TW202140560A - An anti-fcrn antibody, antigen-binding fragments thereof, and pharmaceutical use thereof - Google Patents

Abstract

The present disclosure relates to an anti-FcRn antibody, antigen-binding fragments thereof, and pharmaceutical use thereof. Specifically, this application discloses an anti-FcRn antibody, its antigen-binding fragment, and its pharmaceutical use for treating autoimmune diseases and inflammatory diseases.

Description

Anti-FcRn antibody, its antigen binding fragment and its medical use

This application claims the priority of the Chinese patent application (application number 202010083892.1) filed on February 10, 2020. This application quotes the full text of the aforementioned Chinese patent application.

This application relates to anti-FcRn antibodies, antigen-binding fragments thereof, chimeric antibodies and humanized antibodies containing the CDR regions of the anti-FcRn antibodies, pharmaceutical compositions containing the anti-FcRn antibodies and antigen-binding fragments thereof, and their use in therapy or Prevents autoimmune diseases and inflammatory diseases for medical purposes.

The neonatal Fc receptor (FcRn) is a non-covalent heterodimer, usually present in the endosomes of endothelial cells and epithelial cells. It is a class I major histocompatibility complex (MHC)-like heavy chain and soluble β 2 The intracellular transport integrated membrane Fc receptor formed by the combination of microglobulin (β 2M) light chain. Specifically, FcRn has a molecular weight of about 46kD, is composed of three heavy chain α domains (α 1, α 2 and α 3) and a single β 2M domain. It has a single sugar chain, a single transmembrane, and a relatively short cell. The characteristics of the mass tail.

FcRn was initially recognized as playing an important role in the life of newborns. Further studies have found that FcRn also exerts an extension of the half-life of immunoglobulin G (IgG) and albumin in adults. Role (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996). Under normal conditions, the half-life of most human IgG (excluding the IgG3 isotype) in the serum is about 22-23 days, which is relatively long compared to the serum half-life of other plasma proteins. For this long serum half-life of IgG, the IgG that enters the cell through the endocytosis process can strongly bind to the FcRn in the endosome at a slightly acidic pH (about 6.0) to avoid the degrading lysosome pathway. When the IgG-FcRn complex circulates to the plasma membrane, IgG rapidly dissociates from FcRn at a slightly alkaline pH (approximately 7.4) in the bloodstream. Through this receptor-mediated regeneration mechanism, FcRn effectively rescues IgG from degradation in the lysosome, thereby prolonging the half-life of IgG (Roopenian et al., J. Immunol. 170: 3528, 2003).

Regarding the correlation between FcRn and IgG homeostasis, mouse experiments have proved that blocking the binding of FcRn and IgG with specific drug molecules can effectively reduce the content of IgG in the blood (Vaccaro et al., Nat Biotechnol. 23: 1283, 2005). In addition, mice were engineered to remove at least part of the genes encoding β 2M and FcRn heavy chains so that they did not express the protein. In these mice, the serum half-life and concentration of IgG were greatly reduced, which also suggested the FcRn-dependent mechanism of IgG homeostasis. The production of anti-human FcRn antibodies in these FcRn knockout mice can prevent the binding of IgG to FcRn. Inhibition of the binding of IgG to FcRn prevents the regeneration of IgG, which can treat autoimmune diseases caused by autoantibodies. This possibility is shown in a mouse model of autoimmune bullous skin disease (Li et al., J. Clin. Invest. 115: 3440, 2005). Therefore, drugs that block or antagonize the binding of IgG to FcRn can or are promising for the treatment or prevention of IgG-mediated autoimmune diseases and inflammatory diseases.

Patients with autoimmune diseases attack their normal tissues, organs or other body components due to their immune system abnormalities. Almost all parts of the body, such as the nervous system, digestive system, endocrine system, skin, skeletal system, and blood vessel tissue, may have autoimmune diseases, and most of them have not yet found a specific cause. Studies have revealed that patients with a variety of autoimmune diseases have overexpressed IgG type autoantibodies in the blood body. Autoimmune diseases caused by IgG type autoantibodies, including immune neutropenia, myasthenia gravis (MG), multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), Pemphigus vulgaris, Guillain-Barre syndrome, autoimmune encephalitis, epilepsy, lupus nephritis or membranous nephropathy or others. Intravenous immunoglobulin (IVIG) can be applied to some of the above diseases, but it is accompanied by headaches, dermatitis, allergic reactions, acute renal failure and other medical complications, which are expensive and risk exposure to blood products. In addition, multiple strains of anti-D immunoglobulins, corticosteroids, immunosuppressants (including chemotherapeutics), cytokines, plasma separation, in vitro antibody adsorption (for example, using Prosorba columns), surgical interventions such as splenectomy, etc. are also used For the treatment of autoimmune diseases, these therapies are also complicated, incomplete and expensive.

At present, many multinational pharmaceutical companies and biotechnology companies (such as Shire, UCB, Argenx) are developing monoclonal antibodies or antigen-binding proteins that target the mechanism of FcRn-IgG. Harbour BioMed has cooperated with HanAll biomedical company in South Korea. Start drug development in China. Part of the drug molecules under investigation have reached the clinical stage, and clinical studies have been carried out on the indications of different autoimmune diseases. The clinical data show that the effect of alleviating the symptoms of the disease has been achieved to varying degrees. Therefore, there is a need to develop antibodies or pharmaceutical compositions that can effectively and specifically block the binding of IgG to FcRn, which has the potential to be applied to a variety of autoimmune diseases.

Although anti-FcRn antibodies are disclosed in WO2016/142782, WO2018/083122, WO2014/019727, WO2018/229249, CN106459215B, CN104364265B, WO2009/131702, US7662928B, WO2006/118772, WO2012/167039, etc., there is still a need to develop high levels of FcRn Anti-human FcRn antibody with affinity, low immunogenicity, and even low-dose administration.

The present disclosure provides an FcRn binding protein, an anti-FcRn antibody or an antigen-binding fragment thereof, which encodes a nucleic acid, a vector, a host cell, a pharmaceutical composition, a method for treating or delaying autoimmune diseases, and a detection application thereof.

Anti-FcRn antibody or its antigen-binding fragment

The present disclosure provides anti-FcRn antibodies or antigen-binding fragments, which comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 4, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 5;

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 6, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 7;

HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 8, and LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 9;

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 10, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 11;

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 12, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 13;

HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 14, and LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 15;

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 16, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 17;

HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 18, and LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 19;

The HCDR1, HCDR2, and HCDR3 of the VH contain HCDR1, HCDR2, and HCDR3 in any one of SEQ ID Nos: 96, 99, 103, 104, and 107, respectively. The LCDR1, LCDR2, and LCDR3 of the VL respectively contain those in SEQ ID No: 13 LCDR1, LCDR2, LCDR3;

The HCDR1, HCDR2, and HCDR3 of the VH contain HCDR1, HCDR2, and HCDR3 in any one of SEQ ID Nos: 89, 93, and 94, respectively, and the LCDR1, LCDR2, and LCDR3 of the VL contain LCDR1, LCDR2, and LCDR2 in SEQ ID No: 76, respectively. LCDR3;

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 87, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 84;

The HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in any one of SEQ ID Nos: 87-110, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1 and any one of SEQ ID Nos: 76-86. LCDR2, LCDR3;

The above-mentioned CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; in some specific embodiments, the CDR is defined according to the Kabat numbering system; in other embodiments, the CDR is defined according to the AbM numbering system.

The present disclosure provides an anti-FcRn antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:

The heavy chain variable region comprises a complementarity determining region (CDR) selected from:

The amino acid sequence of _{HCDR1 is shown in GY X 1} FX ₂ (SEQ ID No: 182) or GYX ₁ FX ₂ X ₃ X ₄ X ₅ IA (SEQ ID No: 173), wherein X _{1 is} selected from S, N , D, V, A, P, K, X _{2 is} selected from T, N, K, R, S, A, X _{3 is} selected from G, N, K, S, E, X _{4 is} selected from Y, H, S , N, T, X _{5 are} selected from W, Y, F, M; and/or

The amino acid sequence of HCDR2 is shown in X ₆ IX ₇ PDX ₈ SNTI (SEQ ID No: 174) or X ₆ IX ₇ PDX ₈ SNTIYSPSFRG (SEQ ID No: 175), wherein X _{6 is} selected from I, L, V , X _{7 is} selected from Y, S, T, G, A, X _{8 is} selected from N, R, K, A, F; and/or

The amino acid sequence of HCDR3 is shown in FGGPTFAQWYFDY (SEQ ID No: 40);

The light chain variable region comprises a CDR selected from:

The amino acid sequence of LCDR1 is shown in TGSSGSIASNYVX ₉ (SEQ ID No: 176), wherein X _{9 is} selected from Q, S, N, A, V, T; and/or

The amino acid sequence of _{LCDR2 is shown in X 10} DNQRAS (SEQ ID No: 177), wherein X _{10 is} selected from E, S, A, W, C; and/or

The amino acid sequence of LCDR3 is shown in QSYDSSSHNWV (SEQ ID No: 43).

In some embodiments, the amino acid sequence of HCDR1 of the anti-FcRn antibody or antigen-binding fragment thereof is shown in SEQ ID No: 182, the amino acid sequence of HCDR2 is shown in SEQ ID No: 175, and the amino acid sequence of HCDR3 is shown in SEQ ID No: 175. The sequence is shown in SEQ ID No: 40; the amino acid sequence of LCDR1 is shown in SEQ ID No: 176, the amino acid sequence of LCDR2 is shown in SEQ ID No: 177, and the amino acid sequence of LCDR3 is shown in SEQ ID No. : Shown at 43. This embodiment adopts the Kabat numbering system.

In other embodiments, the amino acid sequence of HCDR1 of the anti-FcRn antibody or antigen-binding fragment thereof is shown in SEQ ID No: 173, the amino acid sequence of HCDR2 is shown in SEQ ID No: 174, and the amino acid sequence of HCDR3 is shown in SEQ ID No: 174. The acid sequence is shown in SEQ ID No: 40; the amino acid sequence of LCDR1 is shown in SEQ ID No: 176, the amino acid sequence of LCDR2 is shown in SEQ ID No: 177, and the amino acid sequence of LCDR3 is shown in SEQ ID. No: Shown at 43. This embodiment adopts the AbM numbering system.

In some specific embodiments, X ₁ is S, X ₂ is T, X ₃ is G, X ₄ is Y, and X ₅ is W.

In some specific embodiments, X ₁ is D, X ₂ is K, X ₃ is K, X ₄ is S, and X ₅ is F.

In some specific embodiments, X ₁ is N, X ₂ is N, X ₃ is K, X ₄ is H, and X ₅ is Y.

In some specific embodiments, X ₁ is S, X ₂ is K, X ₃ is N, X ₄ is Y, and X ₅ is Y.

In some specific embodiments, X ₁ is V, X ₂ is N, X ₃ is K, X ₄ is Y, and X ₅ is F.

In some specific embodiments, X ₁ is D, X ₂ is T, X ₃ is K, X ₄ is T, and X ₅ is M.

In some embodiments, X ₆ is I, X ₇ is Y, X ₈ is N, X ₉ is Q, and X ₁₀ is E.

In some embodiments, X ₆ is L, X ₇ is S, X ₈ is A, X ₉ is S, and X ₁₀ is S.

In some specific embodiments, X ₆ is L, X ₇ is A, X ₈ is R, X ₉ is S, and X ₁₀ is S.

In some embodiments, X ₆ is V, X ₇ is S, X ₈ is R, X ₉ is S, and X ₁₀ is S.

In some specific embodiments, X ₆ is L, X ₇ is T, X ₈ is R, X ₉ is A, and X ₁₀ is S.

In some embodiments, X ₆ is L, X ₇ is S, X ₈ is R, X ₉ is Q, and X ₁₀ is W.

In some embodiments, the amino acid sequence of HCDR1 of the VH of the anti-FcRn antibody or antigen-binding fragment thereof is as SEQ ID No: 20, 26, 32, 38, 44, 50, 56, 62, 68, 129-141, 150 -As shown in one of 165;

The amino acid sequence of HCDR2 is shown in one of SEQ ID No: 21, 27, 33, 39, 45, 51, 57, 63, 69, 121-128, 142-149, 170;

The amino acid sequence of HCDR3 is shown in one of SEQ ID No: 22, 28, 34, 40, 46, 52, 58, and 64;

The amino acid sequence of LCDR1 of VL is shown in one of SEQ ID No: 23, 29, 35, 41, 47, 53, 59, 65, 111, 115-119;

The amino acid sequence of LCDR2 is shown in one of SEQ ID No: 24, 30, 36, 42, 48, 54, 60, 66, 112, 113, 114, 116, 120;

The amino acid sequence of LCDR3 is shown in one of SEQ ID Nos: 25, 31, 37, 43, 49, 55, 61, and 67.

In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the anti-FcRn antibody or antigen-binding fragment thereof are selected from any one of (1) to (15):

(1) As shown in SEQ ID No: 20-22;

(2) As shown in SEQ ID No: 26-28;

(3) As shown in SEQ ID No: 32-34;

(4) As shown in SEQ ID No: 38-40;

(5) As shown in SEQ ID No: 44-46;

(6) As shown in SEQ ID No: 50-52;

(7) As shown in SEQ ID No: 56-58;

(8) As shown in SEQ ID No: 62-64;

(9) As shown in SEQ ID No: 68, 69, 40;

(10) HCDR1 shown in SEQ ID No: 38, HCDR2 shown in one of SEQ ID No: 121-128, HCDR3 shown in SEQ ID No: 40;

(11) HCDR1 shown in one of SEQ ID No: 129-141, HCDR2 shown in SEQ ID No: 39, HCDR3 shown in SEQ ID No: 40;

(12) HCDR1 shown in SEQ ID No: 68, HCDR2 shown in one of SEQ ID No: 142-149, HCDR3 shown in SEQ ID No: 40;

(13) HCDR1 shown in one of SEQ ID No: 150-165, HCDR2 shown in SEQ ID No: 69, HCDR3 shown in SEQ ID No: 40;

(14) As shown in SEQ ID No: 38, 170, 40;

(15) As shown in SEQ ID No: 133, 170, 40;

And the amino acid sequence of LCDR1, LCDR2, LCDR3 is selected from any one of (1')-(19'):

(1') As shown in SEQ ID No: 23-25;

(2') As shown in SEQ ID No: 29-31;

(3') As shown in SEQ ID No: 35-37;

(4’) As shown in SEQ ID No: 41-43

(5') As shown in SEQ ID No: 47-49;

(6') as shown in SEQ ID No: 53-55;

(7') As shown in SEQ ID No: 59-61;

(8') as shown in SEQ ID No: 65-67;

(9') as shown in SEQ ID No: 111, 112, 43;

(10') as shown in SEQ ID No: 111, 113, 43;

(11') as shown in SEQ ID No: 111, 114, 43;

(12') as shown in SEQ ID No: 115, 113, 43;

(13') as shown in SEQ ID No: 115, 112, 43;

(14') as shown in SEQ ID No: 115, 116, 43;

(15') as shown in SEQ ID No: 117, 112, 43;

(16') as shown in SEQ ID No: 118, 42, 43;

(17') as shown in SEQ ID No: 41, 116, 43;

(18') as shown in SEQ ID No: 119, 120, 43;

(19') As shown in SEQ ID Nos: 119, 116, and 43.

In some embodiments, the anti-FcRn antibody or antigen-binding fragment thereof comprises a CDR selected from any of the following:

(a) The amino acid sequence of HCDR1, HCDR2, HCDR3 is shown in SEQ ID No: 20-21, and the amino acid sequence of LCDR1, LCDR2, LCDR3 is shown in SEQ ID No: 23-25;

(b) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID No: 26-28, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 29-31;

(c) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID No: 32-34, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 35-37;

(d) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID No: 38-40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(e) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 44-46, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 47-49;

(f) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID Nos: 50-52, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 53-55;

(g) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID No: 56-58, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 59-61;

(h) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID Nos: 62-64, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 65-67;

(i) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 68, 69, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(j) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID Nos: 38, 170, and 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(k) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID Nos: 133, 170, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(1) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 151, 69, and 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(m) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 154, 69, and 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(n) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 158, 69, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(o) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 159, 69, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(p) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 162, 69, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No: 41-43;

(q) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 38, 123, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID Nos: 111, 112, 43;

(r) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 38, 127, and 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID Nos: 111, 112, and 43;

(s) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 38, 128, and 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID Nos: 111, 112, and 43;

(t) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 38, 124, 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID Nos: 117, 112, 43;

(u) The amino acid sequences of HCDR1, HCDR2, HCDR3 are shown in SEQ ID Nos: 38, 121, and 40, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID Nos: 41, 116, and 43.

In some embodiments, the amino acid sequence of the VH of the anti-FcRn antibody or antigen-binding fragment thereof is as SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 76-86, 166, 168, 169 One is shown or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; and/or

The amino acid sequence of VL is as shown in SEQ ID No: 5, 7, 9, 11, 13, 15, 17, 19, 76-86, 167 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity.

In some specific embodiments, the amino acid sequence of the VHVH of the anti-FcRn antibody or antigen-binding fragment thereof is as shown in SEQ ID No: 4 or has at least 80%, 90%, 95%, 96%, 97%, 98%. %, 99% identity, the amino acid sequence of VL is as shown in SEQ ID No: 5 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or

The amino acid sequence of VH is shown in SEQ ID No: 6 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 6 ID No: 7 or at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or

The amino acid sequence of VH is shown in SEQ ID No: 8 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 8 ID No: 9 or at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or

The amino acid sequence of VH is shown in SEQ ID No: 10 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 10 ID No: 11 or at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or

The amino acid sequence of VH is as shown in SEQ ID No: 12 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is as SEQ ID No: 12 ID No: 13 or at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or

The amino acid sequence of VH is shown in SEQ ID No: 14 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 14 ID No: 15 or at least 90% identical to it; or

The amino acid sequence of VH is shown in SEQ ID No: 16 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 16 ID No: 17 or at least 90% identical to it; or

The amino acid sequence of VH is shown in SEQ ID No: 18 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 18 ID No: 19 or at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or

The amino acid sequence of VH is shown in one of SEQ ID No: 96, 99, 103, 104, 107 or is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to it Sex, the amino acid of VL The sequence is shown in SEQ ID No: 13 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;

The amino acid sequence of VH is shown in one of SEQ ID No: 89, 93, 94 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, VL The amino acid sequence is shown in SEQ ID No: 76 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;

The amino acid sequence of VH is as shown in SEQ ID No: 87 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is as SEQ ID No: 87 ID No: 84 or at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;

The amino acid sequence of VH is shown in one of SEQ ID No: 87-110 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, the amino acid sequence of VL The acid sequence is shown in one of SEQ ID No: 76-86 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it.

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof, wherein:

The heavy chain amino acid sequence is shown in any one of SEQ ID No: 178-180, or has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;

The light chain amino acid sequence is shown in SEQ ID No: 181, or has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it.

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody. It can be a full-length antibody or a fragment thereof.

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof is a murine antibody or fragment thereof. The light chain variable region includes the light chain FR region and/or the light chain constant region of the murine κ, λ chain or a variant thereof. In some specific embodiments, the murine anti-FcRn antibody or antigen-binding fragment thereof comprises a heavy chain FR region and/or a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof is a chimeric antibody or fragment thereof. It comprises the light chain FR region and/or light chain constant region of a human κ, λ chain or a variant thereof, and/or a heavy chain FR region and/or a heavy chain of a human IgG1, IgG2, IgG3 or IgG4 or a variant thereof Chain constant region.

In some embodiments, there is provided an anti-human FcRn antibody or an antigen-binding fragment thereof as described above, which is a humanized antibody or a fragment thereof, a fully human antibody or a fragment thereof.

In some specific embodiments, the heavy chain variable region of the anti-FcRn antibody or antigen-binding fragment thereof can be linked to human CH1 or mouse CH1. In some embodiments, the anti-FcRn antibody or antigen-binding fragment thereof comprises a constant region Fc, which is the Fc of IgG1, IgG2, IgG3, IgG4 or a variant thereof (such as IgG4P, that is, the S241P mutant of IgG4). In some specific embodiments, Fc is the Fc of mouse IgG1 shown in SEQ ID No: 75 in amino acid sequence, or the Fc of human IgG4P shown in amino acid sequence of SEQ ID No: 172.

In some embodiments, the antigen-binding fragment of the anti-FcRn antibody is Fab, Fv, sFv, Fab', F(ab') ₂ , linear antibody, single chain antibody, scFv, sdAb, sdFv, nano antibody, peptide antibody peptibody, Domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), for example specifically scFv, Fv, Fab or Fab' fragments.

In some embodiments, the anti-FcRn antibody or antigen-binding fragment thereof is conjugated to a polymer. In some embodiments, the polymer is selected from starch, albumin, and polyethylene glycol (PEG). When the polymer is PEG, it may be PEG having a molecular weight of 5-50kDa, 5-10kDa, 5-15kDa, 10-20kDa, 10-30kDa, 20-30kDa, 10-40kDa.

In some embodiments, an anti-FcRn antibody or antigen-binding fragment thereof is provided, which binds to the same epitope as the aforementioned anti-FcRn antibody or antigen-binding fragment thereof.

In some embodiments, an anti-FcRn antibody or antigen-binding fragment thereof is provided, which blocks the binding of the aforementioned anti-FcRn antibody or antigen-binding fragment thereof to human FcRn.

In other embodiments, an anti-FcRn antibody or antigen-binding fragment thereof is provided, the binding of which to human FcRn is blocked by the aforementioned anti-FcRn antibody or antigen-binding fragment thereof.

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof binds to human FcRn (or its epitope).

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof blocks the binding of human IgG to human FcRn.

In some embodiments, the aforementioned anti-FcRn antibody or antigen-binding fragment thereof does not bind β2 microglobulin.

In some embodiments, an anti-FcRn antibody or antigen-binding fragment thereof is provided, and the heavy chain and/or light chain of the aforementioned anti-FcRn antibody or antigen-binding fragment thereof has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

In some embodiments, an anti-FcRn antibody or antigen-binding fragment variant thereof is provided, and the heavy chain variable region and/or light chain variable region of the aforementioned anti-FcRn antibody or antigen-binding fragment thereof comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes. The amino acid changes can be conservative substitutions of amino acid residues in the variable region.

FcRn binding protein

The present disclosure also provides an FcRn binding protein, comprising the anti-FcRn antibody or antigen-binding fragment thereof provided in the foregoing disclosure.

In some embodiments, the FcRn binding protein further comprises one or more effector molecules, including but not limited to: anti-tumor agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring Polymers, nucleic acids and their fragments such as DNA, RNA and Fragments, radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (such as fluorescent compounds), or compounds that can be detected by NMR or ESR spectroscopy.

In some embodiments, the effector molecule is conjugated to the anti-FcRn antibody or antigen-binding fragment thereof.

In some embodiments, the effector molecule is a polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or branched or unbranched polysaccharide, such as homopoly Or heteropolysaccharides, such as optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, lactose, amylose, dextran, glycogen Or its derivatives, human serum albumin or its fragments.

Polynucleotides and vectors

The present disclosure provides isolated polynucleotides that encode the anti-FcRn antibodies or antigen-binding fragments thereof of the present disclosure. The polynucleotide can be DNA or RNA.

The present disclosure provides an expression vector containing the polynucleotide as described above. The expression vector can be a eukaryotic expression vector, a prokaryotic expression vector, a viral vector, such as a plasmid, a cosmid, or a phage.

Host cell

The present disclosure provides a host cell transformed with the above expression vector, which may be a eukaryotic cell or a prokaryotic cell.

In some embodiments, the host cell is a bacterium, yeast, or mammalian cell. In some specific embodiments, the host cell is E. coli, Pichia pastoris, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.

Preparation

The present disclosure provides a method for preparing an anti-FcRn antibody or an antigen-binding fragment thereof, comprising: expressing the antibody, its antigen-binding fragment or complex (for example, a fusion protein) in a host cell as described above, and extracting it from the host The antibody, its antigen-binding fragment or complex (such as a fusion protein) is separated from the cell. Optionally, it can also include a purification step, for example, using a Sepharose FF column containing adjusted buffer A or G to wash out non-specifically bound components, and then use a pH gradient method to extract the bound antibody , Use SDS-PAGE to detect and collect. Optionally, it is filtered and concentrated by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

The methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technique Guide (chapters 5-8 and 15). For example, human FcRn or its fragments can be used to immunize mice, and the obtained antibody can be covered and purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. The human FR germline sequence can be obtained from the website of ImmunoGeneTics (IMGT), for example.

The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable pure strains are obtained by expressing antibodies that specifically bind to human antigens. The positive pure strains are expanded in the serum-free medium of the bioreactor to produce antibodies. The antibody-secreted culture medium can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.

Composition

The present disclosure provides a composition, such as a group pharmaceutical composition, which contains a therapeutically effective amount of the anti-FcRn antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, dilution or carrier.

In some embodiments, the unit dose of the pharmaceutical composition of the group may contain 0.01 to 99% by weight of the anti-FcRn antibody or antigen-binding fragment thereof, or the unit dose of the pharmaceutical composition of the group may contain the anti-FcRn antibody or the antigen-binding fragment thereof. The amount is 0.1-2000 mg, and in some embodiments, 1-1000 mg.

Treatment methods and pharmaceutical uses

The present disclosure provides the use of any one or a combination selected from the following in the preparation of a medicine: the anti-FcRn antibody or antigen-binding fragment thereof according to the present disclosure, and the pharmaceutical composition according to the present disclosure. In some embodiments, the drug is used to treat or prevent autoimmune diseases or delay the progression of autoimmune diseases.

The present disclosure provides a method for treating or preventing an autoimmune disease or delaying the progress of an autoimmune disease, the method comprising administering to a subject an effective amount of an anti-FcRn antibody or an antigen-binding fragment thereof according to the present disclosure, or according to the present disclosure. Group of pharmaceutical compositions.

The anti-FcRn antibodies or antigen-binding fragments thereof and pharmaceutical compositions of the present disclosure can be applied to all autoimmune diseases mediated by IgG and FcRn.

The above-mentioned autoimmune diseases include but are not limited to: acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, gammaglobulinemia, alopecia areata, amyloidosis, ANCA correlation Vasculitis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune familial autonomic nervous system Abnormality, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune Thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, Axonal&nal neuropathy, Barlow disease, Behcet disease (Behcet'sdisease), bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, American trypanosomiasis (Chagas disease), chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multiple Focal osteomyelitis (CRMO), allergic granulomatous vasculitis (Churg-Strauss syndrome), cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital Cardiac block, Coxsackie virus myocarditis, CREST disease, idiopathic mixed cryoglobulinemia, demyelinating neuropathy, herpetiform dermatitis, dermatomyositis, Devik disease (Optic neuromyelitis ), sphincter cardiomyopathy, discoid erythematosus, Dressler syndrome, endometriosis, eosinophilic vascular central fibrosis, eosinophilic fasciitis, erythema nodosa, experimental allergic encephalomyelitis, Iraq Vance syndrome, fibrotic alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, pulmonary hemorrhagic nephritis syndrome, granulomatous vasculitis (GPA) see Wegener's disease and Graves' disease, Guerlain-Barr syndrome, Hashimoto encephalitis, Hashimoto thyroiditis, hemolytic anemia, Henno-Schullen's purpura, herpes pregnancy, hypogammaglobulinemia, idiopathic hypocomplement blood tubulointerstitial nephritis (Idiopathic hypocomplementemic tubulointestitial nephritis), idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related diseases, IgG4-related sclerosing diseases, immunomodulatory lipoproteins, inflammatory aortic aneurysms, inflammatory pseudotumors, inclusion bodies Myositis, insulin-dependent diabetes mellitus (type I), interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Kuttner tumor, Lange-Years syndrome, leukocytotic vasculitis, flat coating Ringworm, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic, mediastinal fibrosis, Meniere's disease, microscopic polyangiitis, Mikulitz syndrome Syndrome, mixed connective tissue disease (MCTD), Moren ulcer, Mu-Harbin disease, multifocal fibrous sclerosis, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic’s ), neutropenia, ocular cicatricial pemphigoid, optic neuritis, Ormond's disease (retroperitoneal fibrosis), recurrent rheumatism, PANDAS (associated with streptococcus but autoimmune neuropsychiatric Disorder), paraneoplastic cerebellar degeneration, paraproteinemic polyneuropathy (Paraproteinemic polyneuropathy), paroxysmal nocturnal hemoglobinuria (PNH), Parrot syndrome, Parsonnage-Turner syndrome, ciliary planitis (peripheral uveitis), pemphigus vulgaris, peri-aortic inflammation, periarteritis, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, nodular Polyarteritis, type I, II and III autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary Biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, simple red blood cell aplasia, Raynaud’s phenomenon, reflex sympathetic nerves Nutritional disorders, Reiter's syndrome, recurrent polychondritis, restless legs syndrome, retroperitoneal fibrosis (Ormond disease), rheumatic fever, rheumatoid arthritis, Reed's otitis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiffness syndrome, subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia, high safety Arteritis, temporal arteritis/giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Throw-Hen's syndrome, transverse myelitis, ulcerative noditis, undifferentiated connective tissue disease (UCTD), Uveitis, vasculitis, vesicular bullous skin disease, vitiligo, Waldenstrom macroglobulinemia, warm idiopathic hemolytic anemia, and Wegener's granulomatosis (now called granulomatous vasculitis (GPA)) .

Detect

The present disclosure provides a composition for detecting FcRn, the composition comprising the anti-FcRn antibody or antigen-binding fragment thereof according to the present disclosure. The present disclosure also provides a method, system, or device for detecting FcRn in vivo or in vitro, which includes treating a sample with an anti-FcRn antibody or an antigen-binding fragment thereof of the present disclosure.

In some embodiments, the in vitro detection method, system or device may include, for example:

(1) Contact the sample with an antibody or antigen-binding fragment that binds to FcRn;

(2) Detect the complex formed between the FcRn-binding antibody or its antigen-binding fragment and the sample; and/or

(3) Contacting a reference sample (e.g., control sample) with the antibody; and

(4) Determine the degree of complex formation by comparing with the reference sample. As compared to a control sample or subject, a change in complex formation (e.g., a statistically significant change) in the sample or subject indicates the presence of FcRn in the sample.

In other embodiments, the in vivo detection method, system or device may include:

(1) Administering an antibody or antigen-binding fragment thereof that binds to FcRn to a subject; and

(2) Detect the formation of a complex between the FcRn-binding antibody or antigen-binding fragment thereof and the subject.

Detection can include determining the location or time of formation of the complex. The FcRn antibody is labeled with a detectable substance, and detection of the FcRn antibody binding substance (for example, FcRn) is achieved by detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. The formation of a complex between an antibody that binds to FcRn or an antigen-binding fragment thereof and FcRn can be detected by measuring or visualizing the antibody that binds or does not bind to FcRn. Conventional detection assays can be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. For detection purposes, the anti-FcRn antibodies or fragments thereof of the present disclosure can be labeled with a fluorophore chromophore.

In some embodiments, a kit is also provided. The kit includes an anti-FcRn antibody or an antigen-binding fragment thereof, and may also include diagnostic instructions. The kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent. For in vivo use, the antibody can be formulated as a pharmaceutical composition.

Figure 1 shows the ELISA detection of FcRn antibody binding to human FcRn protein.

Figure 2A shows the binding test of FcRn antibody to cells overexpressing human FcRn under pH 6.0 environment.

Figure 2B shows the binding test of FcRn antibody to cells overexpressing human FcRn under pH 7.4 environment.

Figure 3 shows the detection of FcRn antibody blocking the binding of hIgG to cells overexpressing human FcRn.

Fig. 4 is a blocking test for detecting that FcRn antibody blocks the binding of cells overexpressing human FcRn to HSA.

Figure 5A shows intravenous administration of FcRn antibody (30mpk) in FcRn transgenic mice, and the percentage of IVIG content detected at different time points.

Figure 5B shows the area under the curve (AUC) of intravenously administered FcRn antibody (30 mpk) in FcRn transgenic mice.

the term

To make it easier to understand the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs.

The three-letter codes and one-letter codes of amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

"Human FcRn" refers to the complex between human IgG receptor α chain and β 2 microglobulin (β 2M), also known as neonatal Fc receptor. Its amino acid sequence can be found in, for example, UniProt No. P55899, β 2M The amino acid sequence is found in, for example, UniProt No. P61769. In the context of this application, "human FcRn" also encompasses any natural variant or recombinant product.

"Antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, multi-strain antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments (or antigens). Binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, Alpha chain and epsilon chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of its hinge area and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is classified into a kappa chain or a lambda chain by the difference in the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain. The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (CDR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The sequence from the amino end to the carboxyl end is FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.

For the determination or definition of the CDR, the deterministic description of the CDR and the identification of the residues containing the binding site of the antibody can be completed by distinguishing the structure of the antibody and/or distinguishing the structure of the antibody-ligand complex. This can be done by any of the various techniques known to those skilled in the art, such as X-ray crystals Physical learning to achieve. A variety of analysis methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformational definition.

The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses a computer program integration suite produced by Oxford Molecular Group for modeling antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies, "Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" described). The contact definition is based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of CDRs can be identified as residues that contribute enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a part of Kabat CDR, although they can be shortened or lengthened according to specific residues or residue groups that do not significantly affect the prediction of antigen binding or experimental results. . As used in the present disclosure, CDR may refer to a CDR defined by any method known in the art (including a combination of methods).

The number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment of the present disclosure conform to the known Kabat or AbM numbering system.

A "monoclonal antibody" or "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may be present in small amounts, each antibody contained in the population is the same. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to multi-strain antibody preparations which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristics of an antibody as obtained from a substantially homogeneous antibody population, and is not construed as requiring the production of the antibody by any specific method. For example, the monoclonal antibody used in accordance with the present disclosure can be prepared by the fusionoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by, for example, the recombinant DNA method in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from the generated phage library using the technique described in McCafferty et al., 1990, Nature 348:552-554.

"Fully human antibodies" or "recombinant fully human antibodies" include fully human antibodies prepared, expressed, created or isolated by recombinant methods. The techniques and methods involved are well known in the art, such as:

(1) Antibodies isolated from transgenic human immunoglobulin genes, transchromosomal animals (such as mice) or fusion tumors prepared therefrom;

(2) Antibodies isolated from host cells transformed to express antibodies, such as transfectomas;

(3) Antibodies isolated from the recombinant combinatorial fully human antibody library; and

(4) Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.

Such recombinant fully human antibodies contain variable and constant regions. These regions utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent strains such as rearrangements and mutations that occur during antibody maturation. .

The term "murine antibody" in the present disclosure refers to a monoclonal antibody against human FcRn or its epitope prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with FcRn antigen, and then the fusion tumor expressing the antibody with the desired sequence or functional properties is isolated. In a specific embodiment of this disclosure Wherein, the murine anti-human FcRn antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1, IgG2, IgG3 or IgG4 or variants thereof The constant region of the heavy chain.

The term "fully human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies") .

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences into the framework of a human antibody variable region. It can overcome the strong immune response induced by the chimeric antibody because it carries a large amount of non-human protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the fully human antibody can be subjected to minimal reverse mutations to maintain activity.

The term "chimeric antibody" is an antibody formed by fusing the variable region of an antibody of the first species with the constant region of an antibody of the second species, which can alleviate the immune response induced by the antibody of the first species. As an example, to establish a chimeric antibody, it is necessary to select a fusion tumor that secretes a murine specific monoclonal antibody, and then select the variable region gene from the mouse fusion tumor cell, and then select the constant region of the fully human antibody as needed Gene, the mouse variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a fully human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or without ADCC after using amino acid mutation (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.

"Antigen-binding fragments" include: single-chain antibodies (ie, full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv , Single domain antibodies (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods of producing and preparing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009/040562, and the disulfide bond stabilized Fab-dsFv format was first disclosed in WO2010/035012. The antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171. Multivalent antibodies may comprise multispecificity such as bispecificity or may be monospecific (see for example WO92/22583 and WO05/113605), an example of the latter is the Tri-Fab (or TFM) described in WO92/22583 .

The term "binding to FcRn" in the present disclosure refers to the ability to interact with FcRn or its epitope, and the FcRn or its epitope may be of human origin. The term "antigen-binding site" in the present disclosure refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.

"Antigen" refers to a molecule used to immunize immunocompetent vertebrates to produce antibodies that recognize the antigen, or to screen expression libraries (e.g., phage, yeast, or ribosome display libraries, among others). In the present disclosure, antigens are defined more broadly and include target molecules specifically recognized by antibodies, and include parts or mimetics of molecules used in the immunization process for antibody production or library screening for antibody selection. For the antibodies of the present disclosure that bind to human FcRn, monomers and multimers (such as dimers, trimers, etc.) of human FcRn, as well as truncated variants and other variants of human FcRn are all referred to as antigens.

The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost after treatment with denaturing solvents. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. The method for determining the spatial conformation of an epitope includes the technology in the field and the technology disclosed in the present disclosure, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.

"Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human FcRn or its epitope is used as an analyte and an antibody is used as a ligand, when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody is approximately lower than 10 ^-7 M or even smaller The equilibrium dissociation constant (K _D ) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is non-specific other than the predetermined antigen (or its epitope) or closely related antigens Sex antigens (such as BSA, etc.) bind at least twice the affinity. The term "antibody that recognizes an antigen" can be used interchangeably with the term "antibody that specifically binds" in this disclosure.

"Binding affinity" or "affinity" is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or part thereof and an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K _{D is} related to ka and kd by the equation K _{D =kd/ka.} The value of the dissociation constant can be determined directly by well-known methods, and can be calculated by methods such as those in Caceci et al. (1984, Byte 9:340-362) even for complex mixtures. For example, a double filter nitrocellulose filter binding assay such as Wong & Lohman: that disclosed in (1993, Proc.Natl.Acad.Sci.USA 90 5428-5432) to determine the K _D. Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of antibodies can also be determined by standards known in the art, such as surface plasmon resonance (SPR), for example, by using the Biacore ^™ system or KinExA. _{The K D} value of each antibody/antigen complex can be compared to compare the binding affinities associated with different molecular interactions, for example, the comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of the interaction can be determined and compared by determining and comparing the K _D value of the target interaction (for example, the specific interaction between the antibody and the antigen) and the non-target interaction (for example, a control antibody that is not known to bind FcRn) The K _D value is evaluated.

"Conservative substitution" refers to substitution with another amino acid residue that has similar characteristics to the original amino acid residue. For example, lysine, arginine, and histidine have similar characteristics in that they have basic side chains, and aspartic acid and glutamic acid have similar characteristics in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar characteristics in that they have uncharged polar side chains And alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar characteristics in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan and histidine have similar characteristics in that they have aromatic side chains. Therefore, it will be obvious to those skilled in the art that even when an amino acid residue in the group showing similar properties as described above is replaced, it will not show a specific change in properties.

"Cross-reactivity" refers to the ability of the antibodies of the present disclosure to bind to FcRn from different species. For example, an antibody of the present disclosure that binds to human FcRn can also bind to FcRn of another species. cross Reactivity is measured by detecting specific reactivity with purified antigens in binding assays (such as SPR and ELISA), or by binding or functional interaction with cells that physiologically express FcRn. Methods of determining cross-reactivity include standard binding assays as described in this disclosure, such as surface plasmon resonance analysis, or flow cytometry.

"Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of FcRn preferably reduces or changes the normal level or type of activity that occurs when FcRn binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in binding affinity for FcRn when contacted with an anti-FcRn antibody compared to FcRn not contacted with an anti-FcRn antibody.

"Inhibition of growth" (e.g., referring to cells) is intended to include any measurable decrease in cell growth.

The methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technique Guide (chapters 5-8 and 15). For example, human FcRn or its fragments can be used to immunize mice, and the obtained antibody can be covered and purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. The human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website.

The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect cells. Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable pure strains are obtained by expressing antibodies that specifically bind to human antigens. The positive pure strains are expanded in the serum-free medium of the bioreactor to grow Produce antibodies. The antibody-secreted culture medium can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

Conventional techniques known to those skilled in the art can be used to screen antibodies competitively for binding to the same epitope. For example, competition and cross-competition studies can be conducted to obtain antibodies that compete with each other or cross-compete with antigen binding. A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in International Patent Publication WO03/48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on FcRn.

"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes the contact of the reagent with the cell, and the contact of the reagent with the fluid, where the fluid is in contact with the cell. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.

"Treatment" means the administration of an internal or external therapeutic agent to a subject, such as a composition comprising any one of the antibodies or antigen-binding fragments or conjugates thereof of the present disclosure, the subject has, is suspected of having, Prone to suffer from one or more diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population to be treated, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinical condition. The degree of measurement on the right side of the bed. The amount of therapeutic agent (also referred to as "therapeutically effective amount") that is effective to alleviate the symptoms of any particular disease can vary according to various factors, such as the subject's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the subject . By any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. As far as the embodiments of the present disclosure (such as treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a certain subject, according to any statistical test methods known in the art such as Studentt test, Chi-square test, according to Mann And Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject can vary depending on factors such as the condition to be treated, the general health of the subject, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.

"Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same nucleotide or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by the same nucleotide, then the molecule is at that position. Are homologous. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, in the optimal sequence alignment, if 6 of the 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.

"Cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell.

"Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.

The “FcRn-binding protein” of the present disclosure is explained to the maximum extent, including the anti-FcRn antibody or the antigen-binding fragment thereof of the present disclosure, as long as it can achieve binding to FcRn, it is within the scope of this term. For example, the FcRn binding protein may contain one or more effector molecules, for example in a conjugation manner. The "effector molecule" includes, for example, anti-tumor agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments such as DNA, RNA and fragments thereof, Radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (such as fluorescent compounds), or compounds that can be detected by NMR or ESR spectroscopy. When the effector molecule is a polymer, it can usually be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkylene or polyoxyalkylene polymer or branched Polysaccharides or unbranched polysaccharides, such as homo- or hetero-polysaccharides. Specific optional substituents that may be present on the aforementioned synthetic polymers include one or more hydroxy, methyl or methoxy groups. Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene two Alcohol) such as methoxy poly(ethylene glycol) or its derivatives. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof. In one embodiment, the polymer is albumin or a fragment thereof, such as human serum Albumin or fragments thereof. The method of conjugating the polymer to the anti-FcRn antibody or antigen-binding fragment thereof of the present disclosure can be achieved by conventional methods.

Example

The following examples are used for further description, but these examples are not limited in scope.

The experimental methods that do not indicate specific conditions in the examples or test examples usually follow the conventional conditions or the conditions recommended by the raw material or commodity manufacturers. See Sambrook et al., Molecular Selection, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. The reagents without specific sources are the conventional reagents purchased on the market.

Example 1. Preparation of human FcRn antigen and detection protein

1. Protein design and expression

Human FcRn (Uniprot accession number: P55899) extracellular domain-β 2M (Uniprot accession number: P61769) complex was used as a template for FcRn to design the amino acid sequence of the FcRn antigen and detection protein (the following FcRn antigen is not specified All refer to human FcRn-β 2M complex).

>Human FcRn extracellular region:

(SEQ ID No：1)；

(SEQ ID No: 1);

> People with His tag β 2M:

(SEQ ID No：2)；

(SEQ ID No: 2);

(Note: The italicized part is His label).

Full-length human FcRn will undergo endocytosis on the cell membrane surface, and normal cell binding and blocking experiments cannot be performed. Therefore, the extracellular region and transmembrane region of human FcRn mutant human FcRn _mut (L320A, L321A) were prepared and co-transfected with human β 2M. This mutant can better maintain the FcRn-β 2M complex on the cell membrane surface. Positioning for the construction of stable cell lines for detection.

>Human FcRn _mut (L320A, L321A):

(SEQ ID No：3)

(SEQ ID No: 3)

(Note: The italicized part is the transmembrane region, and the underlined part is the intracellular region).

2. Purification of FcRn recombinant protein

Purification of FcRn recombinant protein with His tag. Equilibrate the Ni-Sepharose affinity column with 20 mM phosphate buffer, pH 8.0 for 5 column volumes. Centrifuge the sample at high speed to remove impurities and combine it on the column. Wash the column with 20mM phosphate buffer until the A280 reading drops to the baseline. Use 20mM phosphate buffer, 0-500mM imidazole gradient to extract, collect protein and identify the target protein. The sample obtained in the first step of purification was changed to PBS and concentrated to a volume of 2 mL. The gel chromatography Superdex200 (GE) equilibrated with PBS was used for further purification, and the target peak fraction was collected for equipment use. After testing, a purified FcRn recombinant protein was obtained.

Example 2. Screening of anti-human FcRn murine monoclonal antibodies

The experimental steps are as follows:

1) Mouse immunization and serum titer detection

Take 4 SJL white mice and 5 Balb/c white mice, mix with 25-50μg antigen and adjuvant for immunization. The time is the 0th, 14th, and 35th days. Day 0 intraperitoneally (IP) after injection of 50 μ g / mouse emulsified antigen. Injection on Day 14, 35 25 μ g / only. The blood was taken on the 21st and 42nd day, and the antibody titer in the mouse serum was determined by ELISA method. After the 4-5th immunization, mice with high antibody titer in the serum and the titer tending to the plateau were selected for spleen cell fusion. Boost immunization performed 3 days before, intraperitoneal (IP) injection of 50 μ g / normal saline antigen solution only spleen cell fusion.

2) Spleen cell fusion

The splenic lymphocytes and the myeloma cells Sp2/0-Ag14 cells were fused using a PEG-mediated fusion step to obtain fusion tumor cells. Good hybridoma fusion cells were incubated in complete medium and supplemented with HAT 100 μ L / number of cells per well bore to 1 × 10 ⁴ -1 × 10 ⁵ were seeded in 96-well _{plates, 37 ℃, 5% CO 2} , 10-14 days Afterwards, ELISA was performed.

3) Screening of fusion tumor cells

According to the growth density of fusion tumor cells, the fusion tumor culture supernatant was detected by the combined ELISA method. The cell supernatants of positive wells detected by ELISA were purified, cell binding experiments and cell blocking experiments were carried out. Cells in wells that are positive for binding and blocking are expanded, cryopreserved, and sequenced in time.

The amino acid sequence of the antibody variable region corresponding to the selected positive pure strain hybri11 is as follows:

>hybri11-VH amino acid sequence:

(SEQ ID No：4)；

(SEQ ID No: 4);

>hybri11-VL amino acid sequence:

(SEQ ID No：5)。

(SEQ ID No: 5).

4) Construction and screening of mouse immune library

The spleen and lymph node cells of immunized mice were resuspended with Trizol reagent (1×10 ⁷ cells/mL Trizol) to lyse the cells, and placed on ice for 5 min; centrifuged at 13000 rpm for 3 min, take the supernatant, and discard the precipitate; add 1/5 of the volume Shake vigorously for 30-60s, and let stand for 2min in an ice bath; centrifuge at 13000rpm for 10min, and transfer the upper aqueous layer to a new 1.5mL tube; add an equal volume of isopropanol, mix well, and let stand at -20°C for 30min; 13000rpm centrifugal 10min, supernatant was removed, the precipitate retained; washed with 75% ethanol prechilled precipitate room temperature for 5-10min; deionized water was added to remove RNA enzyme 600 μ L, reconstitution, obtained RNA, cDNA was obtained by reverse transcription , The construction of single-chain antibody phage library.

By screening the mouse immune single-chain antibody phage library, antibodies with high affinity to FcRn were obtained. With 10 μ g of random biotinylated FcRn binding 1mg Dynabeads MyOne Streptavidin biotin T1, at room temperature for 1 hour. Wash with PBST (0.05% Tween-20) 3 times, dissolve human albumin (Sigma, 126658) in 1×PBS (pH 6.0) with a final concentration of 1 mg/mL as a blocking agent, add to the system, and block at room temperature for 1 hour. Add the fully human single-chain antibody phage display library blocked with 2% milk for 1 hour at room temperature, and act for 1 hour at room temperature. Wash with PBST (0.05% Tween-20), pH 7.4 solution 8 times to remove unbound phage, albumin and phage bound to it. First with 100 μ g mL competitive binding FcRn and IgG antigen / protein was eluted 0.5mL, then 1mg / mL trypsin 0.5mL of specific binding to FcRn remainder was eluted phage were infected at log phase growth E. coli TG1, produced and purified phage for the next round of screening. After the same screening process was repeated for 2-3 rounds, the positive pure strains were enriched.

380 (4×96-well plates) single-strain colonies were picked from the pure strains that were screened and enriched, packaged into single-chain antibody phage, and used for phage ELISA test. ELISA plates were coated with 2μg/mL FcRn protein, placed overnight at 4 degrees, washed 3 times with PBST (0.05% Tween-20), blocked with 2% skimmed milk at room temperature for 1 hour, PBST (0.05% Tween-20) ) After washing 3 times, add the phage supernatant diluted in blocking solution, react for 1 hour at room temperature, wash 6 times with PBST (0.05% Tween-20), add anti-M13 HRP (Yiqiao Shenzhou, 11973-MM05T-H), Warm the reaction for 1 hour, wash with PBST (0.05% Tween-20) 3 times, add 100μL TMB to develop the color substrate, and stop the reaction with 100μL 1M sulfuric acid. Use SpectraMax M5 microplate reader to read the absorbance value at 450nm for detection. The pure strains with an OD450 value greater than 0.5 in the ELISA binding test were sequenced, and 58 specific sequences were obtained.

Example 3. Screening of a phage library of fully human single-chain antibodies for positive antibodies that specifically bind to human FcRn

Antibodies with high affinity to FcRn were obtained by screening a phage library of fully human single-chain antibodies.

With 10 μ g of random biotinylated FcRn binding 1mg Dynabeads MyOne Streptavidin biotin T1, at room temperature for 1 hour. Wash with PBST (0.05% Tween-20) 3 times, dissolve human albumin (Sigma, 126658) in 1×PBS (pH 6.0) with a final concentration of 1 mg/mL as a blocking agent, add to the system, and block at room temperature for 1 hour. Add the fully human single-chain antibody phage display library blocked with 2% milk for 1 hour at room temperature, and act for 1 hour at room temperature. Wash with PBST (0.05% Tween-20), pH 7.4 solution 8 times to remove unbound phage, albumin and phage bound to it. First with 100 μ g / mL and 0.5mL IgG competition binding FcRn protein antigen was eluted, and then 0.5mL of Trypsin 1mg / mL of the remaining specifically bound to FcRn was eluted phage were infected at log phase growth of Escherichia coli TG1, produced and purified phage for the next round of screening. After the same screening process was repeated for 2-3 rounds, the positive pure strains were enriched.

Pick 2185 (33×96) colonies of single-chain antibody phage from the pure strains that have been screened and enriched for phage ELISA test. ELISA plates were coated with 2μg/mL FcRn protein, placed overnight at 4°C, washed 3 times with PBST (0.05% Tween-20), blocked with 2% skimmed milk at room temperature for 1 hour, PBST (0.05% Tween-20) ) After washing 3 times, add the phage supernatant diluted in blocking solution, react for 1 hour at room temperature, wash 6 times with PBST (0.05% Tween-20), add anti-M13 HRP (Yiqiao Shenzhou, 11973-MM05T-H), Warm the reaction for 1 hour, wash with PBST (0.05% Tween-20) 3 times, add 100μL TMB to develop the color substrate, and stop the reaction with 100μL 1M sulfuric acid, use SpectraMax M5 microplate reader reads the absorbance at 450nm for detection. The pure strains with an OD450 value greater than 0.5 in the ELISA binding test were sequenced, and 43 specific sequences were obtained.

Example 4. Construction of a complete anti-human FcRn monoclonal antibody

25 specific sequences were selected from the 58 specific sequences obtained from the screening of the mouse immune phage library in Example 2, and 39 specific sequences were selected from the 43 specific sequences obtained from the screening of the human phage library in Example 3 to construct a complete recombinant antibody.

The method of purifying the recombinant antibody is: collecting the expression supernatant, high-speed centrifugation to remove impurities, and filtering with a 0.45μm PVDF filter membrane, using Protein A or Protein G column for affinity chromatography. Use 1×PBS (pH 7.4) buffer system as the equilibration buffer to equilibrate the chromatography column for 3-5 times the column volume; the cell supernatant is loaded with a low flow rate and combined, and the flow rate is controlled so that the retention time is about 1 min or longer; Wash the chromatography column with 1×PBS (pH 7.4) 3-5 times the column volume until the UV absorption falls back to the baseline; use 0.1M glycine (pH 3.0) buffer for sample extraction, and collect and extract based on UV detection Peak, the extracted product uses 1M Tris-HCl (pH 9.0) to quickly adjust the pH to 5-6 for temporary storage. The extracted sample is properly diluted to a conductivity of <2 mS/cm, combined with an anion column, washed with 20mM Tris-HCl pH 8.0-9.0 to remove impurities, 20mM Tris-HCl, NaCl 0-1M gradient extraction. Collect the peaks and transfer them to the PBS sub-equipment.

For the obtained antibodies, by ELISA binding experiment, ForteBio protein interaction experiment and cell-level competition experiment with IgG, it was determined that 7 of them had strong binding ability and could inhibit the interaction between FcRn and IgG.

The complete variable region sequence is shown in Table 1:

The CDR numbering system of the anti-human FcRn antibody screened in the present disclosure is the Kabat numbering system or the AbM numbering system. The CDR regions of the Kabat numbering system are underlined, and the CDR regions of the AbM numbering system are italicized. In the above sequence, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underlined or italicized parts in the sequence are the CDR1, CDR2, and CDR3 sequences, respectively.

The heavy chain and light chain CDR sequences of each antibody are summarized in Table 2.

In Table 2, there are two numbering systems for h22. When HCDR1-3 is SEQ ID No: 38-40 and LCDR1-3 is SEQ ID No: 41-43, it is the Kabat numbering system; when HCDR1-3 is SEQ ID No: 68, 69, 40, when LCDR1-3 is SEQ ID No: 41-43, it is the AbM numbering system. The numbering system for other antibodies is Kabat.

The above heavy chain variable regions are fused with the corresponding human CH1 (SEQ ID No: 70) or mouse antibody heavy chain CH1 (SEQ ID No: 71) and mouse IgG1 Fc (SEQ ID No: 75). The chain variable region is fused with the human kappa (SEQ ID No: 72) or lamda constant region CL1 (SEQ ID No: 73) or CL2 (SEQ ID No: 74) to form a recombinant antibody, which is then subjected to subsequent detection.

>Fully human antibody heavy chain CH1:

(SEQ ID No：70)；

(SEQ ID No: 70);

>Mouse antibody heavy chain CH1:

(SEQ ID No：71)；

(SEQ ID No: 71);

>Fully human antibody light chain C κ:

(SEQ ID No：72)；

(SEQ ID No: 72);

>Full human antibody light chain CL1:

(SEQ ID No：73)；

(SEQ ID No: 73);

>Full human antibody light chain CL2:

(SEQ ID No：74)；

(SEQ ID No: 74);

>Mouse IgG1 Fc:

(SEQ ID No：75)。

(SEQ ID No: 75).

Example 5. Genetic modification of anti-human FcRn monoclonal antibody

1. Affinity maturation of anti-FcRn monoclonal antibody h22

The antibody molecule is subjected to a three-dimensional simulation structure and a known antigen structure (PDB ID: 4N0U Chain A&B) for combination simulation. With reference to human germline gene mutation hot spots, three-dimensional structure and combined simulation results, some key amino acid residues in the framework and CDR regions were selected, and several random mutant phage libraries were established. Using phage library display technology, functional antibodies with improved affinity were screened out. Combining and verifying new amino acid residues obtained from different libraries, obtaining functional antibodies with improved affinity and function. The obtained antibody molecule light chain variable region and heavy chain variable region sequences are shown in Table 3 and Table 4, respectively. The CDR combinations are shown in Table 5, Table 6, and Table 7, respectively.

The CDR numbering system of the anti-FcRn antibodies screened in Table 3 and Table 4 is Kabat numbering system (underlined) or AbM numbering system (italics). In the above sequence, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underline or italics in the sequence are the CDR1, CDR2, and CDR3 sequences, respectively.

The CDR numbering system in Table 5 is the AbM or Kabat numbering system, the CDR numbering system in Table 6 is the Kabat numbering system, and the CDR numbering system in Table 7 is the AbM numbering system.

2. Humanized transformation of anti-human FcRn monoclonal antibody (Germlining)

By modeling the three-dimensional structure homology of the selected fully human specific antibody molecule, combining the results of the comparison with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, select The heavy chain and light chain variable region germline genes with high homology to the screened antibody are used as templates, and the FR regions and CDR regions of the original monoclonal antibody are modified to maintain the function while making the sequence The columns are closer to human germline genes. The three-dimensional structure of the transplanted single-domain antibody was simulated and analyzed again, and the specific sites in the FR region that affected the structure and morphology of the CDR region were back-mutated. The amino acid residues are identified and annotated by the Kabat numbering system. The modified antibody has higher stability and lower immunogenicity.

The selection of germline gene architecture taking the anti-human FcRn monoclonal antibody h22 as an example:

After analysis, the heavy chain template of antibody h22 is selected from the human germline gene IGHV5-51*01 in the IMGT database, and the light chain template is selected from the human germline gene IGLV6-57*02 in the IMGT database.

The antibody h22 was genetically modified, and its variable region sequence is shown in Table 8.

The CDR numbering system in Table 8 is the Kabat numbering system (shown underlined) or the AbM numbering system (shown in italics). In the above sequence, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and underlined or italicized are the CDR1, CDR2, and CDR3 sequences, respectively.

After analysis, after Germlining, some of the CDRs of the antibodies have changed, as shown in Table 9.

The CDR numbering system in Table 9 is the Kabat numbering system.

Example 6. Preparation of anti-human FcRn antibody

The screened antibody heavy chain variable region was fused with human CH1 and IgG4 (S241P) Fc and cloned into a mammalian cell expression vector. The variable region of the antibody light chain is cloned into a mammalian cell expression vector containing the lamda or kappa constant region of the human antibody light chain. IgG4 (S241P) is abbreviated as IgG4P.

>Human CH1 sequence:

(SEQ ID No：171)；

(SEQ ID No: 171);

>Human IgG4 (S241P) sequence:

(SEQ ID No：172)。

(SEQ ID No: 172).

Transfection of the vector into HEK293E cells or expiCHO cells is as follows:

Transfection into HEK293E cells:

According to 1 μ g DNA / ml in the transfected cells by liposome ratio 293FectinReagent (Invitrogen, Cat.No.12347019) transfection performed according to reagent instructions. After transfection, the cell density is 1.0X10 ⁶ cells/ml, placed in a 37 degree shaker and cultured with shaking (5% CO ₂ ). On the 6th day, the cell culture solution is collected, centrifuged at 4000 rpm, and the supernatant is taken and filtered with a 0.45 μM filter. .

Transfection into expiCHO cells, method:

According to 1 μ g DNA / ml using the ratio of transfected cells ExpiCHO Expression System (Cat.no.A29133) transfection performed according to reagent instructions. Using standard methods, the cell density after transfection is 6.0X10 ⁶ cells/ml, placed in a 37 degree shaker and cultured with shaking (8% CO ₂ ), the cell culture medium is collected on the 8th day, centrifuged at 4000 rpm, the supernatant is taken and 0.45 μM is used The filter membrane is filtered.

After testing, the target antibody is obtained.

Example 7. Affinity detection of anti-human FcRn antibody and antigen

1. ELISA experiment of anti-FcRn antibody binding to FcRn

In order to test the in vitro binding ability of the selected FcRn antibodies to human FcRn protein, a random biotinylated human FcRn-β 2M complex (biotin-hFcRn) was used for in vitro binding detection. The negative control of this test case is PBS, and the positive control uses HEK293 transiently transfected and expressed Argx-113. The sequence of Argx-113 of the positive control is from sequence 2 in WO2015/100299A1.

PBS buffer, pH 6.0 to NeutrAvidin Protein (Thermo Scientific, Lot: 31000) was diluted to 2 μ g / mL, in a volume of 100 μ L / well added to 96-well microtiter plate (Corning, 901825) in at 4 ℃ Let stand overnight for 16-20 hours. After discarding the liquid, with PBST (pH 6.0,0.05% Tween-20 ) After the plate was washed three times with buffer, was added 100 μ L / well was diluted with PBS buffer to 3 μ g / mL biotin or biotin -hFcRn - cynoFcRn, incubate for 1 hour in a 37°C incubator. After the liquid was discarded, the plate was washed three times with the PBST buffer diluted in PBS buffer with 4% BSA blocking solution (300 μ L / hole), 37 [deg.] C incubator for 1 hour for blocking. After blocking, the blocking solution was discarded, and the plates were washed with PBST buffer three times, added to an initial concentration of antibody protein FcRn 40 μ g / mL, the dilution with a gradient of 7 PBS buffer solution three times, incubated at 37 ℃ Incubate in the box for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed 6 times with PBST, and 100 μL of HRP-labeled anti-mouse Fc secondary antibody (Abcam ab197780, 1:5000 dilution) was added to each well, and incubated at 37°C for 1 hour. After washing the plate 6 times with PBST, add 100μL TMB to develop color substrate, incubate at room temperature for 3-5min, add 50μL 1M sulfuric acid to stop the reaction, read the absorbance value at 450nm with SpectraMax M5 microplate reader, and calculate the binding of antibody to antigen EC ₅₀ value. _{The EC 50} results of some antibodies are shown in Figure 1 and Table 10.

2. Binding experiment of anti-human FcRn antibody and FcRn overexpression HEK293 cells

The ability of FcRn antibody to bind to cell surface FcRn was tested by measuring the binding of the antibody to hFcRn _{on the surface of HEK293 cells stably overexpressing hFcRn mutants (human FcRn mut} (L320A, L321A)). After the HEK293 cells stably overexpressing human FcRn _mut (L320A, L321A) were fixed on the bottom of a 96-well plate, the strength of the signal after the addition of the antibody and the corresponding secondary antibody was used to determine the binding activity of the antibody and hFcRn overexpressing HEK293 cells. The specific experimental methods are as follows:

After the HEK293 cells stably transfected with human FcRn _mut (L320A, L321A) were washed twice with PBS, 5×10 ⁵ cells were added to each well of a U-shaped well 96-well plate. After centrifugation and the supernatant, each well was added 100 μ L was diluted with the sample diluent FcRn antibody (sample dilution is added 1% BSA in PBS), incubated on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added 100 μ L samples were diluted to the appropriate dilution of antibody Fc secondary antibody (mouse Fc: Thermo Fisher, Cat No.A16085,1: 500; human Fc: Jackson Immuno Research, Cat No. 109-095-008, 1:200), incubate on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added 200 μ L sample dilution, and the measured value FL1 channel fluorescence with a flow cytometer (BD Biosciences, BD Accuri C6) , using Flowjo software and data For analysis, use Prism6 software to plot the analysis results. The negative control is PBS, and the positive control uses HEK293 transiently transfected and expressed Argx-113. Part of the antibody test results are shown in Figure 2A, Figure 2B and Table 11 below.

The results show that the antibodies of the present disclosure have good binding activity to FcRn at the cell level under neutral conditions (pH 7.4, Figure 2B) and acidic conditions (pH 6.0, Figure 2A), and their affinity is better than that of the positive control Argx- 113.

3. Biacore determination

Biacore, GE instrument was used to determine the affinity of the humanized anti-FcRn antibody to be tested with human FcRn.

Use Protein A biosensor chip (Cat.# 29127556, GE) to affinity capture a certain amount of antibody to be tested, and then flow through a series of human FcRn antigen under a concentration gradient on the surface of the chip, using Biacore instrument (Biacore T200, GE) in real time The reaction signal is detected to obtain the binding and dissociation curves. After the dissociation of each cycle is completed, the biochip is cleaned and regenerated with the regeneration solution configured in the human anti-capture kit or the pH 1.5 glycine-hydrochloric acid regeneration solution (Cat.# BR-1003-54, GE). The buffer used in the experiment is HBS-EP+10× buffer solution (Cat.# BR-1006-69, GE), diluted to 1× (pH 7.4) with DIWater, or diluted to 1× and used 3M HCl Adjust the pH to 5.5 or 6.0.

The data obtained in the experiment was fitted with BIAevaluation version 4.1, and GE software was fitted with a (1:1) Langmuir model to obtain the affinity value. The results are shown in Table 12 and Table 13.

The results show that antibody h22 and its genetically modified molecules have good binding activity to FcRn under neutral conditions (pH 7.4) and acidic conditions (pH 6.0 or 5.5), and their affinity under acidic environment is slightly higher than that in neutral environment.

The heavy chain and light chain mutant sequences obtained by affinity maturation screening were cross-combined, and the antibody molecules with enhanced affinity to FcRn were obtained by SPR screening. Some of the molecular affinity determination results are as follows:

Combining humanized design and affinity maturation, the resulting molecule is subjected to affinity determination under acidic and neutral conditions, as follows.

The results show that the modified antibody molecule maintains an affinity comparable to the parent molecule h22 while reducing immunogenicity.

It has been identified that the above-mentioned important amino acid residues that maintain antibody affinity and reduce immunogenicity include X ₁ -X ₁₀ , and the details are as follows:

HCDR1: as shown in GYX ₁ FX ₂ (SEQ ID No: 182) or GYX ₁ FX ₂ X ₃ X ₄ X ₅ IA (SEQ ID No: 173), wherein X ₁ can be selected from S, N, D, V , A, P, X ₂ can be selected from T, N, K, R, S, A, X ₃ can be selected from G, N, K, S, E, X ₄ can be selected from Y, H, S, N, T, X ₅ can be selected from W, Y, F, M;

HCDR2: as shown in X ₆ IX ₇ PDX ₈ SNTI (SEQ ID No: 174) or X ₆ IX ₇ PDX ₈ SNTIYSPSFRG (SEQ ID No: 175), wherein X ₆ can be selected from I, L, V, X ₇ Can be selected from Y, S, T, G, A, X ₈ can be selected from N, R, K, A, F;

The amino acid sequence of HCDR3 is shown in FGGPTFAQWYFDY (SEQ ID No: 40).

The amino acid sequence of LCDR1 is shown in TGSSGSIASNYVX ₉ (SEQ ID No: 176), wherein X ₉ can be selected from Q, S, N, A, V, T;

The amino acid sequence of _{LCDR2 is shown in X 10} DNQRAS (SEQ ID No: 177), where X ₁₀ can be selected from E, S, A, W, and C;

The amino acid sequence of LCDR3 is shown in QSYDSSSHNWV (SEQ ID No: 43).

The full-length sequence of part of the antibody is as follows:

>The full length of the heavy chain of h22-0011:

(SEQ ID No：178)；

(SEQ ID No: 178);

>The full length of the heavy chain of h22-0014:

(SEQ ID No：179)；

(SEQ ID No: 179);

The full length of the heavy chain of >h22-0015:

(SEQ ID No：180)；

(SEQ ID No: 180);

The full length of the light chain of >h22-0011/0014/0015:

(SEQ ID No：181)。

(SEQ ID No: 181).

Example 8. Detection experiment of blocking the binding of IgG and FcRn by anti-human FcRn antibody

In this example, the anti-human FcRn antibody was tested for the blocking of biotin-hIgG binding to FcRn in cells overexpressing hFcRn in vitro.

The blocking effect of anti-human FcRn antibody on the binding of IgG to FcRn _{was measured in HEK293 cells overexpressing hFcRn mutants (human FcRn mut} (L320A, L321A)). : I4506) method of fluorescence detection. In this experiment _{, HEK293 cells (ie, 293-hFcRn-mut) overexpressing human FcRn mut} (L320A, L321A) were used. The hFcRn mutant can better maintain cell membrane surface localization. After the cells expressing hFcRn mutations were incubated with diluted FcRn antibodies of different concentrations, the secondary antibodies of biotin-hIgG and SA-FITC were added successively to incubate, and the degree of reduction of the fluorescence signal of the secondary antibodies was used as a measure of the effect of FcRn antibody on biotin-hIgG FcRn binding to the strength of the blocking effect. The specific experimental methods are as follows:

After washing the 293-hFcRn-mut cells twice with pH 6.0 PBS, 5×10 ⁵ cells were added to each well of a U-shaped well 96-well plate. After centrifugation and the supernatant, each well was added 100 μ L was diluted with the sample diluent FcRn antibody (dilution sample was added to pH 6.0PBS 1% BSA), incubated on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added to 100 μ L of diluted biotin-hlgG sample diluent (final concentration of 100 μ g / mL), incubated on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added 100 μ L of diluted sample diluent to streptavidin, avidin -FITC secondary antibody (eBioscience, Cat No.11-4317-87), the dilution ratio 1: 200 , Incubate on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added 200 μ L sample dilution, and the measured value FL1 channel fluorescence with a flow cytometer (BD Biosciences, BD Accuri C6) , using Flowjo software and data For analysis, use Prism6 software to plot the analysis results. See Figure 3 and Table 14 and Table 15 for the inhibitory effect test results of some antibodies.

The results show that the antibody obtained in the present disclosure has a good effect of blocking the binding of IgG to FcRn at the cellular level.

Example 9. The blocking experiment of anti-FcRn antibody on the binding of FcRn overexpressing cells to human serum albumin (HSA)

Both IgG and HSA achieve in vivo circulation by binding to FcRn, but the binding FcRn epitope is different. Antibodies that only block the binding of FcRn to IgG without affecting the binding of FcRn to HSA can be specific It can reduce the circulation of pathological IgG without affecting HSA, so it has better specificity and safety.

In order to detect that the anti-FcRn antibody specifically binds to the FcRn-IgG binding epitope, and to detect whether the anti-FcRn antibody does not affect the binding of FcRn-HSA at the same time, this example measures the antibody on the cell surface in HEK293 cells overexpressing the hFcRn mutant. Combine the fluorescence detection method with random biotinylated HSA (Sigma catalog number: 126658).

In this experiment, the HEK293 cell line overexpressing the hFcRn mutant (human FcRn _mut (L320A, L321A)) was used. The hFcRn mutant can better maintain the cell membrane surface localization. After the cells expressing hFcRn mutations are incubated with diluted FcRn antibody and biotin-HSA, SA-FITC secondary antibody is added to incubate, and the degree of increase or decrease of the fluorescence signal of the secondary antibody is used as a measure of the effect of FcRn antibody on biotin-HSA FcRn binding to the strength of the blocking effect. The specific experimental methods are as follows:

After washing the 293-hFcRn-mut cells twice with pH 6.0 PBS, 5×10 ⁵ cells were added to each well of a U-shaped well 96-well plate. After centrifugation and the supernatant, each well was added 100 μ L FcRn antibody diluted in sample diluent (final concentration 200μg / mL) and biotin -HSA (final concentration 250μg / mL) The mixture was incubated on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added 100 μ L of diluted sample diluent to streptavidin, avidin -FITC secondary antibody (eBioscience, Cat No.11-4317-87), the dilution ratio 1: 200 , Incubate on ice for 40 minutes. After washing twice in 200 μ L sample dilution per well was added 200 μ L sample dilution, and the measured value FL1 channel fluorescence with a flow cytometer (BD Biosciences, BD Accuri C6) , and using the data software F1owjo For analysis, use Prism6 software to plot the analysis results. The control group without antibody was set as the zero point. If the antibody reduces the average fluorescence intensity, the antibody blocks the binding of biotin-HSA to FcRn. In this experiment, the positive control is HSA (Sigma product number: 126658), and the negative control is human IgG (Sigma product number: 14506).

The test results of some antibodies are shown in Figure 4. The results showed that HSA exhibited a blockade of HSA binding, and h22 and its genetically modified antibodies had similar effects on the binding of biotin-HSA and hFcRn to the negative control IgG, and did not affect the binding of FcRn to HSA.

Example 10. Examination of the effect of anti-FcRn antibodies in human FcRn transgenic mice

In this experiment, human IVIG was injected intravenously into transgenic mice expressing human FcRn (C57BL/6-Fcgrttm1(FCGRT)/Bcgen, Biocytogen), 24 hours later, anti-FcRn antibody was injected intravenously, and blood was collected at different time points after administration. The purpose is to check whether the antibody affects the metabolism of human IgG.

The hFcRn transgenic mice were randomly divided into groups, three in each group, and intravenous injection of 500mpk IVIG. After 24 hours, the anti-FcRn antibody to be tested or the control human IgG1 isotype control (BioXcell catalog number: BE0297) was intravenously injected with a dose of 30mpk. Blood was collected before administration, that is, 0 hours, and 8, 24, 48 hours after administration. The sample uses a human IgG detection kit (Cisbio) to detect the IgG concentration. The data obtained were processed with GraphPad Prism 9, and analyzed using the one-way ANOVA method. Compared with the negative control hIgG1 isotype, antibodies h22-0011, h22-0014, and h22-0015 significantly accelerated the metabolism of hIgG in hFcRn transgenic mice. Analyzing the area under the curve (AUC) to obtain candidate antibodies h22-0011, h22-0014, h22-0015 compared with hIgG1 isotype can significantly reduce the retention of human IgG in mice. It is concluded from the above data that the candidate antibody can increase the catabolism of IgG in the body by blocking the binding of FcRn. Part of the test results are shown in Figure 5A and Figure 5B.

Although the specific embodiments of the present disclosure are described above, those skilled in the art should understand that these are only examples, and various changes or modifications can be made to these embodiments without departing from the principle and essence of the present disclosure. .

<110> BEIJING TUO JIE BIOPHARMACEUTICAL CO.LTD.

<120> Anti-FcRn antibody, its antigen-binding fragment and its medical use

<130> PCT2021-3BJTJ3

<150> CN202010083892.1

<151> 2020-02-10

<160> 182

<170> PatentIn version 3.5

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<213> Homo sapiens

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<223> People with His tag β 2M

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<223> Mutant of human FcRn Human FcRn mut (L320A. L321A)

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<223> hybri11-VH amino acid sequence

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<223> VH sequence of m14

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<223> VL sequence of m14

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<223> VH sequence of h3

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<223> VL sequence of h3

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<223> VH sequence of h4

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<223> VL sequence of h4

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<223> VH sequence of h22

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<223> VL sequence of h22

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<223> VH sequence of h30

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<223> VL sequence of h30

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<223> VH sequence of h33

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<223> VL sequence of h33

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<223> VH sequence of h35

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<223> VL sequence of h35

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<223> hybri11-HCDR1

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<223> hybri11-HCDR2

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<223> hybri11-HCDR3

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<223> hybri11-LCDR1

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<223> hybri11-LCDR2

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<223> hybri11-LCDR3

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<223> h3-HCDR1

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<223> h3-HCDR2

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<223> h3-HCDR3

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<223> h3-LCDR1

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<223> h3-LCDR2

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<223> h3-LCDR3

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<223> h4-HCDR1

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<223> h4-HCDR2

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<223> h4-HCDR3

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<223> h4-LCDR1

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<223> h4-LCDR2

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<223> h4-LCDR3

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<223> h22-HCDR1

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<223> h22-HCDR2

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<223> h22-HCDR3

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<223> h22-LCDR1

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<223> h22-LCDR2

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<223> h22-LCDR3

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<223> h30-HCDR1

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<223> h30-HCDR2

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<223> h33-HCDR1

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<223> h33-LCDR2

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<223> h35-HCDR1

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<223> h35-HCDR2

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<223> m14-LCDR2

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<223> h22-HCDR1 (AbM number)

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<223> Fully human antibody heavy chain CH1

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<223> Fully human antibody light chain C κ

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<223> Fully human antibody light chain CL2

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<223> Mouse IgG1 Fc

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<223> H22-AM-VL-1

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<223> H22-AM-VL-2

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<223> H22-AM-VL-3

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<223> H22-AM-VL-4

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<223> H22-AM-VL-5

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<223> H22-AM-VL-6

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<223> H22-AM-VL-7

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<223> H22-AM-VL-8

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<223> H22-AM-VL-9

<400> 84

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<223> H22-AM-VL-10

<400> 85

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<223> H22-AM-VL-11

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<223> H22-AM-VH-1

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<223> H22-AM-VH-2

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<223> H22-AM-VH-3

<400> 89

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<223> H22-AM-VH-4

<400> 90

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<223> H22-AM-VH-5

<400> 91

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<223> H22-AM-VH-6

<400> 92

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<223> H22-AM-VH-7

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<223> H22-AM-VH-8

<400> 94

<210> 95

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-9

<400> 95

<210> 96

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-10

<400> 96

<210> 97

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-11

<400> 97

<210> 98

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-12

<400> 98

<210> 99

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-13

<400> 99

<210> 100

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-14

<400> 100

<210> 101

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-15

<400> 101

<210> 102

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-16

<400> 102

<210> 103

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-17

<400> 103

<210> 104

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-18

<400> 104

<210> 105

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-19

<400> 105

<210> 106

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-20

<400> 106

<210> 107

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-21

<400> 107

<210> 108

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-22

<400> 108

<210> 109

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-23

<400> 109

<210> 110

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-24

<400> 110

<210> 111

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-1 LCDR1

<400> 111

<210> 112

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-1 LCDR2

<400> 112

<210> 113

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-2 LCDR2

<400> 113

<210> 114

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-3 LCDR2

<400> 114

<210> 115

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-4 LCDR1

<400> 115

<210> 116

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-6 LCDR2

<400> 116

<210> 117

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-7 LCDR1

<400> 117

<210> 118

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-8 LCDR1

<400> 118

<210> 119

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-10 LCDR1

<400> 119

<210> 120

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VL-10 LCDR2

<400> 120

<210> 121

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-1 HCDR2

<400> 121

<210> 122

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-2 HCDR2

<400> 122

<210> 123

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-3 HCDR2

<400> 123

<210> 124

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-4 HCDR2

<400> 124

<210> 125

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-5 HCDR2

<400> 125

<210> 126

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-6 HCDR2

<400> 126

<210> 127

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-7 HCDR2

<400> 127

<210> 128

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-8 HCDR2

<400> 128

<210> 129

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-9 HCDR1

<400> 129

<210> 130

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-10 HCDR1

<400> 130

<210> 131

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-11 HCDR1

<400> 131

<210> 132

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-12 HCDR1

<400> 132

<210> 133

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-13 HCDR1

<400> 133

<210> 134

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-14 HCDR1

<400> 134

<210> 135

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-15 HCDR1

<400> 135

<210> 136

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-16 HCDR1

<400> 136

<210> 137

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-17 HCDR1

<400> 137

<210> 138

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-18 HCDR1

<400> 138

<210> 139

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-20 HCDR1

<400> 139

<210> 140

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-21 HCDR1

<400> 140

<210> 141

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-24 HCDR1

<400> 141

<210> 142

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-1 HCDR2 (AbM number)

<400> 142

<210> 143

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-2 HCDR2 (AbM number)

<400> 143

<210> 144

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-3 HCDR2 (AbM number)

<400> 144

<210> 145

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-4 HCDR2 (AbM number)

<400> 145

<210> 146

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-5 HCDR2 (AbM number)

<400> 146

<210> 147

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-6 HCDR2 (AbM number)

<400> 147

<210> 148

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-7 HCDR2 (AbM number)

<400> 148

<210> 149

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-8 HCDR2 (AbM number)

<400> 149

<210> 150

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-9 HCDR1 (AbM number)

<400> 150

<210> 151

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-10 HCDR1 (AbM number)

<400> 151

<210> 152

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-11 HCDR1 (AbM number)

<400> 152

<210> 153

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-12 HCDR1 (AbM number)

<400> 153

<210> 154

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-13 HCDR1 (AbM number)

<400> 154

<210> 155

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-14 HCDR1 (AbM number)

<400> 155

<210> 156

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-15 HCDR1 (AbM number)

<400> 156

<210> 157

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-16 HCDR1 (AbM number)

<400> 157

<210> 158

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-17 HCDR1 (AbM number)

<400> 158

<210> 159

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-18 HCDR1 (AbM number)

<400> 159

<210> 160

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-19 HCDR1 (AbM number)

<400> 160

<210> 161

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-20 HCDR1 (AbM number)

<400> 161

<210> 162

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-21 HCDR1 (AbM number)

<400> 162

<210> 163

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-22 HCDR1 (AbM number)

<400> 163

<210> 164

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-23 HCDR1 (AbM number)

<400> 164

<210> 165

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> H22-AM-VH-24 HCDR1 (AbM number)

<400> 165

<210> 166

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> h22-0011 VH

<400> 166

<210> 167

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> h22-0011 VL

<400> 167

<210> 168

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> h22-0014 VH

<400> 168

<210> 169

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> h22-0015 VH

<400> 169

<210> 170

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> h22-0014 HCDR2

<400> 170

<210> 171

<211> 98

<212> PRT

<213> Homo sapiens

<400> 171

<210> 172

<211> 229

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG4 (S241P) sequence

<400> 172

<210> 173

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1 general formula

<220>

<221> Variation (VARIANT)

<222> (3)..(3)

<223> Xaa is selected from S, N, D, V, A, P, K

<220>

<221> Variation (VARIANT)

<222> (5)..(5)

<223> Xaa is selected from T, N, K, R, S, A

<220>

<221> Variation (VARIANT)

<222> (6)..(6)

<223> Xaa is selected from G, N, K, S, E

<220>

<221> Variation (VARIANT)

<222> (7)..(7)

<223> Xaa is selected from Y, H, S, N, T

<220>

<221> Variation (VARIANT)

<222> (8)..(8)

<223> Xaa is selected from W, Y, F, M

<400> 173

<210> 174

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2 general formula

<220>

<221> Variation (VARIANT)

<222> (1)..(1)

<223> Xaa is selected from I, L, V

<220>

<221> Variation (VARIANT)

<222> (3)..(3)

<223> Xaa is selected from Y, S, T, G, A

<220>

<221> Variation (VARIANT)

<222> (6)..(6)

<223> Xaa is selected from N, R, K, A, F

<400> 174

<210> 175

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2 general formula 2

<220>

<221> Variation (VARIANT)

<222> (1)..(1)

<223> Xaa is selected from I, L, V

<220>

<221> Variation (VARIANT)

<222> (3)..(3)

<223> Xaa is selected from Y, S, T, G, A

<220>

<221> Variation (VARIANT)

<222> (6)..(6)

<223> Xaa is selected from N, R, K, A, F

<400> 175

<210> 176

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1 general formula

<220>

<221> Variation (VARIANT)

<222> (13)..(13)

<223> Xaa is selected from Q, S, N, A, V, T

<400> 176

<210> 177

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2 general formula

<220>

<221> Variation (VARIANT)

<222> (1)..(1)

<223> Xaa is selected from E, S, A, W, C

<400> 177

<210> 178

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> The full length of the heavy chain of h22-0011

<400> 178

<210> 179

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> The full length of the heavy chain of h22-0014

<400> 179

<210> 180

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> The full length of the heavy chain of h22-0015

<400> 180

<210> 181

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> The full length of the light chain of h22-0011/0014/0015

<400> 181

<210> 182

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1 general formula

<220>

<221> Variation (VARIANT)

<222> (3)..(3)

<223> Xaa is selected from S, N, D, V, A, P, K

<220>

<221> Variation (VARIANT)

<222> (5)..(5)

<223> Xaa is selected from T, N, K, R, S, A

<400> 182

Claims (22)

An anti-FcRn antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH contains HCDR1-3 selected from: The amino acid sequence of HCDR1 is shown in GYX ₁ FX ₂ (SEQ ID No: 182) or GYX ₁ FX ₂ X ₃ X ₄ X ₅ IA (SEQ ID No: 173), in, X _{1 is} selected from S, N, D, V, A, P, K, X _{2 is} selected from T, N, K, R, S, A, X _{3 is} selected from G, N, K, S, E, X _{4 is} selected from Y, H, S, N, T, X _{5 is} selected from W, Y, F, M; The amino acid sequence of HCDR2 is shown in X ₆ IX ₇ PDX ₈ SNTI (SEQ ID No: 174) or X ₆ IX ₇ PDX ₈ SNTIYSPSFRG (SEQ ID No: 175), in, X _{6 is} selected from I, L, V, X _{7 is} selected from Y, S, T, G, A, X _{8 is} selected from N, R, K, A, F; The amino acid sequence of HCDR3 is shown in FGGPTFAQWYFDY (SEQ ID No: 40); The VL contains LCDR1-3 selected from the following: The amino acid sequence of LCDR1 is shown in TGSSGSIASNYVX ₉ (SEQ ID No: 176), Wherein, X _{9 is} selected from Q, S, N, A, V, T; The amino acid sequence of _{LCDR2 is shown in X 10} DNQRAS (SEQ ID No: 177), Wherein, X _{10 is} selected from E, S, A, W, C; The amino acid sequence of LCDR3 is shown in QSYDSSSHNWV (SEQ ID No: 43). The anti-FcRn antibody or antigen-binding fragment thereof according to claim 1, wherein VH includes HCDR1, HCDR2, HCDR3 of VH as shown in one of SEQ ID No: 12, 87-110, 169, VL includes LCDR1, LCDR2, LCDR3 of VL shown in one of SEQ ID No: 13, 76-86, and 167; Preferably, VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID No: 38-40, respectively, VL includes LCDR1, LCDR2, LCDR3 as shown in SEQ ID No: 41-43, respectively; or VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID No: 68, 69, and 40, respectively, VL includes LCDR1, LCDR2, LCDR3 as shown in SEQ ID No: 41-43, respectively; or VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID Nos: 133, 170, and 40, respectively, VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID Nos: 41-43, respectively. The anti-FcRn antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein VH is shown in one of SEQ ID No: 12, 87-110, 169 or has at least 90% identity with it, VL is shown in one of SEQ ID No: 13, 76-86, 167 or has at least 90% identity with it; Preferably, VH is shown in SEQ ID No: 169 or has at least 90% identity with it, VL is shown in SEQ ID No: 167 or has at least 90% identity with it. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The heavy chain is shown in SEQ ID No: 180 or has at least 80% identity with it; The light chain is shown in SEQ ID No: 181 or has at least 80% identity with it. An anti-FcRn antibody or antigen-binding fragment thereof, comprising VH and VL, wherein: The VH contains HCDR1-3 selected from: The amino acid sequence of HCDR1 is shown in one of SEQ ID No: 20, 26, 32, 38, 44, 50, 56, 62, 68, 129-141, 150-165; The amino acid sequence of HCDR2 is shown in one of SEQ ID No: 21, 27, 33, 39, 45, 51, 57, 63, 69, 121-128, 142-149, 170; The amino acid sequence of HCDR3 is shown in one of SEQ ID No: 22, 28, 34, 40, 46, 52, 58, and 64; The VL contains LCDR1-3 selected from the following: The amino acid sequence of LCDR1 is shown in one of SEQ ID No: 23, 29, 35, 41, 47, 53, 59, 65, 111, 115-119; The amino acid sequence of LCDR2 is shown in one of SEQ ID No: 24, 30, 36, 42, 48, 54, 60, 66, 112, 113, 114, 116, 120; The amino acid sequence of LCDR3 is shown in one of SEQ ID Nos: 25, 31, 37, 43, 49, 55, 61, and 67. The anti-FcRn antibody or antigen-binding fragment thereof according to claim 5, wherein: (a) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 20-21, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 23-25, respectively; (b) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 26-28, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 29-31, respectively; (c) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 32-34, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 35-37, respectively; (d) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 38-40, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 41-43, respectively; (e) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID No: 44-46, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 47-49, respectively; (f) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 50-52, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 53-55, respectively; (g) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 56-58, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 59-61, respectively; (h) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No: 62-64, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 65-67, respectively; (i) VH includes HCDR1, HCDR2, HCDR3 shown in SEQ ID Nos: 68, 69, 40, and VL includes LCDR1, LCDR2, LCDR3 shown in SEQ ID Nos: 41-43, respectively; (j) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 38, 170, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 41-43, respectively; (k) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 133, 170, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 41-43, respectively; (1) VH includes HCDR1, HCDR2, HCDR3 shown in SEQ ID Nos: 151, 69, 40, and VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID Nos: 41-43, respectively; (m) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 154, 69, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 41-43, respectively; (n) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 158, 69, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 41-43, respectively; (o) VH includes HCDR1, HCDR2, HCDR3 shown in SEQ ID Nos: 159, 69, 40, and VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID Nos: 41-43, respectively; (p) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 162, 69, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 41-43, respectively; (q) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 38, 123, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 111, 112, and 43, respectively; (r) VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID Nos: 38, 127, and 40, respectively, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 111, 112, and 43, respectively; (s) VH includes HCDR1, HCDR2, HCDR3 shown in SEQ ID Nos: 38, 128, and 40, and VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID Nos: 111, 112, and 43, respectively; (t) VH includes HCDR1, HCDR2, HCDR3 as shown in SEQ ID Nos: 38, 124, 40, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No: 117, 112, and 43, respectively; (u) VH includes HCDR1, HCDR2, HCDR3 shown in SEQ ID Nos: 38, 121, and 40, and VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID Nos: 41, 116, and 43, respectively. The anti-FcRn antibody or antigen-binding fragment thereof according to claim 5 or 6, wherein: The amino acid sequence of VH is shown in one of SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 87-110, 166, 168, 169 or has at least 90% identity with it; and The amino acid sequence of VL is shown in one of SEQ ID No: 5, 7, 9, 11, 13, 15, 17, 19, 76-86, 167 or has at least 90% identity with it. The anti-FcRn antibody or antigen-binding fragment thereof according to claim 7, wherein: The amino acid sequence of VH is shown in SEQ ID No: 4 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 5 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 6 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 7 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 8 or has at least 90% identity, and the amino acid sequence of VL is shown in SEQ ID No: 9 or has at least 90% identity; The amino acid sequence of VH is shown in SEQ ID No: 10 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 11 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 12 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 13 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 14 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 15 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 16 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 17 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 18 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 19 or has at least 90% identity with it; The amino acid sequence of VH is shown in SEQ ID No: 96, 99, 103, 104, 107 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 13 or is It is at least 90% identical; The amino acid sequence of VH is shown in SEQ ID No: 89, 93, 94 or has at least 90% identity with one of SEQ ID No: 89, 93, 94, and the amino acid sequence of VL is shown in SEQ ID No: 76 or has at least 90% identity with it. % Identity; The amino acid sequence of VH is shown in SEQ ID No: 87 or has at least 90% identity, and the amino acid sequence of VL is shown in SEQ ID No: 84 or has at least 90% identity; or The amino acid sequence of VH is shown in one of SEQ ID No: 87-110 or has at least 90% identity, and the amino acid sequence of VL is shown in or has one of SEQ ID No: 76-86. At least 90% identical. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, wherein: The heavy chain amino acid sequence is shown in any one of SEQ ID No: 178-180, or has at least 80% identity; the light chain amino acid sequence is shown in SEQ ID No: 181, or has at least 80% identity. % Identity. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is a murine antibody, a chimeric antibody, a fully human antibody, a humanized antibody or a fragment thereof. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which is an IgG antibody or an antigen-binding fragment thereof, preferably an IgG1, IgG4 antibody or an antigen-binding fragment thereof. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is a scFv, Fv, Fab or Fab' fragment. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which blocks the binding of human IgG to human FcRn. The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which does not bind β2 microglobulin. An FcRn binding protein, which contains the anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 14. An isolated polynucleotide encoding the FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 15. A vector containing the polynucleotide according to claim 16. A host cell comprising the vector according to claim 17 or the polynucleotide according to claim 16. A method for preparing an anti-FcRn antibody or an antigen-binding fragment thereof, comprising: Expressing an anti-FcRn antibody or an antigen-binding fragment thereof in the host cell according to claim 18, and The anti-FcRn antibody or antigen-binding fragment thereof is isolated from the host cell. A group of pharmaceutical composition comprising the anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the FcRn-binding protein according to claim 15 or the polynucleotide according to claim 16 Acid; and, a pharmaceutically acceptable excipient, diluent or carrier. Use of any one selected from the following or any combination thereof in the preparation of medicines or pharmaceutical compositions: The anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the FcRn-binding protein according to claim 15 or the polynucleotide according to claim 16, wherein: Preferably, the drug or medical composition is used to treat autoimmune diseases or delay the progress of autoimmune diseases; More preferably, the autoimmune disease is selected from: immune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaac syndrome, mole syndrome, pemphigus vulgaris, pemphigus foliaceus , Bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave disease, Goodpasture syndrome, Heavy Myasthenia, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis and membranous nephropathy. A method for treating autoimmune diseases or delaying the progress of autoimmune diseases, the method comprising: Administering to a subject a therapeutically effective amount or a disease-delaying effective amount of the anti-FcRn antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the FcRn-binding protein according to claim 15 or as claimed The polynucleotide of 16, Preferably, the autoimmune disease is selected from: immune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaac syndrome, nevus syndrome, pemphigus vulgaris, pemphigus foliaceus , Bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave disease, Goodpasture syndrome, Myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis and membranous nephropathy.

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