Anti-human LAIR1 antibody

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

[Technical Field]

[0001] (Sequence listing file) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided in a file entitled "30426 Sequence Listing," created on August 10, 2023, and is 38 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to antibodies that bind to human LAIR1 ("anti-human LAIR1 antibodies," "anti-LAIR1 antibodies," or "human LAIR1 antibodies"), compositions comprising such anti-human LAIR1 antibodies, and methods of using such anti-human LAIR1 antibodies. [Background technology]

[0003] Human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1, also known as CD305) is an inhibitory receptor found on peripheral mononuclear cells, 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 by preventing the lysis of cells recognized as self.

[0004] 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 containing two conserved motifs called immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LAIR1 is structurally related to several other inhibitory immunoglobulin superfamily members, including LILRB, which is localized in the leukocyte receptor complex (LRC) on human chromosome 19q13.4, suggesting that these molecules evolved from a common ancestral gene.

[0005] LAIR1 expression is altered in several autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) (see Zhang Y. et al., Clin Exp Immunol. 2018 May;192(2):193-205). Due to the immunoinhibitory function of LAIR1, there is a need for antibodies that modulate LAIR1 activity, which can be used as therapeutic agents to treat autoimmune diseases. Such antibodies can be used to treat autoimmune diseases, including SLE and lupus nephritis. Currently, standard treatments include numerous steroids, which have many undesirable 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

[0006] Provided herein are novel anti-human LAIR1 antibodies or antibody fragments thereof. In some embodiments, the anti-human LAIR1 antibodies or antibody fragments thereof provided herein are LAIR1 agonists. In some embodiments, the anti-human LAIR1 antibodies provided herein are human antibodies, for example, human IgG2 or IgG4 isotypes. In some embodiments, the anti-human LAIR1 antibodies provided herein also bind to cynomolgus monkey LAIR1.

[0007] In some embodiments, provided herein is an antibody that binds to human LAIR1, the antibody comprising 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.

[0008] In some embodiments, provided herein is an antibody that binds to human LAIR1, the antibody comprising 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.

[0009] 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.

[0010] 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.

[0011] In another aspect, provided herein are nucleic acids encoding the heavy or light chain, i.e., VH or VL, of the novel anti-human LAIR1 antibodies described herein, and vectors or cells comprising such nucleic acids.

[0012] In another aspect, provided herein is a pharmaceutical composition comprising an antibody, nucleic acid, or vector described herein.

[0013] 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.

[0014] In another embodiment, the antibody of the present invention is an antibody that does not form a complex with the LAIR1 ligand C1q.

[0015] According to yet another aspect of the invention, the antibody of the invention increases the population of Treg cells in the spleen.

[0016] In a still further aspect of the invention, the antibodies of the invention do not require full receptor occupancy (RO) to induce agonism against human LAIR1. [Brief explanation of the drawings]

[0017] [Figure 1A] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb4 significantly inhibited the increase in plasma human inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F) on days 7 (1A to 1C) and 14 (1D to 1F) in a GvHD model. [Figure 1B] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb4 significantly inhibited the increase in plasma human inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F) on days 7 (1A to 1C) and 14 (1D to 1F) in a GvHD model. [Figure 1C] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb4 significantly inhibited the increase in plasma human inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F) on days 7 (1A to 1C) and 14 (1D to 1F) in a GvHD model. [Figure 1D] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb4 significantly inhibited the increase in plasma human inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F) on days 7 (1A to 1C) and 14 (1D to 1F) in a GvHD model. [Figure 1E] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb4 significantly inhibited the increase in plasma human inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F) on days 7 (1A to 1C) and 14 (1D to 1F) in a GvHD model. [Figure 1F]The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb4 significantly inhibited the increase in plasma human inflammatory cytokines IFN-γ (1A and 1D), TNF-α (1B and 1E), and IL-10 (1C and 1F) on days 7 (1A to 1C) and 14 (1D to 1F) in a GvHD model. [Figure 2A] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb3 significantly reduce circulating immunoglobulins IgM and IgA. [Figure 2B] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb3 significantly reduce circulating immunoglobulins IgM and IgA. [Figure 2C] The figures show that the exemplified anti-human LAIR1 antibodies mAb1 to mAb3 significantly reduce circulating immunoglobulins IgM and IgA. [Figure 3] Figure 1 shows that mAb4 inhibits TCR-stimulated NFAT activation in Jurkat-hLAIR1+ cells in vitro. A. Inhibition of NFAT activity after TCR stimulation reported as % antibody-mediated inhibition relative to NFAT activity in the absence of antibody. mAb4 NFAT IC50 curve calculated and averaged from n=2 independent experiments. [Figure 4] Figure 1 shows that mAb4 agonist antibodies inhibit BCR-stimulated IL-6 responses in human B cells in vitro. A. Inhibition of IL-6 responses after BCR stimulation reported as % antibody-mediated inhibition of IL-6 responses in the absence of antibody. mAb4 B cell IL-6 IC50 curves calculated and averaged from Bn=3 independent experiments. [Figure 5] Figure 1 shows that mAb4 dose-dependently and significantly inhibited serum human cytokine production compared to isotype control measured 8 days after engraftment (7 days after treatment). One-way ANOVA followed by Dunnett's post-hoc test for isotype (mean ± SEM, n = 7–8). [Figure 6] Dose-dependent receptor occupancy of mAb4 in three T cell subsets 7 days after a single SC administration is shown. [Figure 7]Dose-dependent serum drug concentrations of mAb4 are shown 4 days (blue bars) and 7 days (red bars) after a single SC administration. [Figure 8] Figure 1 shows that mAb4 induced Treg proliferation in a dose-dependent manner. [Figure 9] Urinary albumin-to-creatinine ratio (ACR) of IFNα-induced NZB / W F1 mice treated with IgG isotypes, surrogate antibodies, or cyclophosphamide (CP) starting on day 7 (D7) or day 21 (D21) is shown. One-way ANOVA followed by Dunnett's post-hoc test for IFNα induction with IgG isotypes, mean ± SEM, n = 5–10. [Figure 10] Urinary albumin-to-creatinine ratio (ACR) on day 44 of IFNα-induced NZB / W F1 mice treated with IgG isotype, surrogate antibody, or cyclophosphamide (CP) starting on day 7 (D7) or day 21 (D21) is shown. One-way ANOVA followed by Dunnett's post-hoc test for IFNα induction treated with IgG isotype, mean ± SEM, n = 5–10. [Figure 11] Kidney gross histology scores are shown for IFNα-induced NZB / W F1 mice treated with IgG isotype, surrogate antibody, or cyclophosphamide (CP) starting on day 7 (D7) or day 21 (D21). One-way ANOVA followed by Dunnett's post-hoc test for IFNα-induced treated with IgG isotype, mean ± SEM, n = 5–10. [Figure 12] indicates that Ab4 and the humanized IgG4-P isotype control antibody did not bind to complement component C1q. The anti-LAIR1 IgG1 antibody and the human IgG1 isotype control antibody bound to complement component C1q as expected. [Figure 13] mAb4 shows selectivity for LAIR1 and no activity against LAIR2. DETAILED DESCRIPTION OF THE INVENTION

[0018] 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.

[0019] In one aspect, novel anti-human LAIR1 antibodies or antibody fragments thereof are provided herein. In some embodiments, the anti-human LAIR1 antibodies or antibody fragments thereof provided herein are LAIR1 agonists. 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 a Jurkat-NFAT activation assay, inhibition of the IFN-γ response in primary T cells after a TCR stimulation assay, inhibition of the IL-6 response in primary B cells after a BCR stimulation assay, inhibition of the increase in human inflammatory cytokines IFN-γ, IL-10, and TNF-α in plasma, and / or reduction of circulating IgM and IgA in a human PBMC-transplanted GvHD mouse model, as described in the Examples. In some embodiments, the anti-human LAIR1 antibodies provided herein do not block the interaction between LAIR1 and its ligand, such as collagen I.

[0020] In some embodiments, the anti-human LAIR1 antibodies provided herein are human antibodies, for example, human IgG2 or IgG4 isotypes. In some embodiments, the anti-human LAIR1 antibodies provided herein also bind to cynomolgus monkey LAIR1. In some embodiments, the anti-human LAIR1 antibodies provided herein have a low immunogenicity risk.

[0021] In some embodiments, the anti-human LAIR1 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. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 1. In some embodiments, the anti-human LAIR1 antibody comprises a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 1. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising an HCDR1, HCDR2, and HCDR3 selected from Table 1, and / or a VL comprising an LCDR1, LCDR2, and LCDR3 selected from Table 1. In some embodiments, the anti-human LAIR1 antibody comprises a VH comprising a sequence having at least 95% sequence identity to a VH of Table 1. In some embodiments, the anti-human LAIR1 antibody comprises a VL comprising a sequence having at least 95% sequence identity to a VL of Table 1. In some embodiments, the anti-human LAIR1 antibody comprises a VH and / or VL of Table 1.

[0022] [Table 1-1]

[0023] [Table 1-2]

[0024] [Table 1-3]

[0025] [Table 1-4]

[0026] [Table 1-5]

[0027] [Table 1-6]

[0028] In some embodiments, provided herein is an antibody that binds to human LAIR1, the antibody comprising 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.

[0029] In some embodiments, provided herein is an antibody that binds to human LAIR1, the antibody comprising 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.

[0030] 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 containing a C131S mutation (according to EU index numbering) that reduces disulfide bond 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 containing a S228P mutation (according to EU index numbering) that reduces IgG4 Fab arm exchange in vivo (see Labrijn, et al., Nat. Biotechnol. 2009, 27(8):767).

[0031] 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.

[0032] 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.

[0033] In some embodiments, provided herein is an antibody fragment (e.g., Fab or scFv) that binds 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.

[0034] In some embodiments, provided herein is an antibody fragment (e.g., Fab or scFv) that binds 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.

[0035] In another aspect, provided herein are nucleic acids encoding the heavy or light chain, i.e., VH or VL, of the novel anti-human LAIR1 antibodies described herein, and vectors comprising such nucleic acids.

[0036] In some embodiments, provided herein are nucleic acids encoding the heavy or light chain of an anti-human LAIR1 antibody 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 comprising SEQ ID NO: 9, 25, 21, or 27. For example, the nucleic acid can 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 comprising SEQ ID NO: 10 or 22. For example, the nucleic acid can comprise a sequence selected from SEQ ID NO: 12 or 24.

[0037] Also provided herein are vectors comprising a nucleic acid sequence encoding an antibody heavy or light chain. For example, such vectors may comprise a nucleic acid sequence 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.

[0038] 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.

[0039] 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.

[0040] The nucleic acids of the present disclosure can be expressed in host cells, for example, after the nucleic acid is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked nucleic acid are well known in the art. The expression vector may include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. The expression vector containing the nucleic acid of interest (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, the expression vector may include one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to facilitate detection of host cells transformed with the desired nucleic acid sequence.

[0041] In another aspect, provided herein are cells, e.g., host cells, comprising a nucleic acid, vector, or nucleic acid composition described herein. Host cells can be cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of an antibody described herein. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the HC and LC polypeptides of an antibody of the present disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing the HC polypeptide and a second vector expressing the LC polypeptide of an antibody described herein. Such host cells, e.g., mammalian host cells, can express the anti-human LAIR1 antibodies described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0042] In some embodiments, a cell, e.g., a host cell, comprises a vector comprising 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, e.g., a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 21 or 27 and a second nucleic acid sequence encoding SEQ ID NO: 22.

[0043] In some embodiments, a cell, e.g., a host cell, 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, a cell, e.g., a host cell, 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.

[0044] The present disclosure further provides a process for producing the anti-human LAIR1 antibodies described herein by culturing the above-described host cells, e.g., mammalian host cells, under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. The medium into which the antibody is secreted can be purified by conventional techniques. Various methods of protein purification can be used, 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).

[0045] Also provided are antibodies produced by any of the processes described herein.

[0046] In another aspect, provided herein is a pharmaceutical composition comprising the antibody, nucleic acid, or vector described herein. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients, diluents, or carriers. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).

[0047] 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, 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.

[0048] In some embodiments, provided herein are methods of treating an autoimmune disease or fibrotic disease in a subject (e.g., a human patient) in need thereof by administering to the subject a therapeutically effective amount of an anti-human LAIR1 antibody described herein, a nucleic acid encoding such an anti-human LAIR1 antibody, a vector comprising such a nucleic acid, or a pharmaceutical composition comprising such an anti-human LAIR1 antibody, nucleic acid, or vector. The antibody, nucleic acid, vector, or pharmaceutical composition described herein can be administered parenterally (e.g., subcutaneously or intravenously).

[0049] Also provided are the anti-human LAIR1 antibodies, nucleic acids, vectors, or pharmaceutical compositions described herein for use in therapy. Additionally, the present disclosure also provides the anti-human LAIR1 antibodies, nucleic acids, vectors, or pharmaceutical compositions described herein for use in treating autoimmune diseases or fibrotic diseases.

[0050] Also provided herein is the use of an anti-human LAIR1 antibody, nucleic acid, vector, or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of an autoimmune disease or a fibrotic disease.

[0051] Also provided is an antibody or antigen fragment thereof that binds to 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0052] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0053] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0054] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0055] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0056] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0057] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein 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 from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0058] Preferably, the antibody or antigen fragment thereof that binds to human LAIR1 protein binds to an epitope comprising 8, 9, 10, 11, 12, 13, or 14 or more amino acid residues selected from FVCRGPVGVQTFRLER (SEQ ID NO: 32) and 8, 9, 10, 11, 12, 13, or 14 amino acid residues from VSQASPSESEARFRI (SEQ ID NO: 33), wherein the amino acid residues are selected from amino acids 26 to 41 and 53 to 68, and the amino acid positions correspond to SEQ ID NO: 29.

[0059] Preferably, the epitope comprises FVCRGPVGVQTFRLER (SEQ ID NO: 32) and VSQASPSESEARFRI (SEQ ID NO: 33), with amino acid residues selected from amino acids 26 to 41 and 53 to 68, and amino acid positions corresponding to SEQ ID NO: 29.

[0060] Also provided is an antibody or antibody fragment thereof that binds to the above-defined epitope of human LAIR1 protein, 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: 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 antibody has a modified human IgG4 hinge region containing the S228P mutation (according to EU index numbering), which reduces IgG4 Fab arm exchange in vivo (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 a HC having at least 95% sequence identity to SEQ ID NO: 39 and a LC having at least 95% sequence identity to SEQ ID NO: 40.

[0061] Preferably, the antibody that binds to human LAIR1 protein, or an antigenic fragment thereof, is Ab0.

[0062] Preferably, the antibody that binds to the human LAIR1 protein, or an antigenic fragment thereof, is a human antibody.

[0063] Preferably, the antibody or antigen fragment thereof that binds to an epitope according to the present invention is an antibody that stimulates the human LAIR1 protein.

[0064] Also provided is an anti-LAIR1 antibody or antibody fragment thereof that competes with any one of the antibodies defined according to the present invention for binding to an epitope of the present invention.

[0065] Preferably, the epitope-binding anti-LAIR1 antibody or antigenic fragment thereof is Ab0.

[0066] In some embodiments, the anti-LAIR1 antibody or antibody fragment thereof that competes with the anti-LAIR1 antibody defined below for binding to an epitope is Ab1, Ab2, Ab3, or Ab4.

[0067] In some embodiments, the anti-LAIR1 antibody is an antibody or antibody fragment thereof that binds to an epitope of the human LAIR1 protein as defined above, 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: 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 hinge region containing the S228P mutation (according to EU index numbering), which reduces IgG4 Fab arm exchange in vivo (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 a HC having at least 95% sequence identity to SEQ ID NO: 39 and a LC having at least 95% sequence identity to SEQ ID NO: 40.

[0068] The epitope is preferably determined by hydrogen-deuterium exchange (HDX) mapping techniques.

[0069] The advantage of anti-human LAIR1 antibodies or antigenic fragments thereof is that they can engage the body's natural immune inhibitory mechanisms, which may lead to both target cell-specific efficacy and important safety benefits over current immunomodulatory therapies.

[0070] The anti-human LAIR1 antibodies of the present invention preferably have one or more of the following important properties: - Binding to human LAIR1 and cross-reactivity to cynomolgus monkey LAIR1 - Binding to a unique epitope (non-ligand blocking). - Demonstrates LAIR1 agonism. - Antibody-mediated agonism attenuates human primary B cell activation in a dose-dependent manner - Demonstrate in vivo efficacy in a humanized mouse model of graft-versus-host disease. - Preclinical efficacy in a mouse model of lupus nephritis

[0071] As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) should be construed to cover both the singular and the plural unless otherwise indicated or clearly contradicted by context.

[0072] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0073] 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 via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more, which is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region, which is primarily responsible for effector function. 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 may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0074] The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, 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 CDRs can be performed using the methods of 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 standard available at www.imgt.org). This may be done according to well-known schemes, including those described in the ImMunoGeneTics database; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212. The North CDR definitions are used for the anti-human LAIR1 antibodies described herein.

[0075] The present disclosure also includes antibody fragments or antigen-binding fragments that comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, and linear antibodies.

[0076] The term "epitope" refers to the amino acid residues of an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope.

[0077] The term "epitope" may also be used in reference to a structural epitope, which, according to some embodiments, may be used to describe the region of an antigen that is covered by an antibody (e.g., the footprint of the antibody when bound to the antigen).

[0078] Epitopes can be determined according to various experimental techniques, also known as "epitope mapping techniques." It is understood that epitope determination may vary based on the different epitope mapping techniques used and may also vary depending on the different experimental conditions used, for example, due to conformational changes or cleavage of the antibody induced by the specific experimental conditions. Epitope mapping techniques are well known in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays (e.g., Rockberg and Nivebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018).

[0079] As used herein, the terms "competes for binding" or "competes with" refer to two antibodies that cross-compete (i.e., compete with each other) for binding to the same antigen. In some embodiments, two antibodies may compete for binding to the same antigen if 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 blocks binding by the other antibody, e.g., due to steric hindrance or conformational changes in the antigen induced by the first antibody. Many types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays, surface plasmon resonance, biolayer interferometry, or flow cytometry. Epitope binning can be performed using Carterra technology (e.g., PLoS One, 2014 Mar 20; doi:10.137 / journal.pone.0092451, Y. Abdiche et al.).

[0080] As used herein, the term "agonist" or "agonistic" refers to an antibody or antibody fragment that can induce or increase one or more activities or functions associated with human LAIR1, for example, one or more activities or functions associated with human LAIR1 as described in the Examples.

[0081] As used herein, unless otherwise specified, the terms "bind" and "binds" refer to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, bringing the two proteins or molecules into proximity as determined by common methods known in the art.

[0082] An "effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. The effective amount of an antibody may vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.

[0083] As used herein, the term "Fc region" refers to the region of an antibody comprising the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion of or the entire hinge region of the antibody heavy chain.

[0084] As used herein, the term "LAIR1," unless otherwise specified, 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.

[0085] MSPHPTALLGLVLCLAQTIHTQEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERDSRSTYNDTEDVSQASPSESEARFRIDSVREGNAGLYRCIYYKPPKWSEQSDYLELLVKESSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLYILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 29)

[0086] Several shorter isoforms of human LAIR1 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.

[0087] The amino acid sequence of cynomolgus monkey LAIR1 can be found in XP_045236925.1 (isoform X1), XP_045236926.1 (isoform X2), XP_045236927.1 (isoform X3), or XP_045236928.1 (isoform X4).

[0088] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs.

[0089] As used herein, the term "subject" refers to a mammal, including, but not limited to, a human, chimpanzee, ape, monkey, cow, horse, sheep, goat, pig, rabbit, dog, cat, rat, mouse, guinea pig, etc. Preferably, the subject is a human.

[0090] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or halt the progression of a disorder or disease disclosed herein, or may ameliorate the disorder or disease symptoms, but does not necessarily indicate the complete disappearance of all disorder or disease symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition for the treatment of a disease or condition in a patient, particularly a human. [Example]

[0091] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0092] Example 1. Generation of anti-human LAIR1 antibodies Human anti-human LAIR1 antibodies were generated using AlivaMab® human transgenic mice and cloning of the anti-LAIR1 variable region. Mice were immunized with human LAIR1 (SEQ ID NO: 30) fused to human Fc with a His tag and a 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.

[0093] The LAIR1 immunogen has the following amino acid sequence: QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHENLYFQGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ ID NO: 30).

[0094] The LAIR2 immunogen has the following amino acid sequence: QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAPENLYFQGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31)

[0095] The variable regions of the LAIR1-specific antibody were cloned and expressed, and the activity of the recombinant antibody was confirmed by ELISA, demonstrating selectivity for LAIR1 but no activity against LAIR2 (Figure 13). The antibody was then engineered for higher affinity following mutagenesis of CDR residues and identifying and enhancing mutations from combined residues for each CDR. Individual combination 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.

[0096] Anti-human LAIR1 antibodies can be produced by recombinant DNA technology. Such antibodies can be expressed in mammalian cell lines, such as HEK293 or CHO, either transiently or stably transfected with an expression system using an optimal, predetermined HC:LC vector ratio, or with a single vector system encoding both the HC and LC. The clarified medium into which the antibody is secreted can be purified using commonly used techniques.

[0097] Example 2: Characterization of anti-human LAIR1 antibodies Antibody binding affinity and kinetics 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+ (150 mM sodium chloride, 3 mM EDTA, 0.05% (w / v) surfactant P-20, and 10 mM HEPES, pH 7.4) as the running buffer. Binding experiments used the recombinantly produced soluble extracellular domain (ECD) of LAIR1 (SEQ ID NO: 17), which was diluted to a working concentration in HBS-EP+ containing 0.1 mg / mL bovine serum albumin. Goat anti-human kappa (Southern Biotech) was immobilized on all eight flow cells of a CM4 sensor chip using an amine coupling kit.

[0098] The soluble extracellular domain (ECD) of LAIR1 has the following amino acids: QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTPGSSAGPTQRPSDNSHNEHAPASQGLKAEHENLYFQ (SEQ ID NO: 17).

[0099] Binding was assessed using multiple analytical cycles. Each cycle was performed at a flow rate of 30 μL / min and consisted of the following steps: injection of antibody into a different flow cell (25 μL of 0.5 μg / mL antibody at 10 μL / min), injection of 75 μL of each LAIR1-ECD dilution (30 μL / min, 150 seconds) (starting at 1 μM, using a 3-fold serial dilution down to 1.4 nM per cycle, one injection per concentration), followed by a 1200-second delay for dissociation and chip surface regeneration using three 15 μL injections of 10 mM glycine hydrochloride, pH 1.7 (30 μL / min, 30 seconds). on and k off Association 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 rate constants. d =k off / k on Using the relationship of the equilibrium binding constant (K D ) was calculated.

[0100] Table 2 shows the binding affinities and kinetics of anti-human LAIR1 mAbs.

[0101] [Table 2]

[0102] Physical-chemical properties of exemplified LAIR1 antibodies Thermal stability: The stability of the exemplified LAIR1 antibodies to thermal denaturation was evaluated using differential scanning calorimetry (DSC). DSC was performed using a Malvern MicroCal VP-DSC instrument. Samples in PBS buffer were heated from 20°C to 110°C at a constant rate of 60°C / h. The analytical method was performed using a MicroCal VP-Capillary DSC automated analysis program. Baseline correction was performed, and the Tm onset and Tm were determined. The results, shown in Table 3, indicated that the exemplified LAIR1 antibodies were thermally stable, with Tm onsets >60°C.

[0103] [Table 3]

[0104] Solubility: Sufficiently high solubility is desirable to allow convenient administration. Additionally, it is also desirable to maintain the antibody in a monomeric state at high concentrations without high molecular weight (HMW) aggregation. The solubility of the exemplified LAIR1 antibody was analyzed by concentrating 15 mg of the exemplified antibody to a volume of less than 100 μl using a 10K molecular weight cutoff filter (Amicon UC filter, Millipore, catalog number UFC903024). The final concentration of the sample was measured using a SoloVPE spectrophotometer (C Technologies, Inc.). Following substantially the above procedure, the exemplified antibody exhibits a solubility of 150 mg / ml (in 5 mM histidine, pH 6.0) and greater than 200 mg / ml (in PBS buffer, pH 7.4). Concentrated solution samples were stored at 4°C for one week, followed by one week at -5°C. The aggregation profile of the antibody after two weeks of storage was assessed using size exclusion chromatography (SEC). The results shown in Table 4 indicate that at high concentrations, only low levels of high molecular weight (HMW) aggregates are present (approximately 1%) and no phase separation is observed.

[0105] [Table 4]

[0106] Photostability: The photostability of the exemplified antibodies is evaluated at high concentrations (approximately 100 mg / mL) in 5 mM histidine buffer (pH 6.0) containing excipients. The concentrated samples are photoexcited in a photochamber at 25°C and 40 watt hr / m 2 of UV light (10 watts / m 2 The samples were exposed to a 20% ICH Q1B minimum exposure of 4 hours at 80 klux (4 hours at 80 klux) + 240 klux hours (30 hours at 80 klux). A protected concentrated sample (wrapped in aluminum foil) was used as a dark control and placed side-by-side with the authentic sample. Upon exposure, the samples were analyzed for the growth percentage of HMW aggregates (Δ% HMW) using SEC. The results, provided in Table 5, show that the exemplified antibody has a growth percentage of HMW of 5-7% after exposure to the 20% ICH guideline.

[0107] [Table 5]

[0108] Chemical stability: Chemical stability facilitates the development of drug formulations with sufficient shelf life. The chemical stability of the exemplified antibodies is evaluated by formulating the exemplified antibodies at a concentration of 100 mg / ml in a pH 6 buffer solution. The formulated samples are incubated at 4°C for 4 weeks and at 35°C for accelerated degradation studies. Changes in the antibody fragmentation and aggregation profile are evaluated using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and SEC according to standard procedures. Following substantially the procedures described above, the exemplified antibodies exhibit the chemical stability results shown in Table 6.

[0109] [Table 6]

[0110] The results provided in Table 6 show that after 4 weeks of storage at 35° C., the exemplified antibodies have an increase in fragment percentage of approximately 0.2-1.3%. The level of HMW growth for the exemplified antibodies is 0.9-2.5%.

[0111] Chemical stability data indicates that the exemplified antibodies have sufficient chemical stability to facilitate the development of solution formulations with suitable shelf life.

[0112] In summary, the exemplified LAIR1 antibodies exhibit the characteristics of solubility, low aggregation, chemical stability, and physical stability essential for therapeutic administration of the parent drug.

[0113] Example 3: Immunogenicity evaluation of anti-human LAIR1 antibodies A panel of in vitro and ex vivo methods was used to characterize the relative risk of immunogenicity of the exemplified LAIR1 mAbs, as described below.

[0114] Dendritic Cell (DC) Internalization Assay: This assay was performed to investigate the internalization of molecules by CD14+ monocyte-derived dendritic cells. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and cultured and differentiated into DCs according to standard protocols (see Wen, Y., et al., AAPS J 2020 Apr 16;22(3):68). Briefly, PBMCs were isolated from LRS-WBCs using density gradient centrifugation with Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies). CD14+ monocytes were isolated using positive selection with a CD14+ MicroBead Kit (#130-050-201, Miltenyi Biotec) according to the manufacturer's instructions. The cells were then cultured at 1 million / mL in RPMI medium (purchased from Life Technologies) containing L-glutamine and 25 mM HEPES, supplemented with 10% FBS, 1 mM sodium pyruvate, 1x penicillin-streptomycin, 1x non-essential amino acids, and 55 μM 2-mercaptoethanol, along with 1,000 units / mL GM-CSF and 600 units / mL IL-4, for 6 days to induce immature dendritic cells (MDDCs). The medium was changed twice, on days 2 and 5. On day 6, the cells were gently collected with a cell scraper and used for experiments. To obtain mature DCs, the cells were treated with 1 μg / mL LPS for 4 hours.

[0115] Individual test molecules were normalized to 1 mg / mL in 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. Equal volumes of antibody and Fab-TAMRA-QSY7 were mixed and incubated for 30 minutes at 4°C in the dark to allow complex formation. MDDCs were resuspended at 4 million / mL in complete RPMI medium and seeded at 50 μL per well in a 96-well round-bottom plate, to which 50 μL of antibody / probe complex was added. Cells were incubated for 24 hours at 37°C in a CO2 incubator. Cells were washed with 2% FBS PBS and resuspended in 100 μL of 2% FBS PBS containing Cytox Green live / dead dye. Data were collected on a BD LSR Fortessa X-20 and analyzed using FlowJo. Live single cells were gated, and the percentage of TAMRA-fluorescent positive cells was recorded as the readout. A normalized internalization index was used to allow for molecular comparison of data generated from different donors. The internalization signal was normalized to the IgG1 isotype (normalized internalization index = 0) and the internal positive control PC (normalized internalization index = 100) using the following formula:

[0116]

number

[0117] [Table 7]

[0118] As shown in Table 7, the exemplified anti-human LAIR1 antibodies have low-moderate and moderate risk by dendritic cell internalization assay.

[0119] MAPP assay (MHC-associated peptide proteomics): MAPP profiles human leukocyte antigen class II (HLA-II)-presented peptides on human dendritic cells previously treated with a test molecule. Primary human dendritic cells from a panel of 10 normal human donors were prepared from buffy coats by isolation of CD-14+ cells as described 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 number A2596101) at 37°C and 5% CO2 for 3 days (Knierman et al., Cell Rep 2020 Dec 1;33(9):108454). On day 4, 3 micromolar 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 transform the cells into mature dendritic cells. The next day, mature cells were lysed in 1 mL of RIPA buffer containing protease inhibitors and DNAse. The lysates were stored at -80°C until sample analysis.

[0120] Using an automated liquid handling system, HLA-II molecules were isolated from thawed lysates using a biotinylated anti-pan-HLA class II antibody (clone Tu39). Bound receptor-peptide complexes were eluted with 5% acetic acid, 0.1% TFA. The eluted HLA-II peptides were passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The isolated HLA-II peptides were analyzed by nanoLC / MS using a Thermo Easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. The separation used a 75 μm x 7 cm YMC-ODS C18 column at a flow rate of 250 nL / min and a 65-minute gradient of 0.1% formic acid in water as solvent A and 80% acetonitrile with 0.1% formic acid as solvent B. Mass analysis was performed in full scan mode at a resolution of 240,000, followed by a 3 second data-dependent MS / MS cycle consisting of an ion trap rapid scan with HCD and EThcD fragmentation.

[0121] Peptide identifications were generated by an internal proteomics pipeline using multiple search algorithms without enzyme search parameters against a bovine / human database containing test molecule sequences (Higgs et al., Methods Mol Biol. 2008;428:209-30). Peptides identified from the test molecules were aligned to the parent sequences. A summary was generated for all test molecules annotating the percentage of donors presenting peptides with non-germline residues and the number of different regions of the test molecules presenting peptides with non-germline residues. An increased degree of non-germline peptide presentation is associated with an increased risk of immunogenicity.

[0122] [Table 8]

[0123] As shown in Table 8, the exemplified anti-LAIR1 antibodies mAb1 to 4 have a moderate risk according to the MAPP assay.

[0124] T cell proliferation assay This assay evaluates the ability of test molecules to activate CD4+ T cells by inducing cell proliferation (Walsh, RE, et al., MAbs. 2020;12(1):1764829). Cryopreserved PBMCs from 10 healthy donors were used. PBMCs were depleted of CD8+ T cells and labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). PBMCs were seeded at 4 x 106 cells / ml / well in AIM-V medium (Life Technologies, catalog number 12055-083) containing 5% CTS™ Immune Cell SR (Gibco, catalog number A2596101). Tested in triplicate in 2.0 mL of medium containing various test molecules, DMSO control, medium control, and keyhole limpet hemocyanin (KLH; positive control). Cells were cultured and incubated at 37°C with 5% CO for 7 days. On day 7, 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 LSRFortessa™ equipped with a High Throughput Sampler (HTS). Data were analyzed using FlowJo® software (FlowJo, LLC, TreeStar) to calculate the cellular division index (CDI). Briefly, the CDI for each test molecule was calculated by dividing the percentage of proliferating CFSedimCD4+ T cells from molecule-stimulated wells by the percentage of proliferating CFSedimCD4+ T cells in unstimulated wells. A CDI of >2.5 was considered to represent a positive response. Percent donor frequencies across all donors were assessed. Growth in <30% of donors is considered a low risk for immunogenicity, 30-40% a moderate risk, and >40% a high risk.

[0125] [Table 9]

[0126] As shown in Table 9, the exemplified anti-human LAIR1 antibody mAbs 2 to 4 have low risk in the T cell proliferation assay. Anti-human LAIR1 mAb 1 was not tested.

[0127] Existing reactivity assays: This assay was performed to examine the presence of reactivity from pre-existing anti-drug antibodies (PEA) and potentially other cross-reactive proteins in treatment-naive normal human serum (see Bivi, N., et al., MAbs. 2019 Jul;11(5):861-869). Diluted serum from a panel of at least 50 treatment-naive donors was captured overnight on plates coated with biotinylated test molecules. The following day, the captured reactive proteins were eluted with acid and then neutralized in the presence of biotinylated and ruthenylated test molecules. If anti-drug antibodies are present, they cross-link the labeled test antibodies, forming complexes. The complexes are captured by streptavidin-coated mesoscale plates, and the resulting signal is referred to as the Tier 1 signal (expressed as electrochemiluminescence). This signal is confirmed in Tier 2 by adding an excess of unlabeled test molecules in the detection step, which suppresses the Tier 1 signal. The presence of pre-existing anti-drug antibodies is expressed as the 90th percentile of Tier 2 inhibition, with results <30% representing low, 30%-55% representing moderate, and >55% representing high immunogenicity risk.

[0128] [Table 10]

[0129] As shown in Table 10, the exemplified anti-human LAIR1 antibodies mAb1 to 4 have low risk in existing reactivity assays.

[0130] Example 4: In vitro activity of anti-human LAIR1 antibodies Cell-based conjugation: Anti-human LAIR1 antibodies were evaluated for binding to human and cynomolgus monkey (cyno) LAIR1-engineered cell lines and primary human T cells endogenously expressing LAIR1. Jurkat-hLAIR1+ (Jurkat cells overexpressing human LAIR1), Jurkat-LAIR1ko (Jurkat cells knocking out human LAIR1), 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 the antibody ranging from 0.0017 μg / mL to 3.33 μg / mL were incubated with the cells for 20 minutes at 4°C. The cells were then washed and incubated with an anti-human IgG Alexa Fluor 647 secondary antibody for 20 minutes at 4°C. The cells were then washed, and antibody binding was assessed by flow cytometry. For primary human T cells, cells were also stained for CD4 and CD8 to represent CD4+ and CD8+ T cells.

[0131] As shown in Table 11, all of the anti-human LAIR1 test antibodies bound to Jurkat-hLAIR1+ cells and primary human CD4+ and CD8+ T cells with similar binding strength. The EC50 for binding to Jurkat-cyLAIR1+ cells was within two-fold of the EC50 for binding to Jurkat-hLAIR1+ cells. The test anti-LAIR1 antibodies did not show binding to LAIR1ko, a control cell line that does not express LAIR1.

[0132] [Table 11]

[0133] Activation of Jurkat-NFAT: The effect of anti-human LAIR1 antibody on NFAT activation in Jurkat cells was evaluated. Jurkat-NFAT-luciferase cells expressing human LAIR1 (Jurkat-hLAIR1+), cyno LAIR1 (Jurkat-cyLAIR1+), or LAIR1-deficient (Jurkat-LAIR1ko) were stimulated with anti-human CD3 antibody via TCR stimulation in the presence of anti-human LAIR1 antibody. Specifically, CHO-K1 cells were seeded overnight in 96-well flat-bottom tissue culture sterile plates. Upon reaching 85-95% confluence, the cells were washed with RPMI / 5% human serum and incubated with 10 μg / mL anti-human CD3 antibody for 1 hour at 37°C. Unbound CD3 antibody was then removed, and the cells were washed and incubated with the indicated concentrations of anti-human LAIR1 antibody for 20 minutes at 37°C. 1 x 10 Jurkat-hLAIR1+, Jurkat-cyLAIR1+, or Jurkat-LAIR1ko cells were added and incubated at 37°C for 6 hours. Jurkat cells were then transferred to an opaque, flat, clear-bottom 96-well plate, and an equal volume of BrightGlo luciferase was added. After a 2-minute incubation for lysis, NFAT activity (as measured by luciferase) was assessed using a luminometer. Test anti-LAIR1 antibodies were evaluated in the activation assay at concentrations ranging from 0.1 ng / mL to 1 μg / mL, with eight three-fold serial dilutions.

[0134] As shown in Table 12, all anti-human LAIR1 antibodies tested inhibited Jurkat NFAT activation in both human and cynomolgus monkey LAIR1-expressing Jurkat-NFAT cells with inhibition ranging from 60 to 70%, whereas 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 values ​​were obtained for Jurkat-cyLAIR1+ cells. Anti-human LAIR1 antibodies did not affect NFAT activity in Jurkat-LAIR1ko cells. Values ​​for Jurkat-LAIR1ko are not shown because the anti-LAIR1 antibodies showed no inhibitory effect on this control cell line.

[0135] [Table 12]

[0136] Anti-human LAIR1 antibody, mAb4, inhibits NFAT activation in an in vitro cell-based agonism assay. The ability of LAIR1 agonist antibodies to inhibit NFAT activation in human T cell lines overexpressing human LAIR1 was determined as follows.

[0137] Jurkat-NFAT-luciferase reporter cells were engineered to overexpress human LAIR1 (Jurkat-hLAIR1+) via lentiviral transduction. Jurkat-hLAIR1+ cells were TCR-stimulated with anti-human CD3 antibody (clone OKT3) in the presence of cross-linked anti-human LAIR1 mAb4 or hIgG4SP isotype control antibody. Antibodies were cross-linked using a Chinese hamster ovary (CHO) cell line engineered to express human Fc gamma RIIb.

[0138] CHO-K1 cells were seeded overnight at 37°C in 96-well flat-bottom tissue culture sterile plates. When they reached 85-95% confluency, the cells were washed with RPMI / 5% human serum and incubated with anti-human CD3 antibody for 1 hour at 37°C. Unbound CD3 antibody was then removed, and the cells were washed and incubated with mAb4 or hIgG4SP isotype antibody for 20 minutes at 37°C. 1 x 10 Jurkat-hLAIR1+ cells were added to the plate and incubated for 6 hours at 37°C. Jurkat-hLAIR1+ cells were then transferred to an opaque, flat, clear-bottom 96-well plate and an equal volume of BrightGlo luciferase was added. After a 2-minute incubation for cell lysis, NFAT activity (luciferase readout) was assessed using a luminometer. Antibodies were serially diluted for IC50 assessment of NFAT activity and evaluated at concentrations ranging from 0.001 to 6.7 nM (0.2 to 1000 ng / mL).

[0139] As shown in Figure 3, mAb4 inhibited Jurkat-hLAIR1 NFAT activation, with inhibition ranging from 60 to 70% (calculated as % NFAT activity with antibody vs. without antibody) and an IC value of 0.045 nM (6.7 ng / mL), whereas the hIgG4SP isotype control antibody had no effect on NFAT activity. As a parallel control, the antibody was evaluated in LAIR1-deficient Jurkat-NFAT-luciferase cells (Jurkat-hLAIR1ko). mAb4 had no effect on NFAT activity in Jurkat-hLAIR1ko cells (data not shown).

[0140] Primary human B cells: The effect of anti-human LAIR1 antibodies on primary human B cell cytokine responses was evaluated. Primary human B cells were stimulated with BCR-mediated stimulation with anti-human IgM antibodies plus IL4 in the presence of test anti-human LAIR1 antibodies at concentrations ranging from 0.00128 ng / mL to 8 ng / mL. Specifically, CHO-K1 cells were seeded overnight in 96-well flat-bottom tissue culture sterile plates. Upon reaching 85-95% confluency, the cells were washed with RPMI / 5% human serum and incubated with the indicated concentrations of anti-human LAIR1 antibodies for 20 hours 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. Stimulators or controls were then added (20 ng / mL IL-4 plus 5 μg / mL anti-human IgM or medium alone as a non-stimulated control), and the cells were incubated for 72 hours at 37°C. Test antibodies were also evaluated against an anti-human IgG isotype control. The effect of LAIR1 engagement on B cell IL-6 responses was assessed by ELISA at 72 hours and reported as % inhibition compared to no antibody control.

[0141] As shown in Table 13, all anti-human LAIR1 test antibodies inhibited the IL6 response, with inhibition at the highest dose of 8 ng / mL ranging from 20 to 70% depending on the donor, while the isotype control had no effect on the cytokine response.

[0142] [Table 13]

[0143] The anti-human LAIR1 antibody, mAb4, inhibits primary B cell cytokine responses in an in vitro cell-based agonism assay. The ability of LAIR1 agonist antibodies to inhibit BCR stimulation-induced IL-6 responses in primary human B cells was determined as follows.

[0144] Primary human B cells (n = 6 donors) were BCR-stimulated with anti-human IgM antibody plus IL-4 in the presence of cross-linking anti-human LAIR1 mAb4 or hIgG4SP isotype control antibody. Antibodies were cross-linked using a Chinese hamster ovary (CHO) cell line engineered to express human Fc gamma RIIb.

[0145] CHO-K1 cells were seeded overnight at 37°C in 96-well flat-bottom tissue culture sterile plates. When they reached 85-95% confluency, the cells were washed with RPMI / 5% human serum and incubated with mAb4 or hIgG4SP isotype antibodies for 20 minutes at 37°C. 1 x 10 isolated B cells from human PBMCs were added and incubated for an additional 20 minutes at room temperature to allow cell / antibody interaction. Stimulators (anti-human IgM + IL-4) or controls (media only + / - IL-4) were then added, and the cells were incubated for 72 hours at 37°C. The effect of antibodies on B cell IL-6 responses was assessed by ELISA at 72 hours and reported as % inhibition compared to the no-antibody control. Antibodies were serially diluted at concentrations ranging from 0.000013 to 0.0539 nM (0.002 to 8 ng / mL) for IC50 evaluation of inhibition of B cell IL-6 responses.

[0146] As shown in Figure 4, mAb4 inhibited the B cell IL-6 response to BCR stimulation, with an inhibition range of 60-80% (calculated as % IL-6 response with antibody vs. without antibody) and an IC50 value of 0.0002 nM (0.03 ng / mL), whereas the hIgG4SP isotype control antibody had no effect on the IL-6 response.

[0147] Primary human T cells: The effect of anti-human LAIR1 antibody on primary human T cell cytokine responses was evaluated. Primary human T cells were stimulated with anti-human CD3 and CD28 antibodies in the presence of anti-human LAIR1 antibody at concentrations ranging from 1 ng / mL to 1 μg / mL. Specifically, T cells were incubated overnight at 37°C with plate-bound anti-human CD3 and CD28 antibodies at 1 μg / mL and 3 μg / mL, respectively. CHO-K1 cells were seeded overnight in 96-well flat-bottom tissue culture sterile plates. Upon reaching 85-95% confluence, the cells were washed with RPMI / 5% human serum and incubated with the indicated concentrations of anti-human LAIR1 antibody at 37°C for 20 hours. Stimulated T cells were washed and resuspended in fresh RPMI / 5% human serum. 1-1.5 x 105 T cells were then layered on top of CHO-K1 cells and incubated at 37°C for 72 hours. After incubation, supernatants were collected and assessed for IFN-γ secretion by ELISA.

[0148] As shown in Table 14, all anti-human LAIR1 antibodies tested inhibited the IFN-γ response, with inhibition at the highest dose of 1 μg / mL ranging from 20 to 80% depending on the donor, while the isotype control had no effect.

[0149] [Table 14]

[0150] Example 5: In vivo activity of anti-human LAIR1 antibodies in a human PBMC-transplanted mouse model of graft versus host disease (GvHD) To test the immunomodulatory activity of the exemplary antibodies mAb1-mAb4, we utilized a humanized model of xenogeneic GvHD. Human immune cells recognize the mouse as foreign and initiate an immune response, resulting in a significant increase in human inflammatory cytokines, immune cell activation and expansion, and immunoglobulin production. Importantly, the inflammatory response is driven by human cells, and thus, human-specific therapies can be investigated in this model.

[0151] Briefly, female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, stock no. 05557) were housed three per cage at 72°C under a 12-h light:dark cycle and allowed free 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 × 10 cells / mL, and mice were intravenously transplanted with 100 μL of the PBMC suspension on day 0 (1.2 × 10 cells / mouse, n = 36). Mice were divided into five groups and administered isotype control or mAbs 1-4 subcutaneously at 0.3 mg / kg on days 1 and 8 (200 μL / mouse; n = 7-8 mice / group). On day 7, mice were briefly anesthetized with isoflurane, and blood was obtained from the retro-orbital sinus. On day 14, mice were anesthetized again, blood was collected by cardiac puncture, and mice were euthanized. Mice were weighed 2-3 times / week in a BSL2 hood and assessed for clinical signs of distress. Clinical signs common to this model include unkempt hair, a hunched body, weakness, 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 analyzed using the Human Pro-inflammatory 10-Vplex, and IgM, IgA, and IgG were analyzed using the Human Isotyping Panel (Meso Scale). Measurements were performed using a fluoroscopy system (Discovery, Rockville, Maryland) according to the manufacturer's instructions.

[0152] Data were graphed and statistics were calculated using Prism Software (GraphPad, San Diego, CA). Differences in plasma analytes compared to isotype controls were determined by one-way analysis of variance with Dunnett's post-hoc test and were considered significant when p<0.05.

[0153] In experiments performed essentially as described above, the exemplified anti-human LAIR1 antibodies significantly inhibited the marked increase in plasma human inflammatory cytokines (IFN-γ, IL-10, and TNF-α) associated with disease progression in a GvHD model. Furthermore, the exemplified anti-human LAIR1 antibodies mAb1 to mAb3 significantly reduced circulating immunoglobulins IgM and IgA, suggesting an inhibitory effect on B cells.

[0154] The results demonstrate that the exemplified anti-human LAIR1 antibodies have immunomodulatory effects on human immune cells in a humanized mouse disease model.

[0155] Humanized Graft-versus-Host Disease: The antibody described herein as mAb4 was tested in NOD SCID gamma 2 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 transplanted into NSG mice, causing human immune cells to recognize the mice as foreign, initiate an immune response, and induce graft-versus-host disease (GvHD). The objective was to evaluate the ability of mAb4 to stimulate LAIR1 and inhibit T cell activation (measured by inflammatory cytokine production) and 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 transplantation, mice were administered a single subcutaneous dose of mAb4 or human IgG4P isotype control (3 mg / kg) at half-log increments of 0.003 to 3 mg / kg. Mice were then euthanized on day 8. Blood was obtained via retro-orbital sinus on day 5 and cardiac puncture on day 8 and processed for analysis of serum cytokines (MSD human inflammatory cytokine panel) and drug exposure (antigen capture ELISA). Spleens were harvested on day 8, processed into single-cell splenocytes, and analyzed by FACS for immunophenotyping and receptor occupancy (RO). mAb4 dose-dependently inhibited immune cell-associated inflammatory cytokines, indicative of T cell function inhibition (Figure 5). These beneficial activities were mechanistically supported by postmortem 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 post-dose, showed a dose-dependence similar to receptor occupancy. Interestingly, the PD response was more potent than exposure or RO, suggesting that complete RO is not required to elicit the beneficial effects of agonism. Additionally, the increased percentage of Tregs observed in the high-dose group presents another potential therapeutic mechanism by which LAIR1 agonism may prove advantageous in the treatment of autoimmune diseases (Figure 8).

[0156] overview 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 are transplanted with human donor immune cells. After transplantation, the human immune cells initiate an inflammatory attack against the mice, as measured by the production of human cytokines in the mouse peripheral blood. mAb4, an LAIR1 agonist antibody, was able to reduce the production of these cytokines in a dose-dependent manner (Figure 5). This reduction correlated with receptor occupancy (Figure 6) and serum concentration of mAb4 (Figure 7). Furthermore, mAb4 was able to increase the percentage of Treg cells in the spleen (Figure 8), which may explain another mechanism of action in controlling the inflammatory response in this model.

[0157] Example 6: In vivo study of anti-human LAIR1 antibodies in a mouse model of spontaneous lupus nephritis Primary pharmacodynamics Type I Interferon Lupus Nephritis: NZB / W F1 mice are used as a classical model of spontaneous lupus nephritis. To accelerate and synchronize disease induction, we injected mice with adeno-associated virus (AAV) expressing murine IFNα5. Therapies targeting T and B cells in this model have been shown to reduce disease severity. The purpose of this study was to demonstrate whether an alternative LAIR1 agonist antibody could affect disease severity in a preclinical model of lupus nephritis.

[0158] Lupus Model: Female NZB / W F1 mice (Jackson Laboratories) were 10 weeks old upon arrival. All mice were housed five per cage and allowed to acclimate for one week before the start of the study. Mice were fed Teklad Irradiated Global 18% Protein Rodent Diet 2908 (Innotiv) and provided with water ad libitum. Mice were housed in an ambient temperature range of 68-79°F under a 12-h light / dark cycle. Mice were culled based on weight, and one day later (day 0), mice were intravenously injected with LacZ-AAV (non-disease control, 10 genome copies (GC)) or mouse IFNα5-AAV (3 × 10 GC) in 100 μl of PBS. The assigned treatment groups were: (1) LacZ-AAV induction, PBS treatment (sc, starting on day 7, bid; n = 5); (2) IFN-AAV induction, IgG isotype treatment, used as a surrogate antibody (10 mg / kg sc, starting on day 7, bid; n = 10); (3) IFN-AAV induction, surrogate antibody treatment (10 mg / kg sc, starting on day 7, bid; n = 10); (4) IFN-AAV induction, surrogate antibody treatment (10 mg / kg sc, starting on day 21, bid; n = 10); and (5) IFN-AAV induction, cyclophosphamide treatment (15 mg / kg i.p., Q10D, starting on day 7; n = 10). Serum and urine samples were collected at baseline and every 2 weeks throughout the study. 42 days after AAV injection, mice were euthanized, and body weights were collected. Both kidneys were collected and weighed in pairs. The right kidney was fixed in 10% neutral buffered formalin for 24–48 h and then transferred to 70% alcohol.

[0159] Albumin and Creatinine Assays: To monitor kidney function, microalbumin concentrations in urine (dilutions 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. Urinary creatinine was measured using the CREP2 enzyme creatinine assay on a Cobas C501 clinical chemistry analyzer (Roche Diagnostics, USA) according to the manufacturer's instructions.

[0160] Histology: Kidneys from each mouse were embedded in paraffin, sectioned, and stained with hematoxylin and eosin PAS.

[0161] Histological Scoring: Scoring of inflammation, glomerular changes, and tubular proteins was based on the following criteria and summed to give a total score.

[0162] inflammation: A score of 0 to 3 was based on a combination of the number of affected areas and the amount of affected areas.

[0163] Glomerular score: The glomerular score (0–6) was based on assessment of glomeruli in the outer half of the cortex and, because of variability among glomeruli within each kidney, was based on the most frequent grade encountered in this region. · Grade 1 -Minimal increase in cellularity and / or mesangial expansion + / - minimal increase in glomerular size (less than 2-fold). · Grade 2 -Mild increase in cellularity and mesangial expansion (most glomeruli at least two-fold larger in size). · Grade 3 Moderate increase in cellularity in most affected glomeruli and some areas of marked mesangial expansion and / or capillary proliferation (up to a three-fold increase in glomerular size). · Grade 4- Marked increase in cellularity in most affected glomeruli and some areas of marked mesangial expansion and / or capillary proliferation (up to a fourfold increase in glomerular size); rare sclerotic glomeruli; may have mural cell hypertrophy. · Grade 5 - As above, <25% glomerular sclerosis and / or capillary proliferation in the majority of affected glomeruli; up to a 5-fold increase in glomerular size. · Grade 6 ->25% glomerular sclerosis characterized in part by decreased tuft cellularity, + / - juxtaglomerular fibrosis + / - mural cell hypertrophy, together with the above.

[0164] PAS score: A PAS score of 0–3 was based on the presence of increased staining of the glomerular mesangial matrix in the outer half of the cortex compared with control sections cut at the same thickness. · Grade 1 -Minimal increase in scattered glomerular mesangial staining. · Grade 2 -More extensive expansion of the mesangium affecting more of the glomeruli (hence the PAS staining). · Grade 3 - Marked expansion of the mesangium in most of the glomeruli.

[0165] Tubular Protein Score: A score of 0 to 3 was based on the percentage of tubules containing proteinaceous fluid. · Grade 1 -<25% affected tubules. · Grade 2 -25-50% affected tubules. · Grade 3 ->50% affected tubules.

[0166] The sum of the scores for the four parameters was used to calculate a total histology score.

[0167] Statistical analysis: Statistical analysis was performed for IFNα induction with treatment with IgG isotypes using one-way ANOVA followed by Dunnett's post-hoc test comparisons.

[0168] overview Administration of mIFNα-AAV to NZB / W F1 mice induced lupus nephritis, characterized by increased urinary ACR levels and histology scores, in the IgG isotype group compared with mice administered nonpathogenic LacZ-AAV (Figures 5 and 7). Compared with the IgG isotype group, therapeutic treatment with the surrogate antibody starting on day 21 (D21) reduced urinary ACR levels by the end of the study on day 44, but was not as effective as the positive control cyclophosphamide (CP) administered starting on day 7 (D7) (Figures 9 and 10). However, prophylactic treatment with the surrogate antibody starting on D7 did not significantly reduce urinary ACR (Figures 9 and 10). Histological evaluation of kidneys from mice demonstrated that the surrogate antibody reduced histology scores by 40% and 36% when treatment was initiated on D7 and D21, respectively (Figure 11). The effect was not statistically significant compared to the IgG isotype control with p-values ​​of 0.06 and 0.09, respectively (Figure 11).

[0169] The results demonstrate that alternative agonist antibodies can modulate disease in preclinical models of lupus nephritis.

[0170] Example 7: Epitope mapping of parental mAb4 to the LAIR1 extracellular domain (ECD) protein by hydrogen-deuterium exchange mass spectrometry Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) was performed to determine where parental mAb4 binds to the ECD of LAIR1.

[0171] Peptide identification of LAIR1 ECD was performed on a Waters Synapt G2Si (Waters Corporation) instrument using 3.5 μg of LAIR1 ECD protein in zero exchange (1:10 dilution in 0.1x phosphate-buffered saline in HO) and nepenthesin II (Nep II) for digestion. The mass spectrometer was set to HDMSe (mobility ESI+ mode) with a 0.4 s scan time and a mass acquisition range of m / z 255.00–1950.00. Data were processed using PLGS 2.3.03 (Waters Corporation). For exchange experiments, 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) containing 150 mM NaCl (1x PBS buffer). Using a custom TECAN sample preparation system, experiments were initiated by adding 25 μL of DO buffer containing 0.1× PBS to 2.5 μL of LAIR1 ECD (0.7 mg / mL) or LAIR1 ECD + mAb4 complex for various times (0 s, 10 s, 2 min, 10 min, and 60 min) at 15 °C (Espada et al., 2019). The reaction was stopped with an equal volume of 0.32 M TCEP, 0.1 M phosphate, pH 2.5, at 4 °C for 2 min and immediately frozen at -70 °C. The sample injection system consisted of a UR3 robot, a LEAP PAL3 HDX autosampler, and an HPLC system interfaced with a Waters Synapt G2Si (Waters Corporation), modified as described (Espada et al., 2019, https: / / pubmed.ncbi.nlm.nih.gov / 31724102 / ). The LC mobile phase consisted of water (A) and acetonitrile (B) containing 0.2% formic acid, respectively. Each sample was thawed for 1 min with 50 μL of 0.2% formic acid in water, pH 2.5, injected onto the Nep II column, and digested for 2.5 min with mobile phase A at a flow rate of 250 μL / min at 4°C.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% to 85% mobile phase B over 7 min. The peptides were then transferred to a mass spectrometer for mass analysis. The Synapt G2Si was calibrated with Glu-fibrinopeptide (Waters Corporation) prior to use. Mass spectra were acquired in HDMS mode over an m / z range of 255 to 1950, with a lock mass m / z of 556.2771 (Leucine Enkephalin, Waters Corporation). The relative deuterium uptake of each peptide was determined by processing the MS data of the deuterated samples along with undeuterated controls using the identified peptide list in DynamX 3.0 (Waters Corporation). Peptides in the free and bound states of the RBD were compared for differences in deuterium uptake to identify protected regions representing binding epitopes.

[0172] Sequence coverage across the LAIR1 ECD was 77%. For parental mAb4, decreased deuterium incorporation upon binding to the LAIR1 ECD was observed at residues 26-41 (FVCRGPVGVQTFRLER) (SEQ ID NO: 32) and 53-68 (VSQASPSESEARFRI) (SEQ ID NO: 33), suggesting putative epitope regions. The parental mAb4 sequence is shown in Table 1 above.

[0173] Example 6: C1q Binding Results A 96-well microplate was coated with 100 μL / well of each antibody diluted in DPBS (Dulbecco's HyClone) at concentrations ranging from 10 μg / mL to 0.19 μg / mL. Tests were performed in duplicate. 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 three times with wash buffer (1x TBE containing 0.05% Tween 20). 100 μL / well of 10 μg / mL Human C1q (MS Biomedical) diluted in casein blocking reagent was added and incubated for 3 hours at room temperature. The plate was then washed three times with wash buffer, after which 100 μL / well of a 1:800 dilution of sheep anti-human C1q-HRP (Abcam #ab46191) in casein blocker was added and incubated for 1 hour at room temperature. The plate was washed six times with wash buffer, and 100 μL / well of TMB substrate (Pierce) was added to each well and incubated for 7 minutes. The reaction was stopped by adding 100 microliters of 1N HCl to each well. Optical density was immediately measured using a colorimetric microplate reader set at 450 nm. The results show that mAb4, the humanized IgG4-P isotype control antibody, and the human IgG1 isotype control antibody did not bind to complement component C1q. The anti-LAIR1 IgG1 antibody and the human IgG1 isotype control antibody bound to complement component C1q, as expected.

[0174] The results indicated that mAb4 was unlikely to induce Fc-mediated effector function responses in vivo.

[0175] [Table 15-1]

[0176] [Table 15-2]

[0177]

Table 15-3

[0178]

Table 15-4

[0179]

Table 15-5

Claims

1. An antibody that binds to human LAIR1, the antibody comprising 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; 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; An antibody wherein LCDR3 comprises SEQ ID NO:

6.

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

8.

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

6. The antibody of 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 of 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, the antibody comprising a VH and a VL, the VH comprising HCDR1, HCDR2, and HCDR3, and the VL comprising LCDR1, LCDR2, and LCDR3; 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; An antibody wherein LCDR3 comprises SEQ ID NO:

18.

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

20.

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

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

22.

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

22.

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

16. The antibody of any one of claims 1 to 15, wherein the antibody also binds to 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 of claim 17.

19. 19. The vector of 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. 19. The vector of claim 18, comprising 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. 27. A process for producing an antibody, comprising culturing a 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. 28. An antibody produced by the process of claim 27.

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

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

31. 31. The method of 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. 29. The antibody of any one of claims 1 to 16 or claim 28, wherein the antibody does not form a complex with the LAIR1 ligand Clq.

33. 29. The antibody of any one of claims 1 to 16 or claim 28, wherein the antibody does not require full receptor occupancy (RO) to induce agonism.

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

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

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

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

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

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