Antibodies specifically binding to MASP3 and multispecific antibodies specifically binding to MASP3 and MASP2

Abstract

The invention relates to the field of biological medicine, in particular to a specific recognition MASP3 antibody and application thereof, and the antibody has high human MASP3 affinity and alternative pathway inhibitory activity. In addition, the invention also relates to a multispecific antibody which specifically binds to MASP3 and MASP2, as well as a pharmaceutical composition comprising an antibody which specifically binds to MASP3 or a multispecific antibody which specifically binds to MASP3 and MASP2, the antibody or the composition is used for preparing a medicament for treating autoimmune diseases, transplantation-related diseases, inflammatory diseases, hematological diseases, coagulation diseases, angiogenesis-dependent diseases and / or virus infectious diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims priority to Chinese patent application No. 202310680269.8, application date June 9, 2023, and invention name “Antibodies specifically binding to MASP3 and multispecific antibodies specifically binding to MASP3 and MASP2”, and the entire contents of the application are incorporated herein by reference.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (text name: M23-3.xml, recording date: 2023.06.09, size: 74KB) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to antibodies that specifically bind to MASP3, multispecific antibodies that specifically bind to MASP3 and MASP2, and pharmaceutical compositions comprising antibodies or multispecific antibodies that specifically bind to MASP3, as well as preparation methods and uses thereof, including methods for preventing and treating complement disorders using the same. Background Art

[0006] The complement system plays a vital role in both innate and adaptive immune responses. Complement removes immune complexes and cell debris, and rapidly produces highly efficient and strictly regulated inflammatory and cytolytic immune responses to infectious organisms (including bacteria, viruses, and protozoan parasites) (Dunkelberger, Jason R, and Wen-Chao Song. Cell research vol. 20, 1 (2010): 34-50.). Complement activation leads to a proteolytic cascade, ultimately leading to inflammatory cell recruitment, phagocytosis, and cell lysis (Kjaer, Troels R et al. Molecular immunology vol. 56, 4 (2013): 413-22.). It also produces anaphylatoxins (C3a, C4a, C5a) that can effectively promote inflammatory responses, as well as opsonins (C3b and C4b) that cover the surface of pathogens and mediate phagocytosis. The end point of the cascade reaction is the assembly of the membrane attack complex (MAC) on the cell membrane, forming a pore that leads to cell lysis (Dunkelberger, Jason R, and Wen-Chao Song. Cell research vol. 20, 1 (2010): 34-50.).

[0007] The complement system can be activated through three pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The classical pathway starts with C1q. When C1q recognizes some target molecules (such as immune complexes or surface-bound pentraxin), its conformation changes, allowing C1q to interact with C1r and C1s to form activated C1qr2s2 (Carroll, MC. Annual review of immunology vol. 16 (1998): 545-68.). The enzyme active site is on C1s. Subsequently, the activated C1qr2s2 cleaves C4 and subsequent C2 to generate the C3 convertase C4b2a in the classical pathway. C4b2a can induce proteolytic activity and activate the common terminal pathway (Sim, RB, and SA Tsiftsoglou. Biochemical Society transactions vol. 32, Pt 1 (2004): 21-7.).

[0008] The lectin pathway can be triggered by circulating pattern recognition receptors (PRRs) that recognize carbohydrates on the surface of microorganisms, including mannose-binding lectin (MBL), collectins, and ficolins. Ficolins are PRRs with fibrin-like domains, such as M-ficolin (ficolin-1), L-ficolin (ficolin-2), and H-ficolin (ficolin-3). MBL-related serine proteases (MASP-1 and MASP-2) are evolutionarily related to C1r and C1s and have similar functions. MBL forms a complex with MASPs, then cleaves C4 and C2 to form the C3 convertase C4b2a in the lectin pathway (Garred, Peter et al. Immunological reviews vol. 274, 1 (2016): 74-97.).

[0009] When C3b appears on the surface of the activator, the alternative pathway can be initiated (Lachmann, Peter J. Immunobiology vol. 223, 8-9 (2018): 519-523.). After C3b captures FB, the FB bound to C3b can be cleaved by FD into Ba and Bb. In the blood, MASP-3 can cleave pro-PD and continue to provide PD for the alternative pathway, even before any activation signal appears (Oroszlán, Gábor et al. Journal of immunology (Baltimore, Md.: 1950) vol. 196, 2 (2016): 857-65.; Dobó, József et al. Scientific reports vol. 6 31877. 18Aug. 2016). C3b and Bb form the C3 convertase C3bBb in the alternative pathway, which can cleave more C3, thereby producing more C3 convertase complexes. This positive feedback mechanism can amplify complement activation initiated by the classical or alternative pathway (Harboe, M et al. Clinical and experimental immunology vol. 138, 3 (2004): 439-46.). The alternative pathway can also be self-activated by the so-called "slow transport" mechanism (Pangburn, MK et al. The Journal of experimental medicine vol. 154, 3 (1981): 856-67.). C3 is slowly hydrolyzed in the circulation to generate C3 (H2O) similar to C3b. After C3 (H2O) binds to FB, it is cut by FD. C3 (H2O) Bb is a liquid C3 convertase that can generate C3b near any surface. On the surface of its own cells, it can be protected from complement-mediated damage by different complement inhibitors. On unprotected surfaces (such as bacterial surfaces), deposited C3b can initiate the amplification loop in the alternative pathway, leading to complete complement activation.

[0010] When the density of deposited C3b reaches a certain point, C3b forms C5 convertase (C4b2aC3b or C3bBbC3b) with C3 convertase (C4b2a or C3bBb) (Mannes, Marco et al. Blood vol.137, 4 (2021): 443-455.; Roumenina, Lubka T. Blood vol.137, 4 (2021): 431-432.). From this point on, the three activation pathways enter the common terminal pathway (TP). C5 convertase cleaves C5 into C5a and C5b, of which C5a is a very effective anaphylatoxin, while C5b combines with C6 and C7 to form C5b67, exposing the membrane binding site, non-specifically binding to the nearby cell membrane, and capturing C8 and multiple C9 molecules to form the C5b6789n complex, the membrane attack complex (MAC). MAC inserted into the cell membrane can form hydrophilic pores through the membrane, leading to lysis and destruction of target cells (Tegla, Cosmin A et al. Immunologic research vol. 51, 1 (2011): 45-60).

[0011] The complement system is a very effective mechanism for cell killing and initiating inflammation. To prevent overactivation, the complement system is strictly controlled by different inhibitory mechanisms. A delicate balance between activation and inhibition is necessary to maintain inflammatory homeostasis in the human body. When this balance is disrupted for any reason, self-organization is disrupted and serious diseases occur. There are many clinical diseases involving uncontrolled (or sometimes insufficient) complement activation. Often, the etiology of these diseases is complex, and unnecessary complement activation is only one of the pathological factors. However, evidence obtained through various disease models suggests that preventing or inhibiting pathological complement activation may be a promising treatment approach (Dobó, József et al. Frontiers in immunology vol.9 1851.).

[0012] Inappropriate or uncontrolled activation of the complement system has been identified in many diseases. Some of these are rare, mainly complement-mediated diseases, such as paroxysmal nocturnal hemoglobinuria, C3 glomerulonephritis, and atypical hemolytic uremic syndrome. Complement also plays a role in various multifactorial diseases that affect millions of people worldwide, such as ischemia-reperfusion injury (myocardial infarction, stroke) (Hart, Melanie L et al. "Journal of immunology (Baltimore, Md.: 1950) vol. 174, 10 (2005): 6373-80.);), age-related macular degeneration (Geerlings, Maartje Jet al. Molecular immunology vol. 84 (2017): 65-76.) and several neurodegenerative diseases (Hong, Soyon et al. Science (New York, NY) vol. 352, 6286 (2016): 712-716.; Sekar, Aswin et al. Nature vol. 530, 7589 (2016): 177-83.).

[0013] Recently, in addition to AP, LP, as a trigger of complement activation, also plays an important role in the pathogenesis of IRI and AMD (Hart, Melanie L et al. Journal of immunology (Baltimore, Md.: 1950) vol. 174, 10 (2005): 6373-80.; Jordan, J E et al. Circulation vol. 104, 12 (2001): 1413-8.; Rohrer, et al.Molecular immunology vol.48,6-7(2011):e1-8.;Joseph,Kusumam et al.The Journal of biological chemistry vol.288,18(2013):12753-65.)。 In IRI and AMD, the mechanism of complement activation is: first, natural IgM binds to self-antigens and causes LP activation, followed by AP amplification reaction and tissue / organ damage (Joseph,Kusumam et al.The Journal of biological chemistry vol.288,18(2013):12753-65.;Holers,V Michael et al.Seminars in immunology vol.28,3(2016):260-7.)。 Obviously, without the expansion loop provided by AP, CP and LP will not work effectively. In addition, both LP and AP are involved in IgA nephropathy (Daha, Mohamed R, and Cees van Kooten. Journal of nephrology vol. 29, 1 (2016): 1-4.). Another study showed that AP and CP / LP pathways are activated simultaneously in autoimmune glomerulonephritis (Genest, Dominique S et al. Kidney international reports vol. 7, 5 1027-1036. 14 Feb. 2022). In these diseases, dysregulation of LP and AP is believed to play an important pathophysiological role.

[0014] Therefore, a drug or pharmaceutical composition that inhibits the terminal pathway or specifically targets multiple upstream molecules of the complement pathway has become a new research and development direction. Although targeting the downstream terminal pathway may be more effective in controlling excessive complement activation, important complement system functions will also be lost. These measures include fighting infection, connecting innate immunity and adaptive immune responses, clearing debris, apoptotic cells, etc. (Dobó, József et al. Frontiers in immunology vol.91851.8Aug.2018,). Therefore, targeting the upstream molecules of the AP and / or LP signaling pathways can maintain the clearance ability of the immune system, maintain a certain degree of host defense against pathogens, and specifically inhibit the complement pathway.

[0015] At present, there are reports on antibodies targeting MASP3 or MASP2, for example, Chinese patent CN103687620B discloses anti-MASP2 antibody OMS721 and its application; Chinese patent application CN109715209A discloses anti-MASP3 antibody OMS906 (used as a control antibody in the examples of this application) and its application. However, there are no monoclonal antibodies targeting MASP3 and multispecific antibodies (e.g., bispecific antibodies) targeting both MASP3 and MASP2 on the market, so there is an urgent need to develop monoclonal antibodies targeting MASP3 and multispecific antibody drugs targeting both MASP3 and MASP2.

[0016] The disclosures of all publications, patents, patent applications and published patent applications mentioned in this application are incorporated herein by reference in their entirety.

[0017] Application Overview

[0018] In one aspect, the present application provides an isolated antibody or antigen-binding fragment that can specifically bind to MASP3. In some embodiments, the present application provides an isolated antibody or antigen-binding fragment that specifically binds to MASP3, comprising: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:13, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:16, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:19.

[0019] In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds to MASP3 is provided, comprising: (i) H , comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 23; and V L , which comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 33; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 24; and V L, comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 34;

[0020] (iii)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 34; (iv) V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L , comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 34; (v) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; (vi) V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; (vii) V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:35.

[0021] In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds to MASP3 is provided, which competes with any of the above-described isolated antibodies or antigen-binding fragments for specific binding to MASP3. In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds to MASP3 is provided, which specifically binds to the same epitope as any of the above-described isolated antibodies or antigen-binding fragments.

[0022] In some embodiments, any of the isolated antibodies or antigen-binding fragments that specifically bind to MASP3 as described above comprises an Fc region. In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to MASP3 is a full-length IgG antibody. In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to MASP3 is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to MASP3 is a chimeric, fully human, or humanized antibody. In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to MASP3, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody, and linear antibody.

[0023] In some embodiments, an isolated nucleic acid molecule is provided, the nucleic acid molecule encoding any one of the antibodies or antigen-binding fragments that specifically bind to MASP3 as described above. In some embodiments, a vector is provided, the vector comprising any one of the nucleic acid molecules as described above. In some embodiments, a host cell is provided, the host cell comprising any one of the antibodies or antigen-binding fragments that specifically bind to MASP3 as described above, any one of the nucleic acid molecules as described above, or any one of the vectors as described above. In some embodiments, a method for preparing an antibody or antigen-binding fragment that specifically binds to MASP3 is provided, comprising: a) culturing any one of the host cells described above under conditions effective to express the antibody or antigen-binding fragment that specifically binds to MASP3; and b) obtaining the expressed antibody or antigen-binding fragment from the host cell.

[0024] In some embodiments, pharmaceutical compositions, kits, and articles of manufacture are provided that include any of the antibodies or antigen-binding fragments that specifically bind to MASP3 as described above.

[0025] In some embodiments, a method of treating a disease or condition in a desired individual is provided, comprising administering to the individual an effective amount of any one of the antibodies or antigen-binding fragments that specifically bind to MASP3 as described above, or a pharmaceutical composition comprising the same. In some embodiments, use of any one of the antibodies or antigen-binding fragments that specifically bind to MASP3 as described above in the preparation of a pharmaceutical composition for treating a disease or condition in a desired individual is provided. In some embodiments, use of any one of the antibodies or antigen-binding fragments that specifically bind to MASP3 as described above, or a pharmaceutical composition comprising the same, in the preparation of a medicament for treating a disease or condition is provided. In some embodiments, the disease or condition comprises a disease or condition associated with complement dysregulation. In some embodiments, the disease or condition comprises ischemia-reperfusion injury, atherosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), Henoch-Schönlein purpura nephritis, IgA nephropathy, ischemic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), secondary TMA after transplantation, Upshaw-Schulman syndrome, arthritis, traumatic brain injury, respiratory distress syndrome, Invasive pneumonia, neuromyelitis optica, multiple sclerosis, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, myasthenia gravis, Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancers, age-related macular degeneration, retinopathy, proliferative diabetes, vitreous hemorrhage secondary to retinopathy, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection.

[0026] On the other hand, the present application provides a multispecific antibody (e.g., a multispecific antibody) that specifically binds to MASP3 and specifically binds to MASP2, and a pharmaceutical composition comprising a multispecific antibody (e.g., a multispecific antibody) that specifically binds to MASP3 and specifically binds to MASP2. In other embodiments, the present application also provides a method for treating a disease associated with complement disorder using the multispecific antibody (e.g., a multispecific antibody) or a pharmaceutical composition thereof. In other embodiments, the present application also provides a use of the multispecific antibody (e.g., a multispecific antibody) or a pharmaceutical composition thereof in the preparation of a medicament for treating a disease associated with complement disorder.

[0027] In some embodiments, the present application provides a multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2, wherein the first antigen binding domain comprises: (a) V H , which comprises V as shown in the amino acid sequence SEQ ID NO:23 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:33 L or (b) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9: and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the second antigen binding domain comprises: (a) V H , which comprises V as shown in the amino acid sequence SEQ ID NO:48 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:50 L or (b) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:47.

[0028] In some embodiments, the present application provides a multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2, wherein the second antigen binding domain comprises: (a) V H , which comprises V as shown in the amino acid sequence SEQ ID NO:48 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:50 L or (b) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:47.

[0029] In some embodiments, according to any multispecific antibody described herein, the first antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9: and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V LIt comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:47.

[0030] In some embodiments, according to any multispecific antibody described herein, the first antigen binding domain comprises: (a) V H , comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 23; and V L , which comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 33; (b) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 34; (c) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; (d) V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; or (e) V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:35.

[0031] In some embodiments, according to any multispecific antibody described herein, the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO:48; and V L , which comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:50.

[0032] In some embodiments, according to any multispecific antibody described herein, the first antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; and wherein the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO:48; and V L , which comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:50.

[0033] In some embodiments, the multispecific antibodies described herein comprise Fc. In other embodiments, the Fc described herein is selected from the Fc of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. In some embodiments, the Fc comprises an Fc variant. In some embodiments, the Fc is glycosylated. In some embodiments, the Fc is deglycosylated. In some embodiments, the Fc is fucosylated or afucosylated. In some embodiments, the Fc variant comprises a substitution at position 297. In some embodiments, the substitution at position 297 is 297Q. In some embodiments, the variable Fc region comprises a substitution at one or more of positions 239, 282, 289, 297, 312, 324, 330, 335, 337, 339, 356, 359, 361, 383, 384, 398, 400, 440, 422 and 442, numbered according to the EU index.

[0034] In some embodiments, the structure of the multispecific antibody is selected from DVD-Ig, Bs4Ab, Hetero H, CrossMab, CrossMab2+1, IgG-(scFv)2 or scFv-Fab IgG and other structural forms.

[0035] In some embodiments, the multispecific antibody has a Hetero H, CrossMab structure. In some embodiments, the multispecific antibody comprises four polypeptide chains: wherein,

[0036] A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to MASP3; C H 1 is the heavy chain constant region C H 1 domain, wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and

[0037] A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to MASP3, C L is the light chain constant region; and

[0038] A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to MASP2, C L is a light chain constant region, wherein the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and

[0039] A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L 2 is the first light chain variable region that specifically binds to MASP2, C H 1 is the heavy chain constant region C H 1 domain.

[0040] Among them, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to MASP3, V H 2-C L and VL 2-C H 1 constitutes the antigen binding domain (Fab) that specifically binds to MASP2.

[0041] In some embodiments, the multispecific antibody comprises four polypeptide chains:

[0042] A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to MASP2; C H 1 is the heavy chain constant region C H 1 domain, wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and

[0043] A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to MASP2, C L is the light chain constant region; and

[0044] A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to MASP3, C L is a light chain constant region, wherein the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and

[0045] A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L 2 is the first light chain variable region that specifically binds to MASP3, C H 1 is the heavy chain constant region C H 1 domain.

[0046] Among them, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to MASP2, V H 2-C L and V L 2-C H 1 constitutes an antigen binding domain (Fab) that specifically binds to MASP3.

[0047] In some embodiments, the multispecific antibody described herein comprises: an amino acid sequence of SEQ ID NO: 60 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 60; and / or an amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 61; and / or an amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 62; and / or an amino acid sequence of SEQ ID NO: 63 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 63.

[0048] In some embodiments, the multispecific antibody described herein comprises: the amino acid sequence of SEQ ID NO:64 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:64; and / or the amino acid sequence of SEQ ID NO:61 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:61; and / or the amino acid sequence of SEQ ID NO:65 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:65; and / or the amino acid sequence of SEQ ID NO:63 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:63.

[0049] In some embodiments, a method for treating a desired individual disease or condition is provided, comprising administering to the individual an effective amount of any multispecific antibody as described above or a pharmaceutical composition comprising the same. In some embodiments, the use of any multispecific antibody as described above in the preparation of a pharmaceutical composition for treating a desired individual disease or condition is provided. In some embodiments, the use of any multispecific antibody as described above or a pharmaceutical composition comprising the same in the preparation of a medicament for treating a disease or condition is provided. In some embodiments, the disease or condition is associated with complement disorders, including autoimmune diseases, transplant-related diseases, inflammatory diseases, blood diseases, coagulation diseases, angiogenesis-dependent diseases, and / or viral infectious diseases or conditions. In some embodiments, the disease or condition comprises ischemia-reperfusion injury, atherosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), Henoch-Schönlein purpura nephritis, IgA nephropathy, ischemic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), secondary TMA after transplantation, Upshaw-Schulman syndrome, arthritis, traumatic brain injury, respiratory distress syndrome, Invasive pneumonia, neuromyelitis optica, multiple sclerosis, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, myasthenia gravis, Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancers, age-related macular degeneration, retinopathy, proliferative diabetes, vitreous hemorrhage secondary to retinopathy, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection.

[0050] In some embodiments, the present application provides an isolated nucleic acid molecule encoding any multispecific antibody (e.g., a bispecific antibody) as described above. In some embodiments, a vector is provided, comprising any nucleic acid molecule as described above. In some embodiments, a host cell is provided, comprising any multispecific antibody (e.g., a bispecific antibody), any nucleic acid molecule, or any vector as described above. In some embodiments, a method for preparing a multispecific antibody (e.g., a bispecific antibody) that specifically binds to MASP3 and MASP2 is provided, comprising: a) culturing any host cell as described above under conditions effective to express a multispecific antibody (e.g., a bispecific antibody) that specifically binds to MASP3 and MASP2; and b) obtaining the expressed multispecific antibody (e.g., a bispecific antibody) in the host cell.

[0051] Also provided are pharmaceutical compositions, kits, and articles of manufacture comprising any of the multispecific antibodies (eg, bispecific antibodies), nucleic acid molecules, vectors, or host cells described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1A-1B The results shown are the inhibitory activity of M3-C133 and M3-C134 humanized antibodies in the complement alternative pathway-mediated hemolysis assay. Figure 1A )、M3-C134 humanized antibody ( Figure 1B ) can inhibit the hemolytic activity mediated by the alternative complement pathway.

[0053] Figure 2A-2B The results shown are that the M3-C133 and M3-C134 humanized antibodies inhibited the MASP3 enzyme activity. Figure 2A )、M3-C134 humanized antibody ( Figure 2B ) can inhibit the enzymatic activity of MASP3.

[0054] Figure 3A Shown is a schematic diagram of the structure of DVD-Ig (Dual-variable domain-Ig) multispecific antibody; Figure 3B Shown is a schematic diagram of the structure of the Bs4Ab multispecific antibody; Figure 3C Shown is a schematic diagram of the structure of Hetero H, a CrossMab multispecific antibody; Figure 3D Shown is a schematic diagram of the structure of IgG-(scFv)2 multispecific antibody; Figure 3E Shown is a schematic diagram of the structure of scFv-Fab IgG multispecific antibody; Figure 3F Shown is a schematic diagram of the structure of Hetero H, CrossMab2+1 multispecific antibody. Figure 3GShown is a schematic diagram of Hetero H, CrossMab2+1 multispecific antibody with oppositely charged amino acid mutations in the non-exchange Fab region.

[0055] Figure 4A The results shown are that bispecific antibodies that specifically bind to MASP2 and MASP3 inhibit the activity of the lectin pathway in cynomolgus monkeys. Figure 4B Results shown are that bispecific antibodies that specifically bind to MASP2 and MASP3 inhibit the activity of the alternative pathway in cynomolgus monkeys.

[0056] Detailed description of this application

[0057] In one aspect, the present application provides an antibody or antigen-binding fragment that specifically binds to MASP3. In another aspect, the present application provides a multispecific antibody that specifically binds to MASP3 and MASP2. In another aspect, the present application also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to MASP3, and a pharmaceutical composition comprising a multispecific antibody that specifically binds to MASP3 and MASP2. In another aspect, the present application also provides a method for treating a disease or condition in a desired individual, comprising administering to the individual an effective amount of an antibody that specifically binds to MASP3 or a pharmaceutical composition comprising the same, a multispecific antibody that specifically binds to MASP3 and MASP2 or a pharmaceutical composition comprising the same. In another aspect, the present application provides the use of an antibody that specifically binds to MASP3 or a pharmaceutical composition comprising the same, a multispecific antibody that specifically binds to MASP3 and MASP2 or a pharmaceutical composition comprising the same in the preparation of a medicament for preventing or treating a disease or condition in a desired individual.

[0058] Through a combination of scFv phage library screening, antibody humanization, and appropriately designed biochemical and biological experiments, antibodies or antigen-binding fragments that specifically bind to MASP3 and antibodies or antigen-binding fragments that specifically bind to MASP2 were identified. Multispecific antibodies that specifically bind to MASP3 and MASP2 were also prepared. The results described herein indicate that the combination of these antigen-binding portions, either in the form of (i) a pharmaceutical composition, or (ii) a multispecific antibody, or (iii) a combined application, can be used to prevent and / or treat diseases / disorders associated with complement dysregulation by inhibiting both the complement lectin pathway and the alternative pathway, compared to administration of a single antibody molecule alone.

[0059] The present application also provides nucleic acids encoding antibodies or antigen-binding fragments that specifically bind to MASP3, and multispecific antibodies that specifically bind to MASP3 and MASP2, compositions comprising antibodies or antigen-binding fragments that specifically bind to MASP3 and antibodies or antigen-binding fragments that specifically bind to MASP2, and methods for preparing and using antibodies or antigen-binding fragments that specifically bind to MASP3, multispecific antibodies that specifically bind to MASP3 and MASP2, and pharmaceutical compositions comprising any of the above antibodies or antigen-binding fragments.

[0060] definition

[0061] As described herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result, including clinical results. In view of the purpose of this application, the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, alleviating the disease (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying disease progression, improving or improving the quality of life, increasing body weight, and / or prolonging survival. At the same time, "treatment" also includes the reduction of disease pathological results (e.g., reduced immune complex deposition, inhibiting thrombosis). The method of the present application takes into account any one or more aspects of these treatments.

[0062] The term "prevent" and similar words, such as "prevented", "preventing", "prevention" or "prophylactic", etc., refer to a method of preventing, inhibiting or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of a symptom of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms and / or burden of a disease or condition before it occurs or recurs. As used herein, "prevention" and similar words also include reducing the risk and susceptibility of the occurrence or recurrence of a disease or condition.

[0063] As described herein, the term "antibody" is broad and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as multispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three highly variable loops, which are called complementary determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein may be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, characterized by having α, δ, ε, γ and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0064] As used herein, the term "antigen-binding fragment" includes an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody (scFv), an scFv dimer (a bivalent diabody), a multispecific antibody composed of an antibody fragment comprising one or more CDRs, a single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Among them, the Fab (fragment antigen-binding) as described in the present application is a V antibody comprising an antibody. L Domain, V H Domain, C L domain, and CH 1 domain. Antigen binding fragments also include fusion proteins comprising the above antibody fragments. Antigen binding fragments can bind to the same antigen as the parent antibody or parent antibody fragment (such as parent scFv). In some embodiments, the antigen binding fragment may include one or more CDRs from a specific human antibody, which are transplanted to the framework region from one or more different human antibodies.

[0065] As described herein, the term "multispecific antibody" refers to an antibody molecule (e.g., a bispecific antibody) having binding specificity to at least two different antigens or epitopes in one molecule. Preferably, a multispecific antibody is a bispecific antibody. As described in the present application, the term "bispecific antibody" refers to an antibody molecule having binding specificity to two different antigens or epitopes in one molecule. The production process of multispecific antibodies (e.g., bispecific antibodies) includes the design of complete molecules, the synthesis and cloning of the nucleotide sequence of each domain, the expression of mammalian cells, and the purification of the final product. The structure of an exemplary multispecific antibody (e.g., a bispecific antibody) includes structures known in the art, for example, DVD-Ig structure, Bs4Ab structure, Hetero H, CrossMab structure, CrossMab2+1 structure, IgG-(scFv)2 structure or scFv-FabIgG structure, etc. (e.g., see review document Labrijn AF, et al. Nat Rev Drug Discov. 2019Aug; 18(8): 585-608).

[0066] DVD-Ig (Dual-variable domain-Ig) is a multi-specific antibody with a light chain and a heavy chain N-terminus of a full-length IgG antibody connected to the V domain of another antibody. L and V H domain, through V H With V LInteract to form an antigen binding domain (Fv), and at the same time bind to the corresponding antigen to achieve bispecificity. The multispecific antibody of the exemplary DVD-Ig structure is also recorded in the document Wu C, et al.Molecular construction and optimization of anti-human IL-1alpha / beta dual variabledomain immunoglobulin (DVD-Ig) molecules. MAbs. 2009 Jul-Aug; 1 (4): 339-47. The multispecific antibody of the Bs4Ab structure is a bispecific 4-valent antibody, which includes a full-length IgG structure and achieves bispecificity by inserting another binding unit scFv in its hinge region. The multispecific antibody of the Bs4Ab structure is also recorded in the document Bezabeh B, et al. Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties. MAbs. 2017 Feb / Mar; 9 (2): 240-256. Hetero H, a multispecific antibody with CrossMab structure, has a knobs-in-holes (KIH) structure designed in the Fc region, and two Cys residue mutations (S354C on the "knob" side and Y349C on the "hole" side) that can form a stabilizing disulfide bridge are introduced. CrossMab technology is also used to ensure the correct pairing between the light and heavy chains of the antibody. CrossMab technology is based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, which can be the exchange of the entire Fab domain (CrossMAb Fab) or the exchange of only the variable region in the Fab domain (CrossMAb V H -V L) or only the constant region is exchanged (CrossMAb CH1-CL). The multispecific antibodies with Hetero H and CrossMab structures are also described in the literature Klein C, et al. The use of CrossMAb technology for the generation of bi-and multispecific antibodies. MAbs. 2016 Aug-Sep; 8(6): 1010-20. The multispecific antibodies with CrossMab2+1 structure are based on the CrossMab structure and have an Fab portion of one of the antigen binding domains added to form a univalent binding of one of the antigen binding domains and a bivalent binding of the other antigen binding domain. In addition, by adding a Fab portion of the ... H 1 and C L The introduction of charged amino acids with opposite charges at specific amino acid positions in the domains can increase the ratio of the desired multispecific antibody compared to unwanted side products, for example, in the constant domain C of one of the Fab molecules. L The amino acid at position 128 is replaced by arginine (R), and the amino acid at position 129 is replaced by lysine (K), respectively (numbering is according to Kabat); and wherein in the constant domain C of the corresponding Fab molecule H1, the amino acid at position 148 is replaced by glutamic acid (E), and the amino acid at position 214 is replaced by glutamic acid (E), respectively (numbering is in accordance with Kabat) (see patent document CN106661120B). The CrossMab 2+1 structure is also described in documents CN103748114B, CN106661120B, KleinC, et al. Engineering therapeutic bispecific antibodies using CrossMab technology. Methods. 2019 Feb 1; 154: 21-31. IgG-(scFv)2 structure multispecific antibodies are achieved by connecting the scFv fragment of another antibody to the Fc termini of the two heavy chains of an IgG antibody. Multispecific antibodies with IgG-(scFv)2 structure are also described in documents Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63. scFv-Fab IgG structure multispecific antibody is a heterodimeric antibody, an IgG antibody structure, in which one Fab arm is replaced with a scFv structure, wherein the first monomer comprises scFv and IgG Fc, the scFv is connected to the N-terminus of the IgG FcCH2 domain through a connecting peptide, and the second monomer comprises Fab and IgG Fc.

[0067] As described in this application, the term "antigen binding domain" refers to the part of an antigen binding molecule that specifically binds to an antigen. More specifically, the term "antigen binding domain" refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. If it is a large antigen, the antigen binding molecule may only bind to a specific portion of the antigen, which is called an antigen epitope. For example, an antigen binding domain can be provided by one or more variable regions (also called variable domains). Preferably, the antigen binding domain includes the variable region of the antibody light chain (V L ) and antibody heavy chain variable region (V H In one aspect, the antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of the antigen. Antigen binding domains that specifically bind to MASP3 or MASP2 include antibodies and antigen binding fragments as further defined herein.

[0068] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope on the antigen.

[0069] As described herein, a first antibody "competes" for binding to a MASP3 target with a second antibody when the first antibody inhibits binding of the second antibody to the MASP3 target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) at equimolar concentrations, and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0070] As used herein, the terms "specifically bind", "specifically recognize", or "specific for" refer to a measurable and reproducible interaction, such as the binding of a target to an antibody that can determine the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody is able to specifically recognize a target (which may be an epitope) means that the antibody binds to the target with higher affinity, avidity, more readily and / or more persistently than other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its affinity for other targets.

[0071] As used herein, an "isolated" antibody refers to an antibody that (1) is not related to naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not exist in nature.

[0072] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA or synthetic origin or a combination thereof. Depending on its origin, the "isolated nucleic acid" (1) is unrelated to all or part of the polynucleotides in the "isolated nucleic acid" found in nature, (2) can be operably linked to a polynucleotide that is not naturally associated with it, or (3) does not exist as part of a longer sequence in nature.

[0073] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabatet al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These special regions have been described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein these definitions include overlaps or subsets of amino acid residues when compared to one another. However, any definition used to indicate the CDR of an antibody or a transplanted antibody or a variant thereof is included within the scope of the terms defined and used in this application. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the above-cited references for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated into this application by reference in their entirety for use in this application and in one or more claims that may be included in this application.

[0074] Table 1: CDR Definition

[0075] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> <![CDATA[MacCallum 3 ]]> <![CDATA[IMGT 4 ]]> <![CDATA[AHo 5 ]]> <![CDATA[V H CDR1]]> 31-35 26-32 30-35 27-38 25-40 <![CDATA[V H CDR2]]> 50-65 53-55 47-58 56-65 58-77 <![CDATA[V H CDR3]]> 95-102 96-101 93-101 105-117 109-137 <![CDATA[V L CDR1]]> 24-34 26-32 30-36 27-38 25-40 <![CDATA[V L CDR2]]> 50-56 50-52 46-55 56-65 58-77 <![CDATA[V L CDR3]]> 89-97 91-96 89-96 105-117 109-137

[0076] 1 The amino acid residue numbers refer to the nomenclature of Kabat et al.

[0077] 2 The amino acid residue numbers refer to the nomenclature of Chothia et al.

[0078] 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al.

[0079] 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al.

[0080] 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun above.

[0081] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, and the remaining portion of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0082] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable region and a light chain variable region tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains, which provide the antibody with amino acid residues for binding to the antigen and give the antibody specificity for binding to the antigen. However, even a single variable region (or half of an Fv fragment, which contains only 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0083] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv comprising V linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V LThe connecting polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0084] The term "diabodies" refers to double-chain antibodies H and V L A small antibody fragment prepared by constructing an scFv fragment (see above) with a short linker (e.g., 5 to 10 residues) between the chains, so that the variable regions pair between the chains rather than within the chains, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. A multispecific diabody is a heterodimer of two "crossed" scFv fragments, in which the V H and V L The domains are located on different polypeptide chains.

[0085] Diabodies are fully described in 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0086] The "humanized" form of a non-human (such as rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which the hypervariable region (HVR) residues of the receptor antibody are replaced by hypervariable region residues from non-human species such as mice, rats, rabbits or non-human primates and have ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the framework region (FR) of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in either the receptor antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will contain essentially at least one, usually two variable regions, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin sequences. Human antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0087] The "percent (%) amino acid sequence homology" or "homology" of the polypeptide and antibody sequences identified in this application is defined as the percentage of identical amino acid residues in the candidate sequence and the polypeptide sequence to be compared, when conservative substitutions are considered to be part of the sequence homology. The percentage of amino acid sequence homology can be determined by a variety of alignment methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to maximize alignment over the entire length of the compared sequences. However, for the purposes of this application, the percentage of amino acid sequence homology values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0088] The term "Fc (fragment crystallizable)" or "Fc region" refers to a polypeptide comprising the constant region of a complete antibody, excluding the CH1 domain and, in some cases, part of the hinge, whether in monomeric or multimeric form. The original immunoglobulin source of the natural Fc is preferably human, and can be any immunoglobulin, for example, IgG1, IgG2, IgG3 or IgG4. The natural Fc is composed of monomeric polypeptides, which can be linked into dimer or multimer forms by covalent (i.e., disulfide bonds) and non-covalent associations. The Fc region of an immunoglobulin generally comprises the C of the heavy chain constant region. H 2 domains and C H 3 domains, and optionally including C H 4 domains.

[0089] In some embodiments, each of the two Fc monomers in the Fc dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, heterodimerization of the Fc monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole" residue pairs in the two Fc monomers. The "knob-into-hole" technology is also disclosed in U.S. Patent Publication No. 8,216,805. In some embodiments, one Fc monomer comprises a knob mutation T366W, and the other Fc monomer comprises a hole mutation T366S, L358A and Y407V. In some embodiments, two Cys residues (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilized disulfide bridge are introduced.

[0090] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (activating receptor) and FcγRIIB (inhibiting receptor), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15: 203-234 (1997)). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) and Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0091] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins IgGs from mothers to newborns and in regulating serum IgG levels. As a rescue receptor, FcRn can bind and transport endocytosed IgG in an intact form within and between cells, and protect them from the default degradation pathway.

[0092] The “C” of the human IgG heavy chain constant region H The "1 domain" generally extends from amino acid 118 to amino acid 215 (EU numbering system).

[0093] The "hinge region" is generally defined as extending from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same position as IgG1, the hinge regions of other IgG subtypes can be aligned with the IgG1 sequence.

[0094] Human IgG Fc region "C HThe "2 domain" usually extends from amino acid 231 to amino acid 340. H Domain 2 is unique in that it does not pair closely with another region. Instead, it is located between the two C H Two N-terminally linked branched sugar chains are inserted between the 2 domains. It is speculated that the sugars may serve as a substitute for domain-domain pairing, helping to maintain C H 2 domain stabilization. Burton, Molec Immunol. 22: 161-206 (1985).

[0095] “C H The 3" domain includes the C-terminal residues extending from the C H 2 domain (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 of IgG).

[0096] A "functional Fc fragment" has the "effector functions" possessed by a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (such as an antibody variable region) and can be evaluated using a variety of experimental methods known in the art.

[0097] An antibody having an IgG Fc variant with "altered" FcR binding affinity or ADCC activity has increased or decreased FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. An Fc variant exhibiting "enhanced binding" to an FcR has a higher binding affinity (e.g., lower apparent Kd or ICd) to at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. 50 In some embodiments, the binding capacity is enhanced by 3-fold, such as 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500-fold, or the binding capacity is increased by 25% to 1000% compared to the parent polypeptide. An Fc variant that exhibits "reduced binding" to an FcR has a lower affinity (e.g., a higher apparent Kd or ICd) for at least one FcR compared to the parent polypeptide. 50 Compared with the parent polypeptide, its binding capacity is reduced by 40% or more.

[0098] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigens and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Pat. Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, ADCC activity of the target molecule can also be assessed in vivo, for example, as described in an animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0099] The polypeptide comprising the Fc variant exhibits "enhanced ADCC activity" or can more effectively mediate the ADCC effect in the presence of human effector cells compared to the polypeptide comprising the wild-type IgG Fc polypeptide or the parent polypeptide. The polypeptide comprising the Fc variant can more effectively mediate ADCC in vitro or in vivo when the amount of the polypeptide comprising the wild-type IgG Fc polypeptide (or the parent polypeptide) is substantially the same during the experiment. Such variants are generally identified using any in vitro ADCC experimental method known in the art, such as an experiment or method for identifying ADCC activity, such as in an animal model, etc. In some embodiments, such variants mediate ADCC with an efficiency of 5 to 100 times, such as 25 to 50 times, compared to the wild-type Fc (or the parent polypeptide).

[0100] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to a cognate antigen. To assess complement activation, a CDC assay may be performed, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants having altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated into this application by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0101] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein contains introns in some forms.

[0102] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby allowing the latter to be expressed. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to a coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, two protein coding regions can be linked in the same reading frame.

[0103] "Homologous" refers to the sequence similarity or sequence homology between two polypeptides or between two nucleic acid molecules. If the same position of two compared sequences is the same base or amino acid monomer subunit, for example, the same position of two DNA molecules is adenine, then the two DNA molecules are homologous at that position. The homology percentage between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, the homology of the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0104] The "effective amount" of the antibodies (including multispecific antibodies) or compositions disclosed in the present application refers to an amount sufficient to achieve a specific purpose. The "effective amount" can be determined empirically and by known methods related to the purpose.

[0105] The term "therapeutically effective amount" refers to the amount of the antibody (including multispecific antibodies) or composition described herein that can effectively treat an individual's disease or symptoms. That is, an amount sufficient to reduce or improve the severity and / or duration of the disease or one or more of its symptoms; to prevent the development of the disease, cause regression of symptoms, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, detect the disease, or enhance / improve the preventive or therapeutic effect of another therapy (e.g., a preventive or therapeutic agent). In some embodiments, a therapeutically effective amount refers to an amount that can prolong the patient's survival. In some embodiments, a therapeutically effective amount refers to an amount that can improve the patient's progression-free survival.

[0106] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to materials without biological activity or other undesirable properties, such as materials that can be added to a pharmaceutical composition administered to a patient without causing significant adverse biological reactions, or that do not interact in a harmful manner with any other components contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0107] The embodiments of the present application described in this application should be understood to include embodiments that "consist of" and / or "consist essentially of."

[0108] In this application, "about" is mentioned as a value or parameter, including (and describing) variations of the value or parameter itself. For example, the description involving "about X" includes the description of "X".

[0109] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, the method cannot be used to treat type X infection, which means that the method is generally used to treat other types of infection except type X.

[0110] As used in this application and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0111] Antibodies that specifically bind to MASP3

[0112] In one aspect, the present application provides antibodies or antigen-binding fragments that specifically bind to MASP3, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy chain and / or light chain CDRs as described herein. In one aspect, the antibody or antigen-binding fragment is an isolated antibody that binds to MASP3. Antibodies or antigen-binding fragments that specifically bind to MASP3 are expected to include all or fragments of full-length antibodies (such as full-length IgG1, IgG2 or IgG4) that specifically bind to MASP3, single-chain antibodies that specifically bind to MASP3, antibodies that multispecifically (such as bispecifically) bind to MASP3, immunoconjugates that specifically bind to MASP3, and the like. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 is Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody or double-chain antibody (diabody) or linear antibody. In some embodiments, an antibody or antigen-binding fragment that specifically binds to MASP3 means that the affinity of the antibody or antigen-binding fragment to MASP3 is at least 10 times (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 In some embodiments, a non-target is an antigen that is not MASP3.

[0113] Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interferometry (BLI) technology.

[0114] Although the present application generally discusses antibodies or antigen-binding fragments that specifically bind to MASP3 that include human sequences (e.g., human heavy and light chain variable regions that include human CDR sequences), non-human antibodies are also contemplated. In some embodiments, non-human antibodies include human CDR sequences and non-human framework sequences of antibodies or antigen-binding fragments that specifically bind to MASP3 described herein. In some embodiments, non-human framework sequences include any sequence used to generate heavy and / or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc. In some embodiments, non-human antibodies or antigen-binding fragments that specifically bind to MASP3 include antibodies or antigen-binding fragments that specifically bind to MASP3 generated by grafting one or more human CDR sequences described herein into non-human framework sequences (e.g., mouse or chicken framework sequences).

[0115] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 described herein binds to an epitope in human MASP3. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 is completely specific to human MASP3 and has no species cross-reaction or does not cross-react with other types of non-MASP3. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 cross-reacts with MASP3 antigens of species other than humans. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: a heavy chain variable region (V H ), which comprises V as shown in the amino acid sequence SEQ ID NO:23 H HC-CDR1, HC-CDR2 and HC-CDR3; and light chain variable region (V L ), which comprises V as shown in the amino acid sequence SEQ ID NO:33 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0116] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0117] HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 9, or the V HA variant of V comprising substitutions of up to about 5 amino acids in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or the V L A variant comprising substitutions of up to about 5 amino acids in the LC-CDRs.

[0118] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H , which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-26 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs: 23-26; and V L , which comprises an amino acid sequence shown in any one of SEQ ID NOs: 33-35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 33-35.

[0119] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: (i) V H , comprising the amino acid sequence of SEQ ID NO:23 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:23; and V L , which comprises the amino acid sequence of SEQ ID NO:33 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:33; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence of SEQ ID NO:34 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:34; (iii) V H, comprising the amino acid sequence of SEQ ID NO:25 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:25; and V L , which comprises the amino acid sequence of SEQ ID NO:34 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:34; (iv) V H , comprising the amino acid sequence of SEQ ID NO:26 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:26; and V L , comprising the amino acid sequence of SEQ ID NO:34 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:34; (v) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:35; (vi) V H , comprising the amino acid sequence of SEQ ID NO:25 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:25; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:35; or (vii) V H, comprising the amino acid sequence of SEQ ID NO:26 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:26; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:35.

[0120] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H , which comprises V as shown in the amino acid sequence SEQ ID NO: 27 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:36 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0121] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and

[0122] HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 10, or the V H A variant of V comprising substitutions of up to about 5 amino acids in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or the V L A variant comprising substitutions of up to about 5 amino acids in the LC-CDRs.

[0123] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 27-30 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs: 27-30; and V L , which comprises an amino acid sequence shown in any one of SEQ ID NOs:36-39 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:36-39.

[0124] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: (i) V H , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 27; and V L , which comprises the amino acid sequence of SEQ ID NO:36 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:36; (ii) V H , comprising the amino acid sequence of SEQ ID NO:28 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:28; and V L , which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:37; (iii) V H , comprising the amino acid sequence of SEQ ID NO:29 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:29; and V L , which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:37; (iv) V H, comprising the amino acid sequence of SEQ ID NO:30 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:30; and V L , comprising the amino acid sequence of SEQ ID NO:37 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:37; (v) V H , comprising the amino acid sequence of SEQ ID NO:28 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:28; and V L , comprising the amino acid sequence of SEQ ID NO:38 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:38; (vi) V H , comprising the amino acid sequence of SEQ ID NO:29 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:29; and V L , comprising the amino acid sequence of SEQ ID NO:38 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:38; (vii) V H , comprising the amino acid sequence of SEQ ID NO:30 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:30; and V L , comprising the amino acid sequence of SEQ ID NO:38 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:38; (viii) V H , which comprises the amino acid sequence

[0125] SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence homology (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) to the amino acid sequence of SEQ ID NO:28; and V L , comprising the amino acid sequence of SEQ ID NO:39 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:39; (ix) V H , comprising the amino acid sequence of SEQ ID NO:29 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:29; and V L , comprising the amino acid sequence of SEQ ID NO:39 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:39; (x) V H , comprising the amino acid sequence of SEQ ID NO:30 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:30; and V L , which comprises the amino acid sequence of SEQ ID NO:39 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:39.

[0126] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: (i) V H , which comprises V as shown in the amino acid sequence SEQ ID NO:31 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:40 L or (ii) V H , which comprises V as shown in the amino acid sequence SEQ ID NO:32 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L, which comprises V as shown in the amino acid sequence SEQ ID NO:41 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0127] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:7, and

[0128] HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 11, or the V H A variant of V comprising substitutions of up to about 5 amino acids in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21, or the V L A variant of V comprising substitutions of up to about 5 amino acids in its LC-CDRs; or (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or the V H A variant of V comprising substitutions of up to about 5 amino acids in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising substitutions of up to about 5 amino acids in the LC-CDRs.

[0129] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: (i) V H, comprising the amino acid sequence of SEQ ID NO:31 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:31; and V L , which comprises the amino acid sequence of SEQ ID NO:40 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:40; or (ii) V H , comprising the amino acid sequence of SEQ ID NO:32 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:32; and V L , which comprises the amino acid sequence of SEQ ID NO:41 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:41.

[0130] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H , comprising the amino acid sequence of SEQ ID NO:31 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:31; and V L , which comprises the amino acid sequence

[0131] SEQ ID NO:40 or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:40.

[0132] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises: H , comprising the amino acid sequence of SEQ ID NO:32 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:32; and V L , which comprises the amino acid sequence

[0133] SEQ ID NO:41 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:41.

[0134] In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 10 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 10 of the present application.

[0135] In some embodiments, the present application provides antibodies that can competitively bind to MASP3 with any of the above antibodies that specifically bind to MASP3. In some embodiments, the present application provides antibodies that competitively bind to the same epitope as any of the above antibodies that specifically bind to MASP3.

[0136] In some embodiments, competition assays can be used to identify monoclonal antibodies that compete with antibodies that specifically bind to MASP3 as described herein. Competition assays can determine whether two antibodies bind to the same epitope by recognizing the same or spatially overlapping epitopes or by competitively inhibiting the binding of another antibody to the antigen. In certain embodiments, such competing antibodies bind to the same epitope as the antibodies described herein. Some exemplary competition assays include, but are not limited to, routine assays as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, each antibody is said to bind to the same epitope if it blocks 50% or more of the binding of the other antibody. In some embodiments, the antibody that competes with the antibody described herein that specifically binds to MASP3 is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0137] Exemplary antibody sequences that specifically bind to MASP3 are shown in Tables 2 and 3, wherein CDR numbering is performed according to the Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the position of CDRs and defining the variable regions of antibody light and heavy chains. Hand / or V L Sequences, but based on prediction algorithms other than the antibodies exemplified in the table below are also within the scope of the present application.

[0138] Full-length antibody that specifically binds to MASP3

[0139] In some embodiments, the antibody that specifically binds to MASP3 is a full-length antibody. In some embodiments, the full-length antibody that specifically binds to MASP3 is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the full-length antibody that specifically binds to MASP3 comprises an IgG constant region, such as a constant region of IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises an IgG1-type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises an IgG2-type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises an IgG3-type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises an IgG4-type heavy chain constant region. In some embodiments, the IgG refers to human IgG. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 52. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 53. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 54. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 55. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 comprises an antibody heavy chain variable region and an antibody light chain variable region. In some embodiments, the full-length antibody that specifically binds to MASP3 comprises an altered or otherwise modified Fc sequence such that it has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) effector functions.

[0140] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1, IgG2, IgG3 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a) a heavy chain variable region, the heavy chain variable region comprising: HC-CDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-4 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; HC-CDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5-8 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; and HC-CDR3, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9-12 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; and b) a light chain variable region, the light chain variable region comprising: LC-CDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-4 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids. NOs: 13-15 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; LC-CDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 16-18 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 19-22 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 55.

[0141] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1, IgG2, IgG3 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a) a heavy chain variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-4, HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5-8, and HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 9-12, or a variant of the heavy chain variable region comprising up to about 5 (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and b) a light chain variable region, the light chain variable region comprising: LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 13-15, LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 16-18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NOs: NOs: 19-22, or a variant of the light chain variable region, wherein the LC-CDR sequence comprises a substitution of up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acids. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence

[0142] In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the IgG2 is a human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG3 is a human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0143] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a) a heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0144] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1, IgG2, IgG3 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a heavy chain variable region (V H ), the V HThe invention relates to a method for preparing a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 23-26 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs: 23-26; and a light chain variable region (V L ) H Comprising an amino acid sequence as shown in any one of SEQ ID NOs:33-35 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs:33-35. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG3 is a human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55.

[0145] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 23; and V L , which comprises the amino acid sequence SEQ ID NO:33 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:33. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0146] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L, which comprises the amino acid sequence SEQ ID NO:34 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:34. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0147] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L, which comprises the amino acid sequence SEQ ID NO:34 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:34. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0148] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L, which comprises the amino acid sequence SEQ ID NO:34 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:34. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0149] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L, which comprises the amino acid sequence SEQ ID NO:35 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:35. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0150] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L, which comprises the amino acid sequence SEQ ID NO:35 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:35. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0151] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L, which comprises the amino acid sequence SEQ ID NO:35 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:35. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0152] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a) a heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:6, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:10; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0153] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1, IgG2, IgG3 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a heavy chain variable region (V H ), the V HThe invention relates to a method for preparing a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 27-30 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs: 27-30; and a light chain variable region (V L ) H Comprising an amino acid sequence as shown in any one of SEQ ID NOs:36-39 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs:36-39. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG3 is a human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55.

[0154] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 27; and V L , which comprises the amino acid sequence SEQ ID NO:36 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:36. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0155] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 28; and V L, which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:37. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0156] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 29; and V L, which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:37. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0157] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 30; and V L, which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:37. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0158] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 28; and V L, which comprises the amino acid sequence SEQ ID NO:38 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:38. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0159] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 29; and V L, which comprises the amino acid sequence SEQ ID NO:38 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:38. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0160] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 30; and V L, which comprises the amino acid sequence SEQ ID NO:38 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:38. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0161] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 28; and V L, which comprises the amino acid sequence SEQ ID NO:39 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:39. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0162] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 29; and V L, which comprises the amino acid sequence SEQ ID NO:39 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:39. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0163] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 30; and V L, which comprises the amino acid sequence SEQ ID NO:39 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:39. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0164] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a) a heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:7, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:17, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:21. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0165] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 31; and V L, which comprises the amino acid sequence SEQ ID NO:40 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:40. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0166] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: a) a heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:8, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:12; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0167] In some embodiments, a full-length antibody that specifically binds to MASP3 comprising an IgG1 or IgG4 constant region is provided, wherein the antibody that specifically binds to MASP3 comprises: V H , comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 32; and V L, which comprises the amino acid sequence SEQ ID NO:41 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:41. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55.

[0168] Antibodies that specifically bind to MASP2

[0169] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 includes, but is not limited to, a humanized antibody, a chimeric antibody, a mouse antibody, a human antibody, and an antibody molecule comprising heavy chain and / or light chain CDRs as described herein. In one aspect, the antibody or antigen-binding fragment is an isolated antibody that binds to MASP2. Antibodies or antigen-binding fragments that specifically bind to MASP2 include all or fragments of full-length antibodies (such as full-length IgG1, IgG2 or IgG4) that specifically bind to MASP2, single-chain antibodies that specifically bind to MASP2, antibodies that multispecifically (such as bispecifically) bind to MASP2, immunoconjugates that specifically bind to MASP2, and the like. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 is Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody or double-chain antibody (diabody). In some embodiments, an antibody or antigen-binding fragment that specifically binds to MASP2 means that the antibody or antigen-binding fragment binds to MASP2 with an affinity that is at least 10 times greater (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 In some embodiments, a non-target is an antigen that is not MASP2.

[0170] Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interferometry (BLI) technology.

[0171] In certain aspects, the antibody or antigen-binding fragment that specifically binds to MASP2 is capable of (a) promoting, mediating or enhancing the AA activity of the relevant infectious agent; and / or (b) promoting, mediating or enhancing the BB activity of the relevant infectious agent.

[0172] Although the present application generally discusses antibodies or antigen-binding fragments that specifically bind to MASP2 that comprise human sequences (e.g., human heavy and light chain variable regions that comprise human CDR sequences), non-human antibodies are also contemplated. In some embodiments, non-human antibodies comprise human CDR sequences and non-human framework sequences of antibodies or antigen-binding fragments that specifically bind to MASP2 as described herein. In some embodiments, non-human framework sequences comprise any sequence used to generate heavy and / or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc. In some embodiments, non-human antibodies or antigen-binding fragments that specifically bind to MASP2 comprise antibodies or antigen-binding fragments that specifically bind to MASP2 generated by grafting one or more human CDR sequences described herein into non-human framework sequences (e.g., mouse or chicken framework sequences).

[0173] In some embodiments, the antibodies or antigen-binding fragments that specifically bind to MASP2 described herein bind to an epitope in human MASP2. In some embodiments, the antibodies or antigen-binding fragments that specifically bind to MASP2 are completely specific to human MASP2 and have no species cross-reaction or do not cross-react with other types of non-MASP2. In some embodiments, the antibodies or antigen-binding fragments that specifically bind to MASP2 cross-react with MASP2 antigens of species other than human.

[0174] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises an IgG1-type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises an IgG2-type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises an IgG3-type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises an IgG4-type heavy chain constant region. In some embodiments, the IgG refers to human IgG. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:52. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:53. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:54. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 55. In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 comprises an antibody heavy chain variable region and an antibody light chain variable region. In some embodiments, the full-length antibody that specifically binds to MASP2 comprises an altered or otherwise modified Fc sequence such that it has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) effector functions.

[0175] Multispecific antibodies that specifically bind to MASP3 and MASP2

[0176] In one aspect, the present application provides a multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2. In some embodiments, the multispecific antibody can bind to MASP3 or MASP2. In other preferred embodiments, the multispecific antibody can bind to both MASP3 and MASP2. In other more preferred embodiments, the multispecific antibody can bind to both MASP3 and MASP2 simultaneously.

[0177] In some embodiments, the first antigen binding domain that specifically binds to MASP3 comprises: (a) V H , which comprises an amino acid sequence such as

[0178] V shown in SEQ ID NO:23 HHC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:33 L or (b) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0179] HC-CDR3, comprising the amino acid sequence SEQ ID NO:9: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:13, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:16, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:19.

[0180] In some embodiments, the second antigen binding domain that specifically binds to MASP2 comprises: (a) V H , which comprises an amino acid sequence such as

[0181] V shown in SEQ ID NO:48 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:50 L or (b) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:47.

[0182] In some embodiments, the present application provides a multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2, wherein the first antigen binding domain comprises: V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0183] HC-CDR3, comprising the amino acid sequence SEQ ID NO:9: and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:47.

[0184] In some embodiments, the first antigen binding domain that specifically binds to MASP3 comprises: H , which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-26 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence as shown in any one of SEQ ID NOs: 23-26; and V L , which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence as shown in any one of SEQ ID NOs: 33-35. In some embodiments, the first antigen binding domain that specifically binds to MASP3 comprises: V H , the V H comprising an amino acid sequence as shown in any one of SEQ ID NOs: 23-26, and V L , the V L Containing SEQ ID NOs:33-

[0185] The amino acid sequence shown in any one of 35.

[0186] In some embodiments, the first antigen binding domain that specifically binds to MASP3 comprises: (a) V H , which comprises the amino acid sequence

[0187] SEQ ID NO:23 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:23; and V L , comprising the amino acid sequence of SEQ ID NO:33 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:33; (b) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO:34 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:34; (c) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:35; (d) V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L, which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:35; or (e) V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence of SEQ ID NO:35.

[0188] In some embodiments, the second antigen binding domain that specifically binds MASP2 comprises: H , the V H comprising the amino acid sequence shown in SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:48; and V L , the V L comprising the amino acid sequence set forth in SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence set forth in SEQ ID NO:50. In some embodiments, the second antigen binding domain that specifically binds to MASP2 comprises: V H , the V H comprising the amino acid sequence shown in SEQ ID NO:48, and V L , the V L Comprising the amino acid sequence shown in SEQ ID NO:50.

[0189] In some embodiments, the present application provides a multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2, wherein the first antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L, comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; and wherein the second antigen binding domain comprises: V H , which comprises the amino acid sequence SEQ ID

[0190] NO:48 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO:48; and V L , which comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:50.

[0191] Multispecific antibodies with DVD-Ig structures

[0192] In some embodiments, according to any multispecific antibody (e.g., bispecific antibody) described herein, it has a dual variable region immunoglobulin molecule (DVD-Ig) structure, which is a V region of a normal IgG antibody. L and V H The N-terminus of each antibody is connected to the V L and V H domain, through two antibody V H With V L The interaction forms an antigen binding domain, which can bind to the corresponding antigen at the same time to achieve bispecificity. In some embodiments, the DVD-IgG structure is a homodimer structure, composed of two identical monomers, each monomer includes two antigen binding domains, one of which is Fv and the other is Fab, and the two regions are connected in series by a connecting peptide (L). In some embodiments, the DVD-Ig structure further includes Fc. An exemplary DVD-Ig structure is shown in Figure 3A middle.

[0193] In one embodiment of the present application, one antigen binding domain specifically binds to MASP2, and the other antigen binding domain specifically binds to MASP3. In some embodiments, the multispecific antibody can bind to MASP3 and MASP2 simultaneously.

[0194] In some embodiments, the multispecific antibody is composed of two identical monomers, each monomer comprising two polypeptide chains, a heavy chain and a light chain, for a total of four polypeptide chains. The heavy chain comprises V from the N-terminus to the C-terminus. H 1-LV H 2-C H 1. The light chain contains V from N-terminus to C-terminus L 1-LV L2-C L In some embodiments, the heavy chain further comprises an Fc comprising a C H 2 and C H In other embodiments, the heavy chain comprises V from N-terminus to C-terminus. H 1-LV H 2-C H 1-C H 2-C H 3. The light chain contains V from N-terminus to C-terminus L 1-LV L 2-C L . Where V H 1 and V L 1 is the heavy chain variable region and light chain variable region that specifically bind to an antigen; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen; L is the connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; C L Is the light chain constant region. H 1 and V L 1 constitutes an antigen binding domain (Fv) of a multispecific antibody; V H 2-C H 1 and V L 2-C L The other antigen-binding domain (Fab) that makes up the multispecific antibody.

[0195] In some embodiments, the V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 1 and V L 1 constitutes an antigen binding domain (Fv) that specifically binds to MASP2; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 2-C H 1 and V L 2-C L In some embodiments, the V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 1 and V L 1 constitutes an antigen binding domain (Fv) that specifically binds to MASP3; V H 2 and VL 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 2-C H 1 and V L 2-C L It consists of an antigen binding domain (Fab) that specifically binds to MASP2.

[0196] The connecting peptide (or linker) sequence can be a single amino acid or polypeptide sequence. In some embodiments, the connecting peptide (or linker) comprises or consists of a glycine-serine linker. As used herein, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include those of the general formula (Gly4Ser) n The amino acid sequence of n , wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). A preferred glycine-serine linker is (Gly4Ser)2, i.e., GGGGSGGGGS (SEQ ID NO: 66). A preferred glycine-serine linker is (Gly4Ser)4, i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 67). A preferred glycine-serine linker is (Gly4Ser)3, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 68). In other aspects, two or more glycine-serine linkers are connected in series in a connecting peptide. In certain aspects, the connecting peptide includes at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3 or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, such as (Gly4Ser) n In some embodiments, the connecting peptide comprises the amino acid sequence ASTKGP (SEQ ID NO: 69) or the amino acid sequence TVAAP (SEQ ID NO: 70).

[0197] Multispecific antibody with Bs4Ab structure

[0198] In some embodiments, according to the multispecific antibody (e.g., bispecific antibody) described in the present application, it has a Bs4Ab structure, which comprises two identical monomers, each of which comprises two antigen binding domains, one of which is Fab and the other is scFv. In some embodiments, the multispecific antibody further comprises an Fc, which comprises CH2 and CH3 domains. The scFv is connected to the Fab via a first connecting peptide (L1) and to the Fc via a second connecting peptide (L2). An exemplary Bs4Ab structure is shown in Figure 3B middle.

[0199] In some embodiments of the present application, one antigen binding domain (Fab or scFv) specifically binds to MASP2, and the other antigen binding domain (scFv or Fab) specifically binds to MASP3. In some embodiments, the multispecific antibody can bind to MASP3 and MASP2 antigens simultaneously.

[0200] In some embodiments, the multispecific antibody is composed of two identical monomers, each monomer comprising two polypeptide chains, a heavy chain and a light chain, for a total of four polypeptide chains. In some embodiments, the heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H 1-L1-V H 2-L3-V L 2 structure. In other embodiments, the heavy chain of the multispecific antibody comprises from N-terminus to C-terminus: V H 1-C H 1-L1-V L 2-L3-V H 2 structure. In some embodiments, the heavy chain further comprises an Fc, wherein the Fc comprises a CH2 and a CH3 domain. In some embodiments, the heavy chain of the multispecific antibody comprises, from the N-terminus to the C-terminus: V H 1-C H 1-L1-V H 2-L3-V L 2-L2-C H 2-C H 3 structure. In other embodiments, the heavy chain of the multispecific antibody comprises from N-terminus to C-terminus: V H 1-C H 1-L1-V L 2-L3-V H 2-L2-C H 2-C H 3 Structure. In some embodiments, the light chain of the multispecific antibody comprises, from N-terminus to C-terminus: V L 1-C L Structure. Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively. H 1 is the heavy chain constant region C H 1 domain, C L is the light chain constant region, L1, L2 and L3 are connecting peptides. H 1-C H 1 and VL 1-C L One of the antigen-binding domains (Fab) that makes up a multispecific antibody, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes the other antigen-binding domain (scFv) of the multispecific antibody.

[0201] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 1-C H 1 and V L 1-C L Composed of an antigen binding domain (Fab) that specifically binds to MASP2; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to MASP3. H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 1-C H 1 and V L 1-C L Composed of an antigen binding domain (Fab) that specifically binds to MASP3; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to MASP2. In some embodiments, the multispecific antibody comprises C H 1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 58-59. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H3, which comprises one or more substitutions at positions 22, 24, and 26 relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions of M22Y, S24T, T26E relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions at positions 198 and 204 relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions of M198L, N204S relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions at positions 4 and 5 relative to the amino acid sequence shown in SEQ ID NOs: 56. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions in L4A, L5A relative to the amino acid sequence shown in SEQ ID NOs:56.

[0202] In some embodiments, the antigen binding domain scFv that specifically binds to MASP3 or MASP2 comprises genetically engineered cysteine ​​mutations by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-bond-stabilized multispecific antibody.

[0203] Connecting peptides (or linkers) can be used to connect the domains and / or structural regions of the heavy chains of the multispecific antibodies into a continuous molecule. The multispecific antibodies may include additional linkers, such as flexible linkers connecting the variable heavy chains and light chains of the scFv. In some embodiments, the multispecific antibodies may include additional linkers, such as flexible linkers connecting the variable heavy chains and light chains of the scFv and other linkers for connecting other binding units to the core structure of the multispecific antibody.

[0204] A typical, non-limiting example of a connecting peptide (or joint) is a polypeptide chain comprising at least 4 residues. The position of this type of joint may be flexible, hydrophilic, and they themselves rarely or will not form a secondary structure (joint position or flexible joint position). After the molecular assembly is completed, at least 4 amino acid joints can be used to connect domains and / or regions close to each other. Longer joints can also be used. In certain embodiments, the joint can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues. When using multiple joints to connect the various parts of the molecule, the joint can be the same or different (for example, the same or different length and / or amino acid sequence).

[0205] In certain aspects, the connecting peptide comprises or consists of a glycine-serine linker. As used herein, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include those of the general formula (Gly4Ser) n An amino acid sequence of, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). A preferred glycine-serine linker is (Gly4Ser)2, i.e., GGGGSGGGGS (SEQ ID NO: 66). A preferred glycine-serine linker is (Gly4Ser)4, i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 67). A preferred glycine-serine linker is (Gly4Ser)3, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 68). In other aspects, two or more glycine-serine linkers are connected in series in a connecting peptide. In certain aspects, the connecting peptide includes at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3 or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, such as (G4S) n .

[0206] In some embodiments, L1 and / or L2 include a hinge portion and a linker portion, such as a linker portion including a glycine-serine linker. In other aspects, L1 and / or L2 include only a hinge portion or only a linker portion, such as a glycine-serine linker. In some aspects, L1 and L2 include a glycine-serine linker. In some aspects, the length of the glycine-serine linker portion of L1 and L2 is the same, while in other aspects, the length of the glycine-serine linker portion of L1 and L2 is different. When the multispecific antibody comprises an scFv, the heavy chain and light chain of the scFv can be connected by a flexible linker. In some embodiments, this flexible linker does not usually include a hinge portion, but a glycine-serine linker or other flexible linker. The length and amino acid sequence of the flexible linker that interconnects the scFv domains can be selected and optimized.

[0207] In some embodiments, the connecting peptide (e.g., L1 and / or L2) comprises a glycine-serine or all-glycine linker and a portion or a modified portion of the hinge region. In certain aspects, the connecting peptide (L1) connecting one antigen-binding domain (e.g., Fab or scFv) and another antigen-binding domain (e.g., scFv or Fab) in the multispecific antibody comprises the amino acid sequence EPKSDKTGGGGSGGGGS (SEQ ID NO: 71) or EPKSCGKTGGGGSGGGGS (SEQ ID NO: 72) or EPKSCGGGGSGGGGS (SEQ ID NO: 73). In certain aspects, the connecting peptide (L2) connecting the antigen-binding domain scFv and Fc in the multispecific antibody comprises the amino acid sequence GGGGSGGGGSEPKSDKTHTCPPCP (SEQ ID NO: 74) or GGGGSGGGGSCPPCP (SEQ ID NO: 75) or GGGGSGGGGSDKTHTCPPCP (SEQ ID NO: 76).

[0208] In some embodiments, the multispecific antibody may optionally include additional connecting peptides in addition to the connecting peptides connecting one antigen binding domain to another antigen binding domain or connecting one of the antigen binding domains to Fc (e.g., L1 and L2). The length and sequence of these additional connecting peptides are independently selected. For example, the multispecific antibody may further include a flexible connecting peptide (L3) that connects the variable heavy chain and light chain (V HSCFV and V LSCFV ). This flexible connecting peptide may include a glycine-serine linker. Typically, this linker does not include a hinge portion. In some embodiments, the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the amino acid sequence

[0209] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 67).

[0210] Hetero H, a multispecific antibody with CrossMab structure

[0211] In some embodiments, according to any multispecific antibody (e.g., bispecific antibody) described herein, it has a Hetero H, CrossMab structure, which is a bivalent multispecific antibody composed of a heterodimer, comprising two antigen-binding domains Fab. In other embodiments, the multispecific antibody further comprises two Fc, which comprises C H 2 and C H 3 domains. In some embodiments, one of the Fc H The amino acid residues in the 3 domain are replaced with amino acid residues with larger side chain volume to form a "knob" and another C in Fc H The amino acid residues in the 3 domains are replaced with amino acid residues with smaller side chain volumes, forming a "hole" that can promote the mutual binding of heterodimers. L ) and heavy chain constant region C H 1 domain (C H 1) can be interchangeable; or the heavy chain variable region (V H ) and light chain variable region (V L ) can be interchangeable; or the light chain constant region (C L ) and heavy chain constant region C H 1 domain (C H 1) and the heavy chain variable region (V H ) and light chain variable region (V L ) are simultaneously replaced with each other. An exemplary Hetero H, CrossMab structure is shown in Figure 3C middle.

[0212] In some embodiments, the Fc is derived from wild-type human IgG1 Fc. In other embodiments, the C H 2 domains include, but are not limited to, the following amino acid substitutions: L234A, L235A, wherein the numbering is according to the EU index as in Kabat. In other embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0213] In other embodiments of the present application, one of the antigen binding domains specifically binds to MASP3, and the other antigen binding domain specifically binds to MASP2. In some embodiments, the multispecific antibody can bind to MASP3 and MASP2 simultaneously.

[0214] In some embodiments, the multispecific antibody described in the present application has a Hetero H, CrossMab structure, which is a heterodimer composed of two different monomers, each of which contains two polypeptide chains. The first monomer contains a first heavy chain and a first light chain of an antibody that specifically binds to one of the antigens, and the second monomer contains a second heavy chain and a second light chain of an antibody that specifically binds to another antigen. In some embodiments, the first heavy chain of the multispecific antibody includes from N-terminus to C-terminus: V H 1-C H 1 structure; the first light chain from N-terminus to C-terminus includes V L 1-C L In some embodiments, the first heavy chain further comprises an Fc comprising a C H 2 and C H In some embodiments, the first heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3 structure; the first light chain from N-terminus to C-terminus includes V L 1-C L Structure. H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, respectively, C H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 1-C H 1 and V L 1-C L In some embodiments, the second heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 2-C L Structure; The second light chain from N-terminus to C-terminus includes: V L 2-C H 1 structure. In some embodiments, the second heavy chain further comprises an Fc comprising a C H 2 and C H In some embodiments, the second heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 2-CL -C H 2-C H 3 structure; the first light chain from N-terminus to C-terminus includes V L 2-C H 1 Structure. V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively. H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 2-C L and V L 2-C H 1 constitutes an antigen binding domain (Fab) that binds to another antigen. In some embodiments, C in the first monomer or the second monomer of the multispecific antibody L and C H 1 are replaced with each other. In some embodiments, V H 1 and V L 1 can be replaced with each other. In some embodiments, the C H 2 domains include, but are not limited to, the following amino acid substitutions: L234A, L235A, wherein the numbering is according to the EU index as in Kabat. In other embodiments, V H 2 and V L 2 positions can be interchangeable. In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C3 domain of the second heavy chain are replaced with amino acid residues with smaller side chain volumes to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob" and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volumes to form a "hole". H The 3 domains include, but are not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0215] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 2 and V L2 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to MASP2, V H 2-C L and V L 2-C H 1 constitutes an antigen binding domain (Fab) that specifically binds to MASP3. In other embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to MASP3, V H 2-C L and V L 2-C H 1 constitutes the antigen binding domain (Fab) that specifically binds to MASP2.

[0216] In some embodiments, the multispecific antibody described herein comprises the amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 61.

[0217] In some embodiments, the multispecific antibody described herein comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 63.

[0218] In some embodiments, the multispecific antibody described herein comprises the amino acid sequence of SEQ ID NO: 60 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 60.

[0219] In some embodiments, the multispecific antibody described herein comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 62.

[0220] In some embodiments, the multispecific antibody according to the present application comprises: an amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 61; and / or an amino acid sequence of SEQ ID NO: 63 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63; and / or an amino acid sequence of SEQ ID NO: 60 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 60; and / or an amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: NO:62 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0221] In some embodiments, the multispecific antibody according to the present application comprises: an amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 61; and / or an amino acid sequence of SEQ ID NO: 63 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63; and / or an amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 64; and / or an amino acid sequence of SEQ ID NO: 65 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: NO:65 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0222] Multispecific antibodies with IgG-(scFv)2 structure

[0223] In some embodiments, the multispecific antibody described in the present application has an IgG-(scFv)2 structure, which is composed of two identical monomers, each of which contains two antigen binding domains, one of which is a Fab and the other is a scFv. In some embodiments, the multispecific antibody further contains an Fc, which contains a C H 2 and C H The scFv is connected to the carboxyl terminus of Fc via a linker peptide (L). An exemplary IgG-(scFv)2 structure is shown in Figure 3D middle.

[0224] In some embodiments of the present application, one antigen binding domain (Fab or scFv) specifically binds to MASP2, and the other antigen binding domain (scFv or Fab) specifically binds to MASP3. In some embodiments, the multispecific antibody can bind to MASP3 and MASP2 simultaneously.

[0225] In some embodiments, the multispecific antibody disclosed in the present application comprises two identical monomers, each monomer comprising a heavy chain and a light chain. In some embodiments, the heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H1 structure. In some embodiments, the light chain of the multispecific antibody comprises from N-terminus to C-terminus: V L 1-C L In some embodiments, the heavy chain further comprises an Fc comprising a C H 2 and C H In some embodiments, the heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3-LV H 2-L3-V L 2 structure. In other embodiments, the heavy chain of the multispecific antibody comprises from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3-LV L 2-L3-V H 2 structure. In some embodiments, the light chain of the multispecific antibody comprises from N-terminus to C-terminus: V L 1-C L Structure. Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively. H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region, L and L3 are connecting peptides. H 1-C H 1 and V L 1-C L One of the antigen-binding domains (Fab) that makes up a multispecific antibody, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes the other antigen-binding domain (scFv) of the multispecific antibody.

[0226] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 1-C H 1 and V L 1-C L Composed of an antigen binding domain (Fab) that specifically binds to MASP2; V H 2 and VL 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to MASP3. H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 1-C H 1 and V L 1-C L Composed of an antigen binding domain (Fab) that specifically binds to MASP3; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to MASP2. In some embodiments, the multispecific antibody comprises C H 1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 58-59. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions at positions 22, 24, and 26 relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions of M22Y, S24T, T26E relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H 3, which comprises one or more substitutions at positions 198 and 204 relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57. In some embodiments, the multispecific antibody comprises C H 2-C H3, which comprises one or more substitutions of M198L, N204S relative to the amino acid sequence shown in any one of SEQ ID NOs: 56-57.

[0227] Connecting peptides (or linkers) can be used to connect the domains and / or structural regions of the heavy chains of the multispecific antibodies into a continuous molecule. The multispecific antibodies may include additional linkers, such as flexible linkers connecting the variable heavy chains and light chains of the scFv. In some embodiments, the multispecific antibodies may include additional linkers, such as flexible linkers connecting the variable heavy chains and light chains of the scFv and other linkers for connecting other binding units to the core structure of the multispecific antibody.

[0228] A typical, non-limiting example of a connecting peptide (or joint) is a polypeptide chain comprising at least 4 residues. The position of this type of joint may be flexible, hydrophilic, and they themselves rarely or will not form a secondary structure (joint position or flexible joint position). After the molecular assembly is completed, at least 4 amino acid joints can be used to connect domains and / or regions close to each other. Longer joints can also be used. In certain embodiments, the joint can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues. When using multiple joints to connect the various parts of the molecule, the joint can be the same or different (for example, the same or different length and / or amino acid sequence).

[0229] In certain aspects, the connecting peptide comprises or consists of a glycine-serine linker. As used herein, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include those of the general formula (Gly4Ser) nAn amino acid sequence of, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). A preferred glycine-serine linker is (Gly4Ser)2, i.e., GGGGSGGGGS (SEQ ID NO: 66). A preferred glycine-serine linker is (Gly4Ser)4, i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 67). A preferred glycine-serine linker is (Gly4Ser)3, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 68). In other aspects, two or more glycine-serine linkers are connected in series in a connecting peptide. In certain aspects, the connecting peptide includes at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3 or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, such as (G4S) n .

[0230] In some embodiments, the connecting peptide comprises a glycine-serine or all-glycine linker and a portion or a modified portion of the hinge region. H The connecting peptide of 3 (eg, L) comprises the amino acid sequence GGGGSGGGGTGGGGS (SEQ ID NO: 78).

[0231] In some embodiments, in addition to the connecting peptide (e.g., L) connecting the antigen binding domain to the Fc, the multispecific antibody can optionally include additional connecting peptides. The length and sequence of these additional connecting peptides are independently selected. For example, the multispecific antibody can also include a flexible connecting peptide (L3) connecting the variable heavy chain and light chain (V HSCFV and V LSCFV ). This flexible connecting peptide may include a glycine-serine linker. Typically, this linker does not include a hinge portion. In some embodiments, the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the sequence

[0232] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 67).

[0233] scFv-Fab IgG structure of multispecific antibodies

[0234] In some embodiments, any multispecific antibody (e.g., bispecific antibody) described herein has a scFv-Fab IgG structure, which is in the form of a heterodimer. The heterodimeric antibody comprises a first monomer and a second monomer, wherein the first monomer comprises an antigen binding domain (Fab) capable of binding to one of the antigens, and the second monomer comprises an antigen binding domain (scFv) capable of binding to another antigen. In some embodiments, the multispecific antibody further comprises two Fcs, which comprise C H 2 and C H The 3 domains, the two Fc further contain amino acid substitutions, which can promote the mutual binding of heterodimers. An exemplary scFv-Fab IgG structure is shown in Figure 3E middle.

[0235] In some embodiments, one of the antigen binding domains (Fab or scFv) specifically binds to MASP3, and the other antigen binding domain (scFv or Fab) specifically binds to MASP2. In some embodiments, the multispecific antibody can bind to both MASP3 and MASP2 simultaneously.

[0236] In some embodiments, any multispecific antibody described herein has a scFv-Fab IgG structure, which is in the form of a heterodimer. The heterodimer antibody comprises a first monomer and a second monomer, wherein the first monomer comprises two polypeptide chains: a first heavy chain and a light chain, wherein the first heavy chain comprises from the N-terminus to the C-terminus: V H 1-C H 1 structure. The light chain contains V L 1-C L In some embodiments, the first heavy chain further comprises an Fc, the Fc comprising a C H 2 and C H In some embodiments, the first heavy chain comprises, from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3 structures, the light chain contains V L 1-C L Structure. H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, respectively, C H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region. H 1-C H 1 and V L 1-C LThe second monomer comprises a polypeptide chain: a second heavy chain. The second heavy chain comprises V from the N-terminus to the C-terminus. H 2-L3-V L 2 structure or V L 2-L3-V H 2 structure. In some embodiments, the second heavy chain further comprises an Fc, the Fc comprising a CH2 and a CH3 domain. In some embodiments, the second heavy chain comprises from the N-terminus to the C-terminus: V H 2-L3-V L 2-C H 2-C H 3 structures or V L 2-L3-V H 2-C H 2-C H 3 Structure. V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, and L3 is a connecting peptide. H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes another antigen binding domain (scFv).

[0237] In some embodiments, V# H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 1-C H 1 and V L 1-C L Composed of an antigen binding domain (Fab) that specifically binds to MASP2. H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to MASP3. In some embodiments, V# H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 1-C H 1 and V L 1-C L Composed of an antigen binding domain (Fab) that specifically binds to MASP3. H 2 and V L2 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to MASP2.

[0238] In some embodiments, the Fc is derived from human wild-type IgG1. In another embodiment, the Fc in one monomer comprises, but is not limited to, the following amino acid substitutions E357Q, and S364K relative to human wild-type IgG1; the Fc in another monomer comprises, but is not limited to, the following amino acid substitutions Q295E, L368D, K370S, N384D, Q418E, and N421D relative to human wild-type IgG1, wherein the numbering is according to the EU index as in Kabat. In some embodiments, the connecting peptide (e.g., L3) connecting VH1 and VL1 in an exemplary scFv comprises the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 77).

[0239] Hetero H, CrossMab 2+1 multispecific antibody

[0240] In some embodiments, according to any multispecific antibody (e.g., bispecific antibody) described herein, it has a Hetero H, CrossMab 2+1 structure, which is a trivalent multispecific antibody composed of a heterodimer, comprising three antigen-binding domains Fab, wherein the first and second antigen-binding domains specifically bind to one of the antigens, and the third antigen-binding domain specifically binds to another antigen. In other embodiments, wherein the multispecific antibody further comprises two Fc, comprising C H 2 and C H 3 domains. In some embodiments, one of the Fc H The amino acid residues in the 3 domain are replaced with amino acid residues with larger side chain volume to form a "knob" and another C in Fc H The amino acid residues in the 3 domains are replaced with amino acid residues with smaller side chain volumes, forming a "hole" that can promote the mutual binding of heterodimers. In the third antigen-binding domain Fab, the light chain constant region (C L ) and heavy chain constant region C H 1 domain (C H 1) can be interchangeable; or the heavy chain variable region (V H ) and light chain variable region (V L ) can be interchangeable; or the light chain constant region (C L ) and heavy chain constant region CH 1 domain (C H 1) and the heavy chain variable region (V H ) and light chain variable region (V L ) are simultaneously replaced with each other. An exemplary Hetero H, CrossMab2+1 structure is shown in Figure 3F An exemplary Hetero H, CrossMab2+1 structure with oppositely charged amino acid mutations in the non-exchange Fab region is shown in Figure 3G middle

[0241] In some embodiments, the Fc is derived from wild-type human IgG1 Fc. In other embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0242] In other embodiments of the present application, two of the antigen binding domains specifically bind to MASP3, and one of the antigen binding domains specifically binds to MASP2. In some embodiments, two of the antigen binding domains specifically bind to MASP2, and one of the antigen binding domains specifically binds to MASP3. In some embodiments, the multispecific antibody can bind to MASP3 and MASP2 simultaneously.

[0243] In some embodiments, the multispecific antibody described in the present application has a Hetero H, CrossMab 2+1 structure, which is a heterodimer composed of two different monomers, the first monomer comprises two polypeptide chains, and the second monomer comprises three polypeptide chains. The first monomer comprises a first heavy chain and a first light chain of an antibody that specifically binds to one of the antigens, and the second monomer comprises a second heavy chain, a second light chain, and a third light chain of an antibody that specifically binds to two antigens. In some embodiments, the first heavy chain of the multispecific antibody includes from N-terminus to C-terminus: V H 1-C H 1 structure; the first light chain from N-terminus to C-terminus includes V L 1-C L In some embodiments, the first heavy chain further comprises an Fc comprising a C H 2 and C H In some embodiments, the first heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3 structure; the first light chain from N-terminus to C-terminus includes V L1-C L Structure. H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, respectively, C H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 1-C H 1 and V L 1-C L In some embodiments, the second heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H 1-V H 2-C L Structure; The second light chain from N-terminus to C-terminus includes V L 1-C L Structure; The third light chain from N-terminus to C-terminus includes: V L 2-C H 1 structure. In some embodiments, the second heavy chain further comprises an Fc comprising a C H 2 and C H In some embodiments, the second heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 1-C H 1-V H 2-C L -C H 2-C H 3 structure; the second light chain from N-terminus to C-terminus includes V L 1-C L Structure; The third light chain from N-terminus to C-terminus includes V L 2-C H 1 Structure. V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to an antigen, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively. H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 1-C H 1 and V L 1-C L Composed of the antigen binding domain (Fab) that binds one of the antigens, V H 2-C L and V L 2-CH 1 constitutes an antigen binding domain (Fab) that binds to another antigen. In some embodiments, C in the first monomer or the second monomer of the multispecific antibody L and C H 1 are replaced with each other. In some embodiments, V H 1 and V L 1 can be replaced with each other. In other embodiments, V H 2 and V L 2 positions can be interchangeable. In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C3 domain of the second heavy chain are replaced with amino acid residues with smaller side chain volumes to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob" and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volumes to form a "hole". H The 3 domains include, but are not limited to, the following amino acid replacements: S354C, T366W, Y349C, T366S, L368A and Y407V, wherein the numbering is according to the EU index as in Kabat. In some embodiments, the C of the first heavy chain H The amino acid residues in the 1 domain are replaced by negatively charged amino acid residues, and the first light chain C L The amino acid residues in the domain are replaced with positively charged amino acid residues; and the C H The amino acid residues in the 1 domain are replaced by negatively charged amino acid residues, and the second light chain C L In some other embodiments, the amino acid residues in the C domain of the first heavy chain are replaced with positively charged amino acid residues. H The amino acid residues in the 1 domain are replaced by positively charged amino acid residues, and the first light chain C L The amino acid residues in the domain are replaced with negatively charged amino acid residues; and the C H The amino acid residues in the 1 domain are replaced by positively charged amino acid residues, and the second light chain C L In some embodiments, the amino acid residues in the C H 1 domain includes, but is not limited to, the following amino acid substitutions: K148E, K214E, wherein the numbering is according to the EU index as in Kabat; the C LThe domain includes, but is not limited to, the following amino acid substitutions: E128R, Q129K, wherein the numbering is according to Kabat.

[0244] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to MASP3, V H 2-C L and V L 2-C H 1 constitutes an antigen binding domain (Fab) that specifically binds to MASP2. H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to MASP2, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to MASP3, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to MASP2, V H 2-C L and V L 2-C H 1 constitutes an antigen binding domain (Fab) that specifically binds to MASP3. Exemplary antibody sequences are shown in Tables 2 to 9, wherein the CDR numbering is based on the EU index in Kabat. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the position of CDRs and defining the variable regions of antibody light and heavy chains. The CDRs, V H and / or V L Sequences, but based on prediction algorithms other than the antibodies exemplified in the table below are also within the scope of the present application.

[0245] Table 2: Exemplary CDR sequences of antibodies that specifically bind to MASP3

[0246]

[0247]

[0248]

[0249] Table 3: Exemplary antibodies that specifically bind to MASP3 H / V L sequence

[0250]

[0251]

[0252] Table 4: Exemplary CDR sequences of antibodies that specifically bind to MASP2

[0253]

[0254] Table 5: Exemplary antibodies that specifically bind to MASP2 H / V L sequence

[0255]

[0256]

[0257] Table 6: Exemplary antibody constant region sequences

[0258]

[0259] Table 7: Partial heavy and light chain sequences of Hetero H, CrossMab structures, exemplary multispecific antibodies that specifically bind to MASP3 and MASP2

[0260]

[0261]

[0262] Table 8: Hetero H, CrossMab structure Full length heavy and light chain sequences of exemplary multispecific antibodies that specifically bind to MASP3 and MASP2

[0263]

[0264] Table 9: Exemplary connecting peptide (or linker) sequences

[0265]

[0266]

[0267] Binding affinity

[0268] Binding affinity can be expressed as Kd, Koff, Kon or Ka. As used in this application, the term "Koff" refers to the rate constant at which the antigen binding domain dissociates from the antigen binding domain / antigen complex, which is measured by a kinetic selection device. The term "Kon" refers to the binding rate constant of the antibody and antigen to form an antigen binding domain / antigen complex. The dissociation constant "Kd" used in this application refers to the dissociation constant when a specific antibody-antigen interaction occurs, which refers to the antigen concentration required in the antibody molecule solution when the antigen occupies half of all antibody binding domains and reaches equilibrium, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In the case where the antigen binding domain is attached to the cell wall, for example in a yeast expression system, the corresponding dissociation rate constant is expressed using EC 50 It is expressed as , which is a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0269] The equilibrium dissociation constant (Kd) can be used as an indicator of the affinity of the antigen binding domain portion for the antigen. For example, simple analysis can be performed by the Scatchard method using antibodies labeled with various markers and a Biacore instrument (manufactured by Amersham Biosciences) to analyze the interaction between biomolecules by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values ​​obtained using these methods are expressed in units of M. An antibody that specifically binds to a target may have, for example, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0270] The binding specificity of the antibody can be experimentally determined by methods known in the art, including, but not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore assays, and peptide scanning.

[0271] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP3 specifically binds to the MASP3 target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10-12 Thus, in some embodiments, the Kd value of the binding between an antibody or antigen-binding fragment that specifically binds to MASP3 and MASP3 is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 - 7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP3 and MASP3 is 10 -7 M to 10 -13 M.

[0272] In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP3 and the non-target is higher than the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP3 and the target, and in some embodiments cited in the present application, the binding affinity of the antibody or antigen-binding fragment that specifically binds to MASP3 and the target (e.g., MASP3) is higher than the binding affinity of the antibody or antigen-binding fragment that specifically binds to MASP3 and the non-target. In some embodiments, the non-target refers to non-MASP3. In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP3 and the non-MASP3 target is at least 10 times, for example, 10-100 times, 100-10 ... 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0273] In some embodiments, the antibody or antigen-binding fragment that specifically binds to MASP2 specifically binds to the MASP2 target with a Kd value of 10 -7 M to 10-13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 Thus, in some embodiments, the Kd value of the binding between an antibody or antigen-binding fragment that specifically binds to MASP2 and MASP2 is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 - 7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10-10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP2 and MASP2 is 10 -7 M to 10 -13 M.

[0274] In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP2 and the non-target is higher than the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP2 and the target, and in some embodiments cited in the present application, the binding affinity of the antibody or antigen-binding fragment that specifically binds to MASP2 and the target (e.g., MASP2) is higher than the binding affinity of the antibody or antigen-binding fragment that specifically binds to MASP2 and the non-target. In some embodiments, the non-target refers to non-MASP2. In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to MASP2 and the non-MASP2 target is at least 10 times, for example, 10-100 times, 100-10 ... 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -1011 times, 10 11 -10 12 times.

[0275] In some embodiments, any multispecific antibody described in the present application comprises a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2. The first antigen binding domain specifically binds to the MASP3 antigen target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Therefore, in some embodiments, the Kd value of the binding between the first antigen binding domain and MASP3 is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10-12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the first antigen binding domain and MASP3 is 10 -7 M to 10 -13 M. The second antigen binding domain specifically binds to the MASP2 antigen target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Thus, in some embodiments, the Kd value for binding between the second antigen binding domain and MASP2 is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10-7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the second antigen binding domain and MASP2 is 10-7 M to 10 -13 M.

[0276] In some embodiments, any multispecific antibody described in the present application comprises a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2. The Kd value of the binding between the first antigen binding domain and the non-target is higher than the Kd value of the first antigen binding domain and the target, and in some embodiments cited in the present application, the binding affinity of the first antigen binding domain to the target (e.g., MASP3) is higher than the binding affinity of the first antigen binding domain to the non-target. In some embodiments, the non-target refers to a non-MASP3 antigen. In some embodiments, the Kd value of the binding between the first antigen binding domain and the non-MASP3 target is at least 10 times the Kd of the binding between the first antigen binding domain and the MASP3 antigen, for example 10-10 2 times, 10 2 -10 3 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times. Wherein the Kd value of the binding between the second antigen binding domain and the non-target is higher than the Kd value of the second antigen binding domain and the target, and in some embodiments cited in this application, the binding affinity of the second antigen binding domain to the target (e.g., MASP2) is higher than the binding affinity of the second antigen binding domain to the non-target. In some embodiments, the non-target refers to a non-MASP2 antigen. In some embodiments, the Kd value of the binding between the second antigen binding domain and the non-MASP2 antigen target is at least 10 times the Kd of the binding between the second antigen binding domain and the target MASP2, for example 10-10 2 times, 10 2 -10 3 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6-10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0277] Nucleic acids and vectors

[0278] Nucleic acid molecules encoding antibodies or antigen-binding fragments that specifically bind to the MASP3 antigen, antibodies or antigen-binding fragments that specifically bind to MASP2, and multispecific antibodies are also contemplated.

[0279] In some embodiments, the present application provides a nucleic acid (or a group of nucleic acids) encoding an antibody or antigen-binding fragment that specifically binds to MASP3, or a nucleic acid encoding an antibody or antigen-binding fragment that specifically binds to MASP2, or a nucleic acid encoding a multispecific antibody that specifically binds to MASP3 and MASP2, including any antibody or antigen-binding fragment that specifically binds to MASP3, or an antibody or antigen-binding fragment that specifically binds to MASP2, or a multispecific antibody that specifically binds to MASP3 and MASP2 described in the present application. In some embodiments, the nucleic acid (or a group of nucleic acids) of the antibody or antigen-binding fragment or multispecific antibody described in the present application may also include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His-tag, an HA tag).

[0280] At the same time, the present application also provides an antibody or antigen-binding fragment that specifically binds to MASP3, an antibody or antigen-binding fragment that specifically binds to MASP2, or a multispecific antibody that specifically binds to MASP3 and MASP2; or a nucleic acid molecule that encodes the antibody or antigen-binding fragment; or an isolated host cell that contains a vector carrying the nucleic acid molecule. The present application also includes variants of these nucleic acid sequences. For example, the variant includes a nucleotide sequence that hybridizes with a nucleic acid sequence encoding an antibody or antigen-binding fragment or a multispecific antibody of the present application under at least moderately stringent hybridization conditions.

[0281] The present application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to a nucleic acid sequence encoding an antibody or antigen-binding fragment or multispecific antibody of the present application under at least moderately stringent hybridization conditions.

[0282] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0283] In brief, a natural or synthetic nucleic acid encoding an antibody, antigen-binding fragment or multispecific antibody is inserted into a suitable expression vector so that the nucleic acid is operably linked to 5' and 3' regulatory elements, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), and the antibody or antigen-binding fragment or multispecific antibody can be expressed. The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences and promoters that regulate the expression of the target nucleic acid sequence.

[0284] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy by using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0285] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0286] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0287] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and reproduction in daughter cells. Lentiviral vectors have additional advantages over retroviruses derived from tumors, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, it also has the additional advantage of low immunogenicity.

[0288] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. They are usually located 30-110 bp upstream of the start site, although recently it has been discovered that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so that promoter function is maintained when elements are swapped or moved relative to each other. In the thymidine kinase (tk) promoter, the activity begins to decline only when the spacing between promoter elements increases to 50 bp.

[0289] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present application should not be limited to using only constitutive promoters. Inducible promoters are also considered in the present application. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence to which it is operably linked when such expression is desired, and turn off expression when such expression is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0290] In some embodiments, the expression of the antibody or antigen binding fragment or multispecific antibody is inducible. In some embodiments, the nucleic acid sequence encoding the antibody or antigen binding fragment or multispecific antibody is operably linked to an inducible promoter, including any inducible promoter described herein.

[0291] Inducible promoter

[0292] The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when expression is desired, and turn off expression when expression is not desired. Exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21: 377-405. In some embodiments, the inducible promoter system used to express the antibody or antigen-binding fragment or multispecific antibody is the Tet system. In some embodiments, the inducible promoter system used to express the antibody or antigen-binding fragment or multispecific antibody is the E. coli lac repression system.

[0293] An exemplary inducible promoter system used in the present application is the Tet system. The system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the activated state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0294] In one embodiment, TetR is codon optimized for expression in mammalian cells, such as mice or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants, and the amino acid sequence encoded by it does not change at all. However, many organisms have differences in codon usage, also known as "codon preference" (i.e., the preference for using specific codons for a given amino acid). Codon preference is generally related to the presence of a dominant tRNA species for a specific codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0295] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcription activator protein (tTA), which is composed of a fusion of TetR and the strong transcription activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE element consists of a fusion of the TetO sequence in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0296] In contrast, in the Tet-On system, transcription is activated in the presence of Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcription activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transactivation domain. However, changes in four amino acids in the DNA binding region of TetR alter the binding properties of rtTA, allowing it to only recognize the tetO sequence on the target transgene TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can only activate the transcription of the target gene regulated by the TRE in the presence of Dox.

[0297] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising a lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducer, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0298] In order to evaluate the expression of polypeptide or part thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both, so as to identify and select expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection experiment. Either the selectable marker gene or the reporter gene may be flanked by a suitable regulatory sequence to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and similar genes.

[0299] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by receptor organisms or tissues, and encode a polypeptide whose expression is expressed as some easily detectable characteristics, such as enzyme activity. After DNA is introduced into the receptor cells, the expression of the reporter gene is detected at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein (eg, Ui-Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared by known techniques or obtained commercially. Generally, a construct with a minimum 5' flanking region that can show the highest expression level of a reporter gene is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to assess the ability of certain substances in regulating promoter-driven transcription.

[0300] In some embodiments, a nucleic acid encoding any one of the antibodies or antigen-binding fragments or multi-specific antibodies described herein is provided. In some embodiments, the nucleic acid includes one or more nucleic acid sequences encoding antibody or antigen-binding fragment or multi-specific antibody heavy chain and light chain. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses).

[0301] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian cells, bacteria, yeast or insect cells, by any method in the art. For example, the expression vector can be introduced into the host cell by physical, chemical or biological methods.

[0302] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0303] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, etc. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0304] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0305] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, the nucleic acid can be combined with a lipid. Nucleic acids combined with lipids can be wrapped into the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule combined with the liposome and the oligonucleotide, embedded in the liposome, forming a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, suspended in lipids, contained in micelles or mixed with micelles, or otherwise combined with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, in micelles or in a "collapsed" structure. They can also be simply dispersed in a solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0306] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of experiments can be performed to confirm that the recombinant DNA sequence is present in the host cell. Such experiments include, for example, "molecular biology" experiments well known to those skilled in the art. For example, Southern and Northern blotting, RT-PCR and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or by the experiments described in the present application to perform identification are all within the scope of the present application.

[0307] Preparation of antibodies or antigen-binding fragments and multispecific antibodies

[0308] In some embodiments, the antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment that specifically binds MASP3 or MASP2) is a monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment, or multispecific antibody is derived from a monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment, or multispecific antibody comprises a V from a monoclonal antibody. H and V L , or a variant thereof. In some embodiments, the antibody or antigen-binding fragment or multispecific antibody further comprises a C from a monoclonal antibody H 1 and C LRegion, or its variant. Monoclonal antibodies can be prepared using methods known in the art, such as hybridoma cell method, yeast display, phage display method or recombinant DNA method. In addition, exemplary yeast display and phage display methods are described in the present application and in the following examples. Multispecific antibodies can be prepared using methods known in the art, such as chemical coupling, hybridoma method and genetic engineering method.

[0309] In the hybridoma method, hamsters, mice or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are required, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian cells are required, spleen cells or lymph node cells are used. Lymphocytes are fused with immortal cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortal cell lines are typically transformed mammalian cells, especially myeloma cells of rodents, bovines and humans. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortal cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0310] In some embodiments, the immortalized cell line is effectively fused, and the antibody is stably expressed at a high level by the selected antibody production cells, and is sensitive to certain culture media, such as HAT culture media. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for the preparation of human monoclonal antibodies.

[0311] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by, for example, Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0312] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture media. Alternatively, hybridoma cells can be grown in mammals in the form of ascites.

[0313] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0314] In some embodiments, according to any antibody or antigen-binding fragment or multispecific antibody described herein, the antibody or antigen-binding fragment or multispecific antibody comprises a sequence selected from a clone of an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by a method of screening a combinatorial library of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 339(3):341-357 (2005); al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0315] In some phage display methods, V H and V L All components of the gene are randomly recombined in a phage library, and then phages that can bind to the antigen are screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phages of immune origin provide high-affinity antibodies against immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies against a variety of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0316] The antibody or antigen-binding fragment or multispecific antibody is prepared by screening the library for antigen-binding portions that can specifically bind to the target (e.g., MASP3 or MASP2) through phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11) kinds of diverse unique human antibody fragments. In certain embodiments, the library is a human natural library, constructed by DNA extracted from PMBCs and spleen of healthy subjects, comprising all heavy and light chain subfamilies. In certain embodiments, the library is a human natural library, constructed by DNA extracted from PMBCs isolated from various disease patients, such as patients with autoimmune diseases, cancer patients and patients with infectious diseases. In certain embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present in any given position with the same probability. (See, e.g., Hoet, RMet al., Nat.Biotechnol.23 (3): 344-348, 2005). In certain embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0317] Phage clones with high affinity for a target antigen (e.g., MASP3 or MASP2) can be screened by iterative binding of phage to the target antigen, which is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage. Subsequently, bound phage clones are eluted and used to infect appropriate host cells, such as E. coli XL1-Blue, for expression and purification. Phage clones that specifically bind to the target antigen can be enriched by multiple rounds of panning (e.g., 2, 3, 4, 5, 6 or more rounds), such as solution panning, cell panning, or a combination of both. Specific binding of the enriched phage clones to the target antigen can be detected by any method known in the art, including, for example, ELISA and FACS.

[0318] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in the present application can be easily separated and sequenced by conventional methods (e.g., by oligonucleotide probes that can specifically bind to the light and heavy chain genes encoding mouse antibodies). The hybridoma cells described above or the antigen-specific phage clones of the present application can be used as the source of such DNA. After separation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain a monoclonal antibody synthesized in a recombinant host cell. The DNA can also be modified, such as replacing homologous non-human sequences with coding sequences for human heavy and light chain constant structures and / or framework regions (U.S. Patent No. 4,816,567; Morrison et al., supra), or covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. This non-immunoglobulin polypeptide can replace the constant region of the antibody in the present application, or can replace an antigen binding site in the variable region of the antibody in the present application to form a chimeric bivalent antibody. In some embodiments, additional variable regions for different epitopes or antigens can be included to produce chimeric multispecific antibodies.

[0319] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a recombinant expression method involving immunoglobulin light chains and modified heavy chains. The heavy chains are usually truncated at any position in the Fc region to prevent the heavy chains from cross-linking with each other. Alternatively, the relevant cysteine ​​residues are replaced by other amino acid residues or are deleted to prevent cross-linking.

[0320] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0321] Chemical coupling is the earliest technology used to prepare multispecific antibodies. In 1985, Brennan first used the chemical combination of two monoclonal antibody G1 fragments to prepare multispecific antibodies (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments [J]. Science, 1985, 229 (4708): 81-83). There are two main ways of chemical coupling. One is to directly couple two monoclonal antibodies or their derivatives to form a multispecific antibody. The other is to first dissociate the two monoclonal antibodies into free light chains and heavy chains by various physical and chemical methods, and then recombine these light chains and heavy chains. The advantages of chemical coupling are rapidity, simple operation, and high recovery rate, but it is also easy to destroy the antigen binding domain of the antibody, affect the antibody activity, and easily form polymers.

[0322] The use of hybridoma cell lines to prepare multispecific antibodies refers to the fusion of two different hybridoma cell lines through cell fusion technology, and then the identification and separation of cells that can produce specific therapeutic antibodies (Kohler, G, et al. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. J Immunol., 2005, 174 (5): 2453-2455). Because two hybridoma cells can produce two different light-heavy chains, and these light-heavy chains can be randomly combined, the multispecific antibodies prepared by this method have greater randomness and low preparation efficiency.

[0323] Genetic engineering technology is currently also used to prepare a variety of multi-specific antibodies (Roland E K. Antibody-cytokine fusion proteins [J]. Arch Biochem Biophys., 2012, 526 (2): 194-205). Using genetic engineering to edit recombinant antibodies can solve the problem of random combination by limiting the selectivity of the combination of two pairs of light-heavy chains in various ways. KiH (Knob into hole) and CrossMab are two commonly used technologies currently used to improve the light-heavy chain pairing problem. KiH technology is to use C H The asymmetric mutation structure was introduced into the 3 domain (the “knob” mutation refers to the asymmetric mutation in the C HIn the 3 domain, a large amino acid residue is substituted for a smaller residue, while a "hole" mutation refers to the use of a small amino acid residue to replace a larger residue). The Fc region of the engineered multispecific antibody is more inclined to heterodimerization rather than homodimerization due to steric hindrance (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody C H 3 domains for heavy chain heterodimerization[J].Protein Eng.1996,9(7):617-621). In the glycosylated C H Introducing a Y349C mutation into the 3 domain can form a disulfide bond between glycosylated heavy chains and enhance the stability of KiH (Kuglstatter A, et al. Structural differences between glycosylated, disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J]. Protein Eng Des Sel., 2017, 30(9): 649-656).

[0324] In addition to the steric hindrance effect, the charge effect of amino acid residues is also used to improve the heterodimerization between the two heavy chains of multispecific antibodies. Through structural simulation and molecular design modification, "positive charge" is generated in one chain and "negative charge" is generated in the paired chain, and the formation of heavy chain heterodimers is promoted through the mode of like charges repelling each other and heterogeneous charges attracting each other. Mutations in the two chains include K409D and D399K, K409D / K392D and D399K / E356K, or E356K / E357K / D399K and K370E / K409D / K439E, which can improve the formation of heterodimers to a certain extent (IGAWA T, et al. Methods for producing polypeptides by regulating polypeptide; association: US, 20100015133A1[P]. 2006). Combining the steric hindrance effect of KiH with the charge effect is also one of the strategies to improve heterodimerization.

[0325] CrossMab technology is a new antibody pairing technology developed by Roche based on KiH technology. It exchanges the domains of the light chain and heavy chain of one Fab in a multispecific antibody, while the other does not. The exchanged light chain will contain a fragment of a homologous heavy chain, so that it cannot be paired with the unexchanged heavy chain, thereby ensuring the correct combination between the light chain and the heavy chain (Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies [J]. Proc Natl Acad Sci USA, 2011, 108 (27): 11187-11192). The structure includes "CrossMab Fab", "CrossMab V H -V L ” or “CrossMab C H 1-C L " and other forms.

[0326] Antibody variable regions with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant regions. Preferably, the fusion is to an immunoglobulin heavy chain constant region, which includes at least part of the hinge, C H 2 and C H In some embodiments, the heavy chain constant region C that contains the necessary site for light chain binding H 1 domain is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion, and if desired, DNA encoding the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. In some embodiments, antibody variable regions directed against different antigenic epitopes or different antigens can be fused with immunoglobulin constant region sequences to produce chimeric multispecific antibodies.

[0327] Fully human and humanized antibodies

[0328] The antibody or antigen-binding fragment or multispecific antibody can be a humanized antibody or a fully human antibody. The humanized form of a non-human (such as mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv or other antigen-binding subsequences of an antibody), which generally includes a minimum sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins, immunoglobulin chains or fragments thereof (receptor antibodies), wherein the residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues with the desired specificity, affinity and performance, such as the CDR of mice, rats or rabbits. In certain embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies can also include amino acid residues that are neither in the receptor antibody nor in the CDR or framework region sequences introduced. Generally, a humanized antibody includes at least one, generally two variable regions, wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all framework regions are human immunoglobulin consensus sequences.

[0329] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, usually from the "imported" variable region. According to some embodiments, humanization can be performed substantially according to the following method of Winter and his colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences. Therefore, this "humanized" antibody portion (US Patent No. 4,816,567), which is substantially less than a complete human antibody, has its variable region replaced by the corresponding sequence from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0330] Producing human antibody parts is an alternative to humanization. For example, it is currently possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies after immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits the production of endogenous antibodies. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce fully human antibodies under antigen stimulation, see, for example, akobovits et al., PNAS USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al., Year in Immunol., 7: 33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). After antigen stimulation, it can be found that the production of fully human antibodies is very similar to that in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0331] Human antibodies or human antibody portions can also be produced by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147 (1): 86-95 (1991).

[0332] Variants of antibodies or antigen-binding fragments

[0333] In some embodiments, the amino acid sequences of variants of antibodies or antigen-binding fragments (e.g., antibodies that specifically bind to MASP3 or MASP2, or multispecific antibodies that specifically bind to MASP3 and MASP2) provided herein are also under consideration. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody or antigen-binding fragment. The amino acid sequence of the variant antigen-binding entity can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding entity or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antigen-binding entity. The final construction can be completed by any combination of amino acid residue deletion, insertion and substitution to give it the desired characteristics. For example, antigen binding. In some embodiments, variants of antibodies or antigen-binding fragments having one or more amino acid substitutions are provided. The target sites of substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products of desired activity, for example, improved biological activity or antibody affinity. In some embodiments, the amino acid substitutions described herein are limited to the "exemplary substitutions" in Table 10 of this application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" in Table 10 of the present application.

[0334] Conservative substitutions are shown in Table 10 below.

[0335] Table 10: Conservative substitutions

[0336]

[0337]

[0338] Amino acids are divided into different categories based on the properties of their side chains:

[0339] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leu, Ile;

[0340] b. Neutral hydrophilic amino acids: cysteine ​​Cys, serine Ser, threonine Thr, asparagine Asn, glutamine Gln;

[0341] c. Acidic amino acids: Aspartic acid Asp, glutamic acid Glu;

[0342] d. Basic amino acids: histidine His, lysine Lys, arginine Arg;

[0343] e. Amino acids that affect chain direction: glycine Gly, proline Pro;

[0344] f. Aromatic amino acids: tryptophan Trp, tyrosine Tyr, phenylalanine Phe.

[0345] Non-conservative amino acid substitutions include substitutions of one species for another.

[0346] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated, the variant antibody portion is displayed on the phage, and variants with specific biological activity (e.g., based on RBC cell lysis inhibition assay or binding affinity) are screened. Changes (e.g., substitutions) can be made in the HVRs region to obtain improved RBC lysis inhibition assay or antibody affinity. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific determinant residues (SDRs), and the resulting variant V is detected. H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0347] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0348] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided in this application) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0349] A method for identifying amino acids or regions in antibodies that can bind to a target by mutation is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine and glutamic acid) are replaced by neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the position of the amino acid to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or in addition, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0350] Insertions of amino acid sequences include fusions at the amino and / or carboxyl termini ranging in length from 1 residue to polypeptides containing 100 or more residues, and also include insertions of 1 or more amino acid residues within a sequence. Examples of terminal insertions include antigen-binding portions having a methionyl residue at the N-terminus. Other insertion variants of the antigen-binding portion include fusions of an enzyme (e.g., ADEPT) or polypeptides that increase the serum half-life of the antigen-binding portion to the N-terminus or C-terminus of the antigen-binding portion.

[0351] Fc variants

[0352] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody or antigen-binding fragment described herein (e.g., a full-length antibody that specifically binds to MASP3, a full-length antibody that specifically binds to MASP2, a multispecific antibody that specifically binds to MASP3 and MASP2, or a fusion protein comprising the antibody or antigen-binding fragment or multispecific antibody), thereby generating an Fc variant. In some embodiments, the Fc variant has enhanced ADCC potency, which is generally associated with receptors that bind to Fc (FcRs). In some embodiments, the Fc variant has reduced ADCC potency. There are many examples of how changes or mutations in Fc sequences affect their potency, for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0353] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., infected cells) when antigens on the surface of the target cell membrane are bound by specific antigen-binding moieties (e.g., antibodies that specifically bind to MASP3, antibodies that specifically bind to MASP2, or multispecific antibodies that specifically bind to MASP3 and MASP2). Typically, the ADCC effect involves NK cells activated by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules that are bound to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of Fc receptors to the Fc region of antibodies leads to activation of NK cells, release of cytolytic granules, and subsequent apoptosis of target cells.

[0354] In some embodiments, the present application also provides an antibody or antigen-binding fragment that specifically binds to MASP3, an antibody or antigen-binding fragment that specifically binds to MASP2, or a multispecific antibody variant that specifically binds to MASP3 and MASP2 (e.g., a full-length antibody that specifically binds to MASP3, a full-length antibody that specifically binds to MASP2, or a variant of a multispecific antibody that specifically binds to MASP3 and MASP2), which comprises an Fc region having one or more effector functions, which makes it an ideal candidate antibody for application, where the in vivo half-life of the antibody or antigen-binding fragment that specifically binds to MASP3, the antibody or antigen-binding fragment that specifically binds to MASP2, or the multispecific antibody that specifically binds to MASP3 and MASP2 is important, but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. The reduction / elimination of CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Among the main cells that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assessment of ADCC activity of target molecules are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.). Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, ADCC activity of the target molecule is tested in vivo, for example, in an animal model, as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0355] Antibodies with reduced effector function include substitutions of one or more residues at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Pat. No. 6,737,056). These Fc variants include Fc variants with substitutions of two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine (US Pat. No. 7,332,581).

[0356] Such antibody variants with improved or reduced binding to FcRs have been described (see, e.g., US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0357] In some embodiments, alterations in the Fc region result in altered (ie, increased or decreased) opsonization, as described in Moore et al., MAbs. 2(2): 181-189 (2010).

[0358] In some embodiments, a variant of an antibody or antigen-binding fragment that specifically binds to MASP3, an antibody or antigen-binding fragment that specifically binds to MASP2, or a multispecific antibody (e.g., a full-length antibody that specifically binds to MASP3, a full-length antibody that specifically binds to MASP2, or a multispecific antibody that specifically binds to MASP3 and MASP2) is provided, which comprises an Fc variant having one or more amino acid substitutions, which can extend the half-life and / or enhance the binding to the Fc receptor (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). The Fc region of these antibodies comprises one or more amino acid substitutions, which enhances the binding of the Fc region to FcRn. These Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434 in the Fc region, such as a substitution at residue 434 in the Fc region ( US Pat. No. 7,371,826 ).

[0359] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No.

[0360] Examples of other Fc variants are provided in 5,624,821 and WO 94 / 29351.

[0361] Also contemplated are antibodies or antigen-binding fragments that specifically bind to MASP3, antibodies or antigen-binding fragments that specifically bind to MASP2, or multispecific antibodies that specifically bind to both MASP3 and MASP2 (e.g., full-length antibodies that specifically bind to MASP3, full-length antibodies that specifically bind to MASP2, or multispecific antibodies that specifically bind to both MASP3 and MASP2) comprising any one of the Fc variants described herein or a combination thereof.

[0362] Glycosylation variants

[0363] In some embodiments, the antibodies or antigen-binding fragments that specifically bind to MASP3, antibodies or antigen-binding fragments that specifically bind to MASP2, or multispecific antibodies that specifically bind to MASP3 and MASP2 (e.g., full-length antibodies that specifically bind to MASP3, full-length antibodies that specifically bind to MASP2, or multispecific antibodies that specifically bind to MASP3 and MASP2) provided herein are altered to increase or decrease the degree of glycosylation of the antibodies or antigen-binding fragments that specifically bind to MASP3, antibodies or antigen-binding fragments that specifically bind to MASP2, or multispecific antibodies that specifically bind to MASP3 and MASP2. Adding or deleting glycosylation sites on the antibodies or antigen-binding fragments that specifically bind to MASP3, antibodies or antigen-binding fragments that specifically bind to MASP2, or multispecific antibodies that specifically bind to MASP3 and MASP2, or polypeptide portions thereof, can be conveniently achieved by changing the amino acid sequence of the antibodies or antigen-binding fragments that specifically bind to MASP3, antibodies or antigen-binding fragments tha...

Claims

1. An isolated antibody or antigen-binding fragment that specifically binds to MASP3, comprising: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:13, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:16, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

19.

2. The isolated antibody or antigen-binding fragment that specifically binds to MASP3 according to claim 1, comprising: (i)V H , comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 23; and V L , comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 33; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 34; (iii)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L , comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 34; (iv)V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L, comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 34; (v)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 35; (vi)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; or (vii) V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:

35.

3. The isolated antibody or antigen-binding fragment that specifically recognizes MASP3 according to any one of claims 1-2, wherein the antibody or antigen-binding fragment comprises an Fc fragment.

4. The isolated antibody or antigen-binding fragment that specifically recognizes MASP3 according to claim 3, wherein the antibody or antigen-binding fragment is a full-length IgG antibody.

5. The isolated antibody or antigen-binding fragment that specifically recognizes MASP3 according to claim 4, wherein the antibody or antigen-binding fragment is a full-length IgG1, IgG2, IgG3 or IgG4 antibody.

6. The isolated antibody or antigen-binding fragment that specifically recognizes MASP3 according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a chimeric, humanized, or fully human antibody.

7. The isolated antibody or antigen-binding fragment that specifically recognizes MASP3 according to any one of claims 1-2, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, diabody and linear antibody.

8. A multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2, wherein the first antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:13, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:16, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

19.

9. The multispecific antibody of claim 8, wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

47.

10. The multispecific antibody according to any one of claims 8-9, wherein: The first antigen binding domain comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9: and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

47.

11. The multispecific antibody according to any one of claims 8 to 10, wherein the first antigen binding domain comprises: (i)V H , comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 23; and VL , comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 33; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 34; (iii)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 35; (iv)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 35; or (v)V H , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 26; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:

35.

12. The multispecific antibody according to any one of claims 8 to 11, wherein the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO:48; and V L , which comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:

50.

13. The multispecific antibody according to any one of claims 8 to 12, wherein: The first antigen binding domain comprises: H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 35; And wherein the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO:48; and V L , which comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:

50.

14. A multispecific antibody comprising a first antigen binding domain that specifically binds to MASP3, and a second antigen binding domain that specifically binds to MASP2, wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44: and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:45, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:46, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

47.

15. The multispecific antibody of claim 14, wherein the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO:48; and V L , which comprises the amino acid sequence of SEQ ID NO:50 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence of SEQ ID NO:

50.

16. The multispecific antibody according to any one of claims 8 to 15, wherein the structure is selected from DVD-Ig, Bs4Ab, Hetero H, CrossMab, CrossMab2+1, IgG-(scFv)2 or scFv-Fab IgG.

17. The multispecific antibody according to any one of claims 8 to 16, comprising four polypeptide chains: One of the polypeptide chains contains V from N-terminus to C-terminus H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to MASP3, C H 1 is the heavy chain constant region C H 1 domain, wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to MASP3, C L is the light chain constant region; and A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to MASP2, C L is a light chain constant region, wherein the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and One polypeptide chain comprises VL2-CH1 from N-terminus to C-terminus, wherein VL2 is the light chain variable region that specifically binds to MASP2, and C H 1 is the heavy chain constant region C H 1 domain.

18. The multispecific antibody according to any one of claims 8 to 16, comprising four polypeptide chains: A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to MASP2; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising C H 2 and C H 3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to MASP2, C L is the light chain constant region; and A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to MASP3; C L is a light chain constant region; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L2 is the light chain variable region that specifically binds to MASP3, C H 1 is the heavy chain constant region C H 1 domain.

19. The multispecific antibody according to any one of claims 17-18, comprising: The amino acid sequence of SEQ ID NO: 60 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 60; and / or the amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 61; and / or the amino acid sequence of SEQ ID NO: 62 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 62; and / Or the amino acid sequence of SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:

63.

20. The multispecific antibody according to any one of claims 17-18, comprising: The amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 64; and / or the amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 61; and / or the amino acid sequence of SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 65; and / Or the amino acid sequence of SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:

63.

21. A nucleic acid molecule encoding the isolated MASP3-binding antibody or antigen-binding fragment of any one of claims 1-7, or the multispecific antibody of any one of claims 8-20.

22. A vector comprising the nucleic acid molecule of claim 21.

23. An isolated host cell comprising the isolated MASP3-binding antibody or antigen-binding fragment of any one of claims 1-7, the multispecific antibody of any one of claims 8-20, the nucleic acid molecule of claim 21, or the vector of claim 22.

24. A method for preparing the isolated antibody or antigen-binding fragment specifically binding to MASP3 according to any one of claims 1 to 7, or the multispecific antibody according to any one of claims 8 to 20, comprising: a) culturing the host cell of claim 23 under conditions effective to express the antibody; and b) obtaining the expressed antibody from the host cell.

25. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment specifically binding to MASP3 according to any one of claims 1 to 7, the multispecific antibody according to any one of claims 8 to 20, the nucleic acid molecule according to claim 21, the vector according to claim 22, the isolated host cell according to claim 23, or the antibody prepared by the method according to claim 24, and a pharmaceutically acceptable carrier or excipient.

26. Use of the isolated MASP3-binding antibody or antigen-binding fragment of any one of claims 1-7, the multispecific antibody of any one of claims 8-20, the nucleic acid molecule of claim 21, the vector of claim 22, the isolated host cell of claim 23, the antibody produced by the method of claim 24, or the pharmaceutical composition of claim 25 in the preparation of a medicament for treating a disease or condition in an individual in need thereof.

27. The use according to claim 26, wherein the disease or disorder is an autoimmune disease associated with complement dysregulation, a transplantation-related disease, an inflammatory disease, a blood disease, a coagulation disease, an angiogenesis-dependent disease and / or a viral infectious disease.

28. The method of claim 27, wherein the disease or condition is selected from the group consisting of ischemia-reperfusion injury, atherosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinaemic glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), Henoch-Schönlein purpura nephritis, IgA nephropathy, ischemic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), secondary TMA after transplantation, Upshaw-Schulman syndrome, arthritis, trauma The following are some of the following: acute brain injury, aspiration pneumonia, neuromyelitis optica, multiple sclerosis, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, myasthenia gravis, Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancers, age-related macular degeneration, retinopathy, proliferative diabetes, vitreous hemorrhage secondary to retinopathy, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection.

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