Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/35—Valency
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/524—CH2 domain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/526—CH3 domain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a

Definitions

• human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules.
• human C2 binding molecules e.g., nanobodies
• SMVAD single monomeric variable antibody domain
• the SMVAD comprises camelid, human, or humanized framework regions.
• 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 .
• MAC membrane attack complex
• excessive complement activation is a causal factor in many diseases, such as myasthenia gravis 2 , atypical hemolytic uremic syndrome 3 , paroxysmal nocturnal hemoglobinuria (PNH) 4 , and autoimmune hemolytic anemia (AIHA) 5 .
• complement inhibitors As new therapeutics are underway, and inhibitors that target complement components 5 (C5) 6 , 3 (C3) 7,8 and Is (Cis) 9 have been approved for clinical use. Although these inhibitors have been applied successfully to ameliorate different diseases, all of them have drawbacks 10 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/mL 13 (versus -75 pg/mL for C5 14 and -1500 pg/mL for C3 14 ).
• Nanobodies, or single-chain domain antibodies are unique antibodies produced in camelids such as llamas and alpacas 15 . 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 trials 16 . 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 development 17 . Additionally, nanobodies can be easily manipulated using routine molecular biology techniques to generate multivalent antibodies with augmented avidity without negatively affecting their antigen-binding abilities 18 19 . Humanization processes to further reduce the minimal immunogenicity of nanobodies have also been well established 20 .
• AIHA Autoimmune hemolytic anemia
• erythrocytes are produced and cause anemia 5 .
• 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.
• AIHA is induced by injecting a mouse anti-mouse erythrocyte-specific antibody, mAb 34-3C (isotype Ig2a), which was isolated from autoimmune NZB mice 21 .
• mAb 34-3C isotype Ig2a
• 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 anemia 21 , indicating an important role of complement underlying AIHA.
• human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules.
• human C2 binding molecules e.g., nanobodies
• SMVAD single monomeric variable antibody domain
• the SMVAD comprises camelid, human, or humanized framework regions.
• the human C2 binding molecules are human C2a binding molecules.
• 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
• kits for 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.
• the complement-related disease comprises a dysregulated complement activation disease.
• the complement-related disease comprises myasthenia gravis or atypical hemolytic uremic syndrome.
• the complement-related disease comprises paroxysmal nocturnal hemoglobinuria (PNH) or and autoimmune hemolytic anemia (AIHA).
• kits for 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.
• the sample is from a subject that has, or is suspected to develop, a complement-related disease or condition.
• the human C2 binding molecule comprises a detectable label.
• 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.
• the first SMVAD further comprises four Framework regions, wherein the four Framework regions are camelid, humanized, or human Framework regions.
• 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
• the human C2 binding molecule further comprises a linker which is attached to both the first SMVAD and the second SMVAD.
• 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).
• 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.
• the human C2 binding molecule further comprises a CH2 heavy chain constant region and/or a CH3 heavy chain constant region.
• the CH2 and/or CH3 heavy chain constant regions are camelid, humanized, or human.
• the human C2 binding molecule comprises at least an antigen binding portion of Clone IB 10 C2 nanobody.
• compositions, kits, and systems herein further comprise a physiologically tolerable buffer.
• 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.
• the composition comprises the human C2 binding molecule.
• the CDR1 amino acid sequence comprises SEQ ID NO:2
• the CDR2 amino acid sequence comprises SEQ ID NO:3
• the CDR3 amino acid sequence comprises SEQ ID NO:4.
• 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.
• 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.
• NablBlO binds to C2a but not to C2b.
• 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.
• NablBlO does not block C2 cleavage by Cis but prevents C3 convertase C4b2a assembly.
• NablBlO blocks classical complement pathway activation in human and monkey serum but not rat, guinea pig, or mouse serum.
• 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.
• 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.
• 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 Ca 2+ and 0.5 mM Mg 2+ ) 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.
• 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%.
• FIG. 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.
• 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).
• Figure 9 shows the various parts of an exemplary VHH antibody.
• 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).
• 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).
• 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).
• 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).
• 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).
• 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.
• 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.
• Figure 17 shows ELISA binding data for the 20 humanized C20 antibodies described in figures 10-14.
• 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.
• the terms “subject” and “patient” refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.
• 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.
• an oligonucleotide having a nucleotide sequence encoding a polypeptide 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• human complement component 2 (C2) binding molecules and nucleic acid sequences encoding such molecules.
• human C2 binding molecules e.g., nanobodies
• SMVAD single monomeric variable antibody domain
• the SMVAD comprises camelid, human, or humanized framework regions.
• NablBlO is an anti-C2 nanobody that potently and selectively inhibits both the classical and lectin pathways of complement activation.
• 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.
• the nanobodies 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.
• framework sequences or FRs
• CDRs complementary determining regions
• 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.
• 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.
• nanobody and “SMVAD,” in their broadest sense, are not limited to a specific biological source or to a specific method of preparation.
• 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.
• 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.
• 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.
• 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
• 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
• 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.
• position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to the
• 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.
• 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.
• 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.
• the SMVADs may be further modified by one or more other amino substitutions while maintaining their activity as C2 binding molecules.
• substitutions are made in the framework regions and not in the CDR domains.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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:
• hydrophobic norleucine, met, ala, val, leu, ile
• 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.
• 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.
• 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).
• 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).
• 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 2TM 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.
• 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.
• 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.
• the human complement component 2 binding molecules of the present invention are useful for immunoassays which detect or quantify human complement component 2 in a sample (e.g., a purified blood sample from a subject).
• 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.
• a detectably labeled antibody or antibody fragment of the present invention capable of selectively binding to complement component 2
• 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.
• 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
• 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
• a second, detectably labeled, molecule 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.
• 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).
• EIA enzyme immunoassay
• ELISA enzyme-linked immunosorbent assay
• 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.
• human complement component 2 which is detected by the above assays can be present in a biological sample.
• a biological sample such as, for example, blood, brain tissue, serum, lymph, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, a tissue extract or homogenate, and the like.
• 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.
• 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).
• a biological sample e.g., brain tissue
• kits for the detection of complement component 2 that include a human complement component 2 detection molecule.
• 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.
• 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).
• a sample e.g., a container or cartridge for a sample.
• 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.
• a nanobody-based complement inhibitor targeting complement component 2 reduces hemolysis in a humanized mouse model of autoimmune hemolytic anemia
• 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.
• ELISA enzyme-linked immunosorbent assay
• HRP horseradish peroxidase
• 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 (8xlO 8 ) 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 primers 22 :
• 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 protocol 22,23 .
• the nanobody phage display library was cultured and infected with the M13KO7 helper phage (New England Biolabs, MA) to initiate phage production.
• M13KO7 helper phage New England Biolabs, MA
• 1 x 10 12 purified 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.
• 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.
• 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.
• HisPur Cobalt resins Thermo Fisher, MA
• 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.
• glycine buffer pH 1.7
• Origin 7.0 Origin 7.0
• 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.
• 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.
• Sheep erythrocytes were pre-coated with rabbit anti-sheep erythrocyte antiserum (MP Biomedicals, OH) to generate antibody-sensitized ESh (EShA).
• EShA Sheep erythrocytes
• 1 x 10 7 EShA 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 Ca 2+ and Mg 2+ (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.
• 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).
• 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.
• BSA bovine serum albumin
• 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.
• mice erythrocytes were sensitized with the anti-mouse erythrocyte antibody clone 34-3C 21 at 0-60
• 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 Ca 2+ and 0.5 mM Mg 2+ was administered to induce complement-mediated intravascular hemolysis.
• 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.
• NablB lO selectively inhibits the classical and lectin pathways of complement activation without affecting the alternative pathway
• NablB 10 binds to the C2a but not the C2b portion of C2
• C2 is cleaved by activated Cis into C2a and C2b during complement activation 24 .
• 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.
• 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 pathway 25 . C4b2a, the C3 convertase, then activates C3 to initiate the downstream complement activation cascade.
• NablBlO did not inhibit C2 cleavage by Cis, suggesting that NablBlO does not interfere with C2 activation (Figure 3A).
• NablBlO inhibits animal complement activation by cross-reacting with C2, it can be evaluated in vivo using established preclinical models.
• NablBlO inhibited monkey but not rat or guinea pig complement-mediated hemolysis ( Figure 4A).
• Figure 4A As mouse complement hemolytic activity is too weak to lyse EShA 26 , 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.
• 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.
• NablBlO inhibits human classical complement pathway-mediated hemolysis of mouse erythrocytes in vitro
• NablBlO protects mouse erythrocytes from hemolysis in vivo in a humanized AIHA model
• 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.
• nanobodies are their modality 28 .
• 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.
• conventional mAbs are naturally bivalent.
• complement 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.
• C5 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 cells 1 .
• Eculizumab and its longer half-life derivative, ravulizumab, are anti-C5 mAbs that inhibit C5 activation 6 , therefore preventing the assembly of MACs and the release of C5a.
• MACs directly damage tissues in many diseases such as PNH
• anti-C5 mAbs are clinically effective for treating such diseases 30 .
• 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 anemia 4,31 .
• C3 is the most abundant complement protein, present at concentrations of 1000-1500 ug/mL 14 . 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 treatment 10 . 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.
• the C2 concentration in the blood is approximately 100-fold lower than that of C3, making C2 a much easier target to inhibit.
• 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.
• antibody-antigen complexes activate complement through the classical pathway 32 and autoantibodies or alloantibodies are causal factors in many autoimmune diseases 33 and transplant rejection 34 , 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 disease 9 , a subtype of AIHA in which autoantibodies against erythrocyte surface antigens activate complement through the classical pathway, causing hemolysis and anemia 35 .
• Cis is not required to activate the lectin pathway of complement, and Sutimlimab does not affect this pathway at all.
• 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 damage 36 .
• I/R ischemia-reperfusion
• 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.
• TA-TMA transplant- associated thrombotic microangiopathy
• C2-blocking nanobodies could be used to ameliorate these pathological conditions, because C2, but not Cis, is required for lectin pathway complement activation.
• 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.

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