Anti-human complement C5 VHH antibody and its application

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

Technical Field

[0001] The present invention relates to a single domain antibody or heavy chain antibody that specifically binds to complement C5. The present invention also relates to a polynucleotide encoding the antibody, an expression vector and a host cell, and a pharmaceutical composition or combination product thereof, and a method and use for treating C5 protein-related diseases. Background Art

[0002] The complement system is composed of more than 30 soluble protein molecules and is part of the natural immune system. Its components include more than 30 molecules such as complement intrinsic components, multiple regulatory factors and complement receptors. The complement system can be activated through three relatively independent and interconnected pathways, thereby exerting a variety of biological effects such as regulating phagocytosis, lysing cells, mediating inflammation, immunomodulation and clearing immune complexes, including enhancing phagocytosis, enhancing chemotaxis of phagocytes, increasing vascular permeability, neutralizing viruses, cell lysis, and regulating immune responses. Although complement activation provides a valuable first line of defense against potential pathogens, complement activation that promotes protective inflammatory responses may also present a potential threat to the host. Complement activation and its deposition on target structures can also indirectly cause cell or tissue damage. Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement activation in host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases.

[0003] Complement proteins include proteins denoted C1 to C9, and these proteins are activated sequentially through three different pathways (classical pathway, lectin pathway, alternative pathway) to cause immune responses. Complement C5 is the fifth component of complement and plays an important role in inflammation and cell killing. This protein consists of α and β polypeptide chains linked by disulfide bonds. The activated peptide C5a is an anaphylatoxin that induces inflammatory responses in various cells via C5aR (CD88) and C5L2 (GPR77), has potent spasmotogenic and chemotactic activities, and is derived from the α polypeptide by cleavage with C5 convertase. The C5b macromolecular cleavage product can form a complex with the C6 complement component, and this complex is the basis for the formation of the membrane attack complex (MAC), which includes additional complement components. If the complement system is not properly controlled or is overactivated, the complement system may produce strong cytotoxicity to host cells.

[0004] Numerous studies have shown that complement C5 is associated with a variety of diseases, such as a variety of diseases associated with the hemolytic activity of human complement.

[0005] The anti-C5 monoclonal antibody Eculizumab (Soliris (registered trademark)) exhibits high affinity for complement C5 and inhibits complement activation by inhibiting the cleavage of C5 into C5a / C5b and the formation of the accompanying membrane attack complex. Thus, Eculizumab exhibits an inhibitory effect on hemolysis and is therefore used as a therapeutic agent for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. In addition, Eculizumab is known as a therapeutic agent for systemic myasthenia gravis (gMG).

[0006] In the in vitro hemolysis inhibition activity test, although eculizumab showed good classical pathway hemolysis inhibition effect, its alternative pathway hemolysis inhibition effect was weaker, which may be the reason why up to 75% of patients treated with Soliris experienced persistent anemia and anemia-related symptoms.

[0007] In addition, eculizumab is relatively expensive. One of the reasons is that the content of complement C5 protein in the human body is high and its turnover rate is fast. This requires the injection of a large amount of drug in a single treatment to completely block the complement activity. Therefore, the development of single-domain antibodies with better activity, higher stability and smaller molecular size has the potential to improve treatment effects and increase patient compliance.

[0008] Therefore, it is hoped that alternative antibodies suitable for the treatment and prevention of complement C5-related diseases can be developed, especially single-domain antibodies with higher stability and smaller molecular size. Summary of the invention

[0009] The present invention relates to a single domain antibody, such as a VHH antibody, that can efficiently bind to human and monkey complement C5 proteins, and a heavy chain antibody comprising the same. In some embodiments, the antibody can effectively block the hemolytic activity of human complement.

[0010] In some embodiments, the antibodies of the present invention can specifically bind to human C5 protein and / or monkey (e.g., cynomolgus monkey) C5 protein. In some embodiments, the antibodies of the present invention can effectively inhibit 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, and the activity is stronger than known anti-C5 antibodies such as eculizumab. In some embodiments, the antibodies of the present invention can have good human complement inhibition activity and / or good monkey (e.g., cynomolgus monkey) complement inhibition activity, for example, the activity is stronger than known anti-C5 antibodies such as eculizumab. In some embodiments, the antibodies of the present invention have higher thermal stability, for example, they can withstand heat treatment at 70°C, 75°C, 76°C, 77°C, 78°C or 79°C (e.g., heat treatment for 1 hour); preferably, they can withstand heat treatment at 80°C, 81°C, 82°C, 83°C, or 84°C (e.g., heat treatment for 1 hour).

[0011] In some aspects of the present invention, the present invention provides a VHH antibody that specifically binds to complement C5, comprising three complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 contained in the VH shown in any one of SEQ ID NOs: 34, 33, 11-14.

[0012] In some embodiments, the VHH antibody of the present invention comprises CDR1, CDR2 and CDR3, wherein:

[0013] (i) CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21;

[0014] 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;

[0015] 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;

[0016] 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.

[0017] In some embodiments, the VHH antibody of the present invention comprises or consists of a heavy chain variable region, which comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 34, 33, 11-14; or comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 34, 33 and 11-14.

[0018] In some aspects of the present invention, the present invention provides a heavy chain antibody that specifically binds to complement C5, which comprises the VHH antibody of the present invention.

[0019] In some embodiments, the heavy chain antibody of the present invention comprises the VHH antibody of the present invention connected to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, and optionally, the VHH antibody is connected to the Fc region via a hinge region. In some embodiments, the VHH antibody is connected to the Fc region via a hinge region, for example, the hinge region is from human IgG1, for example, a natural 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 shown in SEQ ID NO: 38 or 39.

[0020] In some embodiments, the heavy chain antibody of the present invention comprises the VHH antibody of the present invention connected to the Fc region of an antibody, 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 higher identity thereto.

[0021] In some embodiments, the heavy chain antibodies of the invention:

[0022] (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 27-30;

[0023] Or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 36, 35 and 27-30.

[0024] In some embodiments, the VHH antibody or heavy chain antibody of the present invention is a camelid antibody, a chimeric antibody, or a humanized antibody.

[0025] In some aspects of the present invention, the present invention provides a nucleic acid molecule encoding the VHH antibody of the present invention, or the heavy chain antibody of the present invention; or comprising the nucleic acid sequence of any one of SEQ ID NOs: 32, 31 and 7-10, or consisting of the nucleic acid sequence.

[0026] In some aspects of the present invention, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention. Preferably, the expression vector is pCDNA3.1.

[0027] In some aspects of the present invention, the present invention provides a host cell comprising the nucleic acid molecule of the present invention or the expression vector of the present invention. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells, such as 293FT cells.

[0028] In some aspects of the invention, the invention provides a method for preparing the VHH antibody of the invention, or the heavy chain antibody of the invention, the method comprising culturing the host cell of the invention under conditions suitable for the expression of the VHH antibody or heavy chain antibody or its chain, and optionally recovering the VHH antibody or heavy chain antibody from the host cell (or host cell culture medium).

[0029] In some aspects of the present invention, the present invention provides an immunoconjugate comprising the VHH antibody of the present invention, or the heavy chain antibody of the present invention.

[0030] In some aspects of the present invention, the present invention provides a pharmaceutical composition comprising one or more VHH antibodies of the present invention, or heavy chain antibodies of the present invention, or immunoconjugates of the present invention, and optionally a pharmaceutically acceptable excipient.

[0031] In some aspects of the present invention, the present invention provides a pharmaceutical composition comprising two or three VHH antibodies of the present invention; or two or three heavy chain antibodies of the present invention, and optionally a pharmaceutically acceptable excipient.

[0032] In some aspects of the invention, the invention provides a pharmaceutical combination product comprising the VHH antibody or heavy chain antibody of the invention, and other therapeutic agents.

[0033] In some aspects of the present invention, the present invention provides a pharmaceutical combination product, wherein the pharmaceutical combination product comprises two or three VHH antibodies or heavy chain antibodies described in the present invention.

[0034] In some aspects of the present invention, the present invention provides a method for preventing or treating a complement C5-related 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 present invention, or one or more heavy chain antibodies of the present invention, the immunoconjugate of the present invention; or the pharmaceutical composition of the present invention; or the pharmaceutical combination of the present invention. In some aspects of the present invention, the use of one or more VHH antibodies of the present invention, or one or more heavy chain antibodies of the present invention, the immunoconjugate of the present invention; or the pharmaceutical composition of the present invention; or the pharmaceutical combination of the present invention for preparing a medicament for preventing or treating a complement C5-related disease or disorder or for blocking or inhibiting complement hemolysis in a subject. In some aspects of the present invention, the present invention provides one or more VHH antibodies of the present invention, or one or more heavy chain antibodies of the present invention, the immunoconjugate of the present invention; or the pharmaceutical composition of the present invention; or the pharmaceutical combination of the present invention for preventing or treating a complement C5-related disease or disorder or for blocking or inhibiting complement hemolysis in a subject. In some embodiments, the diseases or conditions described herein include disease phenotypes caused by unregulated C5 function, such as due to dysregulated C5 activation, such as increased C5 activation. In some embodiments, the subject described herein has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein or the disease treatment will benefit from inhibition of 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The anti-C5 antibody titers in the serum of alpacas after immunization are shown.

[0036] Figure 2 The inhibitory activity of the recombinant C5 VHH antibody on human complement CP pathway hemolysis was shown.

[0037] Figure 3 The inhibitory activity of the recombinant C5 VHH antibody on human complement AP pathway hemolysis was shown.

[0038] Figure 4 The SEC-HPLC purity of the recombinant humanized C5 VHH antibody C5006d1-G1 (wavelength = 280 nM) is shown.

[0039] Figure 5 The SEC-HPLC purity of the recombinant humanized C5 VHH antibody C5006e3-G1 is shown (wavelength = 280 nM).

[0040] Figure 6The figure shows the binding of humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 to human CD5 when the coated antibody detects serially diluted antigen human C5.

[0041] Figure 7 It shows the binding of humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 to human C5 when the antigen human C5 protein was coated to detect serially diluted antibodies.

[0042] Figure 8 The inhibitory activity of the recombinant humanized C5 VHH antibody on human complement CP pathway hemolysis was shown.

[0043] Fig. 9 The inhibitory activity of the recombinant humanized C5 VHH antibody on human complement AP pathway hemolysis was shown.

[0044] Fig.10 The inhibitory activity of the recombinant humanized C5 VHH antibody on monkey complement CP pathway hemolysis was shown.

[0045] Fig.11 The inhibitory activity of the recombinant humanized C5 VHH antibody on monkey complement AP pathway hemolysis was shown.

[0046] Fig.12 The inhibitory activity of the recombinant humanized C5 VHH antibody on complement CP pathway hemolysis after heat treatment is shown. DETAILED DESCRIPTION

[0047] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present 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 only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0048] I. Definitions

[0049] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. 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.

[0050] The term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values ​​having a lower limit that is 5% (e.g., 4%, 3%, 2%, or 1%) less than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2%, or 1%) greater than the specified numerical value.

[0051] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0052] As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0053] 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 article, unless otherwise stated, the term also covers any variants of C complement C5, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers species homologs thereof. In addition, it should be understood that the term covers not only CDH17 naturally or recombinantly expressed by cells or CDH17 expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins comprising CDH17 or fragments thereof.

[0054] Human complement C5 (e.g., Uniprot entry P01031) is a secreted multidomain glycoprotein consisting of an α chain (999 amino acids) and a β chain (655 amino acids) linked by a disulfide bridge. The peptide bond between Arg751 and Leu752 of the α chain is cleaved by C5 convertase, generating 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 binding of C6. For example, in some embodiments, human C5 has the sequence shown in SEQ ID NO: 1 and the cynomolgus monkey ( Macaca fascicularis ) C5 has the sequence shown in SEQ ID NO:6.

[0055] "Single domain antibody" (sdAb) is used herein to refer to an antibody polypeptide that recognizes and binds to an antigen of interest through a single variable antibody domain, such as a single VH or a single VL. The single variable antibody domain of a single domain antibody is capable of recognizing and binding to an antigen of interest without being paired with another antibody variable domain. In this article, a single domain antibody comprising a heavy chain variable domain is also referred to as VHH.

[0056] The term "VHH" generally refers to an antibody that contains or consists of only one heavy chain variable region and has the activity of binding to an antigen, i.e., an antibody that contains only one chain from the C-terminus to the N-terminus: FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1. VHH is usually a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, also known as a nanobody. The VHH used in the present invention is preferably from a camelid, such as an alpaca, or a humanized form or sequence optimized form thereof (e.g., an affinity mature form to increase binding affinity). In some embodiments, the VHH of the present invention is a monovalent monospecific polypeptide molecule consisting of or substantially consisting of a single heavy chain variable region (e.g., a heavy chain variable region of a heavy chain antibody).

[0057] The single domain antibody or VHH of the present invention may also be contained in a larger polypeptide / protein. Examples of polypeptides / proteins containing VHH of the present invention include, but are not limited to, heavy chain antibodies (HcAb). The "heavy chain antibody" described in the present invention refers to an antibody without a light chain, for example, it may contain VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from N segment to C segment, or may contain VH-CH1-CH2-CH3; homodimers may be covered, such as heavy chain dimer antibodies without light chains. The heavy chain antibody of the present invention may contain VH from a standard antibody or VH from a single domain antibody. For example, the VH in the heavy chain antibody of the present invention may be VHH. In some embodiments, the heavy chain antibody of the present invention may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from a camelid (llama, camel, especially alpaca), a humanized form thereof or a sequence optimized form thereof (affinity mature form), or a fragment thereof (e.g., a fragment containing at least part of the constant region). The heavy chain antibodies of the present invention also encompass antibodies formed by fusing a heavy chain variable region or VHH with an Fc region (eg, a human IgG Fc region, such as a human IgG1 or IgG4 Fc region).

[0058] The term "antigen-binding fragment" of an antibody is a part or a segment of a full-length or full-length antibody that has fewer amino acid residues than a full-length antibody or a full-length antibody, but that can bind to an antigen or compete with a full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and is a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment consists 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 independent genes, but can also be produced as a single protein chain using recombinant methods, using a synthetic linker peptide to connect the two domains, and in the single protein chain the VL region and the VH region are paired to form a single-chain Fv (scFv).

[0059] In this article, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain that participates in the binding of the antibody to the antigen. In the case of heavy chain antibodies, for example, from camelid heavy chain antibodies, 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 chain and light chain variable regions of conventional IgG antibodies, comprises four conservative framework regions (FRs) and three complementary determining regions (CDRs), and is arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some aspects of the present invention, one or more residues in the variable region of the antibody may be modified, for example, one or more CDR regions and / or one or more framework regions may be modified with residues, especially conservative residue substitutions, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen binding ability) of the parent antibody. In some further aspects, the antibody variable region may be modified by CDR transplantation. Since the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibody variants that simulate the properties of known antibodies may be constructed. In such antibody variants, the CDR sequences from known antibodies are transplanted to the framework regions of different antibodies with different properties, and 1 to several residue mutations, such as back mutations, may be performed 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 the activity of inhibiting hemolysis in the complement pathway, may be assessed in in vitro or in vivo assays.

[0060] The "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" of an antibody is a region of the antibody variable domain (VH or VHH) that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are numbered sequentially from the N-terminus and are usually referred to as CDR1, CDR2 and CDR3. The CDRs located in the variable domain of the antibody heavy chain are also referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located in the variable domain of the antibody light chain 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 schemes known in the art, such as: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0061] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0062] CDR Kabat regimen AbM Protocol Chothia Solution Contact Plan IMGT Program LCDR1 (Kabat and Chothia numbering systems) L24-L34 L24-L34 L26-L32 L30-L36 L27-L32 LCDR2 (Kabat and Chothia numbering system) L50-L56 L50-L56 L50-L52 L46-L55 L50-L52 LCDR3 (Kabat and Chothia numbering system) 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

[0063] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0064] CDRs can also be identified based on having the same Kabat numbering position as a reference CDR sequence. Unless otherwise indicated, in the present invention, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

[0065] In one embodiment, the CDRs in the antibody molecules of the invention are determined by the IMGT rules.

[0066] 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. A native immunoglobulin "Fc domain" or "Fc region" comprises two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a natural antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of the heavy chain derived from IgG, IgA, and IgD class antibodies; or comprises the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise specified herein, the amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) (Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991). In this article, the terms "Fc region", "Fc part" and "Fc fragment" do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulin and the heavy chain constant region CH1 and light chain constant region CL, but may include the 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 carboxyl end of the heavy chain. In addition, the C-terminal terminal lysine (Lys447) of the Fc region may be present or absent. In some embodiments, the human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as the sequence from E216 to P230 or the sequence from D221 to P230 according to EU numbering.

[0067] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region having a different or altered class, effector function and / or species origin, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) or the like that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigen specificity. For example, a camel-derived heavy chain antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced antigenicity in humans compared to the original camel-derived antibody.

[0068] As used herein, a "humanized antibody" is an antibody that retains the antigenic specific reactivity of a non-human antibody (e.g., an alpaca monoclonal antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the rest of the antibody with their human counterparts (i.e., replacing the constant region and the portion of the variable region that is not involved in binding with the corresponding portion of a human antibody).

[0069] As used herein, the term "fusion protein" is used interchangeably with "chimeric polypeptide" and refers to a larger polypeptide formed by the fusion of at least two heterologous polypeptide sequences, optionally via a linker. Fusion proteins can be produced by recombinant expression.

[0070] 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).

[0071] The linker can be a connecting peptide. In this article, 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 connecting peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, 30 amino acids. In one embodiment, the connecting peptide comprises an amino acid sequence (G4S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises an amino acid sequence TS (G4S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises an amino acid sequence G (G4S) n, wherein n is an integer equal to or greater than 1, for example, 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, such as a hinge region amino acid sequence comprising "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP (SEQ ID NO: 38)" or "EPKSSDKTHTCPPCP (SEQ ID NO: 39)" or "ESKYGPPCPPCP (SEQ ID NO: 37)".

[0072] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0073] "Affinity" or "binding affinity" refers to the intrinsic 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 a 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.

[0074] "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 specific amino acid sequence shown in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a considerable degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes.

[0075] For polypeptide sequences, "conservative changes" include replacement, deletion or addition of polypeptide sequences, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative substitutions: 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 change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the target antigen relative to the parent antibody or binding protein.

[0076] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. 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 present 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, and also include cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0077] The term "expression vector" refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; 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) into which the recombinant polynucleotide is incorporated.

[0078] The terms "individual" or "subject" are used interchangeably and refer to mammals. 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.

[0079] The term "treatment" includes administering a composition or antibody to prevent or delay the onset of symptoms, complications, or biochemical indications 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 its clinical or subclinical symptoms) or therapeutic suppression or alleviation of symptoms after the disease has manifested. The term "prevention" includes inhibition of the occurrence or development of a disease or disorder, or symptoms of a particular disease or disorder.

[0080] As used herein, the term "complement C5-associated disease or disorder" refers to a disease or disorder in which unregulated C5 function may lead to a disease or disease phenotype, such as due to deregulated C5 activation, such as increased C5 activation.

[0081] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0082] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0083] The terms "drug combination", "combination product", "drug association" or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to kits and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody molecule of the present invention, and (ii) other therapeutic agents; or two or more antibody molecules of the present invention; or two or more antibody molecules of the present invention and other therapeutic agents) are administered to a patient as separate entities simultaneously, without specific time restrictions or at the same or different time intervals, sequentially, wherein such administration provides preventive or therapeutically effective levels of two or more active agents in the patient. In some embodiments, the antibody molecules of the present invention and other therapeutic agents used in the drug combination are administered at levels no greater than when they are 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. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0084] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents or treatments to treat diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a plurality of or in separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0085] The term "effective amount" refers to an amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses administered to a patient, produces the desired effect in a patient in need of treatment or prevention.

[0086] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome 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 outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter 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.

[0087] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0088] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling) or can catalyze the chemical change of a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to a probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent of a direct label. In some embodiments, the label is His or biotin.

[0089] "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 can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. In some embodiments, the sample is blood or serum.

[0090] II. Single domain antibodies or VHH antibodies

[0091] On the one hand, the present invention relates to a single domain antibody (sdAb) that specifically binds to human complement C5 protein and / or specifically binds to monkey (such as cynomolgus monkey) complement C5 protein (preferably specifically binds to human complement C5 protein and monkey (such as cynomolgus monkey) complement C5 protein at the same time), in particular a VHH antibody.

[0092] Single domain antibodies or VHH antibodies have a molecular weight of about one-tenth that of human IgG molecules, and a physical diameter of only a few nanometers. Due to their small molecular size, single domain antibodies have the following advantages over conventional four-chain antibodies: high stability and solubility, and the ability to recognize hidden antigenic sites. In addition, single domain antibodies are also cheaper to prepare than conventional four-chain antibodies. In addition to being used as individual molecules, single domain antibodies are also suitable components for constructing multispecific molecules.

[0093] In some embodiments, the single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, and the heavy chain variable region generally has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; CDR1 to CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be defined according to any CDR definition scheme described in the "Definition" section, for example, 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.

[0094] In some embodiments, the anti-complement C5 VHH of the present invention comprises:

[0095] (i) three complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 contained in the VH represented by any one of SEQ ID NOs: 34, 33 and 11-14,

[0096] or (ii) three complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 contained in the amino acid sequence encoded by the nucleic acid sequence of any one of SEQ ID NOs: 32, 31 and 7-10;

[0097] or (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three CDR regions relative to the sequence of (i) or (ii);

[0098] Preferably, the CDR sequences are according to the IMGT definition.

[0099] In some embodiments, the anti-complement C5 VHH antibody of the present invention comprises or consists of a heavy chain variable region comprising:

[0100] (i) three complementarity determining regions (CDRs) contained in the VH set forth in any one of SEQ ID NOs: 34, 33, and 11-14,

[0101] or (ii) three complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 contained in the amino acid sequence encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 32, 31 and 7-10;

[0102] or (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three CDR regions relative to the sequence of (i) or (ii),

[0103] Preferably, the CDR sequences are according to the IMGT definition.

[0104] In some embodiments, the CDR1 contained in the anti-complement C5 VHH antibody of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 15-18, or consists of the amino acid sequence, or CDR1 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 any one of SEQ ID NOs: 15-18, or consists of the sequence.

[0105] In some embodiments, the CDR2 contained in the anti-complement C5 VHH antibody of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, or consists of said amino acid sequence, or CDR2 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 SEQ ID NO: 19, or consists of said sequence.

[0106] In some embodiments, the CDR3 contained in the anti-complement C5 VHH antibody of the present invention comprises an amino acid sequence selected from SEQ ID NO: 20 or 21, or consists of the amino acid sequence, 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 SEQ ID NO: 20 or 21, or consists of the sequence.

[0107] In some embodiments, the anti-complement C5 VHH antibody of the present invention comprises CDR1, CDR2 and CDR3, wherein:

[0108] (i) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 NOs: 15-18, or consists of said amino acid sequence;

[0109] (ii) CDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, or CDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 19, or consists of said amino acid sequence; and

[0110] (iii) CDR3 comprises an amino acid sequence selected from the group consisting of 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 SEQ ID NO: 20 or 21, or consists of said amino acid sequence.

[0111] In some embodiments, the anti-complement C5 VHH antibody of the present invention comprises CDR1, CDR2 and CDR3, wherein:

[0112] (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;

[0113] 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;

[0114] 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;

[0115] 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.

[0116] In some embodiments, the anti-complement C5 VHH antibody of the present invention:

[0117] (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: 34, 33 and 11-14;

[0118] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 34, 33, and 11-14;

[0119] (iii) 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 encoded by a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10;

[0120] (iv) comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10;

[0121] Or (v) comprises an amino acid sequence having one 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 conservative amino acid substitutions) compared to the amino acid sequence selected from any one of SEQ ID NOs: 34, 33 and 11-14 or the amino acid sequence encoded by the nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10, preferably, the amino acid changes do not occur in the CDR regions.

[0122] In some embodiments, the anti-complement C5 VHH antibody of the present invention comprises or consists of a heavy chain variable region, wherein:

[0123] (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: 34, 33 and 11-14;

[0124] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 34, 33, and 11-14;

[0125] (iii) 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 encoded by a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10;

[0126] (iv) comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10;

[0127] Or (v) comprises an amino acid sequence having one 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 conservative amino acid substitutions) compared to the amino acid sequence selected from any one of SEQ ID NOs: 34, 33 and 11-14 or the amino acid sequence encoded by the nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10, preferably, the amino acid changes do not occur in the CDR regions.

[0128] Preferably, the anti-complement C5 VHH antibody of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 34, 33 and 11-14, or an amino acid sequence encoded by a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10, or consists of said sequence.

[0129] In some embodiments, the amino acid changes include replacement, insertion or deletion of amino acids. Preferably, the amino acid changes described herein are amino acid replacements, preferably conservative replacements. In preferred embodiments, the amino acid changes described herein occur in regions outside of CDRs (e.g., in FRs). In some embodiments, the replacements are conservative replacements. Conservative replacements refer to replacement of one amino acid with another amino acid within the same class, such as replacement of an acidic amino acid with another acidic amino acid, replacement of a basic amino acid with another basic amino acid, or replacement of a neutral amino acid with another neutral amino acid.

[0130] In some embodiments, the present invention also provides functional variants of single domain antibodies or VHH antibodies of the present invention. The functional variants can be obtained by introducing mutations into the encoding nucleic acid sequence of the exemplary single domain antibody of the present invention, such as into the CDR sequence and / or FR sequence, by methods well known in the present invention, such as by random or site-directed mutagenesis, and then screening (e.g., by phage display library screening) variants that retain the desired properties. Typically, the functional variant retains significant sequence identity with 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), for example, relative to the biological activity of the parent, the variant has a comparable (e.g., at least 50%, 60%, 70%, 80%, preferably more than 90%) biological activity, or improved biological activity (e.g., 110-150% or higher) biological activity. The desired biological properties include, for example, but are not limited to, target antigen binding affinity (such as measured by KD value), activity in blocking target antigen binding to receptor (such as measured by IC50 value), blocking human complement hemolytic activity (such as measured by EC50 value), etc.

[0131] In some embodiments, the present invention provides affinity variants of single domain antibodies or VHH antibodies of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parental single domain antibody from which it is derived, wherein the affinity variants have a changed binding affinity for the target antigen compared to the parental antibody. Typically, affinity variants exhibit improved antigen binding affinity that is superior to 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 non-human species homologous proteins of the target antigen. Affinity variants often have one or more amino acid residue substitutions in CDR compared to the parent. The substitution may be a conservative substitution or a non-conservative substitution. In one embodiment, the affinity variants of the present invention have a total of no more than 10, no more than 6, or 1-5, such as 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 mutating CDRs, chain shuffling, use of E. coli mutator strains, DNA shuffling, phage display, and the like.

[0132] In some embodiments, the VHH antibodies of the present invention against complement C5 comprise CDR amino acid sequences and / or framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunizing camelids (e.g., alpacas). In some embodiments, the VHH monoclonal antibodies of the present invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. Therefore, in one embodiment, the VHH antibodies of the present invention are camelid antibodies.

[0133] Generally, humanization is performed in a manner that retains the favorable binding properties of the single domain antibody. Assays for determining the biological properties of humanized single domain antibodies, such as binding affinity, are well known in the art to determine and select appropriate humanized residue mutations or combinations of mutations.

[0134] In one embodiment, the anti-complement C5 VHH antibody of the present invention is a humanized antibody. For example, the original VHH sequence is humanized using the "best match method", which includes:

[0135] (i) using a human germline V gene database to compare and analyze the amino acid sequences of the VHH framework region and select the best germline sequence, such as the human germline IGHV3-23*01 sequence;

[0136] (ii) replacing the best matching human CDR sequence with a VHH CDR sequence and optionally generating a humanized VHH sequence by backmutating multiple residues in the framework region and optionally removing post-translational modifications (PTMs);

[0137] (iii) optionally sequencing the VHH antibodies.

[0138] III. Heavy chain antibodies

[0139] In another aspect of the present invention, the present invention provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.

[0140] In some embodiments, the single domain antibody or VHH of the present invention (e.g., camel-derived VHH or a humanized form thereof) can be linked to a constant region of a human antibody, such as an Fc region, to produce a heavy chain antibody comprising VHH-Fc. In one embodiment, the heavy chain antibody comprises a single domain antibody of the present invention and an Fc region at its C-terminus. In some embodiments, VHH and Fc are linked via a hinge region, such as a hinge region of IgG (e.g., a hinge region of IgG1, 2, 3, or 4).

[0141] In some embodiments, the anti-C5 protein heavy chain antibody of the present invention comprises a VHH as defined herein or a heavy chain variable region therein, and a heavy chain constant region or an Fc region of a heavy chain constant region. In some embodiments, a connecting peptide is included between the VHH or heavy chain variable region and the heavy chain constant region or the Fc region, such as an antibody hinge region, such as an IgG hinge region. In some embodiments, a hinge region is included between the VHH or heavy chain variable region and the heavy chain constant region or the 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 IgG1. In some embodiments, the hinge region is a hinge region of natural human IgG1, or a variant thereof, such as a C220S variant thereof. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 38 or SEQ ID NO: 39, or consists of the amino acid sequence.

[0142] In one embodiment, the heavy chain antibody comprises an Fc portion from a camelid (e.g., an alpaca). In one embodiment, the heavy chain antibody is produced and isolated by immunizing the camelid, e.g., an alpaca. Various methods are known in the art for immunizing camelids and isolating the VHH antibodies or heavy chain antibodies produced against an antigen of interest.

[0143] In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, such as a constant region from human IgG1, human IgG2, human IgG3, or human IgG4.

[0144] 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 IgG1 Fc region. In some embodiments, when the Fc region is connected to a VHH antibody via a hinge region, the Fc region itself does not contain a hinge region. In some embodiments, the Fc region connected to the VHH antibody via a hinge region is from IgG1, which comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 24.

[0145] In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different another heavy chain antibody) comprising the Fc region through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising the heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably includes a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0146] In some embodiments, the heavy chain antibody:

[0147] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 36, 35 and 27-30; or

[0148] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 36, 35, and 27-30; or

[0149] (iii) comprises an amino acid sequence having one 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 conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 36, 35 and 27-30, preferably, the amino acid changes do not occur in the CDR regions.

[0150] In addition to the above-mentioned single domain antibodies (particularly VHH antibodies) and heavy chain antibodies, the present invention also provides other fusion proteins / chimeric polypeptides comprising at least one single domain antibody or VHH or heavy chain antibody according to the present invention, such as bispecific or multispecific antibodies.

[0151] In some embodiments, the fusion protein / chimeric polypeptide of the present invention comprises a single single domain antibody or VHH or heavy chain antibody according to the present invention. In another embodiment, the fusion protein / chimeric polypeptide of the present invention comprises a plurality of single domain antibodies or VHH or heavy chain antibodies according to the present invention. The fusion protein according to the present invention may be monospecific; or may be multispecific. In some embodiments, the fusion protein / chimeric polypeptide of the present invention comprises only specificity for complement C5. In yet another embodiment, the fusion protein / chimeric polypeptide of the present invention, in addition to comprising a single domain antibody or VHH or heavy chain antibody (one or more) of the present invention for complement C5, also comprises specificity for another antigen.

[0152] In one aspect, the present invention also provides an immunoconjugate comprising a single domain antibody or VHH antibody or heavy chain antibody of the present invention, wherein the single domain antibody or VHH antibody or heavy chain antibody of the present invention is conjugated with other active agents (e.g., anti-hemolytic drugs) or labels to form the immunoconjugate.

[0153] IV. Properties of the VHH antibodies and / or heavy chain antibodies of the invention

[0154] The single domain antibody (particularly VHH antibody) that specifically binds to human or monkey complement C5 of the present invention or the heavy chain antibody comprising the single domain antibody has one or more biological activities selected from the following:

[0155] (i) binds with high affinity to human complement C5, and / or monkey complement C5, such as cynomolgus monkey;

[0156] (ii) exhibit cross-reactivity to human and monkey complement C5, such as cynomolgus monkey complement;

[0157] (ii) inhibiting hemolysis in the classical pathway of complement immunity, for example, inhibiting hemolysis in human serum and / or monkey serum such as cynomolgus monkey serum in the classical pathway of complement immunity, for example, having a stronger activity than known anti-C5 antibodies such as eculizumab;

[0158] (iii) inhibiting hemolysis in the alternative complement immune pathway, for example, inhibiting hemolysis in human serum and / or monkey serum, such as cynomolgus monkey serum, in the alternative complement immune pathway, for example, with greater activity than known anti-C5 antibodies, such as eculizumab;

[0159] (iv) blocking human complement hemolytic activity or blocking monkey, such as cynomolgus monkey, complement hemolytic activity, for example, the activity is stronger than known anti-C5 antibodies such as eculizumab;

[0160] (v) having higher heat stability, for example, being able to withstand heat treatment at 70°C, 75°C, 76°C, 77°C, 78°C or 79°C (for example, heat treatment for 1 hour); preferably, being able to withstand heat treatment at 80°C, 81°C, 82°C, 83°C or 84°C (for example, heat treatment for 1 hour).

[0161] V. Polynucleotides, Vectors and Hosts

[0162] The present invention provides nucleic acids encoding any of the above molecules of the present invention (single domain antibodies or heavy chain antibodies). A vector comprising the nucleic acid is also provided. In one embodiment, the vector is an expression vector (e.g., a pCDNA vector, e.g., pCDNA3.1). A host cell comprising the nucleic acid or the vector is also provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, e.g., 293FT cells). In another embodiment, the host cell is prokaryotic.

[0163] In one aspect, the present invention provides nucleic acids encoding any of the above single domain antibodies or VHHs or heavy chain antibodies against C5 proteins. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit antigen binding ability against human or monkey (e.g., cynomolgus monkey) C5 protein. In some embodiments, the nucleic acid is operably linked in frame with a nucleic acid encoding another peptide / polypeptide, so that when expressed from a suitable expression vector, a fusion protein or chimeric polypeptide comprising the single domain antibody or VHH and another peptide / polypeptide is produced. For example, in some embodiments, the nucleic acid is operably linked in frame with a nucleic acid encoding an Fc region (e.g., a human Fc region), so that when expressed from a suitable expression vector, a heavy chain antibody comprising the single domain antibody or VHH and the Fc region is produced.

[0164] In order to facilitate production and purification, a single domain antibody or a VHH domain or a heavy chain antibody can be fused at the N-terminus with a secretory signal peptide, and / or a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin tag. In some embodiments, the signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.

[0165] As will be apparent to those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0166] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 33-36, 27-30 and 11-14, or a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 33-36, 27-30 and 11-14 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, the nucleic acid of the present invention comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10, or a nucleic acid sequence selected from any one of SEQ ID NOs: 32, 31 and 7-10 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0167] Nucleic acid sequences encoding molecules of the invention can be generated using methods well known in the art, such as by de novo solid phase DNA synthesis, or by PCR amplification.

[0168] In one embodiment, one or more vectors comprising nucleic acid of the present invention are provided. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. The vector includes but is not limited to a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0169] In one embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In some embodiments, a host cell comprising an expression vector of the present invention is provided. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce the antibody molecule of the present invention. Host cells suitable for replication and support of antibody molecule expression of the present invention are well known in the art. As required, such cells can be transfected or transduced with specific expression vectors, and a large number of cells containing vectors can be cultivated for inoculating large-scale fermenters to obtain sufficient amounts of molecules of the present invention for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can 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 (CV1), 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 and Sp2 / 0, etc. Mammalian host cell lines suitable for producing antibodies are known in the art. In a preferred embodiment, the host cell is a CHO or HEK293 cell or a 293FT cell.

[0170] VI. Production and Purification of the Molecules of the Invention

[0171] In another aspect, the present invention provides a method for producing a molecule of the present invention (a single domain antibody, a VHH antibody or a heavy chain antibody), the method comprising: culturing a host cell containing a protein encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chains to produce the molecule under conditions suitable for the assembly of the polypeptide chains into the molecule.

[0172] For recombinant production, the polynucleotide encoding the polypeptide chain of the molecule of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector comprising one or more polynucleotides of the present invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection or other conventional techniques.

[0173] Antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0174] The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0175] VII. Assay

[0176] The molecules provided herein (single domain antibodies, VHH antibodies or heavy chain antibodies) can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0177] The binding of the molecules of the present invention to human complement C5 protein and / or monkey (e.g., cynomolgus monkey) complement C5 protein can be determined by methods known in the art, such as ELISA, Western blotting, etc., or the exemplary methods disclosed in the Examples herein. For example, the binding kinetics (e.g., K) of the molecules can be determined in a biointerferometry assay using human or monkey complement C5 protein. D In some embodiments, the equilibrium dissociation constant of the binding of a molecule to human or monkey complement C5 protein is determined using biointerferometry (e.g., Fortebio Octet system affinity measurement), such as the method described in Example 3.

[0178] The hemolysis-inhibiting effect of the molecules of the present invention can be measured by methods known in the art, such as in vitro tests and / or in vivo animal experiments. For example, a hemolysis test, such as the method shown in Example 4, can be used to detect the hemolysis-inhibiting effect of the molecules on the complement immunity classical pathway and / or the complement immunity alternative pathway.

[0179] VIII. Pharmaceutical Compositions, Drug Combinations, and Kits

[0180] In one aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising one or more (e.g., two or three) molecules of the invention (e.g., single domain antibodies, VHH antibodies or heavy chain antibodies, or immunoconjugates, etc.). In some embodiments, the pharmaceutical composition is a pharmaceutical formulation.

[0181] In one embodiment, the composition of the invention comprises two or three molecules of the invention, such as two or three anti-complement C5 single domain antibodies, VHH antibodies, heavy chain antibodies or immunoconjugates of the invention.

[0182] In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, a pharmaceutical excipient, including a buffer, known in the art.

[0183] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents that are physiologically compatible. For the use of pharmaceutical excipients and their uses, see also the literature ("Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Seskey and SC Owen, Pharmaceutical Press, London, Chicago).

[0184] The compositions or medicaments or formulations of the invention may also comprise, in addition to one or more molecules of the invention, other therapeutic agents that are required for the specific indication being treated and preferably do not adversely affect 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.

[0185] The composition or medicine or preparation of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections), powders or suspensions, liposomes and suppositories. The composition or medicine or preparation of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). The preferred form depends on the expected mode of administration and therapeutic use.

[0186] The present invention also provides a pharmaceutical combination or a pharmaceutical combination product comprising:

[0187] (1) a molecule of the invention such as a single domain antibody or VHH or heavy chain antibody or immunoconjugate and one or more other therapeutic agents;

[0188] (2) two or more molecules of the present invention such as single domain antibodies or VHHs or heavy chain antibodies or immunoconjugates;

[0189] (3) Two or more molecules of the present invention, such as single domain antibodies or VHHs or heavy chain antibodies or immunoconjugates, and one or more other therapeutic agents.

[0190] In some embodiments, the present invention also provides a kit comprising the drug combination, for example, the kit comprises one or more containers containing a pharmaceutical composition comprising two or more molecules of the present invention, such as single domain antibodies or VHH or heavy chain antibodies, in the same package. In some embodiments, two or more molecules of the present invention are in the same container, or in different containers.

[0191] In some embodiments, the present invention also provides a kit comprising the drug combination, for example, the kit comprises in the same package:

[0192] (i) a first container containing a pharmaceutical composition comprising one or more molecules of the invention, such as single domain antibodies or VHHs or heavy chain antibodies (in some embodiments, two or more molecules of the invention are in the same container, or in different containers);

[0193] (ii) a second container containing a pharmaceutical composition comprising one or more additional therapeutic agents (in some embodiments, the two or more additional therapeutic agents are in the same container, or in separate containers).

[0194] IX. Purpose and Method

[0195] In one aspect, the present invention provides a method for preventing or treating a complement C5-related 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 present invention (e.g., a single domain antibody, a VHH antibody or a heavy chain antibody, or an immunoconjugate, etc.), or a composition or a drug or a preparation comprising the same, or a drug combination comprising the same.

[0196] In other aspects, the present invention provides the use of a molecule of the present invention or a composition or pharmaceutical combination comprising the same in the production or preparation of a medicament for the uses described herein, such as for preventing or treating a complement C5-related disease or disorder mentioned herein or for blocking or inhibiting complement hemolysis.

[0197] In other aspects, the invention provides a molecule of the invention or a composition or pharmaceutical combination comprising the same for use in therapy, such as the uses described herein, such as for preventing or treating a complement C5-related disease or disorder mentioned herein or for blocking or inhibiting complement hemolysis.

[0198] In some embodiments, the subject has complement C5 protein (eg, elevated levels, eg, nucleic acid or protein levels) in the subject.

[0199] In some embodiments, the disease treatment would benefit from inhibition of complement C5 protein at the nucleic acid or protein level.

[0200] In some embodiments, the complement C5-related disease or disorder may be a disease requiring hemolysis inhibition, such as a disease requiring inhibition of hemolysis of the classical complement immunity pathway and / or the alternative complement immunity pathway.

[0201] In some embodiments, a molecule of the invention or a composition or medicament or formulation comprising the same delays the onset of a disorder and / or symptoms associated with the disorder.

[0202] In some embodiments, the complement hemolysis is complement classical pathway hemolysis and / or complement alternative pathway hemolysis.

[0203] In some embodiments, the molecules of the invention or compositions or drugs or formulations or drug combinations comprising the same (e.g., drug combinations comprising two or more molecules of the invention) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for preventing and / or treating the relevant diseases or disorders mentioned herein or for blocking or inhibiting complement hemolysis.

[0204] The administration route of the molecule of the present invention or the composition or medicament or formulation comprising the same is according to known methods, for example, injection or infusion.

[0205] X. Diagnosis and testing

[0206] In certain embodiments, the molecules provided herein (eg, single domain antibodies, VHH antibodies, heavy chain antibodies or immunoconjugates against complement C5 protein, etc.) can be used to detect the presence of complement C5 in a biological sample.

[0207] The term "detection" as used herein includes quantitative or qualitative detection, and 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 body fluid.

[0208] In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein under conditions that allow it to bind to complement C5, and detecting whether a complex is formed between the molecule and complement C5. The formation of a complex indicates the presence of complement C5. The method can be an in vitro or in vivo method. In one embodiment, the molecules of the invention are used to select subjects suitable for treatment with an inhibitor against complement C5 (e.g., an antibody against complement C5, such as a molecule of the invention), for example, wherein complement C5 is a biomarker for selecting the subject.

[0209] In certain embodiments, a labeled molecule of the present invention (e.g., a single domain antibody, VHH antibody, heavy chain antibody, or immunoconjugate of the present invention against complement C5 protein, etc.) is provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detected moieties, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions.

[0210] In some embodiments, the label is a label such as biotin or a His tag.

[0211] 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 other therapies. In some embodiments, the sample is obtained during treatment with other therapies, or after treatment with other therapies.

[0212] In some embodiments, complement C5 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0213] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: testing 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 with 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 present invention, or a composition, medicament or formulation comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.

[0214] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the detailed description of the invention and are exemplified in the examples. Any or all of the features discussed throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0215] Example 1: Antigen preparation and alpaca immunization

[0216] Example 1.1 Expression and purification of recombinant human complement C5 protein (hC5)

[0217] The human full-length complement C5 protein includes α chain and β chain (UniProt#P01031), with a relative molecular mass of approximately 188.3KDa. The DNA molecule encoding hC5 (GenScript Biotechnology, NM_001735.2) was recombinantly cloned into a mammalian expression vector (pCDNA3.1), and Avi-tag (GLNDIFEAQKIEWHE) and 10xHistag (HHHHHHHHHH) were added to the C-terminus. The protein was expressed by transfecting suspension 293 cells (Expi293, Thermofisher) using PEI (Polysciences, Cat. No.: 24765-1) transfection reagent. The cell culture supernatant was harvested after 7 days of culture in a cell shaker at 220 rpm, 37°C, 8% CO2, and the hC5 protein was purified using a nickel column (His TrapTM excel catalog number: 17-3712-05, GE Healthcare) to obtain a protein with a purity greater than 85% and dissolved in PBS.

[0218] Human complement C5 amino acid sequence:

[0219]

[0220] Example 1.2 Alpaca Immunization

[0221] The hC5 protein prepared in 1) was emulsified and mixed with Freund's adjuvant, and then 500 μg / time was subcutaneously injected to immunize alpacas (Shanghai Bojin Biological). The immunization interval was once every 10 days, for a total of 4 immunizations. After the 4th immunization, 500 μg / time of protein without adjuvant was subcutaneously injected for shock immunization. After the second and third immunizations, serum was collected, centrifuged, and the supernatant was taken. The anti-hC5 antibody titer (Titer) in the serum was measured by ELISA.

[0222] Example 1.3 ELISA method for determination of anti-hC5 antibody titer in alpaca serum

[0223] Coat 100 μL of 1 μg / mL hC5 prepared in 1.1 on an ELISA plate (Cat. No. 42592, Costar) overnight at 4°C. Wash the plate with PBST the next day and block with PBS containing 2.5% skim milk (hereinafter referred to as PBSM) for 1 hour; take 10uL camel serum and dilute it with PBSM by 103, 104, 105, 106, 107, 108, and 109 times to obtain serum dilutions of different dilution multiples. Take 100uL of serum dilution of each concentration and add it to the blocked ELISA plate. Incubate at 37℃ for 1 hour. Wash each well 3 times with PBS (PBST) containing 0.1% Tween 20. After washing, add 100μL HRP-labeled anti-alpaca antibody (Goat PAb to Llama IgG (HRP), abcam catalog number: ab112786, 1:10000 dilution) to each well and incubate at room temperature for 30 minutes. Wash again with PBST 3 times, add TMB substrate (thermofisher, 002023) to develop color for 5-10 minutes, add stop solution (Shanghai Biotech, E661006-0200) to stop the reaction and read the plate at OD450nm. The detection results are as follows: Figure 1 shown.

[0224] The test results showed that a large amount of anti-hC5 antibodies were produced in the immunized alpaca serum, while no binding signal was detected in the non-immunized control alpaca serum. In addition, the antibody titer against hC5 in the serum after the third immunization was significantly increased compared with the second immunization, and it could still be clearly detected after a 1 million-fold dilution, indicating that the alpaca immunization was successful and subsequent experiments could be carried out.

[0225] Example 2: Construction and screening of anti-hC5 VHH antibody phage library

[0226] Example 2.1 Isolation of alpaca PBMC and preparation of VHH bacterial monoclonal library

[0227] After the fourth immunization, peripheral blood of alpacas was collected, and peripheral mononuclear lymphocytes (PBMCs) of alpacas were separated using lymphocyte separation tubes (Dakoway, catalog number: 7922021). PBMCs were lysed to extract Total RNA (RNeasy plus Minikit, catalog number: 74134, Qiagen), and then Total RNA was reverse transcribed into cDNA (SuperscriptTMIV First-strand synthwsissystem, catalog number: 18091050, Invirogen). Using cDNA as a template, the variable region of heavy chain antibodies in alpaca B cells was amplified by two rounds of polymerase chain reaction (PCR). The primers used in the two rounds of PCR were:

[0228] First-round PCR forward primer: GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO: 2);

[0229] First round PCR reverse primer: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 3);

[0230] Second round PCR forward primer: CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT (SEQ ID NO: 4);

[0231] Second round PCR reverse primer: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 5).

[0232] The amplified product was purified by gel recovery and then connected to the phage display vector pComb3XTT (Miaoling Biotechnology, P0831). The phage display vector with VHH inserted was transferred into TG1 cells (Lucigen) by electroporation to construct a VHH bacterial clone library.

[0233] The VHH bacterial clone library was inoculated into 2xYT medium (Shanghai Bioengineering) containing ampicillin and 2% glucose and cultured on a constant temperature shaker at 37°C. When the bacterial concentration OD600 reached 0.4-0.8, M13K07 (Cat. No. N0315S, NEB) helper phage was added and incubated at 37°C for 30 minutes. The cells were collected by centrifugation and resuspended in 2xYT medium containing kanamycin and 1mM IPTG (Shanghai Bioengineering), and cultured overnight at 30°C 250rpm. The overnight culture was centrifuged at 9000g for 15 minutes at 4°C, the supernatant was collected, sterilized by filtering through a 0.22μm filter (Millipore), and 1 / 5 volume of PEG-NaCl was added to precipitate the phage particles. The mixture was incubated on ice for 1 hour, and the obtained phage suspension was centrifuged at 9000g for 15 minutes at 4°C. The supernatant was discarded as much as possible, the phage particles were resuspended in PBS buffer, and transferred to a new sterilized tube. The obtained phage display library can be stored at 4°C for a short term or added with a final concentration of 20% glycerol and frozen in a -80°C refrigerator for long-term storage.

[0234] Example 2.2 Phage display screening of anti-hC5 antibodies with VHH structure

[0235] Biotin-labeled human complement C5 protein (hC5 protein prepared in Example 1) and biotin-labeled cynomolgus complement C5 protein (Cynomolgus Complement C5 Protein, His Tag, Catalog No.: C05-C52HX, ACRO) were used to screen VHH antibodies that can bind to both hC5 and cynoC5. The biotin labeling method is described in the kit manual, EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit, Thermofisher, 21435, and the ratio of biotin to protein labeling is 10:1.

[0236] Cynomolgus monkey complement C5 amino acid sequence:

[0237]

[0238] In the first round of screening, 50 nM biotin-labeled cynomolgus monkey complement C5 protein (hereinafter referred to as "cynoC5" or "biotin-labeled cynoC5") was incubated with the phage display library prepared in 2.1 in PBS containing 2.5% milk at room temperature for 2 hours, and then M-270 magnetic beads (Cat. No.: 65305, Invitrogen) were added to capture biotin-labeled cynoC5 and phage-displayed VHH antibodies that can bind to cynoC5, and incubated at room temperature for 30 minutes. M-270 magnetic beads were adsorbed on a magnetic stand, washed 10 times with PBST, and then washed 10 times with PBS, and phages were eluted with 0.25 mg / mL Trypsin solution (Trypsin, Cat. No. 25200-072, Gibco) at room temperature for 30 minutes. Then, 4 mg / mL AEBSF solution (AEBSF protease inhibitor, catalog number: 78431, Sigma) was added at a ratio of 20:1 to terminate the enzyme activity.

[0239] The eluted phage library is the first round of phage display library, which was infected with fresh TG1 bacteria (Lucigen) with OD=0.4-0.5 and allowed to stand for 30 minutes. It was added to 50mL of 2xYT medium containing ampicillin and 2% glucose, and shaken at 37℃225rpm for 1-2 hours, centrifuged at 9000g for 15 minutes, and the bacteria were resuspended in 2xYT medium containing kanamycin and 1mM IPTG, and 10-100uL of the suspension was taken for dilution counting, and helper phage M13K07 (NEB) was added, and cultured overnight at 30℃250rpm.

[0240] The second round of screening used 20 nM biotin-labeled hC5, and the method was the same as the first round of screening to obtain the second round of phage display library, and then the third round of screening was performed in sequence to obtain the third round of phage display library.

[0241] After the third round of phage library infection TG1 and mixing, dilute and spread on agar plates containing ampicillin antibiotics, pick a single colony of bacteria and inoculate it into a sterile 96-well deep-well plate in 2xYT medium containing ampicillin antibiotics and 2% glucose at 37°C and 225rpm. When the bacteria grow to the logarithmic phase, take 100uL and add it to a new 96-well plate and add M13K07 helper phage, incubate for 30 minutes, add 100uL of fresh 2xYT medium containing ampicillin and kanamycin, and shake the bacteria at 30°C overnight.

[0242] The next day, the culture supernatant containing phages was verified by phage ELISA to test whether it could specifically bind to hC5 and cynoC5.

[0243] Example 2.3 Phage ELISA screening of positive clones

[0244] StripwellTMMicroplate (Cat. No. 42592, Costar) was coated with hC5 and cynoC5 1μg / mL at 4℃ overnight. The next day, the plate was washed and blocked with PBS containing 2.5% milk at room temperature for 1 hour. After washing the plate with PBST, 100uL of the culture supernatant containing phage obtained in the previous step was added to each well and incubated at room temperature for 1 hour. The plate was washed again and anti-M13HRP detection antibody (M13Bacteriophage antibody (HRP) Cat. No.: 11973MM05T-HH013JA1501, Sino Bio) was added and incubated at room temperature for 30 minutes.

[0245] Wash the plate 5 times with PBST, then add 1-step Ultra TMB-ELISA reagent (ThermoScientific, catalog number: #34029) and observe the color change of each well. When the color is appropriate, stop the reaction with 2M sulfuric acid and read the value at OD450nm. According to the results of the ELISA reaction, pick out the positive clones C5006, C5006-12, C5006-13, C5006-14, C5006-15 and C5006-16 expressing positive phages and send them to Sunny Bio for sequencing. The gene sequence encoding the variable region of the VHH antibody and the amino acid sequence deduced based on the gene sequence are shown below (C5006-15 and C5006-16 sequences are not shown).

[0246] VHH antibody variable region nucleotide sequence

[0247] C5006:

[0248] GACGTTCAGCTGCAAGAGTCTGGCGGAGGACTGGTTCAAGCTGGCGGAAGCCTGACACTGTCTTGTGCCGCCTCTGGCAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACTACGCCAAGAACACCGTGTCTCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTATTACTGCGCCGCCTACAGCGAGTTCGCCAGAATCAGCGACAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCACGTGACCGTTTCTTCT(SEQ ID NO:7)

[0249] C5006-12:

[0250] CAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGGCTGGCGGATCTCTGACACTGAGCTGTGCTGCCTCTCCTGGCACCTTCAGCACAAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGATTACGCCGAGAACACCGTGTCTCTGCAGATGAGCAGCCTGAAGCCTGAGGACACCGCCGTGTATTACTGTGCCGCCACAAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCAAGTGACCGTGTCATCT(SEQ ID NO:8)

[0251] C5006-13:

[0252] CAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGGCTGGCGGATCTCTGACACTGAGCTGTGCCGCCTCTCCTAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGATTACGCCGAGAACACCGTGTCTCTGCAGATGAGCAGCCTGAAGCCTGAGGACACCGCCGTGTATTATTGTGCCGCCACCAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTCGTGACCGTTTCTTCT (SEQ ID NO:9)

[0253] C5006-14:

[0254] CAGGTGCAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGGCTGGCGGATCTCTGACACTGAGCTGTGCCGCCTCTCCTAGAACACTGAGCACCAATGCCATGGGCTGGTTCAGACAGACCCCTGGCAAAGAACGCGAGTTTGTGGCCGCTGTGTCTTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGATTACGCCGAGAACACCGTGTCTCTGCAGATGAGCAGCCTGAAGCCTGAGGACACCGCCGTGTATTATTGTGCCGCCACCAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTCGTGACCGTTTCTTCT (SEQ ID NO:10)

[0255] Amino acid sequence of the variable region of the VHH antibody

[0256] C5006:

[0257] DVQLQESGGGLVQAGGSLTLSCAASGRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVSLQMNSLKPEDTAVYYCAAYSEFARISDSSSYRYWGQGTHVTVSS (SEQ ID NO: 11)

[0258] C5006-12:

[0259] QVQVVESGGGLVQAGGSLTLSCAASPGTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTQVTVSS (SEQ ID NO: 12)

[0260] C5006-13:

[0261] QVQLVESGGGLVQAGGSLTLSCAASPRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSS (SEQ ID NO: 13)

[0262] C5006-14:

[0263] QVQVVESGGGLVQAGGSLTLSCAASPRTLSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSS (SEQ ID NO:14)

[0264] Table 1: VHH antibody CDR amino acid sequences (IMGT)

[0265] serial number 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

[0266] Example 3: Expression and purification of recombinant C5 VHH antibody

[0267] Example 3.1 Antibody Expression

[0268] The nucleic acids encoding the VHH antibodies of C5006, C5006-12, C5006-13, and C5006-14 were synthesized (Qingke Bio) and constructed into a mammalian cell expression vector (pCDNA3.1) containing a signal peptide (SEQ ID NO: 22) and a human IgG1 Fc containing a hinge region (SEQ ID NO: 23). The resulting plasmid DNA was transiently transfected into 293FT cells using Lipofectamine2000 (Thermofisher, Cat. No.: 11668030) transfection reagent and cultured for 96 hours. At the same time, the eculizumab antibody (IMGT / mAb-DB ID: 37, INN number: 8231) was expressed as a positive control for the hC5 antibody.

[0269] Signal peptide amino acid sequence: SEQ ID NO: 22

[0270] MGWSCIILFLVATATGVHS

[0271] Human IgG1 Fc region amino acid sequence containing a mutated hinge region: SEQ ID NO: 23

[0272] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (wherein the bold is the hinge region, and has the C220S mutation)

[0273] Human IgG1 Fc region amino acid sequence (wild type, without hinge region): SEQ ID NO: 24

[0274] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0275] Control antibody sequence:

[0276] >Eculizumab heavy chain:

[0277] QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:25)

[0278] >Eculizumab light chain:

[0279] DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:26)

[0280] Amino acid sequence of VHH antibody linked to human Fc fragment

[0281] C5006-G1:

[0282] DVQLQESGGGLVQAGGSLTLSCAASGRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVSLQMNSLKPEDTAVYYCAAYSEFARISDSSSYRYWGQGTHVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:27)

[0283] C5006-12-G1:

[0284] QVQVVESGGGLVQAGGSLTLSCAASPGTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:28)

[0285] C5006-13-G1:

[0286] QVQLVESGGGLVQAGGSLTLSCAASPRTFSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:29)

[0287] C5006-14-G1:

[0288] QVQVVESGGGLVQAGGSLTLSCAASPRTLSTNAMGWFRQTPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAENTVSLQMSSLKPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:30)

[0289] Example 3.2 Antibody Purification

[0290] The VHH antibody linked to the human Fc fragment obtained by purification using Protein A (sequence see SEQ ID NO: 27-30) was dissolved in PBS buffer.

[0291] Example 4: Detection of hemolytic inhibition activity of C5 VHH antibody

[0292] Example 4.1 Detection of inhibitory activity of classical complement pathway (CP) hemolysis

[0293] This experiment was conducted by testing different concentrations of the test antibodies to determine the degree of inhibition of complement-mediated hemolysis of sheep erythrocytes in 5% normal human serum, and to analyze and compare the inhibitory activity of each antibody on hemolysis mediated by the classical complement pathway. The experimental process is briefly described as follows:

[0294] (1) Antibody gradient dilution: Determine the mass concentration and molecular weight of the antibody to be tested, prepare the antibody into a 1200 nM solution using CP buffer (10 mM HEPES, 150 mM NaCl, 3 mM CaCl2, 3 mM MgCl2, 0.1% Gelatin, pH 7.4), and then dilute the solution 3-fold from 1200 nM using CP buffer for a total of 6 concentration gradients;

[0295] (2) Serum dilution: Normal human serum (NHS, Shanghai Rubai Biotechnology) was taken out from the -70°C freezer, thawed naturally at room temperature, and then the serum stock solution was diluted with CP buffer to a 20% NHS solution;

[0296] (3) Activation of sheep red blood cells: 2% sheep red blood cells (sheep Red Blood Cell, Catalog No.: SBJ-RBC-S002, Sbjbio) were washed with CP buffer until the supernatant was transparent, and then the red blood cells were resuspended with CP buffer. Rabbit anti-sheep red blood cell antibody (hemolysin, Rabbit anti-sheep RBC, Catalog No.: S25861, Yuanye Bio) with a titer of 1:4000 was added to the suspension, and the sheep red blood cells were incubated at 37°C for 30 minutes to activate the sheep red blood cells. The activated sheep red blood cells were washed three times with CP buffer and resuspended with CP buffer.

[0297] (4) Incubation of antibody reaction solution with red blood cells: Add 50 ul of sheep red blood cells activated in step (3) to a 96-well V-bottom plate, then add 25 ul of the antibody diluted in step (1), add 25 μL of the serum diluted in step (2) to the mixture, set up two control groups, the negative control group does not contain antibody and uses serum inactivated by 56°C, and the positive control group does not contain antibody and uses normal serum. Incubate at 37°C for 1 hour;

[0298] (5) Reaction termination: After the incubation, add 100 μL stop buffer (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, 0.1% Gelatin) to each well to terminate the reaction. Centrifuge the 96-well V-bottom plate at 3000 rpm for 5 min in a 4°C centrifuge. Transfer 100 μL of the supernatant to a 96-well flat-bottom plate. Detect the absorbance of the supernatant at OD415 nm in a multi-function plate reader (Molecular Devices) and save the data.

[0299] (6) Data processing: Substitute the OD415nm reading obtained in (5) into the following formula to calculate the hemolysis inhibition rate of red blood cells: Hemolysis inhibition rate (%) = (positive control OD415-experimental group OD415) / (positive control OD415-negative control OD415). Then, with the final antibody concentration as the horizontal axis and the hemolysis inhibition rate as the vertical axis, Graph Pad Prism8 software was used to perform a four-parameter fitting plot and calculate the IC50 value.

[0300] The test results showed that C5 VHH antibodies C5006, C5006-12, C5006-13 and C5006-14 could effectively inhibit the classical pathway (CP)-mediated hemolysis of sheep erythrocytes, and the inhibitory ability gradually increased with the increase of antibody concentration. In comparison, C5006, C5006-13 and C5006-12 had better inhibitory activity, C5006-14 had slightly weaker inhibitory activity, and C5006-15 and C5006-16 had no inhibitory activity. For experimental results, see Figure 2 .

[0301] Example 4.2 Detection of inhibitory activity of the alternative complement pathway (AP) hemolysis

[0302] This experiment measures the degree of inhibition of rabbit erythrocyte hemolysis mediated by complement in 10% normal human serum by different concentrations of the tested antibodies, and analyzes and compares the inhibitory activity of each antibody on hemolysis mediated by the complement alternative pathway. The experimental process is briefly described as follows:

[0303] (1) Antibody gradient dilution: Determine the mass concentration and molecular weight of the antibody to be tested, use AP buffer (10mM HEPES, 150mM NaCl, 3mM MgCl2, 0.1% Gelatin, 5mM EGTA, pH7.4) to prepare the antibody concentration to 1200nM solution, and then start from 1200nM and dilute it 3 times downward with AP buffer, for a total of 6 concentration gradients;

[0304] (2) Serum dilution: Normal human serum (NHS, Shanghai Rubai Biotechnology) was taken out from the -70°C refrigerator, thawed naturally at room temperature, and then the serum stock solution was diluted into 40% NHS solution with AP buffer;

[0305] (3) Preparation of rabbit red blood cells: Take 2% rabbit red blood cells (rabbit Red Blood Cell, catalog number: SBJ-RBC-RAB002, Sbjbio) and wash them with AP buffer until the supernatant is transparent, then resuspend the red blood cells with AP buffer;

[0306] (4) Incubation of antibody reaction solution with red blood cells: Add 50 ul of rabbit red blood cells prepared in step (3) to a 96-well V-bottom plate, then add 25 ul of the antibody diluted in step (1), add 25 ul of the serum diluted in step (2) to the mixture, set up two control groups, the negative control group does not contain antibody and uses serum inactivated by 56°C treatment, and the positive control group does not contain antibody and uses normal serum. Incubate at 37°C for 1 hour;

[0307] (5) Reaction termination: After the incubation, add 100 μL of stop buffer (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, 0.1% Gelatin) to each well to terminate the reaction. Centrifuge the 96-well V-bottom plate at 3000 rpm for 5 min at 4 °C. Transfer 100 μL of the supernatant to a 96-well flat-bottom plate. Detect the absorbance of the supernatant at OD415 nm in a multi-function plate reader (Molecular Devices) and save the data.

[0308] (6) Data processing: Substitute the OD415nm reading obtained in (5) into the following formula to calculate the hemolysis inhibition rate of red blood cells: Hemolysis inhibition rate (%) = (positive control OD415-experimental group OD415) / (positive control OD415-negative control OD415). Then, with the final antibody concentration as the horizontal axis and the hemolysis inhibition rate as the vertical axis, Graph Pad Prism8 software was used to perform a four-parameter fitting plot and calculate the IC50 value.

[0309] The test results showed that C5 VHH antibodies C5006, C5006-12, C5006-13 and C5006-14 could effectively inhibit the alternative pathway (AP)-mediated hemolysis of rabbit erythrocytes, and the inhibitory ability gradually increased with the increase of antibody concentration. In comparison, the inhibitory activity of C5006, C5006-13 and C5006-12 was better, and the inhibitory activity of C5006-14 was slightly weaker. For experimental results, see Figure 3 .

[0310] Example 5: C5 VHH antibody humanization

[0311] Sequence comparison and analysis revealed that the variable region of the C5 VHH antibody had a high degree of homology with the human germline IGHV3-23*01 sequence. The antibody was humanized using the CDR transplantation method with the IGHV3-23*01 sequence as the backbone. The CDR region of the antibody was defined according to the IMGT rules. After several rounds of back mutation screening, two humanized sequences, C5006d1 and C5006e3, were finally obtained. The nucleotide and amino acid sequences of the humanized antibodies are shown below.

[0312] VHH antibody variable region nucleotide sequence

[0313] C5006d1:

[0314] GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCTGGCAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGGCCCCTGGCAAAGAAAGGGAATTCGTGGCCGCTGTGTCCTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACTACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGCGGCCTGAGGATACCGCCGTGTACTACTGTGCCGCTTACAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTGGTCACCGTTTCTTCT (SEQ ID NO:31)

[0315] C5006e3:

[0316] GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCTCCCAGAACCTTCAGCACCAATGCCATGGGCTGGTTCAGACAGGCCCCTGGCAAAGAAAGGGAATTCGTGGCCGCTGTGTCCTGGGGCAATGGCATCCCTTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACTACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGCGGCCTGAGGATACCGCCGTGTACTACTGTGCCGCTACCAGCGAGTTCGCCCGGATCAGCGATAGCAGCAGCTACAGATATTGGGGCCAGGGCACCCTGGTCACCGTTTCTTCT (SEQ ID NO:32)

[0317] Amino acid sequence of the variable region of the VHH antibody

[0318] C5006d1:

[0319] EVQLLESGGGLVQPGGSLRLSCAASGRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAAYSEFARISDSSSYRYWGQGTLVTVSS (SEQ ID NO: 33)

[0320] C5006e3:

[0321] EVQLLESGGGLVQPGGSLRLSCAASPRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSS (SEQ ID NO: 34)

[0322] Table 2: VHH antibody CDR amino acid sequences (IMGT)

[0323] serial number 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

[0324] Example 6: Expression, purification and purity detection of recombinant humanized C5 VHH antibody

[0325] Example 6.1 Humanized C5 VHH Antibody Gene Synthesis, Antibody Expression and Purification

[0326] The gene synthesis, expression and purification of the humanized C5 VHH antibody were the same as described in Example 3, and the complete antibody sequence information is shown below.

[0327] Humanized VHH antibody amino acid sequence linked to human Fc fragment

[0328] C5006d1-G1:

[0329] EVQLLESGGGLVQPGGSLRLSCAASGRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAAYSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:35)

[0330] C5006e3-G1:

[0331] EVQLLESGGGLVQPGGSLRLSCAASPRTFSTNAMGWFRQAPGKEREFVAAVSWGNGIPYYADSVKGRFTISRDYAKNTVYLQMNSLRPEDTAVYYCAATSEFARISDSSSYRYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:36)

[0332] Example 6.2 Detection of the purity of humanized C5 VHH antibody (SEC-HPLC)

[0333] Protein purity analysis was performed using size exclusion chromatography (SEC). The purified protein samples in PBS were applied to a 300×4.6 mm, 5 μm column (TOSOH) of TSKgel Super SW3000. SEC was performed using a U3000 HPLC instrument (DIONEX). Elution was performed isocratically at a flow rate of 0.25 mL / min. All proteins were detected using UV at 280 nm and 214 nm. Component analysis was performed using the instrument's own software.

[0334] The test results showed that the purity of the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 in solution reached 96% and 98% respectively after one-step purification by protein A, which was excellent ( Figure 4 and Figure 5 ).

[0335] Example 7: Detection of binding activity of humanized C5 VHH antibody

[0336] Example 7.1 ELISA for detecting antigens by coated antibodies

[0337] To detect the binding ability of humanized C5 VHH antibody to antigen human C5, the antibody was diluted to 0.5 μg / mL with PBS, 100 μL was taken to coat the ELISA plate (Corning, Cat. No.: 42592), and incubated at 4°C overnight. The next day, after removing the coating solution, the plate was washed 3 times with PBST, and blocked with PBS containing 2.5% skim milk powder at 37°C for 1 hour. The hC5 protein prepared in Example 1.1 was gradiently diluted with PBS containing 2.5% skim milk powder, starting from 1 μg / mL and diluted 5 times, with a total of 7 concentration points, and the negative control point did not contain hC5. After washing the plate 3 times with PBST, the diluted hC5 solution was added and incubated at 37°C for 1 hour. After washing the plate 3 times with PBST, 100 μL of 1:5000 diluted HRP-labeled rabbit anti-HIS secondary antibody (jackson, Cat. No.: 300-035-240) was added and incubated at 37°C for 1 hour. After washing the plate three times with PBST, TMB (Invitrogen™, Catalog No.: 002023) substrate was added to develop color for 5-10 minutes. ELISA stop solution (Sangon, Catalog No.: E661006-0500) was added to terminate the reaction and the OD450 absorbance was read using a microplate reader.

[0338] The test results showed that when the coated antibody detected the gradient dilution of the antigen human C5, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 could bind to human C5, and the binding signal gradually increased with the increase of C5 concentration. In comparison, the binding abilities of the two were similar, and were basically consistent with the binding ability of the control antibody eculizumab. The experimental results can be found in Figure 6.

[0339] Example 7.2 ELISA coated with antigen detection antibody

[0340] To further detect the binding ability of humanized C5 VHH antibody to antigen human C5, human C5 protein was diluted to 1 μg / mL with PBS, and 100 μL was coated on an ELISA plate (Corning, Cat. No. 42592) and incubated overnight at 4°C. The next day, after removing the coating solution, the plate was washed 3 times with PBST and blocked with PBS containing 2.5% skim milk powder at 37°C for 1 hour. The antibody to be detected was diluted with PBS containing 2.5% skim milk powder, starting from 1 μg / mL and diluted 5 times, with a total of 7 concentration points, and the negative control point did not contain antibody. After washing the plate 3 times with PBST, the diluted antibody solution was added and incubated at 37°C for 1 hour. After washing the plate 3 times with PBST, 100 μL of 1:5000 diluted HRP-labeled goat anti-human IgG antibody (Jackson, Cat. No. 109-035-098) was added and incubated at 37°C for 1 hour. After washing the plate three times with PBST, TMB (Invitrogen™, Catalog No.: 002023) substrate was added to develop color for 5-10 minutes. ELISA stop solution (Sangon, Catalog No.: E661006-0500) was added to terminate the reaction and the OD450 absorbance was read using a microplate reader.

[0341] The test results show that when the antigen human C5 protein is coated to detect the gradient dilution of the antibody, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 can bind to human C5, and the binding signal gradually increases with the increase of antibody concentration. In comparison, the binding abilities of the two are similar, and both are stronger than the binding ability of the control antibody eculizumab. The experimental results are shown in Figure 7 .

[0342] Example 8: Detection of hemolytic inhibition activity of humanized C5 VHH antibody

[0343] Example 8.1 Detection of the inhibitory activity of humanized C5 VHH antibody on CP / AP hemolysis in normal human serum (NHS)

[0344] To detect the inhibitory activity of the humanized C5 VHH antibody on complement-mediated hemolysis in human serum, the same method as in Example 4 was used for detection. The antibody was prepared into an 80 μg / mL solution (final concentration 20 μg / mL) and diluted 2-fold to a total of 10 concentration points.

[0345] The results showed that humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 had similar inhibitory activities against CP-mediated hemolysis of sheep erythrocytes, with IC50 values ​​of 0.46 and 0.52 μg / mL, respectively. Their activities were stronger than those of the control antibody eculizumab, whose inhibitory activity IC50 was 0.76 μg / mL ( Figure 8 ); For AP-mediated hemolysis of rabbit erythrocytes, humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 also have similar inhibitory activity, with IC50 of 1.52 and 1.63 μg / mL, respectively. Their activities are also stronger than the control antibody eculizumab, whose inhibitory activity IC50 is 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 the activity is stronger than that of the control antibody eculizumab.

[0346] Example 8.2 Detection of the inhibitory activity of humanized C5 VHH antibody on monkey serum (NMS) CP / AP hemolysis

[0347] As the model animal closest to humans, non-human primates (NHPs) are often used to evaluate the safety and efficacy of drugs during drug development, which can greatly improve the success rate of drug development and reduce the risk of late-stage drug development. To detect the inhibitory activity of humanized C5 VHH antibodies on complement-mediated hemolysis in NHP serum, the same method as in Example 4 and the commonly used model animal cynomolgus monkey serum (Suzhou Xihua New Drug Development Co., Ltd.) were used for detection. The antibody was prepared into a solution of 80 μg / mL (final concentration 20 μg / mL) and diluted 2-fold gradient, with a total of 10 concentration points.

[0348] The results showed that humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 had the same inhibitory activity against CP-mediated hemolysis of sheep erythrocytes, with IC50 values ​​of 0.81 μg / mL. In contrast, the control antibody eculizumab had only weak hemolysis inhibitory activity ( Fig.10 ); For AP-mediated hemolysis of rabbit erythrocytes, humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 had similar inhibitory activity, with IC50 of 3.54 and 4.06 μg / mL, respectively. Similarly, the control antibody eculizumab had only weak hemolysis inhibitory activity ( Fig.11 ). In summary, the humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 have good monkey complement inhibition activity, while the humanized antibody eculizumab has very weak activity.

[0349] Example 9: Thermal stability test of humanized C5 VHH antibody

[0350] The thermal stability of antibodies is an important indicator for evaluating the developability of antibody drugs. Good thermal stability often means that antibody drugs have better storage stability and shelf life. This experiment used a gradient heating method to heat the antibody, and then performed activity tests on samples treated at different temperatures to evaluate the thermal stability of the humanized C5 VHH antibody.

[0351] Example 9.1 Antibody Heat Treatment

[0352] The antibody to be tested was prepared into a 100 nM solution using CP buffer. After packaging, the antibody was incubated at different temperatures for 1 hour. The temperature was set to decrease by 5°C from 85°C to 55°C. A total of 7 temperature points were prepared. Samples that were not heated and incubated at 4°C were used as controls.

[0353] Example 9.2 CP activity detection of heat-treated antibodies

[0354] In order to detect the change in activity of the antibody after treatment at different temperatures, the same CP detection method as in Example 4 was used for detection, and the final antibody concentration was 25 nM.

[0355] The results showed that eculizumab completely lost its complement inhibitory activity after being treated at 70°C or above; C5006d1-G1 completely lost its complement inhibitory activity after being treated at 80°C or above; and C5006e3-G1 completely lost its complement inhibitory activity after being treated at 85°C. In comparison, humanized C5 VHH antibodies C5006d1-G1 and C5006e3-G1 had better heat stability. Fig.12 shown.

Claims

1. A VHH antibody that specifically binds to complement C5, which contains three complementary determining regions (CDRs) CDR1, CDR2 and CDR3. (i) CDR1 consists of the amino acid sequence set forth in SEQ ID NO:17, CDR2 consists of the amino acid sequence set forth in SEQ ID NO:19, and CDR3 consists of the amino acid sequence set forth in SEQ ID NO:21; or (ii) CDR1 consists of the amino acid sequence set forth in SEQ ID NO:15, CDR2 consists of the amino acid sequence set forth in SEQ ID NO:19, and CDR3 consists of the amino acid sequence set forth in SEQ ID NO:20; (iii) CDR1 consists of the amino acid sequence set forth in SEQ ID NO:16, CDR2 consists of the amino acid sequence set forth in SEQ ID NO:19, and CDR3 consists of the amino acid sequence set forth 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 to complement C5, which consists 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 to complement C5, comprising the VHH antibody according to any one of claims 1 to 3.

5. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1 to 3 linked to an antibody constant region or Fc region. The heavy chain antibody of claim 5 , wherein the antibody constant region or Fc region is derived 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 connected to the Fc region via a hinge region.

8. The heavy chain antibody of claim 7, wherein the hinge region consists of the amino acid sequence shown 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 shown in SEQ ID NO:

24.

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

11. A heavy chain antibody that specifically binds to complement C5, which consists of the amino acid sequence shown 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 to 3, or the heavy chain antibody of any one of claims 4 to 11.

13. The nucleic acid molecule of claim 12, which consists of the nucleic acid sequence described in 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 for preparing the VHH antibody of any one of claims 1 to 3, or the heavy chain antibody of any one of claims 4 to 11, the method comprising culturing the host cell of claim 15 under conditions suitable for expression of the VHH antibody or heavy chain antibody or its chain, and recovering the VHH antibody or heavy chain antibody from the 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 pharmaceutically acceptable excipient.

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