Anti-human LAIR1 antibodies

Abstract

The present disclosure relates to antibodies that bind to human LAIR1 ("anti-human LAIR1 antibodies" or "anti-human LAIR1 antibodies"), compositions comprising such anti-human LAIR1 antibodies, and methods of using such anti-human LAIR1 antibodies.

Description

[0001] Sequence Listing File

[0002] This application is filed together with a Sequence Listing in the ST.26 XML format. The Sequence Listing is provided as a file named "Sequence Listing for 30426" created on August 10, 2023, and is 38 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to antibodies that bind to human LAIR1 ("anti-human LAIR1 antibodies" or "anti-LAIR1 antibodies" or "human LAIR1 antibodies"), compositions comprising such anti-human LAIR1 antibodies, and methods of using such anti-human LAIR1 antibodies. Background Art

[0004] Human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1, also known as CD305) is an inhibitory receptor found on peripheral monocytes, including natural killer cells, T cells, B cells, macrophages, dendritic cells, and hematopoietic progenitor cells, including human CD34+ cells. It belongs to the immunoglobulin superfamily and plays a role in regulating the immune response. Inhibitory receptors regulate the immune response to prevent lysis of cells recognized as self.

[0005] Structurally, LAIR1 is a type I transmembrane glycoprotein that contains an extracellular C2-type immunoglobulin-like domain, a stalk region, a single transmembrane domain, and an intracellular domain that contains two conserved motifs called immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LAIR1 is structurally related to several other inhibitory immunoglobulin superfamily members, including LILRB of the leukocyte receptor complex (LRC) located on human chromosome 19q13.4, suggesting that these molecules have evolved from a common ancestral gene.

[0006] In several autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), LAIR1 expression is altered (see Zhang Y. et al., Clin Exp Immunol. May 2018;192(2):193 - 205). Due to the immunosuppressive function of LAIR1, antibodies that modulate LAIR1 activity are needed, which can be used as therapeutic agents for treating autoimmune diseases. Such antibodies can be used to treat autoimmune diseases, including SLE and lupus nephritis. Current standard treatments include numerous steroids, which have many adverse and / or potentially dangerous side effects. Therefore, there is a need to find safe and effective therapeutic treatments for such autoimmune diseases. Summary of the Invention

[0007] The present disclosure provides novel anti-human LAIR1 antibodies or antibody fragments thereof. In some embodiments, the anti-human LAIR1 antibodies or antibody fragments thereof provided herein are agonists of LAIR1. In some embodiments, the anti-human LAIR1 antibodies provided herein are human antibodies, such as human IgG2 or IgG4 isotypes. In some embodiments, the anti-human LAIR1 antibodies provided herein also bind cynomolgus monkey LAIR1.

[0008] In some embodiments, the present disclosure provides antibodies that bind human LAIR1, wherein the antibody comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:3, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising SEQ ID NO:7 and a VL comprising SEQ ID NO:8. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising a sequence having at least 95% sequence identity to SEQ ID NO:7, and a VL comprising a sequence having at least 95% sequence identity to SEQ ID NO:8.

[0009] In some embodiments, the present disclosure provides antibodies that bind human LAIR1, wherein the antibody comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:13, HCDR2 comprises SEQ ID NO:14, HCDR3 comprises SEQ ID NO:15, LCDR1 comprises SEQ IDNO:16, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:18. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising SEQ ID NO:19 and a VL comprising SEQ ID NO:20. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising a sequence having at least 95% sequence identity to SEQ ID NO:19, and a VL comprising a sequence having at least 95% sequence identity to SEQID NO:20.

[0010] In some embodiments, the anti-human LAIR1 antibody has a human IgG2 isotype. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO:9 and a light chain (LC) comprising SEQ ID NO:10. In some embodiments, the antibody comprises a heavy chain comprising SEQ ID NO:21 and a light chain comprising SEQ ID NO:22.

[0011] In some embodiments, the anti-human LAIR1 antibody has a human IgG4 isotype. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO:25 and a light chain (LC) comprising SEQ ID NO:10. In some embodiments, the antibody comprises a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:22.

[0012] In another aspect, provided herein are nucleic acids encoding the heavy or light chain, or VH or VL, of the novel anti-human LAIR1 antibodies described herein, as well as vectors or cells comprising such nucleic acids.

[0013] In another aspect, provided herein are pharmaceutical compositions comprising the antibodies, nucleic acids, or vectors described herein.

[0014] The anti-human LAIR1 antibodies, nucleic acids, vectors, or pharmaceutical compositions described herein can be used to treat autoimmune diseases or fibrotic diseases, such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus (SLE), lupus nephritis, pemphigus vulgaris, systemic sclerosis, idiopathic pulmonary fibrosis, scleroderma, ulcerative colitis, Crohn's disease, hidradenitis suppurativa, atopic dermatitis, multiple sclerosis, scleroderma-associated interstitial lung disease, IgG4-related disease, or chronic fibrotic interstitial lung disease.

[0015] In another aspect, the antibody of the invention is an antibody that does not form a complex with the LAIR1 ligand C1q.

[0016] According to yet another aspect of the invention, the antibody of the invention increases the Treg cell population in the spleen.

[0017] In yet a further aspect of the invention, the antibody of the invention does not require complete receptor occupancy (RO) to elicit agonistic effects on human LAIR1. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A - 1FShows that on day 7 (1A-1C) and day 14 (1D-1F) in the GvHD model, the exemplified anti-human LAIR1 antibodies mAb1-mAb4 significantly inhibited the increase in plasma human pro-inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F).

[0019] Figure 2A - 2C Shows that the exemplified anti-human LAIR1 antibodies mAb1-mAb3 significantly reduced circulating immunoglobulins IgM and IgA.

[0020] Figure 3 Shows that mAb4 inhibits TCR-stimulated NFAT activation in Jurkat-hLAIR1+ cells in vitro. A. Inhibition of NFAT activity after TCR stimulation was reported as the percentage of antibody-mediated inhibition relative to NFAT activity in the absence of antibody. B. The mAb4 NFAT IC50 curve was calculated and averaged from n = 2 independent experiments.

[0021] Figure 4 Shows that the mAb4 agonist antibody inhibits the BCR-stimulated IL-6 response in human B cells in vitro. A. Inhibition of the IL-6 response after BCR stimulation was reported as the percentage of antibody-mediated inhibition relative to the IL-6 response in the absence of antibody. B. The mAb4 B cell IL-6 IC50 curve was calculated and averaged from n = 3 independent experiments.

[0022] Figure 5 Shows that compared to the isotype control measured 8 days after implantation (7 days after treatment), mAb4 inhibited serum human cytokine production in a dose-dependent and significant manner. One-way ANOVA was followed by Dunnett's post hoc test relative to the isotype (mean ± SEM, n = 7-8).

[0023] Figure 6 Shows the dose-dependent receptor occupancy of mAb4 on 3 T cell subsets 7 days after a single SC dose.

[0024] Figure 7 Shows the dose-dependent serum drug concentrations of mAb4 4 days (blue bars) and 7 days (red bars) after a single SC dose.

[0025] Figure 8 Shows the Treg expansion induced by mAb4 in a dose-dependent manner.

[0026] Figure 9Shows the urinary albumin / creatinine ratio (ACR) of IFNα-induced NZB / W F1 mice treated with IgG isotype, alternative antibody, or cyclophosphamide (CP) starting from day 7 (D7) or day 21 (D21). One-way ANOVA was followed by Dunnett's post hoc test relative to IFNα-induced treated with IgG isotype, mean ± SEM, n = 5 - 10.

[0027] Figure 10 : Shows the 44-day urinary albumin / creatinine ratio (ACR) of IFNα-induced NZB / W F1 mice treated with IgG isotype, alternative antibody, or cyclophosphamide (CP) starting from day 7 (D7) or day 21 (D21). One-way ANOVA was followed by Dunnett's post hoc test relative to IFNα-induced treated with IgG isotype, mean ± SEM, n = 5 - 10.

[0028] Figure 11 : Shows the total histological score of the kidneys of IFNα-induced NZB / W F1 mice treated with IgG isotype, alternative antibody, or cyclophosphamide (CP) starting from day 7 (D7) or day 21 (D21). One-way ANOVA was followed by Dunnett's post hoc test relative to IFNα-induced treated with IgG isotype, mean ± SEM, n = 5 - 10.

[0029] Figure 12 : Shows that Ab4 and the humanized IgG4-P isotype control antibody do not bind to complement component C1q. The anti-LAIR1 IgG1 antibody and the human IgG1 isotype control antibody do bind to complement component C1q as expected.

[0030] Figure 13 : Shows the selectivity for LAIR1 by mAb4 and no activity against LAIR2. Detailed Description

[0031] Provided herein are antibodies that bind to human LAIR1 (“anti-human LAIR1 antibodies” or “anti-human LAIR1 antibodies”), compositions comprising such anti-human LAIR1 antibodies, and methods of using such anti-human LAIR1 antibodies.

[0032] In one aspect, the present disclosure provides novel anti-human LAIR1 antibodies or antibody fragments thereof. In some embodiments, the anti-human LAIR1 antibodies or antibody fragments thereof provided herein are agonists of LAIR1. In some embodiments, the anti-human LAIR1 antibodies or antibody fragments thereof provided herein can induce or increase one or more activities or functions associated with human LAIR1, such as one or more activities or functions described in the Examples. Such activities or functions associated with human LAIR1 include, but are not limited to, inhibition of NFAT activation as determined by the Jurkat-NFAT activation assay, inhibition of IFN-γ response in primary T cells after TCR stimulation assay, inhibition of IL-6 response in primary B cells after BCR stimulation assay, inhibition of the increase of plasma human pro-inflammatory cytokines IFN-γ, IL-10, and TNF-α, and / or reduction of circulating IgM and IgA in the GvHD mouse model implanted with human PBMC, as described in the Examples. In some embodiments, the anti-human LAIR1 antibodies provided herein do not block the interaction between LAIR1 and its ligands such as collagen I.

[0033] In some embodiments, the anti-human LAIR1 antibodies provided herein are human antibodies, such as human IgG2 or IgG4 isotypes. In some embodiments, the anti-human LAIR1 antibodies provided herein also bind cynomolgus monkey LAIR1. In some embodiments, the anti-human LAIR1 antibodies provided herein have a low risk of immunogenicity.

[0034] In some embodiments, the anti-human LAIR1 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-human LAIR1 antibody includes a VH comprising HCDR1, HCDR2, and HCDR3 selected from Table 1. In some embodiments, the anti-human LAIR1 antibody includes a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 1. In some embodiments, the anti-human LAIR1 antibody includes a VH comprising HCDR1, HCDR2, and HCDR3 selected from Table 1, and a VL comprising LCDR1, LCDR2, and LCDR3 selected from Table 1. In some embodiments, the anti-human LAIR1 antibody includes a VH comprising a sequence having at least 95% sequence identity to the VH in Table 1. In some embodiments, the anti-human LAIR1 antibody includes a VL comprising a sequence having at least 95% sequence identity to the VL in Table 1. In some embodiments, the anti-human LAIR1 antibody comprises the VH and / or VL in Table 1.

[0035] Table 1. Sequences of Exemplary Anti-LAIR1 Monoclonal Antibodies

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] In some embodiments, antibodies that bind to human LAIR1 are provided herein, wherein the antibodies comprise a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:3, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising SEQ ID NO:7 and a VL comprising SEQ ID NO:8. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising a sequence having at least 95% sequence identity to SEQ ID NO:7, and a VL comprising a sequence having at least 95% sequence identity to SEQ ID NO:8.

[0053] In some embodiments, antibodies that bind to human LAIR1 are provided herein, wherein the antibodies comprise a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:13, HCDR2 comprises SEQ ID NO:14, HCDR3 comprises SEQ ID NO:15, LCDR1 comprises SEQ ID NO:16, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:18. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising SEQ ID NO:19 and a VL comprising SEQ ID NO:20. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising a sequence having at least 95% sequence identity to SEQ ID NO:19, and a VL comprising a sequence having at least 95% sequence identity to SEQ ID NO:20.

[0054] In some embodiments, the anti-human LAIR1 antibody is a human antibody. In some embodiments, the anti-human LAIR1 antibody has a human IgG2 or IgG4 isotype. In some embodiments, the anti-human LAIR1 antibody has a human IgG2 isotype. In some embodiments, the anti-human LAIR1 antibody has a modified human IgG2 Fc region comprising a C131S mutation (according to EU index number), which reduces disulfide heterogeneity in human IgG2 (see Allen, et al., Biochemistry 2009, 48:3755–3766). In some embodiments, the anti-human LAIR1 antibody has a human IgG4 isotype. In some embodiments, the anti-human LAIR1 antibody has a modified human IgG4 hinge region comprising an S228P mutation (according to EU index number), which reduces IgG4 Fab arm exchange in vivo (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767).

[0055] In some embodiments, the anti-human LAIR1 antibody has a human IgG2 isotype. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO:9 and a light chain (LC) comprising SEQ ID NO:10. In some embodiments, the antibody comprises a heavy chain comprising SEQ ID NO:21 and a light chain comprising SEQ ID NO:22.

[0056] In some embodiments, the anti-human LAIR1 antibody has a human IgG4 isotype. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO:25 and a light chain (LC) comprising SEQ ID NO:10. In some embodiments, the antibody comprises a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:22.

[0057] In some embodiments, provided herein are antibody fragments (e.g., Fab or scFv) that bind to human LAIR1, wherein the antibody fragment comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:3, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, the antibody fragment comprises a VH comprising SEQ ID NO:7 and a VL comprising SEQ ID NO:8.

[0058] In some embodiments, provided herein are antibody fragments (e.g., Fab or scFv) that bind to human LAIR1, wherein the antibody fragment comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:13, HCDR2 comprises SEQ ID NO:14, HCDR3 comprises SEQ ID NO:15, LCDR1 comprises SEQ ID NO:16, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:18. In some embodiments, the antibody fragment comprises a VH comprising SEQ ID NO:19 and a VL comprising SEQ ID NO:20.

[0059] In another aspect, provided herein are nucleic acids encoding the heavy or light chain, or VH or VL, of the novel anti-human LAIR1 antibodies described herein, and vectors comprising such nucleic acids.

[0060] In some embodiments, provided herein are nucleic acids encoding the heavy or light chains of the anti-human LAIR1 antibodies described herein. In some embodiments, provided herein are nucleic acids comprising a sequence encoding SEQ ID NO: 9, 25, 10, 21, 27, or 22. In some embodiments, provided herein are nucleic acids comprising a sequence encoding an antibody heavy chain, the antibody heavy chain comprising SEQ ID NO: 9, 25, 21, or 27. For example, the nucleic acid may comprise a sequence selected from SEQ ID NO: 11, 26, 23, or 28. In some embodiments, provided herein are nucleic acids comprising a sequence encoding an antibody light chain, the antibody light chain comprising SEQ ID NO: 10 or 22. For example, the nucleic acid may comprise a sequence selected from SEQ ID NO: 12 or 24.

[0061] Also provided herein are vectors comprising nucleic acid sequences encoding antibody heavy or light chains. For example, such vectors may comprise nucleic acid sequences encoding SEQ ID NO: 9, 25, 10, 21, 27, or 22. In some embodiments, the vector comprises SEQ ID NO: 11, 26, 12, 23, 28, or 24.

[0062] Also provided herein are vectors comprising a first nucleic acid sequence encoding an antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 9 or 25 and a second nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 21 or 27 and a second nucleic acid sequence encoding SEQ ID NO: 22.

[0063] Also provided are compositions comprising a first vector comprising a nucleic acid sequence encoding an antibody heavy chain and a second vector comprising a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 or 25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 21 or 27 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 22.

[0064] For example, after the nucleic acid has been operably linked to an expression control sequence, the nucleic acids of the present disclosure can be expressed in a host cell. Expression control sequences capable of expressing nucleic acids are well known in the art and the nucleic acids are operably linked thereto. Expression vectors can include sequences encoding one or more signal peptides that facilitate the secretion of polypeptides from the host cell. Expression vectors containing a target nucleic acid (e.g., a nucleic acid encoding an antibody heavy or light chain) can be transferred into a host cell by well-known methods such as stable or transient transfection, transformation, transduction, or infection. Additionally, expression vectors can contain one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired nucleic acid sequence.

[0065] In another aspect, the present disclosure provides cells, such as host cells, comprising the nucleic acids, vectors, or nucleic acid compositions described herein. The host cell can be a cell that has been stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express all or a portion of an antibody described herein. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with an expression vector that expresses the HC and LC polypeptides of an antibody of the present disclosure. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with a first vector that expresses the HC polypeptide of an antibody described herein and a second vector that expresses the LC polypeptide. Such host cells, such as mammalian host cells, can express an anti-human LAIR1 antibody described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0066] In some embodiments, a cell, such as a host cell, comprises a vector that comprises a first nucleic acid sequence encoding SEQ ID NO:9 or 25 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, a cell, such as a host cell, comprises a vector that comprises a first nucleic acid sequence encoding SEQ ID NO:21 or 27 and a second nucleic acid sequence encoding SEQ ID NO:22.

[0067] In some embodiments, a cell, such as a host cell, includes a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9 or 25, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, a cell, such as a host cell, includes a first vector comprising a nucleic acid sequence encoding SEQ ID NO:21 or 27, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:22.

[0068] The present disclosure further provides a method for generating the anti-human LAIR1 antibodies described herein, which comprises culturing the above host cells, such as mammalian host cells, under conditions that allow antibody expression, and recovering the expressed antibody from the culture medium. The culture medium in which the antibody has been secreted can be purified by conventional techniques. A variety of protein purification methods can be employed, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd edition, Springer, NY (1994).

[0069] Also provided are antibodies produced by any of the methods described herein.

[0070] In another aspect, the present disclosure provides pharmaceutical compositions comprising the antibodies, nucleic acids or vectors described herein. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients, diluents or carriers. The pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd edition (2012), A. Loyd et al., Pharmaceutical Press).

[0071] The anti-human LAIR1 antibodies, nucleic acids, vectors or pharmaceutical compositions described herein can be used to treat autoimmune diseases or fibrotic diseases. Examples of such autoimmune diseases or fibrotic diseases include rheumatoid arthritis, psoriasis, systemic lupus erythematosus, lupus nephritis, pemphigus vulgaris, systemic sclerosis, idiopathic pulmonary fibrosis, scleroderma, scleroderma-associated interstitial lung disease, IgG4-related disease, chronic fibrotic interstitial lung disease, ulcerative colitis, Crohn's disease, hidradenitis suppurativa, atopic dermatitis or multiple sclerosis.

[0072] In some embodiments, the present disclosure provides a method for treating an autoimmune disease or a fibrotic disease in a subject (e.g., a human patient) in need thereof, which comprises administering to the subject a therapeutically effective amount of an anti-human LAIR1 antibody, a nucleic acid encoding such anti-human LAIR1 antibody, a vector comprising such nucleic acid, or a pharmaceutical composition comprising such anti-human LAIR1 antibody, nucleic acid or vector, as described herein. The antibodies, nucleic acids, vectors or pharmaceutical compositions described herein can be administered by a parenteral route (e.g., subcutaneous or intravenous).

[0073] Also provided are the anti-human LAIR1 antibodies, nucleic acids, vectors or pharmaceutical compositions described herein for use in therapy. In addition, the present disclosure provides the anti-human LAIR1 antibodies, nucleic acids, vectors or pharmaceutical compositions described herein for use in treating autoimmune diseases or fibrotic diseases.

[0074] Also provided herein is the use of the anti-human LAIR1 antibodies, nucleic acids, vectors or pharmaceutical compositions described herein in the manufacture of a medicament for treating autoimmune diseases or fibrotic diseases.

[0075] Also provided are antibodies or antigenic fragments thereof that bind to the human LAIR1 protein, wherein the antibody binds to an epitope comprising one or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and one or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0076] Preferably, the antibody or antigenic fragment thereof that binds to the human LAIR1 protein is an antibody that binds to an epitope comprising two or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and two or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0077] Preferably, the antibody or antigenic fragment thereof that binds to the human LAIR1 protein is an antibody that binds to an epitope comprising three or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and three or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0078] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein is an antibody that binds to an epitope comprising: four or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and four or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0079] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein is an antibody that binds to an epitope comprising: five or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and five or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0080] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein is an antibody that binds to an epitope comprising: six or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and six or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0081] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein is an antibody that binds to an epitope comprising: seven or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and seven or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0082] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein is an antibody that binds to an epitope comprising: eight, nine, ten, eleven, twelve, thirteen, or fourteen or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO:32) and eight, nine, ten, eleven, twelve, thirteen, or fourteen or more amino acid residues selected from VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0083] Preferably, the epitope comprises FVCRGPVGVQTFRLER (SEQ ID NO:32) and VSQASPSESEARFRI (SEQ ID NO:33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and wherein the amino acid positions correspond to SEQ ID NO:29.

[0084] Also provided are antibodies or antibody fragments thereof that bind to the epitope of human LAIR1 protein as defined above, wherein the antibody comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:34, HCDR2 comprises SEQ ID NO:35, HCDR3 comprises SEQ ID NO:36, LCDR1 comprises SEQ ID NO:37, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:38. In some embodiments, the anti-human LAIR1 antibody has a human IgG4 isotype. In some embodiments, the anti-human LAIR1 antibody has a modified human IgG4 isotype. In some embodiments, the anti-human LAIR1 has a modified human IgG4 hinge region comprising the S228P mutation (according to EU index number), and the S228P mutation reduces in vivo IgG4 Fab arm exchange (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767). In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO:39 and a light chain (LC) comprising SEQ ID NO:40. In some embodiments, the anti-human LAIR1 antibody comprises an HC having at least 95% sequence identity with SEQ ID NO:39 and an LC having at least 95% sequence identity with SEQ ID NO:40.

[0085] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein is Ab0.

[0086] Preferably, the antibody or antigen fragment thereof that binds to the human LAIR1 protein is a human antibody.

[0087] Preferably, the antibody or antigen fragment thereof that binds to the epitope according to the present invention is an antibody that agonizes the human LAIR1 protein.

[0088] Also provided are anti-LAIR1 antibodies or antibody fragments thereof that compete with any of the antibodies defined according to the present invention for binding to the epitope of the present invention.

[0089] Preferably, the anti-LAIR1 antibody or antigen fragment thereof that binds to the epitope is Ab0.

[0090] In some embodiments, the anti-LAIR1 antibody or antibody fragment thereof that competes with the anti-LAIR1 antibody defined below for binding to the epitope is Ab1, Ab2, Ab3, or Ab4.

[0091] In some embodiments, the anti-LAIR1 antibody is an antibody or antibody fragment thereof that binds to the epitope of the human LAIR1 protein as defined above, wherein the antibody comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:34, HCDR2 comprises SEQ ID NO:35, HCDR3 comprises SEQ ID NO:36, LCDR1 comprises SEQ ID NO:37, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:38. In some embodiments, the anti-human LAIR1 antibody has a human IgG4 isotype. In some embodiments, the anti-human LAIR1 has a modified human IgG4 hinge region comprising the S228P mutation (according to the EU index number), and the S228P mutation reduces in vivo IgG4 Fab arm exchange (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767). In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO:39 and a light chain (LC) comprising SEQ ID NO:40. In some embodiments, the anti-human LAIR1 antibody comprises an HC having at least 95% sequence identity with SEQ ID NO:39 and an LC having at least 95% sequence identity with SEQ ID NO:40.

[0092] The epitope is preferably determined by hydrogen-deuterium exchange (HDX) mapping technology.

[0093] The advantage of the anti-human LAIR1 antibody or its antigenic fragment is that it can initiate the body's natural immunosuppressive mechanism. This may result in both target cell-specific efficacy and key safety benefits that exceed those of current immunomodulatory therapies.

[0094] The anti-human LAIR1 antibody of the present invention preferably has one or more of the following key properties:

[0095] - Binding to human LAIR1 and cross-reactivity with cynomolgus monkey LAIR1

[0096] - Binding to a unique epitope (non-ligand blocking).

[0097] - Demonstrating agonistic effects of LAIR1.

[0098] - Antibody-mediated agonistic effects resulting in dose-dependent attenuation of human primary B cell activation

[0099] - Demonstrating in vivo efficacy in a humanized mouse model of graft-versus-host disease.

[0100] - Preclinical efficacy in a mouse model of lupus nephritis

[0101] As used herein, the terms "a", "an", "the", and similar terms used in the context of the present disclosure (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context.

[0102] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0103] An exemplary antibody is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked by interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains includes a variable region of about 100 to 125 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector functions. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0104] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The CDRs are exposed on the surface of the protein and are important regions for antibody antigen-binding specificity. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this article, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3", and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to the CDRs can be done according to well-known schemes, including those described in the following: Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the International ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The North CDR definition is used for the anti-human LAIR1 antibodies described herein.

[0105] The present disclosure also includes antibody fragments or antigen-binding fragments that include at least a portion of an antibody that retains the ability to interact specifically with an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, and linear antibodies.

[0106] The term "epitope" refers to the amino acid residues of an antigen that are bound by an antibody. An epitope may be a linear epitope, a conformational epitope, or a mixed epitope.

[0107] The term "epitope" can be used to refer to a structural epitope. According to some embodiments, a structural epitope can be used to describe the region of an antigen covered by an antibody (e.g., the footprint when the antibody binds to the antigen).

[0108] Epitopes can be determined according to different experimental techniques, also referred to as "epitope mapping techniques". It should be understood that the determination of an epitope may vary based on the different epitope mapping techniques used and may also vary due to the use of different experimental conditions, such as conformational changes or cleavage of the antibody induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nivebrant, Epitope mapping Protocols: Methods in Molecular Biology, Humana press, 3rd edition 2018), including but not limited to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species-switching mutagenesis, alanine-scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0109] As used herein, the terms "competitive binding" or "competition" refer to two antibodies cross-competing (i.e., competing against each other) for binding to the same antigen. In some embodiments, two antibodies may compete for binding to the same antigen, where they bind to spatially overlapping regions of the same antigen. In some embodiments, two antibodies may compete for binding to the same antigen, where the antibodies bind to non-overlapping regions of the antigen, but the binding of one antibody is blocked by the binding of the other antibody, for example, due to steric hindrance or conformational changes in the antigen induced by the first antibody. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another antibody, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay, surface plasmon resonance, biolayer interferometry, or flow cytometry methods. Epitope binning can be performed using Carterra technology (e.g., PLoS One, March 20, 2014; doi:10.137 / journal.pone.0092451, Y. Abdiche et al.).

[0110] As used herein, the term "agonist" or "agonistic" refers to an antibody or antibody fragment that is capable of inducing or increasing one or more activities or functions associated with human LAIR1, such as one or more activities or functions associated with human LAIR1 described in the Examples.

[0111] As used herein, unless otherwise specified, the terms "bind" and "binds" are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in the proximity of the two proteins or molecules, as determined by common methods known in the art.

[0112] "Effective amount" means the amount necessary (for a period of time and for a means of administration) to achieve the desired therapeutic result. The effective amount of an antibody may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody to elicit the desired response in the individual. An effective amount is also the amount in which any toxic or harmful effects of the antibody are outweighed by the therapeutic beneficial effects.

[0113] As used herein, the term "Fc region" refers to the region of an antibody that contains the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion or the entire hinge region of the antibody heavy chain.

[0114] As used herein, unless otherwise specified, the term "LAIR1" refers to human leukocyte-associated immunoglobulin-like receptor 1 (also known as CD305). The amino acid sequence of human LAIR1 isoform a (the longest isoform) can be found in NCBI accession number NP_002278.2:

[0115] MSPHPTALLGLVLCLAQTIHTQEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERDSRSTYNDTEDVSQASPSESEARFRIDSVREGNAGLYRCIYYKPPKWSEQSDYLELLVKESSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLYILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO:29)

[0116] Several shorter human LAIR1 isoforms have been reported, including isoform b (NCBI accession number NP_068352.2), isoform c (NCBI accession number NP_001275952.2), isoform e (NCBI accession number NP_001275954.2), isoform f (NCBI accession number NP_001275955.2), and isoform g (NCBI accession number NP_001275956.2). The term "LAIR1" is used herein to collectively refer to all known human LAIR1 isoforms.

[0117] The amino acid sequences of cynomolgus monkey LAIR1 can be found at XP_045236925.1 (isoform X1), XP_045236926.1 (isoform X2), XP_045236927.1 (isoform X3), or XP_045236928.1 (isoform X4).

[0118] As used interchangeably herein, the terms "nucleic acid" or "polynucleotide" refer to polymers of nucleotides, including single-stranded and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules that incorporate natural nucleotides, modified nucleotides, and / or nucleotide analogs.

[0119] As used herein, the term "subject" refers to a mammal, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Preferably, the subject is a human.

[0120] As used herein, "treatment" or "treating" refers to all processes in which there may be a slowing, controlling, delaying, or stopping of the progression of a disorder or disease disclosed herein, or an improvement in the disorder or disease symptoms, but does not necessarily indicate the complete elimination of all disorder or disease symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition for treating a patient, particularly a disease or condition in a human.

[0121] Examples

[0122] The following examples are provided to illustrate, not limit, the invention.

[0123] Example 1. Generation of anti-human LAIR1 antibodies

[0124] Use Human transgenic mice and cloning of anti-LAIR1 variable regions to generate human anti-human LAIR1 antibodies. Mice were immunized with human LAIR1 (SEQ ID NO:30) fused to human Fc with a His tag and TEV cleavage site, with or without co-administration of human LAIR2 (SEQ ID NO:31) fused to human Fc with a TEV cleavage site, using standard procedures, and antigen-specific B cells were isolated by standard sorting methods using fluorophore-labeled LAIR1.

[0125] The LAIR1 immunogen has the following amino acid sequence:

[0126] QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHENLYFQGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ ID NO:30).

[0127] The LAIR2 immunogen has the following amino acid sequence:

[0128] QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAPENLYFQGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31)

[0129] The variable regions of the LAIR1-specific antibody were cloned, expressed, and the activity of the recombinant antibody was confirmed by ELISA, and the selectivity for LAIR1 and the absence of activity against LAIR2 were demonstrated( Figure 13 ). Subsequently, the antibody was engineered for higher affinity by mutagenesis of the CDR residues and identification of enhancing mutations from the combinatorial residues for each CDR. The individual combinatorial clones were sequenced. The heavy and light chain CDRs, VH / VL, and HC / LC sequences of exemplary anti-human LAIR1 antibodies are provided in Table 1.

[0130] Anti-human LAIR1 antibodies can be generated by recombinant DNA technology. Such antibodies can be expressed in mammalian cell lines such as HEK293 or CHO, which are transiently or stably transfected using an expression system with an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC. The clarified medium into which the antibody has been secreted can be purified using techniques commonly known in the art.

[0131] Example 2: Characterization of Anti-Human LAIR1 Antibodies

[0132] Antibody Binding Affinity and Kinetics

[0133] The antibody binding affinity and kinetics were determined by surface plasmon resonance (SPR) using a Biacore 8K (Cytivia Life Sciences). Measurements were performed at 37 °C using HBS-EP+ as the running buffer (150 mM sodium chloride, 3 mM EDTA, 0.05% (w / v) surfactant P-20, and 10 mM HEPES, pH 7.4). The binding experiment used the soluble extracellular domain (ECD) of recombinantly produced LAIR1 (SEQ ID NO:17), which was diluted to the working concentration in HBS-EP+ containing 0.1 mg / mL bovine serum albumin. Using an amine coupling kit, goat anti-human κ (Southern Biotech) was immobilized on all eight flow cells of a CM4 sensor chip.

[0134] The soluble extracellular domain (ECD) of LAIR1 has the following amino acid sequence:

[0135] QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTPGSSAGPTQRPSDNSHNEHAPASQGLKAEHENLYFQ (SEQ ID NO:17).

[0136] Multiple analysis cycles were used to evaluate binding. Each cycle was performed at a flow rate of 30 μL / min and consisted of the following steps: injecting the antibody into different flow cells (25 μL of antibody at 0.5 μg / mL at 10 μL / min), injecting 75 μL (30 μL / min for a total of 150 s) of each LAIR1-ECD dilution (starting from 1 μM and using three-fold serial dilutions down to 1.4 nM for each cycle, with one injection for each concentration), followed by a 1200-s delay for dissociation, and regeneration of the chip surface using three 15-μL injections (30 μL / min for a total of 30 s) of 10 mM glycine hydrochloride, pH 1.7. The binding and dissociation rates for each cycle were determined by fitting the biosensor data to a simple 1:1 association model using the provided instrument analysis software to extract k on and k off rate constants; the equilibrium binding constant K d was calculated using the relationship K off = k on / k D .

[0137] Table 2 shows the binding affinity and kinetics of the anti-human LAIR1 mAb.

[0138] Table 2. Binding affinity and kinetics of anti-human LAIR1 mAb determined by Biacore at 37 °C.

[0139] Antibody ID kon (M-1s-1) koff (sec-1) KD (nM) mAb1 <![CDATA[1.44×10 6 > <![CDATA[1.74×10 -4 > 0.121 mAb2 <![CDATA[1.09×10 6 > <![CDATA[3.41×10 -4 > 0.312 mAb3 <![CDATA[1.34×10 6 > <![CDATA[1.68×10 -4 > 0.126 mAb4 <![CDATA[1.09×10 6 > <![CDATA[3.17×10 -4 > 0.291

[0140] Physicochemical properties of the exemplified LAIR1 antibodies

[0141] Thermal stability:

[0142] Differential scanning calorimetry (DSC) was used to evaluate the stability of the exemplified LAIR1 antibodies against thermal denaturation. DSC was run using a Malvern MircoCal VP-DSC instrument. Samples in PBS buffer were heated from 20 °C to 110 °C at a constant rate of 60 °C / hour. The analysis method was performed using the MicroCal VP-Capillary DSC Automated Analysis program. Baseline correction was performed, and Tm onset and TM were determined. The results shown in Table 3 indicate that the exemplified LAIR1 antibodies have a Tm onset > 60 °C and are thermally stable.

[0143] Table 3. Summary of DSC results

[0144] Sample Tm start (°C) <![CDATA[Tm1(℃)]]> <![CDATA[Tm2(℃)]]> mAb1 65.1 72.4 76.5 mAb2 63.5 70.3 80.8 mAb3 61.9 69.2 75.9 mAb4 61.1 68.8 80.3

[0145] Solubility:

[0146] Sufficiently high solubility is required for ease of administration. Additionally, it is desirable to maintain the antibody in a monomeric state without high molecular weight (HMW) aggregation at high concentrations. The solubility of the exemplified LAIR1 antibodies was analyzed by concentrating 15 mg of the exemplified antibody to a volume of less than 100 μl using a 10K molecular weight cut-off filter (Amicon U.C. filter, Millipore, catalog #UFC903024). The final concentration of the sample was measured using a SoloVPE spectrophotometer (C Technologies, INC). Following a procedure essentially as described above, the exemplified antibodies showed solubilities greater than 150 mg / mL (in 5 mM histidine, pH 6.0) and 200 mg / ml (in PBS buffer at pH 7.4). The concentrated solution samples were stored at 4 °C for 1 week, followed by 1 week of storage at -5 °C. The aggregation profile of the antibody after two weeks of storage was evaluated using size exclusion chromatography (SEC). As the results shown in Table 4, only low levels of high molecular weight (HMW) aggregates (about 1%) were present at high concentrations, and no phase separation was observed.

[0147] Table 4. Summary of solubility results

[0148]

[0149] Photo-stability:

[0150] The photo-stability of the exemplified antibodies was evaluated at high concentration (about 100 mg / mL) in 5 mM histidine buffer (pH 6.0) with excipients. The concentrated samples were exposed to 40 watt-hours / m 2 UV light (4 hours at 10 watts / m 2 )+ 240 klux-hours (8 klux for 30 hours) of the 20% ICH Q1B minimum exposure. Protected concentrated samples (wrapped in aluminum foil) were used as dark controls and placed together with the test samples. After exposure, the percentage of HMW aggregate growth (Δ% HMW) of the samples was analyzed by SEC. The results provided in Table 5 show that after exposure to the 20% ICH guideline, the exemplified antibodies had an HMW growth percentage between 5 and 7%.

[0151] Table 5. Summary of % HMW growth after photo-exposure

[0152] Sample Δ% HMW after light exposure (relative to dark control) mAb1 7 mAb2 6.7 mAb3 5.8 mAb4 5

[0153] Chemical stability:

[0154] Chemical stability facilitates the development of pharmaceutical formulations with sufficient shelf life. The chemical stability of the exemplified antibodies was evaluated by formulating the exemplified antibodies in a buffer solution, pH 6, to a concentration of 100 mg / ml. In an accelerated degradation study, the formulated samples were incubated at 4 °C and 35 °C for four weeks. According to standard procedures, capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and SEC were used to evaluate changes in the fragmentation and aggregation profiles of the antibodies. Following a procedure essentially as described above, the exemplified antibodies showed the chemical stability results presented in Table 6.

[0155] Table 6. Summary of Δ% fragments measured by CE-SDS and Δ% HMW measured by SEC over four weeks at 35 °C relative to samples incubated at 4 °C.

[0156]

[0157] The results provided in Table 6 show that after four weeks of storage at 35 °C, the exemplified antibodies had a percentage increase in fragments between 0.2 and 1.3%. The HMW growth levels of the exemplified antibodies were between 0.9 and 2.5%.

[0158] The chemical stability data indicate that the exemplified antibodies have sufficient chemical stability to facilitate the development of solution formulations with sufficient shelf life.

[0159] In summary, the exemplified LAIR1 antibodies demonstrated good solubility, low aggregation, chemical stability, and physical stability characteristics necessary for parenteral therapeutic administration.

[0160] Example 3: Immunogenicity evaluation of anti-human LAIR1 antibodies

[0161] In vitro and ex vivo methods were used to characterize the relative immunogenic risk of the exemplified LAIR1 mAbs, as described below.

[0162] Dendritic cell (DC) internalization assay:

[0163] This assay was performed to investigate the molecular internalization by dendritic cells derived from CD14+ monocytes. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and cultured and differentiated into DCs following a standard protocol (see Wen, Y., et al., AAPS J April 16, 2020; 22(3):68). Briefly, PBMCs were isolated from LRS-WBC by density gradient centrifugation using Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies). CD14+ monocytes were isolated using positive selection with the CD14+ microbead kit (#130-050-201, Miltenyi Biotec) following the manufacturer's manual. The cells were then cultured at 1 million / mL with 1000 units / mL GM-CSF and 600 units / mL IL-4 for 6 days to be converted into immature dendritic cells (MDDCs) in RPMI medium with L-glutamine and 25 mM HEPES, which was supplemented with 10% FBS, 1 mM sodium pyruvate, 1× penicillin-streptomycin, 1× non-essential amino acids, and 55 μM 2-mercaptoethanol (hereinafter referred to as complete RPMI medium or medium, purchased from Life Technologies). The medium was changed twice on days 2 and 5. On day 6, the cells were gently collected using a cell scraper and used for experiments. To obtain mature DCs, the cells were treated with 1 μg / mL LPS for 4 hours.

[0164] Various test molecules were normalized to 1 mg / mL with PBS and then further diluted to 8 μg / mL in complete RPMI medium. The detection probe Fab-TAMRA-QSY7 was diluted to 5.33 μg / mL in complete RPMI medium. The antibody and Fab-TAMRA-QSY7 were mixed in equal volumes and incubated for 30 minutes at 4 °C in the dark for complex formation. MDDCs were resuspended at 4 million / mL in complete RPMI medium and seeded at 50 μL / well in a 96-well round-bottom plate, to which 50 μL of the antibody / probe complex was added. The cells were incubated at 37 °C in a CO2 incubator for 24 hours. The cells were washed with 2% FBS PBS and resuspended in 100 μL of 2% FBS PBS with Cytox Green live / dead dye. Data were collected on a BD LSRFortessa X-20 and analyzed in FlowJo. Live single cells were gated, and the percentage of TAMRA-fluorescent positive cells was recorded as the readout. To allow comparison of molecules with data generated from different donors, a normalized internalization index was used. The internalization signal was normalized against the IgG1 isotype (normalized internalization index = 0) and an internal positive control PC (normalized internalization index = 100) using the following formula:

[0165]

[0166] where XTAMRA, IgG1 isotype TAMRA, and PCTAMRA are the percentages of the TAMRA-positive populations of test molecule X, IgG1 isotype, and PC, respectively. For the normalized internalization index, 0 - 15 was considered low immunogenic risk, >15 - 30 was considered low to medium immunogenic risk, >30 - 60 was considered medium immunogenic risk, and >60 was considered high immunogenic risk.

[0167] Table 7: Dendritic cell internalization results for the exemplified LAIR1 antibodies

[0168]

[0169]

[0170] As shown in Table 7, the exemplified anti-human LAIR1 antibodies have low to medium risk and medium risk from the dendritic cell internalization assay.

[0171] MAPP assay (MHC-related peptide proteomics):

[0172] MAPP profiles peptides presented by human leukocyte antigen class II (HLA-II) on human dendritic cells previously treated with test molecules. Primary human dendritic cells from a group of experimental subjects from 10 normal human donors were prepared from buffy coats by isolating CD-14 positive cells and differentiated into immature dendritic cells by incubation with 20 ng / ml IL-4 and 40 ng / ml GM-CSF in complete RPMI medium containing 5% serum replacement (Thermo Fisher Scientific, catalog #A2596101) at 37 °C and 5% CO2 for 3 days as described (Knierman et al., Cell Rep December 1, 2020; 33(9):108454). On day 4, 3 μM of the test antibody was added to approximately 5 x 106 cells and fresh medium containing 5 μg / ml LPS was replaced after 5 hours of incubation to convert the cells into mature dendritic cells. The next day, the mature cells were lysed in 1 mL RIPA buffer with protease inhibitors and DNase. The lysates were stored at -80 °C until sample analysis.

[0173] An automated liquid handling system was used to isolate HLA-II molecules from the thawed lysates using a biotinylated anti-pan HLA class II antibody (clone Tu39). The bound receptor-peptide complexes were eluted with 5% acetic acid, 0.1% TFA. The eluted HLA-II peptides were passed through a pre-washed 10 kDa MWCO filter to remove high molecular weight proteins. The isolated HLA-II peptides were analyzed by nano LC / MS using a Thermo easy 1200 nLC-HPLC system with a Thermo LUMOS mass spectrometer. Separation was performed using a 75 μm x 7 cm YMC-ODS C18 column for a 65-minute gradient with a flow rate of 250 nL / min and an aqueous solution of 0.1% formic acid as solvent A and 80% acetonitrile with 0.1% formic acid as solvent B. Mass spectrometry was run in full scan mode with 240,000 resolution, followed by a 3-second data-dependent MS / MS cycle consisting of rapid scans of the ion trap with HCD and EThcD fragmentation.

[0174] Peptide identification was generated by an in-house proteomics pipeline (Higgs et al., Methods Mol Biol. 2008;428:209-30) that used a multiple search algorithm to search a bovine / human database containing the test molecule sequences under enzyme-free search parameters. Peptides identified from the test molecules were aligned against the parental sequences. Summaries were created for all test molecules that annotated the percentage of donors that exhibited peptides with non-germline residues and the number of distinct regions in the test molecules that exhibited peptides with non-germline residues. An increase in the extent of non-germline peptide presentation was associated with an increased immunogenic risk.

[0175] Table 8: MAPP results for the exemplified LAIR antibodies

[0176]

[0177] As shown in Table 8, the exemplified anti-LAIR1 antibodies mAb1 to 4 had a medium risk from the MAPP assay.

[0178] T cell proliferation assay

[0179] This assay evaluates the ability of the test molecule to activate CD4+ T cells by inducing cell proliferation (see Walsh, R.E., et al., MAbs. 2020;12(1):1764829). Cryopreserved PBMC from 10 healthy donors were used and CD8+ T cells were depleted from the PBMC and labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). The PBMC were seeded at 4 x 106 cells / ml / well in AIM-V medium (Life Technologies, catalog #12055-083) containing 5% CTSTM Immune Cell SR (Gibco, catalog #A2596101) and tested in triplicate in 2.0 mL containing different test molecules, DMSO control, medium control, keyhole limpet hemocyanin (KLH; positive control). The cells were cultured and incubated at 37 °C with 5% CO2 for 7 days. At day 7, the samples were stained with the following cell surface markers: anti-CD3, anti-CD4, anti-CD14, anti-CD19, and DAPI for viability detection by flow cytometry using a BD LSRFortessaTM equipped with a High Throughput Sampler (HTS). Using Software (FlowJo, LLC, TreeStar) analyzed the data and calculated the cell division index (CDI). Briefly, the CDI for each test molecule was calculated by dividing the percentage of proliferating CFSEdim CD4+ T cells from the molecule-stimulated wells by the percentage of proliferating CFSEdim CD4+ T cells in the unstimulated wells. A CDI > 2.5 was considered to represent a positive response. The percentage donor frequency across all donors was evaluated. Proliferation in < 30% of donors was considered low immunogenic risk, 30 - 40% of donors was considered moderate immunogenic risk, and > 40% of donors was considered high immunogenic risk.

[0180] Table 9: T cell proliferation results for the exemplified LAIR1 antibodies

[0181]

[0182]

[0183] As shown in Table 9, the exemplified anti-human LAIR1 antibodies mAb 2 to 4 had low risk from the T cell proliferation assay. Anti-human LAIR1 mAb1 was not tested.

[0184] Determination of pre-existing reactivity:

[0185] This assay was performed to investigate the presence of reactivity of pre-existing anti-drug antibodies (PEA) and potentially other cross-reactive proteins from normal human sera used for treatment for the first time (see Bivi, N., et al., MAbs. July 2019;11(5):861 - 869). Diluted sera from a group of subjects from at least 50 donors used for treatment for the first time were captured overnight on plates coated with biotinylated test molecules. On the next day, the captured reactive proteins were acid eluted and then neutralized in the presence of biotinylated and ruthenated test molecules. If anti-drug antibodies were present, they would bridge the labeled test candidates and form complexes. The complexes were captured by streptavidin protein-coated Mesoscale plates, and the resulting signal was referred to as the Layer 1 signal (expressed as electrochemiluminescence). This signal was confirmed in Layer 2 by adding an excess of unlabeled test molecule during the detection step, which led to inhibition of the Layer 1 signal. The presence of pre-existing anti-drug antibodies was expressed as the 90th percentile of Layer 2 inhibition. Results < 30% were low immunogenic risk, 30% - 55% were moderate immunogenic risk, and > 55% were high immunogenic risk.

[0186] Table 10: Results of pre-existing reactivity for the exemplified LAIR1 antibodies

[0187] Exemplary antibody 90th percentile T2 inhibition mAb1 7.1％ mAb2 7.6％ mAb3 8.0％ mAb4 25.8％

[0188] As shown in Table 10, the exemplary anti-human LAIR1 antibodies mAb 1 to 4 have a low risk from pre-existing reactivity assays.

[0189] Example 4: In vitro activity of anti-human LAIR1 antibodies

[0190] Cell-based binding:

[0191] Anti-human LAIR1 antibodies were evaluated for binding to human and cynomolgus monkey (cyno) LAIR1-engineered cell lines and primary human T cells that endogenously express LAIR1. Jurkat-hLAIR1+ (Jurkat cells overexpressing human LAIR1), Jurkat-LAIR1ko (Jurkat cells with human LAIR1 knocked out), Jurkat-cyLAIR1+ cells (Jurkat LAIR1ko cells expressing cyno LAIR1), and primary human T cells were incubated with anti-human LAIR1 test antibodies. Serial dilutions of antibodies ranging from 0.0017 μg / mL – 3.33 μg / mL were incubated with the cells at 4 °C for 20 minutes. The cells were then washed and incubated with anti-human IgG Alexa Fluor 647 secondary antibody at 4 °C for 20 minutes. The cells were then washed and antibody binding was evaluated by flow cytometry. For primary human T cells, the cells were also stained for CD4 and CD8 to delineate CD4+ and CD8+ T cells.

[0192] As shown in Table 11, all anti-human LAIR1 test antibodies bound to Jurkat-hLAIR1+ cells, primary human CD4+ and CD8+ T cells with similar binding strengths. The EC50 for binding to Jurkat-cyLAIR1+ cells was within 2-fold of the EC50 for binding to Jurkat-hLAIR1+ cells. The test anti-LAIR1 antibodies confirmed no binding to LAIR1ko, a control cell line that does not express LAIR1.

[0193] Table 11. EC50 of anti-human LAIR1 antibody binding to Jurkat-hLAIR1+, Jurkat-cyLAIR1+, primary human CD4+ T cells, and primary human CD8+ T cells. (Data are representative of 1 - 5 independent experiments; 2 donors were evaluated for primary T cell binding.)

[0194]

[0195] Jurkat-NFAT activation:

[0196] The effect of anti-human LAIR1 antibodies on NFAT activation in Jurkat cells was evaluated. In the presence of anti-human LAIR1 antibodies, Jurkat-NFAT-luciferase cells expressing human LAIR1 (Jurkat-hLAIR1+), cyno LAIR1 (Jurkat-cyLAIR1+), or LAIR1ko-deficient (Jurkat-LAIR1ko) were stimulated with anti-human CD3 antibody for TCR. Specifically, CHO-K1 cells were seeded overnight in a 96-well flat-bottomed sterile tissue culture plate. When the confluence reached 85-95%, the cells were washed with RPMI / 5% human serum and incubated with anti-human CD3 antibody at 10 μg / mL for 1 hour at 37°C. Then the unbound CD3 antibody was removed, the cells were washed, and incubated with anti-human LAIR1 antibody at the indicated concentrations for 20 minutes at 37°C. 1x105 Jurkat-hLAIR1+, Jurkat-cyLAIR1+, or Jurkat-LAIR1ko cells were added and incubated for 6 hours at 37°C. Then the Jurkat cells were transferred to an opaque, flat, clear-bottomed 96-well plate, and an equal volume of BrightGlo luciferase was added. After a 2-minute incubation for lysis, NFAT activity (via luciferase readout) was evaluated by a luminometer. In the activation assay, anti-LAIR1 antibodies were tested at concentrations ranging from 0.1 ng / mL - 1 μg / mL, with 8 steps of serial dilution with 3-fold decreasing titration.

[0197] As shown in Table 12, all anti-human LAIR1 test antibodies inhibited Jurkat NFAT activation in Jurkat-NFAT cells expressing human and cyno LAIR1, with inhibition ranging from 60 - 70%, while the isotype control had no effect on NFAT activity. Similar IC50 values were observed among the tested anti-human LAIR1 antibodies (Table 12). No IC50 value was available for Jurkat-cyLAIR1+ cells. Anti-human LAIR1 antibodies had no effect on NFAT activity in Jurkat-LAIR1ko cells. Values for Jurkat-LAIR1ko were not shown because anti-LAIR1 antibodies did not exhibit inhibitory effects on this control cell line.

[0198] Table 12. In vitro agonistic activity of anti-human LAIR1 antibodies on NFAT activity in Jurkat cells after TCR stimulation. (Data represent 2 to 5 independent experiments.)

[0199]

[0200] Anti-human LAIR1 antibody mAb4 inhibits NFAT activation in an in vitro cell-based agonistic assay:

[0201] The ability of LAIR1 agonist antibodies to inhibit NFAT activation in a human T cell line overexpressing human LAIR1 was determined as follows.

[0202] Jurkat-NFAT-luciferase reporter cells were engineered to overexpress human LAIR1 (Jurkat-hLAIR1+) via lentiviral transduction. In the presence of cross-linked anti-human LAIR1 mAb4 or hIgG4SP isotype control antibody, Jurkat-hLAIR1+ cells were TCR-stimulated with anti-human CD3 antibody (clone OKT3). Antibody cross-linking was performed using a Chinese hamster ovary (CHO) cell line engineered to express human FcγRIIb.

[0203] CHO-K1 cells were seeded overnight at 37 °C in a 96-well flat-bottomed tissue culture sterile plate. When confluence reached 85–95%, the cells were washed with RPMI / 5% human serum and incubated with anti-human CD3 antibody at 37 °C for 1 hour. Then the unbound CD3 antibody was removed, the cells were washed, and incubated with mAb4 or hIgG4SP isotype antibody at 37 °C for 20 minutes. 1 x 105 Jurkat-hLAIR1+ cells were added to the plate and incubated at 37 °C for 6 hours. Then the Jurkat-hLAIR1+ cells were transferred to an opaque, flat, clear-bottomed 96-well plate, and an equal volume of BrightGlo luciferase was added. After incubation for 2 minutes for cell lysis, NFAT activity (via luciferase readout) was assessed by luminometer. Antibodies were evaluated at concentrations ranging from 0.001–6.7 nM (0.2–1000 ng / mL), with serial dilutions for IC50 assessment of NFAT activity.

[0204] As Figure 3 shown, mAb4 inhibited Jurkat-hLAIR1+ NFAT activation, with inhibition ranging from 60–70% (calculated as % NFAT activity of antibody relative to no antibody) and an IC50 value of 0.045 nM (6.7 ng / mL), while the hIgG4SP isotype control antibody had no effect on NFAT activity. As a parallel control, the antibodies were evaluated in LAIR1-deficient Jurkat-NFAT-luciferase cells (Jurkat-hLAIR1ko). mAb4 had no effect on NFAT activity in Jurkat-hLAIR1ko cells (data not shown).

[0205] Primary human B cells:

[0206] The effect of anti-human LAIR1 antibodies on cytokine responses of primary human B cells was evaluated. In the presence of test anti-human LAIR1 antibodies at concentrations ranging from 0.00128 ng / mL to 8 ng / mL, primary human B cells were stimulated via BCR with anti-human IgM antibody plus IL4. Specifically, CHO-K1 cells were seeded overnight in 96-well flat-bottomed sterile tissue culture plates. When the confluence reached 85 - 95%, the cells were washed with RPMI / 5% human serum and incubated with anti-human LAIR1 antibody at the indicated concentrations for 20 minutes at 37°C. 1 - 1.5 x 105 B cells were added and incubated for an additional 20 minutes at room temperature to allow cell / antibody interaction. Then, stimulants or controls (20 ng / mL IL-4 + 5 μg / mL anti-human IgM or medium alone as an unstimulated control) were added, and the cells were incubated at 37°C for 72 hours. The test antibodies were also evaluated against an anti-human IgG isotype control. The effect of LAIR1 engagement on B cell IL-6 responses was evaluated by ELISA at 72 hours and reported as % inhibition compared to the no-antibody control.

[0207] As shown in Table 13, all anti-human LAIR1 test antibodies inhibited the IL6 response, with inhibition ranging from 20 - 70% depending on the donor at the highest dose of 8 ng / mL, while the isotype control had no effect on cytokine responses.

[0208] Table 13. In vitro agonistic activity of anti-human LAIR1 antibodies on primary B cells after BCR stimulation.

[0209]

[0210]

[0211] The anti-human LAIR1 antibody mAb4 inhibits primary B cell cytokine responses in an in vitro cell-based agonistic assay:

[0212] The ability of LAIR1 agonist antibodies to inhibit BCR-stimulated IL-6 responses in primary human B cells was determined as follows.

[0213] In the presence of cross-linked anti-human LAIR1 mAb4 or hIgG4SP isotype control antibody, primary human B cells (n = 6 donors) were stimulated via BCR with anti-human IgM antibody plus IL-4. Antibody cross-linking was achieved using a Chinese hamster ovary (CHO) cell line engineered to express human FcγRIIb.

[0214] CHO-K1 cells were seeded overnight at 37 °C in a 96-well flat-bottomed sterile tissue culture plate. When the confluence reached 85 - 95%, the cells were washed with RPMI / 5% human serum and incubated with mAb4 or hIgG4SP isotype antibody at 37 °C for 20 minutes. 1x105 isolated B cells from human PBMCs were added and incubated for an additional 20 minutes at room temperature to allow cell / antibody interaction. Then stimulants (anti-human IgM + IL-4) or controls (media alone + / − IL-4) were added, and the cells were incubated at 37 °C for 72 hours. The effect of the antibody on B cell IL-6 response was evaluated by ELISA at 72 hours and reported as % inhibition compared to the no-antibody control. Antibodies were evaluated at concentrations ranging from 0.000013–0.0539 nM (0.002–8 ng / mL), and serial dilutions were used for IC50 assessment of inhibition of B cell IL-6 response.

[0215] As Figure 4 shown, mAb4 inhibited B cell IL-6 response to BCR stimulation, with inhibition ranging from 60 - 80% (calculated as % IL-6 response of antibody relative to no antibody) and an IC50 value of 0.0002 nM (0.03 ng / mL), while the hIgG4SP isotype control antibody had no effect on IL-6 response.

[0216] Primary human T cells:

[0217] The effect of anti-human LAIR1 antibodies on primary human T cell cytokine responses was evaluated. In the presence of anti-human LAIR1 antibodies at concentrations ranging from 1 ng / mL–1 μg / mL, primary human T cells were TCR-stimulated with anti-human CD3 and anti-human CD28 antibodies. Specifically, T cells were incubated overnight at 37 °C with anti-human CD3 antibody at 1 μg / mL and anti-human CD28 antibody at 3 μg / mL bound to the plate. CHO-K1 cells were seeded overnight in a 96-well flat-bottomed sterile tissue culture plate. When the confluence reached 85 - 95%, the cells were washed with RPMI / 5% human serum and incubated with anti-human LAIR1 antibody at the indicated concentrations at 37 °C for 20 minutes. The stimulated T cells were washed and resuspended in fresh RPMI / 5% human serum. Then 1 - 1.5x105 T cells were plated on the CHO-K1 cells and incubated at 37 °C for 72 hours. After incubation, the supernatant was harvested and IFN-γ secretion was evaluated via ELISA.

[0218] As shown in Table 14, all anti-human LAIR1 test antibodies inhibited IFN-γ response, with inhibition depending on the donor ranging from 20 - 80% at the highest dose of 1 μg / mL, while the isotype control had no effect.

[0219] Table 14. In vitro agonistic activity of anti-human LAIR1 antibodies on primary T cells after TCR stimulation.

[0220]

[0221] Example 5: In vivo activity of anti-human LAIR1 antibodies in a murine model engrafted with human PBMCs for graft-versus-host disease (GvHD)

[0222] To test the immunomodulatory activity of the exemplified antibodies mAb1 - mAb4, a humanized model of xenogeneic GvHD was utilized. Human immune cells recognize the mouse as foreign and mount an immune response, resulting in a significant increase in human pro-inflammatory cytokines, activation and expansion of immune cells, and production of immunoglobulins. Importantly, the inflammatory response is driven by human cells and thus human-specific therapies can be investigated in this model.

[0223] Briefly, female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, Stock#05557) were housed three per cage at 72 °C on a 12-hour light:dark cycle and allowed ad libitum access to food and water. Human PBMCs were isolated from LRS tubes obtained from a single anonymous donor (San Diego Blood Bank) using SepMate 50 Ficoll preparation tubes according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC). Freshly isolated PBMCs were suspended in Pedialyte solution at 1.2 x 108 cells / mL and the mice were implanted intravenously on day 0 with 100 μL of the PBMC suspension (1.2 x 107 / mouse, n = 36). The mice were divided into 5 groups and were dosed subcutaneously on days 1 and 8 with an isotype control or mAbs 1-4 at 0.3 mg / kg (200 μL / mouse; n = 7-8 / group). On day 7, the mice were briefly anesthetized with isoflurane and blood was obtained from the retro-orbital sinus. On day 14, the mice were anesthetized again, blood was collected by cardiac puncture, and the mice were euthanized. The mice were weighed in a BSL2 hood and clinical signs of distress were evaluated 2-3 times / week. Common clinical symptoms in this model were ruffled fur, hunched body, wasting, and difficulty breathing or moving. Blood from the two collections was clarified by centrifugation and the resulting plasma was stored at -80 °C for future processing. Plasma cytokines were measured using the Human Pro-inflammatory 10-Vplex and IgM, IgA, and IgG were measured using the Human Isotyping Panel (Meso Scale Discovery, Rockville, Maryland) according to the manufacturer's instructions.

[0224] Data graphs were plotted and statistics were calculated using Prism Software (GraphPad, San Diego, CA). Differences in plasma analytes compared to the isotype control were determined by one-way ANOVA with Dunnett's post-test and the differences were considered significant if p < 0.05.

[0225] In experiments performed essentially as described above, the exemplified anti-human LAIR1 antibodies significantly inhibited the significant increases in plasma human pro-inflammatory cytokines (IFN-γ, IL-10, and TNF-α) associated with disease progression in the GvHD model. Additionally, the exemplified anti-human LAIR1 antibodies mAb1-mAb3 significantly reduced circulating immunoglobulins IgM and IgA, suggesting an inhibitory effect on B cells.

[0226] The results confirmed that the exemplified anti-human LAIR1 antibody has an immunomodulatory effect on human immune cells in a humanized mouse disease model.

[0227] Humanized graft-versus-host disease:

[0228] The antibody described as mAb4 herein was tested in NOD SCIDγ2 chain - / -(NSG) humanized mice to evaluate its ability to inhibit human T cell function in an in vivo setting. Human peripheral blood mononuclear cells (PBMCs) were implanted into NSG mice, where the human immune cells recognize the mice as foreign and mount an immune response, resulting in graft-versus-host disease (GvHD). The aim was to evaluate the ability of mAb4 to agonize LAIR1 and inhibit T cell activation, as measured by proinflammatory cytokine production, and to correlate these effects with drug exposure and immune cell receptor occupancy to aid in human dose prediction. 1.2e7 human PBMCs were injected intravenously into NSG mice. Twenty-four hours after cell implantation, mAb4 or human IgG4P isotype control (3 mg / kg) was administered subcutaneously once at semi-logarithmic increments starting from 0.003 - 3 mg / kg, and the mice were euthanized on day 8. Blood was obtained via retro-orbital sinus on day 5 and via cardiac puncture on day 8, and processed for analysis of serum cytokines (MSD human proinflammatory cytokine panel) and drug exposure (antigen capture ELISA). Spleens were harvested on day 8, processed into single-cell splenocytes, and immunophenotyping and receptor occupancy (RO) were analyzed by FACS. mAb4 inhibited immune cell-related proinflammatory cytokines in a dose-dependent manner, indicating T cell function inhibition ( Figure 5 ). These beneficial activities were mechanistically supported by post-mortem FACS analysis of splenocytes, where dose-dependent receptor occupancy of LAIR1 was observed on regulatory T cells (Tregs), CD4, and CD8 T cells ( Figure 6 ). Drug exposure measured on days 4 and 7 after dosing demonstrated a similar dose-dependence to receptor occupancy. Interestingly, the PD response was more robust than implied by exposure or RO, suggesting that full RO is not required to elicit the beneficial agonistic effect. Additionally, the increase in the percentage of Tregs observed in the high-dose group provides another potential therapeutic mechanism by which LAIR1 agonism may prove beneficial in the treatment of autoimmune diseases ( Figure 8 ).

[0229] Summary

[0230] The pharmacodynamic activity of mAb4 was evaluated in a humanized mouse model of graft-versus-host disease. In this model, mice lacking a complete immune system were implanted with human donor immune cells. After implantation, the human immune cells launched an inflammatory attack on the mice. This was measured by the production of human cytokines in the mouse peripheral blood. The LAIR1 agonist antibody mAb4 was able to reduce the production of these cytokines in a dose-dependent manner( Figure 5 ). This reduction was associated with the amount of receptor occupancy( Figure 6 ) and the serum concentration of mAb4( Figure 7 ). In addition, mAb4 was able to increase the percentage of the Treg cell population in the spleen( Figure 8 ), which could explain another mechanism of action for controlling the inflammatory response in this model.

[0231] Example 6: In Vivo Study of Anti-Human LAIR1 Antibody in a Mouse Model of Spontaneous Lupus Nephritis

[0232] Primary Pharmacodynamics

[0233] Type I Interferon Lupus Nephritis:

[0234] NZB / WF1 mice were used as a classical model of spontaneous lupus nephritis. To accelerate and synchronize disease induction, we injected the mice with adeno-associated virus (AAV) expressing murine IFNα5. Therapies targeting T cells and B cells have been shown to reduce the disease severity in this model. The aim of this study was to confirm whether alternative LAIR1 agonist antibodies could affect the disease severity in a preclinical model of lupus nephritis.

[0235] Lupus Model:

[0236] Female NZB / WF1 mice (Jackson Laboratories) were 10 weeks old at arrival. All mice were housed five per cage and allowed to acclimate for 1 week before the start of the study. Mice were fed Teklad Irradiated Global 18% Protein Rodent Diet 2908 (Innotiv) and given water ad libitum. Mice were maintained on a 12-hour light / dark cycle with an ambient temperature range of 68 - 79°F. Mice were sorted based on body weight and one day later (day 0) mice were injected intravenously with LacZ-AAV (non-diseased control, 10^11 genome copies (GC)) or murine IFNα5-AAV (3x10^12 GC) in 100 μl PBS. The assigned treatment groups were: (1) LacZ-AAV induced, treated with PBS (s.c., BID starting on day 7, n = 5), (2) IFN-AAV induced, treated with IgG isotype used as a surrogate antibody (10 mg / kg s.c., BID starting on day 7, n = 10), (3) IFN-AAV induced, treated with a surrogate antibody (10 mg / kg s.c., BID starting on day 7, n = 10), (4) IFN-AAV induced, treated with a surrogate antibody (10 mg / kg s.c., BID starting on day 21, n = 10), and (5) IFN-AAV induced, treated with cyclophosphamide (15 mg / kg i.p., Q10D starting on day 7, n = 10). During the study, serum and urine samples were collected at baseline and every 2 weeks. At 42 days post-AAV injection, mice were euthanized and body weights were collected. Both kidneys were collected and weighed as a pair. The right kidney was fixed in 10% neutral buffered formalin for 24 - 48 hours and then transferred to 70% ethanol.

[0237] Albumin and creatinine assays:

[0238] To monitor renal function, microalbumin concentrations in urine (dilution 1:500–1:50,000) samples were determined by ELISA (Mouse Microalbumin ELISA kit, Kamiya Biomedical Co, Seattle, WA) according to the manufacturer's instructions. Urine creatinine was measured by using the CREP2 enzymatic creatinine assay with a Cobas C501 clinical chemistry analyzer (Roche Diagnostics, USA) according to the manufacturer's instructions.

[0239] Histology:

[0240] Kidneys from each mouse were embedded in paraffin, sectioned, and stained with hematoxylin and eosin PAS.

[0241] Histological scoring: The scores for inflammation, glomerular changes, and tubular protein are based on the following criteria and, when added together, result in a total score.

[0242] Inflammation: The score of 0 - 3 is based on a combination of the number of affected areas and the amount of affected areas.

[0243] Glomerular scoring: The glomerular score (0 - 6) is based on the evaluation of glomeruli in the outer half of the cortex and the most common grade encountered in this area, due to variability between glomeruli within each kidney: -

[0244] · Grade 1 - Minimal increase in cellular structure and / or mesangial dilation + / - minimal increase in glomerular size (less than 2-fold).

[0245] · Grade 2 - Mild increase in cellular structure and mesangial dilation, accompanied by at least a 2-fold increase in the size of most glomeruli.

[0246] · Grade 3 - Moderate increase in cellular structure in most affected glomeruli and some areas of significant mesangial dilation and / or capillary proliferation, accompanied by a maximum 3-fold increase in glomerular size.

[0247] · Grade 4 - Marked increase in cellular structure in most affected glomeruli and some areas of significant mesangial dilation and / or capillary proliferation, accompanied by a maximum 4-fold increase in glomerular size; rare sclerotic glomeruli; may have parietal cell hypertrophy.

[0248] · Grade 5 - Above, accompanied by <25% glomerular sclerosis and / or capillary proliferation in most affected glomeruli; maximum 5-fold increase in glomerular size.

[0249] · Grade 6 - Above, accompanied by >25% glomerular sclerosis, characterized in part by a reduction in tuft cell structure + / - periglomerular fibrosis + / - parietal cell hypertrophy.

[0250] PAS scoring: The PAS score of 0 - 3 is based on the presence of increased mesangial matrix staining in glomeruli in the outer half of the cortex compared to control sections cut at the same thickness.

[0251] · Grade 1 - Minimal increase in mesangial staining scattered in glomeruli.

[0252] · Grade 2 - More extensive dilation of mesangium (and thus PAS staining) affecting more glomeruli.

[0253] · Grade 3 - Marked dilation of mesangium in most glomeruli.

[0254] Tubular protein scoring: The score of 0 - 3 is based on the percentage of tubules containing proteinaceous fluid.

[0255] · Grade 1 - <25% of the renal tubules are affected.

[0256] · Grade 2 - 25 - 50% of the renal tubules are affected.

[0257] · Grade 3 - >50% of the renal tubules are affected.

[0258] The total histological score is calculated using the sum of scores for 4 parameters.

[0259] Statistical analysis:

[0260] Statistical analysis was performed using one-way ANOVA, followed by Dunnett's post hoc test to compare IFNα-induced relative to treatment with IgG isotype.

[0261] Summary

[0262] Administration of mIFNα-AAV to NZB / WF1 mice induced lupus nephritis, characterized by increased urinary ACR levels and histological scores in the IgG isotype group compared to mice administered non-pathogenic LacZ-AAV ( Figure 5 and 7 ). Therapeutic treatment with alternative antibodies starting from day 21 (D21) reduced urinary ACR levels at the end of the study on day 44 compared to the IgG isotype group, but was less effective than the positive control cyclophosphamide (CP) administered starting from day 7 (D7) ( Figure 9 and 10 ). However, prophylactic treatment with alternative antibodies starting from D7 did not significantly reduce urinary ACR ( Figure 9 and 10 ). Histological evaluation of kidneys from mice showed that alternative antibodies reduced histological scores by 40% and 36% when treatment was started at D7 and D21, respectively ( Figure 11 ). Compared to the IgG isotype control, the effects were not statistically significant, with p-values of 0.06 and 0.09, respectively ( Figure 11 ).

[0263] The results confirmed that alternative agonistic antibodies can modulate the disease in a preclinical model of lupus nephritis.

[0264] Example 7: Epitope mapping of parental mAb4 to the LAIR1 extracellular domain (ECD) protein by hydrogen-deuterium exchange mass spectrometry

[0265] Hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS) was performed to determine the location where parental mAb4 binds to the ECD of LAIR1.

[0266] Use nepenthesin II (Nep II) for digestion. Peptide identification of LAIR1 ECD was performed on a Waters Synapt G2Si (Waters Corporation) instrument using 3.5 μg of LAIR1 ECD protein (diluted 1:10 in 0.1X phosphate-buffered saline in H2O) at zero exchange. The mass spectrometer was set to HDMSe (MobilityESI+ mode) with a mass acquisition range of m / z 255.00–1950.00 and a scan time of 0.4 s. Data were processed using PLGS 2.3.03 (Waters Corporation). For the exchange experiment, a complex of LAIR1 ECD protein and mAb4 was prepared at a molar ratio of 1:1.2 in 10 mM sodium phosphate buffer, pH 7.4 (1xPBS buffer) containing 150 mM NaCl. Using a custom TECAN sample preparation system (Espada et al., 2019), the experiment was initiated by adding various time amounts (0 s, 10 s, 2 min, 10 min, and 60 min) of 25 μL of D2O buffer containing 0.1xPBS to 2.5 μl of LAIR1 ECD (0.7 mg / mL) or LAIR1 ECD + mAb4 complex at 15 °C. The reaction was quenched for two minutes at 4 °C using an equal volume of 0.32 M TCEP, 0.1 M phosphate pH 2.5, and immediately frozen at –70 °C. The sample injection system consisted of a UR3 robot, a LEAP PAL3 HDX autosampler, and an HPLC system interfacing with the Waters Synapt G2Si (Waters Corporation), with modifications as described (Espada et al., 2019, https: / / pubmed.ncbi.nlm.nih.gov / 31724102 / ). The LC mobile phase consisted of water (A) and acetonitrile (B) each containing 0.2% formic acid. Each sample was thawed for 1 minute using 50 μL of 0.2% formic acid aqueous solution, pH 2.5, and then injected onto a Nep II column for digestion at 4 °C with mobile phase A at a flow rate of 250 μL / min for 2.5 minutes. The resulting peptides were captured on a Waters BEH Vanguard Pre-column at 4 °C and chromatographically separated using a Waters Acquity UPLC BEH C18 analytical column at 4 °C with a flow rate of 200 μL / min and a gradient of 3%–85% mobile phase B in 7 minutes, and introduced into the mass spectrometer for mass spectrometry analysis. The Synapt G2Si was calibrated with Glu-fibrinopeptide (Waters Corporation) before use.In the HDMS mode, mass spectra were acquired in the m / z range of 255 to 1950, with a lock mass m / z of 556.2771 (leucine enkephalin, Waters Corporation). Using the peptide list identified in DynamX 3.0 (Waters Corporation), relative deuterium incorporation for each peptide was determined by processing MS data on the deuterated sample along with the non-deuterated control. Peptides from the free and bound states of the RBD were compared for differences in deuterium incorporation to identify protected regions indicative of the binding epitope.

[0267] The sequence coverage across the LAIR1 ECD was 77%. For the parental mAb4, reduced deuterium uptake was observed upon binding to LAIR1 ECD in residues 26 - 41 (FVCRGPVGVQTFRLER) (SEQ ID NO:32) and 53 - 68 (VSQASPSESEARFRI) (SEQ ID NO:33) that point to the likely epitope regions. The parental mAb4 sequence is shown in Table 1 above.

[0268] Example 6: C1q Binding Results

[0269] A 96-well microplate was coated with 100 μL / well of each antibody diluted in DPBS (Dulbecco’s HyClone) in a concentration range from 10 μg / mL to 0.19 μg / mL. The tests were performed in duplicate wells. The plate was sealed and incubated overnight at 4 °C. The coating reagent was removed from each well, and 200 μL / well of casein blocking reagent (Thermo) was added. The plate was sealed and incubated for 2 hours at room temperature (RT). Each well was washed 3 times with wash buffer (1x TBE with 0.05% Tween20). 100 microliters of human C1q (MSBiomedical) diluted in casein blocking reagent at 10 μg / mL was added to each well and incubated for 3 hours at RT. The plate was then washed three times with wash buffer, and then 100 μL / well of sheep anti-human C1q-HRP (Abcam #ab46191) diluted 1:800 in casein blocker was added and incubated for 1 hour at RT. The plate was washed 6 times with wash buffer, and 100 μL / well of TMB substrate (Pierce) was added to each well and incubated for 7 minutes. 100 microliters of 1N HCl was added to each well to terminate the reaction. The optical density was immediately measured using a colorimetric microplate reader set at 450 nm. The results showed that mAb4 and the humanized IgG4-P isotype control antibody as well as the human IgG1 isotype control antibody did not bind to the complement component C1q. The anti-LAIR1 IgG1 antibody and the human IgG1 isotype control antibody did bind to the complement component C1q as expected.

[0270] The results indicated that mAb4 was unlikely to elicit Fc-mediated effector function responses in vivo.

[0271] Sequence Listing

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

Claims

1. An antibody that binds to human LAIR1, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:3, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:

6.

2. The antibody according to claim 1, wherein the VH comprises a sequence having at least 95% sequence identity with SEQ ID NO:7, and the VL comprises a sequence having at least 95% sequence identity with SEQ ID NO:

8.

3. The antibody according to claim 1 or 2, wherein the VH comprises SEQ ID NO:7, and the VL comprises SEQ ID NO:

8.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a human antibody.

5. The antibody according to any one of claims 1 to 4, wherein the antibody has a human IgG2 or IgG4 isotype.

6. The antibody according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain (HC) comprising SEQ ID NO:9 and a light chain (LC) comprising SEQ ID NO:

10.

7. The antibody according to any one of claims 1 to 5, wherein the antibody comprises a HC comprising SEQ ID NO:25 and a LC comprising SEQ ID NO:

10.

8. An antibody that binds to human LAIR1, wherein the antibody comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:13, HCDR2 comprises SEQ ID NO:14, HCDR3 comprises SEQ ID NO:15, LCDR1 comprises SEQ ID NO:16, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:

18.

9. The antibody according to claim 8, wherein the VH comprises a sequence having at least 95% sequence identity with SEQ ID NO:19, and the VL comprises a sequence having at least 95% sequence identity with SEQ ID NO:

20.

10. The antibody according to claim 8 or 9, wherein the VH comprises SEQ ID NO:19, and the VL comprises SEQ ID NO:

20.

11. The antibody according to any one of claims 8 to 10, wherein the antibody is a human antibody.

12. The antibody according to any one of claims 8 to 11, wherein the antibody has a human IgG2 or IgG4 isotype.

13. The antibody according to any one of claims 8 to 12, wherein the antibody comprises an HC comprising SEQ ID NO:21 and an LC comprising SEQ ID NO:

22.

14. The antibody according to any one of claims 8 to 12, wherein the antibody comprises an HC comprising SEQ ID NO:27 and an LC comprising SEQ ID NO:

22.

15. The antibody according to any one of claims 1 to 14, wherein the antibody is an agonist of LAIR1.

16. The antibody according to any one of claims 1 to 15, wherein the antibody further binds cynomolgus monkey LAIR1.

17. A nucleic acid comprising a sequence encoding SEQ ID NO:9, 25, 10, 21, 27 or 22.

18. A vector comprising the nucleic acid according to claim 17.

19. The vector according to claim 18, wherein the vector comprises a first nucleic acid sequence encoding SEQ ID NO:9 or 25 and a second nucleic acid sequence encoding SEQ ID NO:

10.

20. The vector according to claim 18, wherein the vector comprises a first nucleic acid sequence encoding SEQ ID NO:21 or 27 and a second nucleic acid sequence encoding SEQ ID NO:

22.

21. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9 or 25, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

10.

22. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:21 or 27, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

22.

23. A cell comprising the vector according to any one of claims 18 to 20.

24. A cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9 or 25, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

10.

25. A cell comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:21 or 27, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

22.

26. The cell according to any one of claims 23 to 25, wherein the cell is a mammalian cell.

27. A method for producing an antibody, comprising culturing the cell according to any one of claims 23 to 26 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

28. An antibody produced by the method according to claim 27.

29. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 16 or 28, and a pharmaceutically acceptable excipient, diluent or carrier.

30. A method for treating an autoimmune disease or a fibrotic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 16 or 28.

31. The method according to claim 30, wherein the autoimmune disease or fibrotic disease is selected from rheumatoid arthritis, psoriasis, systemic lupus erythematosus, lupus nephritis, pemphigus vulgaris, systemic sclerosis, idiopathic pulmonary fibrosis, scleroderma, ulcerative colitis, Crohn's disease, hidradenitis suppurativa, atopic dermatitis, multiple sclerosis, scleroderma-associated interstitial lung disease, IgG4-related disease or chronic fibrotic interstitial lung disease.

32. The antibody according to any one of claims 1 to 16 or claim 28, wherein the antibody does not form a complex with the LAIR1 ligand Clq.

33. The antibody according to any one of claims 1 to 16 or claim 28, wherein the antibody does not require full receptor occupancy (RO) to elicit agonistic effects.

34. The antibody according to any one of claims 1 to 16, 28, 32 or 33, for use in therapy.

35. The antibody according to any one of claims 1 to 16, 28, 32 or 33, for use in treating an autoimmune disease or a fibrotic disease.

36. The antibody for use according to claim 35, wherein the autoimmune disease or fibrotic disease is selected from rheumatoid arthritis, psoriasis, systemic lupus erythematosus, lupus nephritis, pemphigus vulgaris, systemic sclerosis, idiopathic pulmonary fibrosis, scleroderma, ulcerative colitis, Crohn's disease, hidradenitis suppurativa, atopic dermatitis, multiple sclerosis, scleroderma-associated interstitial lung disease, IgG4-related disease or chronic fibrotic interstitial lung disease.

37. The antibody for use according to claim 35, wherein the autoimmune disease or fibrotic disease is systemic lupus erythematosus or lupus nephritis.

38. Use of an antibody according to any one of claims 1 to 16, 28, 32 or 33 in the manufacture of a medicament for treating an autoimmune disease or a fibrotic disease.

39. The use according to claim 38, wherein the autoimmune disease or fibrotic disease is selected from rheumatoid arthritis, psoriasis, systemic lupus erythematosus, lupus nephritis, pemphigus vulgaris, systemic sclerosis, idiopathic pulmonary fibrosis, scleroderma, ulcerative colitis, Crohn's disease, hidradenitis suppurativa, atopic dermatitis, multiple sclerosis, scleroderma-associated interstitial lung disease, IgG4-related disease or chronic fibrotic interstitial lung disease.

40. The use according to claim 39, wherein the autoimmune disease or fibrotic disease is systemic lupus erythematosus or lupus nephritis.

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