUTION: In some examples, the targeting moiety is an antibody or an antigen binding fragment thereof. Another embodiment is a trivalent heterodimeric fusion protein construct that binds a complement-associated antigen. Also provided are methods of using the fusion protein constructs, for example, in treating complement mediation conditions.SELECTED DRAWING: Figure 14

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 778,014, filed December 11, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on December 11, 2019, is named 53542_707_601_SL.txt, and is 950,242 bytes in size. Summary of the Invention

[0003] In one embodiment, the present specification provides a monomeric fusion protein construct that binds to a complement-related antigen, the monomeric fusion protein construct comprising a first polypeptide comprising Domain A and Domain B arranged in an AB orientation from N-terminus to C-terminus, and a second polypeptide comprising Domain E and Domain F arranged in an EF orientation from N-terminus to C-terminus, wherein at least one of Domain A, Domain B, Domain E, or Domain F is optionally conjugated to Domain R, wherein Domain A may comprise a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, Domain R comprises a complement regulator polypeptide, Domain E may comprise a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and Domain F may comprise a light chain constant region amino acid sequence (CL1), and Domain B of the first polypeptide and Domain F of the second polypeptide are connected via one or more disulfide bonds.

[0004] In some embodiments, a first polypeptide comprises Domain A, Domain B, and Domain R, wherein the domains of the first polypeptide are arranged N-terminally to C-terminally in an R-Ab orientation, with Domain R being conjugated to Domain A. In some embodiments, a first polypeptide comprises Domain A, Domain B, and Domain R, wherein the domains of the first polypeptide are arranged N-terminally to C-terminally in an A-Bb orientation, with Domain B being conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, wherein the domains of the second polypeptide are arranged N-terminally to C-terminally in an R-Fb orientation, with Domain E being conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, wherein the domains of the second polypeptide are arranged N-terminally to C-terminally in an E-Fr orientation, with Domain F being conjugated to Domain R. In some embodiments, the monomeric fusion protein construct further comprises a second complement regulatory polypeptide, wherein the second complement regulatory polypeptide and Domain R are the same or different.

[0005] One embodiment is a fusion protein construct that binds to a complement-related antigen, which may include a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, Domain R, a hinge region, Domain C, and Domain D, wherein Domains A, B, hinge region, C, and D of the first polypeptide are arranged N-terminally to C-terminally in an AB-hinge-CD orientation, and Domain A may comprise a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B may comprise a heavy chain CH1 constant region amino acid sequence, and Domain C comprises a heavy chain CH2 constant region amino acid sequence. the first polypeptide may comprise a heavy chain CH3 constant region amino acid sequence, and the second polypeptide may comprise a domain E and a domain F, wherein domains E and F of the second polypeptide are arranged N-terminally to C-terminally in an E-F orientation, and (i) domain E may comprise a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and (ii) domain F may comprise a light chain constant region amino acid sequence (CL1), and domain B of the first polypeptide and domain F of the second polypeptide are connected via one or more disulfide bonds.

[0006] In some embodiments, the fusion protein construct may be a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the first polypeptides may comprise conjugated domains D and R, and the two first polypeptides are linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct may be a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the first polypeptides may comprise conjugated domains A and R, and the two first polypeptides are linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct may further comprise domain R1, which may comprise a second complement regulatory polypeptide, and wherein domain R1 and domain R may be the same or different.

[0007] In some embodiments, domain A may be conjugated to domain R, and domain D may be conjugated to domain R1. In some embodiments, domain D may be conjugated to domain R, and domain A may be conjugated to domain R1. In some embodiments, the fusion protein construct may be a tetravalent heterodimeric fusion protein construct comprising: c) a third polypeptide comprising: (i) domain A, domain B, hinge region, domain C, and domain D; (ii) domain A, domain B, hinge region, and domain C; or (iii) domain A, domain B, and hinge region, wherein domains A, B, hinge region, C, and D of the third polypeptide are arranged N-terminally to C-terminally in an AB-hinge region-CD orientation; and d) a fourth polypeptide comprising domain E and domain F, wherein domains E and F of the fourth polypeptide are arranged N-terminally to C-terminally in an EF orientation, wherein domain B of the third polypeptide and domain F of the fourth polypeptide are connected via one or more disulfide bonds, and the first and third polypeptides are connected via one or more disulfide bonds at the hinge region. In some embodiments, domain D and domain R may be conjugated in the first polypeptide. In some embodiments, domain A and domain R may be conjugated in the first polypeptide.

[0008] In some embodiments, the fusion protein construct may further comprise domain R1, which comprises a second complement regulatory polypeptide, and domain R1 and domain R may be the same or different. In some embodiments, domain R1 may be conjugated to a first polypeptide. In some embodiments, domain R1 may be conjugated to a second polypeptide. In some embodiments, domain R1 is conjugated to a third polypeptide. In some embodiments, domain R1 is conjugated to a fourth polypeptide.

[0009] Another embodiment is a fusion protein construct that binds to a complement-related antigen, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, Domain R, a hinge region, and Domain C, and wherein Domain A of the first polypeptide the first polypeptide comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, the second polypeptide comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and the second polypeptide comprises a light chain constant region amino acid sequence (CL1), the second polypeptide comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and the second polypeptide comprises a light chain constant region amino acid sequence (CL1), the second polypeptide comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and the second polypeptide comprises a light chain constant region amino acid sequence (CL1), and the second polypeptide comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof.

[0010] In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the first polypeptides may have Domain C conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the first polypeptides may have Domain A conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct further comprises Domain R1, which may comprise a second complement regulatory polypeptide, and Domain R1 and Domain R may be the same or different. In some embodiments, Domain A and Domain R may be conjugated, and Domain C and Domain R1 may be conjugated. In some embodiments, domain C and domain R may be conjugated, and domain A and domain R1 may be conjugated.

[0011] In some embodiments, the fusion protein construct is a tetravalent heterodimeric fusion protein construct comprising: c) a third polypeptide comprising: (i) Domain A, Domain B, a hinge region, Domain C, and Domain D; (ii) Domain A, Domain B, a hinge region, and Domain C; or (iii) Domain A, Domain B, and a hinge region, wherein Domains A, B, the hinge region, C, and D of the third polypeptide are optionally arranged N-terminally to C-terminally in an AB-hinge-CD orientation; and d) a fourth polypeptide comprising Domain E and Domain F, wherein Domains E and F of the fourth polypeptide are optionally arranged N-terminally to C-terminally in an EF orientation, wherein Domain B of the third polypeptide and Domain F of the fourth polypeptide are optionally connected via one or more disulfide bonds, and wherein the first and third polypeptides are optionally connected at the hinge region via one or more disulfide bonds.

[0012] In some embodiments, domain C and domain R may be conjugated in the first polypeptide. In some embodiments, domain A and domain R may be conjugated in the first polypeptide. In some embodiments, the fusion protein construct further comprises domain R1, which comprises a second complement regulatory polypeptide, and domain R1 and domain R may be the same or different. In some embodiments, domain R1 is conjugated to the first polypeptide. In some embodiments, domain R1 is conjugated to the second polypeptide. In some embodiments, domain R1 is conjugated to the third polypeptide. In some embodiments, domain R1 is conjugated to the fourth polypeptide.

[0013] Another embodiment is a fusion protein construct that binds to a complement-related antigen, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, Domain R, and a hinge region, and Domains A, B, and the hinge region of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region orientation, Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, and Domain R comprises a complement regulator polypeptide, wherein: (1) Domain A and Domain R may be conjugated together, or (2) the hinge region and Domain R may be conjugated together, the second polypeptide may comprise Domain E and Domain F, and Domain E and F may be arranged N-terminally to C-terminally in an E-F orientation, Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and Domain F comprises a light chain constant region amino acid sequence (CL1), and Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds.

[0014] In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the first polypeptides may have Domain A conjugated to Domain R, and the two first polypeptides may be linked together at the hinge region via one or more disulfide bonds. In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the first polypeptides may have Domain R conjugated to the hinge region, and the two first polypeptides may be linked together at the hinge region via one or more disulfide bonds. In some embodiments, the fusion protein construct further comprises Domain R1, wherein Domain R1 comprises a second complement regulatory polypeptide, and Domain R1 and Domain R may be the same or different. In some embodiments, Domain A and Domain R may be conjugated to Domain R, and the hinge domain and Domain R1 may be conjugated to Domain R1. In some embodiments, the hinge domain may be conjugated to Domain R, or Domain A may be conjugated to Domain R1. In some embodiments, the fusion protein construct is a tetravalent heterodimeric fusion protein construct comprising: c) a third polypeptide comprising: (i) Domain A, Domain B, hinge region, Domain C, and Domain D; (ii) Domain A, Domain B, hinge region, and Domain C; or (iii) Domain A, Domain B, and hinge region, wherein Domains A, B, hinge region, C, and D of the third polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region-CD orientation; and d) a fourth polypeptide comprising Domain E and Domain F, wherein Domains E and F of the fourth polypeptide may be arranged N-terminally to C-terminally in an EF orientation, wherein Domain B of the third polypeptide and Domain F of the fourth polypeptide are connected via one or more disulfide bonds, and wherein the first and third polypeptides are connected via one or more disulfide bonds at the hinge region.

[0015] In some embodiments, the hinge domain and domain R may be conjugated in the first polypeptide. In some embodiments, domain A and domain R may be conjugated in the first polypeptide. In some embodiments, the fusion protein construct further comprises domain R1, which comprises a second complement regulatory polypeptide, and domain R1 and domain R may be the same or different. In some embodiments, domain R1 is conjugated to the first polypeptide. In some embodiments, domain R1 is conjugated to the second polypeptide. In some embodiments, domain R1 is conjugated to the third polypeptide. In some embodiments, domain R1 is conjugated to the fourth polypeptide.

[0016] Another embodiment is a trivalent heterodimeric fusion protein construct that binds to a complement related antigen, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, Domain D, and Domain R, and wherein Domains A, B, hinge region, C, and D of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region-CD orientation, and wherein (1) Domain A and Domain R may be conjugated, or (2) Domain D and The second polypeptide may comprise domain E and domain F, with domains E and F of the second polypeptide arranged in an E-F orientation from the N-terminus to the C-terminus; the third polypeptide may comprise (i) domain A, domain B, a hinge region, domain C, and domain D, (ii) domain A, domain B, a hinge region, and domain C, or (iii) domain A, domain B, and a hinge region, with domains A, B, a hinge region, C, and D of the third polypeptide arranged in an E-F orientation from the N-terminus to the C-terminus. the fourth polypeptide may comprise Domain E and Domain F, and Domains E and F of the fourth polypeptide may be arranged N-terminally to C-terminally in an EF orientation; Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds; Domain B of the third polypeptide and Domain F of the fourth polypeptide may be connected via one or more disulfide bonds; the first and third polypeptides may be connected to each other via one or more disulfide bonds in the hinge region; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; Domain B comprises a heavy chain CH1 constant region amino acid sequence; Domain R comprises a complement regulator polypeptide; Domain C comprises a heavy chain CH2 constant region amino acid sequence; Domain D comprises a heavy chain CH3 constant region amino acid sequence; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and Domain F comprisesThe trivalent heterodimeric fusion protein construct comprises a light chain constant region amino acid sequence (CL1).

[0017] In some embodiments, domain A of the first polypeptide may be conjugated to domain R. In some embodiments, domain D of the first polypeptide may be conjugated to domain R.

[0018] Another embodiment is a trivalent heterodimeric fusion protein construct that binds to a complement related antigen, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain R, and wherein Domains A, B, hinge region, and C of the first polypeptide are optionally arranged N-terminally to C-terminally in an AB-hinge-C orientation, and wherein (1) Domain A and Domain R are conjugated, or (2) Domain C and Domain R are conjugated, and the second polypeptide comprises Domain E and Domain F, and wherein Domains E and F of the second polypeptide are optionally arranged N-terminally to C-terminally in an EF orientation, and the third polypeptide comprises (i) Domain A, Domain B, hinge region, Domain C, and Domain R, and wherein Domains A, B, hinge region, and Domain R of the first polypeptide are optionally arranged N-terminally to C-terminally in an EF orientation, and wherein (ii) Domain A and Domain R are conjugated, or (iii) Domain C and Domain R are conjugated, The third polypeptide may comprise (i) domain A, domain B, hinge region and domain C, or (iii) domain A, domain B and hinge region, and domains A, B, hinge region, C and D of the third polypeptide may be arranged in an AB-hinge region-CD orientation from the N-terminus to the C-terminus; the fourth polypeptide may comprise domain E and domain F, and domains E and F of the fourth polypeptide may be arranged in an EF orientation from the N-terminus to the C-terminus; domain B of the first polypeptide and domain F of the second polypeptide may be connected via one or more disulfide bonds; domain B of the third polypeptide and domain F of the fourth polypeptide may be connected via one or more disulfide bonds; and the first and third polypeptides may have a structure in which the hinge region is and Domain F comprises a light chain constant region amino acid sequence (CL1).

[0019] In some embodiments, the first polypeptide may comprise a conjugate between Domain A and Domain R. In some embodiments, the first polypeptide may comprise a conjugate between Domain C and Domain R.

[0020] Yet another embodiment is a trivalent heterodimeric fusion protein construct that binds to a complement related antigen, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, and Domain R, and wherein Domains A, B, and the hinge region of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region orientation, and wherein (1) Domain A and Domain R are conjugated, or (2) the hinge region and Domain R are conjugated. The second polypeptide may comprise domain E and domain F, with domains E and F of the second polypeptide arranged in an E-F orientation from the N-terminus to the C-terminus; the third polypeptide may comprise (i) domain A, domain B, a hinge region, domain C, and domain D, (ii) domain A, domain B, a hinge region, and domain C, or (iii) domain A, domain B, and a hinge region, with domains A, B, a hinge region, C, and D of the third polypeptide arranged in an A-F orientation from the N-terminus to the C-terminus. the fourth polypeptide may comprise Domain E and Domain F, and Domains E and F of the fourth polypeptide may be arranged N-terminally to C-terminally in an E-F orientation; Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds; Domain B of the third polypeptide and Domain F of the fourth polypeptide may be connected via one or more disulfide bonds; the first and third polypeptides may be connected to each other at the hinge region via one or more disulfide bonds; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; Domain B comprises a heavy chain CH1 constant region amino acid sequence; Domain R comprises a complement regulator polypeptide; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and Domain F comprises a light chain constant region amino acid sequence (CL1).

[0021] In some embodiments, the first polypeptide may comprise a conjugation between Domain A and Domain R. In some embodiments, the first polypeptide may comprise a conjugation between the hinge region and Domain R.

[0022] Another embodiment is a fusion protein construct that binds to a complement related antigen, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain D, wherein Domains A, B, hinge region, C, and D are optionally arranged N-terminally to C-terminally in an AB-hinge-CD orientation, wherein Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, Domain C comprises a heavy chain CH2 constant region amino acid sequence, and Domain D comprises a heavy chain CH3 constant region amino acid sequence, and the second polypeptide comprises Domain E, Domain F, and Domain R, wherein Domains E and F are arranged N-terminally to C-terminally in an AB-hinge-CD orientation, The fusion protein construct may be arranged in an E-F orientation, and (1) domain E and domain R may be conjugated, or (2) domain F and domain R may be conjugated, wherein domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, domain F comprises a light chain constant region amino acid sequence (CL1), and domain B of the first polypeptide and domain F of the second polypeptide may be connected via one or more disulfide bonds.

[0023] In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the second polypeptides may have Domain E conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the second polypeptides may have Domain F conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct further comprises Domain R1, wherein Domain R1 comprises a second complement regulatory polypeptide, and Domain R1 and Domain R may be the same or different. In some embodiments, Domain E and Domain R may be conjugated, and Domain F and Domain R1 may be conjugated. In some embodiments, Domain F may be conjugated to Domain R, and Domain E may be conjugated to Domain R1. In some embodiments, the fusion protein construct is a tetravalent heterodimeric fusion protein construct comprising: c) a third polypeptide comprising: (i) Domain A, Domain B, a hinge region, Domain C, and Domain D; (ii) Domain A, Domain B, a hinge region, and Domain C; or (iii) Domain A, Domain B, and a hinge region, wherein Domains A, B, the hinge region, C, and D of the third polypeptide are arranged N-terminally to C-terminally in an AB-hinge-CD orientation; and d) a fourth polypeptide comprising Domain E and Domain F, wherein Domains E and F of the fourth polypeptide are arranged N-terminally to C-terminally in an EF orientation, wherein Domain B of the third polypeptide and Domain F of the fourth polypeptide are connected via one or more disulfide bonds, and wherein the first and third polypeptides are connected via one or more disulfide bonds at the hinge region.

[0024] In some embodiments, domain E and domain R may be conjugated in the first polypeptide. In some embodiments, domain F and domain R may be conjugated in the first polypeptide. In some embodiments, the fusion protein construct further comprises domain R1, which comprises a second complement regulatory polypeptide, and domain R1 and domain R may be the same or different. In some embodiments, domain R1 is conjugated to the first polypeptide. In some embodiments, domain R1 is conjugated to the second polypeptide. In some embodiments, domain R1 is conjugated to the third polypeptide. In some embodiments, domain R1 is conjugated to the fourth polypeptide.

[0025] Another embodiment is a fusion protein construct that binds to a complement-related antigen, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, and Domain C, and Domains A, B, hinge region, and C may be arranged N-terminally to C-terminally in an AB-hinge-C orientation, wherein Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, and Domain C comprises a heavy chain CH2 constant region amino acid sequence, and the second polypeptide comprises Domain E, Domain F, Domain G, Domain H, Domain I, Domain I, Domain I, Domain II, Domain III ... and Domain R, wherein Domains E and F may be arranged N-terminally to C-terminally in an E-F orientation; (1) Domain E and Domain R may be conjugated together, or (2) Domain F and Domain R may be conjugated together; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; Domain F comprises a light chain constant region amino acid sequence (CL1); and Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds.

[0026] In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the second polypeptides may have Domain E conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the second polypeptides may have Domain F conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct further comprises Domain R1, wherein Domain R1 comprises a second complement regulatory polypeptide, and Domain R1 and Domain R may be the same or different. In some embodiments, Domain E and Domain R may be conjugated, and Domain F and Domain R1 may be conjugated. In some embodiments, the fusion protein construct may conjugate Domain F and Domain R, or conjugate Domain E and Domain R1. In some embodiments, the fusion protein construct is a tetravalent heterodimeric fusion protein construct comprising: c) a third polypeptide comprising: (i) Domain A, Domain B, a hinge region, Domain C, and Domain D; (ii) Domain A, Domain B, a hinge region, and Domain C; or (iii) Domain A, Domain B, and a hinge region, wherein Domains A, B, the hinge region, C, and D of the third polypeptide may be arranged N-terminally to C-terminally in an AB-hinge-region-CD orientation; and d) a fourth polypeptide comprising Domain E and Domain F, wherein Domains E and F of the fourth polypeptide may be arranged N-terminally to C-terminally in an EF orientation, wherein Domain B of the third polypeptide and Domain F of the fourth polypeptide are connected via one or more disulfide bonds, and wherein the first and third polypeptides are connected via one or more disulfide bonds at the hinge region.

[0027] In some embodiments, domain E and domain R may be conjugated in the first polypeptide. In some embodiments, domain F and domain R may be conjugated in the first polypeptide. In some embodiments, the fusion protein construct further comprises domain R1, where domain R1 comprises a second complement regulatory polypeptide, and domain R1 and domain R may be the same or different. In some embodiments, domain R1 is conjugated to the first polypeptide. In some embodiments, domain R1 is conjugated to the second polypeptide. In some embodiments, domain R1 is conjugated to the third polypeptide. In some embodiments, domain R1 is conjugated to the fourth polypeptide.

[0028] Yet another embodiment is a fusion protein construct that binds to a complement-related antigen, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, and a hinge region, and Domains A, B, and the hinge region may be arranged N-terminally to C-terminally in an AB-hinge region orientation, and Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, and Domain C comprises a heavy chain CH2 constant region amino acid sequence. the second polypeptide comprises a Domain E, Domain F, and Domain R, where Domain E and F may be arranged N-terminally to C-terminally in an E-F orientation, and (1) Domain E and Domain R may be conjugated together, or (2) Domain F and Domain R may be conjugated together, where Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and Domain F comprises a light chain constant region amino acid sequence (CL1), and Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds.

[0029] In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the second polypeptides may have Domain E conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct is a tetravalent homodimeric fusion protein construct comprising two first polypeptides and two second polypeptides, wherein at least one of the second polypeptides may have Domain F conjugated to Domain R, and the two first polypeptides may be linked together via one or more disulfide bonds at the hinge region. In some embodiments, the fusion protein construct further comprises Domain R1, wherein Domain R1 comprises a second complement regulatory polypeptide, and Domain R1 and Domain R may be the same or different. In some embodiments, Domain E and Domain R may be conjugated, and Domain F and Domain R1 may be conjugated. In some embodiments, domain F and domain R may be conjugated, and domain E and domain R1 may be conjugated.

[0030] In some embodiments, the fusion protein construct is a tetravalent heterodimeric fusion protein construct comprising: c) a third polypeptide comprising: (i) Domain A, Domain B, a hinge region, Domain C, and Domain D; (ii) Domain A, Domain B, a hinge region, and Domain C; or (iii) Domain A, Domain B, and a hinge region, wherein Domains A, B, the hinge region, C, and D of the third polypeptide are optionally arranged N-terminally to C-terminally in an AB-hinge-CD orientation; and d) a fourth polypeptide comprising Domain E and Domain F, wherein Domains E and F of the fourth polypeptide are optionally arranged N-terminally to C-terminally in an EF orientation, wherein Domain B of the third polypeptide and Domain F of the fourth polypeptide are optionally connected via one or more disulfide bonds, and wherein the first and third polypeptides are optionally connected at the hinge region via one or more disulfide bonds.

[0031] In some embodiments, Domain E and Domain R may be conjugated in the first polypeptide. In some embodiments, Domain F and Domain R may be conjugated in the first polypeptide.

[0032] In some embodiments, the fusion protein construct further comprises domain R1, which comprises a second complement regulatory polypeptide, and domain R1 and domain R may be the same or different. In some embodiments, domain R1 is conjugated to a first polypeptide. In some embodiments, domain R1 is conjugated to a second polypeptide. In some embodiments, domain R1 is conjugated to a third polypeptide. In some embodiments, domain R1 is conjugated to a fourth polypeptide.

[0033] Another embodiment is a trivalent heterodimeric fusion protein construct that binds to a complement related antigen, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. wherein the first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain D, and Domains A, B, hinge region, C, and D of the first polypeptide may be arranged in an AB-hinge region-CD orientation from the N-terminus to the C-terminus; the second polypeptide comprises Domain E, Domain F, and Domain R, and Domains E and F are arranged in an EF orientation from the N-terminus to the C-terminus; and (1) Domain E and Domain R may be conjugated, or (2) Domain F and Domain R may be conjugated. and may be conjugated, the third polypeptide may comprise (i) domain A, domain B, domain C, domain D and a hinge region, (ii) domain A, domain B, domain C and a hinge region, or (iii) domain A, domain B and a hinge region, and domains A, B, hinge region, C and D of the third polypeptide may be arranged in an AB-hinge region-CD orientation from the N-terminus to the C-terminus, and the fourth polypeptide may comprise domain E and domain F, and domains E and D of the fourth polypeptide may be conjugated, and F may be arranged N-terminally to C-terminally in an E-F orientation; Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds; Domain B of the third polypeptide and Domain F of the fourth polypeptide may be connected via one or more disulfide bonds; the first and third polypeptides may be connected to each other at the hinge region via one or more disulfide bonds; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; Domain B comprises a heavy chain CH1 constant region amino acid sequence; Domain C comprises a heavy chain CH2 constant region amino acid sequence; Domain D comprises a heavy chain CH3 constant region amino acid sequence; Domain R comprises a complement regulator polypeptide; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and Domain F comprises a light chain constant region amino acid sequence (CL1).

[0034] In some embodiments, domain E and domain R of the second polypeptide may be conjugated. In some embodiments, domain F and domain R of the second polypeptide may be conjugated.

[0035] Another embodiment is a trivalent heterodimeric fusion protein construct that binds to a complement related antigen, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, and Domain C, and wherein Domains A, B, the hinge region, and C of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region-C orientation; the second polypeptide comprises Domain E, Domain F, and Domain R, and wherein Domains E and F may be arranged N-terminally to C-terminally in an EF orientation; and wherein (1) Domain E and Domain R are conjugated or (2) Domain F and Domain R are conjugated; and the third polypeptide comprises (i) Domain A, Domain B, Domain C, Domain D and the hinge region; (ii) Domain A, Domain B, Domain C and the hinge region; or (iii) Domain A, Domain B and the hinge region. domains A, B, hinge region, C, and D of the third polypeptide may be arranged in an AB-hinge region-CD orientation from the N-terminus to the C-terminus; the fourth polypeptide may comprise domain E and domain F, and domains E and F of the fourth polypeptide may be arranged in an EF orientation from the N-terminus to the C-terminus; domain B of the first polypeptide and domain F of the second polypeptide may be connected via one or more disulfide bonds; domain B of the third polypeptide and domain F of the fourth polypeptide may be connected via one or more disulfide bonds; the first and third polypeptides may be connected to each other via one or more disulfide bonds in the hinge region; domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; domain B comprises a heavy chain CH1 constant region amino acid sequence; and domain C comprises a heavy chain CH2 constant region amino acid sequence. wherein Domain R comprises a complement regulator polypeptide, Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and Domain F comprises a light chain constant region amino acid sequence (CL1).

[0036] In some embodiments, domain E and domain R of the second polypeptide may be conjugated. In some embodiments, domain F and domain R of the second polypeptide may be conjugated.

[0037] Another embodiment is a trivalent heterodimeric fusion protein construct that binds to a complement related antigen, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, and a hinge region, wherein Domains A, B, and the hinge region of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region orientation; the second polypeptide comprises Domain E, Domain F, and Domain R, wherein Domains E and F may be arranged N-terminally to C-terminally in an EF orientation, wherein (1) Domain E and Domain R are conjugated or (2) Domain F and Domain R are conjugated; and the third polypeptide comprises (i) Domain A, Domain B, Domain C, Domain D, and a hinge region; (ii) Domain A, Domain B, Domain C, and a hinge region; or (iii) Domain A, Domain B, and a hinge region, wherein Domains A, B, the hinge region, C, and D of the third polypeptide are arranged N-terminally to C-terminally in an AB-hinge region orientation. the fourth polypeptide may comprise Domain E and Domain F, and Domains E and F of the fourth polypeptide may be arranged N-terminally to C-terminally in an E-F orientation; Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds; Domain B of the third polypeptide and Domain F of the fourth polypeptide may be connected via one or more disulfide bonds; the first and third polypeptides may be connected via one or more disulfide bonds at the hinge region; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; Domain B comprises a heavy chain CH1 constant region amino acid sequence; Domain R comprises a complement regulator polypeptide; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and Domain F comprises a light chain constant region amino acid sequence (CL1).

[0038] In some embodiments, Domain E and Domain R may be conjugated in the second polypeptide. In some embodiments, Domain F and Domain R may be conjugated in the second polypeptide.

[0039] In another embodiment, there is provided a fusion protein construct that binds to a complement-related antigen, comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises Domain R, a hinge region, Domain C, and Domain D, wherein Domain R, the hinge region, C, and D are optionally arranged N-terminally to C-terminally in an R-hinge-CD orientation; Domain R comprises a complement regulator polypeptide; Domain C comprises a heavy chain CH2 constant region amino acid sequence; Domain D comprises a heavy chain CH3 constant region amino acid sequence, wherein Domain R and the hinge domain are optionally linked; the second polypeptide comprises Domain A, Domain B, the hinge region, Domain C, and Domain D, wherein Domains A, B, the hinge region, C, and D are optionally arranged N-terminally to C-terminally in an AB-hinge-CD orientation; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; and Domain B comprises a heavy chain CH1 constant region amino acid sequence; the domains may be arranged in an E-F orientation from the N-terminus to the C-terminus, wherein domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and domain F comprises a light chain constant region amino acid sequence (CL1), wherein domain B of the second polypeptide and domain F of the third polypeptide may be connected via one or more disulfide bonds, and wherein the first and second polypeptides may be connected via one or more disulfide bonds in the hinge region.

[0040] Another embodiment provides a monomeric fusion protein construct that binds to a complement-related antigen, comprising: a first polypeptide comprising Domain A, Domain B, and a hinge region arranged N-terminally to C-terminally in an AB-hinge orientation; and a second polypeptide comprising Domain E and Domain F arranged N-terminally to C-terminally in an EF orientation, wherein at least one of Domain A, the hinge domain, Domain E, or Domain F is conjugated to Domain R; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; Domain B comprises a heavy chain CH1 constant region amino acid sequence; Domain R comprises a complement regulator polypeptide; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and Domain F comprises a light chain constant region amino acid sequence (CL1); and optionally, Domain B of the first polypeptide and Domain F of the second polypeptide are connected via one or more disulfide bonds.

[0041] In some embodiments, a first polypeptide comprises Domain A, Domain B, a hinge domain, and Domain R, and the domains of the first polypeptide may be arranged N-terminally to C-terminally in an RAB-hinge orientation, with Domain R optionally conjugated to Domain A. In some embodiments, a first polypeptide comprises Domain A, Domain B, a hinge domain, and Domain R, and the domains of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge-R orientation, with Domain R optionally conjugated to Domain A. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, and the domains of the second polypeptide may be arranged N-terminally to C-terminally in an REF orientation, with Domain E optionally conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, and the domains of the second polypeptide may be arranged N-terminally to C-terminally in an EFR orientation, with Domain F optionally conjugated to Domain R. In some embodiments, the fusion protein construct further comprises a second complement regulatory polypeptide, which may be the same as or different from Domain R.

[0042] Another embodiment provides a monomeric fusion protein construct that binds to a complement-related antigen, the monomeric fusion protein construct comprising: a first polypeptide comprising Domain A, Domain B, a hinge region, and Domain C arranged N-terminally to C-terminally in an AB-hinge-C region orientation; and a second polypeptide comprising Domain E and Domain F arranged N-terminally to C-terminally in an EF orientation, wherein at least one of Domain A, Domain C, Domain E, or Domain F is conjugated to Domain R; Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; Domain B comprises a heavy chain CH1 constant region amino acid sequence; Domain C comprises a heavy chain CH2 constant region amino acid sequence; Domain R comprises a complement regulator polypeptide; Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and Domain F comprises a light chain constant region amino acid sequence (CL1); and optionally, Domain B of the first polypeptide and Domain F of the second polypeptide are connected via one or more disulfide bonds.

[0043] In some embodiments, a first polypeptide comprises Domain A, Domain B, a hinge domain, Domain C, and Domain R, and the domains of the first polypeptide may be arranged N-terminally to C-terminally in an RAB-hinge-C orientation, with Domain R optionally conjugated to Domain A. In some embodiments, a first polypeptide comprises Domain A, Domain B, a hinge domain, Domain C, and Domain R, and the domains of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge-CR orientation, with Domain C optionally conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, and the domains of the second polypeptide may be arranged N-terminally to C-terminally in an REF orientation, with Domain E optionally conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, and the domains of the second polypeptide may be arranged N-terminally to C-terminally in an EFR orientation, with Domain F optionally conjugated to Domain R. In some embodiments, the fusion protein construct further comprises a second complement regulatory polypeptide, which may be the same as or different from Domain R.

[0044] Another embodiment is a monomeric fusion protein construct that binds to a complement-related antigen, comprising: a) a first polypeptide comprising Domain A, Domain B, a hinge region, Domain C, and Domain D arranged N-terminally to C-terminally in an AB-hinge-CD region orientation; and b) a second polypeptide comprising Domain E and Domain F arranged N-terminally to C-terminally in an EF orientation, wherein at least one of Domain A, Domain D, Domain E, or Domain F is conjugated to Domain R; and (i) Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof; and (ii) Domain B comprises a heavy chain CH1 constant region amino acid sequence (VH) or an antigen-binding fragment thereof. (iii) Domain C comprises a heavy chain CH2 constant region amino acid sequence; (iv) Domain D comprises a heavy chain CH3 constant region amino acid sequence; (v) Domain R comprises a complement regulator polypeptide; (vi) Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof; and (vii) Domain F comprises a light chain constant region amino acid sequence (CL1), wherein Domain B of the first polypeptide and Domain F of the second polypeptide may be connected via one or more disulfide bonds.

[0045] In some embodiments, a first polypeptide comprises Domain A, Domain B, a hinge domain, Domain C, Domain D, and Domain R, and the domains of the first polypeptide may be arranged N-terminally to C-terminally in an RAB-hinge-CD orientation, with Domain R optionally conjugated to Domain A. In some embodiments, a first polypeptide comprises Domain A, Domain B, a hinge domain, Domain C, Domain D, and Domain R, and the domains of the first polypeptide may be arranged N-terminally to C-terminally in an AB-hinge-CDR orientation, with Domain C optionally conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, and the domains of the second polypeptide may be arranged N-terminally to C-terminally in an REF orientation, with Domain E optionally conjugated to Domain R. In some embodiments, a second polypeptide comprises Domain E, Domain F, and Domain R, and the domains of the second polypeptide may be arranged N-terminally to C-terminally in an EFR orientation, with Domain F optionally conjugated to Domain R. In some embodiments, the fusion protein construct further comprises a second complement regulatory polypeptide, which may be the same as or different from Domain R.

[0046] In some embodiments, conjugation is achieved by joining the two domains with a peptide linker. In some embodiments, the fusion protein construct binds to C3 and C3b with a lower affinity than to C3d. In some embodiments, the fusion protein construct binds to C3 and C3b with a lower affinity than to C3d. In some embodiments, the fusion protein construct binds to C3 and C3b with a lower affinity than to C3d. In some embodiments, the fusion protein construct binds to C3 and C3b with a lower affinity than to C3d. In some embodiments, the fusion protein construct binds to C3 and C3b with a lower affinity than to C3d. -3 In some embodiments, the fusion protein construct binds iC3b, C3dg, or both with a KD affinity of 10 M or greater. -8The fusion protein construct binds with a KD affinity of M or less. In some embodiments, the fusion protein construct modulates alternative complement activity in a subject when the fusion protein construct or a pharmaceutical composition comprising the fusion protein construct is administered to the subject. In some embodiments, the fusion protein construct modulates classical complement activity in a subject when the fusion protein construct or a pharmaceutical composition comprising the fusion protein construct is administered to the subject. In some embodiments, the fusion protein construct modulates lectin complement activity in a subject when the fusion protein construct or a pharmaceutical composition comprising the fusion protein construct is administered to the subject. In some embodiments, the fusion protein construct binds to a domain of a mammalian annexin protein.

[0047] In some embodiments, the fusion protein construct comprises a domain of a mammalian annexin protein. -8 In some embodiments, the domain is an annexin core domain. In some embodiments, the annexin core domain comprises an alpha helical domain. In some embodiments, the annexin core domain comprises a calcium binding site and a membrane binding site. In some embodiments, the annexin core domain comprises at least one annexin repeat. In some embodiments, the fusion protein construct binds to an annexin repeat sequence in the domain. In some embodiments, the fusion protein construct binds to phospholipids. In some embodiments, the fusion protein construct binds to phospholipids with a KD affinity of 10 M or less. In some embodiments, the annexin core domain comprises an alpha helical domain. In some embodiments, the annexin core domain comprises a calcium binding site and a membrane binding site. In some embodiments, the annexin core domain comprises at least one annexin repeat. In some embodiments, the fusion protein construct binds to annexin repeat sequences in the domain. In some embodiments, the fusion protein construct binds to phospholipids. -8In some embodiments, the phospholipid binds with a KD affinity of no greater than M. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylethanolamine (PE), cardiolipin (CL), phosphatidylcholine (PC), phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, and phosphatidic acid and malondialdehyde (MDA). In some embodiments, the complement regulatory peptide comprises domain A of complement receptor 1 (CR1) protein, or a fragment thereof retaining at least three short consensus repeats (SCRs) of domain A. In some embodiments, the fusion protein construct further comprises domain B of CR1 protein, or a fragment thereof retaining at least three SCRs of domain B.

[0048] In some embodiments, the fusion protein construct further comprises Domain C of the CR1 protein, or a fragment thereof retaining at least three SCRs of Domain C. In some embodiments, the fusion protein construct further comprises Domain D of CR1, or a fragment thereof retaining at least three SCRs of Domain D. In some embodiments, the complement regulator polypeptide comprises the first three SCRs of Domain A, the first three SCRs of Domain B, and the first three SCRs of Domain C of the CR1 protein. In some embodiments, the complement regulator polypeptide is CR1(1-10). In some embodiments, the complement regulator polypeptide is CR1(1-17). In some embodiments, the complement regulator peptide comprises the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:91, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator polypeptide comprises the amino acid sequence of SEQ ID NO:42 or SEQ ID NO:92, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is a DNA activating factor (DAF) or a biologically active fragment thereof. In some embodiments, the DAF is human DAF. In some embodiments, the bioactive fragment of human DAF comprises at least one of the short consensus repeat (SCR) domain and the O-glycosylated serine / threonine-rich domain of full-length human DAF. Includes one.

[0049] In some embodiments, the bioactive fragment of DAF comprises SCR1-4 or SCR2-4 of full-length human DAF. In some embodiments, the bioactive fragment of DAF comprises the amino acid sequence of SEQ ID NO: 184, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is Factor H or a bioactive fragment thereof. In some embodiments, Factor H is human Factor H. In some embodiments, a biologically active fragment of human Factor H comprises one or more of the following groups of short consensus repeats (SCRs): SCR1-20, SCR1-2, SCR2-3, SCR3-4, SCR4-5, SCR5-6, SCR6-7, SCR7-8, SCR8-9, SCR9-10, SCR10-11, SCR11-12, SCR12-13, SCR13-14, SCR14-15, SCR15-16, SCR16-17, SCR17-18, SCR19-20 of full-length human Factor H, or any combination of SCR1-20. In some embodiments, a biologically active fragment of human Factor H comprises SCR1-4 or SCR1-5 of full-length human Factor H. In some embodiments, the biologically active fragment of human Factor H comprises a stretch of amino acids selected from the group consisting of amino acids 21 to 266, amino acids 21 to 320, amino acids 21 to 509, or amino acids 19 to 1106 of SEQ ID NO: 9, or a variant thereof having an amino acid sequence that is at least 85% identical to the stretch of amino acids.

[0050] In some embodiments, the Factor H or a biologically active fragment thereof comprises the amino acid sequence of SEQ ID NO:72 or SEQ ID NO:108, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is MCP or a biologically active fragment thereof. In some embodiments, the MCP is human MCP. In some embodiments, the biologically active fragment of human MCP comprises at least one short consensus repeat (SCR) domain of full-length human MCP. In some embodiments, the biologically active fragment of human MCP comprises SCRs 3-4 of full-length human MCP. In some embodiments, the MCP comprises the amino acid sequence of SEQ ID NO:187, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is Map44 or a biologically active fragment thereof. In some embodiments, Map44 is human Map44. In some embodiments, Map44 comprises the amino acid sequence of SEQ ID NO:186, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is CD59 or a biologically active fragment thereof.

[0051] In some embodiments, the CD59 is human CD59.

[0052] In some embodiments, CD59 comprises the amino acid sequence of SEQ ID NO: 185, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the fusion protein construct comprises a human antibody or antigen-binding fragment thereof. In some embodiments, the fusion protein construct comprises a humanized antibody or antigen-binding fragment thereof. In some embodiments, the first and third polypeptides each comprise at least one orthogonal modification that favors heterodimer formation relative to homodimer formation. In some embodiments, the first polypeptide comprises a knob modification and the third polypeptide comprises a hole modification, or the third polypeptide comprises a knob modification and the first polypeptide comprises a hole modification. In some embodiments, the first and third polypeptides comprise modifications that result in charge or surface complementarity.

[0053] In some embodiments, a) the first polypeptide is selected from the group consisting of (i) SEQ ID NOs: 11, 12, and 13; 17, 18, and 19; 23, 24, and 25; 29, 30, and 31; 35, 36, and 37; 147, 148, and 149; 188, 189, and 190; 196, 197, and 198; 204 or 343, 205 and 206; 212, 213, and 214; 220, 221, and 222; 228, 229, and 230; 29, 259, and 31; or 29, 260 and 31 amino acid sequences, or (ii) three heavy chain complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NOs: 11, 12, and 13; 17, 18, and 19; 23, 24, and 25; 29, 30, and 31; 35, 36, and 37; 147, 148, and 149; 188, 189, and 190; 196, 197, and 198; 204 or 343, 205, and 206; 212, 213, and 214; 20, 221 and 222; 228, 229 and 230; 29, 259 and 31; or 29, 260 and 31; and b) a second polypeptide comprises three heavy chain CDRs having amino acid sequences that differ by a single conservative amino acid substitution in one of (i) SEQ ID NOs: 14, 15 and 16; 20, 21 and 22; 26, 27 and 28; 32, 33 and 34; 38, 39 and and 40; 150, 151 and 152; 191, 192 and 193; 199, 200 and 201; 207, 208 and 209; 215, 216 and 217; 223, 224 and 225; or 231, 232 and 233, or (ii) three light chain complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NOs: 14, 15 and 16; 20, 21 and 22; 26, 27 and 28; 32, 33, and 34; 38, 39, and 40; 150, 151, and 152; 191, 192, and 193; 199, 200, and 201; 207, 208, and 209; 215, 216, and 217; 223, 224, and 225; or 231, 232, and 233.

[0054] In some embodiments, the second polypeptide (light chain comprising domains E and F) comprises at least three CDRs, the light chain CDRs being defined as CDR-L1, CDR-L2, and CDR-L3, respectively, where in SEQ ID NOs: 279, 68, 287, and 59 the CDRs comprise residues 27-37 (CDR-L1), 55-57 (CDR-L2), and 94-102 (CDR-L3), and in SEQ ID NO: 289 the CDRs comprise residues 27-38 (CDR-L1), 56-58 (CDR-L2), and 95-102 (CDR-L3). In some embodiments, the first polypeptide (a heavy chain comprising at least domains A and B) comprises at least three CDRs, the heavy chain CDRs being defined as CDR-H1, CDR-H2, and CDR-H3, respectively, and in SEQ ID NOs: 280, 281, 282, 284, 285, 286, 73, and 288, the CDRs comprise residues 26-33 (CDR-H1), 51-58 (CDR-H2), and 97-100 (CDR-H3); in SEQ ID NO: 244, the CDRs comprise residues 26-33 (CDR-H1), 51-58 (CDR-H2), and 97-102 (CDR-H3); and in SEQ ID NO: 290, the CDRs comprise residues 26-33 (CDR-H1), 51-58 (CDR-H2), and 97-110 (CDR-H3). In some embodiments, the first polypeptide (a heavy chain comprising at least domains A and B) comprises at least three CDRs, and the heavy chain CDRs are defined as CDR-H1, CDR-H2, and CDR-H3, respectively, and in SEQ ID NO: 342, comprise SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. In some embodiments, the second polypeptide (a light chain comprising at least domains E and F) comprises at least three CDRs, and in the light chain, the light chain CDRs are defined as CDR-L1, CDR-L2, and CDR-L3, respectively, and comprise SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0055] In some embodiments, the fusion protein construct further comprises at least one amino acid linker, wherein the at least one linker comprises the amino acid sequence of any of SEQ ID NO:138, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:241 or SEQ ID NO:242.

[0056] In some embodiments, the fusion protein construct comprises SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52 , SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, 107, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 and SEQ ID NO:144. In some embodiments, a first polypeptide comprises the sequence of SEQ ID NO:282 (Domain A, Domain B, Domain C, and Domain D) conjugated to a complement regulator polypeptide comprising a sequence (Domain R) selected from the group consisting of SEQ ID NOs:41, 42, and 72, and a second polypeptide comprises the sequence of SEQ ID NO:279 (Domain E and Domain F). In some embodiments, a first polypeptide comprises the sequence of SEQ ID NO:282 (Domain A, Domain B, Domain C, and Domain D) and a second polypeptide comprises the sequence of SEQ ID NO:279 (Domain E and Domain F) conjugated to a complement regulator polypeptide comprising a sequence (Domain R) selected from the group consisting of SEQ ID NO:72.

[0057] Another embodiment provides a pharmaceutical composition comprising a fusion protein construct according to any one of the above embodiments. Yet another embodiment provides a polynucleotide encoding the fusion protein. Another embodiment provides a method of treatment comprising providing a therapeutically effective amount of the pharmaceutical composition to a subject.

[0058] In some embodiments, the subject is suffering from a complement-mediated disease or a complement-mediated inflammation. In some embodiments, the fusion protein construct specifically targets C3d. -8 In some embodiments, the fusion protein construct specifically binds to C2 antibody-reactive phospholipids with a KD affinity of 10 M or less, and the subject is suffering from a complement-mediated disease, the complement-mediated disease being characterized by increased deposition of C3d. -8 The antibody binds with a KD affinity of M or less, and the subject suffers from a complement-mediated disease, characterized by increased deposition of C2 antibody-reactive phospholipids. In some embodiments, the subject suffers from complement-mediated inflammation, including inflammatory fibrotic diseases, including focal segmental glomerulosclerosis, primary sclerosing cholangitis, or membranoproliferative glomerulonephritis. In some embodiments, the subject suffers from a complement-mediated autoimmune disease, including rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, or pemphigus vulgaris. In some embodiments, the subject suffers from a complement-mediated kidney disease, including membranoproliferative glomerulonephritis or complement 3 glomerulopathy. In some embodiments, the subject suffers from a complement-mediated cardiovascular disease. In some embodiments, the cardiovascular disease includes atherosclerosis or thrombosis. In some embodiments, the subject suffers from a complement-mediated skin disease.

[0059] In some embodiments, the skin disease comprises psoriasis, acne inversa, lupus erythematosus, cutaneous small vessel vasculitis, urticaria, urticarial vasculitis, or bullous pemphigoid. In some embodiments, the subject is suffering from complement-mediated inflammation, including ischemia / reperfusion injury, burns, endotoxemia and septic shock, adult respiratory distress syndrome, cardiopulmonary bypass, hemodialysis, anaphylactic shock, asthma, angioedema, Crohn's disease, sickle cell anemia, glomerulonephritis, membranous pemphigoid, or cutaneous small vessel vasculitis. The condition or disease is selected from the group consisting of nephritis, pancreatitis, transplant rejection, hyperacute xenograft rejection, recurrent abortion, preeclampsia, drug allergy, IL-2-induced vascular leak syndrome, radiographic contrast allergy, myasthenia gravis, Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, antiphospholipid syndrome, autoimmune hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, pemphigus, Sjogren's syndrome, Takayasu's arteritis, myocardial infarction, stroke, acute respiratory distress syndrome, sepsis, plasma purification, plateletpheresis, leukapheresis, extracorporeal membrane oxygenation, heparin extracorporeal LDL precipitation, intestinal inflammation, urticaria, vasculitis, and lupus nephritis.

[0060] In some embodiments, the subject is diagnosed with ischemia-reperfusion injury, rheumatoid arthritis (RA), lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical or infectious hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), paroxysmal nocturnal hemoglobinuria (PNH), multiple sclerosis (MS), macular degeneration, hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. group, sepsis, dermatomyositis, diabetic retinopathy, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, microimmune vasculitis, epidermolysis bullosa, habitual abortion, multiple sclerosis (MS), traumatic brain injury, cardiovascular disorders, myocarditis, cerebrovascular disorders, peripheral vascular disorders, renal vascular disorders, mesenteric / intestinal vasculopathy, revascularization of grafts and / or replants, vasculitis, Henoch-Schönlein purpura nephritis, systemic The patient is suffering from a condition or disease selected from the group consisting of lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic vasculopathy, thoracoabdominal aortic aneurysm, Kawasaki disease (arthritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, percutaneous transluminal coronary angioplasty (PTCA), myasthenia gravis, cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma, warm autoimmune hemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture's syndrome, antiphospholipid syndrome (APS), Degos disease, and fulminant APS (CAPS).

[0061] In some embodiments, the subject is suffering from a condition or disease selected from the group consisting of age-related macular degeneration (AMD), membranoproliferative glomerulonephritis type II (MPGN II), hemolytic uremic syndrome (HUS), asthma, amyloidosis, and thrombotic thrombocytopenic purpura. In some embodiments, the hemolytic uremic syndrome (HUS) is atypical hemolytic uremic syndrome (aHUS). In some embodiments, the subject is suffering from a drusen-associated disease or a disease associated with drusen. In some embodiments, the drusen-associated disease is amyloidosis, elastosis, dense deposit disease, glomerulonephritis, atherosclerosis, or a drusen-associated ocular disease.

[0062] Another embodiment provides a pharmaceutical composition comprising a tetravalent fusion protein construct for modulating complement activity, wherein the tetravalent fusion protein construct comprises (i) an antibody or antigen-binding fragment thereof that binds to a complement-related antigen, and (ii) a first complement regulator peptide and a second complement regulator peptide, wherein the first and second complement regulator polypeptides are the same or different.

[0063] In some embodiments, at least one of the first and second complement regulatory peptides is conjugated to the antibody or antigen-binding fragment by a linker.

[0064] Another embodiment provides a pharmaceutical composition comprising a trivalent fusion protein construct for modulating complement activity, wherein the tetravalent fusion protein construct comprises (i) an antibody or antigen-binding fragment thereof that binds to a complement-related antigen, and (ii) one complement regulatory peptide.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof and the complement regulator peptide may be conjugated by a linker.

[0066] Another embodiment provides a pharmaceutical composition comprising a trivalent fusion protein construct for modulating complement activity, wherein the trivalent fusion protein construct comprises (i) a Fab that binds to a complement-related antigen, (ii) an antibody Fc domain, and (iii) a complement regulator peptide conjugated to the Fab or Fc domain.

[0067] In some embodiments, the Fab or Fc domain and the complement regulator peptide may be conjugated by a linker.

[0068] Another embodiment provides a pharmaceutical composition comprising a trivalent fusion protein construct comprising: a) a first polypeptide monomer comprising an antibody CH2 or CH3 domain, which domain has at least one orthogonal modification that favors heterodimer formation compared to homodimers; and b) a second polypeptide monomer comprising an antibody CH2 or CH3 domain, which domain has at least one orthogonal modification that favors heterodimer formation with the first polypeptide monomer compared to homodimers, wherein one of the first and second polypeptides further comprises a complement modulator peptide, and wherein the trivalent fusion protein construct binds to a complement-related antigen.

[0069] In some embodiments, the first polypeptide comprises a knob modification and the second polypeptide comprises a hole modification. In some embodiments, the first and second polypeptides comprise modifications that result in charge or surface complementarity. In some embodiments, the complement modulator peptide is linked to one of the first and second polypeptides by an amino acid linker.

[0070] Another embodiment includes (i) residues 26-33 (CDR-H1) of SEQ ID NOs: 11, 12 and 13; 17, 18 and 19; 23, 24 and 25; 29, 30 and 31; 35, 36 and 37; 147, 148 and 149; 188, 189 and 190; 196, 197 and 198; 204 or 343, 205 and 206; 212, 213 and 214; 220, 221 and 222; 228, 229 and 230; 29, 259 and 31; 29, 260 and 31; SEQ ID NOs: 280, 281, 282, 284, 285, 286, 73 or 288, 51 to 58 (CDR-H2) and 97 to 100 (CDR-H3); residues 26 to 33 (CDR-H1), 51 to 58 (CDR-H2) and 97 to 102 (CDR-H3) of SEQ ID NO: 244; or residues 26 to 33 (CDR-H1), 51 to 58 (CDR-H2) and 97 to 110 (CDR-H3) of SEQ ID NO: 290, or (ii) three heavy chain complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NOs: 11, 12 and 13; 17, 18 and 19; 23, 24 and 25; 29, 30 and 31; 35, 36 and 37; 147, 148 and 149; 188, 189 and 190; 196, 197 and 198; 204 or 343, 205 and 206; 212, 213 and 214; 220, 221 and 222; 228, 229 and 230; 29, 259 and 31; or 29, 260 and 31; residues 26-33 (CDR-H1), 51-58 (CDR-H2) and 97-100 (CDR-H3) of SEQ ID NO: 280, 281, 282, 284, 285, 286, 73 or 288; residues 26-33 (CDR-H1), 51-58 (CDR-H2) and 97-100 (CDR-H3) of SEQ ID NO: 244 102 (CDR-H3); or three heavy chain CDRs having amino acid sequences that differ by a single conservative amino acid substitution within one of residues 26-33 (CDR-H1), 51-58 (CDR-H2), and 97-110 (CDR-H3) of SEQ ID NO: 290; and (ii) SEQ ID NOs: 14, 15, and 16; 20, 21, and 22; 26, 27, and 28; 32, 33, and 34; 38, 39, and 40; 150, 151, and 152; 191, 192, and 193; 199, 200, and 201; 207, 208, and 209; 215, 216 and 217; 223, 224, and 225; or 231, 232, and 233; residues 27-37 (CDR-L1), 55-57 (CDR-L2), and 94-102 (CDR-L3) of SEQ ID NO: 279, 68, 287, or 59; or residues 27-38 (CDR-L1), 56-58 (CDR-L2), and 95-102 (CDR-L3) of SEQ ID NO: 289; or (ii) three light chain complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NOs: 14, 15, and 16; 20, 21, and 22; 26, 27, and 28; 32, 33, and 34; 38, 39, and 40; 150, 151, and 152; 191, 192, and 193; 199, 200, and 201 or 231, 232 and 233; residues 27-37 (CDR-L1), 55-57 (CDR-L2), and 94-102 (CDR-L3) of SEQ ID NO: 279, 68, 287, or 59; or residues 27-38 (CDR-L1), 56-58 (CDR-L2), 95-102 (CDR-L3) of SEQ ID NO: 289; and a fusion protein construct comprising a complement regulator peptide.

[0071] Another embodiment provides (a) SEQ ID NOs: 54, 58, 67, 73, 74, 81, 88, 89, 98, 99, 112, 145, 153, 194, 202, 210, 218, 226, 234, 238, 243, 244, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, 274, 280, 281, 282, 283, 284, 285, 286, 288, 290 and 342, and (b) a heavy chain variable region comprising at least one amino acid sequence selected from SEQ ID NOs: 45, 59, 68, 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, and 278, 279, 287, and 289; or (c) a light chain variable region comprising at least one amino acid sequence selected from SEQ ID NOs: 54, 58, 67, 73, 74, 81, 88, 89, 98, 99, 112, 145 , 153, 194, 202, 210, 218, 226, 234, 238, 243, 244, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, 274, 280, 281, 282, 283, 284, 285, 286, 288, 290, and 342. and (d) a light chain variable region comprising an amino acid sequence that differs by one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 45, 59, 68, 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, and 278, 279, 287, and 289.

[0072] Another embodiment is a sequence encoding SEQ ID NOs: 45, 51, 54, 58, 59, 62, 64, 67, 68, 73, 74, 79, 75, 81, 88, 89, 98, 99, 112, 121, 194, 195, 202, 203, 210, 211, 218, 219, 237, 226, 226, 227, 234, 235, 238, 239, 240, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255 , 256, 257, 258, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277 and 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290 and 342, and a pharmaceutical composition comprising a fusion protein construct comprising a complement regulatory peptide.

[0073] In some embodiments, the complement regulator peptide is linked to the antibody or antigen-binding fragment thereof by an amino acid linker. In some embodiments, the amino acid linker comprises any of the amino acid sequences set forth in SEQ ID NO:138, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:241, or SEQ ID NO:242. In some embodiments, the pharmaceutical composition comprises an antigen-binding fragment, and the antigen-binding fragment comprises an Fv fragment, Fab, Fab', F(ab')2, or scFv. In some embodiments, the fusion protein construct modulates alternative complement activity in a subject when the pharmaceutical composition is administered to a subject. In some embodiments, the antibody or antigen-binding fragment thereof, or Fab binds to a domain of a mammalian annexin protein. In some embodiments, the antibody or antigen-binding fragment thereof, or Fab binds to a domain of a mammalian annexin protein. -8In some embodiments, the domain is an annexin core domain. In some embodiments, the annexin core domain comprises an alpha helical domain. In some embodiments, the annexin core domain comprises a calcium binding site and a membrane binding site. In some embodiments, the annexin core domain comprises at least one annexin repeat sequence. In some embodiments, the antibody or antigen-binding fragment thereof, or Fab binds to at least one annexin repeat sequence.

[0074] In some embodiments, the complement-related antigen comprises a phospholipid, and the antibody or antigen-binding fragment thereof, or Fab binds to the phospholipid. In some embodiments, the antibody or antigen-binding fragment thereof, or Fab binds to the phospholipid. -8 In some embodiments, the phospholipid binds with a KD affinity of 10 M or less. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylethanolamine (PE), cardiolipin (CL), phosphatidylcholine (PC), and malondialdehyde (MDA). In some embodiments, the complement-related antigen comprises a C3 complement protein or a fragment thereof, and the antibody or antigen-binding fragment thereof, or Fab binds to the C3 complement protein or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof, or Fab binds to the C3 complement protein or a fragment thereof with a KD affinity of 10 M or less. -8 In some embodiments, the C3 complement protein fragment is C3d. In some embodiments, the complement regulator peptide comprises a complement receptor 1 (CR1) protein. In some embodiments, the complement regulator peptide comprises domain A of the CR1 protein, or a fragment thereof retaining at least three short consensus repeats (SCRs) of domain A. In some embodiments, the complement regulator peptide comprises domain B of the CR1 protein, or a fragment thereof retaining at least three SCRs of domain B.

[0075] In some embodiments, the complement regulator peptide comprises domain C of the CR1 protein, or a fragment thereof retaining at least three SCRs of domain C. In some embodiments, the pharmaceutical composition further comprises domain D of the CR1 protein, or a fragment thereof retaining at least three SCRs of domain D. In some embodiments, the complement regulator peptide comprises the first three SCRs of domain A, the first three SCRs of domain B, and the first three SCRs of domain C of the CR1 protein. In some embodiments, the CR1 protein is a human CR1 protein. In some embodiments, the complement regulator polypeptide is CR1(1-10). In some embodiments, the complement regulator polypeptide is CR1(1-17). In some embodiments, CR1(1-10) comprises the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:91, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, CR1(1-17) comprises the amino acid sequence of SEQ ID NO:42 or SEQ ID NO:92, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is decay accelerating factor (DAF) or a biologically active fragment thereof. In some embodiments, the DAF is human DAF.

[0076] In some embodiments, the bioactive fragment of human DAF comprises at least one of the short consensus repeat (SCR) domains and the O-glycosylated serine / threonine-rich domain of full-length human DAF. In some embodiments, the bioactive fragment of human DAF comprises SCR1-4 or SCR2-4 of full-length human DAF. In some embodiments, the bioactive fragment of human DAF comprises the amino acid sequence of SEQ ID NO: 184, or a variant thereof having an amino acid sequence at least 85% identical to said sequence. In some embodiments, the complement regulator peptide is Factor H or a bioactive fragment thereof. In some embodiments, the Factor H is human Factor H. In some embodiments, the bioactive fragment of human Factor H comprises a stretch of amino acids selected from the group consisting of amino acids 21-266, amino acids 21-320, amino acids 21-509, or amino acids 19-1106 of SEQ ID NO: 9, or a variant thereof having an amino acid sequence at least 85% identical to said stretch of amino acids. In some embodiments, the biologically active fragment of human Factor H comprises one or more of a group of short consensus repeats (SCRs) comprising SCRs 1-20, SCRs 1-2, SCRs 2-3, SCRs 3-4, SCRs 4-5, SCRs 5-6, SCRs 6-7, SCRs 7-8, SCRs 8-9, SCRs 9-10, SCRs 10-11, SCRs 11-12, SCRs 12-13, SCRs 13-14, SCRs 14-15, SCRs 15-16, SCRs 16-17, SCRs 17-18, SCRs 19-20 of full-length human Factor H, or any combination of SCRs 1-20.

[0077] In some embodiments, the bioactive fragment of human Factor H comprises SCRs 1-4 of full-length human Factor H. In some embodiments, the bioactive fragment of human Factor H comprises SCRs 1-5 of full-length human Factor H. In some embodiments, the bioactive fragment of human Factor H comprises the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 108, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is MCP or a bioactive fragment thereof. In some embodiments, the MCP is human MCP. In some embodiments, the bioactive fragment of human MCP comprises at least one short consensus repeat (SCR) domain of full-length human MCP. In some embodiments, the bioactive fragment of human MCP comprises SCRs 3-4 of full-length human MCP. In some embodiments, the bioactive fragment of human MCP comprises the amino acid sequence of SEQ ID NO: 187, or a variant thereof having an amino acid sequence at least 85% identical.

[0078] In some embodiments, the complement regulator peptide is Map44 or a biologically active fragment thereof. In some embodiments, Map44 is human Map44. In some embodiments, Map44 comprises the amino acid sequence of SEQ ID NO: 186, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the complement regulator peptide is CD59 or a biologically active fragment thereof. In some embodiments, CD59 is human CD59. In some embodiments, CD59 comprises the amino acid sequence of SEQ ID NO: 185, or a variant thereof having an amino acid sequence at least 85% identical. In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

[0079] Another embodiment provides a polynucleotide encoding the fusion protein. Another embodiment provides a method of treatment comprising providing a therapeutically effective amount of the pharmaceutical composition to a subject. In some embodiments, the subject is suffering from complement-mediated inflammation. In some embodiments, the complement-mediated inflammation comprises an inflammatory fibrotic disease, and the inflammatory fibrotic disease comprises focal segmental glomerulosclerosis, primary sclerosing cholangitis, or membranoproliferative glomerulonephritis. In some embodiments, In some embodiments, the subject has a complement-mediated autoimmune disease, including rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, or pemphigus vulgaris. In some embodiments, the subject has a complement-mediated renal disease, including membranoproliferative glomerulonephritis or complement 3 glomerulopathy. In some embodiments, the subject has a complement-mediated cardiovascular disease. In some embodiments, the cardiovascular disease comprises atherosclerosis or thrombosis. In some embodiments, the subject has a complement-mediated skin disease. In some embodiments, the skin disease comprises psoriasis, acne inversus, lupus erythematosus, cutaneous small-vessel vasculitis, urticaria, urticarial vasculitis, and bullous pemphigoid.

[0080] In some embodiments, complement-mediated inflammation is associated with ischemia / reperfusion injury, burns, endotoxemia and septic shock, adult respiratory distress syndrome, cardiopulmonary bypass, hemodialysis, anaphylactic shock, asthma, angioedema, Crohn's disease, sickle cell anemia, glomerulonephritis, membranous nephritis, pancreatitis, transplant rejection, hyperacute xenograft rejection, recurrent abortion, preeclampsia, drug allergies, IL-2 induced vascular leak syndrome, radiographic contrast allergy, myasthenia gravis, Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin resistance, and the like. The condition or disease is associated with a condition or disease selected from the group consisting of insulin-dependent diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, antiphospholipid syndrome, autoimmune hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, pemphigus, Sjogren's syndrome, Takayasu's arteritis, myocardial infarction, stroke, acute respiratory distress syndrome, sepsis, plasma purification, plateletpheresis, leukopheresis, extracorporeal membrane oxygenation, heparin extracorporeal LDL precipitation, intestinal inflammation, urticarial and vasculitis, and lupus nephritis.

[0081] In some embodiments, the subject is diagnosed with ischemia-reperfusion injury, rheumatoid arthritis (RA), lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical or infectious hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), paroxysmal nocturnal hemoglobinuria (PNH), multiple sclerosis (MS), macular degeneration, hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. group, sepsis, dermatomyositis, diabetic retinopathy, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, microimmune vasculitis, epidermolysis bullosa, habitual abortion, multiple sclerosis (MS), traumatic brain injury, cardiovascular disorders, myocarditis, cerebrovascular disorders, peripheral vascular disorders, renal vascular disorders, mesenteric / intestinal vasculopathy, revascularization of grafts and / or replants, vasculitis, Henoch-Schönlein purpura nephritis, systemic The patient is suffering from a condition or disease selected from the group consisting of lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic vasculopathy, thoracoabdominal aortic aneurysm, Kawasaki disease (arthritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, percutaneous transluminal coronary angioplasty (PTCA), myasthenia gravis, cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma, warm autoimmune hemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture's syndrome, antiphospholipid syndrome (APS), Degos disease, and fulminant APS (CAPS).

[0082] In some embodiments, the subject may be suffering from a condition or disease selected from the group consisting of age-related macular degeneration (AMD), membranoproliferative glomerulonephritis type II (MPGN II), hemolytic uremic syndrome (HUS), asthma, amyloidosis, and thrombotic thrombocytopenic purpura. In some embodiments, the hemolytic uremic syndrome (HUS) is atypical hemolytic uremic syndrome (aHUS). In some embodiments, the subject may be suffering from a drusen-associated disease or a disease associated with drusen. In some embodiments, the disease associated with drusen is amyloidosis, elastosis, dense deposit disease, glomerulonephritis, atherosclerosis, or a drusen-associated ocular disease.

[0083] A further embodiment is a fusion protein construct that binds to a complement-related antigen, comprising a first polypeptide comprising Domain A and Domain B arranged in an AB orientation from N-terminus to C-terminus. and a second polypeptide comprising Domain A, Domain B, and Domain R arranged in an E-F orientation from N-terminus to C-terminus, wherein at least one of Domain A and Domain E is optionally conjugated to Domain R, wherein Domain A may comprise a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, Domain R comprises a complement regulator polypeptide, Domain E may comprise a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and Domain F may comprise a light chain constant region amino acid sequence (CL1). In some embodiments, the first polypeptide comprises Domain A, Domain B, and Domain R, wherein the domains of the first polypeptide are arranged in an E-B orientation from N-terminus to C-terminus, and Domain R is conjugated to Domain A. In some embodiments, the second polypeptide comprises Domain E, Domain F, and Domain R, the domains of the second polypeptide being arranged N-terminally to C-terminally in the orientation of REF, and Domain E and Domain R being conjugated.

[0084] Another embodiment provides a fusion protein construct comprising an antibody or antigen-binding fragment thereof that specifically binds to complement protein 3d (c3d) and two molecules of a complement regulator polypeptide, each molecule of the complement regulator polypeptide comprising a biologically active fragment of a complement protein selected from the group consisting of CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, and factor H, wherein the fusion protein construct exhibits a lower half-maximal inhibitory concentration in a complement assay than an identical control protein construct that lacks the antibody.

[0085] In some embodiments, each molecule of complement regulator polypeptide may be conjugated to an antibody heavy chain or antigen-binding fragment thereof, hi some embodiments, each molecule of complement regulator polypeptide may be conjugated to the C-terminus of the heavy chain.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof may comprise a first polypeptide and a second polypeptide, wherein the first polypeptide may comprise a heavy chain sequence comprising the amino acid sequence of at least one of SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:243, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:285, and SEQ ID NO:286; and the second polypeptide may comprise a light chain sequence comprising the amino acid sequence of at least one of SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:135, and SEQ ID NO:27.

[0087] In some embodiments, the first polypeptide may comprise at least two amino acid sequences selected from the group consisting of SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:243, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:285, and SEQ ID NO:286, and the second polypeptide may comprise at least two amino acid sequences selected from the group consisting of SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:135, and SEQ ID NO:279.

[0088] In some embodiments, at least two amino acid sequences of the first polypeptide can be the same amino acid sequence and at least two amino acid sequences of the second polypeptide can be the same amino acid sequence. In some embodiments, at least two amino acid sequences of the first polypeptide can be SEQ ID NO:282 or SEQ ID NO:285, and at least two amino acid sequences of the second polypeptide can be SEQ ID NO:279.

[0089] In some embodiments, the fusion protein construct further comprises a linker. In some embodiments, the linker may comprise any of the amino acid sequences of SEQ ID NO:138, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:241, or SEQ ID NO:242.

[0090] In some embodiments, the complement protein is Factor H or a biologically active fragment thereof. In some embodiments, Factor H or a biologically active fragment thereof may comprise the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 108, or a variant thereof having an amino acid sequence that is at least 85% identical.

[0091] In some embodiments, the complement protein is CR1 or a fragment thereof. In some embodiments, the complement protein may comprise the amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:91, or SEQ ID NO:92, or a variant thereof having an amino acid sequence at least 85% identical.

[0092] In some embodiments, the fusion protein construct is selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, 2, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:279, SEQ ID NO:282, and SEQ ID NO:285.

[0093] Another embodiment is a fusion protein construct comprising an antibody or antigen-binding fragment thereof that specifically binds to a complement related antigen, wherein the antibody comprises a first polypeptide and a second polypeptide, each of the first and second polypeptides comprising a heavy chain and a light chain; and a first and second molecule of a complement regulator polypeptide, each of the first and second molecules comprising a biologically active fragment of a complement protein selected from the group consisting of CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, and Factor H, wherein, upon administration of the fusion protein construct to a subject having a disease, an albumin to creatinine ratio in a urine sample from the subject having a disease is increased compared to a subject administered the fusion protein construct. The fusion protein construct is provided, which has a lower albumin to creatine ratio than a urine sample from the subject, and which is identical except that the equivalent fusion protein construct does not include the antibody or antigen-binding fragment thereof.

[0094] In some embodiments, the disease is a complement-mediated renal disease. In some embodiments, the complement-mediated renal disease is membranoproliferative glomerulonephritis or complement 3 glomerulopathy.

[0095] In some embodiments, the albumin to creatinine ratio in a urine sample from a subject with the disease may be at least about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% lower (compared to a subject administered an equivalent fusion protein construct).

[0096] In some embodiments, SEQ ID NOs: 54, 58, 60, 61, 64, 65, 67, 69, 70, 71, 73, 74, 75, 76, 78, 80, 81, 82, 85, 87, 88, 89, 98, 99, 112, 132, 133, 134, 145, 153, 194, 202, 210, 218, 226, 234, 238, 243, 244, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, 274, 280, 281, 282, 283, 284, 285, 286, 288, 290 and 342, and a light chain comprising at least one amino acid sequence selected from SEQ ID NOs: 45, 59, 68, 77, 79, 84, 86, 135, 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, 278, 279, 287 and 289, or SEQ ID NOs: 54, 58, 60, 61, 64, 65, 67, 69, 70, 71, 73, 74, 75, 76, 77, 79, 84, 86, 135, 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, 278, 279, 287 and 289, or 4, 75, 76, 78, 80, 81, 82, 85, 87, 88, 89, 98, 99, 112, 132, 133, 134, 145, 153, 194, 202, 210, 218, 226, 234, 238, 243, 244, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, 274, 280, 281, 282, 283, 284, 285, 286, 288, 290, and 342 Fusion protein constructs comprising an antibody or antigen-binding fragment thereof comprising a heavy chain comprising an amino acid sequence that differs in that there are conservative amino acid substitutions, and a light chain comprising an amino acid sequence that differs in that there are one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 45, 59, 68, 77, 79, 84, 86, 135, 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, 278, 279, 287, and 289 are provided.

[0097] In some embodiments, the fusion protein construct may further comprise a complement regulator polypeptide, wherein the complement regulator polypeptide comprises at least one of complement receptor 1 (CR1) protein, DAF, MCP, Crry, MAp44, MAp19, CD59, factor H, and biologically active fragments thereof.

[0098] Incorporation by Reference All publications, patents, patent applications, and NCBI accession numbers mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, and to the same extent as if each individual publication, patent, or patent application were indicated to be incorporated by reference in its entirety. In the event of a conflict between a term used herein and a term defined in an incorporated reference, the definition in the present disclosure shall control.

[0099] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0100] [Figure 1] 1 shows an exemplary monomeric fusion protein construct of the present disclosure comprising a linkage between a light chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the N-terminus of the light chain via a linker. [Figure 2] 1 shows an exemplary monomeric fusion protein construct of the present disclosure comprising a linkage between a heavy chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the N-terminus of the heavy chain via a linker. [Figure 3] 1 shows an exemplary monomeric fusion protein construct of the present disclosure comprising a linkage between a heavy chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the C-terminus of the heavy chain via a linker. [Figure 4]1 shows an exemplary monomeric fusion protein construct of the present disclosure comprising a linkage between a light chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the C-terminus of the light chain via a linker. [Figure 5] 1 shows an exemplary tetravalent homodimeric fusion protein construct of the present disclosure comprising a linkage between a heavy chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the C-terminus of the heavy chain via a linker. [Figure 6] 1 shows an exemplary tetravalent homodimeric fusion protein construct of the present disclosure comprising a linkage between a heavy chain of a targeting moiety and a complement regulator polypeptide, wherein the complement regulator polypeptide is linked to the N-terminus of the heavy chain via a linker. [Figure 7] 1 shows an exemplary trivalent heterodimeric fusion protein construct of the present disclosure comprising a linkage between a heavy chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the C-terminus of the heavy chain via a linker. [Figure 8] 1 shows an exemplary trivalent heterodimeric fusion protein construct of the present disclosure comprising a linkage between a heavy chain of a targeting moiety and a complement regulator polypeptide, wherein the complement regulator polypeptide is linked to the N-terminus of the heavy chain via a linker. [Figure 9] 1 shows an exemplary tetravalent homodimeric fusion protein construct of the present disclosure comprising a linkage between the light chain of a targeting moiety and a complement regulator polypeptide, the complement regulator polypeptide being linked to the C-terminus of the light chain via a linker. [Figure 10] 1 shows an exemplary tetravalent homodimeric fusion protein construct of the present disclosure comprising a linkage between the light chain of a targeting moiety and a complement regulator polypeptide, wherein the complement regulator polypeptide is linked to the N-terminus of the light chain via a linker. [Figure 11] 1 shows an exemplary trivalent heterodimeric fusion protein construct of the present disclosure comprising a linkage between a light chain of a targeting moiety and a complement regulator polypeptide, wherein the complement regulator polypeptide is linked to the N-terminus of the light chain via a linker. [Figure 12]1 shows an exemplary trivalent heterodimeric fusion protein construct of the present disclosure comprising a linkage between the light chain of a targeting moiety and a complement regulator polypeptide, wherein the complement regulator polypeptide is linked to the C-terminus of the light chain via a linker. [Figure 13] 1 shows an exemplary fusion protein construct of the disclosure comprising a linkage between the N-terminus of the Fc region of a targeting moiety and a complement regulator polypeptide. [Figure 14] 1 shows an exemplary design of a C2-complement regulator fusion protein construct of the present disclosure. [Figure 15] 1 shows an exemplary design of an anti-C3d-complement regulatory agent fusion protein construct of the present disclosure. [Figure 16] 1 shows an exemplary design of an anti-C3d complement regulatory agent fusion protein construct of the present disclosure. [Figure 17] 1 shows an exemplary design of an anti-C3d complement regulatory agent fusion protein construct of the present disclosure. [Figure 18] 1 shows an exemplary design of an anti-C3d complement regulatory agent fusion protein construct of the present disclosure. [Figure 19] 1 shows an exemplary fusion protein construct of the disclosure that is a tetravalent heterodimer comprising two different complement regulator polypeptides linked to the C-terminus of the heavy chains via a polypeptide linker. [Figure 20] Results from PEGperMAP® linear epitope mapping and five mouse IgG1 antibodies against extended forms of C3dg are shown. Linear C3d epitopes were determined using PEPperPRINT technology (PEPperPRINT GmbH, Heidelberg, Germany). Peptides 15 amino acids long, derived from the C3dg primary sequence and offset by one amino acid, were synthesized in duplicate on PEPperPRINT microarrays. The arrays were then incubated with antibodies (e.g., 3d8b, 3d9a, 3d29) and stained with a secondary DyLight680 dye-labeled goat anti-mouse IgG (H+L) antibody. Microarrays were read using a LI-COR Odyssey imaging system and analyzed by PEPperPRINT. [Figure 21]1 shows the recognition of an epitope on the C-terminus of C3dg by the exemplary anti-C3d antibody 3d29. SEQ ID NOS: 301-341 are listed on the x-axis in order of appearance, with the sequence "NLDVSLQLPS" listed as SEQ ID NOS: 299. [Figure 22] 1 shows the recognition of an epitope on the C-terminus of C3dg by the exemplary anti-C3d antibody 3d8b. SEQ ID NOs: 301-341 are listed on the x-axis in order of appearance, with the sequence "NLDVSLQLPS" listed as SEQ ID NO: 299. [Figure 23] 1 shows the recognition of an epitope on the C-terminus of C3dg by the exemplary anti-C3d antibody 3d9a. SEQ ID NOs: 301-341 are listed on the x-axis in order of appearance, with the sequence "NLDVSLQLPS" listed as SEQ ID NO: 299. [Figure 24] 1 shows epitope mapping using a negative control anti-C3d antibody that did not bind to the linear epitope, tested at various concentrations. SEQ ID NOS: 301-341 are listed on the x-axis of the graph in order of appearance, respectively. [Figure 25] 1 shows epitope mapping using a negative control anti-C4d antibody tested at various concentrations, with SEQ ID NOS: 301-341, respectively, listed in order of appearance on the x-axis of the graph. [Figure 26]A-L show exemplary designs of C2-complement regulator (CR1) fusion protein constructs of the present disclosure. A shows a fusion protein construct comprising a CR1(1-10) polypeptide and an exemplary C2-scFv, where the CR1(1-10) polypeptide is linked to the C-terminus of the light chain variable domain of an exemplary C2-scFv. B shows a fusion protein construct comprising a Crry polypeptide and an exemplary C2-scFv, where the Crry polypeptide is linked to the C-terminus of the light chain variable domain of an exemplary C2-scFv. C shows a fusion protein construct comprising a Crry polypeptide and an exemplary C2-Fab, where the Crry polypeptide is linked to the C-terminus of the heavy chain of an exemplary C2-Fab. D shows a fusion protein construct comprising a CR1(1-10) polypeptide and an exemplary C2-Fab, where the CR1(1-10) polypeptide is linked to the N-terminus of the heavy chain of an exemplary C2-Fab. E shows a fusion protein construct comprising a CR1(1-10) polypeptide and an exemplary C2-Fab, where CR1(1-10) is linked to the C-terminus of the heavy chain of the exemplary C2-Fab. F shows a fusion protein construct comprising a CR1(1-17) polypeptide and an exemplary C2-Fab, where the CR1(1-17) polypeptide is linked to the N-terminus of the heavy chain of the exemplary C2-Fab. G shows a fusion protein construct comprising a CR1(1-17) polypeptide and an exemplary C2-Fab, where the CR1(1-17) polypeptide is linked to the C-terminus of the heavy chain of the exemplary C2-Fab. H shows a fusion protein construct comprising a single CR1(1-10) polypeptide and an exemplary full-length C2 antibody, where CR1(1-10) is linked to the C-terminus of the heavy chain of the exemplary full-length C2 antibody, where the fusion protein construct comprises a knob-into-hole heterodimeric antibody construct. I shows a fusion protein construct comprising two CR1(1-10) polypeptides and an exemplary full-length C2 antibody, in which each C-terminus of each heavy chain of the exemplary full-length C2 antibody is linked to a single CR1(10) polypeptide, and the fusion protein construct comprises a knob-into-hole heterodimeric antibody construct.J represents a fusion protein construct comprising a single CR1(1-10) polypeptide and an exemplary C2 antibody fragment comprising a variable heavy chain region, a variable light chain region, and a constant region, wherein the single CR1(10) polypeptide is linked to the N-terminus of the CH2-CH3 constant region of the exemplary C2 antibody fragment. K represents a fusion protein construct comprising a single CR1(1-10) polypeptide and an exemplary full-length C2 antibody, wherein the single CR1(10) polypeptide is linked to the N-terminus of one of the light chains of the exemplary full-length C2 antibody. L represents a fusion protein construct comprising a single CR1(1-10) polypeptide and an exemplary full-length C2 antibody, wherein the single CR1(10) polypeptide is linked to the N-terminus of one of the heavy chains of the exemplary full-length C2 antibody. [Figure 27] Figures A-E show exemplary designs of anti-C3d(3d29)-complement regulator (CR1 or factor H) fusion protein constructs of the present disclosure. Figure A shows a fusion protein construct comprising a Crry polypeptide and an exemplary anti-C3d Fab, where the Crry polypeptide is linked to the C-terminus of the heavy chain of the exemplary anti-C3d Fab. Figure B shows a fusion protein construct comprising a CR1(1-10) polypeptide and an exemplary anti-C3d Fab, where the CR1(1-10) polypeptide is linked to the C-terminus of the heavy chain of the exemplary anti-C3d Fab. Figure C shows a fusion protein construct comprising factor H and an exemplary anti-C3d Fab, where factor H is linked to the C-terminus of the heavy chain of the exemplary anti-C3d Fab. Figure D shows a fusion protein construct comprising a single factor H polypeptide and an exemplary full-length anti-C3d antibody, where the single factor H polypeptide is linked to the C-terminus of the heavy chain of the exemplary full-length anti-C3d antibody. E shows a fusion protein construct comprising two single factor H polypeptides and an exemplary full-length anti-C3d antibody, in which the C-terminus of each heavy chain of the exemplary full-length anti-C3d antibody is linked to a single factor H polypeptide. [Figure 28]A-H show exemplary designs of anti-C3d(3d8b)-complement regulator (CR1 or factor H) fusion protein constructs of the present disclosure. A shows a fusion protein construct comprising a Crry polypeptide and an exemplary anti-C3d Fab, where Crry is linked to the C-terminus of the heavy chain of the exemplary anti-C3d Fab. B shows a fusion protein construct comprising a CR1(1-10) polypeptide and an exemplary anti-C3d Fab, where the CR1(1-10) polypeptide is linked to the C-terminus of the heavy chain of the exemplary anti-C3d Fab. C shows a fusion protein construct comprising a factor H polypeptide and an exemplary anti-C3d Fab, where the factor H polypeptide is linked to the C-terminus of the heavy chain of the exemplary anti-C3d Fab. D shows a fusion protein construct comprising a single CR1(1-10) polypeptide and an exemplary full-length anti-C3d antibody, where the single CR1(1-10) polypeptide is linked to the C-terminus of one of the light chains of the exemplary full-length anti-C3d antibody. E shows a fusion protein construct comprising a single CR1(1-10) polypeptide and an exemplary full-length anti-C3d antibody, where the single CR1(1-10) polypeptide is linked to the C-terminus of one of the heavy chains of the exemplary full-length antibody. F shows a fusion protein construct comprising two CR1(1-10) polypeptides and an exemplary full-length anti-C3d antibody, where the C-terminus of each heavy chain of the exemplary full-length anti-C3d antibody is linked to a single CR1(1-10) polypeptide. G shows a fusion protein construct comprising a single factor H polypeptide and an exemplary full-length anti-C3d antibody, where the single factor H polypeptide is linked to the C-terminus of the heavy chain of the exemplary full-length anti-C3d antibody. H shows a fusion protein construct comprising two factor H polypeptides and an exemplary full-length anti-C3d antibody, where the C-terminus of each heavy chain of the exemplary full-length anti-C3d antibody is linked to a single factor H polypeptide. [Figure 29]Figures A-C show exemplary designs of anti-C3d (3d8b)-complement regulator (factor H) fusion protein constructs of the present disclosure. A shows a fH1-5-3d8b heavy chain mouse IgG1 (the complement regulator is linked to the N-terminus of the heavy chain via a linker). B shows a 3d8b kappa light chain-fH1-5 (the complement regulator is linked to the C-terminus of the light chain via a linker). C shows a fH1-5-3d8b kappa light chain (the complement regulator is linked to the N-terminus of the light chain via a linker). [Figure 30] 1 shows an exemplary design of an IgG1-complement regulator (Factor H) fusion protein construct of the present disclosure, in which the complement regulator (Factor H) is linked to the hinge region of IgG1 via a linker. [Figure 31]Figures A-F show exemplary designs of anti-C3d (3d8b)-complement regulator (CR1 1-17) fusion protein constructs of the present disclosure. Figure A shows a fusion protein construct comprising one CR1 (1-17) polypeptide and an exemplary Fab fragment of anti-C3d antibody 3d8b, where the C-terminus of the heavy chain of the exemplary Fab fragment is linked to a single CR1 (1-17) polypeptide. Figure B shows a fusion protein construct comprising one CR1 (1-17) polypeptide and an exemplary full-length anti-C3d 3d8b antibody, where the C-terminus of one heavy chain of the exemplary full-length anti-C3d 3d8b antibody is linked to a single CR1 (1-17) polypeptide. Figure C shows a fusion protein construct comprising two CR1 (1-17) polypeptides and an exemplary full-length anti-C3d 3d8b antibody, where the C-terminus of each heavy chain of the exemplary full-length anti-C3d 3d8b antibody is linked to a single CR1 (1-17) polypeptide. D shows a fusion protein construct comprising two CR1(1-17) polypeptides and an exemplary full-length anti-C3d 3d8b antibody, wherein the C-terminus of each light chain of the exemplary full-length anti-C3d 3d8b antibody is linked to a single CR1(1-17) polypeptide. E shows a fusion protein construct comprising two CR1(1-17) polypeptides and an exemplary full-length anti-C3d 3d8b antibody, wherein the N-terminus of each heavy chain of the exemplary full-length anti-C3d 3d8b antibody is linked to a single CR1(1-17) polypeptide. F shows a fusion protein construct comprising two CR1(1-17) polypeptides and an exemplary full-length anti-C3d 3d8b antibody, wherein the N-terminus of each light chain of the exemplary full-length anti-C3d 3d8b antibody is linked to a single CR1(1-17) polypeptide. [Figure 32] A-B show quantification of C3-positive staining expressed as a percentage of total glomerular area (ImageJ software analysis) or semiquantitative scoring. A shows staining of glomerular C3 deposits in harvested kidneys measured by immunofluorescence (IF). B shows C3 deposits in harvested livers using IF. [Figure 33]A-B show quantification of C3-positive staining expressed as a percentage of total glomerular area (ImageJ software analysis) or semiquantitative scoring. A shows staining of glomerular C3 deposits in harvested kidneys measured by IF. B shows C3 deposits in harvested livers measured using IF. [Figure 34] Percent infarct volume in Balb / c mice is shown. [Figure 35] 1 shows the difference in urinary albumin:creatinine ratio (uACR) in adriamycin-injured mice between days 8 and 22 of the study, where day 8 is before treatment and day 22 represents 12 days after dosing with complement inhibitors (post-treatment). DETAILED DESCRIPTION OF THE INVENTION

[0101] The present disclosure provides a fusion protein construct comprising an antibody or antigen-binding fragment thereof and two molecules of a complement regulator polypeptide. The antibody or antigen-binding fragment thereof can specifically bind to a complement protein. The complement protein may be, for example, complement protein 3d (c3d). In some cases, the complement regulator polypeptide molecule may include at least a biologically active fragment of a complement protein. The complement protein may be, for example, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, or factor H. In some cases, the half-maximal inhibitory concentration (e.g., based on a complement assay) of the fusion protein construct is lower than that of an identical control protein construct that does not contain the antibody. In some cases, the complement regulator polypeptide molecule may be conjugated to the heavy chain of the antibody or antigen-binding fragment thereof. In some cases, the complement regulator polypeptide molecule may be conjugated to the light chain of the antibody or antigen-binding fragment thereof. In some cases, the complement regulator polypeptide molecule is conjugated to the C-terminus of the heavy and / or light chain. In some cases, the complement regulator polypeptide molecule is conjugated to the N-terminus of the heavy and / or light chain.

[0102] The present disclosure provides a fusion protein construct comprising an antibody or antigen-binding fragment thereof that specifically binds to a complement-related antigen, and a first and second molecule of a complement regulator polypeptide. In some cases, the antibody may comprise a first and a second polypeptide. In some cases, the first and second polypeptides may each comprise a heavy chain and a light chain. In some cases, the first and second molecules may each comprise a biologically active fragment of a complement protein. The complement protein may be CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, or Factor H. In some cases, the albumin-to-creatine ratio may be measured in a biological sample from a subject. In some cases, the albumin-to-creatine ratio is determined at a time point after administration of a fusion protein construct of the present disclosure or after administration of an equivalent fusion protein construct. In some cases, the albumin-to-creatine ratio is compared between two or more biological samples from the same or different subjects. In some cases, the biological sample is a urine sample. In some cases, the subject has a disease disclosed herein. In some cases, the subject does not have a disease. In some cases, the albumin to creatinine ratio of a urine sample from a subject with a disease (e.g., a kidney disease) after administration of a fusion protein construct of the present disclosure is lower than the albumin to creatine ratio of a urine sample from a subject administered an equivalent fusion protein construct. In some cases, the equivalent fusion protein construct is identical to the fusion protein construct of the present disclosure except that it does not include an antibody or antigen-binding fragment thereof.

[0103] The present disclosure provides fusion protein constructs comprising at least two components fused to each other: (i) a targeting moiety that binds to a complement-related antigen, and (ii) a complement modulator. The components of the construct may be fused to each other via covalent or non-covalent interactions. The fusion construct protein may be engineered to optimize and adapt its target binding valency depending on the target, condition, and desired amount of complement modulator. For example, the fusion protein construct may be designed to be bivalent, trivalent, or tetravalent, with each valency associated with a target binding moiety or a complement modulator. When a fusion protein construct is described using valency, it refers to the sum of the valencies of each component. In some cases, the targeting moiety may be bivalent while the complement modulator is monovalent, resulting in a trivalent fusion protein construct. In other specific examples, the targeting moiety may be monovalent or bivalent while the complement modulator is bivalent, resulting in a trivalent or tetravalent fusion construct, respectively.

[0104] In addition, the targeting moiety in the fusion protein construct may specifically bind to more than one antigen, thereby making the fusion protein construct a bispecific, trispecific, or multispecific molecule. The fusion protein constructs of the present disclosure can bind to multiple targets with their improved specificity and increased valency. In certain instances, the fusion protein constructs may have additional beneficial properties, such as suitability for production and pharmaceutical compositions, due to their improved stability, reduced aggregation, pharmacokinetic and biological properties, or any combination thereof.

[0105] The targeting moiety can be an antibody or antigen-binding fragment thereof capable of specifically binding to one or more complement-related antigens, e.g., antigens locally presented at or near sites associated with complement activation. An example of a targeting moiety can include an antibody or antigen-binding fragment thereof specific for a domain of a mammalian annexin protein. Alternatively, the targeting moiety can include an antibody or antigen-binding fragment thereof specific for a phospholipid. The targeting moiety can include an antibody or antigen-binding fragment thereof specific for a complement protein, e.g., a fragment of C3 associated with complement activation, e.g., C3b, iC3b, C3d, and C3dg. The targeting moiety can also be a bispecific or trispecific antibody or antigen-binding fragment thereof specific for any combination of the following targets: a domain of a mammalian annexin protein, a phospholipid, and a complement protein, e.g., a fragment of C3, e.g., C3d, iC3b, C3d, C3dg, C3a, C3b, C3c, or C3f. In addition, It is also contemplated that targeting moieties that are multivalent, eg, trivalent, tetravalent, etc., may be part of the fusion protein constructs described herein.

[0106] Some examples of fusion protein constructs may be trivalent and bispecific, and include (i) a bivalent antibody or antigen-binding fragment thereof (e.g., an antibody Fab region), (ii) an antibody Fc domain, and (iii) a complement regulator fused to the antibody or antigen-binding fragment thereof or the Fc domain. The bivalent antibody or antigen-binding fragment thereof may be the targeting portion of the trivalent bispecific fusion protein construct and may bind to two target molecules, such as domains of a mammalian annexin protein (e.g., annexin IV, annexin-2), phospholipids, complement proteins, or fragments of complement proteins (e.g., iC3b, C3d, C3dg, C3a, C3b, C3c, or C3f). The complement regulator of the trivalent bispecific fusion protein construct may be an inhibitor of complement activation. Further variants of trivalent bispecific fusion protein constructs may include bispecific trivalent heterodimeric polypeptide constructs comprising: a) a first polypeptide monomer comprising an antibody CH2 or CH3 domain, said domain having at least one amino acid modification that creates a cavity (also referred to as a "hole"); b) a second polypeptide monomer comprising an antibody CH2 or CH3 domain, said domain having at least one amino acid modification that creates a protrusion (also referred to as a "knob"), whereby the knob interacts with the hole upon heterodimerization of the first and second polypeptide monomers; wherein one of the first and second polypeptides is further fused to a complement modulator.

[0107] In another example, the fusion protein construct includes a tetravalent construct for modulating complement activity, the tetravalent construct comprising (i) a bivalent antibody or antigen-binding fragment thereof and (ii) two complement regulatory agents. The bivalent antibody of the tetravalent fusion construct can be a full-length bivalent antibody or an antigen-binding fragment thereof, such as a variable region fragment or a bivalent single-chain variable fragment. The two complement regulatory agents can be the same protein or different, forming a bispecific tetravalent or trispecific tetravalent fusion protein construct. In another example, the fusion protein construct is monomeric and contains one or more amino acid modifications that eliminate disulfide bonding that occurs when forming a homodimeric construct.

[0108] Antibodies consist of four polypeptides: two heavy chains and two light chains. The antigen-binding portion of an antibody is formed by a light chain variable domain (VL) and a heavy chain variable domain (VH). At one end of these domains, six loops form the antigen-binding site, also called the complementarity-determining region (CDR). Three CDRs are located on the VH domain (H1, H2, and H3), and the other three are located on the VL domain (L1, L2, and L3). During B cell development, unique immunoglobulin regions are formed by somatic recombination, known as V(D)J gene rearrangement. The variable regions of immunoglobulin heavy or light chains are encoded by different gene segments. Heavy chains are encoded by three segments called variable (V), diversity (D), and joining (J) segments, while light chain variable regions are formed by only two segments, V and J. Multiple antibody paratopes (also referred to herein as antigen-binding sites) can be generated by recombination between one of the multiple copies of V, D, and J segments present in the genome. The V-segment encodes CDR1 and CDR2, while CDR3 is generated by recombination events. During the course of an immune response, additional diversity is introduced into the antigen-binding site by a process called somatic hypermutation (SHM). During this process, point mutations are introduced into the variable genes of the heavy and light chains, specifically into the regions encoding the CDRs. This additional variability allows for the selection and expansion of B cells expressing antibody variants with improved affinity for the cognate antigen. The majority of immunoglobulins are composed of two identical heavy chain polypeptides and two identical light chain polypeptides, and are therefore bivalent, monospecific molecules possessing the same specificity in both arms. However, very early on in the development of hybridoma technology, hybrid hybridomas were created by fusion events between two hybridomas. It has been recognized that antibody species can be produced by combining two different heavy chains and two different light chains (see, e.g., MRSuresh et al., Methods Enzymol 1986;121:210-228). These "quadromas" express two different heavy chains and two different light chains, thus generating a wide variety of antibody species resulting from random pairing of heavy and light chains. Among these different species, bispecific antibodies (bsAbs) are generated, carrying a different specificity on each arm. Another naturally occurring exception is immunoglobulins of the IgG4 isotype, which can undergo heavy chain exchange due to the less stable dimerization mediated by the hinge region of that isotype (see, e.g., van der Neut Kolfschoten M (See, e.g., Wang et al., Science. 2007 317(5844):1554-7). This exchange appears to occur in vivo, but its biological significance remains unclear. Monoclonal antibodies have emerged as a successful and attractive class of molecules for therapeutic intervention in several areas of human disease. However, targeting or neutralizing a single protein is not always sufficient to achieve efficacy in a particular disease, limiting the therapeutic use of monoclonal antibodies. It is becoming increasingly clear that neutralizing one component of a biological system is not sufficient to achieve efficacy in multiple indications. One solution to this problem is the co-administration of several monoclonal antibodies. However, this approach is complicated by regulatory issues if the combined antibodies have not previously been approved individually. Moreover, combination approaches are also costly from a manufacturing standpoint. Therefore, there is a need for antibodies and therapeutic agents that allow for targeting multiple antigens with a single molecule.

[0109] The present disclosure includes at least bivalent, trivalent and tetravalent fusion protein constructs that provide molecules with improved therapeutic potential to address diseases, disorders or conditions associated with the complement system.

[0110] Complement regulators The complement system is a major effector of humoral and innate immunity. It has three independent pathways of complement activation: the classical pathway, the alternative pathway, and the lectin pathway. Although these three pathways differ in their initiating events, all three pathways converge on the cleavage of complement component C3. Key to complement system activity is the covalent binding of complement components C3 and / or C4, which are processed protein fragments derived from serum proteins, to tissue sites of complement activation. This unique property is due to the presence of a thioester bond in C3, which, upon cleavage during C3 activation, converts C3 to a form called C3b, which can subsequently link to cell- and tissue-binding molecules via ester or amide bonds. Once covalently bound, C3b is rapidly processed into iC3b, C3dg, and C3d forms, each of which remains covalently bound to the target tissue site. This process "marks" the tissue as undergoing inflammatory injury or other complement-related processes.

[0111] Complement can be activated by one of three pathways: classical, lectin, and alternative. The classical pathway is activated by the binding of the complement system protein C1q to antigen-antibody complexes, pentraxins, or apoptotic cells. Pentraxins include C-reactive protein and serum amyloid P component. The lectin pathway is initiated by the binding of carbohydrates to mannose-binding lectins or by the binding of ficolins or collectins to carbohydrates or acetylated molecules.

[0112] The alternative pathway is activated on the surface of pathogens that do not express or contain complement inhibitors. This occurs as a result of a spontaneous process called C3 "tickover," which involves the interaction of conformationally altered C3 with factor B, resulting in the fixation of activated C3b on pathogens or other surfaces. The alternative pathway is activated when specific antibodies override the intrinsic regulatory mechanisms, such as IgA. The alternative pathway can also be initiated when C3b, deposited on the target via the classical or lectin pathways or by the tickover process itself, binds to factor B, a mechanism known as the "amplification loop." See Muller-Eberhard (1988) Ann. Rev. Biochem. 57:321. For example, Holers and colleagues demonstrated that the alternative pathway is amplified at local injury sites when inflammatory cells are recruited following initial complement activation. See Girardi et al., J. Clin. Invest. 2003, 112:1644. Dramatic amplification of complement by the alternative pathway then occurs through mechanisms involving either the further development of complement-fixing damaged cells or the local synthesis of alternative pathway components, or more likely, because infiltrating inflammatory cells bearing preformed C3 and properdin significantly increase activation specifically at the site.

[0113] Alternative pathway amplification is initiated when circulating factor B binds activated C3b. This complex is then cleaved by circulating factor D to generate the enzymatically active C3 convertase complex, C3bBb. C3bBb cleaves additional C3 to generate C3b, which promotes inflammation and further amplifies the activation process, creating a positive feedback loop. Factor H is a critical regulator (inhibitor) of the alternative complement pathway activation and initiation mechanism, competing with factor B for binding to conformationally altered C3 in the tick-over mechanism and C3b in the amplification loop. Binding of C3b to factor H also leads to the cleavage of C3b by factor I to the inactive form iC3b (also called C3bi), thus further inhibiting complement activation. Factor H regulates complement in the fluid phase, circulating at plasma concentrations of approximately >400-600 μg / ml, but its binding to cells is a regulated phenomenon that is enhanced by the presence of negatively charged surfaces and by immobilized C3b, iC3b, C3dg, or C3d. See Jozsi et al., Histopathol. (2004) 19:251-258.

[0114] Complement activation, C3 fragment fixation and complement-mediated inflammation are involved in the pathogenesis and progression of many diseases. Downregulation of complement activation has been shown to be associated with, for example, systemic lupus erythematosus and glomerulonephritis (Y. Wang et al., Proc. Nat'l Acad. Sci. USA (1996) 93:8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Nat'l Acad. Sci. USA (1995) 92:8955-8959), cardiopulmonary bypass and hemodialysis (C. S. Ind., J. Clin. Invest. (1995) 96:1564-1572), hyperacute rejection in organ transplants (T. J. Kroshus et al., Transplantation (1995) 60:1194-1202), myocardial infarction (J. W. Homeister et al., J. Immunol. (1993) 150:1055-1064, H. F. Weisman et al. al., Science (1990) 249:146-151), ischemia / reperfusion injury (EA Amsterdam et al., Am. J. Physiol. (1995) 268:H448-H457), antibody-mediated allograft rejection, for example in the kidney (J.B. Colvin, J. Am. Soc. Nephrol. (2007) 18(4):1046-56), and adult respiratory distress syndrome (R. Rabinovici et al., J. Immunol. (1992) 149:1744-1750). Moreover, other inflammatory conditions and autoimmune / immune complex diseases, including but not limited to burns, severe asthma, anaphylactic shock, intestinal inflammation, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, myocarditis, membranoproliferative glomerulonephritis, atypical hemolytic uremic syndrome, Sjogren's syndrome, renal and pulmonary ischemia / reperfusion, and other organ-specific inflammatory disorders, are also closely associated with complement activation (BPM Organ. Eur. J. Clin. Invest. (1994) 24:219-228). Currently, local tissue C3 activation and inflammatory It is unclear whether complement activation is essential for the pathogenesis and injury of all diseases in which damage occurs, but C3 fragment fixation is nevertheless frequently found as an associated event.

[0115] Integral membrane-bound proteins that regulate alternative pathway activation are decay-accelerating factor (DAF / CD55), membrane cofactor protein (MCP / CD46), and complement receptor 1 (CR1). Other endogenous proteins that regulate alternative pathway activation include factor H, a 155-kDa circulating glycoprotein that regulates alternative pathway activation in the fluid phase and at tissue surfaces. See JJ Alexander et al., Mol. Immunol. (2006) 44:123-132. Unregulated alternative pathway activation has been implicated in the pathogenesis of a diverse group of diseases, including age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), type II membranoproliferative glomerulonephritis (MPGN II), asthma, and renal ischemia / reperfusion (I / R) injury. See JM Thurman et al., J. Immunol. (2006) 176:1305-1310. Damage to host tissues by the alternative pathway suggests insufficient local control of the alternative pathway by the target tissue. Indeed, mutations in CRP are strong risk factors for aHUS (MC Pickering et al., J. Exp. Med. (2007) 204:1249-1256) and MPGN II (RJ Smith et al., J. Am. Soc. Nephrol. (2007) 18:2447-2456), and functional polymorphisms in factor H, a circulating regulator of the alternative pathway, are associated with the progression of AMD (RJ Klein et al., Science (2005) 308:385-389; A0 Edwards et al., Science (2005) 308:421-424; JL Haines et al., Science (2005) 308:419-421; G5 Hageman et al., Proc. Nat'l Acad. Sci USA (2005) 102:7227-7232) has been substantiated by recent studies.

[0116] Ischemic acute kidney injury (AKI) is associated with activation of the alternative pathway at the basolateral surface of injured tubular cells in rodents (JM Thurman et al., J. Immunol. (2003) 170:1517-1523; JM Thurman et al., Am. Soc. Nephrol. (2006) 17:707-715) and humans (JM Thurman et al., Kidney Int. (2005) 67:524-530). It has been found that complement receptor 1-related gene / protein y (Crry, the rodent analog of human MCP and CR1) is the only C-reactive protein (CRP) expressed by proximal tubular epithelial cells in mice, and that ischemia / reperfusion causes a decrease in surface expression of this protein. See JM Thurman et al., J. Clin. Invest. (2006) 116:357-368. Mice congenitally deficient in Crry (Crry+ / -) are more susceptible to ischemic acute renal failure (Id.) than wild-type controls, highlighting the importance of basolateral Crry in regulating this surface alternative pathway. Whether polymorphisms or mutations in CRP may confer an increased risk of AKI development in humans remains unknown. Nevertheless, uncontrolled activation of the alternative pathway in the setting of reduced surface Crry suggests that the protective capacity of circulating factor H on the surface of hypoxic renal tubular epithelial cells is limited.

[0117] Factor H circulates at high concentrations (>400-600 μg / ml) and is a potent inhibitor of the alternative complement pathway. See J. J. Alexander et al., Mol. Immunol. (2006) 44:123-132. However, alternative pathway inhibition by factor H at the cell surface requires that it properly bind to that surface. Several regions in the factor H protein bind to anionic surfaces, e.g., heparin sulfate- or sialic acid-rich surfaces, and to C3b on the surface. See S. Meri et al., Proc. Nat'l Acad. Sci USA (1990) 87:3982-3986, MK See Pangburn et al., Immunol. (2000) 164:4742-4751. Alternative pathway activation on a particular surface is strongly influenced by the affinity of factor H for that surface. The polymorphisms and mutations associated with AMD and aHUS, respectively, most frequently involve the region of factor H required for anionic surface binding rather than the complement regulatory region. See MC Pickering et al., J. Exp. Med. (2007) 204:1249-1256; AP Sjoberg et al., J. Biol. Chem. (2007) 282:10894-10900. Thus, specific tissues or cell types require factor H to regulate alternative pathway activation on their surfaces. Different binding regions of the factor H protein may be required for complement regulation in those tissues or cell types. In some cases, factor H binding to specific tissue surfaces may be influenced by other proteins. Identification of putative tissue-specific binding partners of factor H may provide a potential mechanism for modulating, i.e., stimulating or inhibiting, the activity of the alternative complement pathway in different tissues.

[0118] Thus, a complement regulator of the present disclosure can be a complement regulator protein or polypeptide (e.g., a complement inhibitor polypeptide), such as membrane cofactor protein (MCP) (SEQ ID NO: 1) (UniProtKB / Swiss-Prot accession number P15529), decay-accelerating factor (DAF), CD59, Crry, CR1, CR2, Factor H, or a variant or fragment thereof. Additional complement regulators can include anti-C5 antibodies, eculizumab, pexelizumab, anti-C3b antibodies, anti-C6 antibodies, anti-C7 antibodies, anti-C8 antibodies, anti-C9 antibodies, anti-factor B antibodies, anti-MASP antibodies, anti-factor D antibodies, and anti-properdin antibodies, anti-MBL antibodies, factor I, linear peptides, cyclic peptides, compstatin or an analog thereof, N-acetylaspartylglutamic acid (NAAGA), and biologically active fragments of any of the above. In some cases, the complement inhibitor can be a human complement inhibitor (e.g., human MCP, human DAF, human CD59, human CR1, human factor H, human Map44, human Map19, or another complement inhibitor derived from a human). In a further example, the complement inhibitor can be a mammalian complement inhibitor (e.g., mouse DAF, mouse CD59 (also known as isoform A), mouse CD59 isoform B, mouse Crry, mouse factor H, mouse Map44, mouse Map19, or another complement inhibitor derived from a mouse. Thus, the polypeptide can be a human polypeptide or a polypeptide of a non-human species. For example, the complement regulator polypeptide can be from a non-human primate (e.g., an orangutan, chimpanzee, rhesus monkey, gorilla, lemur, or gibbon), horse, cow, pig, sheep, goat, dog, cat, or rodent (e.g., a mouse, rabbit, hamster, gerbil, guinea pig, or rat).

[0119] The complement regulator of the fusion protein construct can be a mutant complement regulator. A mutant complement regulator polypeptide has one or more amino acid substitutions compared to the corresponding wild-type sequence (e.g., 59 or fewer, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37 ...58, 5 The variant polypeptide may contain one or more amino acid substitutions (e.g., 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution). The amino acid substitutions may be conservative, non-conservative, or a mixture of both. The variant polypeptide, in some embodiments, may contain one or more deletions or additions, or a combination of one or more deletions, additions, and substitutions. In some examples, the variant complement regulator polypeptide may contain one or more amino acid deletions, additions, or substitutions per 100 amino acids of the polypeptide. In some instances where the complement regulator polypeptide comprises a short consensus repeat (SCR), the variant polypeptide comprises: The complement regulator polypeptide may contain no substitutions, deletions, or additions in the SCR.

[0120] A variant complement regulator polypeptide can comprise an amino acid sequence that is at least 70 (e.g., at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99)% identical to the corresponding wild-type amino acid sequence. Functional fragments of the complement regulator polypeptides or variant polypeptides described herein are shorter than the full-length polypeptide. A variant complement regulator polypeptide can comprise an amino acid sequence that is at least 90%, 95%, or 98% identical to one or more functional (bioactive) fragments or domains of the complement regulator polypeptides identified herein. Mutant polypeptides, and functional fragments of wild-type proteins or variants, retain at least 50 (e.g., at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99)% of the complement regulatory activity of the corresponding wild-type polypeptide.

[0121] A variant complement regulator polypeptide, or a functional fragment of a complement regulator polypeptide, can have greater than 100% ability to regulate complement activity relative to the corresponding wild-type protein. Methods for detecting and / or quantifying complement activity are known in the art and described herein.

[0122] Other examples of sequences useful as complement regulators in the present disclosure include one or more short consensus repeat (SCR) domains derived from one or more of the following complement-related proteins: Factor H, complement receptor 1, complement receptor 2, factor B, DAF, etc. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 SCR regions are incorporated into the complement regulators described herein. In some instances, when a complement regulator polypeptide includes "membrane cofactor protein," "MCP," or "CD46," this may refer to a widely distributed C3b / C4b-binding cell-surface glycoprotein, including homologs thereof, that can inhibit complement activation in host cells and act as a cofactor for factor I-mediated cleavage of C3b and C4b. MCP may belong to a family known as regulators of complement activation ("RCA"). Family members may share certain structural features, including varying numbers of short consensus repeat (SCR) domains, typically 60-70 amino acids in length. Starting from its amino terminus, MCP may include four SCRs, a serine / threonine / proline-rich region, a region of unknown function, a transmembrane hydrophobic domain, a cytoplasmic anchor, and a cytoplasmic tail. It is understood that species and strain variations exist among the peptides, polypeptides, and proteins of the present disclosure, and that human MCP or a biologically active fragment thereof may encompass all species and strain variations.

[0123] SEQ ID NO: 1 represents an exemplary sequence of full-length human MCP (see, e.g., UniProtKB / Swiss-Prot accession number P15529). Amino acids 1-34 may correspond to the signal peptide, amino acids 35-343 may correspond to the extracellular domain, amino acids 344-366 may correspond to the transmembrane domain, and amino acids 367-392 may correspond to the cytoplasmic domain. In the extracellular domain, amino acids 35-96 may correspond to SCR1, amino acids 97-159 may correspond to SCR2, amino acids 160-225 may correspond to SCR3, amino acids 226-285 may correspond to SCR4, and amino acids 302-326 may correspond to the serine / threonine-rich domain. It is understood that species and strain diversity exists among the peptides, polypeptides, and proteins of the present disclosure, and that MCP or its biological activity may be expressed or expressed in a variety of ways. It is understood that fragments can encompass all species and strain diversity. As used herein, the term "bioactive" fragment of MCP can refer to any soluble fragment lacking the cytoplasmic and transmembrane domains, including fragments that have some or all of the complement inhibitory activity of the full-length human MCP protein and comprise, consist essentially of, or consist of one, two, three, or four SCR domains, with or without the serine / threonine-rich domain. In some embodiments, the complement inhibitor portion comprises full-length human MCP (amino acids 35-392 of SEQ ID NO: 1), the extracellular domain of human MCP (amino acids 35-343 of SEQ ID NO: 1), or SCRs 1-4 of human MCP (amino acids 35-285 of SEQ ID NO: 1).

[0124] In some cases, the complement regulatory polypeptide comprises decay-accelerating factor (DAF / CD55), also known as CD55 (SEQ ID NO:2 and SEQ ID NO:3), a membrane-bound glycoprotein with a molecular weight of approximately 70 kilodaltons (kDa) that inhibits complement activation in host cells. Like several other complement regulatory proteins, DAF contains several repeating motifs of approximately 60 amino acids called short consensus repeats (SCRs).

[0125] As used herein, the terms "decay-accelerating factor," "DAF," or "CD55" refer to a 70-kilodalton ("kDa") membrane glycoprotein containing four short consensus repeat (SCR) domains followed by a C-terminal heavily O-glycosylated serine / threonine-rich domain that elevates the molecule from the membrane surface, followed by a glycosylphosphatidylinositol ("GPI") anchor. DAF protects the cell surface from complement activation by dissociating membrane-bound C3 convertase, which is required for cleavage of complement protein C3 and amplification of the complement cascade. DAF prevents aggregation or promotes the decay of both the C3 and C5 convertases of the alternative and classical complement pathways.

[0126] SEQ ID NO:2 represents an exemplary sequence of full-length human DAF (see, e.g., UniProtKB / Swiss-Prot accession number P08173), and SEQ ID NO:3 represents an exemplary sequence of full-length mouse DAF (see, e.g., UniProtKB / Swiss-Prot accession number Q61475). In the human DAF sequence, amino acids 1-34 may correspond to a signal peptide, amino acids 35-353 may appear in the mature protein, and amino acids 354-381 may be removed from the polypeptide post-translationally. In the mature protein, amino acids 35-96 may correspond to SCR1, amino acids 96-160 may correspond to SCR2, amino acids 161-222 may correspond to SCR3, amino acids 223-285 may correspond to SCR4, and amino acids 287-353 may correspond to an O-glycosylated serine / threonine-rich domain. The GPI anchor can be attached to human DAF at the serine at position 353. In the mouse DAF sequence, amino acids 1-34 can correspond to the signal peptide, amino acids 35-362 can appear in the mature protein, and amino acids 363-390 can be removed from the polypeptide after translation. In the mature protein, amino acids 35-96 can correspond to SCR1, amino acids 97-160 can correspond to SCR2, amino acids 161-222 can correspond to SCR3, amino acids 223-286 can correspond to SCR4, and amino acids 288-362 can correspond to the O-glycosylated serine / threonine-rich domain. The GPI anchor can be attached to mouse DAF at the serine at position 362. It is understood that species and strain diversity exists among the peptides, polypeptides, and proteins of the present disclosure, and that DAF or its biologically active fragments can encompass all species and strain diversity. As used herein, the term "bioactive" fragment of DAF can refer to any fragment of DAF lacking the GPI anchor, the amino acid to which it is attached (e.g., Ser-353), or both, including full-length DAF protein comprising, consisting essentially of, or consisting of one, two, three, or four SCR domains that have some or all of the complement inhibitory activity of the full-length DAF protein, with or without an O-glycosylated serine / threonine-rich domain. Any fragment of the DAF protein is also included.

[0127] SEQ ID NO: 4 represents an exemplary sequence of full-length human CD59 (see, e.g., UniProtKB / Swiss-Prot accession number P13987), SEQ ID NO: 5 represents an exemplary sequence of full-length mouse CD59 isoform A (see, e.g., UniProtKB / Swiss-Prot accession number O55186), and SEQ ID NO: 6 represents an exemplary sequence of full-length mouse CD59 isoform B (see, e.g., UniProtKB / Swiss-Prot accession number P58019). In the human CD59 sequence, amino acids 1-25 of SEQ ID NO: 4 may correspond to the leader peptide, amino acids 26-102 of SEQ ID NO: 4 may correspond to the mature protein, and amino acids 103-128 of SEQ ID NO: 4 may be removed post-translationally. A GPI anchor may be attached to CD59 at the asparagine at position 102 of SEQ ID NO: 4. In isoform A of mouse CD59, amino acids 1-23 of SEQ ID NO: 5 may correspond to the leader peptide, amino acids 24-96 of SEQ ID NO: 5 may correspond to the mature protein, and amino acids 97-123 of SEQ ID NO: 5 may be removed post-translationally. A GPI anchor may be attached to CD59 at the serine at position 96 of SEQ ID NO: 5. In isoform B of the mouse CD59 sequence, amino acids 1-23 of SEQ ID NO: 6 may correspond to the leader peptide, amino acids 24-104 of SEQ ID NO: 6 may correspond to the mature protein, and amino acids 105-129 of SEQ ID NO: 6 may be removed post-translationally. A GPI anchor may be attached to CD59 at the asparagine at position 104 of SEQ ID NO: 6. It is understood that species and strain variations exist in the peptides, polypeptides, and proteins of the present disclosure, and that CD59 or biologically active fragments thereof may encompass all species and strain variations.As used herein, the term "bioactive" fragment of human CD59 may refer to any fragment of human CD59 lacking the GPI anchor, the amino acid to which it is attached (e.g., Asn-102), or both, including any fragment of full-length human CD59 protein that has some or all of the complement inhibitory activity of the full-length CD59 protein; the term "bioactive" fragment of murine CD59 may refer to any fragment of murine CD59 isoform A or isoform B lacking the GPI anchor and / or the amino acid to which it is attached (e.g., Ser-96 in isoform A, or Asp-104 in isoform B), including any fragment of either full-length murine CD59 protein isoform that has some or all of the complement inhibitory activity of the full-length CD59 protein.

[0128] SEQ ID NO: 7 represents an exemplary sequence of a full-length mouse Crry protein. Amino acids 1-40 may correspond to a leader peptide, and amino acids 41-483 of SEQ ID NO: 7 may correspond to a mature protein comprising amino acids 41-405 of SEQ ID NO: 7, which may correspond to the extracellular domain; amino acids 406-426 of SEQ ID NO: 7, which may correspond to the transmembrane domain; and amino acids 427-483 of SEQ ID NO: 7, which may correspond to the cytoplasmic domain. In the extracellular domain, amino acids 83-143 of SEQ ID NO: 7 may correspond to SCR1, amino acids 144-205 of SEQ ID NO: 7 may correspond to SCR2, amino acids 206-276 of SEQ ID NO: 7 may correspond to SCR3, amino acids 277-338 of SEQ ID NO: 7 may correspond to SCR4, and amino acids 339-400 of SEQ ID NO: 7 may correspond to SCR5. It is understood that species- and strain-specific diversity exists among the peptides, polypeptides, and proteins of the present disclosure, and that mouse Crry proteins or physiologically active fragments thereof may encompass all species- and strain-specific diversity. As used herein, the term "biologically active" fragment of mouse Crry protein may refer to any soluble fragment of mouse Crry lacking the transmembrane and cytoplasmic domains, including fragments comprising, consisting essentially of, or consisting of one, two, three, four, or five SCR domains, including any fragment of full-length mouse Crry protein that has some or all of the complement inhibitory activity of the full-length Crry protein.

[0129] As used herein, the term "complement receptor 1," "CR1," or "CD35" The term CR1 may refer to a human gene encoding a 2,039-amino acid protein with a predicted molecular weight of 220 kilodaltons (kDa), including its homologs. The gene is primarily expressed on erythrocytes, monocytes, neutrophils, and B cells, but may also be present on some T lymphocytes, mast cells, and glomerular podocytes. The CR1 protein is typically expressed at 100–1,000 copies per cell. CR1 may be a key system for processing and clearance of complement-opsonized immune complexes. CR1 can negatively regulate the complement cascade, mediate immune adhesion responses and phagocytosis, and inhibit all complement pathways. The full-length CR1 protein may contain a 42-amino acid signal peptide, a 1,930-amino acid extracellular domain, a 25-amino acid transmembrane domain, and a 43-amino acid C-terminal cytoplasmic domain. The extracellular domain of CR1 may contain 25 potential N-glycosylation signal sequences and 30 short consensus repeat ("SCR") domains, also known as complement control protein (CCP) repeats or sushi domains, each 60-70 amino acids in length. Sequence homology between SCRs may range from 60-99 percent. The 30 SCR domains may be further divided into four longer regions called long homologous repeats ("LHRs"), each encoding an approximately 45 kDa segment of the CR1 protein, designated LHR-A, -B, -C, and -D (see, e.g., Krych-Goldberg et al., 274(44):31160-31168, 1999). The first three LHRs may each contain seven SCR domains, while LHR-D may contain nine SCR domains. Active sites on the extracellular domain of the CR1 protein may include a C4b-binding site in SCR1-3 that includes amino acids 42-234 and has a lower affinity for C3b, a C3b-binding site in SCR8-11 that includes amino acids 490-745 and has a lower affinity for C4b, a C3b-binding site in SCR15-18 that includes amino acids 940-1196 and has a lower affinity for C4b, and a C1q-binding site in SCR22-28 that includes amino acids 1394-1842.

[0130] SEQ ID NO:8 represents an exemplary sequence of full-length human CR1 (see, e.g., UniProtKB / Swiss-Prot accession number P17927). Amino acids 1-41 may correspond to a signal peptide, and amino acids 42-2039 may correspond to a mature protein, including amino acids 42-1971, which may correspond to an extracellular domain, amino acids 1972-1996, which may correspond to a transmembrane domain, and amino acids 1997-2039, which may correspond to a cytoplasmic domain. In the extracellular domain, amino acids 42-101 may correspond to SCR1, 102-163 may correspond to SCR2, amino acids 164-234 may correspond to SCR3, amino acids 236-295 may correspond to SCR4, amino acids 295-355 may correspond to SCR5, amino acids 356-418 may correspond to SCR6, amino acids 419-489 may correspond to SCR7, and amino acids 491-551 may correspond to SCR8. amino acids 552-613 may correspond to SCR9, amino acids 614-684 may correspond to SCR10, amino acids 686-745 may correspond to SCR11, amino acids 745-805 may correspond to SCR12, amino acids 806-868 may correspond to SCR13, amino acids 869-939 may correspond to SCR14, amino acids 941-1001 may correspond to SCR15, and amino acids 1002-1063 may correspond to SCR16. amino acids 1064-1134 may correspond to SCR17; amino acids 1136-1195 may correspond to SCR18; amino acids 1195-1255 may correspond to SCR19; amino acids 1256-1318 may correspond to SCR20; amino acids 1319-1389 may correspond to SCR21; amino acids 1394-1454 may correspond to SCR22; and amino acids 1455-1516 may correspond to SCR23. amino acids 1517-1587 may correspond to SCR24, amino acids 1589-1648 may correspond to SCR25, amino acids 1648-1708 may correspond to SCR26, amino acids 1709-1771 may correspond to SCR27, amino acids 1772-1842 may correspond to SCR28, amino acids 1846-1906 may correspond to SCR29, and amino acids 1907-1967 may correspond to SCR30. The peptides, polypeptides, and proteins of the present disclosure exhibit species and strain diversity. It is understood that there are many SCR domains, and that the CR1 protein or a biologically active fragment thereof can encompass all species and strain diversity. As used herein, the term "biologically active" fragment of the CR1 protein can refer to any soluble fragment of CR1 lacking the transmembrane and cytoplasmic domains, including fragments comprising, consisting essentially of, or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SCR domains, including any fragment of the full-length CR1 protein that has some or all of the complement inhibitory activity of the full-length CR1 protein. Functional fragments may include SCRs 1 and 2; SCRs 1, 2, 3 and 4; SCRs 1, 2, 3, 4, 5, 6, 7; SCRs 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ("CR1 1-10"); SCRs 6, 7, 8, 9, 10, 11 and 12; SCRs 8 and 9; SCRs 8, 9, 10 and 11; SCRs 8, 9, 10, 11, 12, 13 and 14; SCRs 15 and 16; SCRs 12, 13, 14, 15, 16 and 17; SCRs 15, 16, 17, 18 and 19; SCRs 1-17 ("CR1 1-17"); SCRs 1-23, SCRs 1-28. Exemplary variant polypeptides include at least three SCRs from each of domains A and B; at least three SCRs from each of domains A, B, and C, at least the first three SCRs from domains A, B, and C, or an amino acid sequence at least 90% identical to any of the above.

[0131] As used herein, the terms "complement factor H," "factor H," or "FH" can refer to complement factor H, a single-chain polypeptide plasma glycoprotein, including its homologs. The protein can consist of 20 conserved short consensus repeat (SCR) domains of approximately 60 amino acids arranged in a sequential manner resembling beads on a string, separated by short linker sequences of 2 to 6 amino acids each. Factor H can bind to C3b, promote the degradation of alternative pathway C3 convertase (C3bBb) and alternative pathway C5 convertase (C3bBb3b), and act as a cofactor for the proteolytic inactivation of C3b. In the presence of factor H, proteolysis by factor I can result in the cleavage and inactivation of C3b. Factor H can have at least three distinct binding domains for C3b, which can be located within any one of SCRs 1-20, 1-4, 5-8, and 19-20. Each domain can bind to a distinct region in the C3b protein: an N-terminal site can bind native C3b, a second site located in the middle region of factor H can bind the C3c fragment, and sites located within SCRs 19 and 20 can bind the C3d region. In addition, factor H may also contain binding sites for heparin, which may be located within SCRs 7, 5-12, and 20 of factor H and may overlap with those of the C3b-binding site. Structural and functional analyses have indicated that the domains responsible for the complement inhibitory activity of factor H may be located within the first four N-terminal SCR domains.

[0132] SEQ ID NO:9 represents an exemplary amino acid sequence of a full-length human Factor H protein (see, e.g., UniProtKB / Swiss-Prot accession number P08603), and SEQ ID NO:10 represents an exemplary amino acid sequence of a full-length mouse Factor H protein (see, e.g., UniProtKB / Swiss-Prot accession number P06909). In the human Factor H sequence, amino acids 1-18 of SEQ ID NO:9 may correspond to the signal peptide, and amino acids 19-1231 of SEQ ID NO:9 may correspond to the mature protein. Within that protein, amino acids 21-80 of SEQ ID NO:9 may correspond to SCR1, amino acids 85-141 of SEQ ID NO:9 may correspond to SCR2, amino acids 146-205 of SEQ ID NO:9 may correspond to SCR3, amino acids 210-262 of SEQ ID NO:9 may correspond to SCR4, and amino acids 267-320 of SEQ ID NO:9 may correspond to SCR5. In the mouse Factor H sequence, amino acids 1-18 of SEQ ID NO: 10 may correspond to the signal peptide, and amino acids 19-1234 of SEQ ID NO: 10 may correspond to the mature protein. Within that protein, amino acids 19-82 of SEQ ID NO: 10 may correspond to SCR1, and amino acids 83-143 of SEQ ID NO: 10 may correspond to SCR2. amino acids 144-207 of SEQ ID NO: 10 may correspond to SCR3, amino acids 208-264 of SEQ ID NO: 10 may correspond to SCR4, and amino acids 265-322 of SEQ ID NO: 10 may correspond to SCR5. It is understood that species and strain diversity exists in the peptides, polypeptides, and proteins of the present disclosure, and that Factor H or a biologically active fragment thereof may encompass all species and strain diversity. As used herein, the term "biologically active" fragment of Factor H may refer to any portion of the Factor H protein that has some or all of the complement inhibitory activity of the full-length Factor H protein, and may include, but is not limited to, Factor H fragments including SCRs 1-4, SCRs 1-5, SCRs 1-8, SCRs 1-18, SCRs 19-20, or any naturally occurring homologs or fragments thereof described in detail below. In some examples of fusion protein constructs, the biologically active fragment of Factor H may have one or more of the following properties: (1) binding to C-reactive protein (CRP), (2) binding to C3b and / or fragments thereof, (3) binding to heparin, (4) binding to sialic acid, (5) binding to endothelial cell surfaces, (6) binding to cellular integrin receptors, (7) binding to pathogens, (8) C3b cofactor activity, (9) C3 and C5 alternative pathway convertase decay-accelerating activity, and (10) inhibition of the alternative complement pathway.

[0133] In some examples of the fusion protein construct, the complement regulator portion of the construct can include a complement inhibitor or a biologically active fragment thereof. In some examples of the fusion protein construct, the complement inhibitor can be selected from human MCP, human DAF, mouse DAF, human CD59, mouse CD59 isoform A, mouse CD59 isoform B, mouse Crry protein, human CR1, human Factor H, mouse Factor H, biologically active fragments thereof, and variants thereof.

[0134] In some cases, the complement inhibitor portion of the fusion protein construct can comprise full-length human MCP (SEQ ID NO: 1). In some cases, the complement inhibitor portion of the fusion protein construct can comprise a biologically active fragment of human MCP (SEQ ID NO: 1). In some cases, the biologically active fragment of human MCP can be selected from SCR1-4 (amino acids 35-285 of SEQ ID NO: 1), SCR1-4 and the serine / threonine-rich domain (amino acids 35-326 of SEQ ID NO: 1), and the extracellular domain of MCP (amino acids 35-343 of SEQ ID NO: 1), and any combination thereof.

[0135] In some cases, the complement inhibitor portion of the fusion protein construct may comprise full-length human DAF. In some cases, the complement inhibitor portion of the fusion protein construct may comprise a biologically active fragment of human DAF (SEQ ID NO:2). In some cases, the biologically active fragment of human DAF may be selected from SCRs 1-4 (amino acids 25-285 of SEQ ID NO:2), and SCRs 1-4 and an O-glycosylated serine / threonine-rich domain (amino acids 25-353 of SEQ ID NO:2), and any combination thereof. In some cases, the complement inhibitor portion of the construct may comprise full-length murine DAF (SEQ ID NO:3). In some cases, the complement inhibitor portion of the construct may comprise a biologically active fragment of murine DAF. In some cases, the biologically active fragment of murine DAF may be selected from SCRs 1-4 (amino acids 35-286 of SEQ ID NO:3), and SCRs 1-4 and an O-glycosylated serine / threonine-rich domain (amino acids 35-362 of SEQ ID NO:3), and any combination thereof.

[0136] In some cases, the complement inhibitor portion of the fusion protein construct can include full-length human CR1 (SEQ ID NO:8). In some cases, the complement inhibitor portion of the fusion protein construct can include a biologically active fragment of human CR1 (SEQ ID NO:8). In some cases, the biologically active fragment of human CR1 is selected from the group consisting of SCR1 (amino acids 42-101 of SEQ ID NO:8), SCR2 (amino acids 102-163 of SEQ ID NO:8), SCR3 (amino acids 164-234 of SEQ ID NO:8), SCR4 (amino acids 236-295 of SEQ ID NO:8), SCR5 (amino acids 295-355 of SEQ ID NO:8), SCR6 (amino acids 356-418 of SEQ ID NO:8), SCR7 (amino acids 419-489 of SEQ ID NO:8), SCR8 (amino acids 491-551 of SEQ ID NO:8), SCR9 (amino acids 552-613 of SEQ ID NO:8), SCR10 (amino acids 614-684 of SEQ ID NO:8), SCR11 (amino acids 686 to 745 of SEQ ID NO: 8), SCR12 (amino acids 745 to 805 of SEQ ID NO: 8), SCR13 (amino acids 806 to 868 of SEQ ID NO: 8), SCR14 (amino acids 869 to 939 of SEQ ID NO: 8), SCR15 (amino acids 941 to 1001 of SEQ ID NO: 8), SCR16 (amino acids 1002 to 1063 of SEQ ID NO: 8), SCR17 (amino acids 1064 to 1134 of SEQ ID NO: 8), SCR18 (amino acids 1136 to 1195 of SEQ ID NO: 8), SCR19 (amino acids 1195 to 1255 of SEQ ID NO: 8), SCR20 (amino acids 1256 to 1318 of SEQ ID NO: 8), SCR21 (amino acids 1259 to 1319 of SEQ ID NO: 8), SCR22 (amino acids 1320 to 1321 of SEQ ID NO: 8), SCR23 (amino acids 1322 to 1323 of SEQ ID NO: 8), SCR24 (amino acids 1323 to 1324 of SEQ ID NO: 8), SCR25 (amino acids 1324 to 1325 of SEQ ID NO: 8), SCR26 (amino acids 1325 to 1326 of SEQ ID NO: 8), SCR27 (amino acids 1326 to 1327 of SEQ ID NO: 8), SCR28 (amino acids 1327 to 1328 of SEQ ID NO: 8), SCR29 (amino acids 1328 to 1330 of SEQ ID NO: 8), SCR30 (amino acids SCR21 (amino acids 1319-1389 of SEQ ID NO:8), SCR22 (amino acids 1394-1454 of SEQ ID NO:8), SCR23 (amino acids 1455-1516 of SEQ ID NO:8), SCR24 (amino acids 1517-1587 of SEQ ID NO:8), SCR25 (amino acids 1589-1648 of SEQ ID NO:8), SCR26 (amino acids 1648-1708 of SEQ ID NO:8), SCR27 (amino acids 1709-1771 of SEQ ID NO:8), SCR28 (amino acids 1772-1842 of SEQ ID NO:8), SCR29 (amino acids 1846-1906 of SEQ ID NO:8), SCR30 (amino acids 1907-1967 of SEQ ID NO:8), or any combination thereof.

[0137] In some cases, the complement inhibitor portion of the fusion protein construct may comprise full-length human Factor H (SEQ ID NO:9). In some cases, the complement inhibitor portion of the fusion protein construct may comprise a biologically active fragment of human Factor H (SEQ ID NO:9). In some cases, the complement inhibitor portion of the fusion protein construct may comprise full-length mouse Factor H (SEQ ID NO:10). In some cases, the complement inhibitor portion of the fusion protein construct may comprise a biologically active fragment of mouse Factor H (SEQ ID NO:10). The biologically active fragment of Factor H may comprise SCRs 1-4, SCRs 1-5, SCRs 1-8, SCRs 1-18, SCRs 19-20 of Factor H, or any naturally occurring homolog or fragment thereof of Factor H, or any combination thereof.

[0138] Targeting part The targeting moiety of the multivalent construct may be responsible for the directed delivery of a regulator of the complement system to a site of action, e.g., the site of complement activation. The complement regulator may have a therapeutic activity, such as specifically inhibiting complement activation. Thus, the multivalent constructs described herein collectively have the dual function of binding to the epitope recognized by the antibodies described herein and exerting a therapeutic activity by inhibiting complement activation. The targeting moiety may be a human, murine, humanized, or camelized antibody, or an antigen-binding fragment thereof.

[0139] The epitope recognized by the antibody or antigen-binding fragment thereof may be a domain of a mammalian annexin protein, a phospholipid, such as one or more of the C2 antibody-reactive phospholipids described below, or a complement protein, such as C3d, a C3 fragment (e.g., deposited C3 fragments—C3b, iC3b, C3d, C3dg; free or undeposited C3 fragments—C3a, C3b, C3c or C3f).

[0140] The antibody or antigen-binding fragment thereof may specifically bind to a domain of a mammalian annexin protein. Annexins are proteins that bind to Ca 2+ The binding site is similar to most other Ca 2+ Calcium (Ca) binding proteins 2+) and phospholipid-binding proteins. 2+ The binding site has a unique organizational pattern that allows annexin family members to be reversibly tethered to the outer surface of cell and / or organelle membranes. 2+ The binding site is located in the annexin core domain, which contains four annexin repeats, each 70 amino acids long. The annexin core domain is α-helical, forming a small, rounded disk with a convex surface that binds Ca. 2+ and membrane-binding sites, with the concave side facing outwards towards the membrane where it is available for other types of interactions. Annexin family members also typically have an amino-terminal domain of variable length that precedes the annexin core domain and is variable in sequence and structure. In vertebrates, annexin family members include annexin IV and annexin 2. Twelve annexin subfamilies have been characterized, each with a distinct amino-terminal domain and a Ca receptor at a different location. 2+ The binding site has different splice variants arranged differently. The antibody or antigen-binding fragment thereof that specifically binds to or recognizes an epitope within a domain in an annexin IV protein can be B4 mAb or an antigen-binding fragment derived from B4 mAb. The antibody or antigen-binding fragment thereof that specifically binds to or recognizes an epitope within an annexin IV protein (e.g., human annexin IV protein) can be B4 mAb or an antigen-binding fragment derived from B4 mAb, as described in Kulik et al., J. Immunol. 182(9):5363 (2009). Exemplary CDRs of B4 mAb are set forth in SEQ ID NOS: 11-16. The targeting moiety can also be an antibody or antigen-binding fragment thereof that specifically binds to or recognizes an epitope within an annexin 2 protein (e.g., human annexin 2 protein).

[0141] The antibody or antigen-binding fragment thereof may also specifically bind to a phospholipid (e.g., phosphatidylethanolamine (PE), cardiolipin (CL), phosphatidylcholine (PC), phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, or phosphatidic acid) or malondialdehyde (MDA). The antibody or antigen-binding fragment thereof that specifically binds to a phospholipid may be C2 mAb or a derivative thereof. The phospholipid may be present on the surface of cells, basement membranes (e.g., Bruch's membrane), or in pathological structures (e.g., in drusen) in an individual within or adjacent to tissues that have undergone (or are at risk of undergoing) tissue damage (e.g., non-ischemic damage), oxidative damage, or any combination thereof. The phospholipid may be neutral, negatively or positively charged, or oxidized. The antibody or antigen-binding fragment thereof that specifically binds to phospholipids may be C2 mAb or C2 mAb, as described in Elvington et al., J. Immunol., 188(3):1460-1468 (2012). The C2 mAb may be an antigen-binding fragment derived from a mAb. Exemplary CDRs of C2 mAb are set forth in SEQ ID NOS: 17-22. C2 mAb recognizes a subset of phospholipids exposed after complement activation or ischemia, and this subset of phospholipids is referred to herein as "C2 antibody-reactive phospholipids." C2 mAb has been shown to recognize a subset of phospholipids that includes phosphatidylcholine, phosphatidylethanolamine, and cardiolipin, but does not include phosphatidylglycerol or phosphatidylserine.

[0142] In some cases, the targeting moiety can be an antibody or antigen-binding fragment thereof that specifically binds to deposited or opsonized C3 fragments, e.g., C3b, iC3b, C3d, or C3dg, but may or may not bind to free, circulating, or undeposited C3 fragments, e.g., C3a, C3b, C3c, or C3f. In some examples, a fusion protein construct comprising an anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof can bind to deposited C3 fragments with relatively higher affinity than free C3 or C3 fragments. For example, the antibody or antigen-binding fragment thereof can bind to C3 and C3b with about 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower binding affinity than C3d. In some embodiments, the antibody or antigen-binding fragment binds to C3 and / or C3b with about 10-fold lower binding affinity. -4 M or above, 10 -3 M or above or 10 -2 Binds to iC3b, C3dg, or both with a KD of 10 -8 M or less, 10 -9 M or less or 10 -10 It binds with a binding affinity (KD) of M or less.

[0143] In some examples, fusion protein constructs comprising anti-C3d or anti-C3dg antibodies or antigen-binding fragments thereof bind to both deposited C3 fragments and free C3 or C3 fragments. Furthermore, the anti-C3d or anti-C3dg antibodies or antigen-binding fragments thereof of exemplary fusion protein constructs can bind to the complement fragment C3d, distinguishing between tissue-bound C3 fragments and circulating C3 (e.g., C3, C3b, or C3(HO)). It may also be that the targeting moiety has the ability to bind to C3d. Examples of anti-C3d or anti-C3dg antibodies or antigen-binding fragments thereof include, but are not limited to, mAbs 3d9a, 3d29, and 3d8b (see, e.g., US9815890). In some cases, the anti-C3d or anti-C3dg antibodies of the present disclosure can bind to C3d with greater specificity than commercially available anti-C3d antibodies, such as the anti-C3d antibodies represented by Quidel catalog numbers A207 and A250, available from Quidel Corporation (Quidel Corp., San Diego and Santa Clara, Calif.). In some cases, the targeting moiety can include an antibody specific for C3d and / or other C3 fragments (such as C3b, iC3b, C3c, C3dg), such as the antibody C8D3 described in U.S. Patent Application Publication No. 2016 / 0333082. The antibody C8D3 can bind with high affinity to an epitope on C3d (see, for example, Figures 3 and 8 of US2016 / 0333082), which overlaps with the CR2-binding epitope. The heavy chain sequence of C8D3 can be SEQ ID NO: 154, the light chain sequence is SEQ ID NO: 155, and the CDRH1, CDRH2, and CDRH3 of C8D3 are SEQ ID NOs: 156, 157, and 158, respectively, and the CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 159, 160, and 161, respectively. This publication also describes four hybridoma clones (i.e., clones B7, C2, C6, and C8) that produce antibodies against C3d. The heavy chain sequence of C6 is SEQ ID NO: 162 and the light chain sequence is SEQ ID NO: 163, CDRH1, CDRH2 and CDRH3 of C6 are SEQ ID NOs: 164, 165 and 166, respectively, and CDRL1, CDRL2 and CDRL3 are SEQ ID NOs: 167, 168 and 169, respectively.

[0144] In some examples, the C3d antibody can be an antibody that binds to C3d but not C3c ("neo-anti-C3d"), as described in U.S. Patent Application Publication No. 2015 / 0139899. The C3d antibody binds to its epitope with an affinity of about 100 pM to about 500 pM, e.g., 447 pM. The C3d antibody can be an antibody specific for the neo-epitope iC3b ("neo-anti-iC3b"), and in some cases binds to that epitope with an affinity of about 100 pM to about 500 pM, e.g., 262 pM, but the antibody does not bind to C3c or C3d.

[0145] In some examples, the C3d antibody can be monoclonal antibody M130, which is specific for antigenic determinants expressed on C3bi, C3dg, and C3d, which are nearly undetectable on C3 and C3b. M130 has been shown to have higher affinity for C3d and iC3b than for C3(HO) (JD Tamerius et al., J. Immunol. 135(3):2015-19(1985)), and can bind to residues 1209-1236 and 1217-1232 of C3d (Lambris et al., PNAS, 82(12):4235-9(1985)).

[0146] In some examples, the C3d antibody can be the monoclonal antibody C3-12.2 (Hidalgo et al., Eur. J. Immunol. 47(3):504-15(2017)), which binds to human, rat, and mouse C3dg fragments. It was generated using C3-deficient mice immunized with a human C3b, iC3b, and C3dg protein mixture and has a K of approximately 95 nM as measured by BiaCore. D C3-12.2, as well as antibodies 3d29, 3d8b, and 3d9a, may recognize one or more overlapping C3 fragments or variants, and the Fab appears to bind to the same or adjacent epitope as CR2.

[0147] In some examples, the C3d antibody can be monoclonal antibody 15-39-06, which was assembled in wild-type rats using a synthetic peptide derived from human C3dg and conjugated to diphtheria toxin (K. J. Rassmussen et al., J. Immunol. munol. Methods, 444:51-5 (2017)). It may have specificity for the C3dg complement split product.

[0148] In some instances, the C3d antibody may be a commercially available antibody specific for C3d and / or other C3 fragments (i.e., C3b, iC3b, C3c, C3dg, etc.). Examples include antibodies available from Quidel, where monoclonal antibodies A250 and A209 are reported to be specific for the neoepitopes C3d and iC3b, respectively. Antibody A250 has been shown to agglutinate EC3bi, EC3b, and EC3d cells in an indirect hemagglutination assay and has also been shown to bind to radiolabeled purified iC3b, C3b, and C3d but not similarly labeled C3 or C3c. Antibody A209 has been shown to agglutinate EC3bi but not EC3b or EC3d cells in an indirect hemagglutination assay and has also been shown to bind to radiolabeled purified iC3b but not similarly labeled C3, C3b, C3d, or C3c. As further examples, anti-C3d antibody 7C10, which has an unknown epitope, and anti-C3d antibody [E28-P] (ab136916), which is reported to bind to an epitope at the N-terminus of C3d, are available from Abcam. In some cases, commercially available antibodies may be anti-C3d antibody 053A-514.3.1.4 and iC3b antibody 013III-1.16 (formerly MCA2607), available from BioRad, which are reported to be specific for the neoantigens C3d and iC3b, respectively. Sigma offers antibody 3E7, which is reported to recognize C3b and iC3b. Origene offers the monoclonal antibody AM26358PU-N, which reacts with neoantigens on iC3 (C3(H2O)), iC3b, C3dg, and / or C3g, and recognizes iC3b, C3dg, and C3g in plasma, but not C3 or C3b. USBiological Life The antibody C0010-19 rat anti-C3g (which recognizes iC3, iC3b, and C3dg) is available from USBiological Life Sciences. USBiological Life Sciences also offers the inactivated C7850-13V-ML550 mouse anti-complement C3b. This antibody has been reported to recognize the human inactivated complement C3b (iC3b) neoantigen in serum. Several antibodies are available from Hycult, including antibody HM2199, an anti-human C3g mAb 9 (YB2 / 90-5-20) that reacts with neoantigens on iC3, iC3b, C3dg, and C3g and recognizes iC3b, C3dg, and C3g in serum but not C3 or C3b; and monoclonal antibody HM2198, an anti-human C3d mAb that is reported to react with a linear determinant in C3d found on C3, C3b, iC3b, C3dg, and C3d, and recognizes C3, C3b, iC3b, C3dg, and C3d but not C3c. 3 (YB2 / 39-11-1-7); antibody HM2168 is an anti-active human C3, clone bH6, specific for a C3 neoepitope expressed on cleavage fragments of C3b, iC3b, and C3c, but not on C3dg and C3f (P. Garred et al., Scand J Immunol 1988, 27:319); antibody HM2257 is an anti-active human C3, mAb I3 / I5, which recognizes activated complement protein C3, or more specifically, neoepitopes on C3b, iC3b, and C3dg that are not present in native C3. Meridian also offers antibody H54189M, which has been reported to react with the alpha chain of C3b but not C3a or C3d, allowing for demonstration of C3 deposits in tissues, cells, microorganisms, and immune complexes.

[0149] Each of the 3d9a, 3d29, and 3d8b antibodies has the potential to bind to inflamed renal tissue sections when injected intravenously into mice, and to C3-opsonized zymosan, which is known to express iC3b but not C3b. Therefore, these antibodies may be able to distinguish between tissue-bound fragment C3d, circulating naive C3, and fragment C3b. C3-binding antibodies or their antigen-binding properties The fragment may bind to C3d or C3dg from multiple species (species cross-reactivity). An anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof may bind to C3d or C3dg from at least one species selected from humans, non-human mammals (e.g., cynomolgus monkeys or cynomolgus macaques, rhesus monkeys, apes, baboons, chimpanzees, orangutans, or gorillas), rodents (e.g., mice, rats, hamsters, guinea pigs, gerbils, or rabbits), cows, sheep, goats, donkeys, pigs, dogs, cats, horses, and camels. An anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof may bind to cynomolgus monkey C3d or C3dg. The anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof described herein binds to C3d or C3dg from at least two species selected from those listed above. The anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof binds to both human and cynomolgus monkey C3d or C3dg. The anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof may be an antibody selected from 3d8b, 3d9a, 3d29, 3d11, 3d31, 3d3, 3d15, 3d10, and 3d16 (see, e.g., US9815890). The anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof may be an antibody selected from 3d9a, 3d29, and 3d8b. The anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof may be 3d29.

[0150] In some examples, the anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof of the fusion protein construct can compete with CR2 for binding to C3d or C3dg. Such an antibody or antigen-binding fragment thereof can reduce the binding ability of the CR2 protein to human complement component C3d or C3dg by more than 50% (e.g., more than 55, 60, 65, 70, 75, 80, 85, 90, or 95%). For example, CR2-C3d binding can be reduced by at least 60%, at least 40%, or any percentage value therebetween. An anti-C3d or anti-C3dg antibody or antigen-binding fragment thereof can significantly inhibit or block CR2 binding to C3d. In some embodiments, such an antibody is 3d9a, 3d29, or 3d8b. In some cases, exemplary fusion protein constructs comprising an anti-C3d or anti-C3dg targeting domain and a complement regulator may better compete with CR2 for binding to C3d or C3dg compared to the anti-C3d or anti-C3dg targeting domain alone.

[0151] Fusion protein construct design Fusion protein constructs comprising a targeting moiety and a complement regulatory agent may include an antibody or antigen-binding fragment thereof as the targeting moiety. Examples of targeting moieties include, but are not limited to, monoclonal antibodies or antibody fragments, diabodies, chimerized or chimeric antibodies or antibody fragments, humanized antibodies or antibody fragments, deimmunized human antibodies or antibody fragments, fully human antibodies or antibody fragments, bispecific antibodies or antibody fragments, monovalent antibodies or antibody fragments, single-chain antibodies, immunoglobulin G1 (IgG1) heavy chains, single-chain variable fragments (i.e., scFv), sc(fv)2, tandem scFv, diabodies, VHH domains, VH domains, Fv, Fd, Fab heavy chains, Fab light chains, Fab, Fab', and Fab' light chains, Fab' heavy chains, and F(ab')2. The antibody or antigen-binding fragment forming the targeting moiety may be a human antibody, a humanized antibody, or a murine antibody. A fusion protein construct can contain one or more targeting moieties, such as each targeting moiety containing an antibody or antigen-binding fragment thereof specific for a target. In addition, a fusion protein construct can contain more than one complement regulator. When a fusion protein construct contains more than one complement regulator, it can contain multiple molecules of the same complement regulator, or it can contain distinct types of complement regulators. Similarly, a fusion protein construct with multiple targeting moieties can contain several molecules of the same type of targeting moiety, thereby making it multivalent with respect to the targeting moiety, or several molecules of distinct types of targeting moieties, thereby making it multivalent with respect to the targeting moiety. The target of the fusion protein construct may be, for example, a complement protein or fragment thereof, a domain of a mammalian annexin protein, or a phospholipid. Because a fusion protein construct may have the ability to specifically bind to more than one target, it is contemplated that in some instances at least two of the above targets may be specifically bound by the fusion protein constructs described herein. Thus, the fusion protein construct may be bispecific, trispecific, tetraspecific, etc. Furthermore, the fusion protein construct may be multivalent, e.g., bivalent, trivalent, tetravalent, etc. For example, the targeting portion of a bispecific fusion protein construct may be specific for a target, e.g., a domain of a mammalian annexin, a phospholipid, or a C3 complement protein fragment (e.g., C3d), ​​and the complement regulator of the fusion protein construct may be specific for another protein in the complement pathway. A tetravalent fusion protein construct may have a bivalent targeting portion, e.g., a bivalent antibody or antigen-binding fragment thereof having two binding regions for a target protein, and two molecules of a complement regulator. Furthermore, the fusion protein construct can be a bispecific trivalent protein in which the targeting moiety comprises a bivalent antibody or antigen-binding fragment thereof and an antibody constant domain (Fc), and the complement regulatory agent is fused to the bivalent antibody or the Fc domain.

[0152] Other examples of fusion protein constructs include heterodimeric Fc regions containing various modifications that promote the formation of heterodimeric Fc regions over homodimeric Fc regions. See, e.g., JH Ha et al. Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins. Front. Immuno. 7:394 (2016). Traditional IgG antibodies are multivalent and monospecific, and their assembly relies on the homodimerization of two identical heavy chains (HC) in vivo, mediated by homodimeric association between the CH3 domains in B cells and subsequent disulfide linkage between each HC and each light chain (LC). Therefore, development of bsAbs using unmodified IgG formats with wild-type HC and LC requires the use of HC-HC and HC-HC. VH-CH1In some embodiments, the monomeric Fc domains, Fc1 and Fc2, are IgG Fc domains. In some embodiments, the monomeric Fc domains, Fc1 and Fc2, are from other immunoglobulin subclasses, including IgA, IgE, IgD, and IgM. The heterodimeric Fc regions of the targeting moieties described herein comprise a CH2 constant domain and a variant CH3 constant domain containing amino acid mutations that promote the formation of such heterodimers with stability comparable to that of the native homodimeric Fc. Wild-type Fc is naturally homodimeric, a property facilitated by both hydrophobic interactions at the center of the CH3 interface and symmetric electrostatic interactions around the hydrophobic core. The Fc domains described herein can contain amino acid substitutions that prevent them from forming homodimers. The Fc domains described herein can contain amino acid substitutions that favor heterodimer formation over homodimer formation. In certain examples, heterodimeric Fc domains can be engineered using (i) stereocomplementary designs that convert symmetry to asymmetricity (e.g., KiH, HA-TF, and ZW1) (see, e.g., Klein et al., Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies. mAbs 4(6):653-66 (2012); G.L. Moore et al., A novel bispecific Fc domain. The ificial antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. mAbs 3(6)546-557(2011); T. S. von Kreudenstein et al. Improving biophysical properties of a bispecific antibody scaffold to aid developability: Quality by molecular design. mAbs 5(5):646-654(2013); H. J. Choi et al. A heterodimeric Fc-based bispecific antibody simultaneously targeting VEGFR-2 and Met exhibits potent antitumor activity. Mol Cancer Ther 12(12):2748-59(2013)). (ii) charge-to-charge exchange (e.g., DD-KK), (iii) charge-to-stereocomplementary switching with additional long-range electrostatic interactions (e.g., EW-RVT), and (iv) isotype strand exchange [e.g., strand exchange engineering domain (SEED)]. Strand-exchange mutations include, for example, IgA-derived residue 45 on IgG1 CH3-Fc1 and IgG1-derived residue 57 on IgA CH3-Fc2, or vice versa. Examples of stereocomplementary mutations that convert symmetry to asymmetry include HA-TF (S364H / F405A in Fc1-CH3 or CH3A and Y349T / T394F in Fc2-CH3 or CH3B) and ZW1 (T350V / L351Y / F405A / Y407V in Fc1-CH3 or CH3A and T350V / T366L / K392L / T394W in Fc2-CH3 or CH3B).In another example, Fc variants can be created using the "knob-into-hole (KiH)" approach, where Fc1 contains a T366W "knob" mutation in the Fc1-CH3 or CH3A domain, and Fc2 contains T366S / L368A / Y407V "hole" mutations in the Fc2-CH3 or CH3B domain. In a further example, Fc variants can be created using both the "knob-into-hole (KiH)" and disulfide bond approaches, KiH. s-s where Fc1 contains the "knob" mutations T366W / S354C in the Fc1-CH3 or CH3A, and Fc2 contains the "hole" mutations T366S / L368A / Y407V / Y349C in the Fc2-CH3 or CH3B domain. In such instances, heterodimerization is favored by hydrophobic interactions at the core of the interface between Fc1-CH3 or CH3A and F2-CH3 or CH3B. An example of a charge-to-charge exchange mutation that favors interactions where the Fc heterodimer is based on electrostatic complementarity is DD-KK (K409D / K392D in Fc1-CH3 or CH3A and D399K / E356K in Fc2-CH3 or CH3B, or vice versa). Examples of mutations that switch from charge to steric complementarity and result in additional long-range electrostatic interactions include EW-RVT (K360E / K409W in Fc1-CH3 or CH3A and Q347R / D399V / F405T in Fc2-CH3 or CH3B, or vice versa); EW-RVT, which contains an inter-CH3 S-S bond. s-s(K360E / K409W / Y349C in Fc1-CH3 or CH3A and Q347R / D399V / F405T / S354C in Fc2-CH3 or CH3B, or vice versa). In yet another example, Fc variants can be created using hydrophobic or steric complementarity and electrostatic complementarity, e.g., 7.8.60 (K360D / D399M / Y407A in Fc1-CH3 or CH3A and E345R / Q347R / T366V / K409V in Fc2-CH3 or CH3B, or vice versa). Heterodimer-forming Fc variants described herein can also be created by directed evolution in combination with yeast surface display and high-throughput screening. For example, a combinatorial heterodimeric Fc library display system can be developed by pairing two haploid yeast cell lines, where one haploid cell line displays an Fc chain library with mutations in one CH3 domain (CH3-Fc1 or CH3A) on the yeast cell surface. One cell line displays a library of Fc chains (CH3-Fc2 or CH3B) with mutations in the other CH3 domain. In paired cells, the secreted CH3-Fc2 or CH3B can be displayed on the cell surface by heterodimerization with the displayed CH3-Fc1 or CH3A. Fluorescence-based detection of this interaction allows for the screening of heterodimeric Fc variants against the library by flow cytometry. Antibodies or antigen-binding fragments thereof containing a wild-type Fc domain have the ability to interact with the fetal Fc receptor (FcRn) in a pH-dependent manner, and this interaction can result in an extended serum half-life. Residues important for the high-affinity interaction of the Fc domain with FcγR are located in the CH2 domain. Thus, in some cases, the Fc heterodimer of the targeting moiety contains a CH2 domain with a wild-type IgG sequence.

[0153] In some examples, the "CH3 domain" comprising the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG) can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with a "protrusion" introduced in one chain and a corresponding "hole" introduced in the other chain). Such a variant CH3 domain can, in some examples, be part of a heterodimeric Fc domain described herein. Thus, in some examples, the fusion protein construct comprises an antibody, or antigen-binding fragment thereof, having a protrusion and a hole to accommodate it.

[0154] In some examples, the Fc region can comprise a human or mouse IgG1 or human IgG4 sequence. In some examples, the Fc region can comprise a human IgG1 or IgG4 or mouse IgG1 sequence that contains amino acid substitutions compared to the wild-type sequence. Examples of such Fc regions are set forth in SEQ ID NO:247 and SEQ ID NO:248.

[0155] Domains and Structures of Fusion Protein Constructs A fusion protein construct comprises one or more polypeptides each containing a domain and linked, for example, via one or more disulfide bonds. For example, in some cases, a fusion protein construct may comprise a first polypeptide chain comprising Domain A, Domain B, Domain C, Domain D, and Domain R, where Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, Domain C comprises a heavy chain CH2 constant region amino acid sequence, Domain D comprises a heavy chain CH3 constant region amino acid sequence, and Domain R comprises a complement regulator polypeptide. In some embodiments, one or more of Domains B, C, and D are optional. The first polypeptide may further comprise a hinge region, and may further be linked to a second polypeptide comprising additional domains, such as Domain E comprising a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and optional Domain F comprising a light chain constant region amino acid sequence (CL1). The link between the first and second polypeptides may be via one or more disulfide bonds between Domain B and Domain F. In some instances, the first and second polypeptides are combined using various orientations to create fusion protein constructs such as monomeric, trivalent homodimeric, trivalent homodimeric, tetravalent homodimeric, tetravalent heterodimeric, etc.

[0156] In various examples of multivalent fusion protein constructs (e.g., bivalent, trivalent, tetravalent), the multivalency of the fusion protein construct can improve the avidity of the fusion protein construct for a particular target. As used herein, "avidity" can refer to the overall strength of interaction between two or more molecules, for example, a fusion protein construct comprising a bivalent targeting moiety, and avidity can be the cumulative strength of the interaction given by the affinity of the two antigen-binding sites. Avidity can be used, for example, to determine affinity. It can be measured by the same method used. In certain examples, avidity can be the cumulative strength of interaction, as indicated by the affinity of multiple antigen-binding sites for distinct antigens on a common specific target or complex, such as distinct antigens found on individual cells. In certain examples, avidity can be the cumulative strength of interaction, as indicated by the affinity of multiple antigen-binding sites for distinct epitopes on a common individual antigen.

[0157] Domain A (VH) Domain A of the fusion protein constructs described herein can comprise a VH amino acid sequence, e.g., an antibody heavy chain variable domain sequence. In a typical antibody configuration, both naturally occurring and in the fusion protein constructs described herein, a particular VH amino acid sequence can associate with a particular VL amino acid sequence to form an antigen-binding site. In various examples, the VH amino acid sequence is a mammalian sequence, including a human sequence, a synthetic sequence, or a combination of non-human mammalian, mammalian, and synthetic sequences. In various examples, the VH amino acid sequence can be a mutant sequence of a naturally occurring sequence that retains sufficient CDR sequences to retain the desired antigen-binding affinity. In some examples, the VH sequence can be from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In some examples, the VH sequence can be from an IgG1 isotype.

[0158] Domain E(VL) Domain E of the fusion protein constructs described herein can comprise a VL amino acid sequence, e.g., an antibody light chain variable domain sequence. In a typical configuration, a particular VL amino acid sequence can associate with a particular VH amino acid sequence to form an antigen-binding site in both a native antibody and the fusion protein constructs described herein. In various examples, the VL amino acid sequence can be a mammalian sequence, including a human sequence, a synthetic sequence, or a combination of human, non-human mammalian, mammalian, and synthetic sequences. In various examples, the VL amino acid sequence can be a mutant sequence of a naturally occurring sequence that retains at least 70%, 75%, 80%, 85%, 90%, 95%, or more amino acid identity. In certain examples, the VL amino acid sequence can be a lambda (λ) light chain variable domain sequence. In certain examples, the VL amino acid sequence can be a kappa (κ) light chain variable domain sequence. In some examples, the VL sequence can be from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In some examples, the VL sequence may be from an IgG1 isotype.

[0159] Domain B (CH1) The CH1 amino acid sequence of the fusion protein constructs described herein can be the sequence of the second domain of an antibody heavy chain when viewed from the N-terminus to the C-terminus. In certain examples, the CH1 sequence can be an endogenous sequence or a mutant sequence thereof that retains at least 70%, 75%, 80%, 85%, 90%, 95%, or more amino acid identity. In some examples, the CH1 sequence can be a mammalian sequence, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In some examples, the CH1 sequence can be a human sequence. In some examples, the CH1 sequence can be from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In some examples, the CH1 sequence can be from an IgG1 isotype. In some examples, the CH1 sequence can be from an IgG4 isotype.

[0160] Domain F(CL1) The CL amino acid sequence of the fusion protein constructs described herein can be an antibody light chain constant domain sequence. In certain instances, the CL sequence is an endogenous sequence, or at least 70%, 70%, or 70% identical to the endogenous sequence. It may be a mutant sequence thereof that retains 5%, 80%, 85%, 90%, 95% or more amino acid identity. In some examples, the CL sequence may be a mammalian sequence, including but not limited to, a mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequence. In some examples, the CL sequence may be a human sequence. In some examples, the CL amino acid sequence may be a lambda (λ) light chain constant domain sequence. In some examples, the CL amino acid sequence may be a human lambda light chain constant domain sequence. In certain embodiments, the CL amino acid sequence is a kappa (κ) light chain constant domain sequence. In a preferred embodiment, the CL amino acid sequence is a human kappa (κ) light chain constant domain sequence.

[0161] Domain C (CH2) In the fusion protein constructs described herein, domain C can have a CH2 amino acid sequence. The CH2 sequence can be an endogenous sequence or a mutant sequence thereof that retains at least 70%, 75%, 80%, 85%, 90%, 95% or more amino acid identity. In some examples, the CH2 sequence can be a mammalian sequence, including but not limited to a human sequence. In some examples, the CH2 amino acid sequence can have an N-terminal hinge region linking domain C to domain B, e.g., linking the C-terminus of domain B (CL1 sequence) to the N-terminus of domain C (CH2 sequence). In some examples, the CH2 sequence can be from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In some examples, the CH2 sequence can be from an IgG1 isotype. In some examples, the CH2 sequence can be from an IgG4 isotype.

[0162] Domain D (CH3) In the fusion protein constructs described herein, domain D can comprise a constant region amino acid sequence, e.g., a CH3 amino acid sequence. The CH3 sequence can be an endogenous sequence or a mutant sequence thereof that retains at least 70%, 75%, 80%, 85%, 90%, 95%, or more amino acid identity. In some examples, the CH3 sequence can be a mammalian sequence, including but not limited to a human sequence. In some examples, domain D can comprise a constant region sequence that is a CH3 sequence containing knob-hole orthogonal mutations; isoallotypic mutations; and either the S354C or Y349C mutation, or any combination thereof, that forms an engineered disulfide bridge with the CH3 domain containing the orthogonal mutation. In some examples, the knob-hole orthogonal mutations in combination with isoallotypic mutations can include the following mutational changes: D356E, L358M, T366S, L368A, and Y407V. In some examples, the CH3 sequence can be from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In some examples, the CH3 sequence can be from an IgG1 isotype. In some examples, the CH3 sequence can be from an IgG4 isotype. In any of these examples, the CH1, CH2, and CH3 sequences can be from the same isotype, e.g., IgG1 or IgG3.

[0163] Complementarity-determining regions in domain A and domain F The VH (Domain A) and VL (Domain F) amino acid sequences of the various fusion protein constructs described herein can contain highly variable sequences referred to as "complementarity-determining regions" (CDRs), typically three CDRs (CDR1, CDR2, and CDR3). In various examples, the CDRs can be mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In some examples, the CDRs can be human sequences. In some examples, the CDRs can be naturally occurring sequences. In some examples, the CDRs can be naturally occurring sequences that have been mutated to alter the binding affinity of the antigen-binding site for a particular antigen or epitope. In certain examples, naturally occurring CDRs can be mutated in vivo in a host by affinity maturation and somatic hypermutation. In certain instances, the CDRs may be mutated in vitro by methods including, but not limited to, PCR mutagenesis and chemical mutagenesis. In various instances, the CDRs may comprise synthetic sequences, including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries.

[0164] Framework Region and CDR Grafting The VH and VL amino acid sequences may further comprise "framework region" (FR) sequences. FRs are often conserved sequence regions that can serve as a scaffold for the interspersed CDRs and are typically arranged (from N-terminus to C-terminus) as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In various examples, the FRs may be mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In some examples, the FRs may be human sequences. In various examples, the FRs may be naturally occurring sequences. In various examples, the FRs may be synthetic sequences, including but not limited to rationally designed sequences.

[0165] In various examples, both the FRs and CDRs can be the same naturally occurring variable domain sequence. In various embodiments, the FRs and CDRs can be from different variable domain sequences, with the CDRs providing specificity for a particular antigen and the CDRs grafted onto the FR scaffold. In certain examples, all grafted CDRs can be derived from the same naturally occurring variable domain sequence. In certain examples, the grafted CDRs can be derived from different variable domain sequences. In certain examples, the grafted CDRs can be synthetic sequences, including but not limited to CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. In certain examples, the grafted CDRs and FRs can be from the same species. In certain examples, the grafted CDRs and FRs can be from different species. In certain examples, the various domains of the fusion protein construct can be "humanized," with the grafted CDRs being non-human mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, and the FRs being human sequences. In various instances, a portion or specific sequence of a FR from one species can be used to replace a portion or specific sequence of a FR of another species.

[0166] Homodimeric or heterodimeric pairing of two domains D (CH3 domains) In the fusion protein constructs described herein, two domains D can associate to form a dimeric construct. In various examples, the amino acid sequences of all domains D are identical. In certain examples, domain D comprises an endogenous CH3 sequence. In some examples, when the fusion protein construct is a heterodimer or heterodimer, two domains D having different amino acid sequences can associate and each contain an orthogonal modification in the endogenous CH3 sequence, and one domain D interacts with another domain having a different sequence, and neither of the two domains interacts significantly with another domain D (CH3 domain) that does not have an orthogonal modification.

[0167] An "orthogonal modification" or, synonymously, "orthogonal mutation," as used herein, can be one or more engineered mutations in the amino acid sequence of an antibody domain that can increase the affinity of binding between a first domain bearing the orthogonal modification and a second domain bearing the complementary orthogonal modification. In certain instances, the orthogonal modification can decrease the affinity of a domain bearing the orthogonal modification with a domain lacking the complementary orthogonal modification. In certain instances, the orthogonal modification can be a mutation in the endogenous antibody domain sequence. In various instances, the orthogonal modification can be a modification of the N- or C-terminus of the endogenous antibody domain sequence, including, but not limited to, amino acid addition or deletion. In some instances, the orthogonal modification can include, but is not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge pair mutations. In some instances, the orthogonal modification can be a mutation in the endogenous antibody domain sequence, including, but not limited to, an amino acid addition or deletion, at the N- or C-terminus of the endogenous antibody domain sequence. Modifications may include combinations of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knobs-in-holes mutations, and charge-pair mutations. In some instances, orthogonal modifications may be combined with amino acid substitutions that reduce immunogenicity, such as isoallotypic mutations.

[0168] In certain examples, when the fusion protein construct is a heterodimer, the orthogonal modification may include a mutation that creates an engineered disulfide bridge between the first and second domains. As used herein, an "engineered disulfide bridge" may include a mutation in two or more domains that provides a non-endogenous cysteine ​​amino acid such that a non-natural disulfide bond is formed when the two or more domains are associated. In certain examples, the engineered disulfide bridge can improve the orthogonal association between specific domains. In some examples, the mutation that can create the engineered disulfide bridge may include a K392C mutation in one of the first or second CH3 domains and a D399C mutation in the other CH3 domain. In one example, the mutation that can create the engineered disulfide bridge may include a S354C mutation in one of the first or second CH3 domains and a Y349C mutation in the other CH3 domain. In another example, a mutation capable of generating an engineered disulfide bridge can include the 447C mutation in both the first and second CH3 domains, resulting from an extension of the C-terminus of the CH3 domain to incorporate the KSC tripeptide sequence.

[0169] In some examples, the orthogonal modification can include a knob-hole (synonymously, knob-in-hole) mutation. As described herein, a knob-hole mutation can include a mutation that alters the surface conformation of a first domain such that the first domain preferentially associates with a second domain having a complementary conformational mutation compared to association with a domain that does not have the complementary conformational mutation. In various examples, a knob-hole mutation can be combined with an engineered disulfide bridge. In various embodiments, a knob-hole mutation, an isoallotypic mutation, and an engineered disulfide mutation can be combined to create a heterodimeric fusion protein construct. In a particular example, a knob-in-hole mutation can include a T366Y mutation in one CH3 domain and a Y407T mutation in another CH3 domain. In a particular example, a knob-in-hole mutation can include F405A in one CH3 domain and T394W in another CH3 domain. In certain embodiments, knobs-in-hole mutations can include T366Y and F405A mutations in one CH3 domain and T394W and Y407T mutations in another CH3 domain. In particular examples, knobs-in-hole mutations can include T366W mutations in one CH3 domain and Y407A in another CH3 domain. In certain embodiments, combined knobs-in-hole and engineered disulfide mutations can include S354C and T366W mutations in one CH3 domain and Y349C, T366S, L368A, and Y407V mutations in another CH3 domain. In some examples, the combined knobs-into-holes, isoallotypic, and engineered disulfide mutations can include S354C and T366W mutations in one CH3 domain and Y349C, D356E, L358M, T366S, L368A, and Y407V mutations in another CH3 domain. In various embodiments, the orthogonal modifications can be charge-pair mutations.As used herein, a charge-pair mutation can be a mutation that affects the charge of an amino acid within the surface of a domain, such that the domain will preferentially associate with a second domain that has a complementary charge-pair mutation compared to associating with a domain that does not have the complementary charge-pair mutation. In certain embodiments, the charge-pair mutation can improve orthogonal association between certain domains. In certain embodiments, the charge-pair mutation can improve stability between certain domains. In some examples, the charge-pair mutation can be a T366K mutation in one CH3 domain and a T366K mutation in another CH3 domain. The L351D mutation in the CH3 domain of .

[0170] Monomeric fusion protein constructs Exemplary monomeric fusion protein constructs are shown in Figures 2 and 3 and comprise first and second polypeptides, where the first polypeptide comprises the following domains: Domain A, Domain B, and Domain R, and the second polypeptide comprises the following domains: Domain E and Domain F. In the fusion protein construct shown in Figure 2, Domain A comprises a heavy chain variable region amino acid sequence (VH) or an antigen-binding fragment thereof, Domain B comprises a heavy chain CH1 constant region amino acid sequence, Domain R comprises a complement regulator polypeptide, Domain E comprises a light chain variable region amino acid sequence (VL) or an antigen-binding fragment thereof, and Domain F comprises a light chain constant region amino acid sequence (CL1). Furthermore, Domain B and Domain F of the first and second polypeptides may each be connected via one or more disulfide bonds. The domains of the first polypeptide are arranged from the N-terminus to the C-terminus in an ABR or RAB orientation, and the domains of the second polypeptide are arranged from the N-terminus to the C-terminus in an EF orientation. In the example shown in Figure 2, the monomeric fusion protein construct comprises a linkage, e.g., a chemical linkage or a peptide linker, between domain A and domain R. In a further example, a monomeric fusion protein construct is provided that comprises a linkage between domain R and domain B, as shown in Figure 3.

[0171] Further exemplary monomeric fusion protein constructs are shown in Figures 1 and 4 and comprise first and second polypeptides, where the first polypeptide comprises the following domains: Domain A and Domain B, and the second polypeptide comprises the following domains: Domain E, Domain F, and Domain R. Domain B and Domain F of the first and second polypeptides may each be connected via one or more disulfide bonds. The domains of the first polypeptide are arranged N- to C-terminally in an AB orientation, and the domains of the second polypeptide are arranged N- to C-terminally in an EF orientation. In the example shown in Figure 4, the monomeric fusion protein construct comprises a linkage, e.g., a chemical linkage or a peptide linker, between Domain F and Domain R. In a further example, a monomeric fusion protein construct is provided comprising a linkage between Domain E and Domain R, as shown in Figure 1.

[0172] In a further example, a fusion protein construct is provided that contains more complement regulator polypeptides, i.e., more than one domain R. In such cases, the fusion protein construct comprises a first polypeptide having domains A, B and a first complement regulator polypeptide (domain R1); a second polypeptide having domains E, F and a second complement regulator polypeptide (domain R2); the fusion protein construct comprises linkages between domain R1 and domain A, or between domain R1 and domain B; and between domain R2 and domain F, or between domain R2 and domain E.

[0173] In a further example, a fusion protein construct comprises a first polypeptide comprising Domain A, Domain B, a first complement regulator polypeptide (Domain R1), and a second complement regulator polypeptide (Domain R2); and a second polypeptide comprising Domain E, Domain F, a third complement regulator polypeptide (Domain R3), and a fourth complement regulator polypeptide (Domain R4). The fusion protein construct may comprise linkages between Domain R1 and Domain B and between Domain R2 and Domain A, or between Domain R2 and Domain B and Domain R1 and Domain A; and between Domain R3 and Domain E and between Domain R4 and Domain F, or between Domain R4 and Domain E and between Domain R3 and Domain E.

[0174] Homodimeric fusion protein constructs comprising a complement regulator linked to a heavy chain polypeptide - Patent Application 20070122997 An exemplary tetravalent homodimeric fusion protein construct is shown in Figures 5-6 and comprises two polymers. The polypeptides each comprise a first polypeptide comprising Domain A, Domain B, a hinge region, Domain C, Domain D, and Domain R; and a second polypeptide comprising Domain E and Domain F, wherein the first polypeptide is arranged from N-terminus to C-terminus in an AB-hinge domain-CDR orientation and comprises a linkage between Domain D and Domain R; or in an RAB-hinge region-CD orientation and comprises a linkage between Domain R and Domain A. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds. The second polypeptide may be arranged from N-terminus to C-terminus in an EF orientation. The two first polypeptides may be connected via one or more disulfide bonds in the hinge region. Further exemplary tetravalent homodimeric fusion protein constructs include two polypeptides, each of which includes a first polypeptide comprising Domain A, Domain B, a hinge region, Domain C, and Domain R; and a second polypeptide comprising Domain E and Domain F, wherein the first polypeptide is arranged, from N- to C-terminus, in an AB-hinge-domain-CR orientation and includes a linkage between Domain D and Domain R; or in an R-A-B-hinge-region-C orientation and includes a linkage between Domain R and Domain A. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds. The second polypeptide may be arranged, from N- to C-terminus, in an EF orientation. The two first polypeptides may be connected via one or more disulfide bonds in the hinge region.

[0175] Further exemplary tetravalent homodimeric fusion protein constructs comprise two polypeptides, each polypeptide chain comprising a first polypeptide comprising Domain A, Domain B, a hinge region, and Domain R; and a second polypeptide comprising Domain E and Domain F, wherein the first polypeptide is arranged, from N- to C-terminus, in an AB-hinge-domain-R orientation with a linkage between the hinge region and Domain R; or in an R-A-B-hinge-domain orientation with a linkage between Domain R and Domain A. The second polypeptide may be arranged, from N- to C-terminus, in an EF orientation. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds. The two first polypeptides may be connected via one or more disulfide bonds in the hinge region.

[0176] Heterodimeric fusion protein constructs comprising a complement regulator linked to a heavy chain polypeptide - Patent Application 20070122997 Exemplary trivalent heterodimeric fusion protein constructs are shown in Figures 7-8 and comprise a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, Domain D, and Domain R, arranged N-terminally to C-terminally in an R-Ab-hinge-region-CD orientation (as in Figure 8) with a linkage between Domain A and Domain R, or in an AB-hinge-region-CDR orientation (as in Figure 7) with a linkage between Domain D and Domain R; the second polypeptide comprises Domain E and Domain F arranged N-terminally to C-terminally in an E-F orientation; the third polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain D arranged N-terminally to C-terminally in an AB-hinge-region-CD orientation; and the fourth polypeptide comprises Domain E and Domain F arranged N-terminally to C-terminally in an E-F orientation. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds, and Domain B and Domain F of the third and fourth polypeptides may be connected via one or more disulfide bonds. The first and third polypeptides may be connected to each other via one or more disulfide bonds in the hinge region.

[0177] Further exemplary trivalent heterodimeric fusion protein constructs include a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain R, which are arranged N-terminally to C-terminally in an R-Ab-hinge-region-C orientation with a linkage between Domain A and Domain R, or in an AB-hinge-region-C orientation with a linkage between Domain C and Domain R; the second polypeptide comprises Domain E and Domain F arranged N-terminally to C-terminally in an E-F orientation; the third polypeptide comprises Domain A, Domain B, a hinge region, and Domain C arranged N-terminally to C-terminally in an AB-hinge-region-C orientation; and the fourth polypeptide comprises Domain E and Domain F arranged N-terminally to C-terminally in an E-F orientation. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds, and Domain B and Domain F of the third and fourth polypeptides may be connected via one or more disulfide bonds. The first and third polypeptides may be joined together via one or more disulfide bonds in the hinge region.

[0178] Further exemplary trivalent heterodimeric fusion protein constructs include a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, and Domain R, arranged N-terminally to C-terminally in an R-Ab-hinge-region orientation and including a linkage between Domain A and Domain R, or in an AB-hinge-region-R orientation and including a linkage between the hinge region and Domain R; the second polypeptide comprises Domain E and Domain F arranged N-terminally to C-terminally in an E-F orientation; the third polypeptide comprises Domain A, Domain B, and the hinge region arranged N-terminally to C-terminally in an AB-hinge-region orientation; and the fourth polypeptide comprises Domain E and Domain F arranged N-terminally to C-terminally in an E-F orientation. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds, and Domain B and Domain F of the third and fourth polypeptides may be connected via one or more disulfide bonds. The third polypeptide may be linked via one or more disulfide bonds in the hinge region.

[0179] Fusion protein constructs comprising a complement regulatory agent linked to a light chain polypeptide - Patent Application 20070122999 A further exemplary tetravalent homodimeric fusion protein construct shown in Figure 9 comprises two polypeptides, each comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain D; and the second polypeptide comprises Domain E, Domain F, and Domain R, the first polypeptide arranged N-terminally to C-terminally in an AB-hinge-CD orientation, and the second polypeptide arranged N-terminally to C-terminally in either an EFR orientation (as shown in Figure 9) or a REF orientation (as shown in Figure 10) and a linkage between Domain E and Domain R. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds. The two first polypeptides may be connected via one or more disulfide bonds in the hinge region.

[0180] A further exemplary tetravalent homodimeric fusion protein construct comprises two polypeptides, each comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, and Domain C, and the second polypeptide comprises Domain E, Domain F, and Domain R, the first polypeptide arranged N-terminally to C-terminally in an AB-hinge-domain-C orientation, and the second polypeptide arranged N-terminally to C-terminally in an EFR orientation, with a domain F and a domain R intervening. The first polypeptide may comprise a linkage between domain E and domain R, or may be aligned in the orientation of REF and include a linkage between domain E and domain R. Domain B and domain F of the first and second polypeptides may be connected via one or more disulfide bonds. Two first polypeptides may be connected to each other via one or more disulfide bonds in the hinge region.

[0181] Further exemplary tetravalent homodimeric fusion protein constructs include two polypeptides, each comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises Domain A, Domain B, and a hinge region, and the second polypeptide comprises Domain E, Domain F, and Domain R, arranged N-terminally to C-terminally in an AB-hinge orientation, and the second polypeptide may be arranged N-terminally to C-terminally in an EFR orientation and may comprise a linkage between Domain F and Domain R, or in a REF orientation and may comprise a linkage between Domain E and Domain R. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds. The two first polypeptides may be connected via one or more disulfide bonds in the hinge region.

[0182] An exemplary heterodimeric construct comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises domain A, domain B, a hinge region, domain C, and domain D, the second polypeptide comprises domain E, domain F, and domain R, the third polypeptide comprises domain A, domain B, a hinge region, domain C, and domain D, the fourth polypeptide comprises domain E and domain F, the first polypeptide and the third polypeptide are arranged in the orientation of A-B-hinge region-C-D from the N-terminus to the C-terminus, the second polypeptide is arranged in the orientation of E-F-R (as shown in FIG. 12) or R-E-F (as shown in FIG. 11) from the N-terminus to the C-terminus and contains a linkage between domain F and domain R or between domain E and domain R, and the fourth polypeptide is arranged in the orientation of E-F from the N-terminus to the C-terminus. The domain B and domain F of the first polypeptide and the second polypeptide may be connected via one or more disulfide bonds, and the domain B and domain F of the third and fourth polypeptides may be connected via one or more disulfide bonds. The first polypeptide and the third polypeptide may be connected via one or more disulfide bonds in the hinge region.

[0183] The present invention provides an exemplary heterodimeric construct comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, a hinge region, and Domain C; the second polypeptide comprises Domain E, Domain F, and Domain R; the third polypeptide comprises Domain A, Domain B, a hinge region, and Domain C; and the fourth polypeptide comprises Domain E and Domain F, wherein the first and third polypeptides are arranged N-terminally to C-terminally in an AB-hinge region-C orientation; the second polypeptide is arranged N-terminally to C-terminally in an EFR orientation and comprises a linkage between Domain F and Domain R or in a REF orientation and comprises a linkage between Domain E and Domain R; and the fourth polypeptide is arranged N-terminally to C-terminally in an EF orientation. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds, and Domain B and Domain F of the third and fourth polypeptides may be connected via one or more disulfide bonds. The first and third polypeptides may be connected to each other via one or more disulfide bonds in the hinge region.

[0184]

[0013] An exemplary heterodimeric construct is provided, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises Domain A, Domain B, and a hinge region, the second polypeptide comprises Domain E, Domain F, and Domain R, the third polypeptide comprises Domain A, Domain B, and a hinge region, and the fourth polypeptide comprises Domain E and Domain F, the first and third polypeptides arranged N-terminally to C-terminally in an AB-hinge orientation, the second polypeptide arranged N-terminally to C-terminally in an EFR orientation and comprising a linkage between Domain F and Domain R or in a REF orientation and comprising a linkage between Domain E and Domain R, and the fourth polypeptide arranged N-terminally to C-terminally in an EF orientation. Domain B and Domain F of the first and second polypeptides may be connected via one or more disulfide bonds, and Domain B and Domain F of the third and fourth polypeptides may be connected via one or more disulfide bonds. The first and third polypeptides may be connected to each other via one or more disulfide bonds in the hinge region.

[0185] Fusion protein constructs comprising a complement regulator linked to an antibody constant region - Patent Application 20070122999 Provided herein is an exemplary fusion protein shown in FIG. 13 , comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises Domain R, a hinge region, Domain C, and Domain D; the second polypeptide comprises Domain A, Domain B, a hinge region, Domain C, and Domain D; and the third polypeptide comprises Domain E and Domain F, wherein the first polypeptide may be arranged N-terminally to C-terminally in an R-hinge region-CD orientation and include a linkage between Domain R and the hinge region; the second polypeptide may be arranged N-terminally to C-terminally in an AB-hinge region-CD orientation; and the third polypeptide may be arranged N-terminally to C-terminally in an EF orientation. Domain B and Domain F of the second and third polypeptides may each be connected via one or more disulfide bonds. The first and second polypeptides may be connected via one or more disulfide bonds in the hinge region. In some examples, the fusion protein includes a linkage between the complement regulator and the constant region, and the complement regulator can be, for example, CR1(1-10), CR1(1-17), Factor H, MCP, or DAF linked to a constant region of an immunoglobulin molecule comprising two CH3 domains, two CH2 domains, and a hinge region.

[0186] Exemplary Constructs Specific examples of fusion protein constructs include fusions with complement regulators CR1(1-10), CR1(1-17), and C2 antibodies or antigen-binding fragments thereof, such as C2scFv-CR1 1-10, C2scFv-CR1 1-17, C2scFv-Crry, C2 IgG1 heavy chain-CR1 1-10, C2 IgG1 heavy chain-CR1 1-17, C2 IgG1 heavy chain(hole)-CR1 1-10 [paired with Fc domain containing knob], C2 IgG1 heavy chain(hole)-CR1 1-17 [paired with Fc domain containing knob], C2 IgG1 heavy chain (knob) [paired with an Fc domain containing a hole], C2 IgG1 Fab heavy chain-Crry, C2 IgG1 Fab heavy chain-CR1 1-10, C2 IgG1 Fab heavy chain-CR1 1-17, CR1 1-10-IgG1 Fc(hole) [paired with an Fc domain containing a knob], CR1 1-17-IgG1 Fc(hole) [paired with an Fc domain containing a knob], CR1 1-10-C2 IgG1 heavy chain(hole) [paired with a construct comprising an Fc domain with a knob], CR1 1-17-C2 IgG1 heavy chain(hole) [paired with a construct comprising an Fc domain with a knob], CR1 1-10-C2 IgG1 Fab heavy chain, CR1 1-17-C2 IgG1 Fab heavy chain, CR1 1-10-C2 kappa light chain, CR1 1-17-C2 kappa light chain, as shown in FIG. Constructs that are used in the present invention include fusions with the complement regulator factor H, and anti-C3d antibodies (3d29) or antigen-binding fragments thereof, such as 3d29 kappa light chain, 3d29 Fab heavy chain mouse IgG1, 3d29 Fab heavy chain mouse IgG1-Crry, 3d29 Fab heavy chain mouse IgG1-CR1 1-10, 3d29 Fab heavy chain mouse IgG1-CR1 1-17, 3d29 Constructs shown in Figure 31, including Fab heavy chain mouse IgG1-fH1-5, 3d29 heavy chain mouse IgG1(knob), 3d29 heavy chain mouse IgG1(hole)-fH1-5, 3d29 heavy chain mouse IgG1-fH1-5;Fusions with anti-C3d antibody (3d8b) or antigen-binding fragments thereof, 3d8b Fab heavy chain mouse IgG1-Crry, 3d8b Fab heavy chain mouse IgG1-CR1 1-10, 3d8b Fab heavy chain mouse IgG1-fH1-5, 3d8b heavy chain mouse IgG1, 3d8b kappa light chain-CR1 1-10, 3d8b kappa light chain-CR1 1-17, 3d8b heavy chain mouse IgG1(knob) paired with 3d8b heavy chain mouse IgG1(hole), 3d8b heavy chain mouse IgG1-CR1 1-10, 3d8b heavy chain mouse IgG1-CR1 1-17, 3d8b heavy chain mouse IgG1(hole)-fH1-5 paired with 3d8b heavy chain mouse IgG1(knob), 3d8b heavy chain mouse IgG1-fH1-5, CR1 1-10-3d8b heavy chain mouse IgG1, CR1 1-10-3d8b kappa light chain, CR1 1-17-3d8b kappa light chain, 3d29 heavy chain mouse IgG1-CR1 1-10, 3d29 heavy chain mouse IgG1-CR1 1-10 + His tag, 3d29 heavy chain mouse IgG1 (knob)-CR1 1-10 + His tag, 3d29 heavy chain mouse IgG1 (hole), 3d29 heavy chain mouse IgG1-CR1 1-17, 3d29 heavy chain mouse IgG1-CR1 1-17 + His tag, 3d29 heavy chain mouse IgG1 (knob)-CR1 1-17 + His tag, 3d29 heavy chain mouse IgG1 (hole), fH1-5-3d8b heavy chain mouse IgG1, 3d8b kappa light chain-fH1-5, and fH1-5-3d8b kappa light chain, CR1 1-10-mouse IgG1 Fc (knob), 3d8b heavy chain mouse IgG1 (hole), 3d8b kappa light chain (mouse), fH1-5-mouse IgG1 Fc (knob), 3d8b Fab heavy chain mouse IgG1-CR1 1-17, 3d8b heavy chain mouse IgG1 (hole)-CR1 1-17, 3d8b heavy chain mouse IgG1 (knob), 3d8b heavy chain mouse IgG1-CR1 1-17, 3d8b heavy chain mouse IgG1, 3d8b kappa light chain-CR1 1-17, CR1 1-17-3d8b heavy chain mouse IgG1, CR1 These may include, but are not limited to, the constructs shown in Figure 32, which contain the 1-17-3d8b kappa light chain. The amino acid sequences of exemplary constructs are shown in SEQ ID NOs: 103-149, and Figures 30-35 show exemplary diagrams thereof.

[0187] Table 1 lists exemplary constructs comprising an anti-C3d antibody or antigen-binding fragment thereof; and fusion protein constructs comprising an anti-C3d antibody or antigen-binding fragment thereof and a complement regulator or biologically active fragment thereof. The sequences of the antibody heavy and light chains are listed, including the sequences of the complement regulator polypeptides or biologically active fragments thereof conjugated to the anti-C3d antibody (e.g., one or more complement regulators conjugated to the C-terminus or N-terminus of the heavy chain of an exemplary anti-C3d antibody; one or more complement regulators conjugated to the C-terminus or N-terminus of the light chain of an exemplary anti-C3d antibody; one or more complement regulators conjugated to the hinge region of an anti-C3d antibody). Fusion protein constructs comprising an anti-C3d antibody sometimes comprise an anti-C3d antibody comprising two heavy chains and two light chains, sometimes comprise an anti-C3d Fab fragment, and sometimes comprise an anti-C3d antibody. In some cases, the construct comprises two molecules of a first polypeptide (two heavy chains, identified in the Tables as HC1 or HC1+complement regulator, and HC2 or HC2+complement regulator) and two molecules of a second polypeptide (two light chains, identified in the Tables as LC1 or LC1+complement regulator, and LC2 or LC2+complement regulator).

[0188] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0189] Variable domain and CDR sequences in the fusion protein construct In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 11, 12, or 13; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 14, 15, or 16. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0190] In some embodiments, the fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 11, 12, and 13 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 14, 15, and 16. In some embodiments, the fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 11, 12, and 13 with a single conservative amino acid substitution in the heavy chain CDR2 ... a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 11, 12 and 13 with a single conservative amino acid substitution among them, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 14, 15 and 16.

[0191] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 17, 18, and 19, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 17, 18, or 19; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 20, 21, and 22, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 20, 21, or 22. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0192] In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 17, 18, and 19 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 20, 21, and 22. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 17, 18, and 19 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 20, 21, and 22. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 17, 18, and 19 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 20, 21, and 22.

[0193] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 23, 24, and 25, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 23, 24, and 25; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 26, 27, and 28, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 26, 27, and 28. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0194] In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 23, 24, and 25 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 26, 27, and 28. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 23, 24, and 25 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 26, 27, and 28. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 23, 24, and 25 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 26, 27, and 28.

[0195] In any of the fusion protein constructs described herein, the first polypeptide comprises (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 29, 30, and 31; or (i the second polypeptide may comprise (i) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 29, 30, and 31; and the second polypeptide may comprise (i) three light chain CDRs having amino acid sequences of SEQ ID NOs: 32, 33, and 34, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 32, 33, and 34. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0196] In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 30, and 31 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 30, and 31 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 30, and 31 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34.

[0197] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 29, 259, and 31, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 29, 259, and 31; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 32, 33, and 34, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 32, 33, and 34. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0198] In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 259, and 31 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 259, and 31 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 259, and 31 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34.

[0199] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 29, 260, and 31, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 29, 260, and 31; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 32, 33, and 34, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 32, 33, and 34. In some embodiments, one of the CDRs There are single conservative amino acid substitutions in the above. In other embodiments, there are one, two, three or four additional histidine substitutions in the CDRs.

[0200] In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 260, and 31 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 260, and 31 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 29, 260, and 31 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 32, 33, and 34.

[0201] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 35, 36, and 37, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 35, 36, and 37; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 38, 39, and 40, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 38, 39, and 40. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0202] In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 35, 36, and 37 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 38, 39, and 40. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 35, 36, and 37 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 38, 39, and 40. In some embodiments, a fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 35, 36, and 37 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 38, 39, and 40.

[0203] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 147, 148, and 149, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 150, 151, and 152; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 147, 148, and 149, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 150, 151, and 152. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0204] In some embodiments, the fusion protein construct comprises a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 147, 148, and 149 with a single conservative amino acid substitution in the heavy chain CDR1, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 150, 151, and 152. In some embodiments, the fusion protein construct has two polypeptides. In some embodiments, the fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 147, 148, and 149 with a single conservative amino acid substitution in the heavy chain CDR2, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 150, 151, and 152. In some embodiments, the fusion protein construct has a first polypeptide comprising the three heavy chain CDRs of SEQ ID NOs: 147, 148, and 149 with a single conservative amino acid substitution in the heavy chain CDR3, and a second polypeptide comprising the three light chain CDRs of SEQ ID NOs: 150, 151, and 152.

[0205] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 188, 199, and 190, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 188, 199, and 190; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 191, 192, and 193, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 191, 192, and 193. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions (replacement of an original residue with histidine) in the CDRs.

[0206] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 196, 197, and 198, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 196, 197, and 198; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 199, 200, and 201, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 199, 200, and 201. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0207] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 204 or 343, 205, and 206, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 204 or 343, 205, and 206; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 207, 208, and 209, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 207, 208, and 209. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0208] In any of the fusion protein constructs described herein, the first polypeptide can comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 212, 213, and 214, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 212, 213, and 214; and the second polypeptide can comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 215, 216, and 217, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 215, 216, and 217. The light chain may comprise three light chain CDRs having amino acid sequences that differ in one or more respects. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0209] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 220, 221, and 222, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 220, 221, and 222; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 223, 224, and 225, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 223, 224, and 225. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0210] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) three heavy chain CDRs having the amino acid sequences of SEQ ID NOs: 228, 229, and 230, or (ii) three heavy chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 228, 229, and 230; the second polypeptide may comprise (i) three light chain CDRs having the amino acid sequences of SEQ ID NOs: 231, 232, and 233, or (ii) three light chain CDRs having amino acid sequences that differ by one, two, or three conservative amino acid substitutions in one or more of SEQ ID NOs: 231, 232, and 233. In some embodiments, there is a single conservative amino acid substitution in one or more of the CDRs. In other embodiments, there are one, two, three, or four additional histidine substitutions in the CDRs.

[0211] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a heavy chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 194, 202, 210, 218, 226, 234, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, and 274, and (ii) a light chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, and 278; or (i) SEQ ID NO: 194. , 202, 210, 218, 226, 234, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, and 274, and (ii) a heavy chain variable region comprising an amino acid sequence that differs by one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 195, 203, 211, 219, 227, 235, 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, and 278.

[0212] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, and 274, and (ii) a humanized light chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, and 278; or (i) a humanized heavy chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 270, 271, 272, 273, and 274. and (ii) a humanized heavy chain variable region comprising an amino acid sequence that differs by one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 256, 257, 258, 266, 267, 268, 269, 275, 276, 277, and 278.

[0213] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255, and (ii) a light chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 256, 257, and 258, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 256, 257, and 258 by one or more conservative amino acid substitutions.

[0214] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 254, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 254 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 258 by one or more conservative amino acid substitutions.

[0215] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 251, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 251 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 258 by one or more conservative amino acid substitutions.

[0216] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 261, 262, 263, 264, and 265, and (ii) a light chain variable region comprising the amino acid sequence of at least one of SEQ ID NOs: 266, 267, 268, and 269, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 261, 262, 263, 264, and 265 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 266, 267, 268, and 269 by one or more conservative amino acid substitutions.

[0217] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 264 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 269 by one or more conservative amino acid substitutions.

[0218] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 268, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 264 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 268 by one or more conservative amino acid substitutions.

[0219] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 263, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269, or may comprise (i) a humanized heavy chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 263 by one or more conservative amino acid substitutions, and (ii) a humanized light chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 269 by one or more conservative amino acid substitutions.

[0220] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a humanized heavy chain sequence comprising the amino acid sequence of at least one of SEQ ID NOs: 280, 281, 282, 237, 283, 284, 285, and 286, and (ii) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 279, or may comprise (i) a humanized heavy chain sequence comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 280, 281, 282, 237, 283, 284, 285, and 286 by one or more conservative amino acid substitutions, and (ii) a humanized light chain sequence comprising an amino acid sequence that differs in SEQ ID NO: 279 by one or more conservative amino acid substitutions.

[0221] Fusion protein constructs described herein, in some embodiments, comprise a humanized heavy chain sequence comprising the amino acid sequence of at least one of SEQ ID NOs: 280, 281, 282, 283, 284, 285, and 286, or a humanized heavy chain sequence comprising an amino acid sequence that differs by one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 280, 281, 282, 237, 283, 284, 285, and 286. Fusion protein constructs described herein, in some embodiments, comprise a humanized light chain sequence comprising the amino acid sequence of SEQ ID NO: 279, or a humanized light chain sequence comprising an amino acid sequence that differs by one or more conservative amino acid substitutions in SEQ ID NO: 279.

[0222] In any of the fusion protein constructs described herein, the first polypeptide may comprise (i) a mouse heavy chain sequence comprising the amino acid sequence of at least one of SEQ ID NOs: 73, 288, 244, 290, and 342, and (ii) a mouse light chain sequence comprising the amino acid sequence of at least one of SEQ ID NOs: 68, 287, 59, and 289, or may comprise (i) a mouse heavy chain sequence comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 73, 288, 244, 290, and 342 by one or more conservative amino acid substitutions, and (ii) a mouse light chain sequence comprising an amino acid sequence that differs in at least one of SEQ ID NOs: 68, 287, 59, and 289 by one or more conservative amino acid substitutions.

[0223] Fusion protein constructs described herein, in some embodiments, comprise a murine heavy chain sequence comprising the amino acid sequence of at least one of SEQ ID NOs: 73, 288, 244, 290, and 342, or a murine heavy chain sequence comprising an amino acid sequence that differs by one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 73, 288, 244, 290, and 342. Fusion protein constructs described herein, in some embodiments, comprise a murine light chain sequence comprising the amino acid sequence of at least one of SEQ ID NOs: 68, 287, 59, and 289, or an amino acid sequence that differs by one or more conservative amino acid substitutions in at least one of SEQ ID NOs: 68, 287, 59, and 289. The mouse light chain sequence includes the nucleotide sequence.

[0224] Conservative amino acid substitutions are those that change a given amino acid for a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, size). For example, any one of the following classes of amino acids is considered a conservative amino acid substitution: polar (hydrophilic) neutral amino acids: serine (Ser), threonine (Thr), cysteine ​​(Cys), histidine (His), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr); polar, negatively charged amino acids: aspartic acid (Asp), glutamic acid (Glu); polar, positively charged amino acids: histidine (His), lysine (Lys), arginine (Arg); hydrophobic amino acids: glycine (Gly), alanine (Ala), valine (Val). ), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp); hydrophobic amino acids with aliphatic side chains: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); hydrophobic amino acids with aromatic side chains: phenylalanine (Phe), tryptophan (Trp); amino acids of small size: alanine (Ala), cysteine ​​(Cys), glycine (Gly), proline (Pro), serine (Ser), and threonine (Thr).

[0225] Conjugation of targeting moieties with complement modulating agents The linkage between the targeting moiety and the complement modulator can include (1) conjugation by direct fusion of the two protein sequences or (2) fusion through an intervening linker / linker sequence / spacer / tethering sequence. As used herein, the terms "linker," "linker sequence," "spacer," or "tethering sequence" can refer to a molecule or group of molecules (e.g., a monomer or polymer) that connects two molecules and often serves to position the two molecules in a preferred orientation. Several strategies can be used to covalently link molecules together. These include, but are not limited to, polypeptide linkage between the N- and C-termini of a protein or protein domain, linkage via a disulfide bond, and linkage via a chemical cross-linking reagent. In one embodiment, the linker is a peptide bond generated by recombinant technology or peptide synthesis.

[0226] In some examples, the complement regulator and the targeting moiety may be conjugated via a linker peptide, e.g., a linker peptide that can directly connect the targeting moiety and the complement regulator. The linker peptide may contain amino acid residues that provide flexibility. Thus, the linker peptide may include the following amino acid residues: glycine, serine, alanine, or threonine. The linker peptide must be long enough to connect the two molecules in a manner that allows them to adopt the correct conformation relative to each other so that they retain the desired activity. A suitable length for this purpose may include at least 1 to about 100 amino acid residues or more. The linker may be 1 to 30 amino acids in length. The linker may be about 1 to 20 amino acids in length. The linker peptide may be included as a spacer between the two protein moieties. The linker peptide may promote proper protein folding, stability, expression, and biological activity of the constituent protein moieties. A long, flexible linker peptide may consist of glycine, serine, or threonine, and multiple glycine residues may provide significant conformational flexibility. Serine or threonine residues provide a polar surface area that limits hydrophobic interactions with the peptide or with the constituent fusion protein moieties. The amino acid residues selected for inclusion in the linker peptide may exhibit properties that do not significantly interfere with the activity of the polypeptide. Thus, the linker peptide may not exhibit charges that are inconsistent with the activity of the polypeptide or that would interfere with internal folding or form bonds or other interactions with amino acid residues in the targeting moiety or complement modulator that would seriously interfere with binding of the moiety to its target. Non-limiting examples of sequences that can serve as linkers include: Examples may include short peptides of about 2 to about 15 amino acids in length. Among the peptide sequences that can be used as linkers in the present disclosure are (Gly-Ser) where n=0, 1, 2, 3, 4, 5, 6, 7, or 8. n (SEQ ID NO: 292); n = 0, 1, 2, 3, or 4 (GlyGlyGlySer) n (SEQ ID NO: 293); n = 0, 1, 2, 3, or 4 (GlySerSerGly) n(SEQ ID NO: 294). In some fusion protein constructs, the linker sequence is (GlyGlyGlyGlySerGlyGlyGlyGlySer) (SEQ ID NO: 138). In some cases, tethered blockers for shaker potassium channels (see, e.g., TJ Morin and WR Kobertz, "Tethering Chemistry and K+ Channels," J. Biol. Chem. 283(37):25105-25109 (2008)) can be used, including glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, such as a panel of quaternary ammonium (QA) tethered to maleimide by polyglycine tethers of various lengths, and a variety of other flexible linkers. Glycine-serine polymers can be used because both amino acids are relatively unorganized and therefore potentially can serve as neutral tethers between components. Second, serine is hydrophilic and therefore can solubilize what might otherwise be a globular glycine chain. In some cases, the linker may include a sequence set forth in any one of SEQ ID NOs: 171-193, where in some cases n may be at least 4, and in some cases n may be 1-8, or 1-5. For example, in SEQ ID NO: 161, n may be at least 4, in SEQ ID NO: 164, n may be 1-8, in SEQ ID NO: 165, n may be 1-5, and in SEQ ID NO: 166, n may be 1-5. In SEQ ID NO: 168, X may be A (alanine), K (lysine), or E (glutamic acid), and n may be 5-17. In some cases, in SEQ ID NOs: 161, 164, 165, 166, 168, 170, 171, 174, and 179, n may be in the range of 1-17, 1-8, 1-5, at least 4, or 5-17.

[0227] Suitable linkers can also be identified by screening databases of known three-dimensional structures to find linkers derived from naturally occurring motifs, such as naturally occurring multidomain proteins, that can bridge the gap between two polypeptide chains. In some instances, the linker is not immunogenic when administered to a human patient. Thus, a linker can be selected that has low immunogenicity or is thought to have low immunogenicity. For example, a linker that naturally occurs in humans can be selected. In some instances, the linker can have the sequence of an antibody hinge region, which is the sequence connecting the Fab and Fc regions of an antibody, or the linker can have a sequence that includes a portion of an antibody hinge region or a sequence substantially similar to a hinge region. Another method for obtaining a suitable linker is to use a simple linker, such as (Gly4Ser) n (SEQ ID NO:295) by random mutagenesis. Alternatively, once a suitable polypeptide linker is identified, additional linker polypeptides can be engineered to select amino acids that interact more optimally with the domains to be linked. Other types of linkers that can be used in the present invention include artificial polypeptide linkers and inteins. In some cases, complement regulators and targeting moieties can be conjugated using enzymatic site-specific conjugation methods involving the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of large amounts of relatively pure enzyme, its simplicity of use, and its lack of regulation by calcium and guanosine-5'-triphosphate (GTP) have helped mTG become a major cross-linking enzyme used in both the food industry and biotechnology. Currently, mTG is used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. For example, Pavel Strop, Veracity of microbial transglutaminase, Bioconjugate Chem. 25(5) See: 855-862.

[0228] In some examples, fusion protein constructs are provided that include a targeting moiety containing an acceptor glutamine in the constant region, which can then be conjugated to a complement protein via a lysine-based linker (e.g., one containing any primary amine chain that is a substrate for TGase, e.g., an alkylamine, an oxoamine), where conjugation occurs exclusively on one or more acceptor glutamine residues present in the targeting moiety outside the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur on glutamines within the variable region, e.g., at least partially surface-exposed glutamines. The conjugate can be formed by reacting the targeting moiety with the lysine-based linker in the presence of TGase. The lysine-based linker can, for example, contain, in addition to a primary amine, e.g., an alkylamine, an oxoamine, a peptide, a polypeptide, any organic molecule, a drug, or a diagnostic moiety, or can contain a reactive moiety that can then react with a complement modulator.

[0229] In another embodiment, a disulfide bond can be engineered to join the two molecules. In some cases, the linker is a chemical cross-linker. For example, various bifunctional protein coupling agents may be used, including, but not limited to, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., 1971, Science 238:1098. Chemical linkers can allow for isotope chelation. For example, carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see, e.g., WO 94 / 11026). The linker may be cleavable, thereby facilitating release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., 1992, Cancer Research 52:127-131) can be used. Alternatively, various non-proteinaceous polymers, such as, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, can be utilized as linkers, i.e., to connect a targeting moiety of the present disclosure to a fusion or complex partner, such as a complement regulatory agent, to create a fusion protein construct of the present disclosure.

[0230] When the targeting moiety and the active moiety are joined directly, a hybrid vector can be created in which the DNA encoding the targeting moiety and the complement regulatory agent are directly ligated to each other. When a linker is used, a hybrid vector can be created in which the DNA encoding the targeting moiety is ligated to DNA encoding one end of the linker moiety and the DNA encoding the complement regulatory agent is ligated to the other end of the linker moiety. Such ligations can be performed sequentially or as a three-part ligation.

[0231] Methods of production, purification and characterization The fusion protein constructs described herein can be produced using a variety of techniques. Nucleic acids encoding the fusion protein constructs described herein may be inserted into expression vectors containing transcriptional and translational regulatory sequences, including, for example, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional termination signals, polyadenylation signals, and enhancer or activator sequences. Regulatory sequences include promoters and transcriptional start and stop sequences. In addition, expression vectors may contain more than one replication system, allowing their control in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0232] Several suitable vector systems are available for expressing fusion proteins from nucleic acids in mammalian cells. One class of vectors relies on integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be selected by co-introducing a drug resistance gene, such as E. coli gpt (see Mulligan and Berg Proc. Natl. Acad. Sci. USA 78:2072 (1981)) or Tn5 neo (see Southern and Berg Mol. Appl. Genet. 1:327 (1982)). The selectable marker gene can either be linked to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection (Wigler et al. Cell 16:77 (1979)). A second class of vectors utilizes DNA elements that confer autonomous replication to extrachromosomal plasmids. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. Proc. Natl. Acad. Sci. USA, 79:7147 (1982)), polyomavirus (Deans et al. Proc. Natl. Acad. Sci. USA 81:1292 (1984)), or SV40 virus (Lusky and Botchan Nature 293:79 (1981)).

[0233] The expression vector can be introduced into cells by a method suitable for subsequent expression of the nucleic acid. The method of introduction generally depends on the type of cell of interest, as described below. Exemplary methods include calcium phosphate precipitation, liposome fusion, lipofection, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0234] Suitable host cells for expression of fusion proteins include yeast, bacteria, insect, plant, and mammalian cells as described above. Of interest are bacteria, such as E. coli, fungi, such as Saccharomyces cerevisiae and Pichia pastoris, insect cells, such as SF9, mammalian cell lines (e.g., human cell lines), and primate cell lines (e.g., primate mammalian cells). In some embodiments, the fusion protein may be expressed in Chinese hamster ovary (CHO) cells or a suitable myeloma cell line, such as NSO. Suitable cell lines also include, for example, HEK cells (human embryonic kidney 293 cells), BHK-21 (baby hamster kidney) cells, 293 (human embryonic kidney) cells, HMEpC (human mammary epithelial cells), and 3T3 (mouse embryonic fibroblast) cells.

[0235] As will be recognized by those skilled in the art, some complement regulators that can be used in the present disclosure exist naturally as secreted proteins with signal or leader peptides, as propeptides, or both, and undergo further intracellular or extracellular processing. In such cases, the hybrid vectors of the present disclosure can include one or more DNA sequences encoding such signal or leader peptides, one or more DNA sequences encoding such propeptide sequences, or both, depending on whether such secretion, processing, or combined secretion and processing is desired. Alternatively, the hybrid vectors of the present disclosure can include different signal or leader peptides, propeptides, or both. The DNA sequence encoding the complement regulatory agent may include a signal peptide, a targeting moiety, or both, sequences selected to optimize expression and localization of the fusion protein. In most cases, the signal peptide may be omitted, as the targeting moiety will provide sufficient information to direct the complement regulatory agent to the desired tissues and cells in the subject's body.

[0236] In an exemplary production method, the fusion protein constructs described herein can be expressed in and purified from transgenic animals (e.g., transgenic mammals). For example, the fusion proteins described herein can be produced in transgenic non-human mammals (e.g., rodents, sheep, or goats) and isolated from their milk, as described, for example, in Houdebine Curr. Opin. Biotechnol. 13(6):625-629 (2002), van Kuik-Romeijn et al. Transgenic Res. 9(2):155-159 (2000), and Pollock et al. J Immunol. Methods 231(1-2):147-157 (1999).

[0237] The fusion protein constructs described herein can be produced from host cells transformed with an expression vector containing a nucleic acid encoding an antibody or antigen-binding fragment thereof by culturing the cells under conditions and for an amount of time sufficient to allow expression of the protein. For example, polypeptides expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al., Cytokine 10:319-30 (1998)). Bacterial expression systems and methods for their use are well known in the art (Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning-A (See Laboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, suitable expression vectors, and suitable host cells will vary depending on a number of factors and can be easily optimized as needed. The fusion protein constructs described herein can be expressed in mammalian cells or other expression systems, including, but not limited to, yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al., Protein Expression and Purification 18:213-220 (2000)).

[0238] After expression, the fusion protein construct can be isolated. The terms "purified" or "isolated," as applied to any of the proteins described herein (e.g., the fusion protein constructs described herein), can refer to a polypeptide that has been separated or purified from components that naturally accompany it (e.g., proteins or other naturally occurring biomolecules or organic molecules), such as other proteins, lipids, and nucleic acids in the prokaryotic organism in which the protein is expressed. Typically, a polypeptide is purified if it constitutes at least 60% (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99%) by weight of the total protein in a sample. The fusion protein constructs described herein can be isolated or purified in a variety of ways, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, such as ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, a standard anti-fusion protein antibody affinity column can be used to purify the fusion protein. Ultrafiltration and diafiltration techniques are also useful in conjunction with protein concentration. For example, Scopes (1994) “Protein Purification, 3 rdSee, for example, "The American Society for the Promotion of Science," Springer-Verlag, New York City, NY. The degree of purification required will vary depending on the desired use. In some cases, purification of the expressed polypeptide may not be necessary.

[0239] Methods for determining the yield or purity of purified polypeptides may include, for example, Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, Amido Black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (e.g., using protein stains such as Coomassie blue or colloidal silver stain).

[0240] In another exemplary method of production, the fusion protein constructs described herein can be synthesized de novo, in whole or in part, using chemical methods. For example, the constituent amino acid sequences can be synthesized by solid-phase techniques, cleaved from the resin, purified by preparative high performance liquid chromatography, and then chemically linked to form the desired polypeptide. The composition of the synthetic peptides can be confirmed by amino acid analysis or sequencing.

[0241] The fusion protein constructs described herein, once expressed and purified, or after expression and subsequent purification, can be evaluated for any one of several desired properties using in vitro or in vivo assays, such as any of those described herein. For example, the fusion proteins described herein can be evaluated for their ability to inhibit C5 convertase, e.g., as described in Heinen et al., Factor H-related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation. Blood 114(12):2439-47 (2009). Endotoxin can be removed from fusion protein construct preparations using a variety of commercially available reagents, including, but not limited to, the ProteoSpin™ Endotoxin Removal Kit (Norgen Biotek Corporation), Detoxi-Gel Endotoxin Removal Gel (Thermo Scientific, Pierce Protein Research Products), MiraCLEAN® Endotoxin Removal Kit (Mirus), or Acrodisc™-Mustang® E Membrane (Pall Corporation).

[0242] Methods for detecting and / or measuring the amount of endotoxin present in a sample (both before and after purification) can be based on commercially available kits. For example, the concentration of endotoxin in a protein sample can be determined using the QCL-1000 chromogenic kit (BioWhittaker), limulus amebocyte lysate (LAL)-based kits, such as Pyrotell®, Pyrotell®-T, Pyrochrome®, Chromo-LAL, and CSE kits available from Associates of Cape Cod Incorporated.

[0243] After expression and purification, the fusion protein constructs described herein can be modified. Modifications can be covalent or non-covalent. Such modifications can be introduced into the fusion protein, for example, by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be selected using any of a variety of criteria, including, for example, structural or amino acid sequence analysis of the fusion proteins described herein.

[0244] In some exemplary manufacturing methods, the fusion protein constructs described herein may be conjugated to a heterologous moiety. The heterologous moiety may be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or a detectable label, such as, but not limited to, a radioactive, enzymatic, fluorescent, or luminescent label. Suitable heterologous polypeptides include, for example, antigenic tags for use in antibody purification (e.g., FLAG, polyhistidine, hemagglutinin (HA), glutathione-S-transferase (GST), or maltose-binding protein). Heterologous polypeptides can include polypeptides useful as diagnostic or detectable markers, such as luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT). When the heterologous moiety is a polypeptide, the moiety can be incorporated into a fusion protein described herein to form the fusion protein.

[0245] Pharmaceutical preparations Pharmaceutical formulations comprising the fusion protein constructs described herein are also provided in the present disclosure. Pharmaceutical formulations of the fusion protein constructs of the present disclosure are typically prepared in unit injection form for parenteral administration, i.e., bolus, intravenous, intratumoral, or subcutaneous injection, together with a pharmaceutically acceptable parenteral vehicle. The fusion protein constructs may optionally be mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) in the form of a lyophilized formulation or aqueous solution. Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., These include serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG), or any combination thereof.

[0246] The fusion protein constructs may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0247] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fusion protein construct, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0248] Formulations used for in vivo administration are sterile, which may be accomplished by filtration through sterile filtration membranes. Formulations include those suitable for the above-mentioned routes of administration. Formulations may conveniently be provided in unit dosage form and may be prepared by any method commonly practiced in the preparation of unit-dosage pharmaceutical formulations. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately combining the fusion protein construct with liquid carriers or finely divided solid carriers, or both, and then shaking the product, if necessary.

[0249] Aqueous suspensions of the present disclosure contain the fusion protein construct in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, as well as dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0250] Pharmaceutical preparations containing fusion protein constructs may be in the form of sterile injectable preparations, such as sterile aqueous or oleaginous injectable suspensions. These suspensions may be formulated according to the methods described above using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions made with non-toxic parenterally acceptable diluents or solvents, such as solutions made in 1,3-butanediol, or prepared as lyophilized powders. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils may conveniently be employed as solvents or suspending media. For this purpose, any sterile fixed oil may be employed, including synthetic mono- or diglycerides. Additionally, fatty acids, such as oleic acid, may also be used in the preparation of injectable preparations.

[0251] The amount of active ingredient which can be combined with the carrier materials to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of active ingredient per milliliter of solution in order to effect infusion of a suitable volume at a rate of about 30 mL / hour.

[0252] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0253] Although oral administration of protein therapeutics is precluded due to hydrolysis or denaturation in the stomach, formulations of fusion protein constructs suitable for oral administration can be prepared as discrete units, e.g., capsules, cachets, or tablets, each containing a predetermined amount of the fusion protein construct.

[0254] The formulations may be packaged in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier for injection, for example, water, immediately prior to use. In some cases, the formulations may be administered in a controlled delivery system, for example. The composition may also be packaged in an infusion device, such as an infusion pump for delivery. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

[0255] The present disclosure further provides veterinary compositions comprising at least one active ingredient as defined above together with a veterinary carrier therefor. The veterinary carrier is a material useful for the purpose of administering the composition, and may be a solid, liquid, or gaseous material, or it may be inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally, or by any other desired route.

[0256] Methods of Treating ...

Claims

1. 1. A composition for treating a complement-mediated disease characterized by increased deposition of c3d, the composition comprising a fusion protein construct and a pharmaceutically acceptable excipient, wherein the fusion protein is (i) An antibody or antigen-binding fragment thereof that specifically binds to complement protein 3d (C3D), wherein the antibody or antigen-binding fragment thereof is: (a) a heavy chain comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3), wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a light chain comprising three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO:

34. the antibody or antigen-binding fragment thereof comprising: (ii) a complement regulator polypeptide comprising a complement protein selected from the group consisting of Factor H, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, and biologically active fragments thereof. Including, The composition.

2. 2. The composition of claim 1, wherein the complement-mediated disease is selected from the group consisting of focal segmental glomerulosclerosis, glomerulonephritis, complement 3 glomerulopathy (C3G), membranoproliferative glomerulonephritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGN II), membranous nephropathy (MN), IgA nephropathy (IgAN), lupus nephritis (LN), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), diabetic retinopathy, dry age-related macular degeneration, wet age-related macular degeneration, and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

3. 3. The composition according to claim 1 or 2, (a) the light chain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 258; (b) the heavy chain comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 254; (c) the light chain comprises the amino acid sequence of SEQ ID NO: 279; and / or (d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 284, 282, or 285. The composition.

4. 4. The composition of any one of claims 1 to 3, wherein the fusion protein construct further comprises a linker connecting the antibody or antigen-binding fragment thereof to the complement regulator polypeptide.

5. 5. The composition of any one of claims 1 to 4, wherein the complement regulator polypeptide comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 72 and SEQ ID NO: 108; or (b) an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:91, and SEQ ID NO:

92. The composition comprising:

6. 6. The composition of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain and a second heavy chain, each of the first and second heavy chains comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3), wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO:29, the CDR-H2 comprises the amino acid sequence of SEQ ID NO:260, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO:31; and (b) a first light chain and a second light chain, each of the first and second light chains comprising three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO:

34. The composition comprising:

7. 7. The composition of claim 6, (a) the first and second heavy chains each comprise a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 254, and the first and second light chains each comprise a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 258; and / or (b) the first and second heavy chains each comprise the amino acid sequence of SEQ ID NO: 284, 282, or 285, and the first and second light chains each comprise the amino acid sequence of SEQ ID NO: 279; The composition.

8. 8. The composition of claim 6 or 7, wherein the fusion protein construct further comprises a linker that connects the antibody or antigen-binding fragment thereof to one of the complement regulator polypeptides.

9. 9. The composition of claim 8, (c) a first linker attached to the C-terminus of the first heavy chain, the first linker comprising the amino acid sequence of SEQ ID NO: 138; and (d) a second linker attached to the C-terminus of the second heavy chain, the second linker comprising the amino acid sequence of SEQ ID NO:

138. The composition further comprises:

10. 10. The composition of any one of claims 6 to 9, wherein the complement regulator polypeptide is: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 72 and SEQ ID NO: 108; or (b) an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:91, and SEQ ID NO:

92. The composition comprising:

11. 11. The composition of any one of claims 1 to 10, wherein the fusion protein construct comprises: (1) two heavy chain-containing polypeptides, each of which comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO:282, the amino acid sequence of SEQ ID NO:138, and the amino acid sequence of SEQ ID NO:72 or 41; and two light chain-containing polypeptides, each of which comprises the amino acid sequence of SEQ ID NO:279; (2) two heavy chain-containing polypeptides, each of which comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO:285, the amino acid sequence of SEQ ID NO:138, and the amino acid sequence of SEQ ID NO:72 or 41; and two light chain-containing polypeptides, each of which comprises the amino acid sequence of SEQ ID NO:279; (3) A heavy chain-containing polypeptide comprising, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 72 or 41; and a light chain-containing polypeptide comprising, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 279, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 72 or 41; (4) A heavy chain-containing polypeptide comprising, from the N-terminus to the C-terminus, the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 72 or 41; and a light chain-containing polypeptide comprising the amino acid sequence of SEQ ID NO: 279; (5) two heavy chain-containing polypeptides, each of which comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 282, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108 or 42; and two light chain-containing polypeptides, each of which comprises the amino acid sequence of SEQ ID NO: 279; (6) Two heavy chain-containing polypeptides, each of which comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 285, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108 or 42; and two light chain-containing polypeptides, each of which comprises the amino acid sequence of SEQ ID NO: 279; (7) A heavy chain-containing polypeptide comprising, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108 or 42; and a light chain-containing polypeptide comprising, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 279, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108 or 42; and (8) A heavy chain-containing polypeptide comprising, from the N-terminus to the C-terminus, the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108 or 42; and a light chain-containing polypeptide comprising the amino acid sequence of SEQ ID NO:

279. The composition is selected from the group consisting of:

12. 12. The composition of any one of claims 1 to 11, wherein the fusion protein construct comprises: (i) two heavy chain-containing polypeptides, each of which comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO:282, the amino acid sequence of SEQ ID NO:138, and the amino acid sequence of SEQ ID NO:72; and (ii) two light chain-containing polypeptides, each of which comprises the amino acid sequence of SEQ ID NO:

279. The composition comprising:

13. 13. The composition of claim 12, wherein the complement-mediated disease is IgA nephropathy (IgAN), complement 3 glomerulopathy (C3G), or lupus nephritis (LN).

14. The composition of claim 1, wherein the complement regulatory polypeptide comprises CR1 or a biologically active fragment thereof, and the complement-mediated disease is age-related macular degeneration (AMD).

15. 15. The composition of claim 14, wherein the complement-mediated disease is dry age-related macular degeneration or wet age-related macular degeneration.

16. 1. A composition for treating IgA nephropathy (IgAN), complement 3 glomerulopathy (C3G), or lupus nephritis (LN), the composition comprising a fusion protein construct and a pharmaceutically acceptable excipient, wherein the fusion protein construct comprises: (i) An antibody or antigen-binding fragment thereof that specifically binds to complement protein 3d (C3D), wherein the antibody or antigen-binding fragment thereof is: (a) a heavy chain comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3), wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a light chain comprising three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO:

34. the antibody or antigen-binding fragment thereof comprising: (ii) a complement regulator polypeptide comprising a complement protein selected from the group consisting of Factor H, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, and biologically active fragments thereof. The composition comprising:

17. 17. The composition of claim 16, (a) the light chain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 258; and / or (b) the heavy chain comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 254; The composition.

18. 18. The composition of claim 17, (a) the light chain comprises the amino acid sequence of SEQ ID NO: 279; and / or (b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 284, 282, or 285. The composition.

19. 19. The composition of any one of claims 1 to 13 and 16 to 18, wherein the complement regulator polypeptide is Factor H, or a biologically active fragment thereof.

20. The composition of any one of claims 1 to 18, wherein the complement regulator polypeptide is CR1, or a biologically active fragment thereof.

21. 1. A composition for treating anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), the composition comprising a fusion protein construct and a pharmaceutically acceptable excipient, wherein the fusion protein construct comprises: (i) An antibody or antigen-binding fragment thereof that specifically binds to complement protein 3d (C3D), wherein the antibody or antigen-binding fragment thereof is: (a) a heavy chain comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3), wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a light chain comprising three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO:

34. the antibody or antigen-binding fragment thereof comprising: (ii) a complement regulator polypeptide, the complement regulator polypeptide comprising factor H, or a biologically active fragment thereof. Including, The composition.

Images