Constructs, compositions, and methods for reducing hypersensitivity reactions to therapeutic agents

Abstract

Embodiments of the present invention generally relate to novel constructs, compositions, and methods for reducing hypersensitivity reactions. In certain embodiments, constructs, compositions, and methods are disclosed for reducing or eliminating hypersensitivity reactions using targeted complement inhibitors to complement C3 deposited on therapeutically relevant nanoparticles and nanocomponents. In some embodiments, constructs disclosed herein can include at least one of a fusion polypeptide or isolated fusion polypeptide with a) a component comprising a mammalian complement receptor 2 (CR2) or fragment thereof; and b) a component comprising regulatory consensus repeats of a mammalian complement receptor 1 (CR1) for use alone or in a combination composition with at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof.

Description

CONSTRUCTS, COMPOSITIONS, AND METHODS FOR REDUCING HYPERSENSITIVITY REACTIONS TO THERAPEUTIC AGENTS PRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 536,607, entitled “CONSTRUCTS, COMPOSITIONS, AND METHODS FOR REDUCING HYPERSENSITIVITY REACTIONS TO THERAPEUTIC AGENTS” filed September 05, 2023. This provisional application is incorporated herein by reference in its entirety for all purposes. FIELD

[0002] Embodiments of the present invention generally relate to novel constructs, compositions, and methods for reducing hypersensitivity reactions to therapeutic agents. In certain embodiments, constructs, compositions, and methods are disclosed for reducing or eliminating hypersensitivity reactions in a subject using targeted complement inhibitor constructs to complement C3 induced by a therapeutic agent and / or deposited on therapeutic nanoparticles, na and nanocomponents. GOVERNMENT FUNDING

[0003] This invention was made with government funding under grant numbers R01 CA257958 and R01 AI154959 from the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. The XML copy, created on September 05, 2024, is named 106549-818382 CU6208H-02 PCT1_Sequence Listing_xml and is 36864 bytes in size. BACKGROUND

[0005] Complement systems are an important line of defense against, for example, invading pathogens and nanoparticles. Complement systems aid in the clearance of invading or foreign bodies through opsonization with the third complement protein (C3) as well as microbial killing through surface assembly of membrane attack complexes. C3b and its split products (e.g., iC3b, C3c, C3d) are complement opsonins recognized by a variety of complement receptors on 1 98386650.2neutrophils, monocytes, eosinophils, B-lymphocytes, and resident tissue macrophages. However, when uncontrolled, complement activation can lead to proinflammatory responses, immune activation, and tissue damage through multifaceted mechanisms generating adverse health conditions in a subject. Therefore, a need exists to control aberrant complement system reactions. SUMMARY

[0006] Embodiments of the present invention generally relate to novel constructs, compositions, and methods for reducing hypersensitivity reactions. In certain embodiments, constructs, compositions, and methods are disclosed for reducing or eliminating hypersensitivity reactions using targeted complement inhibitors to, for example, complement C3 or other complement molecules deposited on therapeutically relevant nanoparticles and nanocomponents. In certain embodiments, constructs disclosed herein can include short-circulating targeted molecules that inhibit acute complement events with respect to administering nanoparticles and other complement inducing therapeutic agents (e.g., vaccines, therapeutic antibodies) for example, for reducing adverse reactions and improving the therapeutic response of the therapeutic agent, avoiding acute complement interference without prolonged effects. In certain embodiments, this inhibition of an acute complement event is rapid using compositions disclosed herein, such as within minutes to within hours. In some embodiments, constructs and other soluble molecules disclosed herein can be used in methods disclosed herein to inhibit adverse acute complement effects in circulation as well as in serum without inhibition of therapeutic agent uptake, therapeutic effect, and without conjugation to the therapeutic agent.

[0007] In some embodiments and further to paragraph

[0006] above, compositions and methods for reducing nanoparticle, vaccine, therapeutic antibody, and / or biologic complement activation for improved therapeutic response to a therapeutic agent of a nanoparticle and / or vaccine in a subject are disclosed. In certain embodiments, one or more constructs of a composition can include but are not limited to, a fusion polypeptide including, but not limited to, a) at least one component including a mammalian complement receptor 2 (CR2) or fragment thereof; and b) at least one component including regulatory consensus repeats of a mammalian complement receptor 1 (CR1) or other complement inhibitory domains for example, at least one component derived from Factor H and Decay Accelerating Factor and administering the composition at least one of before, during or after administration of a therapeutic-containing nanoparticle, nanoparticle-containing therapeutic, nanoaggregate, and / or vaccine composition to 2 98386650.2the subject and reducing complement activation and improving therapeutic response in the subject compared to a subject not receiving the one or more constructs. In other embodiments, a polypeptide of use herein can include CR2, CR1 or other third complement protein (C3) inhibitor or related molecule thereto alone, in combination, in combined constructs, or in a mixture thereof, to reduce acute adverse complement responses when administering one or more therapeutic-containing nanoparticles, nanoparticle-containing therapeutic, nanoagregate, and / or vaccine compositions to a subject. In some embodiments, constructs disclosed herein can include CR2 as a targeting molecule.

[0008] In certain embodiments and further to paragraphs

[0006] and

[0007] above, the compositions and methods disclosed herein reduce or eliminate the need for one or more commonly used inhibitory agent for reducing allergic reactions to therapeutic nanoparticles and / or vaccines. In accordance with these embodiments, the one more commonly used inhibitory agents include, but are not limited to, at least one of steroids and anti-histamines or other allergy preventive agents. In accordance with these embodiments, constructs disclosed herein reduce or eliminate the need for these commonly used inhibitory agents. In some embodiments, fusion polypeptide constructs, or other constructs disclosed herein have a rapid and / or short-acting, and / or transient inhibition or suppression of complement in the subject to reduce adverse effects of prolonged complement inhibition in the subject while providing other advantages of administering a soluble agent for inhibiting or eliminating adverse complement reactivity in blood and serum of the subject.

[0009] In certain embodiments and further to paragraphs

[0006] -

[0008] above, the nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, and / or vaccine composition for targeted complement inhibition contemplated herein can include, but is not limited to, at least one of an iron-oxide (e.g., carboxymethyl dextran iron oxide, dextran iron oxide or the like), superparamagnetic iron oxide (SPIO) nanoworms (NWs), Cy5-CLIO, pegylated liposomes, gold spheres, pegylated polybeads, liposomal doxorubicin, mRNA containing lipid nanoparticles (LNPs), other lipid nanoparticles (LNPs), or similar agent containing nanoparticle harboring one or more therapeutic agent. In certain embodiments, the nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, or vaccine composition can include, but is not limited to, a therapeutic of at least one of paclitaxel (e.g., Taxol®), patisiran (e.g., Onpattro®), irinotecan liposome injectable (e.g., Onivyde®), other 3 98386650.2chemotherapeutic agent, or other microtubule-stabilizing drug, an anti-microbial (e.g., anti-viral vaccine) or anti-tumor vaccine, or nanomedicine having a short-term circulation or short-term circulation half-life in a subject. In another embodiment, nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, or vaccine composition can include, but is not limited to, nanosized biologics, including therapeutic antibodies, antibody-drug conjugate, and therapeutic proteins including, for example, PEGylated proteins and peptides. In certain embodiments, constructs disclosed herein can target nanoparticle or vaccine adverse complement reactions without systemic inhibition of complement.

[0010] In other embodiments and further to paragraphs

[0006] -

[0009] above, the fusion polypeptides or constructs disclosed herein can further include a modification to increase the circulating half-life of the fusion polypeptide. In accordance with these embodiments, modifications for increasing the half-life of the fusion polypeptides or constructs disclosed herein can include, but are not limited to, PEGylation, albumin binding domains, Fc conjugation, stealth nanoparticle, or other modifications known in the art to increase the circulating half-life of fusion polypeptides disclosed herein. It is contemplated that these modifications can be used to create a modified fusion molecule having a desirable circulating half-life or pre-determined circulating half-life while reducing any long-term complement inhibition, as desired.

[0011] In some embodiments and further to paragraphs

[0006] -

[0010] above, compositions and methods disclosed herein for providing constructs or fusion polypeptides for example, CR2-CR1, CR1, CR2, biologically active fragment of CR2 and / or CR1, or other inhibitory domains, for example, inhibitory domains derived from Factor H and Decay Accelerating Factor (DAF) thereof can reduce therapeutic interference and this effect can reduce dose requirements of therapeutically relevant agents such as reducing dosage amounts or frequency of administration for nanoparticle-containing therapeutics, therapeutic-containing nanoparticles, nanoaggregates, and / or vaccine compositions. In accordance with these embodiments, constructs or fusion polypeptides for example, CR2-CR1, CR2-DAF, CR2-Factor H, CR1, CR2 or biologically active fragment of CR2 and / or CR1, DAF, and Factor H thereof can reduce dose requirements of therapeutically relevant agents such as reducing dosage amounts or frequency of administration needed to prevent onset of a condition or needed to treat a health condition in a subject by at least one of, reducing complement interference; and reducing granulocyte, monocyte, macrophage and / or any other immune cell uptake of the nanoparticle-containing therapeutic, 4 98386650.2therapeutic-containing nanoparticle and / or vaccine composition. In other embodiments, providing fusion polypeptides, constructs, or other molecule contemplated herein, includes providing the fusion polypeptides, constructs, or other molecule systemically or subcutaneously or by site-directed administration to the subject. In certain embodiments, the fusion polypeptides, constructs, or other molecules contemplated herein can be administered simultaneously or in the same composition as the nanoparticle-containing therapeutic agent, therapeutic-containing nanoparticle, construct, or other therapeutic agent contemplated herein to reduce adverse acute complement reactions in a subject. In certain embodiments, the fusion polypeptides, constructs, or other molecules contemplated herein can be administered to a subject before, during, or after administering a therapeutic or vaccine to rapidly reduce or eliminate hypersensitivity reactions, for example, those of complement C3 or other complement molecules and then the fusion polypeptides, constructs, or other molecules dissipate or are eliminated from the subject’s system having little or no adverse effects while improving therapeutic outcome.

[0012] In some embodiments and further to paragraphs

[0006] -

[0011] above, a synthetic molecule including, but not limited to, an isolated or synthetic polypeptide construct including, but not limited to, complement receptor 2 (CR2), complement receptor 1 (CR1), DAF, Factor H a fusion polypeptide including, a first component including at least a fragment of CR2 where the at least a fragment of the complement receptor 2 binds to at least one nanosurface-deposited (vaccine-deposited) fragment of complement protein 3 (C3) or induced acute complement circulating fragment thereof; and at least a second component including regulatory consensus repeats of mammalian (e.g., human) complement receptor 1 (CR1) to generate a CR2-CR1 fusion polypeptide, where the CR2-CR1 fusion polypeptide reduces or blocks complement activation. In some embodiments, a composition contemplated herein includes CR2, CR1, DAF, and Factor H, a fusion polypeptide including a first component including at least a fragment of CR2 where at least a fragment of the complement receptor 2 binds to at least one nanosurface- deposited fragment of complement protein 3 (C3); and at least a second component include regulatory consensus repeats of human complement receptor 1 (CR1) to generate a CR2-CR1 fusion polypeptide, where the CR2-CR1 fusion polypeptide reduces or blocks complement activation; and at least one nanomedicine (e.g., nanoparticle-containing therapeutic, therapeutic- containing nanoparticle), and at least one pharmaceutically acceptable excipient. In some embodiments, the first component does not bind to native or naturally-occurring serum C3. In 5 98386650.2some embodiments, the first component binds to a therapeutic agent induced C3 (e.g., when the C3 is associated with or bound to the therapeutic agent). In other embodiments, the isolated or synthetic polypeptide can be fused to a second molecule to form a fusion polypeptide. In other embodiments, the CR2, CR1, DAF, and Factor H, and / or CR2-CR1, CR2-DAF, CR2-Factor H fusion polypeptide, or another related molecule, can further include an agent for increasing circulating half-life of the fusion polypeptide or construct contemplated herein.

[0013] In some embodiments and further to the preceding paragraphs, a pharmaceutical composition can include, but is not limited to, the fusion polypeptide and / or a construct and a pharmaceutically acceptable excipient and optionally, at least one nanoparticle-containing therapy or vaccine.

[0014] In other embodiments, kits are contemplated of use to create a fusion polypeptide disclosed herein or carry / transport and store compositions disclosed herein and further include at least one container. In some embodiments, a synthetic molecule and / or a pharmaceutical composition disclosed herein is contemplated to be part of a kit, and at least one container. In some embodiments, a device for administering a combination composition or a composition including a synthetic molecule disclosed herein is contemplated to be part of a kit disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] FIGS.1A-1B represent in 1A representative constructs contemplated herein for use in blocking adverse complement activities and 1B is representative of an exemplary Western Blot illustrating effect of a construct on the deposition of complement of different nanoparticles and liposomes in accordance with certain embodiments of the present disclosure.

[0017] FIG.2 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by small iron oxide nanoworms in vitro in accordance with certain embodiments of the present disclosure. 6 98386650.2

[0018] FIG.3 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by large iron oxide nanoworms in vitro in accordance with certain embodiments of the present disclosure.

[0019] FIG.4 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by PEG-polystyrene beads in vitro in accordance with certain embodiments of the present disclosure.

[0020] FIG.5 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by small crosslinked Cy5 labeled iron oxide nanoworms in vitro in accordance with certain embodiments of the present disclosure.

[0021] FIG.6 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by Feraheme® in vitro in accordance with certain embodiments of the present disclosure.

[0022] FIG.7 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by Onivyde® in vitro in accordance with certain embodiments of the present disclosure.

[0023] FIG.8 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by PEGylated liposomal doxorubicin LipoDox in vitro in accordance with certain embodiments of the present disclosure.

[0024] FIG.9 represents an exemplary graph illustrating that an exemplary construct blocks complement activation by gold spheres in vitro in accordance with certain embodiments of the present disclosure.

[0025] FIG.10A represents an exemplary graph illustrating plasma profile of CR2-Crry labeled with IRDye800 in accordance with certain embodiments of the present disclosure.

[0026] FIG.10B represents an exemplary near-infrared fluorescence image of various organs demonstrating location of a tracked exemplary construct in accordance with certain embodiments of the present disclosure.

[0027] FIG.11A represents an exemplary dot blot image in the presence and absence of exemplary constructs over time in accordance with certain embodiments of the present disclosure. 7 98386650.2

[0028] FIG.11B represents an exemplary graph illustrating C3 deposition in an animal model in the presence and absence of exemplary constructs over time in accordance with certain embodiments of the present disclosure.

[0029] FIG.12 represents an exemplary graph illustrating C5a levels in an animal model in the presence and absence of exemplary constructs over time in accordance with certain embodiments of the present disclosure.

[0030] FIG.13A represents exemplary flow cytometry dot plots at two time points in accordance with certain embodiments of the present disclosure.

[0031] FIG.13B represents exemplary flow cytometry dot plots at two time points in accordance with certain embodiments of the present disclosure.

[0032] FIG.14A represents an exemplary graph illustrating a plasma profile of IRDye800- construct in accordance with certain embodiments of the present disclosure.

[0033] FIG.14B represents an exemplary near-infrared fluorescence image of organs represented location of an IRDye800-construct in accordance with certain embodiments of the present disclosure.

[0034] FIG.15A represents an exemplary dot blot image in the presence and absence of an exemplary construct over time in accordance with certain embodiments of the present disclosure.

[0035] FIG.15B represents an exemplary bar graph illustrating C3 deposition in an animal model in the presence or absence of an exemplary construct in accordance with certain embodiments of the present disclosure.

[0036] FIG.16A represents an exemplary flow cytometry plot of small SPIO NWs in accordance with certain embodiments of the present disclosure.

[0037] FIG.16B represents an exemplary flow cytometry plot of large SPIO NWs in accordance with certain embodiments of the present disclosure.

[0038] FIG.17 represents an exemplary bar graph demonstrating residual complement activity in plasma over time in accordance with certain embodiments of the present disclosure.

[0039] FIG.18A represents an exemplary image demonstrating tracks of an animal model with saline in accordance with certain embodiments of the present disclosure.

[0040] FIG.18B represents an exemplary image demonstrating tracks of non-anesthetized animal model injected with SPIO NWs in accordance with certain embodiments of the present disclosure. 8 98386650.2

[0041] FIG.18C represents an exemplary image demonstrating tracks of a non-anesthetized animal model injected with constructs with SPIO NWs in accordance with certain embodiments of the present disclosure.

[0042] FIG.19 represents an exemplary graph demonstrating statistical comparison in mean activity of SPIO NWs in the presence and absence of an exemplary construct in male versus female animals in accordance with certain embodiments of the present disclosure.

[0043] FIG.20A represents exemplary flow cytometry dot plots demonstrating inhibition of uptake of large SPIO NWs in animal leukocytes after co-injection with low dose constructs in accordance with certain embodiments of the present disclosure.

[0044] FIG.20B represents exemplary flow cytometry dot plots demonstrating inhibition of uptake of large SPIO NWs in animal leukocytes after co-injection with constructs in accordance with certain embodiments of the present disclosure.

[0045] FIG.21A represents an exemplary flow cytometry plot demonstrating inhibition of immune uptake of exemplary nanoparticles by constructs in accordance with certain embodiments of the present disclosure.

[0046] FIG.21B represents an exemplary flow cytometry plot demonstrating inhibition of immune uptake of exemplary nanoparticles by constructs in accordance with certain embodiments of the present disclosure.

[0047] FIG.22A represents an exemplary graph illustrating inhibition of complement activation on SPIO NWs using an exemplary construct in accordance with certain embodiments of the present disclosure.

[0048] FIG.22B represents an exemplary graph illustrating inhibition of complement activation for a small molecule using a Factor B construct inhibitor agent in accordance with certain embodiments of the present disclosure.

[0049] FIG.22C represents an exemplary graph illustrating inhibition of complement activation for a small molecule using a Factor D construct inhibitor agent in accordance with certain embodiments of the present disclosure.

[0050] FIG.23A represents an exemplary graph illustrating inhibition of complement activation on PEGylated liposomal doxorubicin using an exemplary construct in accordance with certain embodiments of the present disclosure. 9 98386650.2

[0051] FIG.23B represents an exemplary graph illustrating inhibition of complement activation of a small molecule Factor B inhibitor agent in accordance with certain embodiments of the present disclosure.

[0052] FIG.23C represents an exemplary graph illustrating inhibition of complement activation of a small molecule Factor D inhibitor agent in accordance with certain embodiments of the present disclosure.

[0053] FIG.24A represents an exemplary graph illustrating inhibition of complement activation of an exemplary agent (e.g., liposomal irinotecan (Onivyde®)) in accordance with certain embodiments of the present disclosure.

[0054] FIG.24B represents an exemplary graph illustrating inhibition of complement activation of an exemplary agent (e.g., small molecule Factor B inhibitor iptacopan) in accordance with certain embodiments of the present disclosure.

[0055] FIG.24C represents an exemplary graph illustrating inhibition of complement activation of an exemplary agent (e.g., small molecule Factor D inhibitor danicopan) in accordance with certain embodiments of the present disclosure.

[0056] FIG.25A represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., mRNA lipid nanoparticles) using constructs in accordance with certain embodiments of the present disclosure.

[0057] FIG.25B represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., small molecule Factor B inhibitor iptacopan) in accordance with certain embodiments of the present disclosure.

[0058] FIG.25C represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., small molecule Factor D inhibitor danicopan) in accordance with certain embodiments of the present disclosure.

[0059] FIG.25D represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., mRNA lipid nanoparticles) using constructs in accordance with certain embodiments of the present disclosure.

[0060] FIG.25E represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., small molecule Factor B inhibitor iptacopan) in accordance with certain embodiments of the present disclosure. 10 98386650.2

[0061] FIG.25F represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., small molecule Factor D inhibitor danicopan) in accordance with certain embodiments of the present disclosure.

[0062] FIG.25G represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., mRNA lipid nanoparticles) using constructs in accordance with certain embodiments of the present disclosure.

[0063] FIG.25H represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., small molecule Factor B inhibitor iptacopan) in accordance with certain embodiments of the present disclosure.

[0064] FIG.25I represents an exemplary graph illustrating inhibition of complement activation for an exemplary agent (e.g., small molecule Factor D inhibitor danicopan) in accordance with certain embodiments of the present disclosure.

[0065] FIG.26 represents an exemplary graph illustrating blood pressure in an animal model over time in the presence or absence of an exemplary construct and a complement inducing agent in accordance with certain embodiments of the present disclosure. Definitions

[0066] As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0067] As used herein, the terms “treat,” “treating,” “treatment” and the like, unless otherwise indicated, can refer to reversing, alleviating, inhibiting the process of or expansion of, or preventing the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of any of the compositions, pharmaceutical compositions, or dosage forms described herein, to prevent the onset of the symptoms or the complications, or alleviating the symptoms or the complications, or eliminating the condition, or disorder.

[0068] As used herein, the term “nanoparticle” can refer to a particle with at least one dimension that can range from about 0.1 to about 500.0 nanometers, or about 0.1 to about 250.0 nm, or about 0.1 to about 200 nm, or about 0.1 to about 100.0 nm or about 10 to about 100.0 nm in size. Examples of nanoparticles include, but are not limited to, gold nanoparticles, superparamagnetic iron oxide nanoparticles, lipid nanoparticles, nanopores, nanotubes, quantum 11 98386650.2dots, nanoshells, dendrimers, liposomes, nanorods, fullerenes, metal-coated particles, metallo- organic coated particles, nanospheres, nanowires, nanobelts, nanorings, nanocapsules, nanoworms, and the like that induce complement contemplated herein.

[0069] As used herein, the term “nanoparticle-containing therapeutic” or “therapeutic containing nanoparticle” can refer to a nanoparticle conjugated with at least one therapeutic or carrying or harboring at least one therapeutic. Non-limiting examples of therapeutics include, but are not limited to, small molecule drugs, biologics, large molecules, polynucleotides, nanoaggregates, polypeptides, vaccines (e.g., anti-microbial, anti-tumor) and the like that induce acute complement reactions contemplated herein. DETAILED DESCRIPTION

[0070] In the following sections, certain exemplary compositions and methods are described in order to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times, and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0071] Embodiments of the present invention generally relate to novel constructs, compositions, and methods for reducing hypersensitivity reactions to therapeutic and / or prophylactic agents. In certain embodiments, constructs, compositions, and methods are disclosed for reducing or eliminating hypersensitivity reactions using targeted complement inhibitors to complement C3 or other complement molecules deposited on therapeutically relevant nanoparticles, vaccines and / or nanocomponents. In certain embodiments, constructs or fusion polypeptides disclosed herein include short-circulating targeted molecules that act rapidly (e.g., immediately upon administration to a subject) to inhibit acute complement events which arise when administering nanoparticles, therapeutic-containing nanoparticles, and other therapeutic agents (e.g., vaccines). For example, constructs or fusion polypeptides disclosed herein can reduce adverse reactions and improve therapeutic efficacy and improve therapeutic response of a targeted therapeutic molecule avoiding, reducing, or eliminating acute complement interference without prolonged effects and with reduced adverse allergic responses. In some embodiments, constructs and other soluble molecules disclosed herein can be used in methods disclosed herein to inhibit adverse acute complement effects in circulation as well as serum 12 98386650.2without inhibition of targeted therapeutic agent uptake and without conjugation to the targeted therapeutic agent. In certain embodiments, therapeutic agents include therapeutic proteins and antibodies. The disclosed therapeutic agents can form nano to micron size aggregates upon storage and agitation in a vial. These aggregates can enhance the immunogenicity of the proteins, as well activate complement and potentially cause adverse complement reactions. In certain embodiments, targeting these molecules with compositions and constructs disclosed herein can inhibit complement activation and infusion adverse complement reactions.

[0072] In some embodiments and further to paragraph

[0071] above, compositions and methods for reducing nanoparticle (e.g., therapeutic-containing nanoparticle) and / or vaccine or other foreign body (e.g., non-self) complement activation are disclosed to reduce or eliminate acute adverse effects of complement and / or improved therapeutic response and / or efficacy in a subject. In certain embodiments, compositions and methods can include a fusion polypeptide or construct including, but not limited to, a) a component including a mammalian complement receptor 2 (CR2) or fragment thereof or biologically active fragment thereof; and b) a component including short consensus repeats (regulatory domains, SCRs and the like) of a mammalian complement receptor 1 (CR1), DAF, and Factor H, in a composition and administering the fusion polypeptide or construct-containing composition at least one of before, during, and after a nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, mRNA-LNP (e.g. mRNA vaccine), other polynucleotide-containing nanoparticle, or the like, or other vaccine (e.g. anti-viral, anti-tumor, anti-microbial) composition to the subject. In accordance with these embodiments, the fusion polypeptide or construct of the composition reduces or eliminates complement activation and improves therapeutic efficacy and response in the subject compared to a subject not receiving the fusion polypeptide or construct composition. In other embodiments, a polypeptide of use herein as part of a fusion polypeptide can include CR2 and / or CR1, DAF, Factor H alone or other third complement protein (C3) inhibitor peptide or related molecule thereto, or a mixture of these components to reduce acute complement responses when administering one or more nanoparticles and / or vaccine compositions contemplated herein to a subject. In some embodiments, SCR1 and SCR2 of CR2 can be used in fusion polypeptide constructs alone or in combination with one or more of SCR3 and SCR4.

[0073] In certain embodiments and further to paragraphs

[0071] -

[0072] above, the compositions of use in methods disclosed herein reduce or eliminate need for one or more 13 98386650.2inhibitory agent for reducing allergic reactions to therapeutic-containing nanoparticle, nanoparticle containing therapeutic and / or vaccine (e.g., anti-tumor, anti-microbial vaccine) which typically linger and contribute to adverse therapeutic responses and side effects. In accordance with these embodiments, these one more inhibitory agents typically administered to reduce allergic reactions can include, but are not limited to, at least one of a steroid and anti- histamine or other allergy preventive or reducing agents. In some embodiments, the fusion polypeptide or construct disclosed herein when administered to the subject has a short-acting or transient inhibition or suppression of complement (e.g., specifically, effectively and rapidly targeting the complement protein) in the subject, thereby reducing adverse effects of prolonged complement inhibition while targeting adverse effects of complement on a therapeutic providing advantages of a soluble agent (e.g., having rapid clearance) to inhibit complement in blood and serum and enhance therapeutic efficacy.

[0074] In certain embodiments and further to paragraphs

[0071] -

[0073] above, the nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, and / or vaccine composition for targeted complement inhibition contemplated herein can include, but is not limited to, at least one of an iron-oxide (e.g., carboxymethyl dextran iron oxide, dextran iron oxide or the like), SPIO NWs, Cy5-CLIO, pegylated liposomes, gold spheres, metal-oxide coated particles, pegylated polybeads, liposomal doxorubicin, mRNA containing lipid nanoparticles (LNPs), other lipid nanoparticles (LNPs), or similar agent containing nanoparticle harboring one or more therapeutic agent such as a pharmaceutical agent or biologic. In certain embodiments, the nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, and / or vaccine composition can include, but is not limited to, a therapeutic of at least one of paclitaxel (e.g., Taxol®), patisiran (e.g., Onpattro®), irinotecan liposome injectable (e.g., Onivyde®), other chemotherapeutic agent, or other microtubule-stabilizing drug, an anti- microbial (e.g., anti-viral vaccine) or anti-tumor vaccine, or nanomedicine or therapeutic agent that aggregates before or during delivery to a subject, in combination with a construct contemplated herein having a short-term circulation or short-term circulation half-life in the subject to inhibit complement activation and enhance therapeutic effect of the nanoparticle, nanoaggregate, and / or vaccine.

[0075] In other embodiments and further to paragraphs

[0070] -

[0074] above, the fusion polypeptides or constructs disclosed herein can further include, a modification to increase 14 98386650.2circulating half-life of the fusion polypeptide, if desired. In accordance with these embodiments, modifications for increasing half-life of the fusion polypeptides or constructs disclosed herein can include, but are not limited to, PEGylation, albumin binding domains, Fc conjugation, formation of repeats of a construct (e.g., where 2 or more constructs are linked together, for example) or other modification to increase circulating half-life of the fusion polypeptide. In accordance with these embodiments, fusion polypeptides can be formed into repeats such as 2, 3. 4 or more fusion polypeptides linked together to improve or increase half-life of these molecules and efficacy for inhibiting complement activation. In other embodiments, constructs or fusion polypeptides disclosed herein are PEGylated to prolong half-life of the construct. In yet other embodiment, a desired half-life of a construct or fusion polypeptide disclosed herein can be created using PEGylation, albumin binding domains, Fc conjugation or the like to prolong half- life for a desired effect.

[0076] In some embodiments and further to paragraphs

[0071] -

[0075] above, compositions and methods disclosed herein for providing constructs or fusion polypeptides for example, CR2-CR1, CR1, CR2, DAF, Factor H or biologically active fragment of CR2, DAF, Factor H and / or CR1 thereof can reduce dose requirements of therapeutically relevant agents such as reducing dosage amounts or frequency of administration for nanoparticle-containing therapeutics, therapeutic- containing nanoparticles and / or vaccine compositions. In accordance with these embodiments, constructs or fusion polypeptides for example, CR2-CR1, CR1, CR2, CR2-DAF, CR2-Factor H, DAF, Factor H or biologically active fragment of CR2, DAF, Factor H and / or CR1 thereof can reduce dose requirements of therapeutically relevant agents such as reducing dosage amounts or frequency of administration required to prevent onset of a condition or dosage amounts or frequency of administration required to needed to treat a health condition in a subject by at least one of, reducing complement interference; and reducing granulocyte, monocyte, macrophage and / or any other immune cell uptake of the nanoparticle-containing therapeutic, therapeutic- containing nanoparticle and / or vaccine composition. In other embodiments, providing fusion polypeptides, constructs, or other molecule contemplated herein, includes providing the fusion polypeptides, constructs, or other molecule systemically to the subject. In certain embodiments, the fusion polypeptides, constructs, or other molecules contemplated herein can be administered simultaneously or in the same composition as the nanoparticle-containing therapeutic agent, therapeutic-containing nanoparticle, construct, or other therapeutic agent contemplated herein to 15 98386650.2reduce adverse acute complement reactions in a subject. In certain embodiments, the fusion polypeptides, constructs, or other molecules contemplated herein can be administered to a subject before, during, or after administering a therapeutic or vaccine to rapidly reduce or eliminate hypersensitivity reactions, for example, those of complement C3 or other complement molecules. In accordance with these embodiments, the fusion polypeptides, constructs, or other molecules for reducing acute complement effects dissipate or are eliminated from the subject’s system having little or no adverse effects while improving therapeutic outcome (e.g., processed to the liver). In some embodiments, this clearance process, if desired, can be rapid, within minutes, within an hour, within a few hours, or within 12 hours of providing constructs contemplated herein.

[0077] In some embodiments and further to paragraphs

[0071] -

[0076] above, a synthetic molecule of use herein includes, but is not limited to, an isolated or synthetic polypeptide construct including, but not limited to, complement receptor 2 (CR2), complement receptor 1 (CR1), or fragments thereof. In some embodiments, a fusion polypeptide can include, a first component having at least a fragment of CR2 where the at least a fragment of the CR2 binds to at least one nanosurface-deposited fragment of complement protein 3 (C3); and at least a second component having regulatory consensus repeats of mammalian (e.g., human) CR1 for making a CR2-CR1 fusion polypeptide (or CR2-DAF, CR2-Factor H), where the CR2-CR1 fusion polypeptide reduces, or blocks complement activation. In some embodiments, a composition contemplated herein includes CR2, CR1, a fusion polypeptide including, a first component including at least a fragment of CR2 where at least a fragment of the complement receptor 2 binds to at least one nanosurface-deposited fragment of complement protein 3 (C3); and at least a second component include regulatory consensus repeats of human complement receptor 1 (CR1) to generate a CR2-CR1 fusion polypeptide, where the CR2-CR1 fusion polypeptide reduces or blocks complement activation; and at least one nanomedicine (e.g., nanoparticle-containing therapeutic, therapeutic-containing nanoparticle), and at least one pharmaceutically acceptable excipient. In some embodiments, the first component of a fusion construct does not bind to native or naturally-occurring serum C3. In other embodiments, the isolated or synthetic polypeptide can be fused to a second molecule to form a fusion polypeptide. In other embodiments, the CR2, CR1, DAF, Factor H, and / or CR2-CR1 CR2-DAF, and / or CR2-Factor H (CR2-FH) fusion polypeptide, or other related molecule, can further include an agent for 16 98386650.2increasing circulating half-life of the fusion polypeptide or construct contemplated herein. In some embodiments, an Fc or other molecule can be associated with a construct disclosed herein to increase circulating half-life if needed to reduce complement activation beyond acute complement activation to a therapeutic-containing nanoparticle or vaccine or other therapeutic or prophylactic contemplated herein.

[0078] In some embodiments and further to paragraphs

[0071] -

[0077] above, a pharmaceutical composition can include, but is not limited to, the fusion polypeptide and / or a construct and a pharmaceutically acceptable excipient and optionally, at least one nanoparticle-containing therapy or vaccine. In some embodiment, a composition disclosed herein can include at least a CR2-CR1, CR2-DAF, and / or CR2-FH fusion polypeptide and one or more therapeutic- containing nanoparticle, nanoparticle-containing therapeutic, nanoaggregate and / or vaccine or other therapeutic or prophylactic agent contemplated herein. In further embodiments, the composition is a pharmaceutical composition.

[0079] In certain embodiments and further to paragraphs

[0071] -

[0078] above, constructs and compositions thereof are useful in the methods disclosed herein when used concurrently with at least one additional agent for reducing acute complement when administered a nanoparticle or vaccine composition contemplated herein. In other embodiments, the constructs and compositions are useful in the methods disclosed herein in combination with at least one additional agent useful for eliminating adverse acute complement reactions contemplated herein or other agent for therapeutic purposes.

[0080] In some embodiments and further to paragraphs

[0071] -

[0079] above, these additional agents can include commercially available agents or other available agents known to reduce acute complement reactions. In certain embodiments, compositions containing at least one construct disclosed herein (e.g., CR2-CR1 fusion polypeptide) or a salt thereof, and at least one additional agent useful for preventing, eliminating, or ameliorating acute complement reactions contemplated herein, can have an additive, complementary or synergistic effect on acute complement reactions.

[0081] In other embodiments and further to paragraphs

[0071] -

[0080] above, the present disclosure is directed to polypeptide constructs or fusion molecules thereof alone or as part of a pharmaceutical composition. In accordance with these embodiments, fusion molecules and / or polypeptide constructs disclosed herein can include at least one of the polypeptide represented by 17 98386650.2any one of SEQ ID 29, SEQ ID 30, and / or SEQ ID 31; or at least 85%, at least 90%, at least 95%, at least 99% homology thereof. In some embodiments, the polypeptide construct can be part of a pharmaceutical composition. In other embodiments, methods for making fusion molecules are disclosed herein and / or kits for making, storing or transporting fusion polypeptides disclosed herein.

[0082] In certain embodiments and as represented in Tables 1-3 list polynucleotide sequences and polypeptide sequences are disclosed for creating some fusion constructs contemplated of use herein. Table 1. CR2-Decay Accelerating Factor nucleotide sequence SEQ ID NAME / T C C T A G G C A C G C C C C A G C C C18 98386650.2GCAACACCGGCTACAAGCTGTTCGGCAGCACAAGCAG CTTCTGCCTGATCAGCGGCAGCAGCGTGCAGTGGAGCLP19 98386650.28 Linker FGGGGSGGGGS E L I S L I S L I A CTable 2. CR2CR1 nucleotide sequence E ID AME tt g g g a a a tt c g c c a t c at g a c20 98386650.2aacagagagaattttcactatggatcagtggtgacctaccgctgcaatcctggaagcgga gggagaaaggtgtttgagcttgtgggtgagccctccatatactgcaccagcaatgacgat caagtgggcatctggagcggccccgcccctcagtgcattatacctaacaaatgcacgcct ccaaatgtggaaaatggaatattggtatctgacaacagaagcttattttccttaaatgaagtt gtggagtttaggtgtcagcctggctttgtcatgaaaggaccccgccgtgtgaagtgccag gccctgaacaaatgggagccggagctaccaagctgctccagggtatgtcagccacctcc agatgtcctgcatgctgagcgtacccaaagggacaaggacaacttttcacctgggcagg aagtgttctacagctgtgagcccggctacgacctcagaggggctgcgtctatgcgctgca caccccagggagactggagccctgcagcccccacatgtgaagtgaaatcctgtgatgac ttcatgggccaacttcttaatggccgtgtgctatttccagtaaatctccagcttggagcaaaa gtggattttgtttgtgatgaaggatttcaattaaaaggcagctctgctagttactgtgtcttggc tggaatggaaagcctttggaatagcagtgttccagtgtgtgaacaaatcttttgtccaagtcc tccagttattcctaatgggagacacacaggaaaacctctggaagtctttccctttgggaaaa cagtaaattacacatgcgacccccacccagacagagggacgagcttcgacctcattgga gagagcaccatccgctgcacaagtgaccctcaagggaatggggtttggagcagccctg cccctcgctgtggaattctgggtcactgtcaagccccagatcattttctgtttgccaagttga aaacccaaaccaatgcatctgactttcccattgggacatctttaaagtacgaatgccgtcct gagtactacgggaggccattctctatcacatgtctagataacctggtctggtcaagtcccaa agatgtctgtaaacgtaaatcatgtaaaactcctccagatccagtgaatggcatggtgcatg tgatcacagacatccaggttggatccagaatcaactattcttgtactacagggcaccgactc attggtcactcatctgctgaatgtatcctctcgggcaatgctgcccattggagcacgaagcc gccaatttgtcaacgaattccttgtgggctaccccccaccatcgccaatggagatttcatta gcaccaacagagagaattttcactatggatcagtggtgacctaccgctgcaatcctggaa gcggagggagaaaggtgtttgagcttgtgggtgagccctccatatactgcaccagcaatg acgatcaagtgggcatctggagcggcccggcccctcagtgcattatacctaactga CR2CR1 amino acid sequence LEPP21 98386650.214 CR1 SCR2SCRNPPDPVNGMVHVIKGIQFGSQIKYSCTKGYRLIGSSSATCIISGDTVIWDNETPICDRIVPGSRSL I S L I K S N G F C L C P PTable 3. 22 98386650.2CR2FH nucleotide sequence SEQ ID NAME / SE UENCEtt g g g a a a tt c g c c c ct a tt g at t t a ct tt c LEPL23 98386650.225 FH_SCR2PCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVD L I S L I K KI D Kencod ng a po ypept de, a operab y n ed to ( .e., n a unct ona re at ons p wt ) one or more regulatory sequences, such as a promoter of use to create polypeptides contemplated herein.

[0084] As used herein “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues associated to create a polypeptide.

[0085] Each nucleic acid sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given sequence respectively has an identity or a similarity of at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% identical with disclosed polynucleotide or polypeptide, respectively. In some embodiments, each polynucleotide sequence or polypeptide sequence has by virtue of its identity or similarity percentage with a given amino acid sequence respectively has an identity or a similarity of at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% identical with the given polynucleotide or polypeptide, respectively. In some embodiments, a fusion polypeptide (or encoding polynucleotide thereof) having at least 85%, at least 90%, at least 95% or at least 99% identity to disclosed fusion polypeptides of Tables 1-3 above is contemplated to reduce acute complement reactions targeted herein. 24 98386650.2

[0086] The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length (in amino acids or nucleotides) of two given SEQ ID NOS or based on a portion thereof. A portion of a full-length sequence can be referred to as a fragment, and preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of the length (in amino acids or nucleotides) of a reference sequence. “Identity” also refers to the degree of sequence relatedness between two amino acid sequences, or between two nucleic acid sequences, as the case can be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using any suitable method or algorithm. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm.

[0087] As used herein “fragment” means a portion of a polypeptide that contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of the reference polypeptide. In certain aspects, the fragment as the same or similar biological activity as the reference polypeptide.

[0088] As used herein, the term “fused” or “joined” refers to the combination or attachment of two or more elements, components, or protein domains, e.g., polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., disulfide bonds and amide bonds. For example, two single polypeptides can be joined to form one contiguous protein structure through recombinant expression, chemical conjugation, a chemical bond, a peptide linker, or any other means of covalent linkage.

[0089] As used herein, the term “linker” refers to a linkage between two elements, e.g., polypeptides or protein domains. A linker can be a covalent bond. A linker can also be a molecule of any length that can be used to couple, for example, a factor H fragment and / or a CR2 fragment with an Fc domain, such as an Fc receptor binding domain. A linker also refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid, 1-150 amino acid, 1-100, a 5-50, or a 1-10 amino acid sequence, particularly amino acids with smaller side chains and / or flexible amino acid sequences) occurring between two polypeptides or polypeptide domains to provide space and / or flexibility between the two polypeptides or polypeptide domains. An amino acid linker can be part of the primary sequence 25 98386650.2of a polypeptide (e.g., joined to the linked polypeptides or polypeptide domains via the polypeptide backbone). In certain embodiments, the linker can include an amino acid with SEQ ID 8.

[0090] As used herein, the term “alternative complement pathway dysregulation” refers to any aberration in the ability of the alternative complement pathway to provide host defense against pathogens and clear immune complexes and damaged cells and for immunoregulation. Alternative complement pathway dysregulation can occur in the fluid phase and at the cell surface and can lead to excessive complement activation or insufficient regulation, both causing tissue injury.

[0091] In certain embodiments, constructs or fusion polypeptides or fragment thereof can be used in constructs disclosed herein. As used herein, “Factor H” refers to a protein component of the alternative complement pathway encoded by the complement factor H gene. Factor H is translated as a 1,213 amino acid precursor polypeptide that is processed by removal of an 18 amino acid signal peptide, resulting in the mature factor H protein (amino acids 19-1231). Factor H consists of 20 short complement regulator (SCR) domains. Amino acids 1-18 can include the signal peptide, residues 21-80 can include SCR1 (SEQ ID NO: 1, residues 85-141 can include SCR 2 (SEQ ID NO: 2), residues 146-205 can include SCR3 (SEQ ID NO: 3), residues 201-262 can include SCR 4 (SEQ ID NO: 4), residues 267-320 can include SCR 5 (SEQ ID NO: 5), residues 1107-1165 can include SCR 19 (SEQ ID NO:6), and residues 1167-1230 can include SCR 20 (SEQ ID NO: 7). Factor H regulates complement activation on self-cells by possessing both cofactor activity for the factor I-mediated C3b cleavage, and decay accelerating activity against the alternative pathway C3 convertase, C3bBb.

[0092] In other embodiments, constructs can include Complement receptor 2. As used herein, “Complement receptor 2” or “CR2” refers to human complement receptor 2, also referred to as CD21 (CR2 / CD21), is a 145 kD transmembrane protein of the C3 binding protein family with 15 or 16 short consensus repeat (SCR) domains, structural units characteristic of such proteins. The SCR domains have a typical framework of highly conserved residues including four cysteines, two prolines, one tryptophan, and several other partially conserved glycines and hydrophobic residues. These SCR domains are separated by short sequences of variable length that serve as spacers. Amino acids 1-20 can include the leader peptide, amino acids 23-82 can include SCR1 (SEQ ID NO: 8), amino acids 91-146 can include SCR2 (SEQ ID NO: 9), amino acids 154-210 26 98386650.2can include SCR3 (SEQ ID NO: 10), and amino acids 215-271 can include SCR4 (SEQ ID NO: 11). The active site (C3d binding site) is located in SCR1-2 (the first two N-terminal SCR domains). CR2 is expressed on mature B cells and follicular dendritic cells, and plays an important role in humoral immunity. CR2 protein does not bind intact C3 protein, but binds its breakdown products, including the C3b, iC3b, and C3d cleavage fragments, via a binding site located within the first two amino-terminal SCR domains (“SCRs 1-2”) of the CR2 protein. Consequently, the SCRs 1-2 of CR2 discriminate between cleaved (i.e., activated) forms of C3 generated during complement activation and intact circulating C3. While the affinity of CR2 for C3d is only 620-658 nM, the avidity of CR2 for clustered C3d makes it an effective method of targeting molecules to sites of complement activation. It is contemplated that fragments of use disclosed herein can contain some variability while still be contemplated for uses of the instant application. All components of fusion polypeptides contemplated of use herein can include naturally-occurring variable of the components (e.g., SNPs, mutants, or other variables while preserving disclosed effects contemplated herein)

[0093] As previously disclosed and understood by one of skill in the art, cleavage of C3 can result initially in generation and deposition of C3b on the activating cell surface. The C3b fragment is involved in the generation of enzymatic complexes that amplify the complement cascade. On a cell surface, C3b is rapidly converted to inactive iC3b, particularly when deposited on a host surface containing regulators of complement activation (e.g., most host tissue). Even in absence of membrane-bound complement regulators, substantial levels of iC3b are formed because of the action of serum factor H and serum factor I. iC3b is subsequently digested to the membrane-bound fragments C3dg and then C3d by factor I and other proteases and cofactors, but this process is relatively slow. By this process, C3 ligands for CR2 are relatively long lived once they are generated and are present in high concentrations at sites of complement activation.

[0094] As used herein, a “functional fragment” or a “biologically active fragment” refers to a fragment, or portion, of a protein having some or all of the activities of the full-length protein. For example, a functional or biologically active fragment of factor H, refers to any fragment of a factor H protein having some or all of the activities of factor H, e.g., alternative complement pathway regulatory activity of the full-length factor H protein. Examples include, but are not limited to, factor H fragments, joined from N-terminus to C terminus (or C-terminus to the N- terminus), containing the following SCRs: [1-4], [1-5], [1-7], [1-20], [19-20], [1-4 and 19-20], 27 98386650.2and [1-5 and 19-20]. A “functional fragment” or a “biologically active fragment” of CR2 protein is one having some or all of the activities of CR2, e.g., alternative complement pathway regulatory activity of the full-length CR2 protein. Examples include, but are not limited to, CR2 fragments, from N-terminus to C-terminus, containing the following SCRs: [1-2], [1-3], or [1-4].

[0095] As used herein, the term “fragment” refers to less than 100 % of the amino acid sequence or a full-length reference protein (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, of the full-length sequence etc.), but including, e.g., 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, or more amino acids depending on the full-length of the polypeptide. A fragment can be of sufficient length such that a desirable function of the full-length protein is maintained. For example, the regulation of the alternative complement pathway in the fluid phase by fragments of, for example, factor H, is maintained. Such fragments are “biologically active fragments.”

[0096] As used herein, the terms “short complement regulator”, or “SCR”, also known as “short consensus repeat”, “sushi domains,” or “complement control protein” or “CCP,” include domains found in all regulators of complement activation (RCA) gene clusters that contribute to their ability to regulate complement activation in the blood or on the cell surface to which they specifically bind. SCRs typically are composed of about 60 amino acids, with four cysteine residues disulfide bonded in a 1-3, 2-4 arrangement and a hydrophobic core built around an almost invariant tryptophan residue when configured. SCRs are also found in proteins including, but not limited to, factor H and CR2.

[0097] In other embodiments, kits are contemplated of use to carry out creating compositions disclosed herein or for transporting constructs of use in embodiments disclosed herein. In some embodiments, a synthetic molecule and / or a pharmaceutical composition disclosed herein is contemplated to be part of a kit, and at least one container. In other embodiments, a device for administering a combination composition or a composition including a synthetic molecule disclosed herein is contemplated to be part of a kit disclosed herein. Pharmaceutical Compositions

[0098] In certain embodiments and further to the previous paragraphs, pharmaceutical compositions are provided herein. In accordance with these embodiments, the pharmaceutical composition can contain a pharmaceutically acceptable carrier combined with an CR1, CR2, DAF, FH, CR2-CR1 fusion polypeptides or similar agent as described herein. In other 28 98386650.2embodiments, one or more therapeutic-containing nanoparticle, nanoparticle associated therapeutic, or vaccine or other therapeutic or prophylactic contemplated herein can be included in the pharmaceutical composition. Pharmaceutically acceptable excipients (carriers) are well known in the art.

[0099] In certain embodiments, the pharmaceutical compositions can further include one or more nanomedicine or vaccine including nanoparticles. In accordance with these embodiments, the combination or mixture can reduce or eliminate adverse acute complement reactions to the one or more nanomedicine or vaccine including nanoparticles.

[0100] Pharmaceutically acceptable carriers or excipients suitable for the compositions described herein are well known to one of skill in the art of use for preserving and delivering agents to any mammalian subject including humans and other mammals. It is contemplated that any pharmaceutically acceptable agent can be used in compositions disclosed herein.

[0101] In certain embodiments, the pharmaceutical compositions to be used in the present methods can include pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. In some embodiments, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG).

[0102] In some embodiments, the pharmaceutical composition described herein can have liposomes. In some embodiments, liposomes for use herein can be generated by the reverse phase evaporation method with a lipid composition having phosphatidylcholine, cholesterol and 29 98386650.2PEG-derivatized phosphatidylethanolamine (PEG-PE). In some embodiments, liposomes for use herein can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0103] In other embodiments, the pharmaceutical compositions described herein can be formulated in sustained-release format. In some embodiments, pharmaceutical compositions herein to be used for in vivo administration must be sterile. In some embodiments, this can be readily accomplished by, for example, filtration through sterile filtration membranes. In some embodiments, therapeutic compositions can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0104] In certain embodiments, pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation. In some embodiments, compositions disclosed herein can be administered as a single bolus and not require supplemental administrations. In other embodiments, the single bolus of complement inhibitor contemplated herein can be administered before, during, simultaneously with, or after the nanomedicine or the like to reduce or eliminate acute adverse effects of complement.

[0105] In some embodiments, pharmaceutical compositions herein for inhalation or intranasal administration include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. In some embodiments, liquid or solid compositions herein can contain suitable pharmaceutically acceptable excipients as set out above. In other embodiments, the compositions can be administered by the oral or nasal respiratory route for local or systemic effect.

[0106] In some embodiments, compositions can be in sterile pharmaceutically acceptable solvents can be nebulized by use of gases. In some embodiments, nebulized solutions herein can be breathed directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent or intermittent positive pressure breathing machine. In some embodiments, solution, suspension, or powder compositions herein can be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. 30 98386650.2

[0107] In some embodiments, concentrations of the soluble complement-reducing agents (e.g., CR2-CR1 fusion polypeptide) disclosed herein can be reduced compared to other complement inhibiting agents such as a pre-determined concentration or a standard concentration. In some embodiments, the concentration can be about 0.01-100.0 mg / kg, 0.01-75.0 mg / kg, 0.05-50.0 mg / kg, or 0.05 to 25.0 mg / kg or about 0.1 to 15.0 mg / kg micrograms or other suitable concentration. In some embodiments, the fusion polypeptide is about 1.5 to about 10 times or about 2 to about 8 times more potent than other comparable agents. In certain embodiments, these lower concentrations with increased efficacy can reduce side effects and permit rapid clearing of the agent after inhibition of acute complement reactions. Recombinant Methods - Production of Fusion Proteins

[0108] Disclosed herein are methods for producing a fusion protein using nucleic acid molecules encoding the fusion proteins, such as the fusion polypeptides illustrated in Tables 1-3. In some embodiments, a polynucleotide molecule can be operably linked to a suitable control sequence to form an expression unit encoding the polypeptide of interest. An expression unit is used to transform a suitable host cell, and the transformed host cell is cultured under conditions that allow the production of the recombinant polypeptide. Optionally, the recombinant polypeptide is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities can be tolerated. Additional residues can be included at the N- or C-terminus of the protein-coding sequence to facilitate purification (e.g., a histidine tag).

[0109] The fusion proteins of the present disclosure can include naturally-occurring or a non- naturally-occurring components; preferably at least one component is non-naturally occurring, e.g., with respect to its structure (e.g., sequence) and / or its association (e.g., how it is linked to other components). As used herein, the term “non-naturally occurring” refers to any molecule, e.g., fusion protein, produced with the aid of human manipulation, including, without limitation, molecules produced by genetic engineering using random mutagenesis or rational design and molecules produced by chemical synthesis. Non-limiting examples of non-naturally occurring molecules include, e.g., conservatively substituted variants, non-conservatively substituted variants, and active hybrids (e.g., chimeras) or fragments. Non-natural molecules further include natural molecules that have been modified, e.g., post-translationally, e.g., via addition of 31 98386650.2chemical moieties, tags, ligands. Preferably, non-natural molecules include the fusion proteins of the present disclosure.

[0110] The fusion protein can be expressed from a single polynucleotide that encodes the entire fusion protein or as multiple (e.g., two or more) polynucleotides that can be expressed by suitable expression systems or can be co-expressed. Polypeptides encoded by polynucleotides that are co-expressed can associate through, e.g., disulfide bonds or other means to form a functional fusion protein. For example, the light chain portion of monoclonal antibody can be encoded by a separate polynucleotide from the heavy chain portion of a monoclonal antibody. When co-expressed in a host cell, the heavy chain polypeptides will associate with the light chain polypeptides to form the monoclonal antibody.

[0111] It is envisioned that any and all polynucleotide molecules that can encode the fusion proteins disclosed in the present specification can be useful, including, without limitation naturally-occurring and non-naturally-occurring DNA molecules and naturally-occurring and non-naturally-occurring RNA molecules. Non-limiting examples of naturally-occurring and non- naturally-occurring DNA molecules include single-stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules, cDNA molecules, vector constructs, such as, e.g., plasmid constructs, phagemid constructs, bacteriophage constructs, retroviral constructs and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non- naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA and mRNA. The present disclosure also provides synthetic nucleic acids, e.g., non-natural nucleic acids. The synthetic nucleic acids include nucleotide sequence encoding one or more of the aforementioned fusion proteins. Included herein are nucleic acids encoding the fusion proteins, including the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.

[0112] Typically, a polynucleotide encoding the desired fusion protein is generated using molecular cloning methods, and is generally placed within a vector, such as a plasmid constructs, phagemid constructs, bacteriophage constructs, retroviral constructs, and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non-naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA, and mRNA. The vector is used to transform the nucleic acid into a host cell appropriate for the expression of the fusion polypeptide. Representative methods are known in the art. Many cell types can be used as 32 98386650.2appropriate host cells, although mammalian cells are preferable because they are able to confer appropriate post-translational modifications. Host cells can include, e.g., a Human Embryonic Kidney (HEK) (e.g., HEK 293) cell, Chinese Hamster Ovary (CHO) cell, L cell, C127 cell, 3T3 cell, BHK cell, COS-7 cell, or any other suitable host cell known in the art.

[0113] In addition, prokaryotic cells including, without limitation, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells such as those derived from, e.g., Bacillus subdlis, Bacillus licheniformis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile, Caulobacter crescentus, Lactococcus lacts, Methylobacterium extorquens, Neisseria meningirulls, Neisseria meningitidis, Pseudomonas fluorescens and Salmonella typhimurium; and eukaryotic cells including, without limitation, yeast strains, such as, e.g., those derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as, e.g., those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca Sexta; and mammalian cells and cell- lines derived from mammalian cells, such as, e.g., those derived from mouse, rat, hamster, porcine, bovine, equine, primate and human can be used. Cell lines can be obtained from any suitable sources. In certain embodiments, E. coli cells are unable to be used to produce a desired fusion polypeptide disclosed herein. In some embodiments, human cell lines used resulted in more efficient production of fusion polypeptide disclosed herein.

[0114] It is contemplated herein that codon-optimized sequences can be used. Codon- optimized sequences of the aforementioned nucleic acid sequences and vectors. Codon optimization for expression in a host cell, e.g., bacteria such as insect Hi5 cells, can be performed using Codon Optimization Tool (CODONOPT) readily available. In one embodiment, a nucleic acid or polynucleotide encoding the fusion protein is provided. In one embodiment, a vector including a nucleic acid or polynucleotide encoding the fusion protein is provided. In one embodiment, a host cell including one or more polynucleotides encoding the fusion protein is provided. In certain embodiments, a host cell including one or more fusion expression vectors is provided. The fusion proteins can be produced by expression of a nucleotide sequence in any suitable expression system known in the art. Any expression system can be used, including yeast, bacterial, animal, plant, eukaryotic, and prokaryotic systems. In some embodiments, yeast systems that have been modified to reduce native yeast glycosylation, hyper-glycosylation or 33 98386650.2proteolytic activity can be used. Furthermore, any in vivo expression systems designed for high level expression of recombinant proteins within organisms known in the art can be used for producing the fusion proteins specified herein. In some embodiments, the factor H fusion protein, as described herein, is produced by culturing one or more host cells including one or more nucleic acid molecules capable of expressing the fusion protein under conditions suitable for expression of the fusion protein. In some embodiments, the factor H fusion protein is obtained from the cell culture or culture medium.

[0115] The fusion protein can also be produced using chemical methods to synthesize the desired amino acid sequence, in whole or in part. For example, polypeptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. The composition of the synthetic polypeptides can be confirmed by amino acid analysis or sequencing. Additionally, the amino acid sequence of a fusion protein or any part thereof, can be altered during direct synthesis and / or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant polypeptide.

[0116] In some embodiments, the type of media used may affect the protein expression. In some embodiments, 1% FBS can be used. In other embodiments, 10% FBS can be used. Isolation / Purification of Fusion Proteins

[0117] Secreted, biologically active fusion proteins described herein, such as those described in Tables 1-3, can be purified by techniques such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, e.g., protein A affinity chromatography, size exclusion chromatography, and the like. The conditions used to purify a particular protein depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., as would be apparent to a skilled artisan.

[0118] In certain embodiments, any fusion polypeptide and construct can be expressed in suitable cell lines. Examples of a suitable cell line include but are not limited to HEK293 cells, insect cell, and the like. The constructs can be purified using any suitable technique. For example, over nickel chromatography or by using any other purification tag and expression system, including insect cells. In certain embodiments, the cell line can be HEK293 cells. Kits

[0119] In certain embodiments, kits are provided for storage, transport and use in treating or alleviating a target disease, such preventing a microbial infections or treating a condition where 34 98386650.2nanoparticles are used as nanomedicines as described herein. In some embodiments, kits can include one or more containers.

[0120] In some embodiments, kits can include instructions for use in accordance with any of the methods described herein. In other embodiments, containers, delivery devices and / or instructions can be included and can contain a description of methods for administration of the molecules or combination compositions disclosed herein. EXAMPLES

[0121] The following examples are included to illustrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the claimed methods, compositions, and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes can be made in some embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of embodiments of the inventions. Example 1

[0122] CR2-CR1 is a 120kDa fusion protein consisting of 4 SCRs of human CR2 and the N- terminal 10 SCRs of human CR1. In the CR2 moiety, only SCRs 1–2 bind to C3d, the other SCRs serving as a linker, whereas in the CR1 part, SCRs 1–4 exhibit a convertase decay function, and SCRs 6–10 also have a cofactor function due to binding of complement Factor I. The mouse structural and functional analog of CR2-CR1 is CR2-Crry and contains 4 SCRs of mouse CR2 linked to the extracellular region of Crry. First, the in vitro efficacy of inhibition of C3 opsonization (half maximal inhibitory concentration or IC50) using non-diluted, complement- sufficient human serum was evaluated.

[0123] In some exemplary methods, nanoparticles of different surface properties and sizes were used (Table 4). For example, these included 58 nm superparamagnetic iron oxide nanoworms (hereafter referred to as small SPIO NWs) and 126 nm superparamagnetic iron oxide nanoworms SPIO NWs (hereafter referred to as large SPIO NWs), 67 nm and 102 nm Cy5-labeled crosslinked iron oxide NWs CLIO NWs (hereafter referred to as small and large Cy5-CLIO NWs, derived from the corresponding SPIO NWs), 87 nm PEGylated liposomal doxorubicin LipoDox®, 27 nm carboxymethyl dextran iron oxide nanoparticle Feraheme®, 94 nm Cy5 / carboxylated gold spheres, 120 nm minimally-PEGylated liposome Onivyde®and 219 nm 35 98386650.2PEGylated polystyrene beads (Polybead®). As illustrated in FIG.1, for all the nanoparticle types, Western Blotting confirmed the inhibition of C3 opsonization and lack of complement activation on the nanosurface.

[0124] In some examples, liposomes were prepared from hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and DSPE-PEG2000 (HSPC / Chol / DSPE-PEG2000 (57 / 38 / 5)) with the addition of 0.2% DiD by extrusion through 100 nm polycarbonate membranes. Small and large SPIO NWs (60 nm and 120 nm) were prepared by precipitation of FeCl2 and FeCl3 salts with different ratios of T-20 dextran by ammonia as described previously.

[0125] Corresponding small and large Cy5-labeled CLIO NWs (derived from small and large SPIO NWs) were prepared by dextran crosslinking with epichlorohydrin, amination with ammonia, labeling with Cy5-NHS and blocking residual amines with sulfosuccinimidyl acetate as described previously. Polybead® aminated microspheres (0.2 µm, cat# 15699) were purchased and modified with an excess of PEG2000-SVA. Residual amines were capped with sulfosuccinimidyl acetate. Cy5-labeled carboxylated Gold Spheres (0.1µM, cat#CF11) were obtained and were used as received. Feraheme®, LipoDox®,and Onivyde®were obtained. The nanoparticle size distribution was measured using Malvern Zetasizer Nano ZS (Malvern Instruments, USA).

[0126] In another exemplary method, complement component 3 (C3) deposition was measured as follows. Particles in PBS were added to freshly thawed serum at >75% final serum concentration. After incubation for 30 mins at 37ºC with or without inhibitors, nanoparticles were washed 3 times with PBS at 450,000g at 4 ºC using Beckman Optima TLX ultracentrifuge equipped with a TLA-100.3 rotor. The pellets were resuspended in PBS to a final concentration of 0.5 mg Fe / mL. Proteins were eluted from a washed nanoparticle pellet with 2% SDS in PBS for 1h at room temperature. The particles were pelleted with an ultracentrifuge. The supernatant was mixed with Bio-Rad sample buffer with beta-mercaptoethanol, boiled for 2 min at 95ºC, and separated on a 4-20% Tris-Glycine SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and processed and detected with Goat IgG fraction against human C3. Table 4. Exemplary Example Compositions Diameter, nm )36 98386650.2small Cy5-CLIO NWs Crosslinked 20kDa dextran iron oxide 67 (0.31) large Cy5-CLIO NWs Crosslinked 20kDa dextran iron oxide 102 (0.228), µ p incubation with serum as described in FIG.1 was applied in replicates on a 0.45 µm pore nitrocellulose membrane. The protein was detected with primary goat anti-human C3 and corresponding IRDye800-labeled secondary antibodies for 1h. The membrane was washed with 0.1%Tween-20 / PBS and scanned using an Odyssey infrared imager (Li-COR Biosciences, Lincoln, NE, US), and the integrated intensities of C3 dots were determined from 8-bit grayscale images using Fiji. The normalized data (percent of “no inhibitor” values were plotted with Prism software v.9.0 and fitted into the normalized inhibition curve (variable slope) to calculate IC50. FIGS.2-9 illustrate measurements of IC50 (inhibition of C3 deposition) with dot blot; representative IC50values for nanoparticles in serum from the same donor (mean and s.d. of 4 technical replicates). FIG.2 illustrates CS deposition (%) vs. inhibitor concentration of small 60 nm SPIO NWs. FIG.3 illustrates CS deposition (%) vs. inhibitor concentration of large 120 nm SPIO NWs. FIG.4 illustrates CS deposition (%) vs. inhibitor concentration of PEG-polystyrene beads. FIG.5 illustrates CS deposition (%) vs. inhibitor concentration of small 60nm crosslinked Cy5 labeled iron oxide nanoworms. FIG.6 illustrates CS deposition (%) vs. inhibitor concentration of Feraheme®. FIG.7 illustrates CS deposition (%) vs. inhibitor concentration of Onivyde®. FIG.8 illustrates CS deposition (%) vs. inhibitor concentration of PEGylated liposomal doxorubicin LipoDox. FIG.9 illustrates CS deposition (%) vs. inhibitor concentration of gold spheres. These observations support that targeted inhibitor CR2-CR1 is consistently more efficient than non-targeted inhibitor CR1 in blocking complement activation. Example 2

[0128] In another exemplary method, to determine the pharmacokinetics and biodistribution of systemically injected CR2-Crry in BALB / c mice, the protein was labeled with IRDye800 for 37 98386650.2near infrared detection in plasma and organs. Despite its relatively large molecular weight, the protein had a biexponential half-life with almost 95% of the protein being cleared within the first few minutes, and the remaining fraction circulating with a 97 min half-life (FIG.10A), with the liver being the main accumulation organ as demonstrated in FIG.10B. The inhibitor appeared to localize in the parenchymal regions. Although the reason for the rapid elimination remains unclear, it is not due to binding and elimination of C3 split products, as evidenced by the fact that the half-life was not prolonged in C3 knockout mice.

[0129] Distribution of IRDye800-labeled proteins was determined by injecting mice intravenously with 3 mg / kg. Mice were injected with small SPIO NWs alone or with 3mg / kg CR2-Crry, and nanoparticles were recovered from plasma and analyzed for C3 deposition with dot blot. The same amount of Fe was dotted on the membrane at each time point hence, C3 is normalized to the number of nanoparticles. Quantification of dot blot illustrates that CR2-Crry blocks C3 deposition at all time points (n = 3 mice at 5 min, n = 2 mice at other time points; non- paired t-test *p = 0.05, repeated twice). Blood was collected via periorbital plexus using potassium EDTA as an anticoagulant in glass hematocrit capillaries (ThermoFisher). Blood was separated into plasma and fractions of blood cell by centrifugation at 500g for 5 min. For organ imaging, animals were sacrificed at 48h post-injection. Organs were placed in wells of a 6-well or 12-well cell culture plate and imaged with Li-COR Odyssey at 800 nm wavelength. The plasma of animals injected with IRDye800 labeled proteins was dotted on a nitrocellulose membrane (1:100 and 1:1000 dilution), the dots were scanned at 800 nm with Li-COR Odyssey, and half-life was calculated by fitting in mono- or bi-exponential decay curve in Prism. FIGS. 10A and 10B illustrate exemplary observations of this study. The plasma profile of CR2-Crry labeled with IRDye800 illustrated rapid elimination with a bi-exponential half-life (n = 2 mice per group, repeated twice). As represented in the image in FIG.10B, liver is the main clearance organ for IRDye800-CR2-Crry (n = 2 mice per group, representative near-infrared fluorescence images of organs are illustrated).

[0130] For activation of complement in vivo, particles from plasma of mice injected with 10 mg / kg SPIO NWs with or without 3 mg / kg CR2-Crry were recovered by ultracentrifuge and the concentration was measured with ferrozine assay. Iron concentration was brought to 0.02 mg / mL for all time points before dotting on the membrane (so that C3 levels are normalized to the particle amount). Standard dilutions of human iC3b were dotted on the same membrane. The 38 98386650.2protein was detected with primary goat anti-human C3 and corresponding IRDye800-labeled secondary antibodies for 1 hour. The membrane was washed with 0.1% Tween-20 / PBS and scanned using an Odyssey infrared imager (Li-COR Biosciences, Lincoln, NE, US), and the integrated intensities of C3 dots were determined from 8-bit grayscale images using Fiji. As demonstrated in FIGS.11A and 11B, in the absence of inhibitor, nanoparticle-bound C3 demonstrated a spike, and rapid elimination from the nanoparticle surface and from plasma.

[0131] FIG.12 illustrates some exemplary results of plasma C5a in SPIO NW injected mice. Co-injection with CR2-Crry prevents the spike in C5a. Dotted line indicates baseline levels of C5a in plasma (n = 3 mice at 5 min, n = 2 mice at other time points; non-paired t-test ****p <0.0001, repeated twice).

[0132] It was observed that, in the absence of the inhibitor, nanoparticle-bound C3 and soluble C5a demonstrated a spike, and rapid elimination from the nanoparticle surface and from plasma (FIGS.11A, 11B, and 12) for large NWs). This observation suggests that complement activation in this accepted animal model is transient and that a complement inhibitory effect would be critical within minutes post-injection. It was further observed that mice co-injected with CR2- Crry had minimal immunodetectable C3 on NWs during 60 min post-injection, and plasma C5a was at the baseline level (See FIGS.11A, 11B, and 12 for large NWs).

[0133] Previous data in C3 knockout mice demonstrate that SPIO NWs are predominantly taken up by blood granulocytes, monocytes, macrophage, or any other immune cell via complement. BALB / c mice were injected with 10 mg Fe / kg of small or large SPIO NWs, isolated magnetically labeled leukocytes from blood 30 min and 60 min post-injection, and analyzed the number and type of the cells by flow cytometry.

[0134] EDTA-anticoagulated whole blood was obtained from mice injected with SPIO NWs and the inhibitors. Blood (100µL) was centrifuged at 500g with a tabletop centrifuge for 5 min to remove plasma. Blood cells were diluted in 1% w / v bovine serum albumin (BSA) in PBS and passed through MS MACS column attached to OctoMACS® separator (Miltenyi Biotec). Column was washed 3 times with 1 mL 1% w / v BSA / PBS and the cells were eluted. Cells were centrifuged at 500 g for 4 min, resuspended in 500 µl 1% w / v BSA / PBS and analyzed with Guava EasyCyte HT flow cytometer (Luminex) using constant (30 s) acquisition time for all samples. FSC / SSC dot plot was used to exclude debris and to analyze leukocyte populations 39 98386650.2(granulocytes, lymphocytes and monocytes). The data were analyzed using FlowJo software V10.

[0135] FIGS.13A-B illustrate uptake by leukocytes as measured with flow cytometry. Cells were recovered from blood at 30 min or 60 min post-injection with a magnetic column and analyzed by flow cytometry: L, lymphocytes; M, monocytes; G, granulocytes. There was a significant number of magnetically labeled lymphocytes, granulocytes, and monocytes in the blood of mice injected with NWs only, at both 30 min and 1h (FIGS.13A-13B). The number of magnetically labeled cells in mice injected with large SPIO NWs was higher than that in mice injected with small SPIO NWs (FIGS.13A-13B), likely due to the former’s superior magnetic properties. In mice co-injected with 3mg / kg CR2-Crry, there was a visible inhibition of granulocyte and monocyte uptake for both types of NWs (FIGS.13A-13B).

[0136] Mouse C5a levels in plasma recovered from mice injected with 10 mg / kg SPIO NWs with or without 3 mg / kg CR2-Crry were measured according to manufacturers’ instructions using mouse C5a DuoSet ELISA kit (Cat#DY2150) from R&D Systems (Minneapolis, MN, USA).

[0137] It was observed in these experiments and supported by FIGS.10-13 that despite the agents’ short half-life, co-injection of targeted inhibitor with nanoparticles prevents / eliminates complement activation, anaphylatoxin release, and blood leukocyte uptake. Example 3

[0138] In another exemplary method, distribution of IRDye800-labeled CR2-CR1 was determined by injecting rats intravenously with 0.78 mg / kg. Blood was collected via the tail vein using potassium EDTA as an anticoagulant in glass hematocrit capillaries (ThermoFisher). Blood was separated into plasma and fractions of blood cells by centrifugation at 500g for 5 min. For organ imaging, animals were sacrificed at 48 h post-injection. Organs were placed in wells of a 6-well or 12-well cell culture plate and imaged with Li-COR Odyssey at 800 nm wavelength. The plasma of animals injected with IRDye800 labeled proteins was dotted on a nitrocellulose membrane (1:100 and 1:1000 dilution), the dots were scanned at 800 nm with Li-COR Odyssey, and half-life was calculated by fitting in mono- or bi-exponential decay curve in Prism. As illustrated in FIG.14A and FIG.14B, human targeted inhibitor IRDye800-CR2-CR1 has fast bi- exponential elimination (n = 2 per group, repeated twice); the liver is the main clearance organ 40 98386650.2for IRDye800-CR2-CR1 (n = 2 per group, 48 h post-injection, representative near-infrared images).

[0139] In another method, for SPIO NW complement activation in vivo, rats were injected with 6.6 mg / kg. Particles from plasma were recovered by ultracentrifuge, and the concentration was measured with ferrozine assay as previously disclosed. Iron concentration was brought to 0.02 mg / mL for all time points before dotting on the membrane (so that C3 levels are normalized to the particle amount). Standard dilutions of human iC3b were dotted on the same membrane. The protein was detected with primary goat anti-human C3 and corresponding IRDye800-labeled secondary antibodies for 1h. The membrane was washed with 0.1%Tween-20 / PBS and scanned using an Odyssey infrared imager (Li-COR Biosciences, Lincoln, NE, US), and the integrated intensities of C3 dots were determined from 8-bit grayscale images using Fiji. Rats were injected with large SPIO NWs, alone or with 0.78 mg / kg CR2-CR1. The inhibitor blocks C3 deposition at all time points. The amount of C3 was normalized to the amount of Fe recovered from plasma (n = 2 per group, an average of 3 technical replicates per time point, paired t-test *p = 0.0134. The results are illustrated in representative FIG.15A and FIG.15B.

[0140] In another method, EDTA-anticoagulated whole blood was obtained from rats injected with 6.6 mg / kg SPIO NWs and 0.78 mg / kg CR2-CR1. Blood (100 µL) was centrifuged at 500g with a tabletop centrifuge for 5 min to remove plasma. Blood cells were diluted in 1% w / v bovine serum albumin (BSA) in PBS and passed through MS MACS column attached to OctoMACS® separator (Miltenyi Biotec). Column was washed 3 times with 1 mL 1% w / v BSA / PBS and the cells were eluted. Cells were centrifuged at 500g for 4 min, resuspended in 500 µl 1% w / v BSA / PBS and analyzed with Guava EasyCyte HT flow cytometer (Luminex) using constant (30 s) acquisition time for all samples. FSC / SSC dot plot was used to exclude debris and to analyze leukocyte populations (granulocytes, lymphocytes, and monocytes). The data were analyzed using FlowJo software V10 and are illustrated in FIGS.16A-16B. Uptake of small and large SPIO NWs by leukocytes in vivo was determined by recovering cells at 30 min or 60 min post-injection with a magnetic column (L represents lymphocytes; M represents monocytes; G represents granulocytes).

[0141] In another method, to measure the duration of the residual complement inhibitory activity in plasma at different times post-injection of a high dose of CR2-CR1 (0.83 mg / kg, corresponding to approximately 0.23 µM initial concentration), lepirudin anticoagulated plasma 41 98386650.2was collected, and C3 opsonization of zymosan, a powerful activator of the alternative and lectin pathways, was measured as described in FIG.2. FIG.17 illustrates that there is residual complement activity (zymosan opsonization) in lepirudin plasma after injection of rats with 0.83 mg / kg CR2-CR1. As illustrated in FIG.17, complement activity is restored within about 24 h (n=3).

[0142] Sprague Dawley rats (male or female, weight 250–330g) were restrained in a cone- shape plastic bag and injected i.v with saline, 6.6 mg / kg SPIO NWs or 6.6 mg / kg SPIO NWs with 0.78 mg / kg CR2-CR1. After 5 min, each rat was released into an empty cage with fresh beddings for tracking the exploration activity for 10 min. The cages were placed in dim light, a camera was set above the cage to record the movement, and the recordings were consistently made on the same time of day. Videos were analyzed. The center point of the animal was detected to generate individual tracks. Activity was automatically assessed based on the amount of temporal change in the gray scale value of each pixel in the arena. This allowed for measuring relative activity (activity %) not restricted to locomotion. FIGS.18A-C demonstrate tracks of non-anesthetized male rats after introduction in a new cage over 10 min time. After injection of NWs, rats stopped exploring the cage because they were lethargic, whereas rats co-injected with CR2-CR1 illustrated mobility similar to control (saline) group.

[0143] FIG.19 demonstrates statistical comparison of mean activity over 10 min (n = 3-4 males and 3-4 females per group; two-way ANOVA with Tukey’s multiple comparisons; ****p < 0.0001, ***p = 0.0003, **p = 0.004, *p = 0.034).

[0144] FIGS.14-19 illustrate that despite its short half-life, co-injection of targeted inhibitor with nanoparticles in rats prevents, ameliorates, or eliminates complement activation, anaphylatoxin release, blood leukocyte uptake, and acute infusion reaction caused by nanoparticles.

[0145] In another exemplary method, EDTA-anticoagulated whole blood was obtained from rats injected with 6.6 mg / kg SPIO NWs and 0.14 mg / kg; 1.16 nmole / kg) of CR2-CR1 or low dose CR1 (0.083mg / kg; 1.16nmole / kg). Blood (100 µL) was centrifuged at 500g with a tabletop centrifuge for 5 min to remove plasma. Blood cells were diluted in 1% w / v bovine serum albumin (BSA) in PBS and passed through MS MACS column attached to OctoMACS® separator (Miltenyi Biotec). Column was washed 3 times with 1 mL 1% w / v BSA / PBS and the cells were eluted. Cells were centrifuged at 500g for 4 min, resuspended in 500 µl 1% w / v 42 98386650.2BSA / PBS and analyzed with Guava EasyCyte HT flow cytometer (Luminex) using constant (30 s) acquisition time for all samples. FSC / SSC dot plot was used to exclude debris and to analyze leukocyte populations (granulocytes, lymphocytes, and monocytes). The data were analyzed using FlowJo software V10. Comparison of inhibition of uptake of large SPIO NWs by rat blood leukocytes after co-injection with low dose CR2-CR1 (0.14 mg / kg; 1.16 nmole / kg) or low dose CR1 (0.083 mg / kg; 1.16 nmole / kg). This dose corresponds to approximately 35 nM concentration of the inhibitors in plasma at time 0. The experiment was repeated 2 times. FIGS. 20A-C illustrate that targeted inhibitors are more potent than non-targeted inhibitors at blocking the leukocyte uptake of nanoparticles in vivo. Example 4

[0146] In another exemplary method, mRNA lipid nanoparticles were prepared using eGFP mRMA, and Onpattro® Mix: DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG 2000. The particles were labeled with 0.1% mole of DiD (Biotium). The particles were added to lepirudin human blood at 5 µg / ml mRNA for 1h at 37ºC. Erythrocytes were lysed with BD Biosciences RBC lysis buffer, and leukocytes were analyzed for fluorescence with Guava EasyCyte HT flow cytometer (Luminex). FSC / SSC dot plot was used to exclude debris and to analyze leukocyte populations (granulocytes, lymphocytes, and monocytes). The data were analyzed using FlowJo software V10. Inhibition of immune uptake of mRNA lipid nanoparticles by CR2-CR1. Particles were incubated in whole human blood with (FIG.21B) or without (FIG.21A) the inhibitor CR2- CR1 (10 nM). FIGS.21A and 21B clearly demonstrate utility of complement inhibitors in blocking the immune recognition of nanoparticles in mammals. The data demonstrates utility of complement inhibitors in blocking the immune recognition of nanoparticles in humans.

[0147] For the inhibition curve, 2 µL of nanoparticles after incubation with serum with or without different concentrations of iptacopan and danicopan (Cayman Chemicals) were obtained and washing was applied in replicates on a 0.45 µm pore nitrocellulose membrane. The complement was detected with primary goat anti-human C3 and anti-goat IRDye800-labeled secondary antibodies for 1h. The membrane was washed with 0.1% Tween-20 / PBS and scanned using an Odyssey infrared imager (Li-COR Biosciences, Lincoln, NE, US), and the integrated intensities of C3 dots were determined from 8-bit grayscale images using Fiji. The normalized data (percent of “no inhibitor” value was plotted with Prism software v.9.0 and fitted into the normalized inhibition curve (variable slope) to calculate IC50. 43 98386650.2

[0148] FIGS.22A-C illustrate comparisons of inhibition of complement activation on superparamagnetic iron oxide nanoworms using targeted fusion protein CR2-CR1 (FIG.22A), small molecule Factor B inhibitor iptacopan (FIG.22B) and small molecule Factor D inhibitor danicopan (FIG.22C). It was observed from these results that targeted inhibitor CR2-CR1 as disclosed herein is at least about 50- to about 200 times more potent than small molecules commonly used in the art to achieve acute complement inhibition.

[0149] FIGS.23A-C illustrate a comparison of inhibition of complement activation on PEGylated liposomal doxorubicin using targeted fusion protein CR2-CR1 (FIG.23A), small molecule Factor B inhibitor iptacopan (FIG.23B) and small molecule Factor D inhibitor danicopan (FIG.23C). Surprisingly, as observed from these experiments, targeted inhibitor CR2-CR1 is highly potent and predictably potent, but small molecule inhibitors in comparison failed to demonstrate any effect and were unreliable even at high concentrations.

[0150] FIGS.24A-C illustrate a comparison of inhibition of complement activation on liposomal irinotecan (Onivyde®) using targeted fusion protein CR2-CR1 (FIG.24A), small molecule Factor B inhibitor iptacopan (FIG.24B) and small molecule Factor D inhibitor danicopan (FIG.24C). Surprisingly, as observed from the graphs, CR2-CR1 is highly potent, whereas the small molecule inhibitors demonstrated poor efficacy in this test.

[0151] FIGS.25A-I illustrate comparison of inhibition of complement activation by mRNA lipid nanoparticles using targeted fusion protein CR2-CR1 (FIGS.25A, 25D, and 26E), small molecule Factor B inhibitor iptacopan (FIGS.25B, 25E, and 26H) and small molecule Factor D inhibitor danicopan (FIGS.25C, 25F, and 26G). Surprisingly, as seen from the graphs, CR2- CR1 is highly potent in all 3 donors, whereas the small molecule inhibitors demonstrated efficacy in 2 / 3 donors. Example 5

[0152] In another example, SPIO NWs (10 mg / ml sterile solution) was mixed with iptacopan Particles were administered to isoflurane anesthetized Beagle female dogs via at a dose to result in a dose of 10mg / kg SPIO NWs and 0.5 mg / kg iptacopan as a bolus of ~10 mL via the cephalic vein. An over the needle catheter will be placed into the cephalic vein for administration of the test material. The dogs were monitored for the following (for 1h): pulse, blood pressure, heart rate, body temperature, and other signs of infusion reactions. Beagle dogs were anesthetized and infused with SPIO NWs (bolus injection, 7 ml volume) with or without iptacopan. As illustrated 44 98386650.2in FIG.26, dogs experienced a drop in blood pressure that was completely reversed by iptacopan. The results are proof of concept that complement inhibition can prevent infusion reactions in response to a bolus injection of nanomedicines in dogs and in support of other mammals. Example 6

[0153] DMEM was prepared with L-Glutamine, glucose, and sodium pyruvate and either 10% FBS with 1% pen strep or 1% FBS, 1% pen strep. A 4M valproic acid stock solution was prepared.

[0154] HEK293T cells were grown in 10% FBS medium (or 1% FBS as observed herein) and plated in T-75 flasks until they reached 80% confluency. 200 µg of plasmid DNA and 60 µL of 1 mg / mL PEI were mixed and incubated for 15 minutes. The DNA / PEI complex was added to the HEK293T cells. After 16 hours of incubation, the media was replaced with DMEM 1% FBS and valproic acid and the cells were placed in an incubator at 32°C for 96 hours. The media was collected and spun down to separate from the cells. After the media and cells were separated, the media was filtered through a 0.2 µm filter. The filtered media was concentrated through 30k 15 mL Pall concentrators at 1000g for 10 minutes until only 1-2 mL remained. The remaining media was frozen until protein purification.

[0155] 12 mL of His 60 Ni superflow resin (Ni resin) was centrifuged at 2000× g for 2 minutes. The supernatant was discarded and 30 mL of sterile water was added. The Ni resin was well mixed and centrifuged at 2000× g for 2 minutes. The supernatant was discarded and 30 mL of sterile PBS was added. The Ni resin was well mixed and centrifuged at 2000× g for 2 minutes. The supernatant was discarded and 30 mL of equilibration buffer was added (635665 Takara). The Ni resin was poured into a column. The prepared medium was passed through the column three times. After three times, the column was washed using washing buffer (35 mL of elution buffer (635665 Takara) and Equilibration buffer in a ratio of 1 to 9). 35 mL of elution buffer (635665 Takara) was directly added and the eluent was collected. The eluent was concentrated using a Pierce protein concentrator (88536 Thermo Scientific) with PBS. The concentrated eluent was centrifuged at 4500g for 30 minutes three times. Lastly, the concentration of protein was measured. Surprisingly, as observed herein protein expression of fusion polypeptides was dramatically increased in 1.0% FBS medium in comparison to 10.0% FBS containing media. 45 98386650.2************************************* The foregoing discussion of the disclosure has been presented for purposes of illustration and description. The foregoing is not intended to limit the disclosure to the form or forms disclosed herein. Although the description of the disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the disclosure, e.g., as can be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and / or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and / or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. 46 98386650.2

Claims

WHAT IS CLAIMED:

1. A method for reducing complement activation in response to administration of a foreign substrate for enhanced therapeutic response in a subject, comprising providing at least one of: a fusion polypeptide comprising a) a component comprising a mammalian complement receptor 2 (CR2) or fragment thereof; and b) a component comprising regulatory consensus repeats of a mammalian complement receptor 1 (CR1), Decay Accelerating Factor (DAF), Factor H (FH); a CR1, Decay Accelerating Factor (DAF), Factor H (FH) molecule or biologically active fragment thereof; and a CR2 molecule or biologically active fragment thereof; at least one of before, during or after administration of the foreign substrate to the subject reducing complement activation and improving therapeutic response in the subject compared to a subject not receiving the fusion polypeptide; wherein the foreign substrate comprises at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof.

2. The method according to claim 1, wherein the method reduces or eliminates the need for one or more inhibitory agent to reduce allergic reactions to the at least one nanoparticle- containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof.

3. The method according to claim 2, wherein the one more inhibitory agent comprises at least one of steroid and anti-histamine or another allergic reaction inhibitor.

4. The method according to any one of claims 1-3, wherein the fusion polypeptide has a short-acting or transient inhibition or suppression of complement in the subject.

5. The method according to any one of claims 1-4, wherein the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof comprises at least one of an iron-oxide containing nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof.

6. The method according to any one of claims 1-4, wherein the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof comprises at least one of paclitaxel, patisiran, irinotecan liposome injection, mRNA lipid nanoparticles, or other nanomedicine with a short-term circulation in a subject. 47 98386650.

27. The method according to any one of claims 1-6, wherein the fusion polypeptide further comprises a modification to increase circulating half-life of the fusion polypeptide; optionally, PEGylation, albumin binding domains, Fc conjugation or other modification to increase circulating half-life of the fusion polypeptide or fragment thereof.

8. The method according to any one of claims 1-7, wherein the presence of the fusion polypeptide or molecule reduces the dose of the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof needed to treat the subject by at least one of, reducing complement interference; and reducing granulocyte, monocyte, macrophage, or any other immune cell uptake of the at least one nanoparticle- containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof.

9. The method according to any one of claims 1-8, wherein providing the fusion polypeptide or molecule comprises providing the fusion polypeptide or molecule systemically or subcutaneously to the subject; optionally, in combination with the at least one nanoparticle- containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof.

10. The method according to claim 1, wherein the fusion polypeptide or molecule has a short-acting or transient inhibition or suppression of complement in the subject; optionally 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, 1.5 hours or less, or 1 hour or less, or 30 minutes or less.

11. The method according to claim 1, wherein the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof comprises at least one of an iron-oxide containing at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof.

12. The method according to claim 1, wherein the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof comprises at least one of paclitaxel, patisiran, irinotecan liposome, mRNA lipid nanoparticles, or other nanomedicine with a short-term circulation in a subject.

13. The method according to claim 1, wherein the fusion polypeptide further comprises a modification to increase circulating half-life of the fusion polypeptide; optionally, 48 98386650.2PEGylation, albumin binding domains, Fc conjugation or other modification to increase circulating half-life of the fusion polypeptide.

14. The method according to claim 1, wherein the presence of the fusion polypeptide or molecule reduces the dose of at least one nanoparticle-containing therapeutic, therapeutic- containing nanoparticle, vaccine, or a combination thereof needed to treat the subject by at least one of reducing complement interference; and reducing granulocyte and monocyte uptake of the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, vaccine, or a combination thereof.

15. The method according to any one of claim 1, wherein providing the fusion polypeptide or molecule comprises providing the fusion polypeptide systemically to the subject; optionally, with the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof.

16. A combination composition comprising, a construct comprising at least one of: a fusion polypeptide or isolated fusion polypeptide thereof comprising a) a first component comprising a mammalian complement receptor 2 (CR2) or fragment thereof; and b) a second component comprising regulatory consensus repeats of a mammalian complement receptor 1 (CR1), Decay Accelerating Factor (DAF), Factor H (FH); a CR1, Decay Accelerating Factor (DAF), Factor H (FH) molecule or biologically active fragment thereof; a CR2 molecule or biologically active fragment thereof, wherein the fusion polypeptide, isolated polypeptide, the CR1, DAF, FH molecule or the CR2 molecule or biologically active fragment thereof reduces, or blocks complement activation; and at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof.

17. The composition according to claim 16, wherein the first component does not bind to native or naturally-occurring serum C3.

18. The composition according to claim 16 or 17, wherein the first component and second component are fused to form a fusion polypeptide.

19. The composition according to any one of claims 16-18, wherein the fusion polypeptide or isolated fusion polypeptide or the CR1, DAF, or FH molecule; or the CR2 molecule or biologically active fragment thereof further comprises a modification to increase 49 98386650.2circulating half-life of the fusion polypeptide; optionally, PEGylation, albumin binding domains, Fc conjugation or other modification to increase circulating half-life of the fusion polypeptide.

20. The composition according to claim 16, wherein the fusion polypeptide or isolated fusion polypeptide or the CR1, DAF, or FH molecule; or the CR2 molecule or biologically active fragment thereof has a short-acting or transient inhibition or suppression of complement in the subject; optionally 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, 1.5 hours or less, 1 hour or less, or 30 minutes or less.

21. The composition according to any one of claims 16-20, wherein the at least one nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof comprises at least one of an iron-oxide containing nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof.

22. The composition according to claim 16, wherein the at least one nanoparticle- containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof comprises at least one of paclitaxel, patisiran, irinotecan liposome, mRNA lipid nanoparticles, or other nanomedicine with a short-term circulation in a subject.

23. The composition according claim 16, wherein the fusion polypeptide or isolated fusion polypeptide or the CR1, DAF, or FH molecule; or the CR2 molecule or biologically active fragment thereof further comprises a modification to increase circulating half-life of the fusion polypeptide; optionally, PEGylation, albumin binding domains, Fc conjugation or other modification to increase circulating half-life of the fusion polypeptide.

24. The composition according to claim 16, wherein the presence of the fusion polypeptide or isolated fusion polypeptide or the CR1, DAF, or FH molecule; or the CR2 molecule or biologically active fragment thereof reduces the dose of the nanoparticle-containing therapeutic, therapeutic-containing nanoparticle, nanoaggregate, vaccine, or a combination thereof needed in the combination composition to treat the subject by at least one of, reducing complement interference; and reducing granulocyte and monocyte uptake of the nanoparticle- containing therapeutic or vaccine composition.

25. The composition according to claim 16, wherein the combination composition is formulated for system administration. 50 98386650.226, The composition according to any one of claims 16-25, further comprising at least one additional agent for reducing allergic reactions to the 27. A pharmaceutical composition comprising the composition according to any one of claims 10-26.

28. The pharmaceutical composition according to claim 27, wherein the composition is formulated for systemic or site-specific administration.

29. A polypeptide construct, comprising an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity to the polypeptide represented by the amino acid sequence of SEQ. ID. NO.28, 29, or 30.

30. The construct according to claim 29, wherein the polypeptide construct is part of a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.

31. A kit comprising a composition according to any one of claims 10-26, or the pharmaceutical composition according to claim 27 or 28; or construct of claim 29 or 30; and at least one container. 51 98386650.2

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