Anti-MS4A6A antibodies and methods of use thereof

Abstract

The present disclosure generally relates to compositions comprising antibodies, such as monoclonal antibodies, antibody fragments, etc., that specifically bind to MS4A6A polypeptides, such as mammalian MS4A6A or human MS4A6A, and to the use of such compositions in preventing, reducing risk, or treating individuals in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 624,578, filed on January 31, 2018, and U.S. Provisional Application No. 62 / 646,846, filed on March 22, 2018, each of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to anti-MS4A6A antibodies and therapeutic uses of such antibodies. Background Art

[0004] The transmembrane 4 domain subfamily A (MS4A) gene cluster is present on chromosome 11q12 and contains eighteen genes. The MS4A gene family encodes membrane proteins that usually have a four-transmembrane topology (Ishibashi et al., 2001, Gene, 265: 87-93; Liang and Tedder, 2001, Genomics, 72: 119-127; Efthymiou and Goate, 2017, Molecular Neurodegeneration, 12: 43). The transmembrane domains are interconnected by an intracellular loop and two extracellular loops, and both the N-terminus and the C-terminus are located in the cytosol. Most MS4A proteins share amino acid sequence homology (20-30% similarity) with MS4A1 (CD20), and the highest degree of sequence identity is present in the first three transmembrane domains. In different MS4A proteins, the highly conserved motifs within these transmembrane regions indicate that the transmembrane domain plays an important general role in the function of MS4A proteins. The regions that vary most among MS4A proteins occur in their N-terminal and C-terminal cytoplasmic domains and within a putative second extracellular loop (Ishibashi et al., 2001, Gene, 265:87-93), suggesting that these regions confer unique functional properties.

[0005] Although there is such diversity, the MS4A domain has some common elements. For example, a notable feature conserved in the MS4A protein (except MS4A8B and MS4A12) is the conservation of two cysteine ​​residues in the second extracellular loop of the putative disulfide bond. The N-terminal and C-terminal domains of the MS4A protein are also rich in proline residues, but its functional significance needs to be clarified (Hulett et al., 2001, Genomics, 72: 119-127). However, the proline-rich region is generally involved in various cellular processes, such as cytoskeleton rearrangement, the initiation of transcription, signal transduction cascades, and the association with the SH3 domain as part of the adapter system, thereby facilitating protein-protein interactions (Kay et al., 2000, FASEB J, 14: 231-241).

[0006] The MS4A family of proteins is relatively undefined in function, with some important exceptions: MS4A1 (CD20) is expressed exclusively in B lymphocytes, where the protein functions in signaling through the B cell antigen receptor and calcium influx. CD20 is the target of immunotherapeutic antibodies that can be used to eliminate pathogenic B cells in chronic lymphocytic leukemia, lymphoma, autoimmune diseases, and solid organ transplantation. MS4A2 (FcεRβ) is a signaling subunit of the high-affinity IgE receptor (FcγRI) and low-affinity IgG receptor (FcγRIII) on mast cells, with a key role in hypersensitivity and allergic reactions. MS4A2 is an ITAM domain protein that amplifies signals through a high-affinity IgE receptor complex of four proteins. MS4A3 (Htm4) is expressed on the intracellular membranes of lymphoid and myeloid cells and acts as an adaptor protein in cell cycle regulation.

[0007] Although most MS4A family members have not been characterized, reports indicate that MS4A proteins can act as chemical sensors and chemoreceptors for a variety of exogenous and endogenous ligands (including fatty acids, peptides, and sulfated steroids), and are involved in mediating calcium influx, regulating endocytosis, trafficking, and can serve as adaptors of signal transduction complexes (Cruse et al., 2015, Mol Biol Cell, 26:1711-1727; Greer et al., 2016, Cell, 165:1734-1748; Eon Kuek et al., 2016, Cell, 165:1734-1748; Koslowski et al., 2008, Cancer Res, 68:3458-3466; Bubien et al., 1993; J Cell Biol, 121:1121-1132).

[0008] Certain MS4A genes are genetically associated with a variety of conditions and diseases, particularly neurodegenerative diseases. For example, genome-wide significant association analysis has identified the MS4A gene cluster located on chromosome 11q12 as one of the most significant Alzheimer's disease loci. One gene of particular interest identified is MS4A6A (Lambert et al., 2013, Nat Genet, 45: 1452-1458; Hollingworth et al., 2011, Nat Genet, 43: 429-435; Naj et al., 2011, Nat Genet, 43: 436-441). Various SNPs and associated alleles within the MS4A6A locus can contribute to differential expression (e.g., reduced expression or activity) of MS4A6A protein associated with disease risk (Ma et al., 2015, Mol Neurbiol, 51: 1240-1248; Lacher et al., 2018, Redox Biology, 14: 686-693).

[0009] Therefore, there is a need for therapies that target MS4A6A, including antibodies that specifically bind to MS4A6A, and / or therapies that are capable of modulating (e.g., activating or increasing) the activity of MS4A6A, such as by increasing MS4A6A protein levels or activity, to treat various diseases, disorders and conditions associated with MS4A6A activity.

[0010] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the invention

[0011] The present disclosure generally relates to anti-MS4A6A antibodies and methods of using such antibodies. The methods provided herein can be used to prevent neurodegenerative diseases, disorders or conditions, reduce the risk of neurodegenerative diseases, disorders or conditions, or treat individuals suffering from neurodegenerative diseases, disorders or conditions. In some embodiments, the present disclosure provides a method of preventing a neurodegenerative disease, disorder or condition, reducing the risk of a neurodegenerative disease, disorder or condition, or treating an individual suffering from a neurodegenerative disease, disorder or condition, wherein the disease neurodegenerative disease, disorder or condition is selected from the group consisting of Alzheimer's disease, late-onset Alzheimer's disease, dementia, and cognitive impairment, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments, the present disclosure provides a method for preventing a disease, disorder or condition associated with decreased expression or activity of MS4A6A, reducing the risk of such diseases, disorders or conditions, or treating an individual suffering from such diseases, disorders or conditions, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof.

[0012] In some embodiments, the present disclosure provides a method of preventing, reducing the risk of, or treating a disease, disorder, condition, or injury caused by or associated with decreased activity or expression of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments, the present disclosure provides a method of preventing, reducing the risk of, or treating a disease, disorder, condition, or injury caused by or associated with loss of function of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments, the present disclosure provides a method of preventing, reducing the risk of, or treating a neurodegenerative disease, disorder, condition, or injury in an individual having at least one genetic allele associated with risk or susceptibility to Alzheimer's disease, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments, the genetic allele associated with risk or susceptibility to Alzheimer's disease is selected from the group consisting of: rs583791 (C) allele, rs7232 (T) allele, and rs610932 (G) allele.

[0013] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to a discontinuous MS4A6A epitope. In certain embodiments that may be combined with any of the preceding embodiments, the discontinuous MS4A6A epitope comprises two or more peptides, three or more peptides, four or more peptides, five or more peptides, six or more peptides, seven or more peptides, eight or more peptides, nine or more peptides, or 10 or more peptides. In certain embodiments that may be combined with any of the preceding embodiments, each peptide comprises five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more amino acid residues of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 3; or corresponds to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 3. NO:3. five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more or 30 or more amino acid residues on the mammalian MS4A6A protein with the amino acid sequence of NO:3.

[0014] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to a conformational epitope of MS4A6A. In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to a linear epitope of MS4A6A.

[0015] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure compete with one or more reference anti-MS4A6A antibodies selected from the group consisting of: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, 6A-23. In some embodiments, the anti-MS4A6A antibody comprises a V of an antibody selected from the group consisting of: H and V L (For example, as shown in Table 4B below): 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22 and 6A-23.

[0016] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody of the present disclosure binds to an epitope of human MS4A6A that is identical to or overlaps with an epitope of MS4A6A bound by at least one reference antibody selected from the group consisting of: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, 6A-23. In some embodiments, the anti-MS4A6A antibody comprises a V domain of an antibody selected from the group consisting of: H and V L (For example, as shown in Table 4B below): 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22 and 6A-23.

[0017] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to an epitope of MS4A6A that is substantially the same as an epitope bound by at least one reference antibody selected from the group consisting of 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, 6A-23, and any combination thereof. In some embodiments, the anti-MS4A6A antibody comprises a V domain of an antibody selected from the group consisting of: H and V L (For example, as shown in Table 4B below): 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22 and 6A-23.

[0018] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to a linear epitope on MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 1-46 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 47-67 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 68-84 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 85-105 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 106-116 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 117-137 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 138-185 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 186-206 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 207-248 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to an amino acid residue at position 185 of human MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure bind to the extracellular domain 1 (ECL1) of MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to the extracellular domain 2 (ECL2) of MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to the ECL1 domain and the ECL2 domain of MS4A6A.

[0019] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A of the present disclosure is an isolated antibody that binds to human MS4A6A, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-16; HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-31; and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-45; and the light chain variable region comprises: HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-60; HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-76; and HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-90.

[0020] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A of the present disclosure is an isolated antibody that binds to human MS4A6A, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:91-108.

[0021] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A of the present disclosure is an isolated antibody that binds to human MS4A6A, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 109-126.

[0022] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A of the present disclosure is an isolated antibody that binds to human MS4A6A, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 91-108; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 109-126.

[0023] In certain embodiments that may be combined with any of the preceding embodiments, the MS4A6A protein is a mammalian protein or a human protein. In certain embodiments that may be combined with any of the preceding embodiments, the MS4A6A protein is a wild-type protein. In certain embodiments that may be combined with any of the preceding embodiments, the MS4A6A protein is a naturally occurring variant. In certain embodiments that may be combined with any of the preceding embodiments, the antibody is an agonist antibody, wherein the antibody induces, increases or enhances one or more MS4A6A activities. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody induces, enhances or maintains MS4A6A clusters on the surface of cells.

[0024] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure increase or enhance the interaction or binding of MS4A6A and at least one MS4A6A ligand or binding partner.

[0025] In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of signaling complexes. In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of signaling complexes associated with adapter proteins encoding ITAMs. In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of inhibitory signaling complexes. In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of inhibitory signaling complexes associated with adapter proteins encoding ITIMs.

[0026] In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of signaling complexes. In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of signaling complexes associated with adapter proteins encoding ITAMs. In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of inhibitory signaling complexes. In some embodiments that may be combined with any of the foregoing embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of inhibitory signaling complexes associated with adapter proteins encoding ITIMs.

[0027] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of MS4A6A homo-oligomeric cell surface protein complexes by: a) reducing the effective level of MS4A6A available for MS4A6A homo-oligomeric complex formation; b) blocking one or more of the sites on MS4A6A required for MS4A6A homo-oligomeric complex formation; c) preventing one or more post-translational events on MS4A6A required for MS4A6A homo-oligomeric complex formation and / or correct processing and / or cellular localization of MS4A6A; d) inducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0028] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure increase or enhance the formation of MS4A6A homo-oligomeric cell surface protein complexes by: a) increasing the effective level of MS4A6A available for MS4A6A homo-oligomeric complex formation; b) stabilizing one or more of the sites on MS4A6A required for MS4A6A homo-oligomeric complex formation; c) maintaining cell surface expression of MS4A6A to allow homo-oligomeric complex formation and / or correct processing and / or maintaining correct cellular localization of MS4A6A; d) reducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0029] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of MS4A6A hetero-oligomeric cell surface protein complexes with one or more signal transduction adaptor proteins by: a) reducing the effective level of MS4A6A available for MS4A6A hetero-oligomeric complex formation; b) blocking one or more of the sites on MS4A6A required for MS4A6A hetero-oligomeric complex formation; c) preventing one or more post-translational events on MS4A6A required for MS4A6A hetero-oligomeric complex formation and / or correct processing and / or cellular localization of MS4A6A; d) inducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0030] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure increase or enhance the formation of MS4A6A hetero-oligomeric cell surface protein complexes with one or more signal transduction adaptor proteins by: a) increasing the effective level of MS4A6A available for MS4A6A hetero-oligomeric complex formation; b) stabilizing one or more of the sites on MS4A6A required for MS4A6A hetero-oligomeric complex formation; c) maintaining cell surface expression of MS4A6A to allow MS4A6A hetero-oligomeric complex formation and / or correct processing and / or maintaining correct cellular localization of MS4A6A; d) reducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0031] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody is a human antibody, a humanized antibody, a bispecific antibody, a monoclonal antibody, a multivalent antibody, a conjugated antibody, or a chimeric antibody.

[0032] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody is a bispecific antibody that recognizes a first antigen and a second antigen. In certain embodiments that may be combined with any of the preceding embodiments, the first antigen is MS4A6A, and the second antigen is an antigen that facilitates transport across the blood-brain barrier. In certain embodiments that may be combined with any of the preceding embodiments, the second antigen is selected from the group consisting of MS4A6A, transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM 197, llama single domain antibodies, TMEM 30 (A), protein transduction domains, TAT, Syn-B, transmembrane peptides, polyarginine peptides, angiopep, basic immunoglobulin (basigin), Glut1 and CD98hc and ANG1005.

[0033] In some embodiments that can be combined with any of the preceding embodiments, the antibody is a monoclonal antibody. In some embodiments that can be combined with any of the preceding embodiments, the antibody is a human antibody. In some embodiments that can be combined with any of the preceding embodiments, the antibody is a humanized antibody. In some embodiments that can be combined with any of the preceding embodiments, the antibody is a bispecific antibody. In some embodiments that can be combined with any of the preceding embodiments, the antibody is a multivalent antibody. In some embodiments that can be combined with any of the preceding embodiments, the antibody is a chimeric antibody. In some embodiments that can be combined with any of the preceding embodiments, the antibody belongs to the IgG class, the IgM class, or the IgA class. In some embodiments, the antibody belongs to the IgG class and has an IgG1, IgG2, or IgG4 isotype. In certain embodiments that can be combined with any of the preceding embodiments, the anti-MS4A6A antibody is an antibody fragment that binds to an epitope comprising amino acid residues on a human MS4A6A or mammalian MS4A6A protein. In certain embodiments that can be combined with any of the preceding embodiments, the fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragment.

[0034] Other aspects of the present disclosure relate to an isolated (e.g., monoclonal) anti-MS4A6A antibody, wherein the anti-MS4A6A antibody comprises at least one, two, three, four, five, or six HVRs of an antibody selected from the group consisting of: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23. In some embodiments, an anti-MS4A6A antibody comprises six HVRs of an antibody selected from the group consisting of: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23 (e.g., as shown in Table 4A below). In some embodiments, an anti-MS4A6A antibody comprises six HVRs of an antibody selected from the group consisting of: H and / or V L: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23. In some embodiments, the anti-MS4A6A antibody comprises a V of an antibody selected from the group consisting of: H and V L (For example, as shown in Table 4B below): 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22 and 6A-23.

[0035] Other aspects of the present disclosure relate to an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-MS4A6A antibody of any of the foregoing embodiments. Other aspects of the present disclosure relate to a vector comprising a nucleic acid of any of the foregoing embodiments. Other aspects of the present disclosure relate to an isolated host cell comprising a vector of any of the foregoing embodiments. Other aspects of the present disclosure relate to a method of producing an anti-MS4A6A antibody, the method comprising culturing the host cell of any of the foregoing embodiments, thereby producing the anti-MS4A6A antibody. In certain embodiments, the method further comprises recovering the anti-MS4A6A antibody produced by the host cell. Other aspects of the present disclosure relate to an isolated anti-MS4A6A antibody produced by the method of any of the foregoing embodiments. Other aspects of the present disclosure relate to a pharmaceutical composition comprising the anti-MS4A6A antibody of any of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0036] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody specifically binds to human MS4A6A, mouse MS4A6A, cynomolgus monkey MS4A6A, or a combination thereof.

[0037] It should be understood that one, some or all of the properties of the various embodiments described herein can be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Shown are immunoblots of cells transiently transfected with human or cynomolgus MS4A6A or MS4A4A and probed with two commercially available anti-MS4A6A antibodies from Abcam. Both antibodies detected human and cynomolgus MS4A6A specific bands, indicating that the cells express MS4A6A protein.

[0039] Figure 2A and Figure 2C HEK293 cells transfected with human MS4A6A ( Figure 2A and 2C ) and untransfected HEK293 cells ( Figure 2A and 2C Two independent FAC analyses (shaded trace in ). Figure 2B and Figure 2D HEK293 cells transfected with cynomolgus monkey MS4A6A ( Figure 2B and 2D ) and untransfected HEK293 cells ( Figure 2B and 2D Two independent FAC analyses (shaded trace in ).

[0040] Figure 3A and 3B The results of the hybridoma supernatant containing the anti-MS4A6A of the present disclosure were compared with untransfected U937 cells ( Figure 3A left trace in ) and untransfected K562 cells ( Figure 3B Compared with the left trace in Figure 2, U937 cells transfected with cynomolgus monkey MS4A6A ( Figure 3A right trace in ) or K562 cells ( Figure 3B Cell surface expression of cynomolgus monkey MS4A6A in (right trace in Figure ). DETAILED DESCRIPTION

[0041] The present disclosure relates to anti-MS4A6A antibodies (eg, monoclonal antibodies); and methods of making and using such antibodies; pharmaceutical compositions comprising such antibodies; nucleic acids encoding such antibodies; and host cells comprising nucleic acids encoding such antibodies.

[0042] The techniques and procedures described or referenced herein are well understood and generally adapted by those skilled in the art using conventional methods, such as widely used methods such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

[0043] I. Definitions

[0044] Unless otherwise indicated, the terms "MS4A6A" or "MS4A6A polypeptide" are used interchangeably herein to refer to any native MS4A6A from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). In some embodiments, the term encompasses both wild-type sequences and naturally occurring variant sequences (e.g., splice variants or allelic variants). In some embodiments, the term encompasses "full-length," unprocessed MS4A6A as well as any form of MS4A6A resulting from processing in a cell. In some embodiments, MS4A6A is human MS4A6A. In some embodiments, the amino acid sequence of an exemplary MS4A6A is Uniprot Accession No. Q9H2W1 as of March 1, 2001. In some embodiments, the amino acid sequence of an exemplary human MS4A6A is SEQ ID NO: 1.

[0045] The terms "anti-MS4A6A antibody," "antibody that binds to MS4A6A," and "antibody that specifically binds to MS4A6A" refer to an antibody that is capable of binding to MS4A6A with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting MS4A6A. In one embodiment, the extent of binding of the anti-MS4A6A antibody to an unrelated, non-MS4A6A polypeptide is less than about 10% of the binding of the antibody to MS4A6A, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to MS4A6A has a binding affinity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or smaller, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 In certain embodiments, the anti-MS4A6A antibody binds to an epitope of MS4A6A that is conserved between MS4A6A from different species.

[0046] With respect to the binding of an antibody to a target molecule, the term "specifically binds" to a specific polypeptide or an epitope on a specific polypeptide target or "specifically binds to" a specific polypeptide or an epitope on a specific polypeptide target or "has specificity" for a specific polypeptide or an epitope on a specific polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be measured by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specifically binds" to an epitope on a specific polypeptide or a specific polypeptide target or "specifically binds to" an epitope on a specific polypeptide or a specific polypeptide target or "has specificity" for a specific polypeptide or an epitope on a specific polypeptide target can be measured, for example, by measuring a KD of about 10 for the target. -4 M or smaller, 10 -5 M or smaller, 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller or KD in 10 -4 M to 10 -6 M or 10 -6 M to 10-10 M or 10 -7 M to 10 -9 M. As will be appreciated by those of skill, affinity and KD values ​​are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to the binding of a molecule to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0047] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) (including those formed from at least two intact antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0048] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light ("L") chains and two identical heavy ("H") chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H ), followed by several constant domains. Each light chain has a variable domain at one end (V L ), with a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains.

[0049] For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0050] Light chains from any vertebrate species can be classified into one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of the constant domain of the light chain. Immunoglobulins can be classified into different classes, or isotypes, based on the amino acid sequence of the constant domain of their heavy chains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("λ"), and muon ("μ"), respectively. The λ and α classes can be further divided into subclasses (isotypes) based on relatively minor differences in CH sequence and function, for example humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, for example, Abbas et al., Cellular and Molecular Immunology, 4th edition (WB Saunders Co., 2000).

[0051] The "variable region" or "variable domain" of an antibody (such as the anti-MS4A6A antibodies of the present disclosure) refers to the amino terminal domain of the heavy chain or light chain of the antibody. The variable domains of the heavy chain and light chain can be referred to as "V H ” and “V L These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0052] The term "variable" refers to the fact that certain segments of the variable domain differ widely in sequence between antibodies (such as the anti-MS4A6A antibodies of the present disclosure). The variable domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, in the light and heavy chain variable domains, the variability is concentrated in three segments called hypervariable regions (HVRs). The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three HVRs, which form a loop connecting the β-sheet structure (in some cases forming part of the β-sheet structure). The HVRs in each chain are held together in close proximity by the FR region and, together with the HVRs of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0053] As used herein, the term "monoclonal antibody" refers to an antibody (such as the monoclonal anti-MS4A6A antibody of the present disclosure) obtained from a group of substantially homogeneous antibodies, i.e., each individual antibody constituting the antibody group is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, etc.) that may be present in small amounts. Monoclonal antibodies are highly specific (directed against a single antigenic site). Compared to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be prepared by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, and techniques for producing human or human-like antibodies in animals having part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0054] The terms "full-length antibody", "intact antibody" or "complete antibody" are used interchangeably and refer to an antibody (such as the anti-MS4A6A antibodies of the present disclosure) in substantially complete form, as compared to an antibody fragment. Specifically, complete antibodies include those having heavy and light chains comprising an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the complete antibody may have one or more effector functions.

[0055] "Antibody fragment" refers to a molecule other than an intact antibody, which includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules formed from antibody fragments and multispecific antibodies.

[0056] Papain digestion of antibodies (such as the anti-MS4A6A antibodies of the present disclosure) produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment (a name reflecting the ability to crystallize readily). The Fab fragment consists of the entire light chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H 1) Composition. Each Fab fragment is monovalent with respect to antigen binding, i.e., has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and is still able to cross-link antigen. Fab' fragments differ from Fab fragments in that the C H The carboxyl terminus of the constant domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced in the form of Fab' fragment pairs that had hinge cysteines between the Fab' fragments. Other chemical conjugates of antibody fragments are also known.

[0057] The Fc fragment comprises the carboxyl terminal portions of two heavy chains held together by disulfide bonds.The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcR) present on certain types of cells.

[0058] The "functional fragment" of an antibody (such as the anti-MS4A6A antibody of the present disclosure) includes a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody, or the Fc region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0059] The term "double body" refers to the use of V H Domain and V L Small antibody fragments prepared by constructing sFv fragments (see the preceding paragraph) with short linkers (about 5-10 residues) between the domains to allow interchain, but not intrachain, pairing of the variable domains, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "cross-linked" sFv fragments, in which the V domains of the two antibodies are linked together. H Domain and V L Domains are present on different polypeptide chains.

[0060] As used herein, "chimeric antibody" refers to an antibody (immunoglobulin) (such as the chimeric anti-MS4A6A antibody of the present disclosure) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. The chimeric antibodies of interest herein include Antibodies, wherein the antigen binding region of the antibody is derived from, for example, an antibody generated by immunizing a macaque with an antigen of interest. As used herein, "humanized antibody" is used as a subset of "chimeric antibody".

[0061] A "humanized" form of a non-human (e.g., murine) antibody (such as a humanized form of an anti-MS4A6A antibody of the present disclosure) is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0062] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human (such as the anti-MS4A6A antibodies of the present disclosure) and / or has been prepared using any of the techniques for preparing human antibodies as disclosed herein. This definition of a human antibody expressly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries and yeast display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic challenge, but the endogenous loci of the transgenic animal have been disabled (e.g., an immunized xenomice).

[0063] As used herein, the term "hypervariable region", "HVR" or "HV" refers to regions of an antibody variable domain (such as the variable domains of the anti-MS4A6A antibodies of the present disclosure) whose sequences are hypervariable and / or form structurally defined loops. Generally, an antibody comprises six HVRs; H There are three (H1, H2, H3), and V L There are three of them (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. Naturally occurring camelid antibodies composed only of heavy chains are functional and stable in the absence of light chains.

[0064] Many HVR descriptions are in use and are included in this article. In some embodiments, HVR can be a Kabat complementary determining region (CDR) based on sequence variability, and is the most commonly used (Kabat et al., supra). In some embodiments, HVR can be a Chothia CDR. And Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). In some embodiments, HVR can be an AbM HVR. AbM HVR represents a compromise between Kabat CDR and Chothia structural loops, and is used by the AbM antibody modeling software of Oxford Molecular. In some embodiments, HVR can be a "contact" HVR. "Contact" HVR is based on the analysis of available complex crystal structures. The residues from each of these HVRs are annotated as follows.

[0065]

[0066] An HVR may comprise an "extended HVR" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (preferred embodiments) (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al. (supra).

[0067] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0068] As used herein, an "acceptor human framework" is a V sequence comprising a human immunoglobulin framework or a human consensus framework. L or V H The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may comprise pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. If pre-existing amino acid changes are present in VH, preferably those changes occur only in three, two, or one of the 71H, 73H, and 78H positions; for example, the amino acid residues at those positions may be 71A, 73T, and / or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to the V L The human immunoglobulin framework sequence or the human consensus framework sequence is identical.

[0069] The "human consensus framework" is a L or V H The framework sequences are selected to represent the most common amino acid residues. L or V H The sequence is selected from a subset of variable domain sequences. Generally, the subset of sequences is the subset described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include: for V L , the subgroup may be subgroup κI, κII, κIII or κIV as described by Kabat et al. (supra). H, the subgroup may be subgroup I, subgroup II or subgroup III as described by Kabat et al. (supra).

[0070] An "amino acid modification" at a specified position of, for example, an anti-MS4A6A antibody of the present disclosure refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to a specified residue means an insertion within one to two residues of the specified residue. The insertion may be at the N-terminus or C-terminus of the specified residue. A preferred amino acid modification herein is a substitution.

[0071] An "affinity matured" antibody (such as the affinity matured anti-MS4A6A antibodies of the present disclosure) is one that has one or more alterations in one or more HVRs of the antibody that result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not have one or more of those alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity matured antibodies by V H Domain and V L Affinity maturation by domain shuffling. Random mutagenesis of HVR and / or framework residues is described, for example, in Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0072] "Fv" is the smallest antibody fragment containing complete antigen recognition and binding sites. The fragment consists of a dimer of a heavy chain and a light chain variable region domain that are tightly and non-covalently associated. Six hypervariable loops (3 loops each from H and L chains) are produced by the folding of these two domains, which contribute to the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigens, although the affinity is less than the entire binding site.

[0073] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide also comprises a VH and V L A polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding.

[0074] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.

[0075] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as a sequence segment from the amino acid residue at position Cys226 or at position Pro230 to their carboxyl termini. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed, for example, during the production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include antibody groups in which all K447 residues are removed, antibody groups in which K447 residues are not removed, and antibody groups with and without a mixture of antibodies with K447 residues. The native sequence Fc region suitable for antibodies of the present disclosure includes human IgG1, IgG2, IgG3, and IgG4.

[0076] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, and naturally occurring variants thereof.

[0077] A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by virtue of at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of a parent polypeptide, such as about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, compared to the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, most preferably at least about 90% homology with them, more preferably at least about 95% homology.

[0078] "Fc receptor" or "FcR" describes a receptor that is bound to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are FcRs that bind to IgG antibodies (gamma receptors), and include receptors of FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors, FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibitory receptors"), which have similar amino acid sequences, the main difference of which is their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. Other FcRs (including those identified in the future) are also encompassed in the term "FcR" herein. FcR can also increase the serum half-life of antibodies.

[0079] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0080] When used in the context of antibodies that compete for the same epitope (e.g., neutralizing antibodies), the term "competition" means competition between antibodies as determined by an assay in which the antibody being tested prevents or inhibits (e.g., reduces) specific binding of a reference molecule (e.g., a ligand or a reference antibody) to a common antigen (e.g., MS4A6A or a fragment thereof). Various types of competitive binding assays can be used to determine whether an antibody competes with another antibody, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assay, solid phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1996). Press); solid phase direct labeling RIA using 1-125 labeling (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, this assay involves the use of purified antigens bound to a solid surface or cells carrying any of these unlabeled test antibodies and labeled reference antibodies. Competitive inhibition is measured by determining the amount of labeling bound to a solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are close enough to the epitope bound by the reference antibody to cause steric hindrance. Additional details about methods for determining competitive binding are provided in the Examples herein. Typically, when a competing antibody is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antibody to a common antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5% and / or nearly 100%.

[0081] As used herein, an "interaction" between an MS4A6A polypeptide and a second polypeptide encompasses, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits" an "interaction" between two polypeptides when the antibody destroys, reduces, or completely eliminates the interaction between the two polypeptides. An antibody to a polypeptide of the present disclosure "inhibits" an "interaction" between two polypeptides when the antibody to the polypeptide binds to one of the two polypeptides. In some embodiments, the interaction may be inhibited by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or nearly 100%.

[0082] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting an antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Most commonly, the epitope is located on a polypeptide, but in some cases, it may be located on other types of molecules (such as nucleic acids). An epitope determinant may include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural features and / or specific charge characteristics. Typically, antibodies that are specific for a particular target antigen will preferentially recognize epitopes on a target antigen in a complex mixture of polypeptides and / or macromolecules.

[0083] An "agonistic" antibody or "activating" antibody is an antibody that induces (eg, increases) one or more activities or functions of the antigen after the antibody binds to the antigen.

[0084] An "antagonist" antibody or "blocking" antibody or "inhibitory" antibody is an antibody that reduces, inhibits and / or eliminates (e.g., reduces) the binding of an antigen to one or more ligands after the antibody binds to the antigen, and / or reduces, inhibits and / or eliminates (e.g., reduces) one or more activities or functions of the antigen after the antibody binds to the antigen. In some embodiments, an antagonist, blocking or inhibitory antibody substantially or completely inhibits the binding of an antigen to one or more ligands and / or one or more activities or functions of the antigen.

[0085] An "isolated" antibody (such as an isolated anti-MS4A6A antibody of the present disclosure) is an antibody that has been identified, separated and / or recovered from a component of the production environment (e.g., natural or recombinant) of the antibody. Preferably, the isolated antibody is free of all other contaminating components of the production environment of the antibody. Contaminating components of the production environment of the antibody, such as those produced from recombinant transfected cells, are substances that typically interfere with the research, diagnostic or therapeutic use of the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified to: (1) greater than 95%, in some embodiments greater than 99%, by weight of the antibody as determined by, for example, the Lowry method; (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence as determined by use of a spinning cup sequencer, or (3) homogeneity as determined by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Because at least one component of the antibody's natural environment will not be present, an isolated antibody includes the antibody in situ within a recombinant T cell. Ordinarily, however, isolated polypeptide or antibody will be prepared by at least one purification step.

[0086] An "isolated" nucleic acid molecule encoding an antibody (such as the anti-MS4A6A antibody of the present disclosure) is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule, which is generally associated with the environment in which the isolated nucleic acid molecule is produced. Preferably, the isolated nucleic acid is free of all components associated with the environment in which it is produced. The form of the isolated nucleic acid molecules encoding the polypeptides and antibodies herein is different from the form or background in which the nucleic acid molecules exist in nature. Therefore, isolated nucleic acid molecules are different from nucleic acids encoding the polypeptides and antibodies herein that are naturally present in cells.

[0087] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA, to which additional DNA segments can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which additional DNA segments can be connected to a viral genome. Some vectors can replicate autonomously in the host cells they introduce (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operably connected to them. These vectors are referred to herein as "recombinant expression vectors", or simply as "expression vectors". Typically, useful expression vectors in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms.

[0088] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0089] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny are not necessarily completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with one or more polynucleotides of the present disclosure.

[0090] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed.

[0091] As used herein, the term "preventing" includes providing prevention against the occurrence or recurrence of a particular disease, disorder or condition in an individual. An individual may be predisposed to, susceptible to, or at risk of a particular disease, disorder or condition, but has not yet been diagnosed as having the disease, disorder or condition.

[0092] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable symptoms of the disease or disease, and may or may not display detectable symptoms of the disease or disease prior to the treatment methods described herein. As known in the art, "at risk" means that an individual has one or more risk factors, which are measurable parameters associated with the development of a particular disease, disorder, or condition. An individual who has one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual who does not have one or more of these risk factors.

[0093] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desired therapeutic effects include reduced progression rate, amelioration or alleviation of pathological conditions, and alleviation or improvement of the prognosis of a particular disease, disorder, or condition. For example, an individual is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0094] "Effective amount" refers to the amount that effectively achieves the desired treatment or prevention result at least at the necessary dosage and time period. The effective amount can be provided in one or more administrations. The effective amount herein can vary according to various factors, such as the disease state, age, sex and weight of the individual and the ability of the treatment to cause the desired response in the individual. The effective amount is also the amount in which the beneficial effect of the treatment exceeds any toxic or harmful effect of the treatment. For preventive use, the beneficial or desired results include such as eliminating or reducing risks, reducing severity or delaying the onset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, complications of the disease, and intermediate pathological phenotypes that occur during the generation of the disease. For therapeutic use, the beneficial or desired results include such as reducing one or more symptoms caused by the disease, improving the quality of life of the disease patient, reducing the dosage of other drugs required for treating the disease, and enhancing the clinical results of the effect of another drug, such as via targeting, delaying the progression of the disease and / or prolonging the survival period. The effective amount of a drug, compound or pharmaceutical composition is sufficient to directly or indirectly achieve the amount of preventive or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if it can achieve or has achieved the desired result in conjunction with one or more other agents.

[0095] For treatment, prevention, or risk reduction purposes, a "subject" refers to any animal classified as a mammal, including humans, domestic and farm animals, zoo animals, sports animals, or pets, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, minks, rats, cats, etc. In some embodiments, the subject is a human.

[0096] As used herein, administration "in conjunction" with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or as a separate composition, including administration at different dosing frequencies or intervals, and using the same route of administration or different routes of administration. In some embodiments, administration in conjunction is administration as part of the same treatment regimen.

[0097] As used herein, the phrase "substantially similar" refers to a sufficiently high similarity between two values ​​(usually one value is associated with a molecule and the other value is associated with a reference / comparison molecule) so that one skilled in the art would consider the difference between the two values ​​to be not statistically significant in the case of a biological characteristic measured by the value (e.g., Kd value, protein expression level). The difference between the two values ​​may vary with the reference / comparison value, for example, less than about 20%, less than about 10%, and / or less than about 5%. The phrase "substantially normal" refers to being substantially similar to a reference (e.g., a normal reference).

[0098] The phrase "substantially different" refers to a sufficiently high difference between two values ​​(usually one value associated with a molecule and the other value associated with a reference / comparator molecule) such that one skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the value (e.g., Kd value, protein expression level). The difference between the two values ​​can vary with the value of the reference / comparator molecule, for example, by greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%.

[0099] The "presence," "amount," or "level" of a biomarker associated with an increased clinical benefit in an individual is a detectable level in a biological sample. These can be measured by methods known to those of skill in the art and disclosed herein. The expression level or amount of the assessed biomarker can be used to determine the response to treatment.

[0100] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process of converting information (e.g., genetic coding and / or epigenetic) into a structure present in a cell and operating in a cell. Therefore, as used herein, "expression" can refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Transcribed polynucleotides, fragments of translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered to indicate whether they are derived from transcripts or degraded transcripts produced by alternative splicing, or from post-translational processing of polypeptides, such as by proteolysis."Expressed genes" include those genes that are transcribed into polynucleotides (i.e., mRNA) and then translated into polypeptides, as well as those genes (e.g., transport and ribosomal RNA) that are not translated into polypeptides but are transcribed into RNA.

[0101] "Elevated expression," "elevated expression level," or "elevated level" refers to increased expression or increased levels of a biomarker or polypeptide in an individual relative to a control, such as one or more individuals not suffering from a disease or disorder (e.g., Alzheimer's disease) or an internal control (e.g., a housekeeping biomarker or polypeptide).

[0102] "Decreased expression," "decreased expression level," or "decreased level" refers to decreased expression or decreased levels of a biomarker or polypeptide in an individual relative to a control, such as one or more individuals not suffering from a disease or disorder (e.g., Alzheimer's disease) or an internal control (e.g., a housekeeping biomarker or polypeptide).

[0103] The term "housekeeping biomarker" refers to a biomarker or a group of biomarkers (e.g., polynucleotides and / or polypeptides) that are generally similarly present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene". "Housekeeping gene" herein refers to a gene or a group of genes that encode proteins whose activity is essential for the maintenance of cellular function and is generally similarly present in all cell types.

[0104] As used herein, the term "sample" refers to a composition obtained from or derived from a subject and / or individual of interest, the composition containing cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics. For example, the phrase "disease sample" and its variations refer to any sample obtained from a subject of interest, and it is expected or known that the subject contains cells and / or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, hematogenous cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates and tissue culture media, tissue extracts (such as homogenized tissue), tumor tissue, cell extracts, and combinations thereof.

[0105] By "tissue sample" or "cell sample" is meant a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample can be solid tissue such as from fresh, frozen and / or preserved organs, tissue samples, biopsies and / or aspirates; blood or any blood component (such as plasma); body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time of pregnancy or development of the subject. The tissue sample can also be a primary or cultured cell or cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. The tissue sample can contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0106] As used herein, "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" refers to a sample, cell, tissue, standard or level for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of the same subject or individual. For example, healthy and / or non-diseased cells or tissues adjacent to diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissues and / or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of an individual that is not a subject or a subject. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from untreated tissues and / or cells of the body of an individual that is not a subject or a subject.

[0107] By "correlate" or "associated" is meant comparing the performance and / or results of a first analysis or protocol to the performance and / or results of a second analysis or protocol in any manner. For example, the results of a first analysis or protocol can be used in performing a second protocol, and / or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be performed. With respect to embodiments of polynucleotide analyses or protocols, the results of a polynucleotide expression analysis or protocol can be used to determine whether a specific treatment protocol should be performed.

[0108] As used herein, the term "about" refers to the typical error range for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments involving that value or parameter per se.

[0109] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to an "antibody" refers to reference to one or more antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[0110] It should be understood that the aspects and embodiments of the present disclosure described herein include "comprising aspects and embodiments," "consisting of," and "consisting essentially of aspects and embodiments."

[0111] II. Anti-MS4A6A Antibody

[0112] Anti-MS4A6A antibodies are provided herein. The provided antibodies can be used, for example, for the diagnosis or treatment of MS4A6A-related diseases.

[0113] In one aspect, the present disclosure provides an isolated (e.g., monoclonal) antibody that binds to an epitope within the MS4A6A protein of the present disclosure. The MS4A6A protein of the present disclosure includes, but is not limited to, mammalian MS4A6A protein, human MS4A6A protein, mouse MS4A6A protein, and cynomolgus monkey MS4A6A protein.

[0114] Human MS4A6A is a 248 amino acid protein encoding a membrane glycoprotein. The amino acid sequence of human MS4A6A is shown in SEQ ID NO: 1:

[0115] MTSQPVPNETIIVLPSNVINFSQAEKPEPTNQGQDSLKKHLHAEIKVIGTIQILCGMMVLSLGIILASASFSPNFTQVTSTLLNSAYPFIGPFFFIISGSLSIATEKRLTKLLVHSSLVGSILS ALSALVGFIILSVKQATLNPASLQCELDKNNIPTRSYVSYFYHDSLYTTDCYTAKASLAGTLSLMLICTLLEFCLAVLTAVLRWKQAYSDFPGSVLFLPHSYIGNSGMSSKMTHDCGYEELLTS.

[0116] In addition, the amino acid sequence of the mouse ortholog MS4A6D is shown in SEQ ID NO: 2:

[0117] MTKPLVHSSLALSILSVLSALTGIAILSVSLAALEPALQQCKLAFTQLDTTQDAYHFFSPEPLNS

[0118] CFVAKAALTGVFSLMLISSVLELGLAVLTATLWWKQSSSAFSGNVIFLSQNSKNKSSVSSESL

[0119] CNPTYENILTS

[0120] In addition, the amino acid sequence of cynomolgus monkey (cyno) MS4A6A is shown in SEQ ID NO: 3:

[0121] MTSQPVPNETMIVLPSNVINFSQAEKPEPTNQGQDSLKKRLQAEVKVIGTTQILCGVMVLSLGIMLASASFSPNFTQVTSTLLNSAYPFIGPFFFIISGSLSIATEKKLTKLLVHSSLVGSIL SALSALVGFIILSVELAALNPASLQCELDKNNIPTRSYVSYYYHDSLYTMDCYTVKASLAGPLSLMLICTLLEFCLAVLTAVLRWKQTVSDFPGSVLFLPHSYIDNSGMSSKMTHGPGYEELLS

[0122] In some embodiments, MS4A6A is expressed in cells. In some embodiments, MS4A6A is expressed in bone marrow cells. In some embodiments, MS4A6A is expressed in brain cells. In some embodiments, MS4A6A is expressed in astrocytes, including but not limited to mature astrocytes. In some embodiments, MS4A6A is expressed in oligodendrocytes. In some embodiments, MS4A6A is expressed in microglia. In some embodiments, MS4A6A is expressed in immune cells, including but not limited to macrophages, eosinophils, mast cells, dendritic cells, natural killer cells, neutrophils, and T cells. In some embodiments, MS4A6A is expressed in olfactory cells. In some embodiments, MS4A6A is expressed on the surface of cells.

[0123] The MS4A6A protein disclosed herein includes several domains, including but not limited to a cytoplasmic domain (amino acid residues 1-46 of human MS4A6A; see SEQ ID NO: 1); a transmembrane domain (amino acid residues 47-67 of human MS4A6A); an extracellular domain (extracellular domain 1; ECL1), corresponding to amino acid residues 68-84 of human MS4A6A; a transmembrane domain (amino acid residues 85-105 of human MS4A6A); a cytoplasmic domain (amino acid residues 106-116 of human MS4A6A); a transmembrane domain (amino acid residues 117-137 of human MS4A6A); an extracellular domain ( Extracellular domain 2; ECL2), corresponding to amino acid residues 138-185 of human MS4A6A; transmembrane domain (amino acid residues 186-206 of human MS4A6A); and cytoplasmic domain (amino acid residues 207-248 of human MS4A6A); In addition, the MS4A6A protein of the present disclosure is expressed in many tissues and cells, including but not limited to the brain, neurons, glial cells, endothelial cells, pericytes, pericytes, etc.

[0124] MS4A6A binding partners and adaptor proteins

[0125] Also provided herein are methods for screening for anti-MS4A6A antibodies that bind to MS4A6A and block the interaction between MS4A6A and one or more MS4A6A ligands or binding partners. In some embodiments, a peptide library can be synthesized in which the MS4A6A protein is cleaved into contiguous 15-mer and 25-mer peptides separated by one amino acid residue, and then the peptide library is spotted onto a filter membrane. The ability of the MS4A6A ligand or binding partner to bind to the MS4A6A peptide or to interact with multiple peptides in the presence or absence of anti-MS4A6A antibodies can then be tested by SPOT binding analysis (e.g., Frank, R and Overwin, H (1996) Methods. Mol. Biol. 66, 149-169; Reineke, U et al., (2002) J. Immunol. Methods 267, 13-26; and Andersen, OS et al., (2010) J, BIOLOGICAL CHEMISTRY 285, 12210-12222). In some embodiments, the cellulose support can be prepared as an N-modified cellulose-aminohydroxypropyl ether membrane and all synthetic rounds begin with spot definition by 9-fluorenylmethoxycarbonylalanine-pentafluorophenyl ester (generating an alanine linker between the peptide and the membrane). For example, the automated linear synthesis performed by the stepwise addition of different amino acids is protected by 9-fluorenyl-methoxycarbonyl at the N-terminus, and the growing peptide chain is appropriately protected by the side chain. In some embodiments, the pattern of deprotection, activation and coupling is continued until a 16-mer peptide is produced, thereby producing a uniformly distributed array of covalently anchored peptides and cellulose supports at the C-terminus, and the N-terminus is a free end (Scharn, D et al., (2000) J. Comb. Chem. 2, 361-369). In some embodiments, the removal of the side protecting group can be performed in two steps. First, the membrane can be treated with 90% trifluoroacetic acid (dissolved in dichloromethane, containing 3% triisobutylsilane and 2% H2O); secondly, the membrane is treated with, for example, 60% trifluoroacetic acid (dissolved in dichloromethane, containing 3% triisobutylsilane and 2% H2O). In order to remove trifluoroacetate, the membrane can be washed several times with H2O, ethanol, Tris buffered saline and ethanol, and then the membrane is dried. Finally, the membranes were blocked in blocking buffer swollen in Tris-buffered saline (pH 8.0) and supplemented with 5% sucrose for 2 h prior to preparing the predetermined peptide library for ligand binding analysis. In some embodiments, for binding studies of cellulose-binding peptides, the membrane-bound library can be incubated with a combinatorial S peptide and a polyhistidine tag.In some embodiments, cellulose binding protein can be used to characterize the interaction between MS4A6A and MS4A6A ligands and binding partners (e.g., Andersen et al., 2010, J Biol Chem, 285, 12210-12222). For example, the membrane-bound protein can be incubated with another MS4A6A ligand or binding partner; incubated overnight at 4°C in blocking buffer, and then incubated a second time with 1 μg / ml HRP-conjugated S protein in blocking buffer, but at room temperature for 3 hours. Subsequently, the membrane can be washed three times for 10 minutes with Tris-buffered saline, and then quantitative characterization of the bound ligand can be performed using UptiLight chemiluminescent substrate and Lumilmager instrument to provide spot signal intensity (Boehringer light units). Alternatively, detection of the bound ligand can be performed by an immunochemical assay using an antibody against a histidine tag and an HRP-conjugated anti-mouse secondary antibody. Incubation can be performed after standard Western blot analysis and spot detection.

[0126] In some embodiments, the interaction between MS4A6A and MS4A6A ligands and binding partners can be characterized using proximity ligation assays (e.g., Gustafsen et al., 2013 The Journal of Neuroscience, 33: 64-71). For example, cells expressing or exposed to MS4A6A and its ligands or binding partners can be subjected to proximity ligation assays (PLA) (Duolinkll) using a primary antibody against MS4A6A and an antibody against a binding partner, followed by incubation with a secondary antibody conjugated to an oligonucleotide, which hybridizes to the subsequently added ring-forming oligonucleotide and initiates rolling circle amplification when the antigen is localized in the vicinity of 40 nM. The amplified DNA can be visualized by adding a complementary fluorescently labeled oligonucleotide.

[0127] In some embodiments, alkaline phosphatase-labeled ligands can be used in cell binding assays to characterize the interaction between MS4A6A and MS4A6A ligands and binding partners (e.g., Hu et al., 2005, J. Neurosci. 25, 5298-5304; Fournier et al., 2001, Nature 409, 341-346; Lauren et al., 2009, Nature 457, 1128-1132; and Hu et al., 2010, Neuron 68, 654-667). For example, alkaline phosphatase (AP)-labeled ligands can be prepared to assess binding to MS4A6A on transfected cells or primary neurons. To detect the binding of AP-labeled ligands to cells expressing MS4A6A, the culture can be washed with, for example, Hanks' balanced salt solution (HBH), which contains 20 mM HEPES sodium pH 7.05 and 1 mg / ml bovine serum albumin (BSA). The plate can then be incubated with AP-labeled ligands in the presence or absence of MS4A6A blocking antibodies, for example, in HBH at 23° C. for 2 h. AP-bound ligands can be detected and quantified according to methods well known in the art.

[0128] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody further inhibits the interaction between MS4A6A and its ligands, signaling proteins, or binding proteins by: a) reducing the effective level of MS4A6A available for interaction with one or more ligands or binding proteins; b) blocking one or more of the sites on MS4A6A required for interaction with one or more ligands or binding proteins; c) preventing one or more post-translational events on MS4A6A required for interaction with one or more ligands or binding proteins and / or correcting the processing and / or subcellular localization of MS4A6A; d) inducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0129] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody further increases or enhances the interaction between MS4A6A and its ligands, signaling proteins, or binding proteins by: a) increasing the effective level of MS4A6A available for interaction with one or more ligands or binding proteins; b) increasing one or more of the sites on MS4A6A required for interaction with one or more ligands or binding proteins; c) increasing one or more post-translational events on MS4A6A required for interaction with one or more ligands or binding proteins and / or correcting the processing and / or subcellular localization of MS4A6A; d) preventing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0130] In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody specifically binds to human MS4A6A, mouse MS4A6A, cynomolgus monkey MS4A6A, or a combination thereof. In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody is a human antibody, a humanized antibody, a bispecific antibody, a monoclonal antibody, a multivalent antibody, a conjugated antibody, or a chimeric antibody. In certain embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibody is a bispecific antibody that recognizes a first antigen and a second antigen. In certain embodiments that may be combined with any of the preceding embodiments, the first antigen is MS4A6A, and the second antigen is an antigen that facilitates transport across the blood-brain barrier. In certain embodiments that may be combined with any of the preceding embodiments, the second antigen is selected from the group consisting of: MS4A6A, transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM 197, llama single domain antibody, TMEM 30 (A), protein transduction domain, TAT, Syn-B, transmembrane peptide, polyarginine peptide, angiopep, basic immunoglobulin (basigin), Glut1 and CD98hc and ANG1005.

[0131] Signal transduction adapter proteins are, but are not limited to, auxiliary proteins for other proteins associated with signal transduction pathways. Adapter proteins contain various protein binding modules or motifs that connect protein binding partners together, helping to generate larger signal transduction complexes. Adapter proteins typically contain several domains within their structure that allow specific interactions with one or more other specific proteins. For example, the Src homology 2 (SH2) domain recognizes specific amino acid residue sequences within proteins containing phosphotyrosine residues. The interaction of adapter proteins with other signal transduction proteins allows multiple specific and coordinated protein-protein interactions to occur within cells during and in relation to signal transduction.

[0132] An immunoreceptor tyrosine-based activation motif (ITAM) is a conserved sequence of four amino acid residues that is repeated twice in the cytoplasmic tail of certain cell surface proteins of the immune system. In the C-terminus of certain proteins, the consensus motif is YxxI / Lx (6-12) YxxI / L (SEQ ID NO: 128). The two repeat sequences are usually separated by between 6 and 12 amino acid residues (YxxL / Ix (6.12) YxxL / I (SEQ ID NO: 128). ITAMs are important for signal transduction; tyrosine residues within these motifs are phosphorylated upon interaction of receptor molecules with their ligands and form docking sites for other proteins involved in signal transduction (Barrow and Trowsdale, 2006, Eur J Immunol, 36: 1646-1653).

[0133] Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are conserved sequences of amino acids (S / I / V / LxYxxI / V / L (SEQ ID NO: 127)) that are present in the cytoplasmic tails of certain inhibitory receptors of the immune system. After ITIM-containing inhibitory receptors interact with their ligands, their ITIM motifs are phosphorylated by enzymes of the Src kinase family, allowing them to recruit other enzymes (such as various phosphotyrosine phosphatases) to reduce the activation of molecules involved in signal transduction. (Barrow and Trowsdale, 2006, Eur J Immunol, 36: 1646-1653).

[0134] Or, some receptors do not have intrinsic ITAM motifs, but encode positively charged transmembrane amino acids, such as lysine or arginine. These positively charged amino acid residues mediate the association of negatively charged transmembrane amino acid residues with the adapter protein of the corresponding coding ITAM. After ligand recognition and receptor clustering, the tyrosine amino acid residue is phosphorylated by Src family protein tyrosine kinase (PTK). The ITAM motif of dual phosphorylation serves as the docking site of the series connection SH2 domain of Syk family PTK (such as ZAP-70 or Syk). Syk family PTK phosphorylates a series of intracellular substrates, resulting in the formation of near-membrane end support, thereby resulting in the recruitment of important effector molecules (such as phospholipase Cc (PLCc)), thereby resulting in calcium signal conduction and Ras activation, and the Ras activation stimulates ERK pathway and cell activation.

[0135] Studies support the importance of MS4A proteins in forming signaling complexes with other cell surface membrane proteins that regulate or replicate downstream biochemical signals. Although in most cases, binding partners have not been clearly determined for MS4A proteins other than MS4A1 and MS4A2, predictive protein analysis shows that Src homology 2 (SH2) and SH3 domain binding sites are commonly found in the N-terminal and C-terminal regions of MS4A proteins, which can serve as docking platforms for other signaling molecules (Dinkel et al., 2012, Nucl Acids Res, 40: DD242-D251), and SH domains preferentially bind to proline-rich sequences, which are also commonly found in the cytoplasmic tail of MS4A proteins (Liang and Tedder, 2001, Genomics, 72: 119-127; Kay et al., 2000, FASEB J, 14: 231-241). This feature of the MS4A protein provides ligand in the presence or absence of anti-MS4A6A antibodies, for example, incubated overnight at 4°C in blocking buffer, followed by a second incubation with 1 mg / ml HRP-conjugated S protein in blocking buffer, but for 3 h at room temperature. Subsequently, the membrane can be washed with Tris-buffered saline (e.g., three times for 10 minutes), and then quantitative characterization of bound ligand can be performed using UptiLight chemiluminescent substrate and LumiImager instrument to provide spot signal intensity (Boehringer light units). Alternatively, detection of bound ligand can be performed by immunochemical assays using antibodies to histidine tags and HRP-conjugated anti-mouse secondary antibodies. Standard Western blot procedures and spot detection can be used for incubation.

[0136] Also provided herein are methods of screening for anti-MS4A6A antibodies that block the interaction (eg, binding) of MS4A6A with one or more MS4A6A ligands or binding partners.

[0137] In some embodiments, the interaction between MS4A6A and MS4A6A ligands or binding partners can be characterized using surface plasmon resonance analysis (e.g., Skeldal et al., 2012 J Biol Chem., 287:43798; and Andersen et al., 201, J Biol Chem, 285, 12210-12222). Direct binding of MS4A6A ligands or binding partners to immobilized MS4A6A can be determined, for example, on a Biacore 2000 instrument (Biacore, Sweden) using CaHBS as a standard running buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2 mM CaCl2, 1 mM EGTA, and 0.005% Tween 20). In some embodiments, a biosensor chip from Biacore (CM5) can be activated using the NHS / EDC method and then coated with MS4A6A to 79 fmol / mm 2 of protein density and used for affinity measurements of binding partners. The preparation of the biosensor surface using pro-MS4A6A will follow the same procedure. After each cycle of the ligand binding experiment, the regeneration of the flow cell can be performed by two 10μl buffer (10mM glycine-HCl pH 4.0, 500mM NaCl, 20mM EDTA and 0.005% Tween20) pulses and a single injection of 0.001% SDS. Fitting of the sensorgram for affinity estimation can be performed, for example, by using BIAevaluation version 3.1. Following a similar protocol, the immobilization of HisS-NGFpro or HisS-BDNFpro on a CM5 biosensor chip can also be performed using an NHS / EDC coupling kit to obtain a similar surface density of immobilized protein (~300fmol / mm 2 ). Biosensor chips immobilized with MS4A6A ligands or binding partners can also be used to examine the binding of MS4A6A in the absence or presence of competing MS4A6A antibodies.

[0138] In some embodiments, the interaction between MS4A6A and MS4A6A ligands and binding partners can be characterized using sedimentation assays (e.g., Andersen et al., 2010, J Biol Chem, 285, 12210-12222). For example, the expressed intracellular or extracellular domain of MS4A6A can be incubated with a labeled MS4A6A ligand or binding partner in the absence or presence of an MS4A6A blocking antibody and precipitated using 100 μl of glutathione (GSH)-agarose beads (Amersham Biosciences, catalog number 17-0756-01). The amount of applied receptor domain can be determined by precipitation using Talon beads as a control. Bound proteins can be separated by SDS-PAGE analysis and visualized by standard Western blot analysis using an anti-histidine antibody.

[0139] They are intimately involved in the solid foundation of protein-protein interactions and can also associate with each other to form signaling complexes.

[0140] Thus, in some embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of signaling complexes. In some embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of signaling complexes associated with adapter proteins encoding ITAMs. In some embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of inhibitory signaling complexes. In some embodiments, the anti-MS4A6A antibodies of the present disclosure enhance or increase the formation of inhibitory signaling complexes associated with adapter proteins encoding ITIMs.

[0141] In some embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of signaling complexes. In some embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of signaling complexes associated with adapter proteins encoding ITAMs. In some embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of inhibitory signaling complexes. In some embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of inhibitory signaling complexes associated with adapter proteins encoding ITIMs.

[0142] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of MS4A6A homo-oligomeric cell surface protein complexes by: a) reducing the effective level of MS4A6A available for MS4A6A homo-oligomeric complex formation; b) blocking one or more of the sites on MS4A6A required for MS4A6A homo-oligomeric complex formation; c) preventing one or more post-translational events on MS4A6A required for MS4A6A homo-oligomeric complex formation and / or correct processing and / or cellular localization of MS4A6A; d) inducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0143] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure increase or enhance the formation of MS4A6A homo-oligomeric cell surface protein complexes by: a) increasing the effective level of MS4A6A available for MS4A6A homo-oligomeric complex formation; b) stabilizing one or more of the sites on MS4A6A required for MS4A6A homo-oligomeric complex formation; c) maintaining cell surface expression of MS4A6A to allow homo-oligomeric complex formation and / or correct processing and / or maintaining correct cellular localization of MS4A6A; d) reducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0144] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit (e.g., block) or reduce the formation of MS4A6A hetero-oligomeric cell surface protein complexes with one or more signal transduction adaptor proteins by: a) reducing the effective level of MS4A6A available for MS4A6A hetero-oligomeric complex formation; b) blocking one or more of the sites on MS4A6A required for MS4A6A hetero-oligomeric complex formation; c) preventing one or more post-translational events on MS4A6A required for MS4A6A hetero-oligomeric complex formation and / or correct processing and / or cellular localization of MS4A6A; d) inducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0145] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure increase or enhance the formation of MS4A6A hetero-oligomeric cell surface protein complexes with one or more signal transduction adaptor proteins by: a) increasing the effective level of MS4A6A available for MS4A6A hetero-oligomeric complex formation; b) stabilizing one or more of the sites on MS4A6A required for MS4A6A hetero-oligomeric complex formation; c) maintaining cell surface expression of MS4A6A to allow MS4A6A hetero-oligomeric complex formation and / or correct processing and / or maintaining correct cellular localization of MS4A6A; d) reducing degradation of MS4A6A; e) changing the conformation of MS4A6A, or any combination thereof.

[0146] In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure increase or enhance MS4A6A receptor clustering. In some embodiments that may be combined with any of the preceding embodiments, the anti-MS4A6A antibodies of the present disclosure inhibit or reduce MS4A6A receptor clustering. In some embodiments, the increase or enhancement or inhibition or reduction of MS4A6A receptor clustering is in myeloid cells.

[0147] Agonist antibodies

[0148] The anti-MS4A6A antibodies of the present disclosure generally bind to one or more MS4A6A proteins expressed in a cell. One class of antibodies of the present disclosure is agonistic antibodies. For example, MS4A6A receptors may need to cluster on the cell surface to transduce signals. Therefore, agonistic antibodies may have unique characteristics to stimulate, for example, MS4A6A receptors. For example, they may have the correct epitope specificity compatible with receptor activation, as well as the ability to induce or maintain receptor clustering on the cell surface. The anti-MS4A6A agonistic antibodies of the present disclosure increase or enhance MS4A6A function or activity, for example, by increasing MS4A6A protein expression, by increasing MS4A6A protein levels, by correcting incorrect cellular localization, or by increasing or restoring cell surface expression.

[0149] In vivo, antibodies can cluster receptors through a variety of potential mechanisms. Some isotypes of human antibodies (such as IgG2) have the intrinsic ability to cluster receptors or keep receptors in a clustered configuration due to their unique structure, thereby activating receptors (such as MS4A6A) without binding to Fc receptors (e.g., White et al., (2015) Cancer Cell 27, 138-148).

[0150] Other antibodies cluster receptors (e.g., MS4A6A) by binding to Fcg receptors on neighboring cells. Binding of the constant IgG Fc portion of antibodies to Fcg receptors leads to aggregation of antibodies, which in turn aggregate receptors bound to them through their variable regions (Chu et al. (2008) Mol Immunol, 45: 3926-3933; and Wilson et al., (2011) Cancer Cell 19, 101-113). Binding to inhibitory Fcg receptors FcgR (FcgRIIB) that do not induce cytokine secretion, oxidative burst, increased phagocytosis, and enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) is generally a preferred way to cluster antibodies in vivo, because binding to FcgRIIB is not associated with adverse immune reactions.

[0151] Other mechanisms may also be used to cluster receptors (e.g., MS4A6A). For example, as described above, antibody fragments (e.g., Fab fragments) that are cross-linked together can be used to cluster receptors (e.g., MS4A6A) in a manner similar to antibodies with Fc regions that bind to Fcg receptors. Without wishing to be bound by theory, it is believed that if cross-linked antibody fragments (e.g., Fab fragments) induce receptor clustering on the cell surface and bind to the appropriate epitope on the target (e.g., MS4A6A), they can act as agonistic antibodies.

[0152] Thus, in some embodiments, antibodies that bind to the MS4A6A protein may include agonistic antibodies that bind to MS4A6A due to their epitope specificity and activate one or more MS4A6A activities. Without wishing to be bound by theory, such antibodies may bind to a ligand binding site on a target antigen (e.g., MS4A6A) and mimic the action of a natural ligand, or stimulate target antigen transduction signals by binding to one or more domains that are not ligand binding sites. Such antibodies do not interfere with ligand binding and may act additively or synergistically with a natural ligand.

[0153] In some embodiments, the anti-MS4A6A antibodies of the present disclosure are agonist antibodies that induce or increase one or more MS4A6A activities. In some embodiments, the anti-MS4A6A antibodies induce or increase one or more activities of the MS4A6A protein expressed in a cell. In some embodiments, the anti-MS4A6A antibodies of the present disclosure are agonist antibodies that increase the cell surface expression of MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure are agonist antibodies that correct the inappropriate cellular localization of MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure are agonist antibodies that increase or restore the cell surface targeting or localization of the MS4A6A protein. In some embodiments, the anti-MS4A6A antibodies of the present disclosure are agonist antibodies that increase or restore the cell surface expression of the MS4A6A protein.

[0154] Inert antibodies

[0155] Another class of antibodies disclosed herein includes inert antibodies. As used herein, "inert" antibodies refer to antibodies that specifically bind to their target antigen but do not modulate (e.g., reduce / inhibit or activate / induce) the function of the antigen. For example, with respect to MS4A6A, inert antibodies do not modulate ligand binding and / or MS4A6A activity. Without wishing to be bound by theory, it is believed that antibodies that do not have the ability to cluster MS4A6A on the cell surface may be inert antibodies, even if they have epitope specificity compatible with receptor activation.

[0156] In some embodiments, antibodies that bind to MS4A6A protein may include antibodies that bind to MS4A6A but do not modulate protein function due to their epitope specificity. Such functionally inert antibodies can be used as cargo for transporting toxins, as described for the CD33 antibody Gemtuzumab zogamicin (trade name Mylotarg), which is conjugated to a cytotoxic agent from the calicheamicin class and used to target and kill acute myeloid leukemia tumors (Naito et al., (2000), Leukemia, 14, 1436-1443; Ricart (2011) Clin Cancer Res 17; 6417-6436; Hamann et al., (2002) Journal: Bioconjugate Chemistry, 13, 47-58; and Beitz et al., (2001) Clin Cancer Res 7; 1490-6.). Thus, in some embodiments, the antibodies of the present disclosure are inert antibodies that bind MS4A6A but are unable to induce one or more MS4A6A activities (eg, the MS4A6A activities described herein).

[0157] Calcium influx

[0158] Cell lines transfected with MS4A1 showed increased calcium conductance between plasma membranes, indicating that MS4A1 acts as an important channel for regulating calcium homeostasis. (Parolini et al., 2012, Int J Biochem Cell Biol, 44: 2095-2105; Li et al., 2003, J Biol Chem, 278: 42427-42434) Therefore, MS4A proteins participate in the control of intracellular free calcium concentration by regulating (e.g., increasing) calcium influx and / or by mobilizing calcium from intracellular reservoirs (Ishibashi et al., 2001, Gene, 264: 87-93). Due to the conservation of protein structure in the MS4A family, other MS4A proteins (e.g., MS4A4A and MS4A6A) may have overlapping calcium regulatory functions (Ma et al., 2015, Mol Neurobiol, 51: 1240-1248). Thus, in some embodiments, the anti-MS4A6A antibodies of the present disclosure modulate calcium levels, calcium influx, and / or calcium mobilization from intracellular stores in a cell. In some embodiments, the anti-MS4A6A antibodies of the present disclosure increase calcium influx in a cell and / or increase calcium mobilization in a cell from intracellular stores. In some embodiments, the anti-MS4A6A antibodies of the present disclosure decrease calcium influx in a cell and / or decrease calcium mobilization in a cell from intracellular stores.

[0159] In addition, members of the MS4A protein family are chemoreceptors expressed in olfactory sensory neurons. The results support a model in which the MS4A receptor binds inhaled odors and induces calcium influx into olfactory sensory neurons, supporting the role of MS4A family members in regulating calcium influx in various cells (Greer et al., 2016, Cell, 165: 1734-1748).

[0160] A. Exemplary Antibodies and Certain Other Antibody Embodiments

[0161] In some embodiments, provided herein is an anti-MS4A6A antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; (b) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31; (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45; (d) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: NO:46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60; (e) HVR-L2, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76; and (f) HVR-L3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.

[0162] In some embodiments, provided herein is an anti-MS4A6A antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 17; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 5; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: NO:47; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:62; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:78; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:6; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:19; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:34; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:48; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:63; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:79; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:20; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: NO:35; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:49; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:64; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:80; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:36; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:50; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:82; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:38; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:52; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:66; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:82; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:38; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:52; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: NO:67; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:83; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:39; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:53; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:68; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:84; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:25; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:53; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:68; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:84 NO:54 amino acid sequence; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:69; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:85; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:11; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:26; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:39; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:53; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:70; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:84;(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:17; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:32; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:46; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:61; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:77; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:27; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:55; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: NO:86; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:56; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:87; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:14; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:29; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:57; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: NO:73 amino acid sequence; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:84; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:11; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:24; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:39; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:53; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:68; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:84; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:15;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:44; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:58; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:74; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:88; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:59; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:89; (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:59; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:89 NO:6; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:19; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:34; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:48; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:63; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:79; (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:16; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:31; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:45; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:60; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:76; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: ID NO:90 amino acid sequence; and (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:16; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:31; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:45; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:60; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:76; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:90. ;

[0163] In some embodiments, provided herein are anti-MS4A6A antibodies comprising at least one, at least two, or all three V selected from the group consisting of: H HVR sequences: (a) HVR-H1, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; (b) HVR-H2, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31; and (c) HVR-H3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.

[0164] In some embodiments, provided herein are anti-MS4A6A antibodies comprising at least one, at least two, or all three V selected from the group consisting of: L HVR sequences: (a) HVR-L1, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60; (e) HVR-L2, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76; and (f) HVR-L3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.

[0165] In some embodiments, provided herein is an anti-MS4A6A antibody comprising (a) V H A domain comprising at least one, at least two or all three V selected from the following HHVR sequences: (i) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, (ii) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31, and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45, and (b) V L A domain comprising at least one, at least two or all three V selected from the following L HVR sequences: (i) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60, (ii) HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76, and (iii) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.

[0166] In some embodiments, provided herein is an anti-MS4A6A antibody comprising (a) V H A domain comprising (i) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, (ii) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31, and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45, and (b) V LA domain comprising (i) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60, (ii) HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76, and (iii) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.

[0167] In another aspect, an anti-MS4A6A antibody comprises a heavy chain variable domain (V H ) sequence, the heavy chain variable domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and 108. In certain embodiments, the V domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of HThe sequence contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence: SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and 108, but the anti-MS4A6A antibody comprising the sequence retains the ability to bind to MS4A6A. In certain embodiments, in SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and 108, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted and / or deleted in SEQ ID NOs: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and 108. In certain embodiments, the substitutions, insertions or deletions occur in regions outside of HVRs (i.e., in FRs). Optionally, the anti-MS4A6A antibody comprises V sequences of SEQ ID NOs: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and 108. H In a specific embodiment, V H comprising one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, (b) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31, and (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.

[0168] In another aspect, an anti-MS4A6A antibody is provided, wherein the antibody comprises a light chain variable domain (V L), said light chain variable domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID NO: 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126. In certain embodiments, the V domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of L The sequence contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence: SEQ ID NO: 109, 110, 111, 112, 113, 114, 116, 117, 118, 119, 120, 121, 122, 123, 124 and 126, but the anti-MS4A6A antibody comprising the sequence retains the ability to bind to MS4A6A. In some embodiments, in SEQ ID NO: 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 and 126, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted and / or deleted in SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of HVRs (i.e., in FRs). Optionally, the anti-MS4A6A antibody comprises V sequences of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126. L In a specific embodiment, V Lcomprising one, two or three HVRs selected from the group consisting of: (a) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60, (b) HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76, and (c) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.

[0169] In some embodiments, an anti-MS4A6A antibody is provided, wherein the antibody comprises a V as in any of the embodiments provided above. H and V in any of the embodiments provided above L In some embodiments, provided herein are anti-MS4A6A antibodies, wherein the antibody comprises V as in any of the embodiments provided above. H and V in any of the embodiments provided above L In one embodiment, the antibody comprises V in SEQ ID NO: 91-108 and SEQ ID NO: 109-126, respectively. H and V L sequences, including post-translational modifications of those sequences.

[0170] In some embodiments, provided herein are anti-MS4A6A antibodies comprising a heavy chain variable domain (V H ) and the light chain variable domain (V L ), where V H and V L Selected from the group consisting of: V comprising the amino acid sequence of SEQ ID NO: 91 H and V comprising the amino acid sequence of SEQ ID NO: 109 L ; V comprising the amino acid sequence of SEQ ID NO:92 H and V comprising the amino acid sequence of SEQ ID NO: 110 L ; V comprising the amino acid sequence of SEQ ID NO:93 H and V comprising the amino acid sequence of SEQ ID NO: 111 L ; V comprising the amino acid sequence of SEQ ID NO:94 Hand V comprising the amino acid sequence of SEQ ID NO: 112 L ; V comprising the amino acid sequence of SEQ ID NO:95 H and V comprising the amino acid sequence of SEQ ID NO: 113 L ; V comprising the amino acid sequence of SEQ ID NO:96 H and V comprising the amino acid sequence of SEQ ID NO: 114 L ; V comprising the amino acid sequence of SEQ ID NO:97 H and V comprising the amino acid sequence of SEQ ID NO: 115 L ; V comprising the amino acid sequence of SEQ ID NO: 98 H and V comprising the amino acid sequence of SEQ ID NO: 116 L ; V comprising the amino acid sequence of SEQ ID NO:99 H and V comprising the amino acid sequence of SEQ ID NO: 117 L ; V comprising the amino acid sequence of SEQ ID NO: 100 H and V comprising the amino acid sequence of SEQ ID NO: 118 L ; V comprising the amino acid sequence of SEQ ID NO: 101 H and V comprising the amino acid sequence of SEQ ID NO: 109 L ; V comprising the amino acid sequence of SEQ ID NO: 102 H and V comprising the amino acid sequence of SEQ ID NO: 119 L ; V comprising the amino acid sequence of SEQ ID NO: 103 H and V comprising the amino acid sequence of SEQ ID NO: 120 L ; V comprising the amino acid sequence of SEQ ID NO: 104 H and V comprising the amino acid sequence of SEQ ID NO: 121 L ; V comprising the amino acid sequence of SEQ ID NO: 105 H and V comprising the amino acid sequence of SEQ ID NO: 116 L ; V comprising the amino acid sequence of SEQ ID NO: 106 H and V comprising the amino acid sequence of SEQ ID NO: 122 L ; V comprising the amino acid sequence of SEQ ID NO:93 H and V comprising the amino acid sequence of SEQ ID NO: 123 L; V comprising the amino acid sequence of SEQ ID NO:93 H and V comprising the amino acid sequence of SEQ ID NO: 124 L ; V comprising the amino acid sequence of SEQ ID NO: 107 H and V comprising the amino acid sequence of SEQ ID NO: 125 L ; and V comprising the amino acid sequence of SEQ ID NO: 108 H and V comprising the amino acid sequence of SEQ ID NO: 126 L .

[0171] In some embodiments, an anti-MS4A6A antibody of the present disclosure competitively inhibits binding of at least one reference antibody selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, 6A-23.

[0172] In some embodiments, the anti-MS4A6A antibody reduces binding to MS4A6A of one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof, when compared to binding to MS4A6A in the absence of the anti-MS4A6A antibody. The anti-MS4A6A antibodies of the present disclosure compete for binding to MS4A6A with one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22 and 6A-23, and any combination thereof, when the binding of MS4A6A to MS4A6A ranges from about 50% to 100%.

[0173] In some embodiments, the anti-MS4A6A antibody reduces binding of one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof, to MS4A6A by at least 50%, at least 55%, at least 6A-7, as compared to binding to MS4A6A in the absence of the anti-MS4A6A antibody. 0%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%, the anti-MS4A6A antibodies of the present disclosure compete for binding to MS4A6A with one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof. In some embodiments, the anti-MS4A6A antibodies of the present disclosure reduce the binding of one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof, to MS4A6A. 100%, indicating that the anti-MS4A6A antibody substantially completely blocks the binding of one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22 and 6A-23 and any combination thereof to MS4A6A.In some embodiments, an anti-MS4A6A antibody is combined with one or more antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof, to correspond to the combination of an anti-MS4A6A antibody with one or more antibodies selected from 6A-1, 6A-2 , 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof, of a 10: 1 ratio, a 9: 1 ratio, an 8: 1 ratio, a 7: 1 ratio, a 6: 1 ratio, a 5: 1 ratio, a 4: 1 ratio, a 3: 1 ratio, a 2: 1 ratio, a 1: 1 ratio, a 0 .75:1 ratio, 0.5:1 ratio, 0.25:1 ratio, 0.1:1 ratio, 0.075:1 ratio, 0.050:1 ratio, 0.025:1 ratio, 0.01:1 ratio, 0.0075: ratio, 0.0050:1 ratio, 0.0025:1 ratio, 0.001: ratio, 0.00075:1 ratio, 0.00050:1 ratio, 0.00025:1 ratio, 0.0001: ratio, 1:10 ratio, 1:9 ratio, 1:8 ratio, 1:7 ratio, 1:6 ratio, The amounts are present in a 1:5 ratio, a 1:4 ratio, a 1:3 ratio, a 1:2 ratio, a 1:0.75 ratio, a 1:0.5 ratio, a 1:0.25 ratio, a 1:0.1 ratio, a 1:0.075 ratio, a 1:0.050 ratio, a 1:0.025 ratio, a 1:0.01 ratio, a 1:0.0075 ratio, a 1:0.0050 ratio, a 1:0.0025 ratio, a 1:0.001 ratio, a 1:0.00075 ratio, a 1:0.00050 ratio, a 1:0.00025 ratio, or a 1:0.0001 ratio. In some embodiments, the anti-MS4A6A antibody is present in an excess amount ranging from about 1.5-fold to 100-fold, or greater than 100-fold, compared to the amount of one or more antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof.In some embodiments, with one or more selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23. 3 and any combination thereof, the anti-MS4A6A antibody is present in about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess compared to the amount of a reference antibody comprising:

[0174] In some embodiments, an anti-MS4A6A antibody of the present disclosure binds to an epitope of human MS4A6A that is identical to or overlaps with an epitope of MS4A6A bound by at least one reference antibody selected from the group consisting of: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23.

[0175] In some embodiments, an anti-MS4A6A antibody of the present disclosure binds to an epitope of MS4A6A that is substantially identical to an epitope bound by at least one reference antibody selected from the group consisting of: 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0176] In some embodiments, the anti-MS4A6A antibodies of the present disclosure compete for binding to MS4A6A with one or more reference antibodies selected from 6A-2, 6A-3, 6A-6, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-18, 6A-19, 6A-21, 6A-22, and 6A-23, and any combination thereof. In some embodiments, the anti-MS4A6A antibodies of the present disclosure compete for binding to MS4A6A with one or more reference antibodies selected from 6A-1, 6A-4, 6A-5, 6A-7, 6A-17, and 6A-20, and any combination thereof.

[0177] In some embodiments, the anti-MS4A6A antibodies of the present disclosure competitively inhibit binding of at least one reference antibody selected from 6A-2, 6A-3, 6A-6, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-18, 6A-19, 6A-21, 6A-22, and 6A-23, and any combination thereof, to MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure competitively inhibit binding of at least one reference antibody selected from 6A-1, 6A-4, 6A-5, 6A-7, 6A-17, and 6A-20, and any combination thereof, to MS4A6A.

[0178] In some embodiments, the anti-MS4A6A antibodies of the present disclosure have an epitope on MS4A6A for binding to MS4A6A that is identical or overlapping with at least one reference antibody selected from 6A-2, 6A-3, 6A-6, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-18, 6A-19, 6A-21, 6A-22, and 6A-23, and any combination thereof. In some embodiments, the anti-MS4A6A antibodies of the present disclosure have an epitope on MS4A6A for binding to MS4A6A that is identical or overlapping with at least one reference antibody selected from 6A-1, 6A-4, 6A-5, 6A-7, 6A-17, and 6A-20, and any combination thereof.

[0179] In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 1-46 of human MS4A6A (SEQ ID NO: 1). In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 47-67 of human MS4A6A (SEQ ID NO: 1). In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 68-84 of human MS4A6A (SEQ ID NO: 1). In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 85-105 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 106-116 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 117-137 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 138-185 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 186-206 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 207-248 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to the extracellular domain 1 (ECL1) of MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to the extracellular domain 2 (ECL2) of MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 180-190 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to one or more amino acids within amino acid residues 181-189 of human MS4A6A, within amino acid residues 182-188 of human MS4A6A, within amino acid residues 183-187 of human MS4A6A, or within amino acid residues 184-186 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to amino acid residue 185 of human MS4A6A. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to amino acid residue 185 of human MS4A6A, wherein amino acid residue 185 of human MS4A6A is threonine. In some embodiments, the anti-MS4A6A antibodies of the present disclosure bind to amino acid residue 185 of human MS4A6A, wherein amino acid residue 185 of human MS4A6A is serine.In some embodiments, an anti-MS4A6A antibody of the present disclosure binds to amino acid residue 185 of MS4A6A, wherein amino acid residue 185 of MS4A6A is proline.

[0180] Any suitable competition assay or MS4A6A binding assay known in the art (such as BIAcore analysis, ELISA assay, or flow cytometry) can be used to determine whether an anti-MS4A6A antibody competes for binding to MS4A6A (or competitively inhibits such binding) with one or more reference antibodies selected from 6A-1, 6A-2, 6A-3, 6A-4, 6A-5, 6A-6, 6A-7, 6A-8, 6A-9, 6A-10, 6A-11, 6A-12, 6A-13, 6A-14, 6A-15, 6A-16, 6A-17, 6A-18, 6A-20, 6A-21, 6A-22, and 6A-23, and any combination thereof. In an exemplary competition assay, immobilized MS4A6A or cells expressing MS4A6A on the cell surface are incubated in a solution containing a first labeled antibody (e.g., human or non-human primate) that binds to MS4A6A and a second unlabeled antibody, the ability of the second unlabeled antibody to compete with the first antibody for binding to MS4A6A being tested. The second antibody may be present in the hybridoma supernatant. As a control, immobilized MS4A6A or cells expressing MS4A6A are incubated in a solution containing the first labeled antibody, but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to MS4A6A, excess unbound antibody is removed, and the amount of label associated with immobilized MS4A6A or cells expressing MS4A6A is measured. If the amount of label associated with immobilized MS4A6A or cells expressing MS4A6A is substantially reduced in the test sample relative to the control sample, it indicates that the second antibody is competing with the first antibody for binding to MS4A6A. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0181] Also provided herein is an anti-MS4A6A antibody that competitively inhibits the binding of an anti-MS4A6A antibody and / or competes with the binding of an anti-MS4A6A antibody, the anti-MS4A6A antibody comprising (a) V H A domain comprising at least one, at least two or all three V selected from the following HHVR sequences: (i) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, (ii) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31, and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45, and (b) V L A domain comprising at least one, at least two or all three V selected from the following L HVR sequences: (i) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, (ii) HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and 76, and (iii) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90. In some embodiments, the antibody comprises V sequences of SEQ ID NOs: 91-108 and SEQ ID NOs: 109-126, respectively. H and V L sequence.

[0182] Provided herein are anti-MS4A6A antibodies that bind to an epitope of human MS4A6A that is identical to or overlaps with an epitope that the anti-MS4A6A antibody binds to, comprising (a) V H A domain comprising at least one, at least two or all three V selected from the following HHVR sequences: (i) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, (ii) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31, and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45, and (b) V L A domain comprising at least one, at least two or all three V selected from the following L HVR sequences: (i) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, (ii) HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and 76, and (iii) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90. In some embodiments, the antibody comprises V sequences of SEQ ID NOs: 91-108 and SEQ ID NOs: 109-126, respectively. H and V L In some embodiments, the epitope of human MS4A6A is the same as the epitope bound by the anti-MS4A6A antibody.

[0183] In some embodiments, the anti-MS4A6A antibody according to any of the above embodiments is a monoclonal antibody, including a humanized antibody and / or a human antibody. In some embodiments, the anti-MS4A6A antibody is an antibody fragment, such as a Fv, Fab, Fab', scFv, diabody or F(ab')2 fragment. In some embodiments, the anti-MS4A6A antibody is a substantially full-length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody classes or isotypes as defined herein.

[0184] In some embodiments, an anti-MS4A6A antibody according to any of the above embodiments may include, alone or in combination, any of the features described in Sections 1-7 below:

[0185] (1) Anti-MS4A6A antibody binding affinity

[0186] In some embodiments of any of the antibodies provided herein, the antibody has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 - 13 M, for example 10 -9 M to 10 -13 M). The dissociation constant can be determined by any analytical technique, including any biochemical or biophysical technique, such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, e.g., by ForteBio's Octet system), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analysis. In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In a certain embodiment, an RIA is performed with a Fab version of the antibody of interest and its antigen, e.g., as described in Chen et al. J. Mol. Biol. 293:865-881 (1999). In some embodiments, Kd is measured using a BIACORE surface plasmon resonance assay, such as an assay performed by a BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at about 10 response units (RU). In some embodiments, Kd is determined using a monovalent antibody (e.g., Fab) or a full-length antibody. D In some embodiments, a monovalent form of the full-length antibody is used to determine K D .

[0187] (2) Antibody fragments

[0188] In some embodiments of any of the antibodies provided herein, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, WO 93 / 16185; and U.S. Patent Nos. 5571894 and 5587458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5869046.

[0189] Diabodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific. See, e.g., EP404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9:129-134 (2003). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6248516).

[0190] As described herein, antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or bacteriophage).

[0191] (3) Chimeric and humanized antibodies

[0192] In some embodiments of any one of the antibodies provided herein, the antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switching" antibody, in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include their antigen binding fragments.

[0193] In some embodiments of any one of the antibodies provided herein, the antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibodies. In certain embodiments, humanized antibodies are substantially non-immunogenic in humans. In certain embodiments, the affinity of humanized antibodies to targets is substantially the same as that of antibodies from another species from which humanized antibodies are derived. See, for example, U.S. Patent Nos. 5530101, 5693761; 5693762; and 5585089. In certain embodiments, the amino acids of antibody variable domains that can be modified without reducing the natural affinity of the antigen-binding domain and reducing immunogenicity are identified. See, for example, U.S. Patent Nos. 5766886 and 5869619. In general, humanized antibodies include one or more variable domains, wherein HVR (or portions thereof) are derived from non-human antibodies, and FR (or portions thereof) are derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0194] Humanized antibodies and methods for making them are reviewed, for example, in Almagro et al. Front. Biosci. 13: 1619-1633 (2008), and further described, for example, in U.S. Pat. Nos. 5821337, 7527791, 6982321, and 7087409. Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using a "best fit" approach (see, for example, Sims et al., J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular light chain variable region or heavy chain variable region subset (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993). 993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0195] (4) Human antibodies

[0196] In some embodiments of any of the antibodies provided herein, the antibody is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk et al., Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg Curr. Opin. Immunol. 20: 450-459 (2008).

[0197] Human antibodies can be prepared by applying an immunogen to a transgenic animal, which has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic challenges. The mouse strains that are defective in mouse antibody production can be engineered with large fragments of human Ig loci, in the expectation that such mice can produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can retain larger variable gene diversity and appropriate regulation of antibody production and expression. By utilizing the antibody diversification and selection mechanism of mice and the lack of immune tolerance of human proteins, the human antibody library replicated in these mouse strains can produce high-affinity fully human antibodies for any antigen of interest (including human antigens). Using hybridoma technology, antigen-specific human mAbs with desired specificity can be produced and selected. Some exemplary methods are described in U.S. Patent No. 5545807, EP 546073 and EP 546073. See also, for example, description of XENOMOUSE TM US Patent Nos. 6075181 and 6150584 for the technology; description US Patent No. 5770429 for technology; description KM US Patent No. 7041870, and describes The human variable regions of intact antibodies produced by such animals can also be further modified, for example, by combining with different human constant regions.

[0198] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor. J. Immunol. 133: 3001 (1984) and Boemer et al., J. Immunol. 147: 86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 1 03: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7189826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (trioma technology) is also described in Vollmers et al., Histology and Histopathology 20 (3): 927-937 (2005) and Vollmers et al., Methods and Findings in Experimental and Clinical Pharmacology 27 (3): 185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The technology for selecting human antibodies from antibody libraries is described below.

[0199] In some embodiments of any of the antibodies provided herein, the antibody is a human antibody isolated by in vitro methods and / or screening of combinatorial libraries (to obtain antibodies with one or more desired activities). Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display (Adimab), and the like. In certain phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR), and random recombination is performed in phage libraries, and then antigen-binding phages can be screened, as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). For example, many methods for generating phage display libraries and screening antibodies with desired binding characteristics from such libraries are known in the art. See also Sidhu et al., J. Mol. Biol. 338(2):299-310, 2004; Lee et al., J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(-2):119-132 (2004). Phages usually display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries derived from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the initial library can be cloned (e.g., from humans) to provide antibodies from a single source for a wide range of non-self-antigens and self-antigens without any immunization, as described in Griffiths et al., EMBO J. 12: 725-734 (1993). Finally, the initial library can also be prepared synthetically by cloning unrearranged V gene segments from stem cells using PCR primers containing random sequences to encode highly variable HVR3 regions and achieve rearrangement in vitro, as described in Hoogenboom et al., J. Mol. Biol., 227: 381-388, 1992. Patent disclosures describing human antibody phage libraries include, for example, U.S. Patent No. 5750373 and U.S. Patent Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies separated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0200] (5) Constant region including Fc region

[0201] In some embodiments of any one of the antibodies provided herein, the antibody comprises an Fc. In some embodiments, the Fc is a human IgG1, IgG2, IgG3 and / or IgG4 isotype. In some embodiments, the antibody belongs to the IgG class, the IgM class or the IgA class.

[0202] In certain embodiments of any one of the antibodies provided herein, the antibody has an IgG2 isotype. In some embodiments, the antibody contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region comprises an Fc region. In some embodiments, the antibody induces one or more MS4A6A activities or is unrelated to the binding of an Fc receptor. In some embodiments, the antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is an inhibitory Fc-gamma receptor IIB (FcγIIB).

[0203] In certain embodiments of any of the antibodies provided herein, the antibody has an IgG1 isotype. In some embodiments, the antibody contains a mouse IgG1 constant region. In some embodiments, the antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region comprises an Fc region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is an inhibitory Fc-gamma receptor IIB (FcγIIB).

[0204] In certain embodiments of any one of the antibodies provided herein, the antibody has an IgG4 isotype. In some embodiments, the antibody contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is an inhibitory Fc-gamma receptor IIB (FcγIIB).

[0205] In certain embodiments of any one of the antibodies provided herein, the antibody has a hybrid IgG2 / 4 isotype. In some embodiments, the antibody comprises an amino acid sequence comprising amino acids 118 to 260 according to the EU numbering of human IgG2 and amino acids 261 to 447 according to the EU numbering of human IgG4 (WO 1997 / 11971; WO 2007 / 106585).

[0206] In some embodiments, the Fc region increases clustering but does not activate complement compared to a corresponding antibody comprising the Fc region (not comprising the amino acid substitutions). In some embodiments, the antibody induces one or more activities of the target to which the antibody specifically binds. In some embodiments, the antibody binds to MS4A6A.

[0207] It is also desirable to modify the anti-MS4A6A antibodies of the present disclosure to modify effector function and / or increase the serum half-life of the antibody. For example, the Fc receptor binding site on the constant region can be modified or mutated to remove or reduce binding affinity for certain Fc receptors (such as FcγRI, FcγRII and / or FcγRIII), thereby reducing antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is weakened by removing N-glycosylation of the Fc region of the antibody (e.g., in the CH2 domain of IgG). In some embodiments, effector function is weakened by modifying regions such as 233-236, 297 and / or 327-331 of human IgG, as described in WO 99 / 58572 and Armour et al., Molecular Immunology 40:585-593 (2003); Reddy et al., J. Immunology 164:1925-1933 (2000). In other embodiments, modification of the anti-MS4A6A antibodies of the present disclosure to modify effector functions to increase selectivity for detection of ITIM-containing FcgRIIb (CD32b) without activating humoral responses, increase clustering of MS4A6A antibodies on neighboring cells, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis is also desired.

[0208] For example, to increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into an antibody (particularly an antibody fragment) as described in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. Other amino acid sequence modifications.

[0209] (6) Multispecific Antibodies

[0210] Multispecificity is an antibody that has binding specificity for at least two different epitopes, including those on the same or another polypeptide (e.g., one or more MS4A6A polypeptides of the present disclosure). In some embodiments, the multispecific antibody may be a bispecific antibody. In some embodiments, the multispecific antibody may be a trispecific antibody. In some embodiments, the multispecific antibody may be a tetraspecific antibody. Such antibodies may be derived from full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). In some embodiments, the multispecific antibody comprises a first antigen binding region that binds to a first site on MS4A6A, and comprises a second antigen binding region that binds to a second site on MS4A6A. In a certain embodiment, the multispecific antibody comprises a first antigen binding region that binds to MS4A6A and a second antigen binding region that binds to a second polypeptide.

[0211] Provided herein are multispecific antibodies comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises six HVRs of an antibody described herein and binds to MS4A6A, and the second antigen-binding region binds to a second polypeptide. In some embodiments, the first antigen-binding region comprises V H or V L .

[0212] In some embodiments of any of the multispecific antibodies, the second polypeptide is: a) an antigen that facilitates transport across the blood-brain barrier; (b) an antigen that facilitates transport across the blood-brain barrier, the antigen being selected from: transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, llama single domain antibody, TMEM 30 (A), protein transduction domain, TAT, Syn-B, penetrant peptide, polyarginine peptide, vascular peptide and ANG1005; (c) a pathogenic protein, the pathogenic protein being selected from amyloid β, oligomeric amyloid β, amyloid β plaques, amyloid precursor protein or fragments thereof, τ, IAPP, α-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxia, ataxia 1, ataxia 2, ataxia 3, ataxia 7, ataxia 8, ataxia 10, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, β2 microglobulin, gelsolin, corneal epithelial protein, cystatin, immune Globulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine (PA) repeat peptide, ubiquitin and proline-arginine (PR) repeat peptide; (d) ligands and / or proteins expressed on immune cells, wherein the ligands and / or proteins are selected from: CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA-4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG-3 and phosphatidylserine; and / or (e) proteins, lipids, polysaccharides or glycolipids expressed on one or more tumor cells and any combination thereof.

[0213] Many antigens that facilitate transport across the blood-brain barrier are known in the art (see, e.g., Gabathuler R. Neurobiol. Dis. 37: 48-57 (2010)). Such second antigens include, but are not limited to, transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor (including CRM 197 (a non-toxic mutant of diphtheria toxin)), llama single domain antibodies (such as TMEM 30 (A) (Flippase)), protein transduction domains (such as TAT), Syn-B or transmembrane peptides, polyarginine or generally positively charged peptides, vascular peptides (such as ANG1005) (see, e.g., Gabathuler, 2010) and other cell surface proteins enriched on blood-brain barrier endothelial cells (see, e.g., Daneman et al., PLoS One 5 (10): e13741 (2010)).

[0214] The multivalent antibody may recognize the MS4A6A antigen, as well as, but not limited to, additional antigens Aβ peptide antigens, or α-synuclein antigens, or tau protein antigens, or TDP-43 protein antigens, or prion protein antigens, or huntingtin protein antigens, or RAN, translation product antigens (including dipeptide repeats (DPR peptides) composed of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR), insulin receptor, insulin-like growth factor receptor. Transferrin receptor or any other antigen that facilitates antibody transfer across the blood-brain barrier. In some embodiments, the second polypeptide is transferrin. In some embodiments, the second polypeptide is tau. In some embodiments, the second polypeptide is Aβ. In some embodiments, the second polypeptide is TREM2. In some embodiments, the second polypeptide is α-synuclein.

[0215] The multivalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), wherein one or more polypeptide chains comprise two or more variable domains. For example, one or more polypeptide chains may comprise VD1-(X1) n -VD2-(X2) n -Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, one or more polypeptide chains may include V H -C H 1-Flexible joint-V H -C H 1-Fc region chain; or V H -CH 1-V H -C H 1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for example, comprise about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0216] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello Nature 305:537 (1983), WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). See also WO 2013 / 026833 (CrossMab). Multispecific antibodies can also be prepared by the following techniques: engineering electrostatic steering effects for preparing antibody Fc heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies (see, e.g., U.S. Pat. No. 4,676,980); using leucine; using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., by Tutt et al., J. Immunol. 147:60 (1991).

[0217] Also included herein are engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576). Antibodies herein also include "dual-acting FAbs" or "DAFs," which contain antigen binding sites that bind to multiple MS4A6A (see, e.g., US 2008 / 0069820).

[0218] (7) Antibody variants

[0219] In some embodiments of any of the antibodies provided herein, amino acid sequence variants of the antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibodies.

[0220] (i) Substitution, insertion and deletion variants

[0221] In some embodiments of any one of the antibodies provided herein, antibody variants with one or more amino acid substitutions are provided. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody.

[0222] Table 1: Amino Acid Substitutions

[0223]

[0224] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​substitution (e.g., a sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into several groups based on common side chain properties:

[0225] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0226] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0227] (3) Acidic: Asp, Glu;

[0228] (4) Basic: His, Lys, Arg;

[0229] (5) Residues that affect chain orientation: Gly, Pro; and

[0230] (6) Aromatic: Trp, Tyr, Phe.

[0231] For example, non-conservative substitutions may involve replacing a member of one of these classes with a member of another class. For example, such substituted residues may be introduced into regions of human antibodies that are homologous to non-human antibodies, or into non-homologous regions of the molecule.

[0232] According to certain embodiments, when changing the polypeptide or antibody described herein, the hydropathic index of the amino acid can be considered. According to the hydrophobicity and charge characteristics of the amino acid, each amino acid has been assigned a hydropathic index. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0233] The importance of the hydropathic amino acid index in conferring a biological function to a protein interaction is understood in the art. Kyte et al., J. Mol. Biol., 157: 105-131 (1982). It is known that certain amino acids can replace other amino acids with similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, replacement of amino acids with hydropathic indices within ± 2 is included. In certain embodiments, those amino acids within ± 1 are included, and in certain embodiments, those amino acids within ± 0.5 are included.

[0234] It is also understood in the art that similar amino acid substitutions can be made effectively on the basis of hydrophilicity, especially where the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as is the case in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of the protein's neighboring amino acids, correlates with the immunogenicity and antigenicity of the protein, i.e., the biological properties of the protein.

[0235] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0 ± 1); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When changes are made based on similar hydrophilicity values, in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included, in certain embodiments, substitutions of those amino acids within ±1 are included, and in certain embodiments, substitutions of those amino acids within ±0.5 are included. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions."

[0236] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as these changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be, for example, outside the antigen contact residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, or contains no more than one, two or three amino acid substitutions.

[0237] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide comprising one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

[0238] Any cysteine ​​residues not involved in maintaining the proper conformation of the antibody may also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine ​​bonds may be added to the antibody to improve the stability of the antibody (especially where the antibody is an antibody fragment, such as an Fv fragment).

[0239] (ii) Glycosylation variants

[0240] In some embodiments of any one of the antibodies provided herein, the antibody is altered to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0241] Glycosylation of antibodies is usually N-connected or O-connected. N-connection refers to the attachment of the carbohydrate moiety to the side chain of asparagine residues. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine are recognition sequences for enzymatic attachment of the carbohydrate moiety to the side chain of asparagine, wherein X is any amino acid except proline. Therefore, any of these tripeptide sequences present in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a sugar (i.e., one of N-acetylgalactosamine, galactose or xylose) to a hydroxyamino acid (most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used).

[0242] The addition of glycosylation sites to antibodies is conveniently achieved by altering the amino acid sequence so that the antibody contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The sequence of the original antibody may also be altered by addition of one or more serine or threonine residues or by replacement of one or more serine or threonine residues (for O-linked glycosylation sites).

[0243] In the case where the antibody comprises an Fc region, the carbohydrate connected thereto can be changed. The natural antibody produced by mammalian cells generally comprises a biantennary oligosaccharide of a branched chain, and the oligosaccharide is generally connected to the Asn297 (according to Kabat numbering) of the CH2 domain of the Fc region by N-connection. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the antibody of the present disclosure can be modified to produce antibody variants with certain improved properties.

[0244] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region.See, e.g., U.S. Patent Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of disclosures concerning "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng 87:614 (2004) and Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006)).

[0245] (iii) Modified constant region

[0246] In some embodiments of any of the antibodies provided herein, the antibody Fc is an antibody Fc isotype and / or modification. In some embodiments, the antibody Fc isotype and / or modification is capable of binding to an Fcγ receptor.

[0247] In some embodiments of any of the antibodies provided herein, the modified antibody Fc is an IgG1 modified Fc. In some embodiments, the IgG1 modified Fc comprises one or more modifications. For example, in some embodiments, the IgG1 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from N297A (Bolt S et al., (1993) Eur J Immunol 23:403-411), D265A (Shields et al., (2001) RJ Biol. Chem. 276, 65916604), L234A, L235A (Hutchins et al., (1995) Proc Natl Acad Sci USA, 92: 11980-11984; Alegre et al., (1994) Transplantation 57: 1537-1543.31; Xu et al., (2000) Cell Immunol, 200: 16-26), G237A (Alegre et al., (1994) Transplantation 57: 1537-1543.31; Xu et al., (2000) Cell Immunol, 200: 16-26). Immunol, 200: 16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109: 1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105: 20167-20172), S267E, L328F, A330L, M252Y, S254T and / or T256E, wherein the amino acid positions are according to the EU numbering convention.

[0248] In some embodiments of any one of the Fc modified by IgG1, the Fc comprises an N297A mutation according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, the Fc comprises an D265A and N297A mutation according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, the Fc comprises an D270A mutation according to EU numbering. In some embodiments, the Fc modified by IgG1 comprises an L234A and L235A mutation according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, the Fc comprises an L234A and G237A mutation according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, the Fc comprises an L234A, L235A and G237A mutation according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc includes one or more (including all mutations) of P238D, L328E, E233, G237D, H268D, P271G and A330R mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc includes one or more of S267E / L328F mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc includes one or more of P238D, L328E, E233D, G237D, H268D, P271G and A330R mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc includes one or more of P238D, L328E, G237D, H268D, P271G and A330R mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc comprises P238D, S267E, L328E, E233D, G237D, H268D, P271G and A330R mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc comprises P238D, S267E, L328E, G237D, H268D, P271G and A330R mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc comprises C226S, C229S, E233P, L234V and L235A mutations according to EU numbering. In some embodiments of any one of the Fc modified by IgG1, Fc comprises L234F, L235E and P331S mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc's, the Fc comprises S267E and L328F mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc's, the Fc comprises a S267E mutation according to EU numbering.In some embodiments of any of the IgG1 modified Fcs, the Fc comprises replacement of the constant heavy chain 1 (CH1) and hinge region of IgG1 with CH1 and replacement of the hinge region of IgG2 (amino acids 118-230 of IgG2 according to EU numbering) with a kappa light chain.

[0249] In some embodiments of any one of the Fc modified by IgG1, compared with the corresponding antibody with Fc region (not comprising two or more amino acid replacements), Fc comprises two or more amino acid replacements, and the amino acid replacement increases antibody clustering but does not activate complement. Therefore, in some embodiments of any one of the Fc modified by IgG1, the Fc modified by IgG1 is an antibody comprising Fc region, wherein the antibody is included in the amino acid replacement at position E430G and one or more amino acid replacements in the Fc region, and the residue position of the amino acid replacement is selected from: according to EU numbering L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S and any combination thereof. In some embodiments, the Fc modified by IgG1 comprises amino acid replacements at positions E430G, L243A, L235A and P331S according to EU numbering. In some embodiments, the Fc modified by IgG1 comprises amino acid replacements at positions E430G and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid replacements at positions E430G and K322A according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid replacements at positions E430G, A330S, and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid replacements at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid replacements at positions E430G, K322A, and A330S according to EU numbering. In some embodiments, the IgG1 modified Fc comprises amino acid replacements at positions E430G, K322A, and P331S according to EU numbering.

[0250] In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise an A330L mutation (Lazar et al., Proc Natl Acad Sci USA, 103:4005-4010 (2006)) according to the EU numbering convention, or one or more of L234F, L235E and / or P331S mutations (Sazinsky et al., Proc Natl Acad Sci USA, 105:20167-20172 (2008)) combined herein to eliminate complement activation. In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise one or more of A330L, A330S, L234F, L235E and / or P331S according to EU numbering. In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise one or more mutations to enhance antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S254T, and T256E mutations according to the EU numbering convention). In some embodiments of any of the IgG1 modified Fcs, the IgG1 modified Fc may further comprise one or more of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and / or S440W according to EU numbering.

[0251] Other aspects of the present disclosure relate to antibodies with modified constant regions (i.e., Fc regions). If the antibody is engineered to eliminate FcgR binding, the antibody that activates the targeted receptor based on binding to the FcgR receptor can lose agonist activity (see, e.g., Wilson et al., Cancer Cell 19: 101-113 (2011); Armour et al., Immunology 40: 585-593 (2003); and White et al., Cancer Cell 27: 138-148 (2015)). Therefore, it is believed that when the antibody has an Fc domain (CH1 and hinge region) from the human IgG2 isotype or another type of Fc domain that can preferentially bind to the inhibitory FcgRIIBr receptor or its variants, the anti-MS4A6A antibodies of the present disclosure with the correct epitope specificity can activate the target antigen with minimal adverse reactions.

[0252] In some embodiments of any one of the antibodies provided herein, the modified antibody Fc is an IgG2 modified Fc. In some embodiments, the IgG2 modified Fc comprises one or more modifications. For example, in some embodiments, the IgG2 modified Fc comprises one or more amino acid replacements (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments of any of the IgG2 modified Fc, one or more amino acid substitutions are selected from V234A according to the EU numbering convention (Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol, 200:16-26 (2000)); G237A (Cole et al., Transplantation, 68:563-571 (1999)); H268Q, V309L, A330S, P331S (US 2007 / 0148167; Armour et al., Eur J Immunol 29:2613-2624 (1999); Armour et al., The Haematology Journal 1(Suppl 1):27 (2000); Armour et al., The Haematology Journal 1(Suppl 1):27(2000)), C219S and / or C220S (White et al., Cancer Cell 27, 138-148(2015)); S267E, L328F (Chu et al., Mol Immunol, 45:3926-3933(2008)); and M252Y, S254T and / or T256E. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises amino acid replacements at positions V234A and G237A according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises amino acid replacements at positions C219S or C220S according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises amino acid replacements at positions A330S and P331S according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering.

[0253] In some embodiments of any of the IgG2 modified Fcs, the Fc comprises a C127S amino acid substitution according to the EU numbering convention (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., Protein Sci. 19: 753-762 (2010); and WO 2008 / 079246). In some embodiments of any of the IgG2 modified Fcs, the antibody has an IgG2 isotype having a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention (White et al., Cancer Cell 27: 138-148 (2015); Lightle et al., Protein Sci. 19: 753-762 (2010); and WO 2008 / 079246).

[0254] In some embodiments of any of the IgG2 modified Fcs, the Fc comprises a C220S amino acid substitution according to the EU numbering convention. In some embodiments of any of the IgG2 modified Fcs, the antibody has an IgG2 isotype having a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention.

[0255] In some embodiments of any of the IgG2 modified Fcs, the Fc comprises a C219S amino acid substitution according to the EU numbering convention. In some embodiments of any of the IgG2 modified Fcs, the antibody has an IgG2 isotype having a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention.

[0256] In some embodiments of any one of the IgG2 modified Fcs, the Fc comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and a hinge region (White et al., Cancer Cell 27: 138-148 (2015)). In certain embodiments of any one of the IgG2 modified Fcs, the IgG2 isotype CH1 and hinge region comprise an amino acid sequence of 118-230 according to EU numbering. In some embodiments of any one of the IgG2 modified Fcs, the antibody Fc region comprises an S267E amino acid replacement, an L328F amino acid replacement, or both thereof, and / or an N297A or N297Q amino acid replacement according to the EU numbering convention.

[0257] In some embodiments of any of the IgG2 modified Fcs, the Fc further comprises one or more amino acid substitutions at positions E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W according to EU numbering. In some embodiments of any of the IgG2 modified Fcs, the Fc may further comprise one or more mutations to enhance antibody half-life in human serum (e.g., one or more (including all mutations) of M252Y, S254T, and T256E mutations according to EU numbering convention). In some embodiments of any of the IgG2 modified Fcs, the Fc may further comprise A330S and P331S.

[0258] In some embodiments of any of the IgG2 modified Fcs, the Fc is an IgG2 / 4 hybrid Fc. In some embodiments, the IgG2 / 4 hybrid Fc comprises IgG2 aa 118 to 260 and IgG4 aa 261 to 447. In some embodiments of any of the IgG2 modified Fcs, the Fc comprises one or more amino acid substitutions at positions H268Q, V309L, A330S, and P331S according to EU numbering.

[0259] In some embodiments of either of the IgG1 and / or IgG2 modified Fc, the Fc comprises one or more additional amino acid substitutions selected from A330L, L234F, L235E, or P331S according to EU numbering; and any combination thereof.

[0260] In certain embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises one or more amino acid replacements at residue positions selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G and P331S according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G and K322A according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G, A330S and P331S according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G, K322A, A330S and P331S according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G, K322A and A330S according to EU numbering. In some embodiments of any one of the Fc modified by IgG1 and / or IgG2, the Fc comprises amino acid replacements at positions E430G, K322A and P331S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fcs, the Fc comprises amino acid replacements at positions S267E and L328F according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fcs, the Fc comprises amino acid replacements at positions C127S according to EU numbering. In some embodiments of any of the IgG1 and / or IgG2 modified Fcs, the Fc comprises amino acid replacements at positions E345R, E430G, and S440Y according to EU numbering.

[0261] In some embodiments of any one of the antibodies provided herein, modified antibody Fc is an Fc modified by IgG4. In some embodiments, the Fc modified by IgG4 includes one or more modifications. For example, in some embodiments, the Fc modified by IgG4 includes one or more amino acid replacements (for example, relative to the wild-type Fc region of the same isotype). In some embodiments of any one of the Fc modified by IgG4, one or more amino acid replacements are selected from L235A, G237A, S229P, L236E (Reddy et al., J Immunol 164: 1925-1933 (2000)), S267E, E318A, L328F, M252Y, S254T and / or T256E according to EU numbering convention. In some embodiments of any one of the Fc modified by IgG4, Fc may additionally include L235A, G237A and E318A according to EU numbering convention. In some embodiments of any of the IgG4 modified Fcs, the Fc may further comprise S228P and L235E according to the EU numbering convention. In some embodiments of any of the IgG4 modified Fcs, the IgG4 modified Fc may further comprise S267E and L328F according to the EU numbering convention.

[0262] In some embodiments of any of the IgG4 modified Fcs, the IgG4 modified Fc comprises can be combined with an S228P mutation according to the EU numbering convention (Angal et al., Mol Immunol. 30: 105-108 (1993)) and / or with one or more mutations described in (Peters et al., J Biol Chem. 287(29): 24525-33 (2012)) to enhance antibody stability.

[0263] In some embodiments of any of the IgG4 modified Fcs, the IgG4 modified Fc may further comprise one or more mutations to enhance antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S254T, and T256E mutations according to the EU numbering convention).

[0264] In some embodiments of any one of the Fc modified by IgG4, Fc includes L235E according to EU numbering. In certain embodiments of any one of the Fc modified by IgG4, Fc includes one or more amino acid replacements at residue positions selected from C127S, F234A, L235A, L235E, S267E, K322A, L328F, E345R, E430G, S440Y and any combination thereof according to EU numbering. In some embodiments of any one of the Fc modified by IgG4, Fc includes amino acid replacements at positions E430G, L243A, L235A and P331S according to EU numbering. In some embodiments of any one of the Fc modified by IgG4, Fc includes amino acid replacements at positions E430G and P331S according to EU numbering. In some embodiments of any one of the Fc modified by IgG4, Fc includes amino acid replacements at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises an amino acid replacement at position E430 according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc region comprises an amino acid replacement at position E430G and K322A according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises an amino acid replacement at position S267E and L328F according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises an amino acid replacement at position C127S according to EU numbering. In some embodiments of any of the IgG4 modified Fcs, the Fc comprises an amino acid replacement at position E345R, E430G, and S440Y according to EU numbering.

[0265] (8) Other antibody modifications

[0266] In some embodiments of any one of the antibodies, the antibody is a derivative. The term "derivative" refers to a chemically modified molecule comprising an insertion, deletion or replacement of an amino acid (or nucleic acid). In certain embodiments, the derivative comprises a covalent modification, including but not limited to chemical bonding with a polymer, a lipid or other organic or inorganic moieties. In certain embodiments, the chemically modified antigen-binding proteins may have a longer circulation half-life than the antigen-binding proteins that are not chemically modified. In certain embodiments, the chemically modified antigen-binding proteins may have improved targeting capabilities for desired cells, tissues and / or organs. In some embodiments, the derivative antigen-binding proteins are covalently modified to include one or more water-soluble polymer attachments, including but not limited to polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. See, for example, U.S. Patent Nos. 4640835, 4496689, 4301144, 4670417, 4791192 and 4179337. In certain embodiments, the derivative antigen binding protein comprises one or more polymers, including but not limited to monomethoxy-polyethylene glycol, dextran, cellulose, copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of such polymers.

[0267] In certain embodiments, derivatives are covalently modified by polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are bonded at one or more specific positions of derivatives (e.g., at the amino terminal). In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of derivatives. In certain embodiments, PEG is used to improve the therapeutic ability of antigen-binding proteins. In certain embodiments, PEG is used to improve the therapeutic ability of humanized antibodies. Some such methods are discussed, for example, in U.S. Patent No. 6133426, which is incorporated by reference for any purpose.

[0268] Peptide analogs are commonly used as non-peptide drugs in the pharmaceutical industry, and their properties are similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics or peptidomimetics". Fauchere, J. Adv. Drug Res., 15: 29 (1986); and Evans et al., J. Med. Chem., 30: 1229 (1987), which are incorporated herein by reference for any purpose. Such compounds are usually developed by means of computerized molecular modeling. Peptide mimetics that are structurally similar to peptides used for treatment can be used to produce similar therapeutic or preventive effects. Typically, mimetics are structurally similar to paradigm polypeptides (i.e., polypeptides having biochemical properties or pharmacological activity), such as human antibodies, but have one or more peptide bonds, which are optionally replaced by a bond selected from the following by methods well known in the art: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. Systematic replacement of one or more amino acids of a consensus sequence with D-amino acids of the same type (e.g., D-lysine replaces L-lysine) can be used in certain embodiments to produce more stable peptides. In addition, constrained peptides comprising a consensus sequence or substantially identical consensus sequence variations can be produced by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem., 61: 387 (1992), incorporated herein by reference for any purpose); for example, by adding internal cysteine ​​residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0269] Drug conjugation involves coupling a biologically active cytotoxic (anticancer) payload or drug to an antibody that specifically targets a tumor marker (e.g., a polypeptide that is ideally present only in or on tumor cells). The antibodies track these proteins in vivo and attach themselves to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cancer. Due to this targeting, the drug ideally has lower side effects and provides a wider therapeutic window than other chemotherapeutic agents. The technology of conjugating antibodies has been disclosed and is known in the art (see, for example, Jane de Lartigue OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al. Bioconjugate Chemistry 21(1):5-13 (2010).

[0270] III. Nucleic Acids, Vectors and Host Cells

[0271] The anti-MS4A6A antibodies of the present disclosure can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, an isolated nucleic acid having a nucleotide sequence encoding any of the anti-MS4A6A antibodies of the present disclosure is provided. Such nucleic acid can encode a V sequence comprising an anti-MS4A6A antibody. L The amino acid sequence and / or comprising V H In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In some embodiments, host cells comprising such nucleic acids are also provided. In some embodiments, the host cell comprises (e.g., has been transduced with): (1) a vector comprising a nucleic acid encoding a V or V sequence of an antibody; L The amino acid sequence of the antibody V H (2) a first vector comprising a V encoding an antibody L A nucleic acid having an amino acid sequence of H In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp20 cell). The host cell of the present disclosure also includes, but is not limited to, isolated cells, cells cultured in vitro, and cells cultured in vitro.

[0272] Methods for preparing the anti-MS4A6A antibodies of the present disclosure are provided. In some embodiments, the methods include culturing a host cell of the present disclosure comprising a nucleic acid encoding an anti-MS4A6A antibody under conditions suitable for antibody expression. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0273] For recombinant production of the anti-MS4A6A antibodies of the present disclosure, nucleic acids encoding the anti-MS4A6A antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0274] Suitable vectors comprising a nucleic acid sequence encoding any of the anti-MS4A6A antibodies disclosed herein, or a cell surface expressed fragment or polypeptide thereof (including an antibody) as described herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary depending on the host cell desired to be used, useful cloning vectors are generally self-replicating, can have a single target for a specific restriction endonuclease, and / or can carry a gene for a marker that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (such as pSA3 and pAT28). These and many other cloning vectors can be obtained from commercial suppliers (such as BioRad, Strategene, and Invitrogen).

[0275] Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells. For example, the anti-MS4A6A antibodies of the present disclosure can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. Regarding the expression of antibody fragments and polypeptides in bacteria (e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523). After expression, the antibodies can be separated from the bacterial cytoplasm as a soluble fraction and can be further purified.

[0276] In addition to prokaryotes, eukaryotic microbes (such as filamentous fungi or yeast) are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partial or full human glycosylation patterns (e.g., Gemgross Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006)).

[0277] Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0278] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed by SV40; human embryonic kidney cell lines (293 or 293 cells, such as Graham et al. J. Gen Virol. 36: 59 (1977) described); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, such as Mather, Biol. Reprod. 23: 243-251 (1980) described); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al. Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0279] IV Pharmaceutical Compositions / Formulations

[0280] Provided herein are pharmaceutical compositions and / or pharmaceutical formulations comprising an anti-MS4A6A antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0281] In some embodiments, the pharmaceutically acceptable carrier is preferably non-toxic to the recipient at the dosage and concentration adopted. The antibodies described herein can be formulated as solid, semi-solid, liquid or gaseous preparations. Examples of such preparations include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Depending on the desired preparation, a pharmaceutically acceptable carrier may include a pharmaceutically acceptable non-toxic carrier of a diluent, which is a vehicle commonly used to prepare a pharmaceutical composition for animal or human administration. In certain embodiments, the pharmaceutical composition may include a formulation material for changing, maintaining or maintaining, for example, pH, osmotic pressure volume molar concentration, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition.

[0282] In certain embodiments, pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight the polypeptide; a salt-forming counterion (such as sodium); a preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); a solvent (such as glycerol, propylene glycol or polyethylene glycol); a sugar alcohol (such as mannitol or sorbitol); a suspending agent; a surfactant or wetting agent (such as pluronics, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80), triton, trishydroxymethylaminomethane, lecithin, cholesterol, tyloxapol); a stability enhancer (such as sucrose or sorbitol); a tonicity enhancer (such as an alkali metal halide, preferably sodium chloride or potassium chloride, mannitol, sorbitol); a delivery vehicle; a diluent; an excipient and / or a pharmaceutical adjuvant. Additional examples of formulations suitable for various types of administration can be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press 22nd edition (2013). For a brief review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0283] Suitable preparations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

[0284] The formulation can be optimized for retention and stability in the brain or central nervous system. When the agent is administered into the cranial cavity, it is desirable to retain the agent in the cavity and not diffuse or cross the blood-brain barrier. Stabilization techniques include cross-linking, polymerizing or connecting to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. to achieve an increase in molecular weight.

[0285] Other strategies for increasing retention include embedding antibodies, such as the anti-MS4A6A antibodies of the present disclosure, in biodegradable or bioerodible implants. The release rate of the therapeutically active agent is controlled by the transport rate through the polymer matrix and the biodegradation of the implant. The implant can be a particle, a sheet, a patch, a plaque, a fiber, a microcapsule, and the like, and can have any size or shape compatible with the selected insertion site. The biodegradable polymer composition that can be used can be an organic ester or ether that produces physiologically acceptable degradation products (including monomers) when degraded. Anhydrides, amides, orthoesters, and the like can be used alone or in combination with other monomers. The polymer will be a condensation polymer. The polymer can be cross-linked or non-cross-linked. Of particular interest are polymers, homopolymers or copolymers of hydroxyaliphatic carboxylic acids and polysaccharides. Among the polyesters of interest, polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof are included. The polysaccharides of interest are calcium alginate and functionalized cellulose, especially carboxymethyl cellulose esters, which are characterized by being water insoluble, having a molecular weight of about 5 kD to 500 kD, etc. Biodegradable hydrogels can also be used in the implants disclosed in the subject matter. Hydrogels are generally copolymer materials, which are characterized by being able to absorb liquids.

[0286] V. Therapeutic Uses

[0287] As disclosed herein, the anti-MS4A6A antibodies of the present disclosure can be used to prevent diseases and disorders, reduce the risk of various diseases and disorders, or treat diseases and disorders. In some embodiments, the anti-MS4A6A antibodies of the present disclosure are effective in preventing, reducing the risk of, or treating Alzheimer's disease, late-onset Alzheimer's disease, and cognitive impairment.

[0288] MS4A6A as a disease target

[0289] Genome-wide association studies have identified various members of the MS4A family as being associated with Alzheimer's disease. These members are MS4A2, MS4A3, MS4A4A, MS4A4E, MS4A6A, and MS4A6E. Certain SNPs associated with Alzheimer's disease risk are located in noncoding regions, including rs610932, rs583791, and rs667897; the intergenic region between MS4A4E and MS4A6A (rs670139); and rs7232 located in the coding region of MS4A6A. Three SNPs in the MS4A gene cluster are associated with an increased risk of late-onset Alzheimer's disease, including rs4938933 in MS4A4A, rs670139 in MS4A4E, and rs610932 in MS4A6A (Hollingworth et al., 2011, Nat Genetics, 43:429-435; Naj et al., 2011, Nature Genetics, 43:436-441; Antunez et al., 2011, Genome Medicine, 3, article 33; Ma et al., 2015 Mol Neurobiol 51:1240-1248). In addition, the MS4A4A locus SNPs (rs2304933 and rs2304935) are associated with higher levels of MS4A4A and increased risk of Alzheimer's disease, including late-onset Alzheimer's disease (LOAD) (Allen et al., 2012, Neurology, 79: 221-228). SNPs can serve as valuable genetic biomarkers. For example, SNPs can be associated with disease risk or susceptibility, wherein a specific allele or more than one specific allele of the SNP is associated with increased disease risk or susceptibility (e.g., susceptibility alleles). Alternatively, the SNP can be associated with a reduced risk of disease (e.g., protective alleles).

[0290] Three SNPs associated with Alzheimer's disease are in strong linkage disequilibrium: the rs583791 (T) allele, the rs7232 (T) allele, and the rs610932 (G) allele are associated and are major (most common) alleles associated with increased risk or susceptibility to Alzheimer's disease; the rs583791 (C) allele, the rs7232 (A) allele, and the rs610932 (T) allele are associated and are minor (less common) alleles associated with reduced risk of Alzheimer's disease. In some embodiments that may be combined with any of the preceding embodiments, the present disclosure provides a method of preventing, reducing the risk of, or treating a neurodegenerative disease, disorder, or condition in an individual having at least one genetic allele associated with risk or susceptibility to Alzheimer's disease, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with increased risk or susceptibility to Alzheimer's disease is the rs583791 (C) allele. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with increased risk or susceptibility to Alzheimer's disease is the rs7232 (T) allele. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with increased risk or susceptibility to Alzheimer's disease is the rs610932 (G) allele. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with increased risk or susceptibility to Alzheimer's disease is associated with decreased or reduced expression or activity of MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with increased risk or susceptibility to Alzheimer's disease is associated with decreased or reduced cell surface expression or activity of MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with increased risk or susceptibility to Alzheimer's disease is associated with loss of function or reduced function of MS4A6A. In some embodiments that may be combined with any of the preceding embodiments, the individual having at least one genetic allele associated with a neurodegenerative disease, disorder, or condition is homozygous for the at least one genetic allele. In some embodiments that may be combined with any of the preceding embodiments, the individual having at least one genetic allele associated with a neurodegenerative disease, disorder, or condition is heterozygous for the at least one genetic allele.

[0291] In certain aspects of the present disclosure, the methods provided herein of administering an anti-MS4A6A antibody to an individual in need thereof increases the expression or activity of MS4A6A, increases or restores proper cell surface expression or localization of MS4A6A, or corrects a biochemical or physiological defect associated with an individual having a neurodegenerative risk allele, such as an rs583791 (T) allele, an rs7232 (T) allele, or an rs610932 (G) allele. In some embodiments that may be combined with any of the preceding embodiments, the present disclosure provides a method for increasing MS4A6A expression or activity in an individual in need thereof, wherein the individual has at least one Alzheimer's disease risk or susceptibility allele selected from the group consisting of rs583791 (T), rs7232 (T), or rs610932 (G), the method comprising administering an anti-MS4A6A antibody to the individual, thereby increasing MS4A6A expression or activity. In some embodiments that may be combined with any of the preceding embodiments, the present disclosure provides a method for increasing or restoring cell surface expression or localization of MS4A6A in an individual in need thereof, wherein the individual has at least one Alzheimer's disease risk or susceptibility allele selected from the group consisting of: rs583791 (T), rs7232 (T), and rs610932 (G), the method comprising administering an anti-MS4A6A antibody to the individual, thereby increasing or restoring cell surface expression or localization of MS4A6A.

[0292] In some embodiments, the present disclosure provides a method of selecting an individual for treatment with an anti-MS4A6A antibody, the method comprising: a) obtaining a sample (e.g., a blood sample) from an individual; b) determining which genetic alleles associated with an increased risk of a neurodegenerative disease, disorder, or condition are present in the individual; and c) selecting an individual for treatment with an anti-MS4A6A antibody, wherein the individual has one or more genetic alleles associated with an increased risk of a neurodegenerative disease, disorder, or condition. In some embodiments, the genetic allele associated with an increased risk of a neurodegenerative disease, disorder, or condition is the rs583791 (C) allele. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with an increased risk of a neurodegenerative disease, disorder, or condition is the rs7232 (T) allele. In some embodiments that may be combined with any of the preceding embodiments, the genetic allele associated with an increased risk of a neurodegenerative disease, disorder, or condition is the rs610932 (G) allele. Any suitable method known to those of skill in the art for obtaining a sample (such as a blood sample) from an individual may be used. Furthermore, it will be appreciated that any method known to those skilled in the art for determining which genetic risk or susceptibility alleles an individual possesses, such as by SNP analysis, may be used.

[0293] The methods provided herein can be used to prevent, reduce the risk of, or treat a subject having a neurodegenerative disease, disorder, or condition. In some embodiments, the disclosure provides a method for preventing, reducing the risk of, or treating a subject having a neurodegenerative disorder, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to a subject in need thereof.

[0294] In some embodiments, the disclosure provides a method for preventing, reducing the risk of, or treating an individual with Alzheimer's disease, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof.

[0295] In some embodiments, the disclosure provides a method for preventing, reducing the risk of, or treating an individual with late-onset Alzheimer's disease, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof.

[0296] In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating an individual with mild cognitive impairment, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof.

[0297] In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating a disease, disorder, or condition associated with decreased or reduced expression or activity of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating a disease, disorder, or condition associated with decreased or reduced cell surface expression or activity of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual in need thereof. In some embodiments, the disease, disorder, or condition associated with decreased or reduced expression or activity of MS4A6A is a neurodegenerative disease, disorder, or condition, such as Alzheimer's disease.

[0298] In certain embodiments, loss of function of MS4A6A, reduction of function of MS4A6A, decreased or reduced expression or activity of MS4A6A, or decreased or reduced cell surface expression of MS4A6A is associated with an increased risk of having or developing a neurodegenerative disease, disorder, or condition, such as Alzheimer's disease.

[0299] In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating a disease, disorder, or condition associated with decreased or reduced activity of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual with decreased or reduced activity of MS4A6A. In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating a disease, disorder, or condition associated with loss of function of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual with decreased or reduced activity of MS4A6A. In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating a disease, disorder, or condition associated with decreased or reduced expression of MS4A6A, comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to an individual with decreased or reduced expression of MS4A6A. In some embodiments, the present disclosure provides a method for preventing, reducing the risk of, or treating a subject having a disease, disorder, or condition associated with inappropriate or incorrect cellular localization of MS4A6A, the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody to a subject having inappropriate or incorrect cellular localization of MS4A6A. In certain aspects of the present disclosure, administering a therapeutically effective amount of an anti-MS4A6A antibody to a subject in need thereof results in increased expression of MS4A6AJ, increased activity of MS4A6A, appropriate or correct cellular localization of MS4A6A, increased cell surface expression of MS4A6A, and / or a gain of function of MS4A6A in the subject.

[0300] In some aspects, a disease, disorder, or condition associated with reduced or decreased activity of MS4A6A, associated with loss of function of MS4A6A, associated with reduced or decreased expression of MS4A6A, or associated with inappropriate or incorrect cellular localization of MS4A6A is at least in part the result of a genetic defect or deficiency associated or linked to the MS4A6A gene.

[0301] In some aspects, a disease, disorder, or condition associated with reduced or decreased activity of MS4A6A, associated with loss of function of MS4A6A, associated with reduced or decreased expression of MS4A6A, or associated with inappropriate or incorrect cellular localization of MS4A6A is at least in part a result of normal aging.

[0302] Other aspects of the present disclosure relate to a method of preventing, reducing the risk of, or treating a subject having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer's disease, mild cognitive impairment, vascular dementia, vascular dementia, seizures, retinal dystrophy, traumatic brain injury, spinal cord injury, chronic depression, atherosclerotic vascular disease, adverse symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, degenerative disc disease, Shy-Drager syndrome, Progressive supranuclear palsy, corticobasal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatous disease, sarcoidosis, diseases of aging, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disease, arthritis, multiple sclerosis, metabolic disorders, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, injury, inflammatory cell debris or protein aggregates, abnormalities in circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-related traits (including but not limited to inflammation), neuronal loss and cognitive deficits (such as cognitive deficits in the absence of known brain diseases, including cognitive deficits in the frontal cerebral cortex of elderly individuals and one or more adverse symptoms of normal aging), the method comprising administering a therapeutically effective amount of an anti-MS4A6A antibody of any one of the preceding embodiments to the individual.Other aspects of the present disclosure relate to an anti-MS4A6A antibody of any of the preceding embodiments for use in preventing, reducing the risk of, or treating a subject having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer's disease, vascular dementia, seizures, retinal dystrophy, traumatic brain injury, spinal cord injury, long-term depression, atherosclerotic vascular disease, adverse symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, Lewy body dementia, polymyalgia, systemic atrophy, degenerative disc disease, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous disease, sarcoidosis, diseases of aging, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disease, arthritis, multiple sclerosis, metabolic disorders, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-related traits (including but not limited to inflammation), neuronal loss and cognitive deficits (such as cognitive deficits in the absence of known brain diseases, including cognitive deficits in the frontal cerebral cortex of elderly individuals and one or more adverse symptoms of normal aging). Other aspects of the present disclosure relate to an anti-MS4A6A antibody of any of the preceding embodiments for use in preventing or reducing metastasis. Other aspects of the present disclosure relate to an anti-MS4A6A antibody of any of the preceding embodiments for use in preventing cancer, reducing the risk of cancer, or treating an individual suffering from cancer.

[0303] Other aspects of the disclosure relate to the use of an anti-MS4A6A antibody of any of the foregoing embodiments for the manufacture of a medicament for preventing, reducing the risk of, or treating a subject having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer's disease, late-onset Alzheimer's disease, vascular dementia, seizures, retinal dystrophy, traumatic brain injury, spinal cord injury, long-term depression, atherosclerotic vascular disease, adverse symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, degenerative disc disease, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous diseases, sarcoidosis, diseases of aging, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infections, sepsis, eye infections, systemic infections, inflammatory diseases, arthritis, multiple sclerosis, metabolic disorders, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, injury, inflammatory cell debris or protein aggregates, abnormalities of circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-related traits (including but not limited to inflammation), neuronal loss and cognitive deficits (such as cognitive deficits in the absence of known brain diseases, including cognitive deficits in the frontal cerebral cortex of elderly individuals and one or more adverse symptoms of normal aging). Other aspects of the disclosure relate to a method of preventing, reducing the risk of, or treating a subject having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, progressive supranuclear palsy, Alzheimer's disease, late-onset Alzheimer's disease, mild cognitive impairment, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, Parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, and osteoarthritis, comprising administering to the subject a therapeutically effective amount of an anti-MS4A6A antibody of any one of the preceding embodiments.Other aspects of the disclosure relate to an anti-MS4A6A antibody of any of the preceding embodiments for use in preventing, reducing the risk of, or treating a subject having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, progressive supranuclear palsy, Alzheimer's disease, late-onset Alzheimer's disease, mild cognitive impairment, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, Parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, and osteoarthritis. Other aspects of the disclosure relate to the use of an anti-MS4A6A antibody of any of the foregoing embodiments for the manufacture of a medicament for preventing, reducing the risk of, or treating a subject having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, progressive supranuclear palsy, Alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, Parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, and osteoarthritis.

[0304] Several genes in the MS4A locus are associated with inflammatory responses, including MS4A6A. (Karch and Goate, 2015, Biol Psychiatry, 77: 43-51). In addition, overexpression of MS4A family genes increases T cell activation and promotes T cell transport across the blood-brain barrier. Activated T cells interact with microglia, leading to microglial activation, resulting in the release of proinflammatory cytokines, leading to neuronal damage. Overexpression of MS4A4B reduces T cell apoptosis, while knockdown of MS4A4B promotes T cells to undergo apoptosis. (Ma et al., 2015, Mol Neurobiol, 51: 1240-1248.)

[0305] Autoinflammatory diseases are a group of clinical conditions that are distinct from autoimmune syndromes. Autoinflammatory diseases are characterized by episodes of unexplained inflammation due to dysregulation of the innate immune system, the absence of autoreactive T lymphocytes and autoantibodies, and are therefore distinct from classic autoimmune diseases. Autoinflammatory diseases are divided into two groups: monogenic autoinflammatory diseases and multifactorial autoinflammatory diseases. In some embodiments, the autoinflammatory disorders prevented or treated by the methods of the present disclosure include, but are not limited to, monogenic autoinflammatory diseases (such as familial Mediterranean fever (FMF)), mevalonate kinase deficiency-associated periodic fever (hyperimmunoglobulin D syndrome), TNF receptor-associated periodic syndrome (TRAPS), cold inflammation-associated periodic syndrome (CAPS), NLRP12-associated autoinflammatory disorders (e.g., NALP12-associated periodic fever), interleukin-1 receptor antagonist deficiency (DIRA), pyogenic arthritis-pyoderma gangrenosum and acne (PAPA) syndrome, Magid syndrome, Blau syndrome, hyperimmunoglobulinemia W with periodic fever syndrome (HIDS), familial cold autoinflammatory syndrome (FCAS), Mueller syndrome (MWS), and neonatal onset of multisystem inflammatory disorder (NOMID). In some embodiments, the autoinflammatory disorders prevented or treated by the methods of the present disclosure include, but are not limited to, multifactorial autoinflammatory diseases (such as those including periodic fevers), ulcerative stomatitis, pharyngitis and adenopathy syndrome (PFAPA), Behcet's disease, systemic juvenile idiopathic arthritis (sJIA), Still's disease, adult-onset Still's disease (AOSD), Crohn's disease, Schnitzler syndrome, Sweet syndrome, chronic relapsing multifocal osteomyelitis (CRMO), synovitis acne pustular hyperostotic osteitis syndrome (SAPHO), and adult-onset Still's disease. (See Ciccarelli et al., 2013, Curr Med Chem, 21:261-269.)

[0306] Although MS4A genes are not well characterized, several members of this cluster, including MS4A1, MS4A2, and MS4A4B, have been shown to play important roles in immunity (Zuccolo et al., 2010, PLoS One; Zuccolo et al., 2013, Front Immunol, 4:195).

[0307] Autoimmune diseases are derived from abnormal immune responses to normal human tissues. In some embodiments, the autoimmune diseases or conditions prevented or treated by the methods of the present disclosure include, but are not limited to, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Barlow's disease, Behcet's disease, benign mucous membrane pemphigus, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic Inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Zug-Strauss, cicatricial pemphigus, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, DeVick's disease (neuromyelitis optica), discoid lupus, De Lacy syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Cubastier syndrome, Wegener's granulomatosis, Graves' disease Stokes disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigus (PG), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, xylem conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Moor's ulcers, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigus, optic neuritis, palindromic rheumatic disease (PR), Pandas, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parrot syndrome, pars plana cyclitis (peripheral uveitis), Parsenager-Turner syndrome, pemphigus, peripheral neuropathy, venous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome,Primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell dysplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff man syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunter syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0308] In some embodiments, the subject or individual is a mammal. Mammals include, but are not limited to, livestock (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates (such as monkeys)), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject or individual is a human.

[0309] Antibodies provided herein (and any other therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intralesional administration, intracerebral spinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical or inhalation routes. Parenteral infusion includes intramuscular, intravenous administration with bolus or continuous infusion for a period of time, intraarterial, intraarticular, intraperitoneal or subcutaneous administration. In some embodiments, administration is intravenous administration. In some embodiments, administration is subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations at various time points, push administration and pulse infusion.

[0310] The antibodies provided herein will be formulated, administered and used in a manner consistent with good medical practice. Factors considered in this context include the specific illness treated, the specific mammal treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the medicament, the method of administration, the administration schedule, and other factors known to practitioners. The antibody is not required but optionally formulated with one or more medicaments currently used to prevent or treat the illness in question. The effective amount of such other medicaments depends on the amount of the antibody present in the preparation, the type of illness or treatment, and other factors discussed above. These medicaments are generally used in the same dose as described herein and by the administration route as described herein, or with about 1% to 99% of the dosage as described herein, or with any dosage determined to be appropriate empirically / clinically and by any approach determined to be appropriate empirically / clinically.

[0311] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the present disclosure (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0312] Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be used as an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. According to the factors mentioned above, a typical daily dose can be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administrations of several days or longer, according to the condition, treatment should generally continue until the desired inhibition of disease symptoms occurs. An exemplary dose of an antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg can be administered to a patient. These doses can be intermittent, for example, administered weekly or every three weeks (e.g., so that the patient receives about two doses to about twenty doses, or, for example, about six doses of antibody). In certain embodiments, the dosing frequency is three times a day, twice a day, once a day, every other day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once a month, once every two months, once every three months, or longer. An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may also be available. The progress of the therapy may be easily monitored by conventional techniques and assays.

[0313] VI. Diagnostic Uses

[0314] In some embodiments of any of the antibodies, any of the anti-MS4A6A antibodies provided herein can be used to detect the presence of MS4A6A in a sample or individual. As used herein, the term "detection" encompasses quantitative or qualitative detection. Provided herein are methods of using the antibodies of the present disclosure for diagnostic purposes, such as detection of MS4A6A in an individual or a tissue sample derived from an individual. In some embodiments, the individual is a human.

[0315] The detection method may involve quantification of antigen-binding antibodies. Antibody detection in biological samples may be performed by any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micropositron emission tomography. In certain embodiments, for example, using 18 F The antibody is radiolabeled and subsequently detected by micropositron emission tomography analysis. Antibody binding in patients can also be quantified by non-invasive techniques such as positron emission tomography (PET), X-ray computed tomography, single photon emission computed tomography (SPECT), computed tomography (CT), and computed axial tomography (CAT).

[0316] VII. Products

[0317] Provided herein are articles (e.g., kits) comprising an anti-MS4A6A antibody described herein. The article may include one or more containers comprising an antibody described herein. The container may have any suitable packaging, including but not limited to vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The container may be a unit dose, bulk packaging (e.g., multi-dose packaging), or a subunit dose.

[0318] In some embodiments, the kit may additionally include a second reagent. In some embodiments, the second reagent is a pharmaceutically acceptable buffer or diluent, including but not limited to such as antibacterial water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. In some embodiments, the second reagent is a pharmaceutically active agent.

[0319] In some embodiments of any of the articles of manufacture, the article of manufacture further comprises instructions for use according to the methods of the present disclosure. The instructions typically include information about the dosage, dosing schedule, and route of administration for the intended treatment. In some embodiments, these instructions include a description of administering an isolated antibody of the present disclosure (e.g., an anti-MS4A6A antibody described herein) to prevent, reduce the risk of, or treat an individual with a disease, disorder, or injury selected from the group consisting of: frontotemporal dementia, Alzheimer's disease, late-onset Alzheimer's disease, cognitive decline or impairment, mild cognitive impairment, vascular dementia, vascular dementia, epileptic seizures, retinal dystrophy, Malnutrition, traumatic brain injury, spinal cord injury, long-term depression, atherosclerotic vascular disease, adverse symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, Lewy body dementia, polymyositis system atrophy, degenerative disc disease, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous disease, sarcoidosis, diseases of aging, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disease, arthritis, multiple sclerosis, metabolic disorders, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-related traits (including but not limited to inflammation), neuronal loss and cognitive deficits (such as cognitive deficits in the absence of known brain diseases, including cognitive deficits in the frontal cerebral cortex of elderly individuals and one or more adverse symptoms of normal aging), the description comprising administering a therapeutically effective amount of an anti-MS4A6A antibody of any one of the preceding embodiments to an individual.

[0320] In some embodiments, the instructions include instructions for use of the anti-MS4A6A antibody and a second agent (eg, a second pharmaceutically active agent).

[0321] The present disclosure will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the present disclosure. All references throughout the present disclosure are hereby expressly incorporated by reference.

[0322] Example

[0323] Example 1: Construction of MS4A6A expression plasmid for DNA immunization

[0324] DNA immunization method was used to develop antibodies against MS4A6A. The cDNA sequences encoding human and cynomolgus monkey (cyno) MS4A6A (SEQ ID NO: 1 and 3, respectively) were cloned into the pCAGGS expression vector (KeraFAST EH1017) for DNA immunization. The expression of MS4A6A protein was confirmed by the following steps: the expression construct was transiently transfected into HEK293T cells, and then western blotting was performed using commercially available anti-MS4A6A antibodies (rabbit polyclonal antibodies from Abcam, Cat#156278 and Cat#189983). To this end, cells that had been transfected for 24 hours were lysed in lysis buffer (RIPA lysis buffer (ThermoFischerScientific Cat#89900) + 1:100 HALT protease inhibitor cocktail (ThermoFischerScientific Cat#87786)) and insoluble debris was removed by centrifugation at 14,000×g for 15 minutes. Soluble fractions were determined using bicinchoninic acid (BCA) reagent for protein quantification. Equal amounts of protein from each sample were loaded onto 4-12% Bis-Tris Plus polyacrylamide gels (ThermoFisherScientific NW04120) and separated by electrophoresis, and then the proteins in the gel were transferred to polyvinylidene fluoride (PVDF) membranes using iBlot2 (ThermoFisher Scientific IM21001) and transfer layers (ThermoFisher Scientific IB24002). The membranes were blocked with 1% bovine serum albumin or 5% skim milk to prevent nonspecific binding. The membranes were then incubated with polyclonal anti-MS4A6A antibodies (Abcam, Cat#156278 and Cat#189983), washed, and then incubated with HRP-conjugated secondary antibodies (rabbit, Abcam#205718; mouse, Abcam#205719). Binding was visualized by developing the membrane with SuperSignal WestPico Plus Chemiluminescent Substrate (ThermoFisher Scientific Cat#34577) and digitally recorded with iBright FL1000 (ThermoFisher A32752) or other compatible system.

[0325] like Figure 1As shown, cells transfected with expression constructs for human MS4A6A or cynomolgus MS4A6A showed a reactive band at approximately 27 kDa as probed by a polyclonal anti-MS4A6A antibody (top: Abcam #156278, bottom: Abcam #189983). Untransfected HEK293 cells or HEK293 cells transfected with an irrelevant MS4A4A expression construct showed very low levels of MS4A6A reactivity, which may be due to some endogenous expression.

[0326] Example 2: Generation of anti-MS4A6A hybridoma antibodies

[0327] To obtain antibodies against MS4A6A, the following procedure was used to generate hybridomas. Balb / c or SJL mice (Jackson Laboratory, Bar Harbor, ME) were co-immunized once a week with 50 μg of each of plasmid DNA encoding full-length human or cynomolgus MS4A6A diluted in lactated Ringer's solution, with or without mFlt3 ligand (DNA) and mGM-CSF (DNA) (Invitrogen, San Diego, CA). A total of 8 injections of MS4A6A expression plasmids were performed per mouse for DNA immunization. Spleens were harvested from mice three days after the final DNA immunization. The reactivity of sera from mice to MS4A6A was analyzed by FACS analysis using HEK293 cells overexpressing human and / or cynomolgus MS4A6A. Splenocytes from mice whose sera showed strong binding to HEK293 cells overexpressing human and / or cynomolgus macaque MS4A6A by FACS were fused with P3X63Ag8.653 mouse myeloma cells (CRL-1580, American Type Culture Collection, Rockville, MD) via electrofusion (ECM2001, BTX, Holliston, MA) and incubated overnight at 37° C., 5% CO2 in Clonacell-HY medium C (StemCell Technologies, Vancouver, BC, Canada).

[0328] The next day, the fused cells were centrifuged and resuspended in 10 ml of ClonaCell-HY medium C with anti-mouse IgG Fc-FITC (Jackson Immunoresearch, West Grove, PA), and then gently mixed with 90 ml of methylcellulose-based ClonaCell-HY medium D (Stemcell Technologies) containing HAT components. The cells were plated into Nunc OmniTrays (Thermo Fisher Scientific, Rochester, NY), and the cells were grown for eight days at 37°C, 5% CO2. Fluorescent colonies were selected and transferred to 96-well plates containing Clonacell-HY medium E (StemCell Technologies) using Clonepix2 (Molecular Devices, Sunnyvale, CA). After five days, HEK293 cells overexpressing full-length cynomolgus monkey MS4A6A were screened from tissue culture supernatants from hybridomas by FACS as described below. A total of 960 hybridoma clones were generated from two independent rounds of fusion.

[0329] Example 3: Transfection of HEK293 cells with MS4A6A gene variants

[0330] HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) + 10% FBS (Gibco) until >80% confluence. Then, 24 hours before transfection, the cells were dissociated with non-enzymatic cell dissociation buffer (CellStripper, Corning) and plated in T150 flasks (ThermoFischerScientific Cat#08-772-48) at 40-50% confluence. Transfection was performed with Lipofectamine3000 (ThermoFischerScientific) according to the manufacturer's protocol. Cells were harvested 24 hours after transfection to show any toxic effects of MS4A6A overexpression in HEK293 cells in advance. Harvested cells can be used for FACS analysis immediately or frozen in 10% DMSO for subsequent use.

[0331] Transiently transfected HEK293 cells expressing human or cynomolgus MS4A6A were generated. MS4A6A protein expression in cells was confirmed by Western blotting as follows. HEK293 cells that had been transfected for 24 hours were lysed in lysis buffer (RIPA lysis buffer (ThermoFischerScientific Cat#89900) + 1:100 HALT protease inhibitor cocktail (ThermoFischerScientific Cat#87786)) and insoluble debris was cleared by centrifugation at 14,000×g for 15 minutes. The soluble fraction was determined with bicinchoninic acid (BCA) reagent for protein quantification. Equal amounts of protein from each sample were loaded onto 4-12% Bis-Tris Plus polyacrylamide gels (ThermoFisherScientific NW04120) and separated by electrophoresis, and then the proteins in the gel were transferred to polyvinylidene fluoride (PVDF) membranes using iBlot2 (ThermoFisher Scientific IM21001) and transfer layers (ThermoFisher Scientific IB24002). The membranes were blocked with 1% bovine serum albumin or 5% skim milk to prevent nonspecific binding. The membranes were then incubated with polyclonal antibodies (Abcam, Cat#156278 and 189983), washed, and then incubated with HRP-conjugated secondary antibodies (rabbit, Abcam#205718; mouse, Abcam#205719). Binding was visualized by developing with SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoFisher Scientific #34577) and digitally recorded with iBrightFL1000 (ThermoFisher Scientific #A32752) or other compatible systems.

[0332] Since there is no commercially available reagent for MS4A6A that can be used for flow cytometry, sera from immunized mice were used to detect surface expression of MS4A6A on cells. Transiently transfected cells were labeled with a Live / Dead Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific L34957) on ice for 30 minutes. After washing with PBS, 2×10 5Each cell was aliquoted into each well of a 96-well U-bottom plate and incubated on ice for 30 minutes with 50 μl diluted serum. After the initial incubation, the supernatant was removed via centrifugation, and the cells were washed twice with 175 μL ice-cold FACS buffer (PBS+1% FBS+2mM EDTA), then incubated on ice for 20 minutes with anti-mouse IgG Fc-APC (Jackson ImmunoResearch Labs, West Grove, PA, Cat#115-136-071) (1:200 dilution). After the secondary incubation, the cells were washed twice again with ice-cold FACS buffer, and resuspended in a final volume of 200 μl FACS buffer. Analyzed on FACS Canto system (BD Biosciences), and gated to exclude dead cells (Aqua positive).

[0333] The results showed that cells transiently transfected with cynomolgus monkey MS4A6A were positive for cell surface expression of MS4A6A. Although MS4A6A protein was detected in these cells using Western blot analysis, cells transiently transfected with human MS4A6A using this method did not show cell surface expression of MS4A6A (see Figure 1 ). Figure 2A and 2C Results of binding of two independent hybridoma supernatants to human MS4A6A-transfected HEK293 cells (clear trace) and to non-transfected HEK293 cells (shaded trace), respectively, are shown. Figure 2B and 2D Results of binding of two independent hybridoma supernatants to HEK293 cells transfected with cynomolgus monkey MS4A6A (clear trace) and to untransfected HEK293 cells (shaded trace) are shown, respectively. Figure 2B and 2D As shown, cells transfected with cynomolgus MS4A6A showed strong binding of anti-MS4A6A antibodies.

[0334] Since the amino acid sequence of cynomolgus monkey MS4A6A is approximately 89% identical to that of human MS4A6A, it can be expected that most clones have cross-reactivity between human and cynomolgus monkey MS4A6A. This was confirmed by the peptide binding studies described below. Therefore, subsequent screening of hybridoma supernatants was performed using cells transfected with cynomolgus monkey MS4A6A.

[0335] Example 4: Transfection of other cell lines with MS4A6A expression plasmid

[0336] Given the difficulty in generating stably transfected HEK293 cell lines expressing human MS4A6A or cynomolgus MS4A6A, other DNA vectors and cell lines were tested to determine if they were more compatible with MS4A6A expression. For stable transfection, the MS4A6A coding sequence was introduced into expression vectors pD2533-G418 or pD3539-puro (Atum, Newark, CA, USA).

[0337] MS4A6A is naturally expressed in bone marrow cells in vivo. Therefore, in order to overcome the toxicity issues observed using the cells described above, several bone marrow-derived cell lines were used for transient expression of recombinant MS4A6A. The cell line panel included THP-1 cells (ATCC TIB202), U937 cells (ATCC CRL-1593.2), K562 cells (ATCC CCL243), HL60 cells (ATCC CCL240), and Kasumi-1 cells (ATCC CRL-2724). The mouse pre-B cell line 300.19 cells (Tufts University T000710) were also tested because they are commonly used for recombinant protein expression purposes. The antibiotic susceptibility of each of these cell lines was screened to determine the appropriate dose of G418 or puromycin for selection and transfection efficiency. After transfection of the MS4A6A expression plasmid and antibiotic selection, transfectants from U937 cells, K562 cells, and 300.19 cells were found to be viable. Following cloning by limiting dilution, individual clones were generated and screened for human MS4A6A protein expression by Western blot as described above.

[0338] In addition, a cynomolgus monkey MS4A6A expression construct was generated and transfected into the cell lines described above. Since the cynomolgus monkey MS4A6A protein on the cell surface can be detected by flow cytometry, this method was used to confirm the surface expression of MS4A6A in stable U937 cell and K562 cell transfectants. The results are shown in Figure 3A (U937 cells) and Figure 3B (K562 cells) as shown. Figure 3A Shown is the binding of cynomolgus MS4A6A-transfected U937 cells (right trace) compared to the binding of untransfected U937 cells (left trace). Figure 3B Binding of cynomolgus MS4A6A transfected K562 cells (right trace) compared to binding of untransfected K562 cells (left trace) are shown. These results show that anti-MS4A6A hybridoma supernatants are able to bind to cell surface expressed cynomolgus MS4A6A in transfected cells.

[0339] Example 5: Preliminary screening of anti-MS4A6A hybridomas

[0340] Initial screening of anti-MS4A6A hybridomas was performed as follows. Tissue culture supernatants obtained from 960 hybridomas were initially screened for their ability to differentially bind to HEK293 cells transfected with cynomolgus MS4A6A by comparing the extent of binding to parental (untransfected) HEK293 cells with that of transfected cells. Cells expressing MS4A6A were generated via transient transfection of HEK293 cells using the lipofectamine system according to the manufacturer's protocol (modified as described above). To ensure reproducibility throughout the screening experiment, large pools of transfected cells (approximately 1×10 9 ), and were aliquoted and frozen for all further screening experiments.

[0341] To screen the hybridoma cell culture supernatant, HEK293 cells transfected with cynomolgus monkey MS4A6A were equally divided into 96-well U-bottom plates (2 × 10 5 Cells) and incubated on ice for 30 minutes with 50 μL hybridoma cell culture supernatant. After the initial incubation, the supernatant was removed via centrifugation, and the cells were washed twice with 175 μL ice-cold FACS buffer (PBS+1% FBS+2mM EDTA), and then further incubated on ice for 20 minutes with anti-mouse IgG Fc-allophycocyanin (APC) (Jackson Labs, Cat#115-136-071) (1:200 dilution). After this secondary incubation, the cells were washed twice again with ice-cold FACS buffer and resuspended in a final volume of 30 μL of FACS buffer + 0.25 μl / well of propidium iodide (BD Biosciences Cat#556463). The binding intensity on the cells was analyzed by the FACS Canto system (BD Biosciences), and the sorting gate was set to exclude dead cells, which can be judged by the lack of propidium iodide exclusion. For each hybridoma supernatant, the ratio of APC mean fluorescence intensity (MFI) on MS4A6A transfectants versus HEK293 parental cells was calculated. A total of 55 clones were identified that showed greater than 1.5-fold difference in binding to MS4A6A transiently transfected HEK293 cells compared to binding to parental (untransfected) HEK293 cells.

[0342] Positive clones were amplified and then rescreened for specificity against HEK293 cells overexpressing cynomolgus macaque MS4A6A and parental HEK293 cells. Briefly, previously prepared cryopreserved transiently transfected cells were labeled on ice for 30 min using the Live / Dead Fixable Aqua Dead Cell Stain Kit (ThermoFisherScientific L34957). After washing with PBS, 2 × 10 5 Each cell was aliquoted into each well of a 96-well round-bottom plate, and incubated on ice for 30 minutes with 50 μl culture supernatant. After the initial incubation, the supernatant was removed via centrifugation, and the cells were washed twice with 175 μl ice-cold FACS buffer (PBS+1% FBS+2mM EDTA), then incubated on ice for 20 minutes with anti-mouse IgG Fc-APC (Jackson ImmunoResearch Labs, West Grove, PA, Cat#115-136-071) (1:200 dilution). After the secondary incubation, the cells were washed twice again with ice-cold FACS buffer, and resuspended in a final volume of 200 μl FACS buffer. Analyzed on FACS Canto system (BD Biosciences), and sorting gate was set to exclude dead cells (Aqua positive). The ratio of the APC MFI on MS4A6A+ / HEK parental cells was calculated for each hybridoma; these results are shown below in Table 2. The screening was repeated twice, and the numbers reported here represent the ratio of the mean fluorescence levels of transfected cells to parental cells.

[0343] Table 2

[0344] clone Filter 1 Filter 2 Mouse strains 6A-1 7.5 7.6 Balb / c 6A-2 25.7 75.5 Balb / c 6A-3 24.1 63.7 Balb / c 6A-4 5.4 16.2 Balb / c 6A-5 24.3 188.7 Balb / c 6A-6 13.5 62.1 Balb / c 6A-7 3.6 8.3 Balb / c 6A-8 26.7 69.5 Balb / c 6A-9 12.6 84.0 Balb / c 6A-10 18.0 95.2 Balb / c 6A-11 8.6 61.7 Balb / c 6A-12 40.3 45.5 Balb / c 6A-13 24.8 30.3 Balb / c 6A-14 20.2 59.1 Balb / c 6A-15 3.8 8.4 Balb / c 6A-16 14.4 28.5 Balb / c 6A-17 19.3 178.4 Balb / c 6A-18 4.1 20.3 Balb / c 6A-19 2.8 35.8 Balb / c 6A-20 2.7 0.2 SJL 6A-21 71.8 136.0 Balb / c 6A-22 175.3 68.8 Balb / c 6A-23 155.5 82.0 Balb / c

[0345] Example 6: Anti-MS4A6A antibody ELISA screening

[0346] Anti-MS4A6A hybridoma supernatants were tested for binding to MS4A6A peptides derived from the sequences of ECL1 (amino acid residues 68-85 of human MS4A6A of SEQ ID NO: 1) and ECL2 (amino acid residues 138-185 of human MS4A6A of SEQ ID NO: 1) using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well polystyrene plates were coated overnight at 4°C with 1-10 μg / mL of synthetic free or BSA-conjugated peptides dissolved in coating buffer (0.05 M carbonate buffer, pH 9.6, Millipore Sigma C3041). The coated plates were then blocked with ELISA diluent (PBS + 0.5% BSA + 0.05% Tween20) for 1 hour, washed in 3 × 300 μL PBST (PBS + 0.05% Tween20, Thermo 28352), and then antibodies were added in ELISA diluent at various dilutions (100-1000×). After 30 minutes of incubation (room temperature, shaking), the plates were washed with 3 × 300 μL of PBST. Anti-mouse HRP secondary antibody (Jackson Immunoresearch Cat#115-035-003) was added to ELISA diluent (50 μl / well) at a dilution of 1:1000 and incubated at room temperature with shaking for 30 minutes. After the last set of washes (3×300 μL of PBST), 50 μL of TMB substrate (BioFx TMBW-1000-01) was added, and then 50 μL of stop solution (BioFx BSTP-1000-01) was used to quench the reaction after 5-10 minutes. The absorbance of the quenched reaction wells was measured at 650 nm using a BioTek Synergy microplate reader using GEN5 2.04 software.

[0347] Of the 23 anti-MS4A6A positive hybridoma clones identified, supernatants from 17 hybridoma clones showed strong binding to the BSA-MS4A6A-ELC2 peptide compared to an irrelevant control peptide, BSA-mouse DAP12. Although these clones were originally selected based on their ability to bind to cynomolgus monkey MS4A6A protein expressed on the cell surface, many clones still showed binding to the human M4A6A ECL2 peptide, suggesting that these clones have some cross-reactivity between these two closely related MS4A6A sequences. Hybridoma clones that did not show binding could be due to a variety of factors, for example, these antibodies could bind to conformational epitopes that were not modeled by this method, these antibodies could recognize epitopes composed of both ECL1 and ECL2, or these antibodies may not cross-react with the human MS4A6A protein.

[0348] On the other hand, no binding to ECL1 was detected for any of the hybridoma clones screened. This may be due to the smaller size of ECL1, which is 18 amino acid residues in length, while ECL2 is 48 amino acid residues in length. If MS4A6A forms a barrel-like structure similar to CD81, i.e., a molecule that similarly spans the cell membrane four times, the larger ECL2 domain may occupy an exposed surface that can be used for antibody binding. It is possible that some of the antibodies identified herein recognize epitopes formed by a combination of amino acid residues located in ECL1 and ECL2 and therefore cannot be detected by this method. It is noteworthy that the amino acids of ECL1 of human MS4A6A and cynomolgus monkey MS4A6A are identical.

[0349] The results of the ELISA binding experiments are shown in Table 3 below.

[0350] Table 3

[0351]

[0352] Example 7: Molecular cloning of anti-MS4A6A hybridoma antibodies

[0353] The anti-MS4A6A antibody obtained from the hybridoma described above was cloned as follows. 5 × 10 5 Each hybridoma cell was resuspended in 0.5 ml Trizol solution (Thermo Fisher Scientific, cat#15596026). Total RNA was extracted from the cells by chloroform extraction and ethanol precipitation. cDNA was generated using the RACE 5' / 3' kit (Takara Bio USA Inc, Cat. No. 634859). The variable heavy chain and light chain immunoglobulin regions were cloned by step-down PCR using the 5'UPM primer provided in the RACE kit with a heavy chain constant region primer (5'-AGCTGGGAAGGTGTGCACA-3') (SEQ ID NO: 127) and a light chain constant region primer (5'-CCATTTTGTCGTTCACTGCCA-3') (SEQ ID NO: 128). The PCR product was purified by the QIAquick PCR purification kit (QIAGEN, Cat. No. 28106) and ligated to Cloning vector ( TA cloning kit, Invitrogen) and transformed into TOP10 competent cells. Transformed Escherichia coli (E. coli) colonies were isolated and the variable heavy chain (VH) and variable light chain (VL) nucleic acids of each corresponding hybridoma cell line were sequenced. After sequencing, primers containing endonuclease restriction sites (for HV, BsrGI and BstEII, and for LV, BssHII and BsiWI) were used to amplify the variable heavy chain region and the variable light chain region by PCR and subcloned into pJG mammalian expression vectors (Alector Inc.) encoding human IgG1 and IgGK, respectively.

[0354] Example 8: Production of recombinant anti-MS4A6A antibodies

[0355] After culturing the hybridomas in low IgG or chemically defined media, hybridoma-derived anti-MS4A6A antibodies were purified using protein A from the hybridoma supernatants. Some anti-MS4A6A antibodies were also produced via direct cloning of variable gene regions obtained from hybridomas into recombinant expression plasmids, generating chimeric antibodies (human IgG1) containing a human Fc domain. The expression plasmids were transiently transfected into Expi293 cells, and the resulting anti-MS4A6A antibodies were purified via protein A.

[0356] Recombinant production of anti-MS4A6A antibodies was performed as described below. Expression plasmids (containing nucleic acids encoding anti-MS4A6A antibody VH and VL chains) were used for recombinant antibody expression in Expi293 cells. Transfection of expression plasmids was performed using the Expifectamine-293 system (ThermoFischerScientific Cat#A14524) according to the manufacturer's protocol. In brief, for each anti-MS4A6A antibody, 12 μg of light chain plasmid DNA and 18 μg of heavy chain plasmid DNA were diluted into 1.5 mL OptiMEM (ThermoFischerScientific Cat#31985070), and 80 μL of Expifectamine reagent was added thereto. The resulting solution was mixed and incubated at room temperature for 30 minutes and then added to 30 mL of Expi293 cells (ThermoFischerScientific A14527) in Expi293 expression medium (ThermoFischerScientificCat#A1435101) in a 125 mL flask (Fischer Scientific FIS#PBV12-5). Prior to transfection, the cells were cultured to approximately 3×10^ 6Expi293 cells were cultured at 37°C / 8% CO2 and orbitally shaken at 125 rpm. 16-24 hours after transfection, 150 μL of ExpiFectamine TM 293 Transfection Enhancer 1 and 1.5mL ExpiFectamine TM 293 Transfection Enhancer 2 was added to each flask to increase recombinant antibody yield. Culture supernatants were harvested 5-7 days after transfection, filtered (0.2 micron), and purified via protein A chromatography.

[0357] Example 9: Antibody heavy and light chain variable domain sequences

[0358] For 22 of the 23 identified antibodies, the amino acid sequences encoding the light chain variable region and heavy chain variable region of the generated antibodies were determined using standard techniques. The Kabat light chain CDR sequences and heavy chain CDR sequences of the antibodies are shown in Table 4A below. The light chain variable region and heavy chain variable region sequences of the antibodies are shown in Table 4B below.

[0359] Table 4A: Antibody CDR sequences

[0360]

[0361]

[0362] Table 4B: V H and V L sequence

[0363]

[0364]

[0365]

[0366] Example 10: Kinetic Characterization of Anti-MS4A6A Antibodies

[0367] The binding kinetics of the purified antibodies to the MS4A6A ECL1 and ECL2 peptides were characterized as follows by Carterra (South San Francisco, CA) using a proprietary array surface plasmon resonance (SPR) instrument (MX-96). Antibodies were printed on CMD500D chips (Xantec #SPMX CMD500D lot #SCCMD500D0117.a Exp. 31.12.18) using a continuous flow microsampler (CFM). First, the chip was activated for 7 minutes with 100 mM MES pH 5.5, 100 μL EDC (final 133 mM), 100 μL S-NHS (final 33.3 mM). Injection of anti-mouse IgG-Fc (Jackson ImmunoResearch cat#115-005-071 lot#130410) was performed for 15 minutes to establish a surface density of 10,000-12,000 RU, and then the chip surface was inactivated for 10 minutes with 1 M ethanolamine at pH 8.5. The anti-MS4A6A antibody in question was then printed in the form of hybridoma supernatant diluted 2:1 with HBS-EP+ buffer (Teknova Cat#H8022), in duplicate, for 20 minutes and 5 minutes from the same sample solution, respectively. The control antibody was diluted to 20 μg / ml for printing.

[0368] For kinetic analysis, the peptides in question were prepared in HBS-EP+ buffer with 1 mg / ml BSA at final assay concentrations of 2000 nM, 400 nM, 80 nM, 16 nmM and 3.2 nM. The peptides were then injected onto the chip for five minutes and then dissociated for seven minutes at 8 uL / s in a non-regenerative kinetic series. Repeated measurements were performed for each anti-MS4A6A antibody to ensure reproducibility.

[0369] For each of the 23 anti-MS4A6A antibodies tested, the K of the MS4A6A ECL-2 peptide was calculated. a (Association rate constant; K on ), K d (Dissociation rate constant; K off ) and K D (equilibrium constant), the calculation results are shown in Table 5 below (clones that bound too weakly to determine these values ​​are indicated as N / A in Table 5). No binding to the ECL-1 peptide was observed for any of the anti-MS4A6A antibodies tested.

[0370] Table 5

[0371]

[0372]

[0373] Fifteen of the 23 anti-MS4A6A antibodies bound to the MS4A6A ECL2 peptide and produced an overall KD value of less than 10e-07. This largely corresponds to the ELISA data in Example 7 above, except that two anti-MS4A6A antibodies (6A-6 and 6A-11) were positive for binding as determined by ELISA, but did not show binding as determined by Carterra. Of these two antibodies, 6A-6 showed binding on the SPR trace, although the excess signal was weak and therefore the KD value was not determined. None of the antibodies tested bound to an unrelated control peptide. The specificity of the assay was confirmed using an isotype control antibody, an unrelated hybridoma clone, an anti-HIS control antibody, and a medium alone control. None of these control reagents showed any binding to the cells.

[0374] The lack of binding to the ECL1 peptide was consistent in both methods used. This may be due to the smaller size of ECL1, which is 18 amino acids in length compared to 48 amino acids for ECL2. If MS4A6A forms a barrel-like structure similar to CD81, i.e. a molecule that similarly spans the cell membrane four times, then the much larger ECL2 would be expected to occupy exposed surfaces available for antibody binding. It is possible that some of the anti-MS4A6A antibodies identified here recognize epitopes formed by a combination of ECL1 and ECL2 amino acid residues and therefore could not be detected by the methods used. It is noteworthy that human and cynomolgus monkey MS4A6AECL1 have the same amino acid sequence; therefore, the lack of cross-reactivity is not relevant.

[0375] Example 11: Measurement of affinity of MS4A6A antibody for transiently and naturally expressing cell lines

[0376] Purified anti-MS4A6A antibodies were evaluated for binding affinity to various MS4A6A expressing cell lines, including HEK293 transiently transfected with human or cynomolgus macaque MS4A6A expression plasmids, as well as HeLa cells and A549 cells. The antibodies tested were mouse IgG purified from hybridoma supernatants or human IgG1 Fc chimeras recombinantly produced in Expi293 cells. Affinity binding to cells was determined as follows. Briefly, cells were harvested, washed and labeled with Aqua Live / Dead for viability identification. After washing with PBS, 2×10^ 5Cells were aliquoted into each well of a 96-well U-bottom plate and incubated with 50 μL of purified anti-MS4A6A antibody at various concentrations (starting from 10 μg / mL, 3× dilution) in FACS buffer (PBS + 2% FBS + 1 mM EDTA). After the initial incubation, the supernatant was removed by centrifugation, washed twice with 150 μL of ice-cold FACS buffer, and incubated on ice for 30 minutes with the appropriate secondary antibody. After the secondary incubation, the cells were washed 2× again with ice-cold FACS buffer and resuspended in a final volume of 200 μL of FACS buffer. Flow cytometric analysis was performed on a FACSCanto system (BD Biosciences). Binding data were analyzed using mean fluorescence intensity, and curves were fitted in Prism (nonlinear regression with four parameters: log inhibitor versus dose response) to determine EC50 values.

[0377] Example 12: Epitope determination of anti-MS4A6A antibodies by peptide binding

[0378] Additional binding characterization of the anti-MS4A6A antibodies was performed as follows. Specifically, epitope mapping of the anti-MS4A6A antibodies was performed by Carterra using a premixed epitope binning method. The epitope binding characteristics of the anti-MS4A6A antibodies were determined by two complementary methods. First, a set of overlapping peptides derived from the extracellular loops were synthesized by JPT Peptides (Berlin, Germany) or Elim Biopharm (Hayward, CA, USA). These were then transferred to Carterra (South San Francisco, CA, USA), where Carterra determined the binding characteristics using a proprietary array surface plasmon resonance (SPR) instrument (MX-96) using a protocol similar to that described above, as described below.

[0379] The peptide library was printed onto a CMD500D chip using a continuous flow microsampler (CFM). First, the chip was activated with 100 mM MES pH 5.5, 100 μL EDC (final concentration 133 mM), 100 μL S-NHS (final 33.3 mM). The peptide library was immobilized on the chip and the chip surface was then inactivated for 10 minutes with 1 M ethanolamine at pH 8.5. The anti-MS4A6A antibodies to be tested were prepared at various final assay concentrations in HBS-EP+ buffer containing 1 mg / ml BSA. The peptides were then injected onto the chip for five minutes and then dissociated for seven minutes at 8 μL / s in a non-regenerative kinetic series. Repeated measurements were performed for each anti-MS4A6A antibody to ensure reproducibility. The binding characteristics of each peptide-antibody combination were determined, allowing the linear peptide region for each antibody interaction to be mapped.

[0380] Some anti-MS4A6A antibodies bind to non-continuous epitopes. Pepscan (Lelstad, The Netherlands) has developed a technology called chemically linked peptides to scaffolds (CLIPS) to resolve such epitopes. In this technology, a library of tertiary structure mimics is designed and synthesized onto a solid support. These mimics can mimic secondary structure elements in proteins, such as loops, alpha helices, and beta strands. The binding of antibodies to each peptide construct is determined and quantified. The degree of binding to the library indicates the region with which a given antibody interacts, thereby indicating its epitope.

[0381] Example 13: Epitope Framing of Anti-MS4A6A Antibodies by Cell Binding

[0382] Since MS4A6A is a multi-transmembrane protein, recombinantly produced soluble proteins may not be appropriate reagents for studying Ab: protein interactions. Anti-MS4A6A antibodies were framed using a cell-based variation of the classic sandwich method. To determine, for example, whether antibodies A and B are in the same frame, cells expressing MS4A6A are first incubated with saturating levels of antibody A. After washing with FACS buffer, antibody B labeled with the fluorophore allophycocyanin (APC) is added to the cells and incubated on ice for 15 minutes. Cells are analyzed by a FACS Canto system (BD Biosciences) and gates are set to exclude dead cells. Compare the level of antibody B binding in the presence or absence of antibody A. A reduction or disappearance of binding in the presence of antibody A indicates competition between the two antibodies. Repeat the procedure for other antibodies to frame and frame the entire group.

[0383] Example 14: Downregulation of MS4A6A by anti-MS4A6A antibodies in cell lines

[0384] The ability of anti-MS4A6A antibodies to reduce cell surface and total cellular protein levels of MS4A6A in various cell lines was evaluated as follows: A decrease in MS4A6A protein in each compartment indicates a decrease in MS4A6A activity in the cell.

[0385] Cell lines expressing MS4A6A were incubated with the anti-MS4A6A antibodies of the present disclosure for various time periods, and the remaining MS4A6A levels associated with the cells were then determined by FACS (cell surface) or Western blotting (total protein levels). For FACS analysis, detection of remaining MS4A6A was performed using a non-competitive antibody conjugated directly to allophycocyanin (APC). For Western blot detection, cells were lysed by adding 50uL of lysis buffer (RIPA lysis buffer (ThermoFischerScientific Cat#89900) + 1:100 HALT protease inhibitor cocktail (ThermoFischer Scientific Cat#87786)), and insoluble debris was cleared by centrifugation at 14,000×g for 15 minutes. The soluble fraction was determined with bicinchoninic acid (BCA) reagent for protein quantification. Equal amounts of protein from each sample were loaded onto 4-12% Bis-Tris Plus polyacrylamide gels (ThermoFisher Scientific NW04120) and separated by electrophoresis, and then the proteins in the gel were transferred to polyvinylidene fluoride (PVDF) membranes using iBlot2 (ThermoFisher Scientific IM21001) and transfer layers (ThermoFisherScientific IB24002). The membranes were blocked with 1% bovine serum albumin or 5% skim milk to prevent nonspecific binding. The membranes were then incubated with in-house or commercially available detection antibodies, washed, and incubated with HRP-conjugated secondary antibodies (rabbit, Abcam#205718; mouse, Abcam#205719). Binding was visualized by developing with SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoFisher Scientific #34577) and digitally recorded with iBright FL1000 (ThermoFisher Scientific A32752) or other compatible systems.

[0386] Example 15: Downregulation of MS4A6A by anti-MS4A6A antibodies in primary cell culture

[0387] The ability of the anti-MS4A6A antibodies of the present disclosure to reduce or downregulate cell surface / cellular expression in primary cell cultures was evaluated as follows. Mouse primary cortical neurons were harvested from early postnatal days (days 0-3) and cultured according to standard methods in the art (Maximov et al., (2007), J. Neu. Meth., 161 75-87). The cultured neurons were then incubated with anti-MS4A6A antibodies under various conditions (1-20 ug / mL, 2-48 hours), harvested, and total MS4A6A levels were quantified by FACS (for cell surface levels) or Western blotting (for total protein levels).

[0388] Primary cortical neurons were isolated as follows. Briefly, cells in the cortex, hippocampus or striatum of P0 mouse pups were incubated at 37°C for 7 minutes in a digestion solution containing 6 mg / ml trypsin (Sigma, Cat#T1005-1G), 0.5 mg / ml DNA enzyme (Sigma, Cat#D5025) and (in mM) 137 NaCl, 5 KCl, 7 Na2HPO4 and 25 HEPES-NaOH pH7.2. The isolated cells containing neurons were then washed once with Hank's balanced salt solution (HBS) containing 20% ​​fetal bovine serum (FBS), then washed twice in serum-free HBS, and further isolated by gently aspirating in HBS containing 12 mM MgSO4 and 0.5 mg / ml DNA enzyme. The cell suspension was centrifuged at 160 g for 10 min and then plated on round glass coverslips coated with Matrigel (Collaborative Biomedical Products, Cat# 871-275-0004) in MEM (Invitrogen). The MEM was supplemented with B27 (Invitrogen, Cat#17504-044), glucose, transferrin and 5% fetal bovine serum. For cortical cultures, cell suspensions obtained from the cortex of a single brain were used to coat 12 wells in a 24-well plate. For all cultures, the initial cell density (including glial cells) at the time of coating varied between 1500 and 2500 per square millimeter. When the confluence of glial cells in the culture reached ~40-50% (usually 2 days after coating), 50% of the conditioned medium was replaced with fresh medium containing 4mM Ara-C (Sigma). The culture was maintained in a medium containing 2mM Ara-C at 37°C and 5% CO2 until the experiment was performed (13-18DIV).

[0389] Primary neurons in culture were then incubated with the anti-MS4A6A antibodies of the disclosure for various time periods, and the remaining MS4A6A levels associated with the cells were then determined by FACS (cell surface) or Western blotting (total protein levels), as described above in Example 14.

[0390] Example 16: Intracellular signal transduction triggered by anti-MS4A6A antibodies

[0391] As a membrane-bound molecule, MS4A6A can directly or indirectly participate in the interaction of cells with extracellular signals. Other molecules in the MS4A family have been shown to be components of surface receptor complexes and regulate signal transduction. The topological structure of MS4A6A shows that it has the ability to act as an ion channel by forming a bundled barrel structure across the plasma membrane. Calcium is a common second messenger for such ion channels. The potential of anti-MS4A6A antibodies to induce or increase calcium flux in cell lines and primary cells was studied as follows. Calcium-sensitive dyes such as Fura-2, Indo-1 or Fluo-4 (ThermoFisher Scientific) were added to the cells. The cells were then incubated with anti-MS4A6A antibodies for up to thirty minutes, and the level of fluorescence was monitored by flow cytometry, fluorescence microscopy or fluorescence spectrometer.

[0392] MS4A6A can also directly or indirectly regulate other signal transduction events. Specifically, protein phosphorylation / dephosphorylation is the main mechanism of intracellular signal transduction. Although MS4A6A does not contain a clear protein motif associated with phosphorylation activity, MS4A6A does contain some tyrosine residues in the intracellular C-terminal region. The farthest end of these tyrosine residues is similar to the consensus sequence of half ITAM, which is a motif used by many receptors to engage adapter molecules containing Src homology 2 (SH2) to mediate downstream signal transduction. MS4A6A can use the half ITAM motif to transmit downstream signals. Alternatively, MS4A6A can regulate downstream phosphorylation or dephosphorylation events by interacting with other signal transduction molecules.

[0393] To examine this, cell lines and primary cells expressing MS4A6A were incubated with anti-MS4A6A antibodies. At different treatment time points, cells were harvested and analyzed by Western blotting for their intracellular protein serine / threonine phosphorylation or tyrosine phosphorylation. For Western blot detection, cells were lysed by adding 50uL lysis buffer (RIPA lysis buffer (ThermoFischerScientific Cat#89900) + 1:100 HALT protease inhibitor cocktail (ThermoFischerScientific Cat#87786)) and insoluble debris was removed by centrifugation at 14,000×g for 15 minutes. The soluble fraction was determined with bicinchoninic acid (BCA) reagent for protein quantification. Equal amounts of protein from each sample were loaded onto 4-12% Bis-Tris Plus polyacrylamide gels (ThermoFisherScientific NW04120) and separated by electrophoresis, and then iBlot2 (ThermoFisher ScientificIM21001) and transfer layers (ThermoFisher Scientific IB24002) were used to transfer the proteins in the gel to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 1% bovine serum albumin or 5% skim milk to prevent nonspecific binding. The membranes were then incubated with internal or commercially available detection antibodies for serine / threonine phosphorylation or tyrosine phosphorylation. The membranes were then washed and incubated with HRP-conjugated secondary antibodies (rabbit, Abcam#205718; mouse, Abcam#205719). Binding was visualized by developing with SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoFisher Scientific #34577) and digitally recorded with iBright FL1000 (ThermoFisher A32752) or other compatible systems.

[0394] It is possible that MS4A6A does not directly bind extracellular ligands, but can act on other cellular receptors in receptor protein complexes on the plasma membrane. For example, MS4A6A can form protein complexes with toll-like receptors (TLRs), cytokine receptors, chemokine receptors, scavenger receptors, triggering receptors expressed on myeloid cells 1 or 2, inhibitory receptors (such as CD33, Siglec family or CD200), Fc receptors, or other cell surface receptors that regulate myeloid cell or microglia function. To examine this, cell lines and primary cells expressing MS4A6A are co-engaged with both the cognate ligands of these receptors and anti-MS4A6A antibodies. Downstream signaling events, such as calcium mobilization and protein phosphorylation, are examined as described above. In addition, changes in gene expression are analyzed by RT-PCR or global profiling methods (such as RNASeq or microarrays).

[0395] Example 17: Characterization of the interaction between MS4A6A and binding partners

[0396] Anti-MS4A6A antibodies can be tested for their use in blocking the interaction of MS4A6A with various binding partners. Little is known about which proteins interact with MS4A6A in vivo. To identify such binding partners, MS4A6A is immunoprecipitated from bone marrow cell lines and primary cells. The immunoprecipitated complexes are then subjected to tandem mass spectrometry to identify proteins that co-precipitate with MS4A6A. Once identified, the interaction can be further confirmed by Western blot or fluorescence resonance energy transfer (FRET) analysis. Alternatively, anti-MS4A6A antibodies can be administered to cell lines expressing MS4A6A, and then MS4A6A and potential binding partners can be stained, with the readout being co-localization or fluorescence resonance energy transfer (FRET).

[0397] Example 18: Characterization of the effects of anti-MS4A6A antibodies on myeloid cell metabolism, biology and function

[0398] Using cultures of myeloid cell lines and primary cells (such as, for example, monocyte-derived macrophages and dendritic cells) as model systems, the downstream consequences of engagement of anti-MS4A6A antibodies bound to their targets on myeloid cells, including microglia in the CNS, are examined in detail. These cells are widely used to study the biological properties of myeloid cells because their functions require routine in vitro studies. These functions include, but are not limited to, energy metabolism, cytokine production, phagocytosis, cell surface molecule expression, polarization, migration, and antigen presentation. Briefly, cells expressing MS4A6A are incubated with anti-MS4A6A antibodies in a plated, soluble, or pre-complexed form, which simulates different ways of presenting antibodies to their in vivo cellular targets. Incubation conditions and times depend on the readout to be analyzed, but are generally in the range of from about 1 hour to about 7 days. After incubation, cells are harvested and lysed for mRNA or protein analysis. Cells are also subjected to flow cytometric analysis for cell surface molecule expression.

[0399] For metabolic studies, cells are incubated with CellTiter-Glo (Promega) or other compatible reagents, which can measure intracellular ATP content or other parameters of cell viability. Culture supernatants are harvested and subjected to ELISA or other similar methods to determine the secretion of cytokines...

Claims

1. An isolated antibody that binds to human MS4A6A, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HVR-H1 of SEQ ID NO:14; HVR-H2 of SEQ ID NO:29; and HVR-H3 of SEQ ID NO:43; and The light chain variable region comprises: HVR-L1 of SEQ ID NO:57; HVR-L2 of SEQ ID NO:73; and HVR-L3 of SEQ ID NO:

84.

2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 104, and wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

121.

3. The antibody of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody according to claim 1 or 2, wherein the antibody belongs to the IgG class, the IgM class or the IgA class.

5. The antibody of claim 4, wherein the antibody is of the IgG class and has an IgG1, IgG2 or IgG4 isotype.

6. The antibody of claim 1 or 2, wherein the antibody is an antibody fragment.

7. The antibody of claim 6, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv or scFv fragment.

8. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody of any one of the preceding claims.

9. A vector comprising the nucleic acid according to claim 8.

10. An isolated host cell comprising the vector of claim 9.

11. A method for producing an antibody that binds to human MS4A6A, the method comprising culturing the cell according to claim 10, thereby producing the antibody.

12. The method of claim 11, wherein the method further comprises recovering the antibody produced by the cell.

13. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

14. Use of the antibody of any one of claims 1-7 in the preparation of a medicament for preventing, reducing the risk of, or treating a subject suffering from a disease, disorder, or injury selected from the group consisting of Alzheimer's disease and cognitive impairment.

15. Use of the antibody of any one of claims 1 to 7 in the preparation of a medicament for preventing late-onset Alzheimer's disease, reducing the risk of late-onset Alzheimer's disease, or treating an individual suffering from late-onset Alzheimer's disease.

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