HUMAN COMPLEMENT C1r BINDING MOLECULES

Abstract

Provided herein are human complement C1r binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human C1r binding molecules (e.g., nanobodies) having a first, and optionally second, third, and fourth single monomeric variable antibody domains (SMVADs, aka VHHs) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, C1r binding molecules comprise a plurality of SMVADs and an antibody Fc region.

Description

[0001] Attorney Docket Number: CCF-43776.601

[0002] HUMAN COMPLEMENT Clr BINDING MOLECULES

[0003] The present application claims priority to U.S. Provisional application serial number 63 / 736,377, filed December 19, 2024, which is herein incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under EY032458 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] Provided herein are human complement Clr binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein arc human Clr binding molecules (e.g., nanobodies) having a first, and optionally second, third, and fourth single monomeric variable antibody domains (SMVADs, aka VHHs) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, Clr binding molecules comprise a plurality of SMVADs and an antibody Fc region.

[0008] BACKGROUND OF THE INVENTION

[0009] Complement is a crucial component of the innate immune system that requires activation to effectively combat infections and maintain homeostasis. The classical pathway represents one of the three primary complement activation pathways, and is initiated when antibodies bind to their targets, forming antibody-antigen complexes that are recognized by Clq. This leads to the activation of Clr, a serine protease with its only function known to cleave and activate Cis. Activated Cis then cleaves C2 and C4 to assemble the classical pathway C3 convertase, C4b2a, which activates C3 to release C3a, an anaphylatoxin, and produce C3b. C3b and its derivatives, including iC3b and C3d, opsonize target cells to facilitate phagocytosis. C3b also binds to C4b2a to form the classical pathway C5 convertase, C4b2aC3b, which activates C5 to release C5a, another anaphylatoxin, and produce C5b. Upon its production, C5b binds to C6, C7, C8, and multiple units of C9 to assemble membrane attack complexes (MACs) that fomr "pores" on the cell membrane, directly damaging the target cells such as the invading pathogens. Attorney Docket Number: CCF-43776.601

[0010] Despite its beneficial roles as part of the innate immune system in eliminating invading pathogens and maintaining body homeostasis, under certain circumstances, excessive activation of complement occurs and causes disorders especially when autoantibodies or alloantibodies are present and vigorously activate complement through the classical pathway. For example, in patients with autoimmune hemolytic anemia (AIHA), autoantibodies against their red blood cells (RBCs) are produced, and some of these autoantibodies bind to their antigens on the surface of RBCs, initiating classical pathway complement activation. The resulting anaphylatoxins promote unwanted inflammatory responses and phagocytosis, C3b / iC3b-mediated opsonization causes extravascular hemolysis, and MAC-mediated cellular damage results in intravascular hemolysis, leading to anemia, fatigue, thrombosis, and other life-threatening complications. Inhibiting the classical pathway of complement activation would be effective in treating these diseases, and indeed, sutimlimab, an anti-Cls monoclonal antibody (mAb) that blocks Cis function to inhibit the classical pathway, has been successfully developed and approved for treating cold agglutinin disease (CAD), a subtype of AIHA. However, sutimlimab does not seem to be very potent, as it showed no apparent effect in an antibody-mediated kidney allograft rejection clinical trial where 25% of the patients treated with sutimlimab did not meet the primary composite endpoint, and many of the treated patients developed adverse effects in a Phase III clinical trial involving CAD patients. Besides, the recommended effective dose of sutimlimab for treating CAD is 6.5 grams in patients weighing less than 75 kg and 7.5 grams in those weighing 75 kg or more, and it requires weekly infusion for the first two weeks then once every two weeks thereafter. Finally, the cost of treatment was $259,200-$315,000 annually for each patient in the USA as of June 2022. There is thus a clinical demand for a more effective and economic complement inhibitor that selectively inhibits the classical pathway of complement activation as a new drug.

[0011] SUMMARY OF THE INVENTION

[0012] Provided herein are human complement Clr binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human Clr binding molecules (e.g., nanobodies) having a first, and optionally second, third, and fourth single monomeric variable antibody domains (SMVADs, aka VHHs) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, Clr binding molecules comprise a plurality of SMVADs and an antibody Fc region. Attorney Docket Number: CCF-43776.601

[0013] In some embodiments, provided herein are compositions comprising a human complement Clr binding molecule, or one or more nucleic acid molecules encoding the human Clr binding molecule, wherein the human Clr binding molecule comprises a first single monomeric variable antibody domain (SMVAD, aka VHH) that comprises: A) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservative amino acid changes, B) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NO:3 or 7 with one or two conservative amino acid changes, and C) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservative amino acid changes.

[0014] In particular embodiments, provide herein are methods of treating or preventing a complement-related disease or condition comprising: treating a subject with a composition comprising a human complement Clr binding molecule, or an expression vector comprising one or more nucleic acid molecules encoding the Clr binding molecule, as recited above or anywhere herein, and wherein the subject has, or is suspected of having or developing, a complement-related disease or condition. In some embodiments, the complement-related disease comprises a dysregulated complement activation disease. In other embodiments, the complement-related disease comprises: myasthenia gravis, atypical hemolytic uremic syndrome, cold agglutinin disease (CAD), Lupus Nephritis, Neuromyelitis optica spectrum disorder, Guillain-Barre syndrome, Multi-focal motor neuropathy, Myasthenia gravis, Miller Fisher syndrome. Chronic inflammatory demyelinating syndrome, Acute hemolytic transfusion reaction, Cold agglutinin disease, Warm antibody hemolytic anemia, Immune thrombocytopenic purpuraor, and autoimmune hemolytic anemia (AIHA).

[0015] In certain embodiments, the first SMVAD further comprises four Framework regions, wherein the four Framework regions are camelid, humanized, or human Framework regions. In other embodiments, the human Clr binding molecule further comprises a second SMVAD that comprises: D) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservative amino acid changes, E) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NOG or 7 with one or two conservative amino acid changes, and F) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservative amino acid changes. In additional embodiments, the human Clr binding molecule further comprises a linker which is attached to both the first SMVAD and the second SMVAD.

[0016] In other embodiments, the human Clr binding molecule further comprises a third SMVAD that comprises: G) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or Attorney Docket Number: CCF-43776.601

[0017] 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservative amino acid changes, H) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NO:3 or 7 with one or two conservative amino acid changes, and I) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservative amino acid changes.

[0018] In further embodiments, the human Clr binding molecule further comprises a fourth SMVAD that comprises: J) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservative amino acid changes, K) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NO:3 or 7 with one or two conservative amino acid changes, and L) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservative amino acid changes.

[0019] In certain embodiments, the human Clr binding molecule further comprises a linker which is attached to both the third SMVAD and the fourth SMVAD, or attached a constant region of an antibody. In other embodiments, the human Clr binding molecule further comprises a CH2 (or CHI) heavy chain constant region and / or a CH3 heavy chain constant region. In further embodiments, the CH2 and / or CH3 heavy chain constant regions are camelid, humanized, or human. In other embodiments, the human Clr binding molecule further comprises a CH2 (or CHI) heavy chain constant region and / or a CH3 heavy chain constant region. In further embodiments, the human Clr binding molecule further comprises a CH2 (or CHI) heavy chain constant region and / or a CH3 heavy chain constant region, or further comprises at least part of a CH2 and / or CH3 heavy chain constant region.

[0020] In some 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 (or at least part of a CH2 region) and / or a CH3 heavy chain constant region (or at least part of a CH3 region) and ii) a second nucleic acid sequence encoding the second SMVAD, and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

[0021] In additional embodiments, the first SMVAD comprises the amino acid sequence shown in SEQ ID NO: 1, 5, 14, 16, or 18-96; or SEQ ID NO: 1, 5, 14, 16, or 18-96 with one, two, three, or four deletions and / or conservative amino acid changes. In other embodiments, the human complement Clr binding molecule comprises the amino acid sequence in SEQ ID NO: 9, or SEQ ID NO:9 with a different linker sequence, and then zero, one, two, or three conservative amino acid changes in the remainder of the sequence. In certain embodiments, Attorney Docket Number: CCF-43776.601 the human Clr binding molecule comprises at least an antigen binding portion of Clone 3 A3 or 1A12 nanobody.

[0022] In further embodiments, the compositions further comprises a physiologically tolerable buffer. In additional embodiments, the composition comprises the one or more 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 additional embodiments, the composition comprises the human Clr binding molecule. In further 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. In other embodiments, the CDR1 amino acid sequence comprises SEQ ID NO:6, the CDR2 amino acid sequence comprises SEQ ID NO:7, and the CDR3 amino acid sequence comprises SEQ ID NO:8.

[0023] In some embodiments, provided herein are methods of detecting human Clr in a sample comprising: a) contacting a sample with the human Clr binding molecule as described above and herein, wherein the sample is suspected of containing human Clr, and wherein the human Clr binding molecule forms a complex with the human Clr if present in the sample; and b) detecting the presence or absence of the complex in the sample. In particular 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 Clr binding molecule comprises a detectable label. In certain embodiments, the methods further comprise contacting the sample with a conjugate molecule capable of binding to the human Clr binding molecule, wherein the conjugate molecule comprises a detectable label.

[0024] DESCRIPTION OF THE FIGURES

[0025] Figure 1 A shows the VHH sequence (SEQ ID NO: 1) of nanobody 3 A3 (SEQ ID NO:1), which includes: CDR1: GFRFSSYT (SEQ ID NO:2), CDR2: VGSGGRTL (SEQ ID NO:3), and CDR3: NAPSLSS (SEQ ID NO:4). Figure IB shows the VHH sequence (SEQ ID NO:5) of nanobody 1A12, which includes: CDR1: GIISSIYT (SEQ ID NO:6), CDR2: ITSGDRT (SEQ ID NO:7), and CDR3: NSLGSFKYD (SEQ ID NO:8). Figure 1C shows the VHH sequence (SEQ ID NO:14) of humanized nanobody ClrO2HHlT, including CDR1 (SEQ ID NO:6), CDR2 (SEQ ID NO:7), and CDR3: NTLGSFKYD (SEQ ID NO: 15). Figure ID shows the VHH sequence (SEQ ID NO: 16) of humanized nanobody Clr01HH41, including CDR1 GFtFSSYT (SEQ ID NO: 17), CDR2 (SEQ ID NO:3), and CDR3 (SEQ ID NO:4). Attorney Docket Number: CCF-43776.601

[0026] Figure 2 shows anti-Clr nanobody 3A3 (NAb3A3) binds to Clr and inhibits classical pathway activation in a dose-dependent manner. (A) Binding kinetics of Clr to immobilized anti-Clr NAb3A3 via SPR. The binding fits 1:1 binding model and the binding affinity of Clr to NAb3A3 is 1.4nM. Raw data and the kinetic fits are shown in black and red lines, respectively. (B) O-lOOOnM NAb3A3 or NAblAl (binding but non-functional Clr nanobody) were incubated with antibody-coated sheep erythrocytes and 1 % normal human serum in GVB++ for 30 minutes at 37°C. The hemolysis was determined by reading absorbance at 414nM and the IC50 (concentration of nanobody required for 50% inhibition of hemolysis) was calculated.

[0027] Figure 3. Anti-Clr NAb3A3 selectively inhibits classical pathway activation by inhibiting Clr cleavage of Cis pro. (A) Normal human serum (NHS) with or without NAb3A3, heat-inactivated NHS (HI-NHS, negative control) were added into activator precoated plate for 1 hour at 37°C as instructed by the manufacturer (Wieslab). Complement activation was detected using an antibody that recognizes C5b-9 neoepitope, followed by the measurement of absorbance at 405nm (OD405). (B) Ipg Clr was incubated with 500ng Cis pro in the presence or absence of 0-50pM NAb3A3 (or lOpM NAblAl) for 4 hour at 37°C. The Cis pro cleavage was detected with goat anti-Cls polyclonal antibody, followed by an HRP-conjugated donkey anti-goat antibody. Cis active enzyme was used as control.

[0028] Figure 4. Anti-Clr NAb3A3 inhibits human Clr but not rodent Clr activity. (A) Sera from human, rat, and guinea pig were incubated with O-lOOOnM NAb3A3 and antibody- coated sheep erythrocytes in GVB++ for 30 minutes at 37°C. Relative hemolysis was calculated. (B) Due to weak hemolytic activity of mouse serum, the cross-reactivity of NAb3A3 to mouse Clr was done by incubating 5% mouse serum, antibody-coated sheep erythrocytes with lOOOnM NAb3A3 in GVB++ buffer. The C3 deposition was evaluated by staining the cells with FITC anti-mouse C3 polyclonal antibody, followed by flow cytometry analysis.

[0029] Figure 5. Anti-Clr NAb3A3 tetramer enhances the potency in inhibiting classical pathway activity. (A) Inhibition activity of monomer and tetramer NAb3A3 were compared by incubating 0-125nM NAb3A3, NAblAl, or NAb3A3-tetramer with 1% normal human serum and antibody-coated sheep erythrocytes in GVB++ for 30 minutes at 37°C. Relative hemolysis was calculated. (B) lOnM NAb3A3, or 0-10nM NAblAl, or NAb3 A3 -tetramer were incubated with 1 % normal human serum and antibody-coated sheep erythrocytes in GVB++ for 30 minutes at 37°C. Relative hemolysis was determined and IC50 was evaluated by calculating the concentration of nanobody required for 50% inhibition of hemolysis. Attorney Docket Number: CCF-43776.601

[0030] Figure 6. Anti-Clr NAb3A3 preferably binds to Clr active enzyme and does not bind to either Cis pro or active enzyme. (A) lOnM Clr pro or active enzyme were coated onto a high binding affinity plate. After washing, O-lOOOnM anti-Clr NAb3A3 were added and the binding was examined using a HRP conjugated anti-VHH antibody. Goat anti-Clr polyclonal antibody, followed by a donkey anti-goat was used as the positive control (Pos. Ctrl). (B) lOnM Cis pro or active enzyme were coated and the binding was examined as the same procedure as (A), except a goat anti-Cls polyclonal antibody was used as the positive control (Pos. Ctrl).

[0031] Figure 7A shows a schematic of construct 3A3-3A3 h!gG4 (aka TNabClr or Clr3A3NAb). This exemplary construct contains four 3A3 VHH (SMVAD) regions and an Fc constant region from an IgG4 antibody. Figure 7B shows the amino acid sequence of the Clr3A3NAb construct (SEQ ID NO:9) shown in Figure 7A.

[0032] Figure 8A shows a schematic of an exemplary construct that contains two 3A3 VHHs and two 1 A12 VHHs, as well an Fc constant region from an IgG4 antibody. Figure 8B shows a schematic of an exemplary construct that contains two 3A3 VHHs and two 1A12 VHHs (on opposite sides from each other), as well an Fc constant region from an lgG4 antibody. Figure 8C shows a schematic of an exemplary construct that contains four 3A3 VHHs, an Fc constant region from an IgG4 antibody, and four 1 A12 VHHs at the other end of the Fc constant region.

[0033] Figure 9 shows the VHH amino acid sequences of humanized AlClrO2VHH nanobodies.

[0034] Figure 10 shows the VHH amino acid sequences of humanized AlClrOl VHH nanobodies.

[0035] Figure 11 shows various VHH constructs. Any of the VHHs described herein may be part of any of the constructs shown in Figures 11 A-l ID. Figure HA shows an exemplary construct that contains two copies of one VHH and two copies of a second VHH, as well as an Fc constant region (e.g., from an IgG4 antibody). Figure 11B shows an exemplary construct similar to 11 A, except the first and second pairs of VHHs are reversed in position. Figure 11C shows an exemplary construct that contains four (first) VHHs, an Fc constant region (e.g., from an IgG4 antibody), and four (second) VHHs at the other end of the Fc constant region. Figure 1 ID shows a schematic of an exemplary construct that contains two (first) VHHs, an Fc constant region (e.g., from an IgG4 antibody) and two (second) VHHs at the other end of the Fc constant region. Attorney Docket Number: CCF-43776.601

[0036] DEFINITIONS

[0037] To facilitate an understanding of the invention, a number of terms are defined below.

[0038] 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.

[0039] 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.

[0040] 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 (e.g., the encode the amino acid sequences shown in Figures 1 and 7).

[0041] As used herein, the temrs "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, Attorney Docket Number: CCF-43776.601 poly adenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0042] DESCRIPTION OF THE INVENTION

[0043] Provided herein are human complement Clr binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human Clr binding molecules (e.g., nanobodies) having a first, and optionally second, third, and fourth single monomeric variable antibody domains (SMVADs, aka VHHs) that comprises certain CDRs, and methods for using such molecules to treat complement-related diseases (e.g., dysregulated complement activation diseases). In certain embodiments, Clr binding molecules comprise a plurality of SMVADs and an antibody Fc region.

[0044] 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.

[0045] The terms “nanobody,” " VHH" 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 Singledomain Antibody and Identification of a Universal Humanized Nanobody Scaffold, The J. of Bio. Chem. Vol. 284, No. 5, pp. 3273-3284, January 30, 2009; both of which are herein incorporated in their entirities and particularly for methods of humanizing nanobodies); (4) Attorney Docket Number: CCF-43776.601 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.

[0046] 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 (e.g., see Figures 7 and 8) 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.

[0047] 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

[0048] 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. It 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 Attorney Docket Number: CCF-43776.601 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

[0049] Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the

[0050] 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.

[0051] 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 arc composed of a VH domain covalently linked to a VL, domain).

[0052] In certain embodiments, the SMVADs (nanobodies) herein may be further modified by one or more other amino substitutions while maintaining their activity as Clr binding molecules. In certain embodiments, substitutions arc 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±l); glutamate (+3.0±l); 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); try ptophan (-3.4). These values can be used as a guide and thus Attorney Docket Number: CCF-43776.601 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.

[0053] In certain embodiments, the human Clr binding molecules comprise one or more of the CDRs or variable regions shown SEQ ID NOS: 1-9 and 14-96 with one or more conservative or non-conservative amino acid changes, and nucleic acid sequences encoding SEQ ID NOs: 1-9 and 14-96. 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 Attorney Docket Number: CCF-43776.601 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, nanobody, variable region, or CDR, such as in SEQ ID NOS: 1-9 and 14-96.

[0065] For expression of human Clr 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 Clr binding molecules of the present disclosure are viral vectors, such as retro-viral vectors. Such viral Attorney Docket Number: CCF-43776.601 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, NJ) is employed to generate Clr binding molecules (and stable cell lines expressing the Clr 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.

[0067] Mammalian host cells for expressing the human Clr 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 Clr 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 Clr 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 Clr binding molecules can be recovered from the culture medium using standard protein purification methods.

[0068] In certain embodiments, the Clr binding molecules of the present invention (e.g., nanobodies or dual nanobodies) are useful for immunoassays which detect or quantify human Clr in a sample (e.g., a purified blood sample from a subject). In some embodiments, an immunoassay for Clr typically comprises incubating a biological sample in the presence of a delectably labeled antibody or antibody fragment of the present invention capable of selectively binding to Clr, and detecting the labeled peptide or antibody which is bound in a sample. Various clinical assay procedures are well known in the art.

[0069] The present disclosure provides immunoassay methods for determining the presence, amount or concentration of human Clr 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), Attorney Docket Number: CCF-43776.601 enzyme multiplied immunoassay technique (EMIT), an ARCHITECT assay (ABBOTT), a bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.

[0070] EXAMPLES

[0071] 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 constmed as limiting the scope thereof.

[0072] EXAMPLE 1

[0073] Nanobody-based complement classical pathway inhibitor targeting Clr

[0074] In this example, anti-Clr nanobodies were developed that preferably bound to different sites of the activated Clr to inhibit its serine protease activity that is required for activating Cis, and consequently, the classical pathway of complement activation. We also engineered a novel bispecific and bivalent anti-Clr antibody as a human IgG4 Ec fusion protein (TNbClr, aka Clr3A3NAb) containing the anti-Cls nanobody modules and showed that it inhibited hemolysis caused by the classical pathway of complement activation in vitro with an IC50 of O.lnM in contrast to an IC50 of 4.4 nM of Sutimlimab. In addition, TNbClr also reduced both C3b and MAC depositions on antibody-sensitized human endothelial cells significantly more efficiently than Sutimlimab in the same assays. These results suggest that the anti-Clr antibody provides a therapeutic in treating diseases in which the classical pathway of complement activation is causally involved in the pathogenesis.

[0075] In this example, we developed a novel anti-Clr nanobody by immunizing an alpaca with activated Clr. This nanobody preferably recognized activated Clr and was shown to neutralize its enzymatic function, inhibiting the classical pathway of complement activation by preventing the activation of Cis. We also produced a human IgG4 Fc fusion protein containing tetra-copies of the anti-Clr VHH modules (Clr3A3NAb), which demonstrated potent inhibition of classical pathway complement activation-mediated cell damage, approximately 10-fold better than sutimlimab.

[0076] Methods and Materials immunization and PBMC isolation Attorney Docket Number: CCF-43776.601

[0077] An alpaca was immunized via four subcutaneous injections of 150 pg of purified human activated Clr enzyme (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 C1R (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- Clr 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).

[0078] Nanobody phage-displav library construction and screening

[0079] 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 (8x10s) 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:

[0080] 5 '-GTCCTGGCTGCTCTTCT AC AAGG-3 ' (SEQ ID NO:10),

[0081] 5 '-GGTACGTGCTGTTGAACTGTTCC-3 ' (SEQ ID NO: 11); and

[0082] 5 '-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3 ' (SEQ ID NO: 12), 5 -CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3' (SEQ ID NO: 13).

[0083] 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. coli cells (Agilent Technologies, CA) to generate the nanobody phage display library following an established protocol22,23.

[0084] Panning and screening to identify high- affinity and functionally blocking Clr nanobodies

[0085] 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 Clr 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 Attorney Docket Number: CCF-43776.601 high Clr binding against 1 nM purified Clr and inhibitory activity against the classical complement pathway were selected for further characterization.

[0086] Expression and purification of the anti-Clr nanobody clone Nab3A3 and the derived bivalent anti-Clr nanobodv

[0087] Twenty high-binding anti-ClR nanobody candidates were selected and sequenced. Among them, clone IB 10 (Nab IB 10) was selected as the most potent inhibitory nanobody against C1R, 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. NablB 10 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-ClR 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.

[0088] Affinity measurement by surface plasmon resonance (SPR)

[0089] The interaction of C1R with the anti-ClR nanobody clone NablB 10 was studied by SPR using a Biacore T200 system (Cytiva, MA). The surface of an S series CM5 sensor chip was activated by NIIS / EDC for 420 s, and 25 pg / mL of NablBlO in acetate buffer (pll 4.5) was injected over the activated surface for 24 s to a final density of 1000 RU. Then, human C1R (Complement Tech, TX) in a series of concentrations ranging from 2.97 to 248 nM were injected over the C1R surface at a rate of 30 L / rnL for 300 s, 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).

[0090] Complement pathway inhibition assays

[0091] To determine which complement pathways are inhibited by NablBlO, 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 Attorney Docket Number: CCF-43776.601 evaluate complement pathway activation, NHS in the presence or absence of NablBlO 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 Nabl BlO was used as the positive control.

[0092] To determine the EC50 of mono- and bivalent NablBlO in the classical complement pathway, a different ELISA-based assay, MicroVue CH50 (Quidel, CA), was perfomred 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 NablBlO for 1 hour at 37°C. The activated scrum 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.

[0093] Complement classical pathway activation-mediated hemolysis and C3 deposition assays

[0094] 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 NablBlO 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).

[0095] To generate the C3 convertase C4b2a, 1 x 107EShA were incubated with 10% human C3-depleted serum in the presence or absence of NablBlO or EDTA for 10 minutes at 37°C in GVB++buffer. The EShA were washed with GVB° (without Ca2+and Mg2+) containing 10 Attorney Docket Number: CCF-43776.601 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 lO on the enzymatic activity of pre-assembled C4b2a, Nab IB 10 was incubated with C4b2a-coated EShA during incubation with Guinea pig serum.

[0096] Western blotting and ELISA to evaluate C1R cleavage and nanobodv binding

[0097] To evaluate the effect of Nab IB 10 on C1R activation, 2 pg of C1R were incubated with or without 2 pg / mL Ci s 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-ClR antibody (Complement Tech, TX), followed by a secondary HRP donkey anti-goat antibody (Jackson ImmunoRcscarch, PA).

[0098] To determine the C1R activation fragment to which NablB 10 binds, C1R was cleaved as described above to generate CIRa and CIRb. 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 CIRa and CIRb, as described above. An ELISA was performed to evaluate whether anti-ClR 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.

[0099] Modified in vitro human complement classical pathway-mediated mouse hemolysis assay

[0100] Mouse erythrocytes were sensitized with the anti-mouse erythrocyte antibody clone 34-3C21at 0-60 jag / 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 Attorney Docket Number: CCF-43776.601

[0101] NablBlO for 30 minutes at 37°C. Hemolysis was determined by determining the OD414 in each well.

[0102] In vivo studies of the Cl r nanobod y using a modified AIHA 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 of Anti-Clr Nanobodies

[0105] After immunizing an alpaca with purified human Clr in its active form, we constructed a nanobody phage-display library using RNAs isolated from PBMCs of the animal. After three rounds of panning with 5, 2, and 1 nM of Clr in sequence, we identified 20 clones that showed strong binding signals in a Clr-specific ELISA. Among the 20 clones, sequencing analyses showed 5 distinct nanobodies that matched the alpaca VHH sequences.

[0106] Screening of the Functionally Blocking Anti-Clr Nanobody

[0107] To identify functionally blocking anti-Clr nanobodies, we tested crude preparations from each of the 5 identified nanobodies in a classical complement pathway activation- mediated hemolytic assay. We found that among the identified high-binding anti-Clr nanobodies, clone Nab3A3, markedly protected EShA from complement-mediated lysis, suggesting its potential as a functionally blocking anti-Clr nanobody. We also picked a high- binding but non-function neutralizing anti-Clr nanobody, clone NablAl, as a control for future studies. Measurement and Validation of Nab3A3 as a

[0108] Functional Anti-Clr Attorney Docket Number: CCF-43776.601

[0109] To validate the screening results, we expressed Nab3A3 and Nabl Al in E. coll and purified them by affinity chromatography using a nickel column. We then measured the affinity of Nab3A3 against Clr using SPR and found that the affinity between Clr and Nab3A3 is 1.62+0.34 nM with the on-rate (1.29+0.20) X105 M-ls-1 and the off-rate (2.08+0.19) X 10-4 s-1. We also analyzed the inhibitory activities of both Nab3A3 and NablAl again in the same conventional classical pathway complement activation-mediated hemolysis assay. We confirmed that Nab3A3, but not NablAl, inhibited the complement- mediated hemolysis in a concentration-dependent manner with an IC50 of 34nM.

[0110] Nab3A3 Selectively Inhibits the Classical Pathway of Complement Activation Without Affecting the Lectin and Alternative Pathways

[0111] To demonstrate the selectivity of Nab3A3 inhibiting the classical pathway of complement activation, we used a commercial kit to examine all the pathways in the presence of 1000 nM of Nab3A3. These results showed that the classical pathway of complement activation was almost completely inhibited at the concentration tested. In contrast, even at this high concentration of Nab3A3 tested (lOOOnM), the lectin and alternative pathway of complement activation were minimally affected, demonstrating the selectivity of the Nab3A3 in inhibiting the classical pathway of complement activation.

[0112] Nab3A3 preferably binds to the activated Clr

[0113] Since we immunized the animal with activated Clr and screened the library using activated Clr, it is possible that Nab3A3 selectively binds to activated Clr. To examine its binding selectivity, we coated plates with 5-50 nM pro-Clr or activated Clr, then detected bindings of 0-1000 nM of Nab3A3 to each of these proteins by ELISA, using a polyclonal anti-Clr Ab as an internal control. These assays showed that at almost all the concentrations of antigens and nanobodies tested, Nab3A3 bound twice as much to the activated Clr than the pro-Clr at the same time, suggesting that Nab3A3 preferably but not selectively binds to the activated Clr.

[0114] Nab3A3 Does Not Recognize Cis

[0115] The genes for Clr and Cis are closely linked on chromosome 12pl 3 as a possible result of gene duplication during evolution, and activated Clr and Cis are highly homologous serine proteases. To exclude the possibility that Nab3A3 might cross-react with Cis, we coated plates with 5-50 nM of pro-Cls or activated Cis, then detected bindings of 0-1000 nM Attorney Docket Number: CCF-43776.601 of Nab3A3 to each of these proteins by ELISA, using a polyclonal anti-Cls Ab as an internal control. These assays showed that while the polyclonal anti-Cls Ab showed high binding to both forms of Cis, Nab3A3 displayed no or nominal binding to either form of Cis, even at the highest concentration combination tested. These results demonstrated that Nab3A3 does not cross-react with Cis at all.

[0116] Nab3A3 Inhibits the Cleavage (Activation) of Cis by Cl r

[0117] As a serine protease, Clr's only known function is to cleave Ci s for its activation in the classical pathway of complement activation. To elucidate the mechanism by which Nab3A3 inhibits the classical pathway, we incubated pro-Cls with or without Clr in the absence or presence of different concentrations of Nab3A3. We then detected pro-Cls cleavage (activation) by western blot using a polyclonal anti-Cls antibody. These studies showed that Clr cleaved pro-Cls in the absence or even presence of as high as 0.4 pM of Nab3 A3 in the assays. However, in the presence of as little as 2 pM of Nab3 A3, the cleavage of pro-Cls by Clr was completely inhibited. These data indicate that Nab3A3 neutralizes the enzymatic activity of Clr, which is required for pro-Cls activation.

[0118] Nab3A3 Does Not Cross-React with Rodent Clr

[0119] We examined the potential cross-reactivity of Nab3A3 with Clr from rat and guinea pig sera using a classical pathway complement-mediated hemolysis inhibition assay, and Clr from mouse serum using a C3b deposition inhibition assay. These studies showed that while Nab3A3 potently inhibits human serum-induced hemolysis, it had minimal effect on rat or guinea pig serum-induced hemolysis, even at the highest concentration tested (1 pM). In addition, at the same high concentration of 1 pM, Nab3A3 did not inhibit mouse C3b deposition on EshA in flow cytometric assays at all. All of these data indicated that Nab3A3 does not cross-react with these rodent Clr proteins.

[0120] Production of a Tetravalent Anti-Clr Antibody (TNabClr, aka Clr3A3NAb) as a Human

[0121] 34 Fc-Fusion Protein Using Nab3A3

[0122] We designed and produced a tetravalent anti-Clr antibody as a human IgG4 Fc-fusion protein based on Nab3A3. The expression construct was synthesized and used to transfect CHO cells for transient expression. The resultant Fc-fusion protein (TNabClr, aka Clr3A3NAb) was then purified by protein A / G affinity-chromatography (Fig. 7). Attorney Docket Number: CCF-43776.601

[0123] TNabClr Shows Significantly Augmented Activity in Inhibiting Complement Classical Pathway Activation That Is 10 Times More Potent Than Sutimlimab

[0124] We then did a direct comparison of TNabClr against its monomeric form (Nab3A3) as well as sutimlimab in inhibiting complement-mediated cell damage using the same EshA- based hemolysis assay. We found that compared to Nab3A3, which showed an IC50 of 4 nM in inhibiting complement-mediated hemolysis in this assay, the IC50 of TNabClr was 0.5 nM. In contrast, sutimlimab had an IC50 of 5.6 nM in the same assays. TNabClr showed significantly augmented activity in inhibiting complement classical pathway activation that is > 10 times more potent than sutimlimab in this assay.

[0125] TNabClr efficiently inhibited complement-mediated damage of RBCs from a CAD patient, with 20 times stronger efficacy than sutimlimab.

[0126] Results from the in vitro functional assays using EshA strongly suggest that TNabClr could be potent in treating classical pathway-mediated diseases such as CAD, the indication approved for sutimlimab. We thus obtained RBCs from a diagnosed RBC IgM+ CAD patient, incubated them with NHS (the source of complement) together with the same concentrations of serial dilutions of TNabClr or sutimlimab. We assessed the levels of complement- mediated hemolysis and found that TNabClr potently protected the patient RBCs in a concentration-dependent manner with a calculated IC50 of InM. Sutimlimab also showed concentration-dependent inhibition of complement-mediated hemolysis of the CAD patient RBCs, but its calculated IC50 was 10 nM, which is 10-fold less potent than TNabClr.

[0127] TNabClr efficiently inhibited complement-mediated damage of antibody-sensitized human endothelial cells, with 20 times stronger efficacy than sutimlimab.

[0128] Endothelial cells are the primary target of alloantibodies during transplantation rejections. To examine the potential of TNabClr in attenuating alloantibody-initiated, complement-mediated endothelial cell damage, HUVEC were first sensitized with anti-HLA IgGs, then subjected to a conventional complement-mediated cell damage assay in the presence of serial dilutions of TNabClr and sutimlimab. Similar to the results from cell damage assays using EshA and CAD patient RBCs, these studies showed that TNabClr efficiently inhibited complement-mediated damage of these antibody-sensitized human endothelial cells in a concentration-dependent manner with a calculated IC50 of 2nM versus an IC50 of 20nM from sutimlimab in the same assays. Attorney Docket Number: CCF-43776.601

[0129] Targeting the classical pathway of complement activation is a validated therapeutic approach and the recently approved sutimlimab targeting Cis is the first man-made drug for this purpose but its efficacy does not seem to be impressive. In this Example, we developed a new nanobody Nab3A3 targeting Clr, and showed that it works by preferably binding to the activated Clr and inhibiting its serine protease activity required for Cis activation during classical pathway complement activation. We also developed a human IgG4 Fc-fusion protein containing tetrameric modules of Nab3A3 and demonstrated that this new antibody (TNabClr) protected antibody-sensitive RBCs and antibody-sensitized human endothelial cells from complement-mediated damage with 10-times better potency than sutimlimab.

[0130] Inhibiting complement activation is a clinically validated therapeutic approach for many diseases. Inhibitors targeting different complement components are under extensive development and a few of them have been successfully used in clinic: eculizumab and ravelizumab are anti-C5 mAbs inhibiting MAC formation, thus protecting host tissue from MAC-mcdiatcd damage; pcgcctacoplan is a pcptidc-bascd inhibitor targeting C3, the central component of all complement activation pathways thus completely blocking complement activation. Even though these drugs are effective in treating many complement-mediated diseases, they also significantly increase the risk of opportunistic infections because either C5 or C3 is shared by all three complement activation pathways. Since different activation pathways play different roles in various diseases, selectively targeting the pathogenic pathway would result in a better safety profile as the remaining complement activation pathways are still functional to control the invading pathogens. Sutimlimab targets Cis to inhibit the classical pathway, and it is so far the first and only FDA-approved man-made complement inhibitor that selectively targets one complement activation pathway without affecting the rest of the pathways for treating complement-mediated diseases. However, as mentioned above, Sutimlimab does not seem to be very potent because large doses of Sutimlimab are required to be effective for treating CAD patients and it also failed to show any efficacy in a renal transplantation rejection clinical trial. Besides, adverse effects such as bladder / chest pain, blurred vision, diarrhea, nausea and dizziness were observed in patients receiving the treatment of sutimlimab, all of which could be ameliorated if a different therapeutic that can inhibit the classical pathway complement activation with a significantly lower effective dosage could be used instead.

[0131] Clr is upstream of Cis that sutimlimab targets in the classical pathway of complement activation, and it has a blood concentration very similar to Cis. After Clq binds to antibody-antigen complexes, Clr proenzyme breaks into two chains with conformational Attorney Docket Number: CCF-43776.601 changes to become Clr enzyme, which is the first protease that initiates the cascade. Activation of Cis is the only known function of Clr enzyme, in contrast, Cis cleaves other proteins including Insulin-like Growth Factor-binding Protein-5 in addition to C4 and C2 in the complement system. All these features make Clr a superior therapeutic target to Cis. However, to the best of our knowledge, no Clr- targeted therapeutic inhibitor has been reported previously.

[0132] As described above, when Clr is activated, there are only conformational changes of the protein without any amino acid sequence change. The results that our anti-Clr nanobody preferably binds to the activated Clr over the pro-Clr protein suggest that the nanobody is recognizing a conformational epitope that emerged after Clr activation. However, this confomrational epitope does not seem to be a complete neoepitope for the activated Clr, as our nanobody still be able to bind to the pro-Clr protein with a reduced efficiency.

[0133] 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

Attorney Docket Number: CCF-43776.601CLAIMS:We claim:

1. A composition comprising a human complement Clr binding molecule, or one or more nucleic acid molecules encoding said human Clr binding molecule, wherein said human Clr binding molecule comprises a first single monomeric variable antibody domain (SMVAD) that comprises:A) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservative amino acid changes,B) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7: or SEQ ID NO:3 or 7 with one or two conservative amino acid changes, andC) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 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 Clr binding molecule further comprises a second SMVAD that comprises:D) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservalivc amino acid changes,E) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NO: 3 or 7 with one or two conservative amino acid changes, andF) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservalivc amino acid changes.

4. The composition of claim 3, wherein said human Clr 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 human Clr binding molecule further comprises a third SMVAD that comprises:Attorney Docket Number: CCF-43776.601G) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQID NO:2 or 6 or 17 with one with one or two conservative amino acid changes,H) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NO:3 or 7 with one or two conservative amino acid changes, andI) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservative amino acid changes.

6. The composition of claim 5, wherein said human Cl r binding molecule further comprises a fourth SMVAD that comprises:J) a CDR1 amino acid sequence comprising SEQ ID NO: 2 or 6 or 17; or SEQ ID NO:2 or 6 or 17 with one with one or two conservative amino acid changes,K) a CDR2 amino acid sequence comprising SEQ ID NO: 3 or 7; or SEQ ID NO:3 or 7 with one or two conservative amino acid changes, andL) a CDR3 amino acid sequence comprising SEQ ID NO: 4 or 8 or 15; or SEQ ID NO:4 or 8 or 15 with one with one or two conservative amino acid changes.

7. The composition of claim 6, wherein said human Clr binding molecule further comprises a linker which is attached to both said third SMVAD and said fourth SMVAD.

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

9. The composition of claim 8, wherein said CII2 and / or CII3 heavy chain constant regions are camelid, humanized, or human.

10. The composition of claim 3, wherein said human Clr binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

11. The composition of claim 6, wherein said human Clr binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

12. 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 andAttorney Docket Number: CCF-43776.601 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.

13. The composition of claim 1, wherein said first SMVAD comprises the amino acid sequence shown in SEQ ID NOT, 5, 14, 16, or 18-96; or SEQ ID NO: 1, 5, 14, 16, or 18-96 with one, two, three, or four deletions and / or conservative amino acid changes.

14. The composition of claim 6, wherein said human complement Cl r binding molecule comprises the amino acid sequence in SEQ ID NO: 9, or SEQ ID NO:9 with a different linker sequence, and then zero, one, two, or three conservative amino acid changes in the remainder of the sequence.

15. The composition of claim 1, wherein said human Clr binding molecule comprises at least an antigen binding portion of Clone 3A3 or 1A12 nanobody.

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

17. 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.

18. The composition of claim 1, wherein said composition comprises said human Clr binding molecule.

19. The composition of claim 1, wherein said CDR1 amino acid sequence comprises SEQ ID NO:2, said CDR2 amino acid sequence comprises SEQ ID NOT, and said CDR3 amino acid sequence comprises SEQ ID NOT.

20. The composition of claim 1, wherein said CDR1 amino acid sequence comprises SEQ ID NO:6, said CDR2 amino acid sequence comprises SEQ ID NO:7, and said CDR3 amino acid sequence comprises SEQ ID NOT.Attorney Docket Number: CCF-43776.60121. A method of treating or preventing a complement- related disease or condition comprising: treating a subject with a composition comprising a human complement Clr binding molecule, or an expression vector comprising one or more nucleic acid molecules encoding said Clr binding molecule, as recited in any of Claims 1-20, and wherein said subject has, or is suspected of having or developing, a complement- related disease or condition.

22. The method of claim 21, wherein said complement-related disease comprises a dysregulated complement activation disease.

23. The method of claim 21, wherein said complement-related disease comprises: myasthenia gravis, atypical hemolytic uremic syndrome, cold agglutinin disease (CAD), Lupus Nephritis, Ncuromyclitis optica spectrum disorder, Guillain-Barre syndrome, Multifocal motor neuropathy, Myasthenia gravis, Miller Fisher syndrome, Chronic inflammatory demyelinating syndrome, Acute hemolytic transfusion reaction, Cold agglutinin disease, Warm antibody hemolytic anemia, Immune thrombocytopenic purpuraor, and autoimmune hemolytic anemia (AIHA).

24. The method of claim 21, wherein said first SMVAD further comprises four Framework regions, wherein said four Framework regions are camelid, humanized, or human Framework regions.

25. The method of claim 21, wherein said human Clr binding molecule further comprises said second SMVAD recited in claim 3.

26. The method of claim 25, wherein said human Clr binding molecule further comprises said third SMVAD recited in claim 5.

27. The method of claim 26, wherein said human Clr binding molecule further comprises said third SMVAD recited in claim 6.

28. The method of claim 25, wherein said human Clr binding molecule further comprises a linker which is attached to both said first SMVAD and said second SMVAD.Attorney Docket Number: CCF-43776.60129. The method of claim 21, 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.

30. The method of claim 21 , wherein said first SMVAD comprises the amino acid sequence shown in SEQ ID NO: 1, 5, 14, 16, or 18-96, or SEQ ID NO: 1, 5, 14, 16, or 18-96 with one, two, three, or four deletions and / or conservative amino acid changes.

31. The method of claim 21, wherein said human Clr binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

32. The method of claim 31, wherein said CH2 and / or CH3 heavy chain constant regions are camelid, humanized, or human.

33. The method of claim 21, wherein said human Clr binding molecule comprises at least an antigen binding portion of Clone 3 A3 or 1 Al 2 nanobody.

34. The method of claim 21, wherein said composition further comprises a physiologically tolerable buffer.

35. The method of claim 21, wherein said composition comprises said expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.

36. The method of claim 21, wherein said composition comprises said human Clr binding molecule.

37. The method of claim 21, 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.Attorney Docket Number: CCF-43776.60138. A method of detecting human Clr in a sample comprising: a) contacting a sample with the human Clr binding molecule of any of Claims 1- 20, wherein said sample is suspected of containing human Clr, and wherein said human Clr binding molecule forms a complex with said human Clr if present in said sample; and b) detecting the presence or absence of said complex in said sample.

39. The method of Claim 38, wherein said sample is from a subject that has, or is suspected to develop, a complement-related disease or condition.

40. The method of Claim 38, wherein said human Clr binding molecule comprises a detectable label.

41. The method of Claim 38, further comprising contacting said sample with a conjugate molecule capable of binding to said human Clr binding molecule, wherein said conjugate molecule comprises a detectable label.

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