CN119409815B

CN119409815B - Anti-human complement C5 VHH antibodies and uses thereof

Info

Publication number: CN119409815B
Application number: CN202510022205.8A
Authority: CN
Inventors: 马海立; 蔡逢春; 刘运华; 董梦飞; 刘恒
Original assignee: Tianchen Biomedical Suzhou Co ltd
Current assignee: Tianchen Biomedical Suzhou Co ltd
Priority date: 2025-01-07
Filing date: 2025-01-07
Publication date: 2025-04-08
Anticipated expiration: 2045-01-07
Also published as: CN119409815A

Abstract

The present invention relates to a single domain antibody or heavy chain antibody that specifically binds to complement C5, a polynucleotide encoding the antibody, an expression vector and a host cell, and a pharmaceutical composition thereof, and a method and use for treating C5 protein-related diseases.

Description

Anti-human complement C5 VHH antibodies and uses thereof

Technical Field

The present invention relates to a single domain antibody or heavy chain antibody that specifically binds complement C5. The invention also relates to polynucleotides encoding the antibodies, expression vectors and host cells, and pharmaceutical compositions or combinations thereof, and methods and uses for treating C5 protein-related diseases.

Background

The complement system is composed of more than 30 soluble protein molecules, is a part of the natural immune system, and comprises more than 30 molecules such as complement intrinsic components, various regulatory factors, complement receptors and the like. The complement system can be activated by 3 relatively independent and interrelated pathways, thereby exerting a variety of biological effects that regulate phagocytosis, lyse cells, mediate inflammation, immunomodulation, and clear immune complexes, including enhancing phagocytosis, enhancing chemotaxis of phagocytes, increasing vascular permeability, neutralizing viruses, cytolysis, modulation of immune responses, and the like. While complement activation provides a valuable first line defense against potential pathogens, complement activation that promotes a protective inflammatory response may also represent a potential threat to the host. Complement activation and its deposition on target structures can also indirectly cause cell or tissue destruction. Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement activation on host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases.

Complement proteins include proteins denoted C1 to C9, and these proteins are activated sequentially by three different pathways (classical pathway, lectin pathway, alternative pathway) to elicit immune responses. Complement C5 is the fifth component of complement and plays an important role in inflammation and cell killing processes. Such proteins consist of alpha and beta polypeptide chains linked by disulfide bonds. The activating peptide C5a is an anaphylatoxin, induces inflammatory responses in various cells via C5aR (CD 88) and C5L2 (GPR 77), has potent spasticity and chemotactic activity, and is derived from alpha polypeptides by cleavage with C5 convertase. The C5b macromolecule cleavage product may form a complex with the C6 complement component, which is the basis for the formation of a Membrane Attack Complex (MAC), including additional complement components. If the complement system is not properly controlled or is overactivated, the complement system may be strongly cytotoxic to the host cell.

Numerous studies have shown that complement C5 is associated with a variety of diseases, such as those associated with human complement hemolytic activity.

Anti-C5 monoclonal antibodies elkuzumab (Eculizumab, soliris (registered trademark)) exhibit high affinity for complement C5 and suppress complement activation by inhibiting cleavage of C5 to C5a/C5b and formation of a complex accompanying membrane attack. Thus, eculizumab exhibits an inhibitory effect on hemolysis and is therefore a therapeutic agent for paroxysmal sleep hemoglobinuria and atypical hemolytic uremic syndrome. In addition, eculizumab is known as a therapeutic for systemic myasthenia gravis (gMG).

While eculizumab exhibits good classical pathway hemolytic inhibition, it has a weaker alternative pathway hemolytic inhibition in vitro assays, which may also be responsible for up to 75% of patients treated with Soliris who experience persistent anemia and symptoms associated with anemia.

In addition, eculizumab is expensive, one of the reasons for this is that it is highly abundant in human complement C5 protein and rapid in turnover, which requires injection of large amounts of drugs for complete blocking of complement activity in a single treatment, thus developing a single-domain antibody with better activity, higher stability and smaller molecular size has the potential to improve therapeutic effects and patient compliance.

It is therefore desirable to develop alternative antibodies, particularly the more stable smaller molecular size single domain antibodies, suitable for the treatment and prevention of complement C5-related disorders.

Disclosure of Invention

The present invention relates to a single domain antibody, such as a VHH antibody, capable of binding human and monkey complement C5 proteins with high efficiency, and a heavy chain antibody comprising the same. In some embodiments, the antibody is capable of effectively blocking human complement hemolytic activity.

In some embodiments, the antibodies of the invention are capable of specifically binding to human C5 protein and/or monkey (e.g., cynomolgus monkey) C5 protein. In some embodiments, the antibodies of the invention are capable of effectively inhibiting human and/or monkey (e.g., cynomolgus monkey) complement classical pathway hemolysis and human and/or monkey (e.g., cynomolgus monkey) complement alternative pathway hemolysis, with greater activity than known anti-C5 antibodies, such as eculizumab. In some embodiments, the antibodies of the invention are capable of having good human complement inhibitory activity and/or good monkey (e.g., cynomolgus monkey) complement inhibitory activity, e.g., greater activity than known anti-C5 antibodies, e.g., eculizumab. In some embodiments, the antibodies of the invention have a higher thermostability, e.g., are capable of withstanding heat treatment at 70 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, or 79 ℃ (e.g., heat treatment for 1 hour), preferably are capable of withstanding heat treatment at 80 ℃, 81 ℃, 82 ℃, 83 ℃, or 84 ℃ (e.g., heat treatment for 1 hour).

In some aspects of the invention, the invention provides a VHH antibody that specifically binds complement C5 comprising three Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 contained in a VH as set forth in any one of SEQ ID NOs 34, 33, 11-14.

In some embodiments, a VHH antibody of the invention comprises CDR1, CDR2 and CDR3, wherein,

(I) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 17, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 21;

Or (ii) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 20;

or (iii) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 16, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 21;

Or (iv) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 18, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 21.

In some embodiments, a VHH antibody of the invention comprises or consists of a heavy chain variable region comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOS: 34, 33, 11-14, or comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOS: 34, 33 and 11-14.

In some aspects of the invention, the invention provides heavy chain antibodies that specifically bind complement C5, comprising a VHH antibody of the invention.

In some embodiments, the heavy chain antibody of the invention comprises a VHH antibody of the invention linked to an antibody constant region or Fc region, preferably from human IgG1, human IgG2, human IgG3 or human IgG4, optionally linked to the Fc region by a hinge region. In some embodiments, the VHH antibody is linked to the Fc region via a hinge region, e.g., the hinge region is from a human IgG1, e.g., a native human IgG1 hinge region or a human IgG1 hinge region comprising C220S. In some embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO. 38 or 39.

In some embodiments, the heavy chain antibody of the invention comprises a VHH antibody of the invention linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, preferably the Fc region comprises the amino acid sequence shown in SEQ ID No. 24 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

In some embodiments, the heavy chain antibodies of the invention:

(i) Comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID nos. 36, 35 and 27 to 30;

Or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOS.36, 35 and 27-30.

In some embodiments, a VHH antibody or heavy chain antibody of the invention is a camelid, chimeric or humanized antibody.

In some aspects of the invention, the invention provides a nucleic acid molecule encoding or consisting of a VHH antibody of the invention, or a heavy chain antibody of the invention, or a nucleic acid sequence comprising or consisting of any one of SEQ ID NOs 32, 31 and 7-10.

In some aspects of the invention, the invention provides an expression vector comprising a nucleic acid molecule of the invention, preferably the expression vector is pcdna3.1.

In some aspects of the invention, the invention provides a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention, preferably the host cell is prokaryotic or eukaryotic, e.g. a 293 cell, e.g. a 293FT cell.

In some aspects of the invention, the invention provides a method of making a VHH antibody of the invention, or a heavy chain antibody of the invention, comprising culturing a host cell of the invention under conditions suitable for expression of the VHH antibody or heavy chain antibody or chain thereof, and optionally recovering the VHH antibody or heavy chain antibody from the host cell (or host cell culture medium).

In some aspects of the invention, the invention provides an immunoconjugate comprising a VHH antibody according to the invention, or a heavy chain antibody according to the invention.

In some aspects of the invention, the invention provides a pharmaceutical composition comprising one or more VHH antibodies of the invention, or heavy chain antibodies of the invention, or immunoconjugates of the invention, and optionally a pharmaceutical excipient.

In some aspects of the invention, the invention provides a pharmaceutical composition comprising two or three VHH antibodies of the invention, or two or three heavy chain antibodies of the invention, and optionally a pharmaceutical excipient.

In some aspects of the invention, the invention provides pharmaceutical combination products comprising a VHH antibody or heavy chain antibody according to the invention, as well as other therapeutic agents.

In some aspects of the invention, the invention provides a pharmaceutical combination product, wherein the pharmaceutical combination product comprises two or three VHH antibodies or heavy chain antibodies according to the invention.

In some aspects of the invention, the invention provides a method for preventing or treating a complement C5-associated disease or disorder or for blocking or inhibiting complement hemolysis in a subject, comprising administering to the subject an effective amount of one or more VHH antibodies of the invention, or one or more heavy chain antibodies of the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention, or a pharmaceutical combination of the invention. In some aspects of the invention, the use of one or more VHH antibodies of the invention, or one or more heavy chain antibodies of the invention, immunoconjugates of the invention, or pharmaceutical compositions of the invention, or pharmaceutical combination products of the invention, for the preparation of a medicament for the prevention or treatment of a complement C5-associated disease or disorder or for blocking or inhibiting complement hemolysis in a subject. In some aspects of the invention, one or more VHH antibodies of the invention, or one or more heavy chain antibodies of the invention, immunoconjugates of the invention, or pharmaceutical compositions of the invention, or pharmaceutical combination products of the invention, are used for preventing or treating a complement C5-associated disease or disorder or for blocking or inhibiting complement hemolysis in a subject. In some embodiments, the disease or disorder of the invention includes disease phenotypes caused by unregulated C5 function, e.g., due to deregulated C5 activation, e.g., increased C5 activation. In some embodiments, the subject of the invention has (e.g., elevated levels of, e.g., nucleic acid or protein levels of) complement C5 protein or the disease treatment would benefit from inhibiting nucleic acid or protein levels of complement C5 protein. In some embodiments, the complement hemolysis is complement classical pathway hemolysis and/or complement alternative pathway hemolysis.

Drawings

Figure 1 shows anti-C5 antibody titers in serum following alpaca immunization.

FIG. 2 shows the inhibitory activity of recombinant C5 VHH antibodies on human complement CP pathway hemolysis.

FIG. 3 shows the inhibitory activity of recombinant C5 VHH antibodies on human complement AP pathway hemolysis.

Figure 4 shows the SEC-HPLC purity (wavelength=280 nM) of recombinant humanized C5 VHH antibody C5006d 1-G1.

Figure 5 shows the purity (wavelength=280 nM) of recombinant humanized C5 VHH antibody C5006e3-G1 SEC-HPLC.

FIG. 6 shows the binding of humanized C5 VHH antibodies C5006d1-G1, C5006e3-G1 to human CD5 when coated antibodies detect gradient diluted antigen human C5.

FIG. 7 shows the binding of humanized C5 VHH antibodies C5006d1-G1, C5006e3-G1 to human C5 when the antigen human C5 protein is coated to detect gradient diluted antibodies.

FIG. 8 shows the inhibitory activity of recombinant humanized C5 VHH antibodies on human complement CP pathway hemolysis.

FIG. 9 shows the inhibitory activity of recombinant humanized C5 VHH antibodies on human complement AP pathway hemolysis.

FIG. 10 shows the inhibitory activity of recombinant humanized C5 VHH antibodies on monkey complement CP pathway hemolysis.

FIG. 11 shows the inhibitory activity of recombinant humanized C5 VHH antibodies on monkey complement AP pathway hemolysis.

FIG. 12 shows the inhibitory activity of recombinant humanized C5 VHH antibodies on complement CP pathway hemolysis after heat treatment.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

I. Definition of the definition

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) less than the specified numerical value and an upper limit of 5% (e.g., 4%, 3%, 2%, or 1%) greater than the specified numerical value.

As used herein, the term "and/or" means any one of the selectable items or two or more or all of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

The terms "complement C5" or "C5 protein" or "complement C5 protein" or "C5 complement protein" are used interchangeably and also refer to complement C5 proteins in different species. In this context, unless otherwise indicated, the term also encompasses any variant of C complement C5, particularly naturally occurring variants, allelic variants, as well as post-translationally modified variants and conformational variants, and encompasses species homologs thereof. Furthermore, it is understood that the term encompasses not only CDH17 naturally or recombinantly expressed by a cell or CDH17 expressed on a natural or recombinant cell, but also recombinantly expressed fusion proteins comprising CDH17 or fragments thereof.

Human complement C5 (e.g., uniprot entry P01031) is a secreted multidomain glycoprotein consisting of the alpha chain (999 amino acids) and the beta chain (655 amino acids) linked by disulfide bridges. The peptide bond between Arg751 and Leu752 of the alpha chain is cleaved by C5 convertase, resulting in a small 74 amino acid long C5a fragment and a large C5b fragment (1580 amino acids). The conversion of C5 to C5b involves a large conformational change and results in subsequent C6 binding. For example, in some embodiments, human C5 has the sequence set forth in SEQ ID NO. 1 and cynomolgus monkey (Macaca fascicularis) C5 has the sequence set forth in SEQ ID NO. 6.

"Single-domain antibody" (single domain antibody, sdAb) is used herein to refer to an antibody polypeptide that recognizes and binds an antigen of interest through a single variable antibody domain, e.g., a single VH or a single VL. A single variable antibody domain of a single domain antibody is capable of recognizing and binding to an antigen of interest without pairing with another antibody variable domain. In this paper, the heavy chain variable domain containing single domain antibodies also called VHH.

The term "VHH" generally refers to antibodies comprising or consisting of only one heavy chain variable region, having antigen binding activity, i.e.antibodies comprising only one chain from the C-terminus to the N-terminus, FR4-CDR3-FR3-CDR2-FR2-CDR1-FR 1. VHH are typically heavy chain variable domains derived from heavy chain antibodies that lack light chains, also known as nanobodies. The VHH used in the present invention is preferably derived from a camelid, such as alpaca, or a humanized or sequence optimised form thereof (e.g. an affinity matured form to increase binding affinity). In some embodiments, a VHH of the invention is a monovalent monospecific polypeptide molecule consisting of or consisting essentially of a single heavy chain variable region (e.g., a heavy chain variable region of a heavy chain antibody).

The single domain antibodies or VHHs of the invention may also be comprised in larger polypeptides/proteins. Examples of polypeptides/proteins comprising a VHH of the invention include, but are not limited to, heavy chain antibodies (HcAb). "heavy chain antibody" as used herein refers to an antibody that does not have a light chain, e.g., it may comprise a VH-Fc or a VH-CH2-CH3 or a VH-hinge region-CH 2-CH3 from the N-to the C-segment, or may comprise a VH-CH1-CH2-CH3, and may encompass homodimers, e.g., heavy chain dimer antibodies that do not have a light chain. The heavy chain antibody of the present invention may contain VH derived from a standard antibody or VH derived from a single domain antibody. For example, the VH in the heavy chain antibody of the invention may be VHH. In some embodiments, the heavy chain antibodies of the invention may be heavy chain antibodies having framework regions and/or heavy chain constant regions derived from camelids (llamas, camels, especially alpacas), humanized forms thereof or sequence-optimized forms thereof (affinity matured forms), or fragments thereof (e.g., fragments comprising at least a portion of the constant regions). Heavy chain antibodies of the invention also encompass antibodies formed upon fusion of a heavy chain variable region or VHH to an Fc region (e.g., a human IgG Fc region, such as a human IgG1 or IgG4Fc region).

The term "antigen-binding fragment" of an antibody is a portion or segment of a full length or full antibody that has fewer amino acid residues than the full antibody or full length antibody, but which is capable of binding or competing with the full length antibody (i.e., with the full length antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen binding fragments may be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, fab ', F (ab') 2, fv, single chain Fv, diabody (diabody), single domain antibody (sdAb), nanobody. For example, fab fragments can be obtained by papain digestion of full length antibodies. In addition, complete antibody production F (ab ') 2, which is a dimer of Fab', is a bivalent antibody fragment by pepsin digestion under the disulfide bonds of the hinge region. F (ab ') 2 can be reduced under neutral conditions by breaking disulfide bonds in the hinge region, thereby converting F (ab ') 2 dimers to Fab ' monomers. The Fab' monomer is essentially a Fab fragment with a hinge region. Fv fragments consist of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but recombinant methods can also be used, using a synthetic linker peptide to join the two domains so that they are produced as a single protein chain in which the VL and VH regions pair to form a single chain Fv (scFv).

Herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. In the case of heavy chain antibodies, e.g. from a camelidae heavy chain antibody, a single VH domain (also referred to herein as a VHH domain) may be sufficient to confer antigen binding specificity. The VHH domain, like the heavy and light chain variable regions of conventional IgG antibodies, comprises four conserved Framework Regions (FRs) and three Complementarity Determining Regions (CDRs) and is arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. In some aspects according to the invention, one or more residues in the variable region of the antibody may be modified, e.g., residue modifications, especially conservative residue substitutions, to one or more CDR regions and/or to one or more framework regions, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen binding capacity) of the parent antibody. In still other aspects, the antibody variable region may be modified by CDR grafting. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants can be constructed that mimic the properties of known antibodies. In such antibody variants, CDR sequences from known antibodies are grafted onto framework regions of different antibodies having different properties, and may be subjected to 1 to several residue mutations, such as back mutations, as needed to refine the desired properties of the antibody. The properties of the mutated and/or modified antibodies, such as target antigen binding properties or other desired functional properties, such as hemolysis inhibiting activity in the complement pathway, can be evaluated in vitro or in vivo assay.

"Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" of an antibody are regions of the antibody variable domain (VH or VHH) that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are numbered sequentially from the N-terminus, and are generally referred to as CDR1, CDR2, and CDR3. CDRs located within the antibody heavy chain variable domain are also referred to as HCDR1, HCDR2 and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, its CDR sequence can be determined using a variety of protocols well known in the art, such as Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops, kabat based on antibody sequence variability (Kabat et al Sequences of Proteins of ImmunologicalInterest, 4 th edition , U.S. Department of Health and Human Services, National Institutes of Health(1987)), AbM (University of Bath),Contact (University College London), International ImMunoGeneTics database (IMGT) (International immunogenetic information System, world Wide Web IMGT. Circuits. Fr /), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures (North et al, "ANew Clustering of Antibody CDR Loop Con formats", journal of Molecular Biology,406,228-256 (2011)).

The following is the range of regions of CDRs defined using kabat, abM, chothia, contact and IMGT schemes.

CDR	Kabat protocol	AbM protocol	Chothia protocol	Contact scheme	IMGT scheme
						LCDR1 (Kabat and Chothia numbering systems)	L24-L34	L24-L34	L26-L32	L30-L36	L27-L32
LCDR2 (Kabat and Chothia numbering systems)	L50-L56	L50-L56	L50-L52	L46-L55	L50-L52
						LCDR3 (Kabat and Chothia numbering systems)	L89-L97	L89-L97	L91-L96	L89-L96	L89-L96
HCDR1 (Kabat numbering system)	H31-H35B	H26-H35B	H26-H32	H30-H35B	H26-H33
						HCDR1 (Chothia numbering system)	H31-H35	H26-H35	H26-H32	H30-H35	H26-H33
HCDR2 (Kabat and Chothia numbering systems)	H50-H65	H50-H58	H53-H55	H47-H58	H51-H57
						HCDR3 (Kabat and Chothia numbering systems)	H95-H102	H95-H102	H96-H101	H93-H101	H93-H102

In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.

CDRs can also be determined based on having the same Kabat numbering positions as the reference CDR sequences. In the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues) it is meant a numbering position according to the Kabat numbering system (Kabat et al ,Sequences of Proteins of Immunological Interest, 5^thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)）.

In one embodiment, the CDRs in the antibody molecules of the invention are determined by IMGT rules.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The native immunoglobulin "Fc domain" or "Fc region" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in natural antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH 2 domain and CH3 domain) derived from the heavy chain of antibodies of the IgG, igA and IgD classes, or the second, third and fourth constant domains (CH 2 domain, CH3 domain and CH4 domain) derived from the two heavy chains of antibodies of the IgM and IgE classes. Unless otherwise indicated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) (Kabat et al Sequences of Proteins of Immunological Interes, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD, 1991). Herein, the terms "Fc region", "Fc portion" and "Fc fragment" do not include the heavy chain variable region VH and light chain variable region VL and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but may include a hinge region at the N-terminus of the heavy chain constant region in some cases. In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Asn231 to the carboxy terminus of the heavy chain. Furthermore, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. In some embodiments, the human IgG heavy chain Fc region carries a hinge sequence or a portion of a hinge sequence of a native immunoglobulin at the N-terminus, e.g., a sequence from E216 to P230 or a sequence from D221 to P230 according to EU numbering.

The term "chimeric antibody" is an antibody molecule in which (a) a constant region or portion thereof is altered, substituted, or exchanged such that an antigen binding site is linked to a constant region of different or altered class, effector function, and/or species origin, or to an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), etc., that confers novel properties to the chimeric antibody, or (b) a variable region or portion thereof is altered, substituted, or exchanged with a variable region of different or altered antigen specificity. For example, camelid heavy chain antibodies may be modified by replacing their constant regions with those derived from human immunoglobulins. Due to the replacement with human constant regions, the chimeric antibody can retain its specificity in recognizing antigen while having reduced antigenicity in humans as compared to the original camelid antibody.

As used herein, a "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., alpaca monoclonal antibody) while being less immunogenic when administered to a human as a therapeutic. This can be accomplished, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterpart (i.e., replacing the constant region and the portion of the variable region that does not participate in binding with the counterpart of a human antibody).

As used herein, the term "fusion protein" is used interchangeably with "chimeric polypeptide" and refers to a larger polypeptide formed by fusion of at least two heterologous polypeptide sequences, optionally through a linker. Fusion proteins may be produced by recombinant expression.

As used herein, the term "monospecific" refers to a polypeptide/protein molecule having one or more antigen binding sites, each of which binds to the same epitope of the same antigen. As used herein, the term "multispecific" refers to a polypeptide/protein molecule having at least two antigen binding sites that bind to different epitopes (different epitopes on the same antigen or different epitopes on different antigens).

The linker may be a connecting peptide. In this context, the term "connecting peptide" refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and/or serine (S) and/or threonine residues (T), used alone or in combination, or from the hinge region of an immunoglobulin. In one embodiment, the linker peptide has a length of 5-50 amino acids, e.g., 10,15,20,25,30 amino acids in length. In one embodiment, the connecting peptide comprises the amino acid sequence (G4S) n, where n is an integer equal to or greater than 1, e.g., n is an integer of 2,3,4,5,6, or 7. In one embodiment, the connecting peptide comprises the amino acid sequence TS (G4S) n, where n is an integer equal to or greater than 1, e.g., n is an integer of 2,3,4,5,6, or 7. In one embodiment, the connecting peptide comprises the amino acid sequence G (G4S) n, where n is an integer equal to or greater than 1, e.g., n is an integer of 2,3,4,5,6, or 7. In yet another embodiment, the connecting peptide is a hinge region from an immunoglobulin, e.g., a hinge region amino acid sequence comprising "CPPC", e.g., the amino acid sequence "EPKSCDKTHTCPPCP (SEQ ID NO: 38)" or "EPKSSDKTHTCPPCP (SEQ ID NO: 39)" or "ESKYGPPCPPCP (SEQ ID NO: 37)".

As used herein, the term "anti," "binding," or "specific binding" means that the binding is selective for an antigen and distinguishable from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen may be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by Radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or Surface Plasmon Resonance (SPR).

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

"Percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in the present specification, after aligning the candidate sequence to the particular amino acid sequence shown in the present specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have substantial identity, e.g., at least 80%,85%,90%,95%, 97%, 98% or 99% or more identity, to the antibody molecules specifically disclosed herein and sequences thereof. The variant may comprise a conservative change.

For polypeptide sequences, "conservative changes" include substitutions, deletions, or additions to the polypeptide sequence, but do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following list 8 groups contain amino acids with conservative substitutions for each other, 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine (C), methionine (M). In some embodiments, the term "conservative sequence changes" is used to refer to amino acid modifications that do not significantly affect or alter the antigen binding characteristics of interest of an antibody molecule or binding protein molecule of the invention that contains an amino acid sequence. For example, conservatively modified variants retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100-110% or more, of the binding affinity for the antigen of interest relative to the parent antibody or binding protein.

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells, and prokaryotic cells, e.g., E.coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide that comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression, and the other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporated into the recombinant polynucleotide.

The terms "individual" or "subject" are used interchangeably to refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

The term "treating" includes administration of a composition or antibody to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, to alleviate symptoms, or to prevent or inhibit further development of a disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of a disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic inhibition or alleviation of symptoms after manifestation of a disease. The term "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder.

As used herein, the term "complement C5-associated disease or disorder" refers to a disease or disorder in which unregulated C5 function may result in a disease or disease phenotype, for example, due to deregulated C5 activation, for example, increased C5 activation.

The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. for administration with the active substance.

The term "pharmaceutical composition" refers to a composition that exists in a form that is effective to allow the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered.

The terms "pharmaceutical combination", "combination product", "pharmaceutical combination" or "combination product" refer to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-immobilized combination" means that the active ingredients (e.g., (i) an antibody molecule of the invention, and (ii) other therapeutic agent, or two or more antibody molecules of the invention, and other therapeutic agents) are administered to a patient simultaneously, without specific time constraints, or sequentially at the same or different time intervals, in separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. In some embodiments, the antibody molecules of the invention and other therapeutic agents used in the pharmaceutical combination are administered at levels that do not exceed their levels when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and/or time intervals of the two or more active agents are preferably selected so that the combined use of the parts will produce an effect in the treatment of a disease or condition that is greater than that achieved by either component alone. The components can be in the form of separate preparations, and the preparations can be the same or different.

The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities to treat the diseases described herein. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, e.g., in a single capsule with a fixed ratio of active ingredients. Or such administration includes co-administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the pharmaceutical combination in treating the disorders or conditions described herein.

The term "effective amount" refers to an amount or dose of an antibody or fragment or composition or combination of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention.

"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are less than the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100% relative to an untreated subject.

"Prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, since the prophylactic dose is administered in the subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to and facilitates detection of an agent (such as a polynucleotide probe or antibody) to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or in the case of enzymatic labels may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. In some embodiments, the tag is His or biotin.

"Subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, like a tissue sample or biopsy or puncture from a fresh, frozen and/or preserved organ or tissue, blood or any blood component, body fluids such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid, cells from a subject at any time of gestation or development. In some embodiments, the sample is blood or serum.

Single domain antibodies or VHH antibodies

In one aspect, the invention relates to a single domain antibody (single domain Antibody, sdAb), particularly a VHH antibody, that specifically binds to a human complement C5 protein and/or specifically binds to a monkey (e.g., cynomolgus monkey) complement C5 protein, preferably simultaneously specifically binds to a human complement C5 protein and a monkey (e.g., cynomolgus monkey) complement C5 protein.

Single domain antibodies or VHH antibodies have a molecular weight of about one tenth of that of human IgG molecules and a physical diameter of only a few nanometers. Due to the small molecular size, single domain antibodies have the advantage of high stability and solubility, as well as the ability to recognize hidden antigenic sites, over conventional four-chain antibodies. In addition, single domain antibodies are also cheaper to prepare than conventional four-chain antibodies. In addition to application as a single molecule, single domain antibodies are also suitable components for constructing multispecific molecules.

In some embodiments, the single domain antibodies of the invention are VHH antibodies comprising or consisting of a heavy chain variable region, typically having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 through FR4 refer to framework regions 1 through 4 and CDR1 through CDR3 refer to complementarity determining regions 1-3. The CDR sequences in the VHH variable can be defined according to any CDR definition scheme described in the "definition" section, e.g. according to AbM, chothia, kabat, IMGT or any combination thereof, preferably the boundaries of the three CDRs in the VHH sequence can be defined by IMGT.

In some embodiments, the VHH of the invention against complement C5 comprises:

(i) Three Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 contained in the VH set forth in any one of SEQ ID NOS 34, 33 and 11-14,

Or (ii) three Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 contained in an amino acid sequence encoded by the nucleic acid sequence of any one of SEQ ID NOs 32, 31 and 7 to 10;

Or (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) together in said three CDR regions relative to the sequence of (i) or (ii);

Preferably, CDR sequences are defined according to IMGT.

In some embodiments, the VHH antibody of the invention against complement C5 comprises or consists of a heavy chain variable region comprising:

(i) Three Complementarity Determining Regions (CDRs) contained in the VH set forth in any one of SEQ ID NOs 34, 33 and 11-14,

Or (ii) three Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 contained in an amino acid sequence encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs 32, 31 and 7 to 10;

Or (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in total over the three CDR regions relative to the sequence of (i) or (ii),

Preferably, CDR sequences are defined according to IMGT.

In some embodiments, the CDR1 comprised by the VHH antibody of the invention against complement C5 comprises or consists of an amino acid sequence selected from any one of SEQ ID NOS: 15-18, or the CDR1 comprises or consists of an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from any one of SEQ ID NOS: 15-18.

In some embodiments, the CDR2 comprised by the VHH antibody of the invention against complement C5 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 19, or the CDR2 comprises or consists of an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 19.

In some embodiments, the CDR3 comprised by the VHH antibody of the invention against complement C5 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 20 or 21, or the CDR3 comprises or consists of an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 20 or 21.

In some embodiments, the anti-complement C5 VHH antibodies of the invention comprise CDR1, CDR2 and CDR3, wherein,

(I) CDR1 comprises or consists of an amino acid sequence selected from any one of SEQ ID NO. 15-18, or CDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from any one of SEQ ID NO. 15-18;

(ii) CDR2 comprises or consists of an amino acid sequence as shown in SEQ ID NO. 19, or CDR2 comprises or consists of an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO. 19, and

(Iii) CDR3 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 20 or 21, or CDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 20 or 21.

(I) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 20;

Or (ii) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 16, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 21;

Or (iii) CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 17, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 21;

In some embodiments, the VHH antibodies of the invention against complement C5:

(i) Comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any of SEQ ID NOs 34, 33 and 11-14;

(ii) Comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs 34, 33 and 11-14;

(iii) An amino acid sequence comprising or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence encoded by a nucleic acid sequence selected from any one of SEQ ID NOs 32, 31 and 7 to 10;

(iv) Comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of any one of SEQ ID NOs 32, 31 and 7 to 10;

or (v) comprises an amino acid sequence having 1 or more (preferably NO more than 10, more preferably NO more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence set out in any one of SEQ ID NOS: 34, 33 and 11-14 or to the amino acid sequence encoded by the nucleic acid sequence set out in any one of SEQ ID NOS: 32, 31 and 7-10, preferably the amino acid changes do not occur in the CDR regions.

In some embodiments, the VHH antibody of the invention against complement C5 comprises or consists of a heavy chain variable region that:

Preferably, the VHH antibody against complement C5 of the invention comprises or consists of an amino acid sequence selected from the amino acid sequences set out in any one of SEQ ID NOS: 34, 33 and 11-14 or a nucleic acid sequence selected from the nucleic acid sequences set out in any one of SEQ ID NOS: 32, 31 and 7-10.

In some embodiments, the amino acid change comprises a substitution, insertion, or deletion of an amino acid. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., in the FR). In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid.

In some embodiments, the invention also provides functional variants of the single domain antibodies or VHH antibodies of the invention. The functional variants may be obtained by introducing mutations into the nucleic acid sequences encoding the exemplary single domain antibodies of the invention, e.g., into CDR sequences and/or FR sequences, using methods well known in the invention, e.g., by random or site-directed mutagenesis, and subsequently screening (e.g., by phage display library screening) for variants that retain the desired properties. Typically, the functional variant retains significant sequence identity to the parent single domain antibody (or VHH antibody). Preferably, the functional variant retains the desired biological properties of the parent single domain antibody (or VHH antibody), e.g., the variant has comparable (e.g., at least 50%,60%,70%,80%, preferably 90% or more) biological activity, or improved biological activity (e.g., 110-150% or more) relative to the biological activity of the parent. Such desirable biological properties include, for example, but are not limited to, antigen binding affinity of interest (as measured by KD values), blocking activity of antigen of interest binding to a receptor (as measured by IC50 values), blocking human complement hemolytic activity (as measured by EC50 values), and the like.

In some embodiments, the invention provides affinity variants of the single domain antibodies or VHH antibodies of the invention. Preferably, the affinity variant exhibits one or more amino acid changes in amino acid sequence relative to the parent single domain antibody from which it is derived, wherein the affinity variant has an altered binding affinity for the antigen of interest as compared to the parent antibody. Typically, the affinity variant exhibits improved antigen binding affinity over the parent. The improvement may be, but is not limited to, a lower KD value, a faster dissociation rate, or an increase (or decrease) in cross-reactivity with a non-human species homologous protein to the antigen of interest. Affinity variants tend to have one or more amino acid residue substitutions in the CDRs compared to the parent. The substitution may be a conservative substitution or a non-conservative substitution. In one embodiment, the affinity variants of the invention have a total of no more than 10, no more than 6, or 1-5, e.g., 1,2, 3, 4, or 5 amino acid substitutions in CDR1-3 relative to the parent. Affinity variants can be obtained by a variety of affinity maturation methods known in the art, including mutant CDRs, chain shuffling, use of e.coli mutator strains, DNA shuffling, phage display, and the like.

In some embodiments, the VHH antibodies of the invention against complement C5 comprise CDR amino acid sequences and/or Framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunization of camelids (e.g., alpaca). In some embodiments, VHH monoclonal antibodies of the invention that are derived from camelidae heavy chain antibodies may be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelidae mammalian species. Thus, in one embodiment, the VHH antibody of the invention is a camelid antibody.

Typically, humanization is performed in a manner that maintains the favorable binding properties of single domain antibodies. Assays for determining the biological properties of humanized single domain antibodies, such as binding affinity and the like, are well known in the art to determine and select the appropriate humanized residue mutation or combination of mutations.

In one embodiment, the VHH antibody of the invention that is anti-complement C5 is a humanized antibody. For example, the original VHH sequences are humanized using a "best match approach" which includes:

(i) The amino acid sequence of the VHH framework region is compared and analyzed by utilizing a human germline V gene database, and the optimal germline sequence, such as human GERMLINE IGHV-23 x 01 sequence, is selected;

(ii) Replacing the best matched human CDR sequence with a VHH CDR sequence, and optionally generating a humanized VHH sequence by back mutating a plurality of residues of the framework region, optionally also performing Post-translational modification (Post-translational modification, PTM) removal;

(iii) Optionally sequencing the VHH antibody.

Heavy chain antibodies

In another aspect of the invention, the invention provides a heavy chain antibody comprising a heavy chain variable region of a VHH antibody of the invention.

In some embodiments, a single domain antibody or VHH of the invention (e.g., a camelid VHH or humanized form thereof) can be linked to a constant region, e.g., an Fc region, of a human antibody to produce a heavy chain antibody comprising VHH-Fc. In one embodiment, the heavy chain antibody is an Fc region comprising a single domain antibody of the invention and located at the C-terminus thereof. In some embodiments, the VHH and Fc are linked by a hinge region, such as the hinge region of an IgG (e.g., the hinge region of IgG1, 2, 3, or 4).

In some embodiments, an anti-C5 protein heavy chain antibody of the invention comprises a VHH or a heavy chain variable region therein as defined herein, and a heavy chain constant region or an Fc region of a heavy chain constant region. In some embodiments, a linking peptide, such as an antibody hinge region, e.g., an IgG hinge region, is included between the VHH or heavy chain variable region and the heavy chain constant region or Fc region. In some embodiments, a hinge region is included between the VHH or heavy chain variable region and the heavy chain constant region or Fc region. In some embodiments, the hinge region is a hinge region derived from human IgG1, 2, 3, or 4. In some embodiments, the hinge region is a hinge region of human IgG 1. In some embodiments, the hinge region is a native human IgG1 hinge region, or a variant thereof, e.g., a variant of C220S thereof. In some embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO. 38 or SEQ ID NO. 39.

In one embodiment, the heavy chain antibody comprises an Fc portion from a camelid (e.g., alpaca). In one embodiment, the heavy chain antibody is produced and isolated by immunization of the camelid, e.g., alpaca. Various means are known in the art for immunizing camelids and isolating produced VHH antibodies or heavy chain antibodies directed against an antigen of interest.

In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (e.g., cynomolgus monkey) antibody, e.g., a constant region from human IgG1, human IgG2, human IgG3, or human IgG 4.

In some embodiments, the heavy chain antibody comprises an Fc portion from a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3 or human IgG4 Fc region, preferably a human IgG1Fc region. In some embodiments, when the Fc region is linked to a VHH antibody via a hinge region, the Fc region itself is free of hinge regions. In some embodiments, the Fc region linked to the VHH antibody via a hinge region is derived from IgG1 comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 24.

In one embodiment, a heavy chain antibody according to the invention may dimerize via an Fc region with another polypeptide chain comprising an Fc region (e.g., another heavy chain antibody that is the same or different). Thus, in one embodiment, the invention also provides a homologous or heteromultimeric protein comprising a heavy chain antibody of the invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

In some embodiments, the heavy chain antibody:

(i) Comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 36, 35 and 27 to 30, or

(Ii) Comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs 36, 35 and 27 to 30, or

(Iii) Comprising an amino acid sequence having 1 or more (preferably NO more than 10, more preferably NO more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence set out in any one of SEQ ID NOs 36, 35 and 27 to 30, preferably said amino acid changes do not occur in the CDR regions.

In addition to the above-described single domain antibodies (in particular VHH antibodies) and heavy chain antibodies, the invention also provides other fusion proteins/chimeric polypeptides, such as bispecific or multispecific antibodies, comprising at least one single domain antibody or VHH or heavy chain antibody according to the invention.

In some embodiments, the fusion protein/chimeric polypeptide of the invention comprises a single domain antibody or VHH or heavy chain antibody according to the invention. In another embodiment, the fusion protein/chimeric polypeptide of the invention comprises a plurality of single domain antibodies or VHH or heavy chain antibodies according to the invention. The fusion proteins according to the invention may be monospecific or may be multispecific. In some embodiments, the fusion protein/chimeric polypeptide of the invention comprises only specificity for complement C5. In yet another embodiment, the fusion protein/chimeric polypeptide of the invention comprises, in addition to the single domain antibody or VHH or heavy chain antibody(s) of the invention directed against complement C5, a specificity for another antigen.

In one aspect, the invention also provides an immunoconjugate comprising a single domain antibody or VHH antibody or heavy chain antibody of the invention conjugated to another active agent (e.g., an anti-hemolytic drug) or a label to form the immunoconjugate.

Properties of the VHH antibodies and/or heavy chain antibodies of the invention

The single domain antibodies (particularly VHH antibodies) of the invention that specifically bind to human or monkey complement C5 or heavy chain antibodies comprising the single domain antibodies have one or more biological activities selected from the group consisting of:

(i) Binding human complement C5 with high affinity, and/or monkey, e.g., cynomolgus monkey complement C5;

(ii) Exhibits cross-reactivity to human and monkey, e.g., cynomolgus monkey complement C5;

(ii) Inhibiting hemolysis in the classical pathway of complement immunity, e.g., inhibiting hemolysis in human serum and/or monkey, e.g., cynomolgus monkey serum in the classical pathway of complement immunity, e.g., greater activity than known anti-C5 antibodies, e.g., eculizumab;

(iii) Inhibiting hemolysis in the alternative complement immune pathway, e.g., inhibiting hemolysis in human serum and/or monkey, e.g., cynomolgus monkey serum in the alternative complement immune pathway, e.g., greater activity than known anti-C5 antibodies, e.g., eculizumab;

(iv) Blocking human complement hemolytic activity or blocking monkey complement hemolytic activity, e.g., greater than known anti-C5 antibodies, e.g., eculizumab;

(v) With a higher heat stability, for example being able to withstand heat treatment at 70 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃ or 79 ℃ (for example heat treatment for 1 hour), preferably being able to withstand heat treatment at 80 ℃, 81 ℃, 82 ℃, 83 ℃, or 84 ℃ (for example heat treatment for 1 hour).

V. Polynucleotide, vector and host

The invention provides nucleic acids encoding any of the above molecules of the invention (single domain antibodies or heavy chain antibodies). Vectors comprising the nucleic acids are also provided. In one embodiment, the vector is an expression vector (e.g., a pCDNA vector, such as pCDNA 3.1). Host cells comprising the nucleic acids or the vectors are also provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., CHO cell or 293 cell, e.g., 293FT cell). In another embodiment, the host cell is prokaryotic.

In one aspect, the invention provides nucleic acids encoding a single domain antibody or VHH or heavy chain antibody to any of the above anti-C5 proteins. The polypeptide encoded by the nucleic acid is capable of exhibiting antigen binding capacity against human or monkey (e.g., cynomolgus monkey) C5 protein when expressed from a suitable expression vector. In some embodiments, the nucleic acid is operably linked in-frame to a nucleic acid encoding another peptide/polypeptide, such that when expressed from a suitable expression vector, a fusion protein or chimeric polypeptide comprising the single domain antibody or VHH and the other peptide/polypeptide is produced. For example, in some embodiments, the nucleic acid is operably linked in-frame to a nucleic acid encoding an Fc region (e.g., a human Fc region) such that when expressed from a suitable expression vector, a heavy chain antibody comprising the single domain antibody or VHH and Fc region is produced.

For ease of production and purification, single domain antibodies or VHH domains or heavy chain antibodies may be fused at the N-terminus to a secretory signal peptide, and/or a tag peptide such as a hexahistidine tag or biotin tag to facilitate purification. In some embodiments, the signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO. 22.

As will be apparent to those of skill in the art, because of the degeneracy of the codons, each antibody or polypeptide amino acid sequence may be encoded by a variety of nucleic acid sequences.

In some embodiments, the nucleic acids of the invention comprise nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs 33-36, 27-30 and 11-14, or encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 33-36, 27-30 and 11-14. In some embodiments, a nucleic acid of the invention comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth in any one of SEQ ID NOs 32, 31 and 7-10, or comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence set forth in any one of SEQ ID NOs 32, 31 and 7-10.

The nucleic acid sequences encoding the molecules of the invention may be produced using methods well known in the art, for example by de novo solid phase DNA synthesis, or by PCR amplification.

In one embodiment, one or more vectors comprising a nucleic acid of the invention are provided. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). In a preferred embodiment, the expression vector is pCDNA, e.g., pCDNA3.1.

In one embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In some embodiments, host cells comprising the expression vectors of the invention are provided. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce an antibody molecule of the invention. Host cells suitable for replication and supporting expression of the antibody molecules of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, as desired, and a large number of vector-containing cells can be incubated for inoculation of a large-scale fermentor to obtain a sufficient amount of the molecules of the invention for clinical use. Suitable host cells include prokaryotic microorganisms, such as E.coli, eukaryotic microorganisms, such as filamentous fungi or yeast, or various eukaryotic cells, such as Chinese hamster ovary Cells (CHO), insect cells, and the like. Mammalian cell lines suitable for suspension culture may be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK 293 or 293F cells or 293FT cells), baby hamster kidney cells (BHK), monkey kidney cells (CV 1), african green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, sp2/0, and the like. Mammalian host cell lines suitable for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO or HEK293 cell or 293FT cell.

VI production and purification of the molecules of the invention

In a further aspect, the invention provides a method for producing a molecule of the invention (a single domain antibody, a VHH antibody or a heavy chain antibody) comprising culturing a host cell comprising a polypeptide chain encoding said polypeptide chain under conditions suitable for expression of said polypeptide chain, optionally further comprising assembling the polypeptide chain to produce said molecule under conditions suitable for assembly of said polypeptide chain to said molecule.

For recombinant production, polynucleotides encoding the polypeptide chains of the molecules of the invention may be inserted into one or more vectors for further cloning and/or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). Once an expression vector comprising one or more polynucleotides of the invention has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. Various techniques may be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

Antibody molecules prepared as described herein may be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., protein a affinity chromatography), size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art.

The purity of the antibody molecules of the invention may be determined by any of a variety of well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activity of the antibody molecules provided herein can be identified, screened, or characterized by a variety of assays known in the art.

VII assay

The molecules provided herein (single domain antibodies, VHH antibodies, or heavy chain antibodies) can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art.

For binding of the molecules of the invention to human complement C5 protein and/or monkey (e.g., cynomolgus monkey) complement C5 protein, it can be determined by methods known in the art, such as ELISA, western blot, etc., or exemplary methods disclosed in the examples herein. For example, the binding kinetics of the molecule (e.g., K _D values) can be determined in a biological optical interferometry using human or monkey complement C5 protein. In some embodiments, the binding equilibrium dissociation constant of a molecule to human or monkey complement C5 protein is determined using a biological optical interferometry (e.g., fortebio oct system affinity measurement) such as the method shown in example 3.

The hemolysis inhibiting effect of the molecules of the invention may be measured by methods known in the art, such as in vitro assays and/or in vivo animal experiments. For example, a hemolysis assay, such as the method shown in example 4, can be used and the hemolysis inhibition of the classical pathway of complement immunity and/or the alternative pathway of complement immunity by the molecule is detected.

Pharmaceutical composition, pharmaceutical combination and kit

In one aspect, the invention provides compositions, e.g., pharmaceutical compositions, comprising one or more (e.g., two or three) molecules of the invention (e.g., a single domain antibody, a VHH antibody, or a heavy chain antibody, or an immunoconjugate, etc.). In some embodiments, the pharmaceutical composition is a pharmaceutical formulation.

In one embodiment, the compositions of the invention comprise two or three molecules of the invention, e.g., two or three single domain antibodies, VHH antibodies, heavy chain antibodies, or immunoconjugates of the invention that are anti-complement C5.

In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients as known in the art, including buffers.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. For the use of pharmaceutical excipients and their use, see also the literature ("Handbook of Pharmaceutical Excipients", eighth edition, R.C.Rowe, P.J.Seskey and s.c. Owen, pharmaceutical Press, london, chicago).

The compositions or medicaments or formulations of the present invention may contain, in addition to one or more molecules of the present invention, other therapeutic agents as required for the particular indication being treated, preferably without adversely affecting the activity of each other. Thus, in one embodiment, a composition or formulation or medicament, e.g., a pharmaceutical composition, comprises a combination of one or more molecules of the invention, and optionally one or more other therapeutic agents.

The composition or medicament or formulation of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection), powders or suspensions, liposomes, and suppositories. The compositions or medicaments or formulations of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). The preferred form depends on the intended mode of administration and the therapeutic use.

The invention also provides a pharmaceutical combination or pharmaceutical combination product comprising:

(1) Molecules of the invention such as single domain antibodies or VHH or heavy chain antibodies or immunoconjugates and one or more other therapeutic agents;

(2) Two or more molecules of the invention, such as a single domain antibody or VHH or heavy chain antibody or immunoconjugate;

(3) Two or more molecules of the invention, such as a single domain antibody or VHH or heavy chain antibody or immunoconjugate, and one or more other therapeutic agents.

In some embodiments, the invention also provides a kit comprising the pharmaceutical combination, e.g., the kit comprises one or more containers comprising a pharmaceutical composition comprising two or more molecules of the invention, e.g., a single domain antibody or a VHH or heavy chain antibody, within the same package. In some embodiments, two or more molecules of the invention are in the same container, or in separate containers.

In some embodiments, the invention also provides a kit of parts comprising the pharmaceutical combination, e.g. the kit of parts comprising, in the same package:

(i) A first container containing a pharmaceutical composition comprising one or more molecules of the invention, e.g., a single domain antibody or a VHH or heavy chain antibody (in some embodiments, two or more molecules of the invention are in the same container, or in separate containers);

(ii) A second container containing a pharmaceutical composition comprising one or more other therapeutic agents (in some embodiments, two or more other therapeutic agents are in the same container, or in separate containers).

IX. uses and methods

In one aspect, the invention provides a method for preventing or treating a complement C5-associated disease or disorder or for blocking or inhibiting complement hemolysis in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., a single domain antibody, a VHH antibody or a heavy chain antibody, or an immunoconjugate, etc.), or a composition or a medicament or formulation comprising the same or a pharmaceutical combination comprising the same.

In other aspects, the invention provides the use of a molecule of the invention or a composition or pharmaceutical combination comprising the same in the manufacture or preparation of a medicament for use as described herein, e.g. for the prevention or treatment of a complement C5-associated disease or disorder as referred to herein or for blocking or inhibiting complement hemolysis.

In other aspects, the invention provides a molecule of the invention or a composition or pharmaceutical combination comprising the same for use in therapy, e.g. for use as described herein, e.g. for preventing or treating a complement C5-associated disease or disorder as referred to herein or for blocking or inhibiting complement hemolysis.

In some embodiments, the subject has (e.g., elevated levels of, e.g., nucleic acid or protein levels of) complement C5 protein therein.

In some embodiments, the disease treatment will benefit from inhibiting complement C5 protein at the nucleic acid or protein level.

In some embodiments, the complement C5-associated disease or disorder can be a disease requiring hemolysis inhibition, e.g., a disease requiring inhibition of hemolysis of the classical pathway of complement immunity and/or the alternative pathway of complement immunity.

In some embodiments, the molecules of the invention or compositions or medicaments or formulations comprising the same delay the onset of the disorder and/or symptoms associated with the disorder.

In some embodiments, the complement hemolysis is complement classical pathway hemolysis and/or complement alternative pathway hemolysis.

In some embodiments, a molecule of the invention or a composition or a drug or formulation or a combination of drugs comprising the same (e.g., a combination of drugs comprising two or more molecules of the invention) can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for preventing and/or treating a related disease or disorder mentioned herein or for blocking or inhibiting complement hemolysis.

The route of administration of the molecules of the invention or of the compositions or medicaments or formulations comprising them is according to known methods, for example injection or infusion.

X.diagnosis and detection

In certain embodiments, the molecules provided herein (e.g., single domain antibodies to complement C5 proteins, VHH antibodies, heavy chain antibodies, or immunoconjugates, etc.) can be used to detect the presence of complement C5 in a biological sample.

The term "detection" as used herein, including quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a bodily fluid.

In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein under conditions that allow it to bind complement C5, and detecting whether a complex is formed between the molecule and complement C5. The formation of the complex indicates the presence of complement C5. The method may be an in vitro or an in vivo method. In one embodiment, the molecules of the invention are used to select a subject suitable for treatment with an inhibitor against complement C5 (e.g., an antibody against complement C5, e.g., a molecule of the invention), e.g., wherein complement C5 is a biomarker for selecting the subject.

In certain embodiments, a labeled molecule of the invention (e.g., a single domain antibody of the anti-complement C5 protein of the invention, a VHH antibody, a heavy chain antibody, or an immunoconjugate, etc.) is provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radiolabels), as well as moieties that are detected indirectly, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions.

In some embodiments, the tag is a tag such as a biotin or His tag.

In some embodiments provided herein, the sample is obtained prior to treatment with a molecule of the invention or a composition, medicament or formulation comprising the same. In some embodiments, the sample is obtained prior to use with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

In some embodiments, complement C5 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a certain treatment after a treatment interval.

In some embodiments, a method of treating a disease of the invention is provided, the method comprising assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of complement C5, thereby determining a complement C5 value, comparing the complement C5 value to a control value (e.g., a value in a normal individual), and if the complement C5 value is greater than the control value, administering to the subject a therapeutically effective amount of a molecule of the invention, or a composition, medicament, or formulation comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.

These and other aspects and embodiments of the application are described in, and exemplified by, the accompanying drawings (which are immediately following the brief description of the drawings) and detailed description of the application. Any or all of the features discussed throughout this application may be combined in various embodiments of the application. The following examples further illustrate the application, however, it is to be understood that the examples are presented by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

Example 1 antigen preparation and alpaca immunization

EXAMPLE 1.1 expression and purification of recombinant human complement C5 protein (hereinafter abbreviated as hC 5)

Human full-length complement C5 protein comprising alpha and beta chains (UniProt#P01031), has a relative molecular mass of about 188.3kDa, and is expressed by recombinant cloning of the hC 5-encoding DNA molecule (Kirschner, NM-001735.2) into a mammalian expression vector (pCDNA3.1) and adding Avi-tag (GLNDIFEAQKIEWHE) and 10xHistag (HHHHHHHHHH) to the C-terminus, using PEI (Polysciences, cat# 24765-1) transfection reagents, and transfection of suspension 293 cells (Expi 293, thermofisher). Cell culture supernatants were harvested after 7 days of culture in a cell shaker at 220rpm,37℃at 8% CO2, and hC5 protein was purified using a nickel column (HIS TRAPTM excel cat# 17-3712-05, GEHealthcare) to obtain protein with a purity of greater than 85% dissolved in PBS.

Human complement C5 amino acid sequence:

QEQTYVISAPKIFRVGASENIVIQVYGYTEAFDATISIKSYPDKKFSYSSGHVHLSSENKFQNSAILTIQPKQLPGGQNPVSYVYLEVVSKHFSKSKRMPITYDNGFLFIHTDKPVYTPDQSVKVRVYSLNDDLKPAKRETVLTFIDPEGSEVDMVEEIDHIGIISFPDFKIPSNPRYGMWTIKAKYKEDFSTTGTAYFEVKEYVLPHFSVSIEPEYNFIGYKNFKNFEITIKARYFYNKVVTEADVYITFGIREDLKDDQKEMMQTAMQNTMLINGIAQVTFDSETAVKELSYYSLEDLNNKYLYIAVTVIESTGGFSEEAEIPGIKYVLSPYKLNLVATPLFLKPGIPYPIKVQVKDSLDQLVGGVPVTLNAQTIDVNQETSDLDPSKSVTRVDDGVASFVLNLPSGVTVLEFNVKTDAPDLPEENQAREGYRAIAYSSLSQSYLYIDWTDNHKALLVGEHLNIIVTPKSPYIDKITHYNYLILSKGKIIHFGTREKFSDASYQSINIPVTQNMVPSSRLLVYYIVTGEQTAELVSDSVWLNIEEKCGNQLQVHLSPDADAYSPGQTVSLNMATGMDSWVALAAVDSAVYGVQRGAKKPLERVFQFLEKSDLGCGAGGGLNNANVFHLAGLTFLTNANADDSQENDEPCKEILRPRRTLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLGRLHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQGVGISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTSGMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSICNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKAFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKREALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVIKWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRLSMDIDVSYKHKGALHNYKMTDKNFLGRPVEVLLNDDLIVSTGFGSGLATVHVTTVVHKTSTSEEVCSFYLKIDTQDIEASHYRGYGNSDYKRIVACASYKPSREESSSGSSHAVMDISLPTGISANEEDLKALVEGVDQLFTDYQIKDGHVILQLNSIPSSDFLCVRFRIFELFEVGFLSPATFTVYEYHRPDKQCTMFYSTSNIKIQKVCEGAACKCVEADCGQMQEELDLTISAETRKQTACKPEIAYAYKVSITSITVENVFVKYKATLLDIYKTGEAVAEKDSEITFIKKVTCTNAELVKGRQYLIMGKEALQIKYNFSFRYIYPLDSLTWIEYWPRDTTCSSCQAFLANLDEFAEDIFLNGC（SEQ ID NO:1）.

EXAMPLE 1.2 alpaca immunization

Mixing hC5 protein prepared in 1) with Freund's adjuvant, emulsifying, injecting subcutaneously to immunize alpaca (Shanghai primary organism) at a time interval of once every 10 days, injecting subcutaneously to immunize 4 times, taking serum after the second and third immunization, centrifuging, collecting supernatant, and measuring anti-hC 5 antibody Titer (Titer) in serum by ELISA method.

Example 1.3 ELISA determination of alpaca serum anti-hC 5 antibody titres

100. Mu.L of hC5 prepared in 1.1 at 1. Mu.g/mL was coated on an ELISA plate (cat# 42592, costar) at 4℃overnight. The next day after PBST plate washing, PBS containing 2.5% skimmed milk (hereinafter referred to as PBSM) was used for blocking for 1 hour, 10uL camel serum was taken and diluted 103, 104, 105, 106, 107, 108 and 109 times by PBSM, respectively, to obtain serum dilutions with different dilution factors. 100uL of each concentration serum dilution is added into a sealed ELISA plate, incubated for 1 hour at 37 ℃, each well is washed 3 times by PBS (PBST) containing 0.1% Tween 20, 100uL of HRP-labeled anti-alpaca antibody (Goat PAb to Llama IgG (HRP), abcam product number: ab112786, diluted 1:10000) is added into each well after washing, incubation is carried out for 30 minutes at room temperature, the mixture is washed 3 times by PBST again, TMB substrate (thermofisher, 002023) is added for color development for 5-10 minutes, and an end solution (Shanghai, E661006-0200) is added for stopping the reaction, and the detection result is shown in figure 1.

The detection result shows that a large amount of anti-hC 5 antibodies are generated in immunized alpaca serum, a binding signal is not detected in non-immunized control alpaca serum, the antibody titer against hC5 in serum after the third immunization is obviously improved compared with that of serum after the second immunization, the antibody can be obviously detected after 100-ten thousand times dilution, and the alpaca immunization is successful and the subsequent experiment can be carried out.

Example 2 construction and screening of anti-hC 5 VHH antibody phage library

EXAMPLE 2.1 alpaca PBMC isolation and preparation of VHH monoclonal library

Alpaca peripheral blood after 4 th immunization is collected, alpaca peripheral mononuclear lymphocytes (PBMC) are obtained by separation through a lymphocyte separation tube (Daidae, cat# 7922021), total RNA (RNeasy plus Mini kit, cat# 74134, qiagen) is extracted by splitting the PBMC, the Total RNA is reversely transcribed into cDNA (SuperscriptTMIV First-STRAND SYNTHWSISSYSTEM, cat# 18091050, invirogen), the heavy chain antibody variable region in alpaca B cells is amplified through two rounds of Polymerase Chain Reaction (PCR) by taking the cDNA as a template, and primers used for the two rounds of PCR are respectively:

first round PCR forward primer GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO: 2);

first round PCR reverse primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 3);

second round PCR forward primer CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT (SEQ ID NO: 4);

the second round PCR reverse primer GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 5).

The amplified product was gel recovered and purified and then connected to phage display vector pComb3XTT (vast. RTM. Organism, P0831), and phage display vector inserted with VHH was transferred into TG1 cells (Lucigen) by electrotransformation to construct VHH bacteria clone library.

The VHH bacterial clone library was inoculated into 2XYT medium (Shanghai) containing ampicillin and 2% glucose for cultivation on a 37℃constant temperature shaker, M13K07 (accession number N0315S, NEB) was added to assist phage until the bacterial concentration OD600 reached 0.4-0.8, the cells were incubated at 37℃for 30 minutes, the cells were collected by centrifugation, and the cells were resuspended in 2XYT medium containing kanavidine and 1mM IPTG (Shanghai) for overnight cultivation at 30℃at 250 rpm. Overnight cultures were centrifuged at 4℃for 15 min at 9000g, supernatants were collected, sterilized with 0.22 μm filter (Millipore) and 1/5 volume of PEG-NaCl precipitated phage particles were added, incubated on ice for 1h, the phage suspension obtained was centrifuged at 4℃for 15 min at 9000g, the supernatant was discarded as clean as possible, phage particles were resuspended in PBS buffer and transferred to a fresh sterilized tube, and the phage display library obtained was placed in 4℃for short term storage or 20% glycerol final concentration was added, frozen in a-80℃refrigerator for long term storage.

Example 2.2 phage display screening of VHH Structure for anti-hC 5 antibodies

The biotin-labeled human complement C5 Protein (hC 5 Protein prepared in example 1) and the biotin-labeled cynomolgus complement C5 Protein (Cynomolgus Complement C Protein, his Tag, cat# C05-C52HX, ACRO) were used to screen for VHH antibodies capable of binding both hC5 and cynoC5, the biotin-labeling method being as described in the kit instructions, EZ-LinkTMSulfo-NHS-LC-Biotinylation Kit, thermofisher, 21435, biotin to Protein labeling ratio of 10:1.

Cynomolgus monkey complement C5 amino acid sequence:

QEQTYVISAPKIFRVGASENIVIQVYGYTEAFDATISIKSYPDKKFSYSSGHVHLSSENKFQNSAVLTIQPKQLPGGQNQVSYVYLEVVSKHFSKSKKIPITYDNGFLFIHTDKPVYTPDQSVKVRVYSLNDDLKPAKRETVLTFIDPEGSEIDMVEEIDHIGIISFPDFKIPSNPRYGMWTIQAKYKEDFSTTGTAFFEVKEYVLPHFSVSVEPESNFIGYKNFKNFEITIKARYFYNKVVTEADVYITFGIREDLKDDQKEMMQTAMQNTMLINGIAEVTFDSETAVKELSYYSLEDLNNKYLYIAVTVIESTGGFSEEAEIPGIKYVLSPYKLNLVATPLFLKPGIPYSIKVQVKDALDQLVGGVPVTLNAQTIDVNQETSDLEPRKSVTRVDDGVASFVVNLPSGVTVLEFNVKTDAPDLPDENQAREGYRAIAYSSLSQSYLYIDWTDNHKALLVGEYLNIIVTPKSPYIDKITHYNYLILSKGKIIHFGTREKLSDASYQSINIPVTQNMVPSSRLLVYYIVTGEQTAELVSDSVWLNIEEKCGNQLQVHLSPDADTYSPGQTVSLNMVTGMDSWVALTAVDSAVYGVQRRAKKPLERVFQFLEKSDLGCGAGGGLNNANVFHLAGLTFLTNANADDSQENDEPCKEIIRPRRMLQEKIEEIAAKYKHLVVKKCCYDGVRINHDETCEQRAARISVGPRCVKAFTECCVVASQLRANNSHKDLQLGRLHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSVTTWEIQGVGISNSGICVADTIKAKVFKDVFLEMNIPYSVVRGEQVQLKGTVYNYRTSGMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSNHLVTFTVLPLEIGLQNINFSLETSFGKEILVKSLRVVPEGVKRESYSGITLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSREGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKRNLEKKLKEGMVSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVHKYVEQNQNSICNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKAFDICPLVKINTALIKADTFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKREALVKGNPPIYRFWKDSLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPIIKWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRLNMDIDVAYKHKGPLHNYKMTDKNFLGRPVEVLLNDDLVVSTGFGSGLATVHVTTVVHKTSTSEEVCSFYLKIDTQDIEASHYRGYGNSDYKRIVACASYKPSKEESSSGSSHAVMDISLPTGINANEEDLKALVEGVDQLFTDYQIKDGHVILQLNSIPSSDFLCVRFRIFELFEVGFLSPATFTVYEYHRPDKQCTMFYSTSNIKIQKVCEGATCKCIEADCGQMQKELDLTISAETRKQTACNPEIAYAYKVIITSITTENVFVKYKATLLDIYKTGEAVAEKDSEITFIKKVTCTNAELVKGRQYLIMGKEALQIKYNFTFRYIYPLDSLTWIEYWPRDTTCSSCQAFLANLDEFAEDIFLNGC（SEQ ID NO:6）

The first round of screening was incubated with 50nM biotin-labeled cynomolgus complement C5 protein (hereinafter referred to as "cynoC" or "biotin-labeled cynoC") with phage display library prepared in 2.1 for 2 hours at room temperature in PBS containing 2.5% milk, followed by the addition of M-270 magnetic beads (cat# 65305, invitrogen) to capture biotin-labeled cynoC and phage display VHH antibody capable of binding to cynoC for 30 minutes at room temperature. M-270 beads were adsorbed with a magnetic rack, washed 10 times with PBST, 10 times with PBS, and phage eluted using a 0.25mg/ML TRYPSIN solution (Trypsin, cat.25200-072, gibco) incubated for 30 minutes with shaking at room temperature. A4 mg/mL solution of AEBSF (AEBSF protease inhibitor, cat# 78431, sigma) was then added at a 20:1 ratio to terminate enzyme activity.

The eluted phage library was the first round of phage display library, which was infected with fresh TG1 bacteria (Lucigen) with od=0.4-0.5, and left for 30 minutes. It was added to 50mL of 2XYT medium containing ampicillin and 2% glucose, shaking at 37℃at 225rpm for 1-2 hours, centrifuging 9000g for 15 minutes, re-suspending the cells with 2XYT medium containing kana antibiotic and 1mM IPTG, and taking 10-100uL of suspension for dilution counting, adding helper phage M13K07 (NEB), and culturing overnight at 30℃at 250 rpm.

The second round of screening was performed using 20nM biotin-labeled hC5, and the method was the same as that of the first round of screening to obtain a second round of phage display library, followed by a third round of screening in sequence to obtain a third round of phage display library.

After mixing evenly the third round of phage library infection TG1, diluting and coating on an agar plate containing ampicillin, picking a bacterial monoclonal on the next day, and inoculating into a sterile 96-well deep-well plate containing 2XYT culture medium containing ampicillin and 2% glucose, wherein the temperature is 37 ℃ and 225rpm. After the bacteria grew to log phase, 100uL was added to a new 96-well plate and M13K07 helper phage was added, incubated for 30 min, supplemented with 100uL of fresh 2xYT medium containing ampicillin and kana antibiotics, and shaken overnight at 30 ℃.

The following day, the resulting phage-containing culture supernatant was validated by phage ELISA (Phage ELISA) to verify whether it specifically bound hcs 5 and cynoC.

EXAMPLE 2.3 screening of phage ELISA (Phage ELISA) for Positive clones

StripwellTMMicroplate (cat# 42592, costar) was coated with hC5 and cynoC 51 μg/mL at 4℃overnight. The next day the plates were blocked with PBS containing 2.5% mill for 1 hour at room temperature. 100uL of phage-containing culture supernatant obtained in the previous step was added to each well after washing the plate with PBST, and incubated at room temperature for 1 hour. The plates were again washed and added with anti-M13 HRP detection antibody (M13 Bacteriophage antibody (HRP) cat# 11973MM05T-HH013JA1501, sino Bio) and incubated for 30 minutes at room temperature.

The plate was washed 5 times with PBST, then 1-step Ultra TMB-ELISA REAGENT (Thermo Scientific, cat# 34029) was added and the color change per well was observed. When the color is appropriate, the reaction is quenched with 2M sulfuric acid and the reading is taken at OD450 nm. Based on the results of ELISA reactions, positive clones C5006, C5006-12, C5006-13, C5006-14, C5006-15 and C5006-16 expressing positive phages were picked and subjected to Morganin biological sequencing to obtain the gene sequences encoding the VHH antibody variable region and the deduced amino acid sequences based on the gene sequences as shown below (C5006-15 and C5006-16 sequences are not shown).

VHH antibody variable region nucleotide sequences

C5006:

GACGTTCAGCTGCAAGAGTCTGGCGGAGGACTGGTTCAAGCTGGCGGAAGCCTGACACTGTCTTGTGCCGCCTCTGGCAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACTACGCCAAGAACACCGTGTCTCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTATTACTGCGCCGCCTACAGCGAGTTCGCCAGAATCAGCGACAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCACGTGACCGTTTCTTCT（SEQ ID NO:7）

C5006-12:

CAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGGCTGGCGGATCTCTGACACTGAGCTGTGCTGCCTCTCCTGGCACCTTCAGCACAAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGATTACGCCGAGAACACCGTGTCTCTGCAGATGAGCAGCCTGAAGCCTGAGGACACCGCCGTGTATTACTGTGCCGCCACAAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCAAGTGACCGTGTCATCT（SEQ ID NO:8）

C5006-13:

CAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGGCTGGCGGATCTCTGACACTGAGCTGTGCCGCCTCTCCTAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGATTACGCCGAGAACACCGTGTCTCTGCAGATGAGCAGCCTGAAGCCTGAGGACACCGCCGTGTATTATTGTGCCGCCACCAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTCGTGACCGTTTCTTCT（SEQ ID NO:9）

C5006-14:

CAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGGCTGGCGGATCTCTGACACTGAGCTGTGCCGCCTCTCCTAGAACACTGAGCACCAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGATTACGCCGAGAACACCGTGTCTCTGCAGATGAGCAGCCTGAAGCCTGAGGACACCGCCGTGTATTATTGTGCCGCCACCAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTCGTGACCGTTTCTTCT（SEQ ID NO:10）

Variable region amino acid sequence of VHH antibody

C5006:

DVQLQESGGGLVQAGGSLTLSCAASGRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVSLQMNSLKPEDTAVYYCAAYSEFARISDSSSYRYWGQGTHVTVSS（SEQ ID NO:11）

C5006-12:

QVQVVESGGGLVQAGGSLTLSCAASPGTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTQVTVSS（SEQ ID NO:12）

C5006-13:

QVQLVESGGGLVQAGGSLTLSCAASPRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSS（SEQ ID NO:13）

C5006-14:

QVQVVESGGGLVQAGGSLTLSCAASPRTLSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSS （SEQ ID NO:14）

TABLE 1 CDR amino acid sequences of VHH antibodies (IMGT)

Numbering device

SEQ ID NO

CDR1

SEQ ID NO

CDR2

SEQ ID NO

CDR3

C5006

15

GRTFSTNA

19

VSWGNGIP

20

AAYSEFARISDSSSYRY

C5006-12

16

PGTFSTNA

19

VSWGNGIP

21

AATSEFARISDSSSYRY

C5006-13

17

PRTFSTNA

19

VSWGNGIP

21

AATSEFARISDSSSYRY

C5006-14

18

PRTLSTNA

19

VSWGNGIP

21

AATSEFARISDSSSYRY

EXAMPLE 3 expression and purification of recombinant C5 VHH antibodies

Example 3.1 antibody expression

Nucleic acids encoding VHH antibodies of C5006, C5006-12, C5006-13, C5006-14 (Optimus Praeparatus) were synthesized and constructed on a mammalian cell expression vector (pCDNA3.1) comprising a signal peptide (SEQ ID NO: 22) and a human IgG1 Fc (SEQ ID NO: 23) comprising a hinge region, and the resulting plasmid DNA was transiently cultured in 293FT cells for 96 hours using Lipofectamine2000 (Thermofisher, cat No. 11668030) transfection reagent. While the Evaporation antibody (IMGT/mAb-DB ID: 37, INN number: 8231) was expressed as a positive control for hC5 antibody.

The amino acid sequence of the signal peptide is SEQ ID NO. 22

MGWSCIILFLVATATGVHS

Human IgG1 Fc region amino acid sequence comprising mutant hinge region SEQ ID NO. 23

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK( Wherein the blackbody is the hinge region and has a C220S mutation

The amino acid sequence of the Fc region of human IgG1 (wild-type, non-hinge region) SEQ ID NO. 24

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Control antibody sequence:

Exku bead Shan Kangchong chain:

QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK（SEQ ID NO:25）

exkuzhudan (Exkuzhudan) anti-light chain:

DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQ ID NO:26）

VHH antibody amino acid sequence linked to human Fc fragment

C5006-G1:

DVQLQESGGGLVQAGGSLTLSCAASGRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVSLQMNSLKPEDTAVYYCAAYSEFARISDSSSYRYWGQGTHVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO:27）

C5006-12-G1:

QVQVVESGGGLVQAGGSLTLSCAASPGTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:28)

C5006-13-G1:

QVQLVESGGGLVQAGGSLTLSCAASPRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:29)

C5006-14-G1:

QVQVVESGGGLVQAGGSLTLSCAASPRTLSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:30)

EXAMPLE 3.2 antibody purification

The resulting VHH antibody (SEQ ID NO: 27-30) linked to the human Fc fragment was purified using Protein A and dissolved in PBS buffer.

EXAMPLE 4 detection of hemolytic inhibitory Activity of C5 VHH antibodies

Example 4.1 detection of inhibitory Activity of complement Classical Pathway (CP) hemolysis

The experiment is to analyze and compare the inhibition activity of each antibody on the hemolysis mediated by the classical complement pathway by detecting the inhibition degree of the hemolysis of sheep erythrocytes mediated by complement in 5% normal human serum by detecting antibodies with different concentrations. The experimental procedure is briefly described as follows:

(1) Antibody gradient dilution, namely determining the mass concentration and molecular weight of an antibody to be detected, preparing the antibody into a 1200nM solution by using a CP buffer (10 mM HEPES, 150mM NaCl, 3mM CaCl2, 3mM MgCl2, 0.1% Gelatin, pH 7.4), and then starting from 1200nM, carrying out gradient dilution by using the CP buffer 3 times downwards for 6 concentration gradients;

(2) Dilution of serum, namely taking normal human serum (NHS, shanghai hundred organisms) out of a refrigerator at-70 ℃, naturally thawing at room temperature, and diluting a serum stock solution into a 20% NHS solution by using a CP buffer solution;

(3) Activation of sheep erythrocytes 2% sheep erythrocytes (sheep Red Blood Cell, cat# SBJ-RBC-S002, sbjbio) were washed clear with CP buffer, then resuspended in CP buffer, and rabbit anti-sheep erythrocytes antibody (hemolysin, rabbitanti-sheep RBC, cat# S25861, source leaf organism) at 1:4000 cost effective was added to the suspension, sheep erythrocytes were activated by incubation at 37℃for 30 minutes, the activated sheep erythrocytes were washed 3 times with CP buffer, and erythrocytes were resuspended with CP buffer;

(4) Adding 50 ul sheep red blood cells activated in the step (3) into a 96-hole V bottom plate, then adding 25-ul of the diluted antibody in the step (1), adding 25-mu L of the diluted serum in the step (2) into the mixed solution, setting two control groups, wherein the negative control group does not contain the antibody and uses the serum deactivated by 56 ℃, the positive control group does not contain the antibody and uses normal serum, and incubating for 1 hour at 37 ℃;

(5) After the incubation is finished, 100 mu L stop buffer (10 mM HEPES, 150mM NaCl,10mM EDTA, 0.1% gelatin) is added into each hole to terminate the reaction, a 96-hole V bottom plate is placed in a 4 ℃ centrifuge for 5 minutes at 3000rpm, 100 mu L supernatant is taken and transferred into the 96-hole flat bottom plate, the absorbance of the supernatant at OD415nm is detected in a multifunctional plate reader (Molecular Devices), and data are stored;

(6) And (3) data processing, namely substituting the OD415nm read value obtained in the step (5) into a formula to calculate the hemolysis inhibition rate of the red blood cells, wherein the calculation formula is that the hemolysis inhibition rate (%) = (positive control OD 415-experimental group OD 415)/(positive control OD 415-negative control OD 415). Four-parameter fitting plots were then made using GRAPH PAD PRISM software with final antibody concentration on the abscissa and inhibition of hemolysis on the ordinate, and IC50 values were calculated.

The detection results show that the C5 VHH antibodies C5006, C5006-12, C5006-13 and C5006-14 can effectively inhibit Classical Pathway (CP) mediated sheep erythrocyte hemolysis, and the inhibition capacity is gradually enhanced along with the increase of the concentration of the antibodies, compared with the C5006, C5006-13 and C5006-12, the inhibition activity of the C5006-14 is better, the inhibition activity of the C5006-14 is slightly weaker, and the C5006-15 and the C5006-16 have no inhibition activity, and the experimental results are shown in figure 2.

Example 4.2 detection of inhibitory Activity of alternative complement pathway (AP) hemolysis

The test is to analyze and compare the inhibition activity of each antibody on complement alternative pathway mediated hemolysis by measuring the inhibition degree of the antibody to be tested on complement mediated rabbit erythrocyte hemolysis in 10% normal human serum at different concentrations. The experimental procedure is briefly described as follows:

(1) Antibody gradient dilution, namely determining the mass concentration and molecular weight of an antibody to be detected, preparing the antibody concentration into a 1200nM solution by using an AP buffer solution (10 mM HEPES, 150mM NaCl, 3mM MgCl2, 0.1% Gelatin, 5mM EGTA, pH 7.4), and then starting from 1200nM, carrying out gradient dilution with the AP buffer solution from 3 times downwards for 6 concentration gradients;

(2) Dilution of serum, namely taking normal human serum (NHS, shanghai hundred organisms) out of a refrigerator at-70 ℃, naturally thawing at room temperature, and diluting a serum stock solution into 40% NHS solution by using an AP buffer solution;

(3) Preparation of rabbit red blood cells by washing 2% rabbit red blood cells (rabbit Red Blood Cell, cat# SBJ-RBC-RAB002, sbjbio) with AP buffer to clear the supernatant, and then re-suspending the red blood cells with AP buffer;

(4) The antibody reaction solution is incubated with red blood cells, 50ul of rabbit red blood cells in the step (3) are added into a 96-well V bottom plate, then 25 ul of the diluted antibodies in the step (1) are added, 25 ul of the diluted serum in the step (2) is added into the mixed solution, two control groups are arranged, the negative control group does not contain the antibodies and uses the serum which is treated and deactivated by 56 ℃, the positive control group does not contain the antibodies and uses normal serum, and the positive control group is incubated for 1 hour at 37 ℃;

(5) After the incubation is finished, 100 mu L of stop buffer (10 mM HEPES, 150mM NaCl,10mM EDTA, 0.1% Gelatin) is added into each well to stop the reaction, a 96-well V bottom plate is placed in a 4 ℃ centrifuge for 5 minutes at 3000rpm, 100 mu L of supernatant is taken and transferred into the 96-well flat bottom plate, the absorbance of the supernatant at OD415nm is detected in a multifunctional plate reader (Molecular Devices), and data are stored;

The detection results show that the C5 VHH antibodies C5006, C5006-12, C5006-13 and C5006-14 can effectively inhibit bypass pathway (AP) -mediated rabbit erythrocyte hemolysis, and the inhibition capacity is gradually enhanced along with the increase of the concentration of the antibodies, compared with the C5006, C5006-13 and C5006-12, the inhibition activity of the C5006-14 is better, and the inhibition activity of the C5006-14 is slightly weaker, and the experimental results are shown in figure 3.

EXAMPLE 5 humanization of the C5 VHH antibody

Sequence comparison analysis shows that the variable region of the C5 VHH antibody has higher homology with human GERMLINE IGHV X3-23X 01, the humanized antibody is carried out by adopting a CDR grafting method and using IGHV 3-23X 01 as a framework, the CDR region of the antibody is defined by the rule of IMGT, and two humanized sequences of C5006d1 and C5006e3 are finally obtained after repeated mutation screening for several rounds, wherein the nucleotide and amino acid sequences of the humanized antibody are shown as follows.

VHH antibody variable region nucleotide sequences

C5006d1:

GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCTGGCAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGGCCCCTGGCAAAGAAAGGGAATTCGTGGCCGCTGTGTCCTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACTACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGCGGCCTGAGGATACCGCCGTGTACTACTGTGCCGCTTACAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTGGTCACCGTTTCTTCT（SEQ ID NO:31）

C5006e3:

GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCTCCCAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGGCCCCTGGCAAAGAAAGGGAATTCGTGGCCGCTGTGTCCTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACTACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGCGGCCTGAGGATACCGCCGTGTACTACTGTGCCGCTACCAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTGGTCACCGTTTCTTCT（SEQ ID NO:32）

Variable region amino acid sequence of VHH antibody

C5006d1:

EVQLLESGGGLVQPGGSLRLSCAASGRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAAYSEFARISDSSSYRYWGQGTLVTVSS（SEQ ID NO:33）

C5006e3:

EVQLLESGGGLVQPGGSLRLSCAASPRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSS（SEQ ID NO:34）

TABLE 2 CDR amino acid sequences of VHH antibodies (IMGT)

Numbering device

SEQ ID NO

CDR1

SEQ ID NO

CDR2

SEQ ID NO

CDR3

C5006d1

15

GRTFSTNA

19

VSWGNGIP

20

AAYSEFARISDSSSYRY

C5006e3

17

PRTFSTNA

19

VSWGNGIP

21

AATSEFARISDSSSYRY

EXAMPLE 6 expression, purification and purity detection of recombinant humanized C5 VHH antibodies

EXAMPLE 6.1 humanized C5 VHH antibody Gene synthesis, antibody expression and purification

The humanized C5 VHH antibody gene synthesis, expression and purification were as described in example 3, and the complete antibody sequence information is shown below.

Humanized VHH antibody amino acid sequences linked to human Fc fragments

C5006d1-G1:

EVQLLESGGGLVQPGGSLRLSCAASGRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAAYSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO:35）

C5006e3-G1:

EVQLLESGGGLVQPGGSLRLSCAASPRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:36)

EXAMPLE 6.2 humanized C5 VHH antibody purity detection (SEC-HPLC)

Protein purity analysis purified protein samples in PBS were applied to a 300 x 4.6mm,5 μm column (TOSOH) of TSKgel Super SW3000 using Size Exclusion Chromatography (SEC), SEC using a U3000 type HPLC instrument (DIONEX), elution being isocratic elution at a flow rate of 0.25 mL/min. All proteins were measured using UV detection at 280nm and 214 nm. Component analysis was performed using the instrument's own software.

The detection results show that the monomer purities of the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 purified by the protein A in one step respectively reach 96% and 98% in the solution state, and the purities are excellent (figures 4 and 5).

EXAMPLE 7 humanized C5 VHH antibody binding Activity assay

EXAMPLE 7.1 ELISA of coated antibody detection antigen

To test the binding capacity of the humanized C5 VHH antibody to the antigen human C5, the antibody was diluted to 0.5. Mu.g/mL with PBS and 100. Mu.L of coated ELISA plate (Corning, cat# 42592) was incubated overnight at 4 ℃. The following day, the coating solution was removed and washed 3 times with PBST, and blocked with PBS containing 2.5% nonfat milk powder at 37 ℃ for 1 hour. The hC5 protein prepared in example 1.1 was diluted in a gradient with PBS containing 2.5% nonfat milk powder, 5-fold from 1. Mu.g/mL for a total of 7 concentration points, and the negative control point was free of hC5. After 3 PBST washes, diluted hC5 solution was added and incubated at 37 ℃ for 1 hour. After 3 washes of PBST, 100. Mu.L of HRP-labeled rabbit anti-HIS secondary antibody (jackson, cat# 300-035-240) was added and incubated at 37℃for 1 hour. After washing the PBST plate 3 times, TMB (InvitrogenTM, cat# 002023) substrate was added for 5-10 minutes, ELISA stop solution (Bio-technology, cat# E661006-0500) was added to stop the reaction, and then the OD450 absorbance was read by using an ELISA reader.

The detection results show that when the coated antibody detects the antigen human C5 subjected to gradient dilution, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 can be combined with the human C5, and the combined signal is gradually enhanced along with the increase of the concentration of the C5. In contrast, the binding capacity of both was similar and substantially identical to that of the control antibody eculizumab, and the experimental results are shown in fig. 6.

EXAMPLE 7.2 ELISA of coated antigen-detecting antibodies

To further examine the binding capacity of the humanized C5 VHH antibody to the antigen human C5, human C5 protein was diluted to 1. Mu.g/mL with PBS and 100. Mu.L of coated ELISA plate (Corning, cat# 42592) was incubated overnight at 4 ℃. The following day, the coating solution was removed and washed 3 times with PBST, and blocked with PBS containing 2.5% nonfat milk powder at 37 ℃ for 1 hour. The antibody to be detected was diluted in a gradient with PBS containing 2.5% nonfat milk powder, 5-fold from 1. Mu.g/mL for 7 concentration points, and the negative control point was free of antibody. After 3 PBST washes, diluted antibody solution was added and incubated at 37 ℃ for 1 hour. After 3 washes of PBST, 100. Mu.L of HRP-labeled goat anti-human IgG antibody (jackson, cat# 109-035-098) diluted 1:5000 was added and incubated at 37℃for 1 hour. After washing the PBST plate 3 times, TMB (InvitrogenTM, cat# 002023) substrate was added for 5-10 minutes, ELISA stop solution (Bio-technology, cat# E661006-0500) was added to stop the reaction, and then the OD450 absorbance was read by using an ELISA reader.

The detection results show that when the antigen human C5 protein is coated, the antibody subjected to gradient dilution is detected, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 can be combined with human C5, and the combined signal is gradually enhanced along with the increase of the concentration of the antibody. In contrast, the binding capacity of both was similar and was stronger than that of the control antibody eculizumab, and the experimental results are shown in fig. 7.

Example 8 detection of the hemolytic inhibitory Activity of humanized C5 VHH antibodies

EXAMPLE 8.1 detection of inhibitory Activity of humanized C5 VHH antibodies against Normal Human Serum (NHS) CP/AP hemolysis

To examine the complement-mediated hemolysis inhibitory activity of the humanized C5 VHH antibody in human serum, the same procedure as in example 4 was used to prepare an 80. Mu.g/mL solution (final concentration 20. Mu.g/mL) and the antibody was subjected to 2-fold gradient dilution for 10 concentration points.

The results showed that the humanized C5 VHH antibodies C5006d1-G1, C5006e3-G1 had similar inhibitory activity for CP-mediated hemolysis of sheep erythrocytes, IC50 was 0.46 and 0.52 μg/mL, respectively, which were stronger than that of the control antibody elkuzumab, the latter having an inhibitory activity IC50 of 0.76 μg/mL (FIG. 8), and that the humanized C5 VHH antibodies C5006d1-G1, C5006e3-G1 had similar inhibitory activity for AP-mediated hemolysis of rabbit erythrocytes, IC50 was 1.52 and 1.63 μg/mL, respectively, which were also stronger than that of the control antibody elkuzumab, the latter having an inhibitory activity IC50 of 2.08 μg/mL (FIG. 9). In summary, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 have good human complement inhibitory activity and are more active than the control antibody eculizumab.

EXAMPLE 8.2 detection of inhibitory Activity of humanized C5 VHH antibodies against monkey serum (NMS) CP/AP hemolysis

As the model animal closest to humans, non-human primate (NHP) is often used for evaluation of drug safety and efficacy during drug development, which can greatly improve the success rate of drug development and reduce the risk of drug post-development. To examine the complement-mediated hemolysis inhibitory activity of the humanized C5 VHH antibody in NHP serum, animal cynomolgus monkey serum (Xihua New drug development Co., ltd.) was assayed using the same method and common mode as in example 4, and 80. Mu.g/mL of the solution (final concentration 20. Mu.g/mL) was prepared as the antibody, and 2-fold gradient dilution was performed for 10 concentration points in total.

The results showed that the humanized C5 VHH antibodies C5006d1-G1, C5006e3-G1 had the same inhibitory activity for CP-mediated hemolysis of sheep erythrocytes and the IC50 was 0.81. Mu.g/mL. In contrast, the control antibody eculizumab had only weak hemolytic inhibitory activity (FIG. 10), and the humanized C5 VHH antibodies C5006d1-G1, C5006e3-G1 had similar inhibitory activity for AP-mediated hemolysis of rabbit erythrocytes with IC50 of 3.54 and 4.06 μg/mL, respectively. Similarly, the control antibody eculizumab had only weak hemolytic inhibitory activity (fig. 11). In summary, humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 have good monkey complement inhibitory activity, whereas the antibody eculizumab is weak.

Example 9 thermostability assay of humanized C5 VHH antibodies

The thermal stability of an antibody is an important index for evaluating the developability of an antibody drug, and good heat resistance stability often means that the antibody drug has better storage stability and shelf life. The experiment uses a gradient heating method to heat the antibody, and then carries out activity detection on samples treated at different temperatures to evaluate the heat-resistant stability of the humanized C5 VHH antibody.

Example 9.1 antibody Heat treatment

The antibody to be tested was prepared as a 100nM solution with CP buffer, and after sub-packaging, the antibody was incubated at different temperatures for 1 hour, the temperature was set to decrease from 5℃to 55℃starting at 85℃and a total of 7 temperature points were prepared, and the sample incubated at 4℃was used as a control.

EXAMPLE 9.2 detection of CP Activity of heat-treated antibodies

To detect changes in the activity of the antibodies after treatment at different temperatures, the same CP detection method as in example 4 was used, with a final concentration of 25nM.

The results showed that the eculizumab treated at 70 ℃ and at the upper temperature completely lost complement inhibitory activity, the C5006d1-G1 treated at 80 ℃ and at the upper temperature completely lost complement inhibitory activity, and the C5006e3-G1 treated at 85 ℃ completely lost complement inhibitory activity. In contrast, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 had better thermostability and the results are shown in FIG. 12.

Claims

1. A VHH antibody that specifically binds complement C5 comprising three Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 wherein

(I) CDR1 consists of the amino acid sequence shown in SEQ ID NO. 17, CDR2 consists of the amino acid sequence shown in SEQ ID NO. 19, CDR3 consists of the amino acid sequence shown in SEQ ID NO. 21, or

(Ii) CDR1 consists of the amino acid sequence shown in SEQ ID NO. 15, CDR2 consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 consists of the amino acid sequence shown in SEQ ID NO. 20;

(iii) CDR1 consists of the amino acid sequence shown in SEQ ID NO. 16, CDR2 consists of the amino acid sequence shown in SEQ ID NO. 19, CDR3 consists of the amino acid sequence shown in SEQ ID NO. 21, or

(Iv) CDR1 consists of the amino acid sequence shown in SEQ ID NO. 18, CDR2 consists of the amino acid sequence shown in SEQ ID NO. 19, and CDR3 consists of the amino acid sequence shown in SEQ ID NO. 21.

2. The VHH antibody of claim 1 comprising or consisting of a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs 34, 33 and 11-14.

3. A VHH antibody that specifically binds complement C5, consisting of the amino acid sequence shown in any one of SEQ ID NOs 34, 33 and 11-14.

4. A heavy chain antibody that specifically binds complement C5 comprising the VHH antibody of any one of claims 1-3.

5. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1-3 linked to an antibody constant region or Fc region.

6. The heavy chain antibody of claim 5, wherein the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3, or human IgG4.

7. The heavy chain antibody of claim 5 or 6, wherein the VHH antibody is linked to the Fc region by a hinge region.

8. The heavy chain antibody of claim 7, wherein the hinge region consists of the amino acid sequence set forth in SEQ ID NO. 38 or 39.

9. The heavy chain antibody of claim 5 or 6, wherein the Fc region consists of the amino acid sequence set forth in SEQ ID No. 24.

10. The heavy chain antibody of any one of claims 4-6 comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs 36, 35 and 27-30.

11. A heavy chain antibody that specifically binds complement C5, consisting of the amino acid sequence set forth in any one of SEQ ID NOs 36, 35 and 27-30.

12. A nucleic acid molecule encoding the VHH antibody of any one of claims 1-3, or the heavy chain antibody of any one of claims 4-11.

13. The nucleic acid molecule of claim 12 consisting of the nucleic acid sequence of any one of SEQ ID NOs 32, 31 and 7-10.

14. An expression vector comprising the nucleic acid molecule of claim 12 or 13.

15. A host cell comprising the nucleic acid molecule of claim 12 or 13 or the expression vector of claim 14.

16. A method of preparing a VHH antibody according to any one of claims 1 to 3, or a heavy chain antibody according to any one of claims 4 to 11, comprising culturing a host cell according to claim 15 under conditions suitable for expression of said VHH antibody or heavy chain antibody or chain thereof, and recovering said VHH antibody or heavy chain antibody from said host cell or host cell culture medium.

17. A pharmaceutical composition comprising one or more VHH antibodies according to any one of claims 1 to 3, or heavy chain antibodies according to any one of claims 4 to 11.

18. The pharmaceutical composition of claim 17, further comprising a pharmaceutical excipient.