Human complement component 2 binding molecules

Abstract

Provided herein are human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human C2 binding molecules (e.g., nanobodies) having a first, and optionally a second, single monomeric variable antibody domain (SMVAD) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, the SMVAD comprises camelid, human, or humanized framework regions.

Description

[0001] HUMAN COMPLEMENT COMPONENT 2 BINDING MOLECULES

[0002] The present application claims priority to U.S. provisional application serial number 63 / 488,825, filed March 7, 2023, which is herein incorporated by reference in its entirety.

[0003] This invention was made with government support under EY032458 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] Provided herein are human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human C2 binding molecules (e.g., nanobodies) having a first, and optionally a second, single monomeric variable antibody domain (SMVAD) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, the SMVAD comprises camelid, human, or humanized framework regions.

[0006] BACKGROUND OF THE INVENTION

[0007] Complement is a key part of the innate immune system, with a primary role of fighting infections. Complement is activated via the classical, lectin, or alternative pathway and consequently promotes inflammation by releasing anaphylatoxins, enhances phagocytosis by opsonizing target cells, and directly kills pathogens via assembling membrane attack complex (MAC)1. However, excessive complement activation is a causal factor in many diseases, such as myasthenia gravis2, atypical hemolytic uremic syndrome3, paroxysmal nocturnal hemoglobinuria (PNH)4, and autoimmune hemolytic anemia (AIHA)5. Therefore, intensive efforts to develop complement inhibitors as new therapeutics are underway, and inhibitors that target complement components 5 (C5)6, 3 (C3)7,8and Is (Cis)9have been approved for clinical use. Although these inhibitors have been applied successfully to ameliorate different diseases, all of them have drawbacks10 12, and more effective, selective, and economical therapeutics that target different complement components are in clinical demand. Complement component 2 (C2) is an excellent therapeutic target, as it is essential to both the classical and lectin pathways of complement activation, and its concentration in the blood is only 11-35 pg / mL13(versus -75 pg / mL for C514and -1500 pg / mL for C314). Nanobodies, or single-chain domain antibodies, are unique antibodies produced in camelids such as llamas and alpacas15. Nanobodies are emerging as the next generation of antibody-based therapies: one nanobody has already been approved by the FDA, and many others are in clinical trials16. As drug candidates, nanobodies are superior to conventional monoclonal antibodies (mAbs) in many aspects. Their high thermal and chemical stability, strong antigen-binding affinity, ability to access difficult epitopes, capacity for easy and economical manufacturing, and excellent tissue penetration make them ideal antibodies for therapeutic development17. Additionally, nanobodies can be easily manipulated using routine molecular biology techniques to generate multivalent antibodies with augmented avidity without negatively affecting their antigen-binding abilities18 19. Humanization processes to further reduce the minimal immunogenicity of nanobodies have also been well established20.

[0008] Autoimmune hemolytic anemia (AIHA) comprises a group of disorders in which autoantibodies against surface antigens on erythrocytes are produced and cause anemia5. In some patients with AIHA, especially those with cold-reactive autoantibodies, antibodyantigen complex formation on the erythrocyte surface leads to complement activation through the classical pathway to cause complement-mediated extravascular hemolysis and, in some cases, intravascular hemolysis, leading to anemia and other severe complications (e.g., thrombosis)5. Many animal models have been developed to study the pathogenesis of AIHA and test novel therapeutics. In one commonly used mouse model, AIHA is induced by injecting a mouse anti-mouse erythrocyte-specific antibody, mAb 34-3C (isotype Ig2a), which was isolated from autoimmune NZB mice21. Injection of mAb 34-3C induces complement-mediated hemolysis and consequent anemia in wild-type mice, whereas C3 knockout model mice were significantly protected from anemia21, indicating an important role of complement underlying AIHA.

[0009] SUMMARY OF THE INVENTION

[0010] Provided herein are human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human C2 binding molecules (e.g., nanobodies) having a first, and optionally a second, single monomeric variable antibody domain (SMVAD) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, the SMVAD comprises camelid, human, or humanized framework regions. In some embodiments, the human C2 binding molecules are human C2a binding molecules. In some embodiments, provided herein are compositions comprising a human complement component 2 (C2) binding molecule, or one or more nucleic acid molecules encoding the human C2 binding molecule, wherein the human C2 binding molecule comprises a first single monomeric variable antibody domain (SMVAD) that comprises: A) a CDR1 amino acid sequence comprising SEQ ID NO: 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86; or SEQ ID NO:2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86 with one with one or two conservative amino acid changes, B) a CDR2 amino acid sequence comprising SEQ ID NO: 3, 11 , 15, 19, 23, 27, 31 , 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87; or SEQ ID NO:3, 11, 15, 19, 23, 27,

[0011] 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87 with one or two conservative amino acid changes, and C) a CDR3 amino acid sequence comprising SEQ ID NO: 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88; or SEQ ID NO:4, 12,

[0012] 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88 with one with one or two conservative amino acid changes.

[0013] In certain embodiments, provided herein are methods of treating or preventing a complement-related disease or condition comprising: treating a subject with a composition comprising a human complement component 2 (C2) binding molecule, or an expression vector comprising the one or more nucleic acid molecules encoding the C2 binding molecule, as recited above and herein, and wherein the subject has, or is suspected to develop, a complement-related disease or condition. In particular embodiments, the complement-related disease comprises a dysregulated complement activation disease. In other embodiments, the complement-related disease comprises myasthenia gravis or atypical hemolytic uremic syndrome. In further embodiments, the complement-related disease comprises paroxysmal nocturnal hemoglobinuria (PNH) or and autoimmune hemolytic anemia (AIHA).

[0014] In other embodiments, provided herein are methods of detecting human complement component 2 (C2) in a sample comprising: a) contacting a sample with the human C2 binding molecule as described above and herein, wherein the sample is suspected of containing human C2, and wherein the human C2 binding molecule forms a complex with the human C2 if present in the sample; and b) detecting the presence or absence of the complex in the sample. In some embodiments, the sample is from a subject that has, or is suspected to develop, a complement-related disease or condition. In other embodiments, the human C2 binding molecule comprises a detectable label. In additional embodiments, the methods further comprise contacting the sample with a conjugate molecule capable of binding to the human C2 binding molecule, wherein the conjugate molecule comprises a detectable label. In certain embodiments, the first SMVAD further comprises four Framework regions, wherein the four Framework regions are camelid, humanized, or human Framework regions. In additional embodiments, the human C2 binding molecule further comprises a second SMVAD that comprises: D) a CDR1 amino acid sequence comprising SEQ ID NO: 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86; or SEQ ID NO:2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86 with one with one or two conservative amino acid changes, E) a CDR2 amino acid sequence comprising SEQ ID NO: 3, 1 1 , 15, 19, 23, 27, 31 , 35, 39, 43, 47, 51 , 55, 59, 63, 67, 71 , 75, 79, 83, or 87; or SEQ ID NO:3, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87 with one or two conservative amino acid changes, and F) a CDR3 amino acid sequence comprising SEQ ID NO: 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88; or SEQ ID NO:4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88 with one with one or two conservative amino acid changes.

[0015] In additional embodiments, the human C2 binding molecule further comprises a linker which is attached to both the first SMVAD and the second SMVAD. In other embodiments, the one or more nucleic acid molecules comprise: i) a first nucleic acid sequence encoding the first SMVAD, and optionally further encoding a CH2 heavy chain constant region (e.g., which is human or humanized) and / or a CH3 heavy chain constant region (e.g., which is human or humanized) and ii) a second nucleic acid sequence encoding the second SMVAD, and optionally further encoding a CH2 heavy chain constant region (e.g., which is human or humanized) and / or a CH3 heavy chain constant region (e.g., which is human or humanized). In some embodiments, the first SMVAD comprises the amino acid sequence shown in SEQ ID NO:1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, or 85; or SEQ ID NO:1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, or 85 with one, two, three, or four deletions and / or conservative amino acid changes. In other embodiments, the human C2 binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region. In some embodiments, the CH2 and / or CH3 heavy chain constant regions are camelid, humanized, or human. In further embodiments, the human C2 binding molecule comprises at least an antigen binding portion of Clone IB 10 C2 nanobody.

[0016] In some embodiments, the compositions, kits, and systems herein further comprise a physiologically tolerable buffer. In certain embodiments, the compositions herein comprise the one or more nucleic acid molecules (e.g., first and second nucleic acid molecules), and optionally the composition further comprises an expression vector, and wherein the first and / or second nucleic acid sequences are present in the expression vector. In particular embodiments, the composition comprises the human C2 binding molecule. In some embodiments, the CDR1 amino acid sequence comprises SEQ ID NO:2, the CDR2 amino acid sequence comprises SEQ ID NO:3, and the CDR3 amino acid sequence comprises SEQ ID NO:4.

[0017] DESCRIPTION OF THE FIGURES

[0018] Figure 1 : The anti-C2 nanobody Nabl BIO binds to C2, selectively inhibits classical and lectin complement pathway activation, and inhibits classical pathway activation in a dose-dependent manner. (A) SPR was used to determine the binding kinetics for C2 and the immobilized anti-C2 NablBlO. Black lines indicate the raw data, and the kinetics fit a 1 :1 binding model, as shown by colored lines. (B) Normal human serum (NHS) with or without NablB 10 was added to an assay plate pre-coated with activator for 1 hour at 37°C according to the manufacturer’ s instructions (Wieslab). Complement activation was detected using an antibody that recognizes the C5b-9 neoepitope, followed by measuring the absorbance at 405 nm. The conditions without serum and with heat-inactivated NHS (HI-NHS) were used as the background control and negative control, respectively. (C) Antibody-coated sheep erythrocytes (EShA) were incubated with 1% NHS in the presence or absence of NablBlO at 0-20 nM in GVB++for 30 minutes at 37°C. Hemolysis was evaluated by measuring the absorbance at 414 nm, and the IC50 was determined by calculating the concentration of nanobody required for 50% inhibition of hemolysis.

[0019] Figure 2: NablBlO binds to C2a but not to C2b. (A) C2 was incubated for 1 hour at 37 °C in the presence or absence of Cis at 2 pg / mL to generate the C2 cleavage products C2a and C2b. The reaction mixture was then incubated with cobalt resin overnight at 4°C in the presence or absence of NablBlO. Both the cobalt resin and supernatants were assessed using western blotting. The C2 fragments were detected using a goat anti-C2 polyclonal antibody, followed by an HRP-conjugated donkey anti-goat antibody. (B) C2 or C2b at 20 nM was coated onto a high-binding 96-well plate and detected with NablBlO at 0.01-30 nM, followed by an HRP conjugated anti-VHH antibody. Goat anti-C2 polyclonal antibody (pAb) was used as a positive control to detect C2 and C2b on the plate.

[0020] Figure 3: NablBlO does not block C2 cleavage by Cis but prevents C3 convertase C4b2a assembly. (A) 2 pg C2 were incubated with 2 pg / mL Cis in the presence or absence of NablBlO (1:15 antigen: antibody molar ratio) for 1 hour at 37°C. C2 cleavage was detected using a goat anti-C2 polyclonal antibody, followed by an HRP-conjugated donkey anti-goat antibody. (B) The C3 convertase C4b2a was generated on the surfaces of EShA during incubation with 10% C3-depleted serum (C3-dpl) for 10 minutes at 37°C, and followed by adding 2% Guinea pig serum (GPS) plus 10 mM EDTA to induce erythrocyte lysis. NablBlO (100 nM) was added during C3-dpl incubation prevented downstream lysis to a similar extent as 10 mM EDTA, but did not interfere with generated C4b2a function during incubation with 2% GPS. p*** < 0.0005, ns: no significance.

[0021] Figure 4: NablBlO blocks classical complement pathway activation in human and monkey serum but not rat, guinea pig, or mouse serum. (A) EShA were incubated with 1 % normal human, monkey, guinea pig or rat serum in the presence of 0-40 nM NablBlO for 30 minutes at 37°C in GVB++buffer. Hemolysis was evaluated by measuring the absorbance at 414 nm. (B) Due to the weak hemolysis activity in mouse serum, C3 fragment deposition was evaluated by incubating EShA with 5% serum from Factor D KO mice in the presence (dotted line) or absence (black line) of 1 pM NablBlO, followed by labeling with a FITC- conjugated anti-C3 fragment antibody and flow cytometry analysis. EDTA (10 mM) was used as the positive control (gray line).

[0022] Figure 5: NablBlO inhibits complement-mediated mouse erythrocyte lysis in vitro and in vivo. (A) Mouse erythrocytes were coated with anti-mouse erythrocyte antibody (clone 34-3C) at 0-60 pg / mL on ice for 30 minutes. Classical complement pathway-mediated hemolysis in vitro was evaluated in the presence of 10% Factor B-depleted serum with or without 400 nM NablB lO for 30 minutes at 37°C in PBS++buffer. (B) Mice were injected intravenously with 34-3C at 12 pg / mL to sensitize erythrocytes in vivo. Factor B-depleted human serum-mediated hemolysis (in the presence of 0.15 mM Ca2+and 0.5 mM Mg2+) and the inhibitory effect of 400 nM NablBlO on hemolysis were evaluated at 30 minutes postinjection by measuring the OD414 of plasma samples. Normal mouse plasma was used as the negative control, p* < 0.05, ns: no significance.

[0023] Figure 6: A bivalent form of NablBlO with doubled potency in inhibiting the classical complement pathway activation. (A) Design of the monovalent (His-tagged) and bivalent anti-C2 nanobodies. (B) A commercially available MicroVue CH50 Eq EIA kit (Quidel) was used to measure the effectiveness of monovalent and bivalent anti-C2 NablB lO at 2-200nM in terms of inhibiting C5b-9, which is generated during classical pathway activation. The EC 50 was determined by calculating the concentration needed to effectively reduce activation by 50%.

[0024] Figure 7: Injection of human serum induces intravascular hemolysis in mice through complement alternative pathway. (A) Image representation of intravascular hemolysis in mice after injecting 10% normal human serum (NHS) for 30 minutes, or 10% Factor B- depleted serum (FB-dpl) for 1 hour. The normal mouse serum serves as the baseline control. (B) Intravascular hemolysis was evaluated by reading the plasma at OD414.

[0025] Figure 8 shows the amino acid sequence (SEQ ID NO: 1) of Clone 1B10 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:3), and CDR3 (SEQ ID NO:4).

[0026] Figure 9 shows the various parts of an exemplary VHH antibody.

[0027] Figure 10A shows the amino acid sequence (SEQ ID NO:9) of humanized clone HH1 C2 nanobody VHH sequence, including CDR1 (SEQ ID NOTO), CDR2 (SEQ ID NO:11), and CDR3 (SEQ ID NO: 12). Figure 10B shows the amino acid sequence (SEQ ID NO: 13) of humanized clone HH2 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 16). Figure 10C shows the amino acid sequence (SEQ ID NO: 17) of humanized clone HH3 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:18), CDR2 (SEQ ID NO:19), and CDR3 (SEQ ID NO:20). Figure 10D shows the amino acid sequence (SEQ ID NO:21) of humanized clone HH4 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:22), CDR2 (SEQ ID NO:23), and CDR3 (SEQ ID NO:24).

[0028] Figure 11 A shows the amino acid sequence (SEQ ID NO:25) of humanized clone HH5 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:26), CDR2 (SEQ ID NO:27), and CDR3 (SEQ ID NO:28). Figure 1 IB shows the amino acid sequence (SEQ ID NO:29) of humanized clone HH6 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:31), and CDR3 (SEQ ID NO:32). Figure 11C shows the amino acid sequence (SEQ ID NO:33) of humanized clone HH7 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:34), CDR2 (SEQ ID NO:35), and CDR3 (SEQ ID NO:36). Figure HD shows the amino acid sequence (SEQ ID NO:37) of humanized clone HH8 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:38), CDR2 (SEQ ID NO:39), and CDR3 (SEQ ID NO:40).

[0029] Figure 12A shows the amino acid sequence (SEQ ID NO:41) of humanized clone HH9 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:42), CDR2 (SEQ ID NO:43), and CDR3 (SEQ ID NO:44). Figure 12B shows the amino acid sequence (SEQ ID NO:45) of humanized clone HH10 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:46), CDR2 (SEQ ID NO:47), and CDR3 (SEQ ID NO:48). Figure 12C shows the amino acid sequence (SEQ ID NO:49) of humanized clone HH11 C2 nanobody VHH sequence, including CDR1 (SEQ ID NQ:50), CDR2 (SEQ ID NO:51), and CDR3 (SEQ ID NO:52). Figure 12D shows the amino acid sequence (SEQ ID NO:53) of humanized clone HH12 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:55), and CDR3 (SEQ ID NO:56).

[0030] Figure 13A shows the amino acid sequence (SEQ ID NO:57) of humanized clone HH13 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO: 58), CDR2 (SEQ ID NO: 59), and CDR3 (SEQ ID NO: 60). Figure 13B shows the amino acid sequence (SEQ ID NO:61) of humanized clone HH14 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:62), CDR2 (SEQ ID NO:63), and CDR3 (SEQ ID NO:64). Figure 13C shows the amino acid sequence (SEQ ID NO:65) of humanized clone HH15 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:66), CDR2 (SEQ ID NO:67), and CDR3 (SEQ ID NO:68). Figure 13D shows the amino acid sequence (SEQ ID NO:69) of humanized clone HH16 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:70), CDR2 (SEQ ID NO:71), and CDR3 (SEQ ID NO:72).

[0031] Figure 14A shows the amino acid sequence (SEQ ID NO:73) of humanized clone HH17 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:74), CDR2 (SEQ ID NO:75), and CDR3 (SEQ ID NO:76). Figure 14B shows the amino acid sequence (SEQ ID NO:77) of humanized clone HH18 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:78), CDR2 (SEQ ID NO:79), and CDR3 (SEQ ID NO:80). Figure 14C shows the amino acid sequence (SEQ ID NO:81) of humanized clone HH19 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:82), CDR2 (SEQ ID NO:83), and CDR3 (SEQ ID NO:84). Figure 14D shows the amino acid sequence (SEQ ID NO:85) of humanized clone HH20 C2 nanobody VHH sequence, including CDR1 (SEQ ID NO:86), CDR2 (SEQ ID NO:87), and CDR3 (SEQ ID NO:88).

[0032] Figure 15 shows direct functional comparison of TNabC2 with ARGX-117. TNabC2 and ARGX-117 were produced by the same CRO as recombinant proteins using a CHO cell transient expression system. A classical pathway-mediated hemolysis assay was used to compare the potencies of TNabC2 and ARGX-117. These experiments showed that the IC50 and IC95 of ARGX-117 were 30.5nM and 6800nM, while the IC50 and IC95 of TNabC2 were 0.35 nM and 1.6nM, respectively.

[0033] Figure 16 the protein sequences for ARGX-117, produced as a IgGl with TM / YTE mutations as disclosed in US Pat. Pub. 20200239554. The variable region are underlined.

[0034] Figure 17 shows ELISA binding data for the 20 humanized C20 antibodies described in figures 10-14. DEFINITIONS

[0035] To facilitate an understanding of the invention, a number of terms are defined below.

[0036] A “nanobody,” or “single variable domain” (“VHH”) or “single monomeric variable antibody domain” (“SMVAD”) as used herein, refer to the smallest antigen binding fragment originally derived from a naturally occurring heavy chain antibody and is known to the person skilled in the art. Such nanobodies can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Nanobodies may also be synthetically produced, such as by overexpression in bacteria. Single domain antibodies are antibodies whose complementary determining regions (CDRs) are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.

[0037] As used herein, the terms "subject" and "patient" refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.

[0038] As used herein, the term "codon" or "triplet" refers to a group of three adjacent nucleotides which specify one of the naturally occurring amino acids found in polypeptides. The term also includes codons which do not specify any amino acid. It is also noted that, due to the degeneracy of the genetic code, there are many codons that code for the same amino acid. As such, many of the bases of the nucleic acid sequences of the present invention can be changed without changing the actual amino acid sequence that is encoded. The present disclosure is intended to encompass all such nucleic acid sequences.

[0039] As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a polypeptide," "polynucleotide having a nucleotide sequence encoding a polypeptide," and "nucleic acid sequence encoding a peptide" means a nucleic acid sequence comprising the coding region of a particular polypeptide. The coding region may be, for example, present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0040] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" or "isolated nucleic acid sequence encoding an complement component 2 binding molecule" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated (e.g. host cell proteins).

[0041] As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, complement component 2 binding molecules may be purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulins that do not bind to the same antigen. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind the particular antigen results in an increase in the percentage of antigen specific immunoglobulins in the sample. In another example, recombinant antigen-specific polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percentage of recombinant antigen- specific polypeptides is thereby increased in the sample.

[0042] DESCRIPTION OF THE INVENTION

[0043] Provided herein are human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human C2 binding molecules (e.g., nanobodies) having a first, and optionally a second, single monomeric variable antibody domain (SMVAD) (aka a “nanobody”) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, the SMVAD comprises camelid, human, or humanized framework regions.

[0044] In work conducted during development of embodiments of the present disclosure, NablBlO has been developed, which is an anti-C2 nanobody that potently and selectively inhibits both the classical and lectin pathways of complement activation. Mechanistically, NablB 10 binds to the C2a portion of C2 and inhibits the assembly of C3 convertase C4b2a. NablBlO cross-reacts with monkey but not rodent C2, and inhibits classical pathway complement activation-mediated hemolysis. Using a humanized mouse model of autoimmune hemolytic anemia, it was demonstrated that NablB lO efficiently prevented classical pathway complement activation-mediated hemolysis in vivo. These data suggest that this novel the anti-C2 nanobodies herein could be employed as a therapeutic for many complement- mediated diseases such as AIHA, in which pathogenesis is dependent on the classical and / or lectin pathway of complement activation.

[0045] The nanobodies (SMVADs), according to the present disclosure, in certain embodiments, generally comprise a single amino acid chain that can be considered to comprise 4 “framework sequences” or FRs and 2 or 3 “complementary determining regions” or CDRs, preferably in a sequence FRl-CDRl-FR2-CDR2-FR3-(optionally CDR3)-FR4. Non-limiting examples of nanobodies of the disclosure are described in more detail further herein. It should be clear that framework regions of nanobodies may also contribute to the binding of their antigens. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a nanobody are not particularly limited as to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.

[0046] The terms “nanobody” and “SMVAD,” in their broadest sense, are not limited to a specific biological source or to a specific method of preparation. For example, the nanobodies of the disclosure can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain (see, e.g., Sulea, Humanization of Camelid Single Domain Antibodies, Methods Mol Biol. 2022; 2446:299-312 and Vincke et al., General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold, The J. of Bio. Chem. Vol. 284, No. 5, pp. 3273-3284, lanuary 30, 2009; both of which are herein incorporated in their entirities and particularly for methods of humanizing nanobodies); (4) by “camelization” of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “Dab,” as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0047] The small size and unique biophysical properties of nanobodies generally exceed conventional antibody fragments for the recognition of uncommon or hidden epitopes and for binding into cavities or active sites of protein targets. Further, nanobodies herein can be designed as bispecific and bivalent antibodies or attached to reporter molecules. Nanobodies are stable and rigid single domain proteins that can generally be easily be manufactured and survive the gastro-intestinal system.

[0048] The amino acid residues of a nanobody are generally numbered according to the general numbering for VH domains given by Kabat et al., as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J Immunol Methods

[0049] 1999 Dec 10;23 l(l-2):25-38, herein incorporated by reference. According to this numbering, FR1 of a Nanobody comprises the amino acid residues at positions 1-30, CDR1 of a Nanobody comprises the amino acid residues at positions 31-35, FR2 of a Nanobody comprises the amino acids at positions 36-49, CDR2 of a Nanobody comprises the amino acid residues at positions 50-65, FR3 of a Nanobody comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody comprises the amino acid residues at positions 103-1.13. Tt should be noted that it is well known in the art for VH domains and for VHH domains that the total number of amino acid residues in each of the CDR's may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR's, position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to the

[0050] Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the

[0051] Kabat numbering corresponds to the start of FR3 and vice versa, and position. 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.

[0052] Nanobodies have a number of unique structural characteristics and functional properties which make isolated SMVADs, and proteins containing the same, highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, SMVADs, which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain, can function as a single, relatively small, functional antigenbinding structural unit, domain or protein. This distinguishes the nanobodies from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which are composed of a VH domain covalently linked to a VL, domain).

[0053] In certain embodiments, the SMVADs (nanobodies) may be further modified by one or more other amino substitutions while maintaining their activity as C2 binding molecules. In certain embodiments, substitutions are made in the framework regions and not in the CDR domains. For example, amino acid substitutions can be made at one or more positions wherein the substitution is for an amino acid having a similar hydrophilicity. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Thus such conservative substitution can be made in a SMVADs of the embodiments and will likely only have minor effects on their activity. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (0.5); histidine -0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be used as a guide and thus substitution of amino acids whose hydrophilicity values are within 2 are preferred, those that are within 1 are particularly preferred, and those within 0.5 are even more particularly preferred. Thus, any of the SMVADs described herein may be modified by the substitution of an amino acid, for different, but homologous amino acid with a similar hydrophilicity value. Amino acids with hydrophilicities within + / -1.0, or + / -0.5 points are considered homologous. Furthermore, it is envisioned that SMVAD sequences may be modified by amino acid deletions, substitutions, additions or insertions while retaining its binding activity. In certain embodiments, the human complement component 2 binding molecules comprise one or more of the CDRs or variable regions shown SEQ ID NOS: 2-4 and 10-88, with one or more conservative or non-conservative amino acid changes, and nucleic acid sequences encoding SEQ ID NOs:l-4 and 10-88. Changes to the amino acid sequences of the CDRs or variable regions may be generated by changing the nucleic acid sequence encoding the amino acid sequence. A nucleic acid sequence encoding a variant of a given CDR or variable region may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid encoding the CDR or variable region.

[0054] Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

[0055] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting CDR (see, e.g., Vallette et. al., (1989) Nucleic Acids Res. 17: 723-733, hereby incorporated by reference). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

[0056] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., (1985) Gene 34: 315-323, hereby incorporated by reference. The starting material is the plasmid (or other vector) comprising the starting CDR or variant region DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There should be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double- stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

[0057] Alternatively, or additionally, the desired amino acid sequence encoding a CDR variant, or variable region variant, can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically. Conservative modifications in the amino acid sequences of the CDRs or variable region may also be made. Naturally occurring residues are divided into classes based on common side-chain properties:

[0058] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0059] (2) neutral hydrophilic: cys, ser, thr;

[0060] (3) acidic: asp, glu;

[0061] (4) basic: asn, gin, his, lys, arg;

[0062] (5) residues that influence chain orientation: gly, pro; and

[0063] (6) aromatic: trp, tyr, phe.

[0064] Conservative substitutions will entail exchanging a member of one of these classes for another member of the same class in a particular antibody, variable region, or CDR, such as in SEQ ID NOS: 1-4 and 10-88.

[0065] For expression of human C2 binding molecules disclosed herein, the expression vector(s) encoding the SMVADs may be transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

[0066] In certain embodiments, the expression vector used to express the human C2 binding molecules of the present invention are viral vectors, such as retro-viral vectors. Such viral vectors may be employed to generate stably transduced cell lines (e.g. for a continues source of the complement component 2 binding molecules). In some embodiments, the GPEX gene product expression technology (from Catalent, Somerset, NI) is employed to generate complement component 2 binding molecules (and stable cell lines expressing the complement component 2 binding molecules). In particular embodiments, the expression technology described in W00202783 and W00202738 (both of which are herein incorporated by reference in their entireties) is employed. Mammalian host cells for expressing the human C2 binding molecules of the invention include, for example, PER. complement component 2™ cells (Crucell, The Netherlands), Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding the human C2 binding molecules are introduced into mammalian host cells, the antibodies are generally produced by culturing the host cells for a period of time sufficient to allow for expression of the human C2 binding molecules in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Human C2 binding molecules can be recovered from the culture medium using standard protein purification methods.

[0067] In certain embodiments, the human complement component 2 binding molecules of the present invention (e.g., nanobodies or dual nanobodies) are useful for immunoassays which detect or quantify human complement component 2 in a sample (e.g., a purified blood sample from a subject). In some embodiments, an immunoassay for complement component 2 typically comprises incubating a biological sample in the presence of a detectably labeled antibody or antibody fragment of the present invention capable of selectively binding to complement component 2, and detecting the labeled peptide or antibody which is bound in a sample. Various clinical assay procedures are well known in the art.

[0068] The present disclosure provides immunoassay methods for determining the presence, amount or concentration of human complement component 2 in a test sample. Any suitable assay known in the art can be used in such a method. Examples of such assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal- polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), an ARCHITECT assay (ABBOTT), a bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.

[0069] A human complement component 2 binding molecule can be captured on beads or nitrocellulose, or on any other solid support which is capable of immobilizing soluble proteins (e.g., magnetic beads). A human complement component 2 containing sample is then added to the support which is subsequently washed with suitable buffers to remove unbound proteins. A second, detectably labeled, molecule (e.g., antibody or peptide) that can bind to the human complement component 2 binding molecule is added to the solid phase support that can then be washed with the buffer a second time to remove unbound molecules. The amount of bound label on the solid support can then be detected by known methods.

[0070] Detectably labeling the human complement component 2 binding molecules can be accomplished by coupling to an enzyme for use in an enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to generate a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the human complement component 2 binding molecules of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0071] In some embodiments of the present invention, human complement component 2 which is detected by the above assays can be present in a biological sample. Any sample containing human complement component 2 can be used. Preferably, the sample is a biological fluid such as, for example, blood, brain tissue, serum, lymph, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, a tissue extract or homogenate, and the like. However, the invention is not limited to assays using only these samples, as it is possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.

[0072] In situ detection can be accomplished by removing a histological specimen from a patient, and providing the combination of labeled human complement component 2 binding molecules of the present disclosure to such a specimen. The human complement component 2 binding molecule is preferably provided by applying or by overlaying the labeled complement component 2 binding molecule to a biological sample (e.g., brain tissue). Through the use of such a procedure, it is possible to determine not only the presence of complement component 2, but also the distribution of complement component 2 in the examined tissue.

[0073] In certain embodiments, provided here are kits for the detection of complement component 2 that include a human complement component 2 detection molecule. Such kits may include any of the immunodiagnostic reagents described herein and may further include instructions for the use of the immunodiagnostic reagents in immunoassays for determining the presence of human complement component 2 in a test sample. The kits may also include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and / or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.

[0074] The various components of the kit may be provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instrument for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.

[0075] EXAMPLES

[0076] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

[0077] EXAMPLE 1

[0078] A nanobody-based complement inhibitor targeting complement component 2 reduces hemolysis in a humanized mouse model of autoimmune hemolytic anemia

[0079] In this example, we developed a panel of anti-human C2 nanobodies by immunizing an alpaca with purified human C2 protein, and identified a clone that potently inhibited both the classical and lectin pathways of complement activation. We determined that, mechanistically, the nanobody inhibited complement activation by binding to the C2a portion of C2 to prevent the assembly of the C3 convertase C4b2a. For in vivo studies, we developed a new human complement-mediated model of AIHA in mice and demonstrated the potential of the anti-C2 nanobody to protect the erythrocytes from human classical pathway complement activation-mediated hemolysis.

[0080] Materials and Methods:

[0081] Alpaca immunization and PBMC isolation

[0082] An alpaca was immunized via four subcutaneous injections of 150 pg of purified human C2 protein (Complement Tech, TX) administered once every 3 weeks. At 12 weeks post-immunization, the successful antibody response was confirmed by enzyme-linked immunosorbent assay (ELISA). Briefly, a high-affinity binding 96-well plate was coated with human C2 (2 nM), after which alpaca serum was serially diluted, added to the wells, and incubated for 2 hours at room temperature. The wells were washed and the anti-C2 IgG titers were detected using a horseradish peroxidase (HRP)-conjugated anti-llama IgG (Bethyl Laboratories, MA) and quantitated using a plate reader (Molecular Devices, CA).

[0083] Nanobody phage-display library construction and screening

[0084] At 18-week post-immunization, a 300 mL blood sample was collected from the immunized alpaca, and the PBMCs were isolated using Ficoll-Paque Plus (Cytiva, MAj.RNA was isolated from alpaca PBMCs (8xlO8) using a RNeasy Midi kit (Qiagen, Germany). cDNA was produced via reverse transcription using SuperScript II Reverse Transcriptase (Thermo Fisher, MA). Genes encoding the alpaca VHH fragments were amplified using two sets of primers22:

[0085] 5 -GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO:5), 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO:6); and 5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3' (SEQ ID NO:7), 5'-CTAGTGCGGCCGC TGGAGACGGTGACCTGGGT-3' (SEQ ID NO:8).

[0086] The amplification products were cloned into the pMES4 vector at the PstI and EcoR91I sites following digestion with restriction enzymes (New England Biolabs, MA). The ligated constructs were transformed into TGI competent E. coll cells (Agilent Technologies, CA) to generate the nanobody phage display library following an established protocol22,23.

[0087] Panning and screening to identify high-affinity and functionally blocking C2 nanobodies

[0088] The nanobody phage display library was cultured and infected with the M13KO7 helper phage (New England Biolabs, MA) to initiate phage production. Next, 1 x 1012purified displaying phages were panned against 1 , 2, or 5 nM purified human C2 coated on an ELISA plate. The binding phages were eluted with trypsin and transfected into TGI cells for amplification. The TGI cells containing the phagemids were plated onto LB agar plates, and single colonies were picked to determine nanobody expression. The clones that exhibited high C2 binding against 1 nM purified C2 and inhibitory activity against the classical complement pathway were selected for further characterization.

[0089] Expression and purification of the anti-C2 nanobody clone NablBlO and the derived bivalent anti-C2 nanobody

[0090] Twenty high-binding anti-C2 nanobody candidates were selected and sequenced. Among them, clone 1B10 (NablBlO) was selected as the most potent inhibitory nanobody against C2, and the expression construct was transformed into BL21 competent cells (Agilent Technologies, CA) for expression and purification. Briefly, a single BL21 colony that contained NablBlO was picked and grown in LB media overnight at 37°C, then diluted 1:100 into 100 mL LB media and grown until the ODeso reached 0.5. NablBlO expression was induced by incubation with 1 mM IPTG overnight at 37°C. On the second day, the pellet was collected and lysed via freezing and thawing, and the nanobody was purified by affinity chromatography using HisPur Cobalt resins (Thermo Fisher, MA) and dialysis against PBS. A bivalent anti-C2 nanobody with a C-terminal 6xHis tag was also produced by expressing two NablBlO sequences in tandem, interspersed by a flexible Gly-Ser linker. Nanobodies to be used in in vivo studies were treated with high-capacity endotoxin removal column (Thermo Fisher, MA) following manufacturer provided protocols.

[0091] Affinity measurement by surface plasmon resonance (SPR)

[0092] The interaction of C2 with the anti-C2 nanobody clone NablBlO was studied by SPR using a Biacore T200 system (Cytiva, MA). The surface of an S series CM5 sensor chip was activated by NHS / EDC for 420 s, and 25 pg / mL of NablBlO in acetate buffer (pH 4.5) was injected over the activated surface for 24 seconds to a final density of 1000 RU. Then, human C2 (Complement Tech, TX) in a series of concentrations ranging from 2.97 to 248 nM were injected over the C2 surface at a rate of 30 pL / mL for 300 seconds, and the interaction signals were recorded. To regenerate the surface, glycine buffer (pH 1.7) was injected at a rate of 30 pl / min for 30 s. Experimental data were analyzed using BIAevaluation 3.3 software and redrawn using Origin 7.0 (OriginLab, MA).

[0093] Complement pathway inhibition assays To determine which complement pathways are inhibited by NablB 10, an ELISA- based assay (Wieslab AB, Sweden) was used according to the manufacturer’s protocol. Briefly, the assay plate was precoated with activators of each complement pathway. To evaluate complement pathway activation, NHS in the presence or absence of NablB 10 was diluted at 1:100 for the classical and lectin pathways, and 1:18 for the alternative pathway. The diluted sera were added to the corresponding activator wells and incubated for 1 hour at 37 °C. After washing the plate, an alkaline phosphatase-conjugated antibody against the C5b- 9 neoantigen was added to the wells; after a 30-minute incubation, alkaline phosphatase substrate solution was added to the wells. The complement activation levels were quantitated by measuring the absorbance in each well at 450 nm. Heat-inactivated serum was used as the negative control, and normal human serum without NablB 10 was used as the positive control.

[0094] To determine the EC 50 of mono- and bivalent NablB 10 in the classical complement pathway, a different ELISA-based assay, MicroVue CH50 (Quidel, CA), was performed per the manufacturer’s protocol. Briefly, 14% NHS was incubated with activator particles in the presence or absence of 0-400 nM mono- or bivalent NablB 10 for 1 hour at 37 °C. The activated serum was diluted 1:200 and added to an assay plate for C5b-9 detection. The amount of nanobody that effectively reduced C5b-9 generation by 50% was defined as the EC50.

[0095] Complement classical pathway activation-mediated hemolysis and C3 deposition assays

[0096] Sheep erythrocytes (ESh) were pre-coated with rabbit anti-sheep erythrocyte antiserum (MP Biomedicals, OH) to generate antibody-sensitized ESh (EShA). Next, 1 x 107EShA were incubated with NHS (or normal monkey / rat / mouse serum) in the presence or absence of 0-40 nM Nab IB 10 for 30 minutes at 37°C in gelatin veronal buffer with Ca2+and Mg2+(GVB++) buffer. The reaction was stopped with 10 mM EDTA, and the extent of hemolysis was evaluated by reading the absorbance at 414 nm (OD414). The percent hemolysis was calculated using the formula: [(OD414 - background) / (maximum OD414 determined by water lysis - background)] x 100. The IC50 value was determined as the concentration of nanobody required for 50% inhibition of hemolysis. As mouse complement does not lyse EShA, C3 deposition was evaluated after the incubation by staining the erythrocytes with a FITC-conjugated polyclonal goat anti-mouse C3 fragment antibody (MP Biomedical, OH); the reaction was detected using an LSRFortessa Cell Analyzer (BD Biosciences, CA). To generate the C3 convertase C4b2a, 1 x 107EShA were incubated with 10% human C3-depleted serum in the presence or absence of NablB 10 or EDTA for 10 minutes at 37°C in GVB++buffer. The EShA were washed with GVB° (without Ca2+and Mg2+) containing 10 mM EDTA, and C4b2a generation was evaluated by incubating the cells with 2% guinea pig serum supplemented with 10 mM EDTA for 30 minutes at 37°C. To evaluate the effect of NablB 10 on the enzymatic activity of pre-assembled C4b2a, NablB 10 was incubated with C4b2a-coated EShA during incubation with Guinea pig serum.

[0097] Western blotting and ELISA to evaluate C2 cleavage and nanobody binding

[0098] To evaluate the effect of NablB 10 on C2 activation, 2 pg of C2 were incubated with or without 2 pg / mL Cis enzyme (Complement Tech, TX) for 1 hour at 37°C in PBS++(0.5 mM Ca2+and 0.5 mM Mg2+)24in the presence or absence of 6 pg of NablBlO. The reaction was stopped by adding 2 x Laemmli sample buffer (Bio-Rad, CA) and boiling for 10 minutes, and the sample was loaded onto a polyacrylamide gel for electrophoresis (Genscript, NJ). The cleavage products were detected using a polyclonal anti-C2 antibody (Complement Tech, TX), followed by a secondary HRP donkey anti-goat antibody (Jackson ImmunoResearch, PA).

[0099] To determine the C2 activation fragment to which NablBlO binds, C2 was cleaved as described above to generate C2a and C2b. The cleavage products were incubated with NablBlO for 1 hour on ice and then with 1% bovine serum albumin (BSA) pre-blocked HisPur Cobalt Resin overnight at 4°C. The supernatants were collected, and the resin was washed four times with 0.05% Tween-20. Both the supernatants and resin were boiled for 10 minutes prior to western blotting to detect C2a and C2b, as described above.

[0100] An ELISA was performed to evaluate whether anti-C2 NablBlO binds to the smaller fragment C2b. Full-length C2 or C2b fragments (20 nM; Complement Tech, TX) were coated on a 96-well high-binding plate overnight at 4°C. The plate was washed with 0.05% Tween- 20 to remove the coating proteins and blocked with 1% BSA for 1 hour. Next, NablBlO was added to the wells at 0.01-30 nM and incubated for 2 hours at room temperature. Bound nanobodies were then detected using an HRP-conjugated anti-VHH antibody (Jackson ImmunoResearch, PA). To demonstrate successful coating with C2 and C2b, a polyclonal anti-C2 antibody (1 :10,000 dilution; Complement Tech, TX) was used as the positive control.

[0101] Modified in vitro human complement classical pathway-mediated mouse hemolysis assay Mouse erythrocytes were sensitized with the anti-mouse erythrocyte antibody clone 34-3C21at 0-60 |ig / mL for 30 minutes at 4°C, then washed with PBS. The antibody- sensitized mouse erythrocytes were then incubated with 10% Factor B -depleted human serum (Quidel, CA) in 0.15 mM Ca2+and 0.5 mM Mg2+in the presence or absence of 400 nM NablBlO for 30 minutes at 37°C. Hemolysis was determined by determining the OD414 in each well.

[0102] In vivo studies of the C2 nanobody using a modified A1HA model

[0103] To establish the in vivo animal model of human complement classical pathway- mediated AIHA, mAb 34-3C (1 mg / kg) was intravenously injected into WT C57BL / 6 mice (male and female, age 10-12 weeks) through the tail vein to sensitize the mouse erythrocytes; subsequently, 5% Factor B-depleted human serum plus 0.15 mM Ca2+and 0.5 mM Mg2+was administered to induce complement-mediated intravascular hemolysis. To evaluate the treatment effect of NablBlO, 0.48 mg / kg NablBlO was injected after AIHA induction via tail vein injection. The mice were sacrificed 30 minutes post- injection; plasma samples were collected, and the degree of hemolysis was determined by measuring the OD414.

[0104] Results

[0105] Development of anti-C2 nanobodies

[0106] After immunizing an alpaca with purified human C2, we constructed a nanobody phage-display library using RNAs isolated from PBMCs of the animal. After 3 rounds of panning with 5, 2, and 1 nM of C2 in sequence, we identified 20 clones that showed strong binding signals in a C2-specific ELISA. Among the 20 clones, sequencing analyses showed 5 distinct nanobodies that matched the alpaca VHH sequences.

[0107] Screening of the functionally blocking anti-C2 nanobody

[0108] To identify functionally blocking anti-C2 nanobodies, we tested each of the 5 identified nanobodies in a classical complement pathway activation-mediated hemolytic assay. We found that despite the strong C2-binding capacities of all nanobodies, only one, clone NablBlO, markedly protected EShA from complement-mediated lysis, suggesting its potential as a functionally blocking anti-C2 nanobody. Expression, purification, and validation of Nab IB 10 as a functional anti-C2 nanobody

[0109] We then transformed BL21 cells with the NablBlO expression construct and bulk- purified NablBlO by affinity chromatography. The purified NablBlO appeared as a single 15 kDa band following SDS-PAGE (data not shown). We first used SPR to determine the affinity of this clone for C2 by fixing the purified nanobody on a sensor chip and running purified C2 protein in the fluid phase. The binding data revealed that NablBlO and C2 were globally fitted to a 1 : 1 Langmuir binding model. The on-rate of binding (Ka) was 1.306E+5 ± 0.029 (1 / M- s), while the off-rate (Kd) was 3.95 IE-4 ± 0.012 (1 / s), yielding a KD of 3.03 ± 0.055 nM (Figure 1 A). We repeated the classical complement pathway-mediated hemolytic assay using purified NablBlO and demonstrated inhibition of hemolysis in a concentrationdependent manner, with an ICso of ~3 nM (Figure IB).

[0110] NablB lO selectively inhibits the classical and lectin pathways of complement activation without affecting the alternative pathway

[0111] We used a commercially available assay to determine the selectivity of NablBlO as an inhibitor of complement pathway activation. The results showed that NablB lO selectively inhibited the classical and lectin pathways but had no effect on the alternative pathway (Figure 1C). This result indicates that the C2 nanobody specifically suppresses complement activation by impairing the classical and lectin pathways.

[0112] NablB 10 binds to the C2a but not the C2b portion of C2

[0113] C2 is cleaved by activated Cis into C2a and C2b during complement activation24. To determine which C2 fragment is the target of NablBlO, we conducted a pull-down assay by incubating cobalt resin with His-tagged NablBlO and Cls-cleaved C2, then detected C2 fragments in the supernatant and on the resin using a polyclonal anti-C2 Ab. Western blotting revealed that both the whole C2 molecule and C2a were detectable in the NablB 10 pulldown samples, whereas C2b was only detectable in the supernatants (Figure 2A), suggesting that NablBlO binds to C2a but not C2b.

[0114] To further validate this result, we performed an ELISA using purified C2 and C2b fragments and found that while the polyclonal anti-C2 Ab detected both C2 and C2b coated on the plates, as expected, NablBlO only detected intact C2 but not C2b (Figure 2B). Taken together, these studies established that NablBlO binds to the C2a portion of C2.

[0115] NablB lO inhibits C4b2a assembly without affecting C2 cleavage by Cis Assembly of the C3 convertase C4b2a occurs when C4 and C2 are cleaved by the active serine protease Cis after initiation of the classical or lectin pathway25. C4b2a, the C3 convertase, then activates C3 to initiate the downstream complement activation cascade. We found that even a high concentration of NablBlO did not inhibit C2 cleavage by Cis, suggesting that NablBlO does not interfere with C2 activation (Figure 3A). In a classical pathway-mediated hemolytic assay, we found while adding NablBlO before C4b2a assembly nearly abolished complement- mediated hemolysis, adding the same concentration of NablB 10 after C4b2a assembly had no significant effect on hemolysis. These results suggest that NablBlO interrupts C4b2a formation by binding to C2a but does not impair the function of the assembled C4b2a (Figure 3B).

[0116] NablB 10 cross-reacts with monkey but not rodent C2

[0117] Animal models are crucial for the pre-clinical characterization of any therapeutic candidate. If NablBlO inhibits animal complement activation by cross-reacting with C2, it can be evaluated in vivo using established preclinical models. We tested NablBlO in an EShA-based hemolytic assay using rat, guinea pig, or monkey serum as the source of complement. NablBlO inhibited monkey but not rat or guinea pig complement-mediated hemolysis (Figure 4A). As mouse complement hemolytic activity is too weak to lyse EShA26, we used flow cytometry to detect mouse C3 fragment deposition on EShA incubated with sera from Factor D KO mice in the presence or absence of 1 pM NablB 10. Notably, NablB 10 did not inhibit mouse C3b deposition on EShA even at this excessively high concentration (Figure 4B), suggesting a lack of cross-reaction with mouse C2.

[0118] NablBlO inhibits human classical complement pathway-mediated hemolysis of mouse erythrocytes in vitro

[0119] The lack of cross-reactivity of our anti-C2 nanobody with rodent C2 increases the difficulty of preclinical evaluation in a model species other than non-human primates, which are extremely expensive and difficult to obtain. To address this issue, we attempted to use an established human complement-mediated hemolysis model in mice. However, in pilot studies, we found that whereas injection with 1% NHS induced massive intravascular hemolysis in mice as reported before1827, injection of 10% Factor B-depleted human serum did not (Figure 7), suggesting that the alternative pathway of complement activation plays a dominant role in this model. To develop a humanized model of classical complement pathway-mediated AIHA for in vitro studies, we sensitized mouse erythrocytes with the anti- mouse erythrocyte antibody 34-3C (0-60 pg / mL), then incubated the erythrocytes with 10% Factor B-depleted human serum in PBS supplemented with 0.15 mM Ca2+and 0.5 mM Mg2+in the presence or absence of 400 nM Nab IB 10. Measurement of the hemolysis levels showed that, under these conditions, Factor B-depleted human serum induced mouse erythrocyte hemolysis, and NablBlO completely abolished the hemolysis (Figure 5A). These data suggest that this new in vitro model can be translated in vivo and used to test the anti-C2 nanobody.

[0120] NablBlO protects mouse erythrocytes from hemolysis in vivo in a humanized AIHA model Encouraged by the in vitro results, we injected WT mice with mAb 34-3C to sensitize the host erythrocytes, then administered 5% Factor B-depleted human serum to induce human classical complement pathway activation-mediated hemolysis. Half of the mice were treated with 0.48 mg / kg NablBlO or an equal volume of PBS, and hemolysis was assessed by measuring the plasma hemoglobin levels. We found that, as suggested by the in vitro results, Factor B-depleted human serum induced intravascular hemolysis in mAb 34-3C-sensitized mice, and administration of the anti-C2 NablBlO nanobody significantly reduced classical complement pathway-mediated hemolysis (Figure 5B).

[0121] Development of a bivalent anti-C2 nanobody based on NablBlO that shows augmented potency

[0122] One of the advantages of nanobodies is their modality28. Multiple nanobody VHH domains can be produced using conventional molecular biology techniques to generate multivalent nanobodies that usually show augmented affinity and bioactivity due to increase avidity. In addition, conventional mAbs are naturally bivalent. To compare the potency of our anti-C2 nanobody with the anti-C2 mAb already in a clinical trial, we synthesized a gene coding for two copies of the VHH domain of NablBlO in tandem with a flexible Gly-Ser link in between, then expressed and purified the bivalent anti-C2 nanobody (Figure 6A). We then determined the EC50 of both our mono- and bivalent anti-C2 nanobody using the same commercial kit used to evaluate the previously anti-C2 mAb29. We found that the bivalent anti-C2 nanobody has an almost doubled potency in inhibiting complement activation compared with its monovalent form, with an EC50 of 28nM vs 53nM in this assay (Figure 6B).

[0123] As described above, we developed a panel of anti-C2 nanobodies and demonstrated that one clone, NablBlO, bound to C2 at an affinity of 3 nM and efficiently inhibited both the classical and lectin pathways of complement activation without affecting the alternative pathway. This new anti-C2 nanobody was shown to protect erythrocytes from classical complement activation-mediated cell damage, such as hemolysis. Mechanistically, this anti- C2 nanobody directly binds to the C2a fragment of C2 to suppress assembly of the C3 convertase C4b2a, thus blocking C3 activation and the downstream complement activation cascade. We showed that Nab IB 10 also effectively protected erythrocytes from human classical complement pathway-mediated hemolysis in a humanized mouse model of AIHA.

[0124] To be functional, complement must be activated through the classical, lectin, and / or alternative pathway. Activation of either the classical or the lectin pathway results in the cleavage of C4 and C2 and assembly of the C3 convertase, a proteolytic C4b2a complex. C4b2a activates C3 to generate C3b, which opsonizes targets to promote phagocytosis; it also initiates the self- amplifying alternative pathway and triggers the release of the inflammatory anaphylatoxin C3a. In the downstream cascade, C5 is activated, leading to the production of C5a, another anaphylatoxin that enhances inflammation, and the assembly of MACs to directly damage target cells1. Eculizumab and its longer half-life derivative, ravulizumab, are anti-C5 mAbs that inhibit C5 activation6, therefore preventing the assembly of MACs and the release of C5a. As MACs directly damage tissues in many diseases such as PNH, anti-C5 mAbs are clinically effective for treating such diseases30. However, in diseases like PNH, erythrocytes are not only damaged by MAC (intravascular hemolysis) but also removed from circulation by phagocytosis after complement-mediated opsonization (extravascular hemolysis), both of which cause anemia4,31. Because blocking C5 only inhibits MAC formation but does not control opsonization, these anti-C5 mAbs only prevent intravascular but not extravascular hemolysis31. Consequently, many patients still require blood transfusions to treat anemia31. This limitation was addressed by the development and approval of pegcetacoplan, a C3 inhibitor, for the treatment of PNH10. C3 is upstream of C5 in the complement activation cascade, and thus pegcetacoplan-mediated C3 inhibition blocks both intra- and extravascular hemolysis in PNH patients10. This therapeutic has shown superior treatment effects when compared with anti-C5 mAbs, increasing the proportion of transfusion-independent patients from 15% (with eculizumab) to 85%10.

[0125] However, targeting C3 presents challenges. In the blood, C3 is the most abundant complement protein, present at concentrations of 1000-1500 ug / mL14. Consequently, large doses of C3 inhibitors are needed to efficiently inhibit the complement activation cascade, e.g., in PNH treatment, twice-weekly infusions of more than 1 g of pegcetacoplan per dose are required to achieve effective treatment10. Therefore, it would be substantially better to achieve the same effects, namely inhibiting both C3b / iC3b-mediated opsonization and M AC- mediated cell damage, by targeting a less-abundant and upstream complement protein.

[0126] The C2 concentration in the blood, at only 11-35 pg / mL3, is approximately 100-fold lower than that of C3, making C2 a much easier target to inhibit. Because C2 is upstream of C3 in the complement activation cascade and is essential for assembly of C4b2a, blocking C2 would inhibit C3a / C5a-promoted inflammation, C3b / iC3b-mediated opsonization, and MAC- mediated tissue damage when complement activation is initiated through either the classical or lectin pathway. As antibody-antigen complexes activate complement through the classical pathway32and autoantibodies or alloantibodies are causal factors in many autoimmune diseases33and transplant rejection34, blocking this pathway could effectively ameliorate such diseases. Sutimlimab, a mAb specific for Cis, which is upstream of C2 in the classical pathway of complement activation, showed good efficacy in treating cold agglutinin disease9, a subtype of AIHA in which autoantibodies against erythrocyte surface antigens activate complement through the classical pathway, causing hemolysis and anemia35.

[0127] In addition, Cis is not required to activate the lectin pathway of complement, and Sutimlimab does not affect this pathway at all. In contrast, C2 blockade could be used to treat diseases in which the lectin pathway is integrally involved. For example, in a preclinical model of ischemia-reperfusion (I / R) injury, the binding of mannose-binding lectin to natural IgMs that recognize neoepitopes exposed on apoptotic or necrotic cells activates complement through the lectin pathway and leads to tissue damage36. Besides, narsoplimab, a mAb inhibits MASP-2 that is required for the lectin pathway, is effective in treating transplant- associated thrombotic microangiopathy (TA-TMA) in a Phase III clinical trial. Unlike sutimlimab, C2-blocking nanobodies could be used to ameliorate these pathological conditions, because C2, but not Cis, is required for lectin pathway complement activation. Besides, since C2 is not required to activate the alternative pathway; thus, inhibiting C2 would not impact complement activation through this pathway, which is important for host defense against infections.

[0128] REFERENCES

[0129] 1. Merle, et al., Complement system part I-molecular mechanisms of activation and regulation. Frontiers in immunology 6, 262 (2015).

[0130] 2 Ttiziin, E. & Christadoss, P. Complement associated pathogenic mechanisms in myasthenia gravis. Autoimmunity reviews 12, 904-911 (2013). 3. Noris, M. & Remuzzi, G. Atypical hemolytic-uremic syndrome. New England Journal of Medicine 361, 1676-1687 (2009).

[0131] 4. Hill, et al., Paroxysmal nocturnal haemoglobinuria. Nature reviews Disease primers 3, 1-14 (2017).

[0132] 5. Berentsen, S. & Barcellini, W. Autoimmune Hemolytic Anemias. N Engl J Med 385, 1407-1419 (2021).

[0133] 6. Thomas, T. C. et al. Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv. Mol Immunol 33, 1389-1401 (1996).

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[0135] 8. Sahu, et al., Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library. J Immunol 157, 884-891 (1996).

[0136] 9. Shi, J. et al. TNT003, an inhibitor of the serine protease Cis, prevents complement activation induced by cold agglutinins. Blood 123, 4015-4022 (2014).

[0137] 10. Hillmen, P. et al. Pegcetacoplan versus Eculizumab in Paroxysmal Nocturnal Hemoglobinuria. N Engl J Med 384, 1028-1037 (2021).

[0138] 11. Risitano et al., Discovering C3 targeting therapies for paroxysmal nocturnal hemoglobinuria: Achievements and pitfalls. Semin Immunol 59, 101618 (2022).

[0139] 12. Ye et al., Complement Cis as a diagnostic marker and therapeutic target: Progress and propective. Frontiers in Immunology 13 (2022).

[0140] 13. Oglesby et al., Radioassays for quantitation of intact complement proteins C2 and B in human serum. Journal of immunological methods 110, 55-62 (1988).

[0141] 14. Kohler, P. F. & Muller-Eberhard, H. J. Immunochemical quantitation of the third, fourth and fifth components of human complement: concentrations in the serum of healthy adults. The Journal of Immunology 99, 1211-1216 (1967).

[0142] 15. De Meyer et al., Nanobody-based products as research and diagnostic tools. Trends Biotechnol 32, 263-270 (2014).

[0143] 16. Yang, E. Y. & Shah, K. Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics. Front Oncol 10, 1182 (2020).

[0144] 17. Hultberg, A. et al. Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PloS one 6, el7665 (2011).

[0145] 18. Ino, et al., Inhibitory effects of FUT-175, a new synthetic protease inhibitor, on intravascular hemolysis by human serum in mice. International journal of immunopharmacology 9, 533-537 (1987). 19. Schoof, et al. An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike. Science 370, 1473-1479 (2020).

[0146] 20. Vincke, C. et al. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem 284, 3273-3284 (2009).

[0147] 21. Shibata, et al. Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause autoimmune hemolytic anemia by two distinct pathogenic mechanisms. Int Immunol 2, 1 133-1 141 (1990).

[0148] 22. Pardon, et al. A general protocol for the generation of Nanobodies for structural biology. Nature protocols 9, 674-693 (2014).

[0149] 23. Chow et al. Immunization of alpacas (Lama pacos) with protein antigens and production of antigen-specific single domain antibodies. JoVE (Journal of Visualized Experiments), e58471 (2019).

[0150] 24. Nagasawa, S. & Stroud, R. M. Cleavage of C2 by Cis into the antigenically distinct fragments C2a and C2b: demonstration of binding of C2b to C4b. Proc Natl Acad Sci U S A 74, 2998-3001 (1977).

[0151] 25. Kerr, M. The human complement system: assembly of the classical pathway C3 convertase. Biochemical Journal 189, 173-181 (1980).

[0152] 26. Brown, G. C. The complementary activity of mouse-serum. The Journal of Immunology 46, 319-323 (1943).

[0153] 27. Lin, K. et al. Development of an anti-human complement C6 monoclonal antibody that inhibits the assembly of membrane attack complexes. Blood advances 4, 2049-2057 (2020).

[0154] 28. Jovcevska, I. & Muyldermans, S. The therapeutic potential of nanobodies. BioDrugs 34, 11-26 (2020).

[0155] 29. Van de Walle, I. et al. ARGX-117, a therapeutic complement inhibiting antibody targeting C2. J Allergy Clin Immunol 147, 1420-1429 el427 (2021).

[0156] 30. Rother et al., Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nature biotechnology 25, 1256-1264 (2007).

[0157] 31. Hill, A. et al. Eculizumab prevents intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low-level extravascular hemolysis occurring through C3 opsonization. Haematologica 95, 567 (2010). 32. Porter, R. & Reid, K. Activation of the complement system by antibody- antigen complexes: the classical pathway. Advances in protein chemistry 33, 1-71 (1979).

[0158] 33. Tobon, G. J. et al. Are autoimmune diseases predictable? Autoimmunity reviews 11, 259-266 (2012).

[0159] 34. Rocha et al., Effector mechanisms in transplant rejection. Immunological reviews 196, 51-64 (2003).

[0160] 35. Berentsen, S. Cold agglutinin disease. Hematology 2014, the American Society of Hematology Education Program Book 2016, 226-2 1 (2016).

[0161] 36. Zhang, M. et al. Activation of the lectin pathway by natural IgM in a model of ischemia / reperfusion injury. J Immunol 177, 4727-4734 (2006).

[0162] 37. Pestronk, A. & Choksi, R. Multifocal motor neuropathy: serum IgM anti-GMl ganglioside antibodies in most patients detected using covalent linkage of GM1 to ELISA plates. Neurology 49, 1289-1292 (1997).

[0163] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, medicine, and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS:We claim:

1. A composition comprising a human complement component 2 (C2) binding molecule, or one or more nucleic acid molecules encoding said human C2 binding molecule, wherein said human C2 binding molecule comprises a first single monomeric variable antibody domain (SMVAD) that comprises:A) a CDR1 amino acid sequence comprising SEQ ID NO: 2, 10, 14, 18, 22, 26,30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86; or SEQ ID NO:2, 10, 14, 18, 22,26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86 with one with one or two conservative amino acid changes,B) a CDR2 amino acid sequence comprising SEQ ID NO: 3, 11, 15, 19, 23, 27,31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87; or SEQ ID NO:3, 11, 15, 19, 23,27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87 with one or two conservative amino acid changes, andC) a CDR3 amino acid sequence comprising SEQ ID NO: 4, 12, 16, 20, 24, 28,32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88; or SEQ ID NO:4, 12, 16, 20, 24,28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88 with one with one or two conservative amino acid changes.

2. The composition of claim 1, wherein said first SMVAD further comprises four Framework regions, wherein said four Framework regions are camelid, humanized, or human Framework regions.

3. The composition of claim 1, wherein said human C2 binding molecule further comprises a second SMVAD that comprises:D) a CDR1 amino acid sequence comprising SEQ ID NO: 2, 10, 14, 18, 22, 26,30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86; or SEQ ID NO:2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86 with one with one or two conservative amino acid changes,E) a CDR2 amino acid sequence comprising SEQ ID NO: 3, 11, 15, 19, 23, 27,31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87; or SEQ ID NO:3, 11, 15, 19, 23,27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87 with one or two conservative amino acid changes, andF) a CDR3 amino acid sequence comprising SEQ ID NO: 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88; or SEQ ID NO:4, 12, 16, 20, 24,28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88 with one with one or two conservative amino acid changes.

4. The composition of claim 3, wherein said human C2 binding molecule further comprises a linker which is attached to both said first SMVAD and said second SMVAD.

5. The composition of claim 3, wherein said one or more nucleic acid molecules comprise: i) a first nucleic acid sequence encoding said first SMVAD, and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region and ii) a second nucleic acid sequence encoding said second SMVAD, and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

6. The composition of claim 1, wherein said first SMVAD comprises the amino acid sequence shown in SEQ ID NO:1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, or 85; or SEQ ID NO:1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, or 85 with one, two, three, or four deletions and / or conservative amino acid changes.

7. The composition of claim 1, wherein said human C2 binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

8. The composition of claim 1, wherein said CH2 and / or CH3 heavy chain constant regions are camelid, humanized, or human.

9. The composition of claim 1 , wherein said human C2 binding molecule comprises at least an antigen binding portion of Clone IB 10 C2 nanobody.

10. The composition of claim 1, further comprising a physiologically tolerable buffer.

11. The composition of claim 1 , wherein said composition comprises said one or more nucleic acid molecules, and optionally the composition further comprises an expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.

12. The composition of claim 1, wherein said composition comprises said human C2 binding molecule.

13. The composition of claim 1, wherein said CDR1 amino acid sequence comprises SEQ ID NO:2, said CDR2 amino acid sequence comprises SEQ ID NO:3, and said CDR3 amino acid sequence comprises SEQ ID NO:4.

14. A method of treating or preventing a complement-related disease or condition comprising: treating a subject with a composition comprising a human complement component 2 (C2) binding molecule, or an expression vector comprising said one or more nucleic acid molecules encoding said C2 binding molecule, as recited in any of Claims 1-13, and wherein said subject has, or is suspected to develop, a complement-related disease or condition.

15. The method of claim 14, wherein said complement-related disease comprises a dysregulated complement activation disease.

16. The method of claim 14, wherein said complement-related disease comprises myasthenia gravis or atypical hemolytic uremic syndrome.

17. The method of claim 14, wherein said complement-related disease comprises paroxysmal nocturnal hemoglobinuria (PNH) or and autoimmune hemolytic anemia (AIHA).

18. The method of claim 14, wherein said first SMVAD further comprises four Framework regions, wherein said four Framework regions are camelid, humanized, or human Framework regions.

19. The method of claim 14, wherein said human C2 binding molecule further comprises a second SMVAD that comprises:D) a CDR1 amino acid sequence comprising SEQ ID NO: 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86; or SEQ ID NO:2, 10, 14, 18, 22,26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, or 86 with one with one or two conservative amino acid changes,E) a CDR2 amino acid sequence comprising SEQ ID NO: 3, 11, 15, 19, 23, 27,31 , 35, 39, 43, 47, 51 , 55, 59, 63, 67, 71 , 75, 79, 83, or 87; or SEQ ID NO:3, 1 1 , 15, 19, 23,27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, or 87 with one or two conservative amino acid changes, andF) a CDR3 amino acid sequence comprising SEQ ID NO: 4, 12, 16, 20, 24, 28,32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88; or SEQ ID NO:4, 12, 16, 20, 24,28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, or 88 with one with one or two conservative amino acid changes.

20. The method of claim 19, wherein said human C2 binding molecule further comprises a linker which is attached to both said first SMVAD and said second SMVAD.

21. The method of claim 19, wherein said one or more nucleic acid molecules comprise: i) a first nucleic acid sequence encoding said first SMVAD, and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region and ii) a second nucleic acid sequence encoding said second SMVAD, and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

22. The method of claim 14, wherein said first SMVAD comprises the amino acid sequence shown in SEQ ID NO: 1 , or SEQ ID NO: 1 with one, two, three, or four deletions and / or conservative amino acid changes.

23. The method of claim 14, wherein said human C2 binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

24. The method of claim 14, wherein said CH2 and / or CH3 heavy chain constant regions are camelid, humanized, or human.

25. The method of claim 14, wherein said human C2 binding molecule comprises at least an antigen binding portion of Clone IB 10 C2 nanobody.

26. The method of claim 14, wherein said composition further comprises a physiologically tolerable buffer.

27. The method of claim 14, wherein said composition comprises said expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.

28. The method of claim 14, wherein said composition comprises said human C2 binding molecule.

29. The method of claim 14, wherein said CDR1 amino acid sequence comprises SEQ ID NO:2, said CDR2 amino acid sequence comprises SEQ ID NO:3, and said CDR3 amino acid sequence comprises SEQ ID NO:4.

30. A method of detecting human complement component 2 (C2) in a sample comprising: a) contacting a sample with the human C2 binding molecule of any of Claims 1 -13, wherein said sample is suspected of containing human C2, and wherein said human C2 binding molecule forms a complex with said human C2 if present in said sample; and b) detecting the presence or absence of said complex in said sample.

31. The method of Claim 30, wherein said sample is from a subject that has, or is suspected to develop, a complement-related disease or condition.

32. The method of Claim 30, wherein said human C2 binding molecule comprises a detectable label.

33. The method of Claim 30, further comprising contacting said sample with a conjugate molecule capable of binding to said human C2 binding molecule, wherein said conjugate molecule comprises a detectable label.

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