ANTI-MS4A4A ANTIBODIES WITH AMYLOID-BETA THERAPIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Appl. No. 63/380,346, filed October 20, 2022, which is herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 4503_023PC01_SequenceListing_ST26.xml; Size: 62,416 bytes; and Date of Creation: October 16, 2023) is herein incorporated by reference in its entirety.

FIELD OF THE PRESENT DISCLOSURE

[0003] The present disclosure relates to anti-MS4A4A antibodies and therapeutic uses of such antibodies, e.g., in combination with amyloid-beta therapies.

BACKGROUND OF THE PRESENT DISCLOSURE

[0004] The membrane-spanning 4-domain subfamily A (MS4A) gene cluster is present on chromosome 1 Iq 12 and includes eighteen genes. The MS4A gene family encodes membrane proteins typically having tetra-spanning 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 membrane spanning domains are interconnected by one intracellular loop and two extracellular loops with both N- and C- termini residing within the cytosol. Most MS4A proteins share amino acid sequence homology to that of MS4A1 (CD20) (20-30% similarity), with the highest degree of sequence identity occurring in the first three transmembrane domains. The highly conserved motifs within these transmembrane regions across different MS4A proteins suggest that the membrane spanning domains have an important general role in MS4A protein function. The regions of greatest variation between MS4A proteins occur within their N- and C-terminal cytoplasmic domains and the putative second extracellular loop (Ishibashi et al, 2001, Gene, 265:87-93), suggesting that these regions impart unique functional properties.

[0005] Despite this diversity, the MS4A domains possess some shared elements. For instance, one notable feature conserved in MS4A proteins (with the exception of MS4A8B and MS4A12) is the conservation of two cysteine residues in the putative second extracellular loop that may form a disulfide bridge. The N- and C-terminal domains of MS4A proteins are also rich in proline residues, although the functional significance of this remains to be elucidated (Hulett et al, 2001, Genomics, 72: 119-127).

Proline rich regions are, however, commonly involved in various cellular processes such as cytoskeletal rearrangement, initiation of transcription, signaling cascades, and association with SH3 domains as part of an adaptor system to facilitate protein-protein interactions (Kay et al, 2000, FASEB J, 14:231-241).

[0006] The MS4A protein family is relatively uncharacterized functionally, with some important exceptions: MS4A1 (CD20) is expressed exclusively in B lymphocytes, where the protein has a function in signaling by the B cell antigen receptor, and calcium influx. CD20 is the target of immunotherapeutic antibodies used to deplete pathogenic B cells in chronic lymphocytic leukemia, lymphomas, autoimmune diseases, and in solid organ transplantation. MS4A2 (FCERP) is a signaling subunit of the high affinity IgE receptor (FcsRI) and the low affinity IgG receptor (FCERIII) on mast cells, having a key role in hypersensitivity and allergic reactions. MS4A2 is an ITAM-domain protein that amplifies signals through a 4-protein high affinity IgE receptor complex. MS4A3 (Htm4) is expressed on intracellular membranes of lymphoid and myeloid cells, and functions as an adaptor protein in cell cycle regulation.

[0007] While the majority of MS4A family members are uncharacterized, reports suggest MS4A proteins act as chemosensors and chemoreceptors for a variety of exogenous and endogenous ligands, including fatty acids, peptides, and sulfated steroids, and have been implicated in mediating calcium influx, regulating endocytosis, trafficking, and may act as adapters for 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 have been genetically linked to various disorders and diseases, in particular neurodegenerative disorders. For example, genome-wide significance association analyses have identified the MS4A gene cluster, located on chromosome 1 lql2, as one of the most significant Alzheimer’s disease loci. One gene of particular interest identified is MS4A4A (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).

[0009] Other targets have also been identified for the potential treatment of Alzheimer's disease. Evidence indicates that deposition of aggregates containing misfolded amyloid beta peptides is a critical event in Alzheimer's disease pathogenesis. Therefore, preventing or reversing amyloid beta misfolding and/or aggregation has been considered an attractive therapeutic strategy. (Estrada et al, 2007, Curr Top Med Chem, 7: 115-126).

[0010] Amyloid beta can be removed by phagocytosis, which involves both internalization and degradation. Multiple findings in culture, animal models, and humans support the idea that internalization of phagocytosed substrates such as amyloid beta might not be the only rate limiting factor in the removal of misfolded proteins and other debris. Instead, some evidence suggest that degradation of the phagocytosed material plays a crucial role as well. Indeed, once cargo is phagocytosed, microglia need to promptly degrade it to avoid becoming dysfunctional and inflammatory.

[0011] For at least twenty years, intensive efforts have been dedicated to identifying methods of decreasing brain amyloid beta levels (e.g., by decreasing production, antagonizing aggregation, or increasing brain clearance of amyloid beta), but their success has been limited. Accordingly, there is a long standing need for therapies that minimize accumulation of misfolded and/or aggregated amyloid beta.

SUMMARY OF THE PRESENT DISCLOSURE

[0012] Provided herein are methods of using anti-MS4A4A antibodies to enhance amyloid beta (A-beta) therapies and combinations of anti-MS4A4A antibodies and A-beta therapies.

[0013] Accordingly, provided herein is method of preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of Alzheimer’s disease, early Alzheimer’s disease, late onset Alzheimer’s disease, and cognitive impairment, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody (a) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:22; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:23; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:24; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:25; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:26; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:27; (b) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:29; (c) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON; (d) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; (e) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:39; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:40; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:41; an HVR-L2 comprising the amino acid sequence of SEQ ID N0:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON; or (f) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:43.

[0014] Also provided herein is a method of preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of Alzheimer’s disease, early Alzheimer’s disease, late onset Alzheimer’s disease, and cognitive impairment, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:9.

[0015] Also provided herein is a method of preventing, reducing risk, or treating an individual having a disease, disorder, condition, or injury caused by or associated with over expression or increased activity of MS4A4A, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NON; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0016] Also provided herein is a method of preventing, reducing risk, or treating an individual having a macrophage colony-stimulating factor 1 receptor (CSFIRj-deficient disease or disorder, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NON; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON. [0017] Also provided herein is a method of treating early Alzheimer's disease, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NON; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:9.

[0018] Also provided herein is a method of preventing, reducing risk, or treating an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR- H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0019] Also provided herein is a method of improving cognitive function in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR- H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0020] Also provided herein is a method of increasing the removal of non-aggregated toxic amyloid beta in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0021] Also provided herein is a method of slowing down the accumulation of tau protein or tau peptides in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of (1) an antibody that binds to a MS4A4A protein, and (2) an amyloid beta targeting therapeutic, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0022] Also provided herein is a method of reducing the use or dosage of an amyloid beta targeting therapeutic administered to an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount an antibody that binds to a MS4A4A protein, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR- H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON, optionally wherein the method further comprises administering the amyloid beta targeting therapeutic.

[0023] In some aspects of the methods provided herein, the individual has Alzheimer's disease. In some aspects, the individual has early Alzheimer's disease. In some aspects, the individual has late onset Alzheimer's disease

[0024] Also provided herein is a method of reducing an adverse effect of an amyloid beta targeting therapeutic administered to an individual, the method comprising administering to an individual in need thereof a therapeutically effective amount an antibody that binds to a MS4A4A protein, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID N0:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:9, optionally wherein the method further comprises administering the amyloid beta targeting therapeutic.

[0025] Also provided herein is a method of increasing phagocytosis in a cell, the method comprising contacting the cell with an antibody that binds to a MS4A4A protein, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:9. The method can further comprise contacting the cell with an amyloid beta targeting therapeutic. The contacting can be in vitro or can be in an individual. [0026] Also provided herein is a method of promoting lysosomal activity in a cell, the method comprising contacting the cell with an antibody that binds to a MS4A4A protein, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR- H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON. The method can further comprise contacting the cell with an amyloid beta targeting therapeutic. The contacting can be in vitro or can be in an individual.

[0027] Also provided herein is a method of reducing amyloid beta plaque density in a cell, the method comprising contacting the cell with an antibody that binds to a MS4A4A protein, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; an HVR- H2 comprising the amino acid sequence of SEQ ID NO:5; and an HVR-H3 comprising an amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and an HVR-L3 comprising the amino acid sequence of SEQ ID NON. The method can further comprise contacting the cell with an amyloid beta targeting therapeutic. The contacting can be in vitro or can be in an individual.

[0028] In some aspects of the methods provided herein, the amyloid beta targeting therapeutic is an antiamyloid beta antibody. In some aspects, the anti-amyloid beta antibody is selected from the group consisting of lecanemab, donanemab, crenezumab, solanezumab, bapineuzumab, aducanumab, gantenerumab, and combinations thereof. [0029] In some aspects of the methods provided herein, the amyloid beta targeting therapeutic is an amyloid beta aggregation inhibitor. In some aspects, the amyloid beta aggregation inhibitor is selected from the group consisting of ELND-005, tramiprosate, PTI-80, and combinations thereof.

[0030] In some aspects of the methods provided herein, the amyloid beta targeting therapeutic is selected from the group consisting of ACU103, CT1812, ALZT-OP1, blarcamesine, and combinations thereof. [0031] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the antibody that binds to a MS4A4A protein comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

[0032] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein increases amyloid-beta uptake in a cell, optionally wherein the cell is a microglial cell or an induced pluripotent stem cell derived (IPSC-derived) microglia. In some aspects, the antibody that binds to a MS4A4A protein decreases amyloid-beta plaque density in a cell, optionally wherein the cell is a microglial cell or an IPSC-derived microglia. In some aspects, the antibody that binds to a MS4A4A protein increases lysosomal activity in macrophage. In some aspects, the antibody that binds to a MS4A4A protein increases membrane TREM2 in primary human macrophages and/or increases soluble TREM2 (sTREM) levels in primary human macrophages. In some aspects, the antibody that binds to a MS4A4A protein increases ATP levels in primary human macrophages, optionally wherein the increase is in a TREM2 -independent manner. In some aspects, the antibody that binds to a MS4A4A protein increases secreted levels of Sphingosine- 1 -phosphate phosphatase 1 (SPP1) and/or Interleukin- 1 receptor antagonist protein (IL1RN) in primary human macrophages. In some aspects, the antibody that binds to a MS4A4A protein activates microglia. In some aspects, the antibody that binds to a MS4A4A protein increases brain osteopontin levels in cynomolgus monkeys.

[0033] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein is humanized. In some aspects, the antibody that binds to a MS4A4A protein is of the IgG class and has an IgGl, IgG2, or IgG4 isotype. In some aspects, the antibody that binds to a MS4A4A protein is of the IgG class and has an IgGl isotype. In some aspects, the antibody that binds to a MS4A4A protein comprises a modified Fc comprising the N325S and L328F mutations according to EU numbering.

[0034] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein is an antibody fragment. In some aspects, the antibody fragment is a Fab, Fab’, Fab’-SH, F(ab’)2, Fv or scFv fragment.

[0035] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein is a bispecific antibody recognizing the MS4A4A protein and a second antigen. In some aspects, the second antigen is an antigen facilitating transport across the blood-brain-barrier, optionally wherein the antigen facilitating transport across the blood-brain-barrier is selected from the group consisting of 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), LRP8, diphtheria toxin receptor, CRM 197, a llama single domain antibody, TMEM 30(A), a protein transduction domain, TAT, Syn-B, penetratin, a poly-arginine peptide, an angiopeptide, Basigin, Glucose Transporter Type 1 (Glutl), CD98, MfsD2a, and ANG1005. 1 some aspects, the second antigen is a disease-causing agent selected from the group consisting of diseasecausing peptides or proteins or, disease-causing nucleic acids, wherein the disease-causing nucleic acids are antisense GGCCCC (G2C4) repeat-expansion RNA, the disease-causing proteins are selected from the group consisting of amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein Al, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine (PR) repeat peptides. In some aspects, the second antigen is a ligands and/ or a protein expressed on an immune cell, wherein the ligand and/or protein is selected from the group consisting of CD40, 0X40, 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. In some aspects, the second antigen is a protein, lipid, polysaccharide, or glycolipid expressed on one or more tumor cells.

[0036] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein comprises a full length heavy chain comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some aspects, the antibody that binds to a MS4A4A protein comprises a full length light chain comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21. In some aspects, the antibody that binds to a MS4A4A protein comprises a full length heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a full length light chain comprising the amino acid sequence of SEQ ID NO:21.

[0037] In some aspects of the methods provided herein, the antibody that binds to a MS4A4A protein and the amyloid beta targeting therapeutic are administered sequentially. In some aspects, the antibody that binds to a MS4A4A protein is administered prior to the amyloid beta targeting therapeutic. In some aspects, the amyloid beta targeting therapeutic is administered prior to the antibody that binds to a MS4A4A protein. In some aspects, the antibody that binds to a MS4A4A protein and the amyloid beta targeting therapeutic are administered simultaneously. In some aspects, the antibody that binds to a MS4A4A protein and the amyloid beta targeting therapeutic are administered is the same pharmaceutical composition. In some aspects, the antibody that binds to a MS4A4A protein and the amyloid beta targeting therapeutic are administered in separate pharmaceutical compositions.

[0038] Also provided herein is an antibody that binds to a MS4A4A protein for use in any method provided herein. Also provided herein is use of an antibody that binds to a MS4A4A protein in any method provided herein.

[0039] Also provided herein is an amyloid beta targeting therapeutic for use any method provided herein. Also provided herein is use of an amyloid beta targeting therapeutic in any method provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

[0041] FIG. 1 shows the time course of A-beta uptake by iPSC microglia pre-treated with 3 pm 4A-450 NSLF IgGl antibody, control (3 pM NSLF IgGl antibody), or media only as an increase in the area of the pHrodo™ label normalized to cell number and as the integrated intensity (mean intensity/area) per cell number.

[0042] FIG. 2 shows (i) the A-beta uptake at a 2-hour time point quantified as an increase in the area of the pHrodo™ label normalized to cell number and as the integrated intensity (mean intensity/area) per cell number for iPSC microglia pre-treated with 4A-450 NSLF IgGl antibody, control (NSLF IgGl antibody), or with media only and (ii) images of cells after incubation with pHrodo™-labeled A-betal-42.

[0043] Fig. 3A shows the time course of uptake of a low concentration of A-beta by iPSC microglia pretreated with 0.3 pM 4A-450 NSLF IgGl antibody, control (0.3 pM of NSLF IgGl antibody), or media only as an increase in the area of the pHrodo™ label normalized to cell number and as the integrated intensity (mean intensity/area) per cell number.

[0044] Fig. 3B shows the time course of uptake of a low concentration of A-beta by iPSC microglia pretreated with 0.3 pM 4A-450 NSLF IgGl antibody and 10 pM CSF1R inhibitor (BLZ), control (0.3 pM NSLF IgGl antibody and 10 pM BLZ), or media and 10 pM BLZ only as an increase in the area of the pHrodo™ label normalized to cell number and as the integrated intensity (mean intensity/area) per cell number.

[0045] Fig. 3C shows the time course of uptake of a high concentration of A-beta by iPSC microglia pretreated with 3 pM 4A-450 NSLF IgGl antibody and 10 pM BLZ, control (3 pM NSLF IgGl antibody and 10 pM BLZ), or media and 10 pM BLZ only as an increase in the area of the pHrodo™ label normalized to cell number and as the integrated intensity (mean intensity/area) per cell number. [0046] Fig. 4 shows increased GCase activity in macrophages after treatment with 4A-450 NSLF antibody as compared to treatment with control (NSLF IgGl antibody).

[0047] Fig. 5A shows images taken over a period of 120 minutes of unfixed macrophages treated with 4A-450 NSLF antibody or control (NSLF IgGl antibody).

[0048] Fig. 5B focuses on the data at 1.5 hours.

[0049] Fig. 5C quantifies the change in Gcase signal over a period of 120 minutes in unfixed macrophages treated with 4A-450 NSLF antibody, control (NSLF IgGl antibody), progranulin, and conduritol-P-epoxide (CBE).

[0050] Fig. 6 shows the effect of 4A-450 NSLF antibody and control (NSLF IgGl antibody) on A-beta plaque density in human iPSC microglia treated with A-beta.

DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0051] The present disclosure relates to methods of using anti-MS4A4A antibodies (e.g., monoclonal antibodies) and combinations of anti-MS4A4A antibodies (e.g., monoclonal antibodies) with A-beta targeting therapeutics.

[0052] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

I. Definitions

[0053] The terms "MS4A4A" or “MS4A4A polypeptide” are used interchangeably herein refer herein to any native MS4A4A from any vertebrate source, including mammals such as primates (e.g., humans and cynos) and rodents (e.g., mice and rats), unless otherwise indicated. In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses "full-length," unprocessed MS4A4A as well as any form of MS4A4A that results from processing in the cell. In some aspects, the MS4A4A is human MS4A4A. In some aspects, the amino acid sequence of an exemplary MS4A4A is Uniprot Accession No. Q96JQ5 as of December 1, 2001. In some aspects, the amino acid sequence of an exemplary human MS4A4A is SEQ ID NO: 1.

[0054] The terms "anti-MS4A4A antibody," an "antibody that binds to MS4A4A," and "antibody that specifically binds MS4A4A" refer to an antibody that is capable of binding MS4A4A with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting MS4A4A. In one embodiment, the extent of binding of an anti-MS4A4A antibody to an unrelated, non-MS4A4A polypeptide is less than about 10% of the binding of the antibody to MS4A4A as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to MS4A4A has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., IO’ ⁸ M or less, e.g. from 10" ⁸ M to 10" ¹³ M, e.g., from 10" ⁹ M to 10" ¹³ M). In certain aspects, an anti-MS4A4A antibody binds to an epitope of MS4A4A that is conserved among MS4A4A from different species.

[0055] The terms "amyloid beta" or "A-beta" are used interchangeably herein and refer to any native amyloid beta from any vertebrate source, including mammals such as primates (e.g., humans and cynos) and rodents (e.g., mice and rats), unless otherwise indicated. These terms can refer to amyloid beta present as monomers, oligomers, in aggregates, and/or in plaques.

[0056] “Early Alzheimer’s Disease” or “early AD” as used herein refers to patients who have mild cognitive impairment (MCI) due to AD and are positive for an AD biomarker (e.g., amyloid beta positivity), or have mild AD dementia (with unknown or positive amyloid beta). In some aspects, early AD patients have MCI due to AD or mild AD dementia, with evidence of amyloid beta, tau, or neurodegeneration .

[0057] The term “Api-40 monomer” or “Api-42 monomer” as used herein refers to the direct product of the enzymatic cleavage, i.e., aspartic protease activity, by P-secretase and y-secretase on the amyloid protein precursor (APP) in a cell-free or cellular environment. Cleavage of APP by P-secretase generates the Ap species beginning at Asp 1 (numbering as to Ap peptide sequence after cleavage), while y-secretase liberate the C-terminus of Ap, predominantly either at residues 40 or 42.

[0058] The term "amyloid beta aggregation inhibitor" means any agent that blocks or interferes with the aggregation of amyloid beta. Agents that block or interfere with aggregation of amyloid beta include small organic molecules, peptides and peptidomimetics, and nanoparticles. In some aspects, an amyloid beta aggregation inhibitor binds to amyloid beta.

[0059] The terms "anti-A-beta antibody," an "antibody that binds to A-beta," and "antibody that specifically binds A-beta" refer to an antibody that is capable of binding A-beta with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting A-beta. In one embodiment, the extent of binding of an anti-A-beta antibody to an unrelated, non-A-beta polypeptide is less than about 10% of the binding of the antibody to A-beta as measured, e.g. , by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to A-beta has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10" ⁸ M or less, e.g. from 10" ⁸ M to 10’ ¹³ M, e.g., from 10’ ⁹ M to 10’ ¹³ M). In certain aspects, an anti-A-beta antibody binds to an epitope of A- beta that is conserved among A-beta from different species.

[0060] With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of about any of 10" ⁴ M or lower, 10" ⁵ M or lower, 10" ⁶ M or lower, 10" ⁷ M or lower, 10" ⁸ M or lower, 10" ⁹ M or lower, IO" ¹⁰ M or lower, 10" ¹¹ M or lower, 10" ¹² M or lower or a KD in the range of 10" ⁴ M to 10" ⁶ M or 10" ⁶ M to IO" ¹⁰ M or 10" ⁷ M to 10" ⁹ M. As will be appreciated by the skilled artisan, affinity and KD values are inversely related. A high affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0061] The term “immunoglobulin” (Ig) is used interchangeably with "antibody' herein. The term “antibody” herein is used in the broadest sense and specially 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.

[0062] “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 among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0063] For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0064] The light chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“ ”), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (“a”), delta (“8”), epsilon (“e”), gamma (“y”), and mu (“p”), respectively. The y and a classes are further divided into subclasses (isotypes) on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al., Cellular and Molecular Immunology, 4 ^th ed. (W.B. Saunders Co., 2000).

[0065] The “variable region” or “variable domain” of an antibody, such as an anti-MS4A4A antibody of the present disclosure, refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL,” respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

[0066] The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, such as anti-MS4A4A 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 across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (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 the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody -dependent-cellular toxicity.

[0067] The term “monoclonal antibody ” as used herein refers to an antibody, such as a monoclonal anti- MS4A4A antibody of the present disclosure, obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations, etc.) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0068] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody, such as an anti-MS4A4A antibody of the present disclosure, in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those with heavy and light chains including 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 intact antibody may have one or more effector functions.

[0069] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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. Patent 5641870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0070] Papain digestion of antibodies, such as anti-MS4A4A antibodies of the present disclosure, produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire light chain along with the variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigenbinding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain 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 originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0071] The Fc fragment comprises the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0072] “Functional fragments” of antibodies, such as anti-MS4A4A antibodies of the present disclosure, comprise 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 which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. [0073] The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the variable domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0074] As used herein, a “chimeric antibody” refers to an antibody (immunoglobulin), such as a chimeric anti-MS4A4A antibody of the present disclosure, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, “humanized antibody” is used a subset of “chimeric antibodies.”

[0075] “Humanized” forms of non-human (e.g., murine) antibodies, such as humanized forms of anti- MS4A4A antibodies of the present disclosure, are chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of each HVR (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of each FR correspond to those 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.

[0076] A “human antibody” is an antibody, such as an anti-MS4A4A antibody of the present disclosure, possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phagedisplay libraries and yeast-display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice as well as generated via a human B-cell hybridoma technology.

[0077] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody-variable domain, such as that of an anti-MS4A4A antibody of the present disclosure, that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display 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 came lid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain.

[0078] A number of HVR delineations are in use and are encompassed herein. In some aspects, the HVRs may be Kabat complementarity-determining regions (CDRs) based on sequence variability and are the most commonly used (Kabat et al., supra). In some aspects, the HVRs may be Chothia CDRs. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some aspects, the HVRs may be AbM HVRs. The AbM HVRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody-modeling software. In some aspects, the HVRs may be “contact” HVRs. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact

LI L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)

Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101

[0079] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (Hl), 50-65 or 49-65 (a preferred embodiment) (H2), and 93- 102, 94-102, or 95-102 (H3) in the VH. The variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-HVR definitions.

[0080] “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

[0081] An “acceptor human framework” as used herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An 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 aspects, the number of pre-existing amino acid changes are 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. Where pre-existing amino acid changes are present in a VH, preferable those changes occur at only three, two, or one of positions 71H, 73H and 78H; for instance, the amino acid residues at those positions may by 71 A, 73T and/or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0082] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH framework sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0083] An “amino-acid modification” at a specified position, e.g., of an anti-MS4A4A antibody of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. “Adjacent” to a specified residue means insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue. A preferred amino acid modification herein is a substitution.

[0084] An “affinity-matured” antibody, such as an affinity matured anti-MS4A4A antibody of the present disclosure, is one with one or more alterations in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In one embodiment, an 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/T echnology 10:779-783 (1992) describes affinity maturation by VH- and VL- domain shuffling. Random mutagenesis of HVR and/or framework residues is described by, for example: 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).

[0085] “Fv” is the minimum antibody fragment which comprises a complete antigen-recognition and - binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute 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 comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0086] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0087] 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.

[0088] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native -sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl -terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4.

[0089] 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 a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0090] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0091] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (“ITAM”) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (“ITIM”) in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. FcRs can also increase the serum half-life of antibodies.

[0092] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (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.

[0093] The term “compete” when used in the context of antibodies (e.g., neutralizing antibodies) that compete for the same epitope means competition between antibody 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. , MS4A4A or a fragment thereof). Numerous types of competitive binding assays can be used to determine if antibody competes with another, 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 labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antibody and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually the test antibody is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, 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 near 100%.

[0094] As used herein, an “interaction” between a MS4A4A polypeptide and a second polypeptide encompasses, without limitation, protein-protein interaction, a physical interaction, a chemical interaction, binding, covalent binding, and ionic binding. As used herein, an antibody “inhibits interaction” between two polypeptides when the antibody disrupts, reduces, or completely eliminates an interaction between the two polypeptides. An antibody of the present disclosure, thereof, “inhibits interaction” between two polypeptides when the antibody thereof binds to one of the two polypeptides. In some aspects, the interaction can be inhibited by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and/or near 100%.

[0095] The term “epitope” includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Most often, epitopes reside on polypeptides, but in some instances, can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of polypeptides and/or macromolecules.

[0096] An “agonist” antibody or an “activating” antibody is an antibody that induces (e.g., increases) one or more activities or functions of the antigen after the antibody binds the antigen.

[0097] An “antagonist” antibody or a “blocking” antibody or an “inhibitory” antibody is an antibody that reduces, inhibits, and/or eliminates (e.g., decreases) antigen binding to one or more ligand after the antibody binds the antigen, and/or that reduces, inhibits, and/or eliminates (e.g. , decreases) one or more activities or functions of the antigen after the antibody binds the antigen. In some aspects, antagonist antibodies, or blocking antibodies, or inhibitory antibodies substantially or completely inhibit antigen binding to one or more ligand and/or one or more activities or functions of the antigen.

[0098] An “isolated” antibody, such as an isolated anti-MS4A4A antibody of the present disclosure, is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly). Preferably, the isolated antibody is free of association with all other contaminant components from its production environment. Contaminant components from its production environment, such as those resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred aspects, the antibody will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some aspects, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant T-cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0099] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide (s) of the present disclosure.

[0100] The terms “membrane TREM2,” “plasma membrane TREM2,” and “cell surface TREM2,” as used herein, are used interchangeably.

[0101] “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. [0102] As used herein, the term “preventing” includes providing prophylaxis with respect to occurrence or recurrence of a particular disease, disorder, or condition in an individual. An individual may be predisposed to, susceptible to a particular disease, disorder, or condition, or at risk of developing such a disease, disorder, or condition, but has not yet been diagnosed with the disease, disorder, or condition.

[0103] As used herein, an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.

[0104] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated,” for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0105] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. An effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the 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 given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0106] An “individual” for purposes of treatment, prevention, or reduction of risk refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some aspects, the individual is human.

[0107] As used herein, administration “in conjunction” or “in combination” 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 administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration. In some aspects, administration in conjunction is administration as a part of the same treatment regimen.

[0108] As provided herein, the combination of an anti-MS4A4A antibody and an A-beta targeting therapy can provide “synergy” and prove “synergistic,” i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can include a significantly reduced effective dose for the combination of the two active ingredients as compared to the effective dose of each active ingredient when administered separately. A synergistic effect can also be an effect that cannot be achieved by administration of any of the active ingredients as single agents. A synergistic effect can be attained, for example, when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered serially, by alternation, or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially.

[0109] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se.

[0110] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise. For example, reference to an “antibody” is a reference to from one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[OHl] It is understood that aspects of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects.

II. Use of Anti-MS4A4A Antibodies, e.g., in Combination with A-Beta Targeting Therapeutics [0112] Genome wide association studies have identified various members of the MS4A family are associated with Alzheimer’s disease. These are MS4A2, MS4A3, MS4A4A, MS4A4E, MS4A6A, and MS4A6E. The associated SNPs are found in the 3' UTR of MS4A6A (rs610932) and the intergenic region between MS4A4E and MS4A6A (rs670139). There are three SNPs in the MS4A gene cluster that have been associated with an increased risk of late-onset Alzheimer’s disease. These include 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). Additionally, MS4A4A locus SNPs (rs2304933 and rs2304935) associated with higher levels of MS4A4A and increased Alzheimer’s disease risk, including late-onset Alzheimer’s disease (LOAD) (Allen et al, 2012, Neurology, 79:221-228).

[0113] As disclosed herein, anti-MS4A4A antibodies can be used, for example, to increase phagocytosis of A-beta, promote lysosomal activity, and/or decrease amyloid beta plaque density. In some aspects, the anti-MS4A4A antibodies are used in combination with A-beta targeting therapies. In some aspects, the anti-MS4A4A antibodies are administered to a subject before or after treatment with A-beta targeting therapies. In some aspects, increasing phagocytosis of A-beta, promoting lysosomal activity, and/or decreasing amyloid beta plaque density can be performed in vitro. In some aspects, administration of an anti-MS4A4A antibody to a subject increases phagocytosis of amyloid beta, promotes lysosomal activity, and/or decreases amyloid beta plaque in the subject. In some aspects, administration of an anti-MS4A4A antibody and an A-beta targeting therapy to a subject increases phagocytosis of amyloid beta, promotes lysosomal activity, and/or decreases amyloid beta plaque in the subject. In some aspects, the increasing phagocytosis, promoting lysosomal activity, and/or decreasing amyloid beta plaque density can be performed in a subject, e.g., for preventing, reducing risk, or treating diseases and disorders.

[0114] Some aspects of the present disclosure relate to a method of increasing phagocytosis in a cell. In some aspects, the present disclosure relates to a method of increasing phagocytosis of A-beta in a cell. The method can comprise contacting the cell with an anti-MS4A4A antibody, and optionally further with an A-beta targeting therapeutic. In some aspects, the phagocytosis in the cell is increased by about 5% to about 95%. In some aspects, the phagocytosis in the cell is increased by about 10% to about 90%. In some aspects, the phagocytosis in the cell is increased by about 20% to about 80%. In some aspects, the phagocytosis in the cell is increased by about 30% to about 70%. In some aspects, the phagocytosis in the cell is increased by about 30% to about 60%. In some aspects, the phagocytosis in the cell is increased by about 40% to about 50%. In some aspects, the phagocytosis in the cell is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some aspects, the phagocytosis in the cell is increased by about 1.5 fold. In some aspects, the phagocytosis in the cell is increased by about 2 fold. In some aspects, the phagocytosis of A-beta in the cell is increased by about 1.5 fold. In some aspects, the phagocytosis of A-beta in the cell is increased by about 2 fold. [0115] In some aspects, the present disclosure relates to a method of promoting lysosomal activity in a cell. The method can comprise contacting the cell with an anti-MS4A4A antibody, and optionally further with an A-beta targeting therapeutic. In some aspects, the lysosomal activity in the cell is increased by about 5% to about 95%. In some aspects, the lysosomal activity in the cell is increased by about 10% to about 90%. In some aspects, the lysosomal activity in the cell is increased by about 20% to about 80%. In some aspects, the lysosomal activity in the cell is increased by about 30% to about 70%. In some aspects, the lysosomal activity in the cell is increased by about 30% to about 60%. In some aspects, the lysosomal activity in the cell is increased by about 40% to about 50%. In some aspects, the lysosomal activity in the cell is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some aspects, the lysosomal activity in the cell is increased by about 1.5 fold. In some aspects, the lysosomal activity in the cell is increased by about 2 fold. In some aspects, the lysosomal activity in the cell is increased by about 3 fold.

[0116] In some aspects, the present disclosure relates to a method of promoting decrease of amyloid beta plaque density in a cell. The method can comprise contacting the cell with an anti-MS4A4A antibody, and optionally further with an A-beta targeting therapeutic. In some aspects, the amyloid beta plaque density is reduced by about 5% to about 95%. In some aspects, the amyloid beta plaque density is reduced by about 10% to about 90%. In some aspects, the amyloid beta plaque density is reduced by about 15% to about 85%. In some aspects, the amyloid beta plaque density is reduced by about 20% to about 80%. In some aspects, the amyloid beta plaque density is reduced by about 30% to about 70%. In some aspects, the amyloid beta plaque density is reduced by about 40% to about 60%. In some aspects, the amyloid beta plaque density is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. [0117] In some aspects, a combination of an anti-MS4A4A antibody and an A-beta targeting therapeutic is effective for preventing, reducing risk, or treating diseases and disorders. In some aspects, a combination of an anti-MS4A4A antibody and an A-beta targeting therapeutic is effective at preventing, reducing risk, or treating Alzheimer’s disease, early Alzheimer's disease, and cognitive impairment.

[0118] The methods provided herein find use in preventing, reducing risk, or treating an individual having a neurodegenerative disease, disorder, or condition. In some aspects, the present disclosure provides a method for preventing, reducing risk, or treating an individual having a neurodegenerative disorder, the method comprising administering to the individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic.

[0119] In some aspects, the present disclosure provides a method for preventing, reducing the risk, or treating an individual having Alzheimer’s disease, the method comprising administering to the individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some embodiments, the present disclosure provides a method for preventing, reducing the risk, or treating an individual having early Alzheimer’s disease, the method comprising administering to the individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some embodiments, the present disclosure provides a method for preventing, reducing the risk, or treating an individual having late onset Alzheimer’s disease, the method comprising administering to the individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic.

[0120] In some aspects, the present disclosure provides a method for preventing, reducing the risk, or treating an individual having mild cognitive impairment, the method comprising administering to the individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic.

[0121] In some aspects, the present disclosure provides a method for preventing, reducing risk, or treating an individual having a disease, disorder, or condition associated with over expression or increased activity of MS4A4A, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic.

[0122] In some aspects, the present disclosure provides a method for preventing, reducing the risk, or treating an individual having a CSFlR-deficient disease or disorder, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some aspects, the CSFlR-deficient disease or disorder is adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) or hereditary diffuse leukoencephalopathy with spheroids (HDLS).

[0123] In some embodiments, the present disclosure provides a method for preventing, reducing risk, or treating an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic.

[0124] In some embodiments, the present disclosure provides a method for improving cognitive function in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some embodiments, the present disclosure provides a method for improving cognitive function in an individual with Alzheimer’s disease, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some aspects, the individual has early Alzheimer’s disease or late onset Alzheimer’s disease.

[0125] In some embodiments, the present disclosure provides a method for removing amyloid beta plaques in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some embodiments, the present disclosure provides a method for removing amyloid beta plaques in an individual with Alzheimer’s disease, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some aspects, the individual has early Alzheimer’s disease or late onset Alzheimer’s disease.

[0126] In some embodiments, the present disclosure provides a method for increasing the removal of nonaggregated toxic amyloid beta in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some embodiments, the present disclosure provides a method for increasing the removal of nonaggregated toxic amyloid beta in an individual with Alzheimer’s disease, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some aspects, the individual has early Alzheimer’s disease or late onset Alzheimer’s disease.

[0127] In some embodiments, the present disclosure provides a method for slowing down the accumulation of tau protein or tau peptides in an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic. In some embodiments, the present disclosure provides a method for slowing down the accumulation of tau protein or tau peptides in an individual with Alzheimer’s disease, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti- MS4A4A antibody and an amyloid beta targeting therapeutic. In some aspects, the individual has early Alzheimer’s disease or late onset Alzheimer’s disease. [0128] In some embodiments, the present disclosure provides a method for reducing the use or dosage of an amyloid beta targeting therapeutic administered to an individual having a disease, disorder, or condition associated with an accumulation of amyloid beta, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody. In some embodiments, the present disclosure provides a method for reducing the use or dosage of an amyloid beta targeting therapeutic administered to an individual with Alzheimer’s disease, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody. In some aspects, the individual has early Alzheimer’s disease or late onset Alzheimer’s disease.

[0129] In some embodiments, the present disclosure provides a method for removing amyloid beta plaques and reducing adverse effects of an amyloid beta targeting therapeutic administered to an individual, the method comprising administering to the individual in need thereof a therapeutically effective amount of an anti-MS4A4A antibody.

[0130] Other aspects of the present disclosure relate to a method of preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer’s disease, early Alzheimer's disease, late onset Alzheimer's disease, mild cognitive impairment, vascular dementia, vascular dementia, seizures, retinal dystrophy, a traumatic brain injury, a spinal cord injury, long-term depression, atherosclerotic vascular diseases, undesirable symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington’s disease, taupathy disease, 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, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatous disorders, Sarcoidosis, diseases of aging, age related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disorders, arthritis, multiple sclerosis, metabolic disorder, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, an injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age- related brain atrophy, age-associated traits, including without limitation inflammation, neuronal loss, and cognitive deficits, such as cognitive deficits in the absence of known brain disease, including cognitive deficits of the frontal cerebral cortex of an older individual and, one or more undesirable symptoms of normal aging, comprising administering to the individual a therapeutically effective amount of an anti- MS4A4A antibody and an amyloid beta targeting therapeutic.

[0131] Other aspects of the present disclosure relate to a combination of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic for use in preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer’s disease, early Alzheimer's disease, late onset Alzheimer's disease, vascular dementia, seizures, retinal dystrophy, a traumatic brain injury, a spinal cord injury, long-term depression, atherosclerotic vascular diseases, undesirable symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington’s disease, taupathy disease, 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, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, age related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disorders, arthritis, multiple sclerosis, metabolic disorder, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, an injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-associated traits, including without limitation inflammation, neuronal loss, and cognitive deficits, such as cognitive deficits in the absence of known brain disease, including cognitive deficits of the frontal cerebral cortex of older individual, and one or more undesirable symptoms of normal aging.

[0132] Other aspects of the present disclosure relate to use of an anti-MS4A4A antibody in the manufacture of a medicament for use in combination with an amyloid beta targeting therapeutic for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer’s disease, late-onset Alzheimer’s disease, early Alzheimer's disease, mild cognitive impairment, vascular dementia, seizures, retinal dystrophy, a traumatic brain injury, a spinal cord injury, long-term depression, atherosclerotic vascular diseases, undesirable symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington’s disease, taupathy disease, 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, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, age related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disorders, arthritis, multiple sclerosis, metabolic disorder, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, an injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-associated traits, including without limitations inflammation, neuronal loss, and cognitive deficits, such as cognitive deficits in the absence of known brain disease, including cognitive deficits of the frontal cerebral cortex of older individual and one or more undesirable symptoms of normal aging. Other aspects of the present disclosure relate to use of an amyloid beta targeting therapeutic in the manufacture of a medicament for use in combination with an anti-MS4A4A antibody for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, Alzheimer’s disease, late-onset Alzheimer’s disease, early Alzheimer's disease, mild cognitive impairment, vascular dementia, seizures, retinal dystrophy, a traumatic brain injury, a spinal cord injury, long-term depression, atherosclerotic vascular diseases, undesirable symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington’s disease, taupathy disease, 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, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, age related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disorders, arthritis, multiple sclerosis, metabolic disorder, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, an injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age- related cognitive impairment, age-related brain atrophy, age-associated traits, including without limitations inflammation, neuronal loss, and cognitive deficits, such as cognitive deficits in the absence of known brain disease, including cognitive deficits of the frontal cerebral cortex of older individual and one or more undesirable symptoms of normal aging.

[0133] Other aspects of the present disclosure relate to a method of preventing, reducing risk, or treating an individual 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, early Alzheimer's disease, mild cognitive impairment, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, a 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 individual a therapeutically effective amount of the anti-MS4A4A antibody and an amyloid beta targeting therapeutic. Other aspects of the present disclosure relate to the combination of an anti-MS4A4A antibody and an amyloid beta targeting therapeutic for use in preventing, reducing risk, or treating an individual 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, early Alzheimer's disease, mild cognitive impairment, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, a 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. [0134] Other aspects of the present disclosure relate to use of an anti-MS4A4A in the manufacture of a medicament for use in combination with an amyloid beta targeting therapeutic for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, progressive supranuclear palsy, Alzheimer’s disease, early Alzheimer's disease, late onset Alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, a 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.

[0135] Other aspects of the present disclosure relate to use of an amyloid beta targeting therapeutic in the manufacture of a medicament for use in combination with an anti-MS4A4A antibody for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of frontotemporal dementia, progressive supranuclear palsy, Alzheimer’s disease, early Alzheimer's disease, late onset Alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, a 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. In some aspects, a subject or individual is a mammal. Mammals include, without limitation, domesticated animals (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 aspects, the subject or individual is a human.

[0136] An anti-MS4A4A antibody and/or an A-beta targeting therapeutic can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intralesional administration, intracerobrospinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusions include intramuscular, intravenous administration as a bolus or by continuous infusion over a period of time, intraarterial, intra-articular, intraperitoneal, or subcutaneous administration. In some aspects, the administration is intravenous administration. In some aspects, the administration is subcutaneous. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0137] Anti-MS4A4A antibodies and A-beta targeting therapeutics would be formulated, dosed, and administered (together in a single pharmaceutical composition or individually in separate pharmaceutical compositions) in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0138] For the prevention or treatment of disease, the appropriate dosage of an anti-MS4A4A antibody, optionally in combination with an A-beta targeting therapeutic will depend on the type of disease to be treated, the type of antibody (or antibodies), the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody and/or A-beta targeting therapeutic, and the discretion of the attending physician. The anti-MS4A4A antibody, optionally in combination with an A-beta targeting therapeutic, is suitably administered to the patient at one time or over a series of treatments.

[0139] The dosage, frequency and mode of administration of each component of the combination (i.e., the anti-MS4A4A antibody and the A-beta targeting therapeutic) can be controlled independently.

III. Anti-MS4A4A Antibodies

[0140] Uses of anti-MS4A4A antibodies, optionally in combination with A-beta targeting therapeutics, e.g., for treatment of MS4A4A-associated disorders and/or A-beta-associated disorders are provided herein. Exemplary anti-MS4A4A antibodies are disclosed in WO 2019/152715 and WO 2021/1022083, each of which is herein incorporated by reference in its entirely.

[0141] In one aspect, an anti-MS4A4A antibody (e.g., monoclonal antibody) binds to an epitope within a MS4A4A protein. MS4A4A proteins include, without limitation, a mammalian MS4A4A protein, human MS4A4A protein, mouse MS4A4A protein, and cynomolgus MS4A4A protein. MS4A4A proteins of the present disclosure include naturally -occurring variants of MS4A4A.

[0142] Human MS4A4A is a 239-amino acid protein that encodes a membrane glycoprotein. The amino acid sequence of human MS4A4A is set forth in SEQ ID NO: 1: MHQTYSRHCRPEESTFSAAMTTMQGMEQAMPGAGPGVPQLGNMAVIHSHLWKGLQEKFLK GEP KVLGVVQILTALMSLSMGITMMCMASNTYGSNPISVYIGYTIWGSVMFIISGSLSIAAGI RTTKGLV RGSLGMNITSSVLAASGILINTFSLAFYSFHHPYCNYYGNSNNCHGTMSILMGLDGMVLL LSVLEF CIAVSLSAFGCKVLCCTPGGVVLILPSHSHMAETASPTPLNEV

[0143] Additionally, the amino acid sequence of mouse MS4A4A is set forth in SEQ ID NO:2: MLVIQGTEQSALEAGYGAQQNGQPLYVNSHSWKRMTEKFLKGEPKILGIVQIVIAIMNLS IGIMMII ATVSTGEIPPSSVYIGYPIWGSLMFIISGSFSIVAGRRTTKGLVRSSLGLNITSSVFAFS GIVISSLSPGI YSFHVYYCTYRGSSEGCHMTLSILMGLDIVVVVLSVLEFCIGVSLSAFGCRVMCCNPGGV MIIMPS NPTKAETANPVTLQSGLMPPEHQERNVPENMH

[0144] Additionally, the amino acid sequence of cynomolgus (cyno) MS4A4A is set forth in SEQ ID NO:3:

[0145] HQTYRRHCRPEESTFSAAMTTMQGMEQATPGAGPGVPQLGNMAVVHSHLWKGLQEKFL KGEPKVLGVVQILIALMSLSMGITMMCVAFSAYGHYPISVYIGYTIWGSVMFIISGSLSI AAGIRTTK GLVRGSLGMNITSSVLAVSAILINTISLTIYSFYHRYCNYYGNPNNCHGTVSILMGMDGM VLLLSV LEFCIAVSLSAFGCKAICCTPGGVVLIIPSNSHMAEAAPLTPLNEV

[0146] In some aspects, MS4A4A is expressed in a cell. In some aspects, MS4A4A is expressed in myeloid cells. In some aspects, MS4A4A is expressed in brain cells. In some aspects, MS4A4A is expressed in astrocytes, including without limitation mature astrocytes. In some aspects, MS4A4A is expressed in oligodendrocytes. In some aspects, MS4A4A is expressed in microglial cells. In some aspects, MS4A4A is expressed in immune cells, including without limitation, macrophages, eosinophils, mast cells, dendritic cells, natural killer cells, neutrophils, and T cells. In some embodiment, MS4A4A is expressed in olfactory cells. In some aspects, MS4A4A is expressed on the cell surface.

[0147] MS4A4A proteins of the present disclosure include several domains, including without limitation, a cytoplasmic domain (amino acid residues 1-64 of human MS4A4A; see SEQ ID NO: 1); a transmembrane domain (amino acid residues 65-85 of human MS4A4A); an extracellular domain (extracellular domain 1; ECL1), corresponding to amino acid residues 86-98 of human MS4A4A; a transmembrane domain (amino acid residues 99-119 of human MS4A4A); a cytoplasmic domain (amino acid residues 120-137 of human MS4A4A); a transmembrane domain (amino acid residues 138-158 of human MS4A4A); an extracellular domain (extracellular domain 2; ECL2), corresponding to amino acid residues 159-179; a transmembrane domain (amino acid residues 180-200 of human MS4A4A); and a cytoplasmic domain (amino acid residues 201-239 of human MS4A4A). Additionally, MS4A4A proteins of the present disclosure are expressed in a number of tissues and cells, including without limitation, the brain, neurons, glial cells, endothelial cells, perivascular cells, pericytes, etc.

MS4A4A in macrophage and microglial cell function

[0148] Macrophages and myeloid cells of the central nervous system (CNS), such as microglia, are inherently plastic in their phenotype and function. Macrophages in vitro can be divided into Ml macrophages and M2 macrophages, which have differing phagocytic and inflammatory potentials, phenotypes, and activities. For example, in peripheral organs, macrophages having an Ml phenotype are considered to have pro-inflammatory and anti-microbial phenotype and function, while macrophages having an M2 phenotype are considered to be in a more homeostatic state, having an anti-inflammatory phenotype and function.

[0149] Microglia associated with healthy, homeostatic conditions express more M2 markers on their cell surface (e.g., CD200R, CD163 and CD115) compared to that of Ml markers (e g., CD16, MHC Class II, CD86) (Ginhoux and Prinz, 2015, Cold Spring Harb Perspect Biol, 7:a020537). However, disease associated microglia (DAM) in both mouse models of Alzheimer’s disease and in human Alzheimer’s disease are in a proinflammatory or activated state. Disease associated microglia in proinflammatory or activated states are considered beneficial by playing an active role in reducing the pathology associated with Alzheimer’s disease and other neurodegenerative disorders. [0150] MS4A4A expression is elevated in M2 macrophages in vitro, and it has been suggested that MS4A4A is a novel cell surface marker for M2 macrophages. Additionally, MS4A4A has also been shown to regulate cell surface transport of cKit on mast cells, suggesting a role of MS4A4A in modulating mast cell degranulation and survival (Cruse et al, 2015, Molecular Biol Cell, 26: 1711-1727). Taken together, these reported findings suggest that targeting MS4A4A may affect the recycling, expression, and/or degradation of various macrophage cell surface receptors associated with Ml and M2 macrophage phenotypes, thus affecting their functions and activities.

[0151] Additionally, anti-MS4A4A antibodies of the present disclosure can increase mRNA levels of microglia activation markers IL1RN and SPP1.

MS4A4A and TREM2 expression

[0152] Neurodegenerative diseases are characterized, in part, by defective immune function in the central nervous system (CNS). For example, a decrease in viability and function in the CNS myeloid cell compartment, including but not restricted to microglia, is thought to contribute to susceptibility to neurodegenerative disorders, such as Alzheimer’s disease. Pharmacological intervention that enhances viability and/or function of myeloid cells would provide effective treatment to ameliorate the onset, severity, or progression of such neurodegenerative diseases and disorders.

[0153] Triggering receptor expressed on myeloid cells-2 (TREM2) is an immunoglobulin-like receptor that is expressed primarily on myeloid cells, such as macrophages, dendritic cells, monocytes, Langerhans cells of skin, Kupffer cells, osteoclasts, and microglia. TREM2 is highly expressed on microglia and infiltrating macrophages in the CNS during experimental autoimmune encephalomyelitis and Alzheimer’s disease (Piccio et al, 2007, Eur J Immunol, 37: 1290-1301; Wang, 2015, Cell, 160: 1061-1071). The TREM2 pathway is considered a key modulator of CNS myeloid cell viability and function.

[0154] Data from human genetics studies have suggested strong genetic links between MS4A4A and TREM2 and with susceptibility to Alzheimer’s disease (Piccio et al., 2016, Acta Neuropathol, 131:925- 9330). In particular, MS4A4A alleles protective for Alzheimer’s disease are linked to increased sTREM2 levels in the cerebrospinal fluid in patients.

[0155] Anti-MS4A4A antibodies of the present invention increase cellular ATP levels in macrophages, indicating that anti-MA4A4A antibodies are effective at increasing, maintaining, or enhancing cell (e.g., macrophages, myeloid cells, microglia) viability and function. Additionally, anti-MS4A4A antibodies of the present invention increased sTREM2 and mTREM2 levels in macrophages, in contrast to that previously reported in which commercially available anti-MS4A4A antibody 5C12 reduced sTREM2 levels in supernatants of cultured human macrophages (Deming et al, 2018, bioRxiv, doi: dx doi org / 10.1101/352179). As MS4A4A protective alleles for Alzheimer’s disease are linked to increased sTREM2 levels, the results provided herein indicated that anti-MS4A4A antibodies of the present invention mimic or replicate a protective phenotype in neurodegenerative diseases and disorders, such as Alzheimer’s disease, by increasing sTREM2 and mTREM2 levels. In some aspects, an anti-MS4A4A antibody of the present disclosure increases cell surface expression of TREM2 in myeloid cells (e.g., macrophages, human macrophages, microglia) by at least 10%, by at least 20%, by at least 25%, by at least 50%, by at least 75%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 200%, or by at least 250%. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in myeloid cells (e.g., macrophages, human macrophages, microglia) by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100%.

[0156] In some aspects, an anti-MS4A4A antibody of the present disclosure increases mTREM protein levels in myeloid cells (e.g., macrophages, human macrophages, microglia) with an EC50 of about 0.039 g/ml. In some aspects, an anti-MS4A4A antibody of the present disclosure increases sTREM protein levels in myeloid cells (e.g., macrophages, human macrophages, microglia) with an EC50 of about 0.069 g/ml. In yet other aspects, an anti-MS4A4A antibody of the present disclosure increases ATP levels in myeloid cells (e.g., macrophages, human macrophages, microglia) with an EC50 of about 0.021 g/ml. In some aspects, an anti-MS4A4A antibody of the present disclosure increases ATP levels in myeloid cells (e.g., macrophages, human macrophages, microglia) by about 1.2-fold or about 1.4-fold over the level of ATP in such cells in the absence of anti-MS4A4A antibody.

[0157] In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in vivo (e.g. , in a non-human primate or in a human). In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo by at least 10%, by at least 20%, by at least 25%, by at least 50%, by at least 75%, by at least 90%, by at least 100%, by at least 125%, or by at least 150% from the baseline soluble TREM2 levels in serum in vivo prior to administration of an anti- MS4A4A antibody of the present disclosure. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo by about 50% from the baseline soluble TREM2 levels in serum in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure.

[0158] In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo by at least 1.1-fold, by at least 1.2-fold, by at least 1.3-fold, by at least 1.4-fold, by at least 1.5-fold, by at least 1.6-fold, by at least 1.7-fold, by at least 1.8-fold, by at least 1.9-fold, or by at least 2-fold relative to the soluble TREM2 levels in serum in vivo prior to administration of an anti- MS4A4A antibody of the present disclosure. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo by about 1.5-fold relative to the soluble TREM2 levels in serum in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure. [0159] In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo compared to the soluble TREM2 levels in serum in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at lead 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo compared to the soluble TREM2 levels in serum in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 20 days. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo compared to the soluble TREM2 levels in serum in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 192 hours, at least 216 hours, at least 240 hours, at least 264 hours, at least 288 hours, at least 312 hours, at least 336 hours, at least 360 hours, at least 384 hours, at least 408 hours, at 432 hours, at least 456 hours, or at least 480 hours. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in serum in vivo compared to the soluble TREM2 levels in serum in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 480 hours.

[0160] In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo by at least 10%, by at least 20%, by at least 25%, by at least 50%, by at least 75%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 200%, by at least 225%, by at least 250%, by at least 275%, by at least 300%, by at least 325%, by at least 350%, by at least 375%, or by at least 400% from the baseline soluble TREM2 levels in CSF in vivo prior to administration of an anti- MS4A4A antibody of the present disclosure. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo by about 300% from the baseline soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure.

[0161] In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo by at least 1.4-fold, by at least 1.6-fold, by at least 1.8-fold, by at least 2.0-fold, by at least 2.2-fold, by at least 2.4-fold, by at least 2.6-fold, by at least 2.8-fold, by at least 3.0-fold, by at least 3.2-fold, by at least 3.4-fold, by at least 3.6-fold, by at least 3.8-fold, or by at least 4.0-fold relative to the soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo by between about 2-fold to about 4-fold relative to the soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure.

[0162] In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo compared to the soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 1 day, at least 2 days, at least 3 days, or at least 4 days. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo compared to the soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 4 days. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo compared to the soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours. In some aspects, an anti-MS4A4A antibody of the present disclosure increases soluble TREM2 levels in CSF in vivo compared to the soluble TREM2 levels in CSF in vivo prior to administration of an anti-MS4A4A antibody of the present disclosure for at least 96 hours.

[0163] The evidence provided herein demonstrates that the anti-MS4A4A antibodies of the present disclosure affect TREM2, but does not exclude the antibodies acting through other pathways.

Antagonist antibodies

[0164] In some aspects, antibodies that bind a MS4A4A protein may include antagonist antibodies that bind MS4A4A inhibit one or more MS4A4A activities, either by preventing interaction between MS4A4A and its ligand(s), or by preventing the transduction of signal of MS4A4A into the cell cytoplasm in the presence of ligand. In some aspects, antagonist antibodies of the present disclosure may have the epitope specificity of an agonist antibody of the present disclosure, but have an Fc domain that is not capable of binding Fey receptors and thus is unable to, for example, cluster MS4A4A receptor.

[0165] In some aspects, an antibody of the present disclosure is an antagonist antibody. In some aspects, the antagonist antibody inhibits one or more MS4A4A activities. In some aspects, the antagonist antibody decreases activity of one or more MS4A4A-dependent genes. In some aspects, the antagonist antibody inhibits interaction between MS4A4A and one or more MS4A4A ligands. In some aspects, the antagonist antibody inhibits MS4A4A signal transduction. In some aspects, the antagonist antibody inhibits interaction between MS4A4A and one or more MS4A4A ligands and inhibits MS4A4A signal transduction.

[0166] In some aspects, down-regulation of MS4A4A protein levels or reducing MS4A4A activity is accomplished by an anti-MS4A4A antibody that down-regulates or reduces MS4A4A protein levels in a cell. In some aspects, down-regulation of MS4A4A protein levels or reducing MS4A4A activity is accomplished by down-regulation of MS4A4A nucleic acid expression or levels, by, e.g., use of antisense methodologies, gene therapy, etc., using methods known and available to one of skill in the art. Accordingly, in some aspects, reducing MS4A4A protein levels or activity is accomplished with an anti- MS4A4A antibody of the present disclosure or by reducing MS4A4A nucleic acid (e.g., mRNA) expression or levels.

[0167] In some aspects, antibody cross-linking is required for agonist antibody function. Antibody crosslinking can occur through binding to a secondary antibody in vitro or through binding to Fc receptors in vivo. For example, antagonistic antibodies can be converted to agonistic antibodies via biotin/streptavidin cross-linking or secondary antibody binding in vitro (see for example Gravestein et al., 1996, J. Exp. Med. 184:675-685; Gravestein etal., 1994, International Immunol, 7:551-557). Agonistic antibodies may exert their activity by mimicking the biological activity of the receptor ligand or by enhancing receptor aggregation, thereby activating receptor signaling. In some aspects, the absence of antibody cross-linking is required for antagonistic activity. Antagonistic antibodies may exert their activity by blocking receptorligand interactions.

Osteopontin, and phenocopy of Alzheimer ’s disease protective allele

[0168] There are three SNPs in the MS4A gene cluster that have been associated with an increased risk of late-onset Alzheimer’s disease. These include 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). Additionally, MS4A4A locus SNPs (rs2304933 and rs2304935) are associated with higher levels of MS4A4A and increased Alzheimer’s disease risk, including late-onset Alzheimer’s disease (LOAD) (Allen et al, 2012, Neurology, 79:221-228). [0169] Alzheimer’s disease-associated genetic variants (SNPs) associated with the MS4A gene cluster have been identified. One of those variant alleles is rsl582763, which is associated with elevated CSF sTREM2 levels and with reduced Alzheimer’s disease risk and delayed age-at-onset, and thus considered a protective allele. (Deming et al, 2018, bioRxiv, doi: dx doi org / 10.1101/352179). The rsl582763 protective allele is associated with decreased MS4A4A mRNA levels in blood. These findings further suggest that the rsl582763 allele performs a protective role by reducing MS4A4A levels, and decreasing Alzheimer’s disease risk or severity. The protective rsl582763 is associated also with increased expression levels of osteopontin. Anti-MS4A4A antibodies of the present invention are effective at phenocopying these aspects of the protective alleles, at least with respect to decreasing MS4A4A expression, increasing osteopontin expression, and/or increasing sTREM levels. In some aspects of the present disclosure, anti- MS4A4A antibodies are provided wherein the antibodies phenocopy one or more alleles of MS4A that are associated with reduced Alzheimer’s disease risk and/or delayed age-at-onset of Alzheimer’s disease.

[0170] Anti-MS4A4A antibodies of the present disclosure increased mRNA levels of osteopontin (SPP1) in human peripheral blood mononuclear cell-derived macrophages. In some aspects, anti-MS4A4A antibodies of the present disclosure increase mRNA and/or protein levels of osteopontin. In some aspects, an anti-MS4A4A antibody of the present disclosure increases mRNA and/or protein levels of osteopontin in myeloid cells (e.g., macrophages, human macrophages, microglia) by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 200%, or by at least 250%.

[0171] Anti-MS4A4A antibodies of the present disclosure increased osteopontin (SSP1) levels in nonhuman primates. In some aspects, anti-MS4A4A antibodies of the present disclosure increase mRNA and/or protein levels of osteopontin in vivo (e.g., in non-human primates or in humans). In some aspects, an anti-MS4A4A antibody of the present disclosure increases mRNA and/or protein levels of osteopontin in serum and/or in CSF. In some aspects, an anti-MS4A4A antibody of the present disclosure increases mRNA and/or protein levels of osteopontin in serum and/or in CSF by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 200%, or by at least 250%. In some aspects, an anti-MS4A4A antibody of the present disclosure increases protein levels of osteopontin in the brain. In some aspects, an anti-MS4A4A antibody of the present disclosure increases protein levels of osteopontin in the frontal cortex and/or in the hippocampus.

[0172] In some aspects, anti-MS4A4A antibodies of the present disclosure phenocopy the protective rsl582763 allele with respect to increasing expression of osteopontin. In other aspects, anti-MS4A4A antibodies of the present disclosure are effective at increasing expression of osteopontin and /or of sTREM2 and are biologically active in decreasing Alzheimer’s disease risk and/or severity, similar to that of the protective rs 1582763 allele. The data indicated that SPP1 and GSN are pharmacodynamic markers for the protective biological activity associated with the rsl58273 allele.

CSF1R and IL1RN expression

[0173] Anti-MS4A4A antibodies of the present disclosure increased CSF1R levels in non-human primates. In some aspects, anti-MS4A4A antibodies of the present disclosure increase mRNA and/or protein levels of CSF1R in vivo (e.g., in non-human primates or in humans). In some aspects, an anti- MS4A4A antibody of the present disclosure increases mRNA and/or protein levels of CSF1R in serum and/or in CSF. In some aspects, an anti-MS4A4A antibody of the present disclosure increases mRNA and/or protein levels of CSF1R in serum and/or in CSF by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 200%, or by at least 250%. In some aspects, an anti-MS4A4A antibody of the present disclosure increases protein levels of CSF1R in the brain. In some aspects, an anti-MS4A4A antibody of the present disclosure increases protein levels of CSF1R in the frontal cortex and/or in the hippocampus.

[0174] CSF1R deficiency negatively impacts the development of microglia in the brain (Swerdlow et al (2000) Neurology, 111:300-311; Baba et al (2006) Acta Neuropath, 111:300-311) and recent research has linked mutations in the CSF1R gene to various disorders, including adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) and hereditary diffuse leukoencephalopathy with spheroids (HDLS) (Oosterhof et al (2019) Am J Hum Genet, 104:936-947; Rademaker et al (2011) Nat Genet, 44:200-205; Nicholson et al (2013) Neurology, 80: 1033-1040). Anti-MS4A4A antibodies of the present disclosure reduced cell death and sustained survival of human macrophages following CSF1R inhibition. Accordingly, in some aspects, anti-MS4A4A antibodies of the present disclosure are useful for treating an individual having a CSFIR-deficient disease or disorder, such as ALSP or HDLS. [0175] Anti-MS4A4A antibodies of the present disclosure increased IL1RN levels in human peripheral blood mononuclear cell-derived macrophages. In some aspects, anti-MS4A4A antibodies of the present disclosure increase mRNA and/or protein levels of IL1RN. In some aspects, anti-MS4A4A antibodies of the present disclosure increase mRNA and/or protein levels of IL1RN. In some aspects, an anti-MS4A4A antibody of the present disclosure increases mRNA and/or protein levels of IL1RN in myeloid cells (e.g., macrophages, human macrophages, microglia) by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 200%, or by at least 250%.

GPNMB expression

[0176] GPNMB (glycoprotein nonmetastatic melanoma protein B); is a surface glycoprotein expressed in multiple cell types including tissue macrophages and microglia. Several genetic variants have been associated with Parkinson’s disease (PD) risk. GPNMB protein levels are elevated in the substantia nigra of PD patients and GPNMB levels are increased following lysosomal stress (Moloney et.al., 2018, Neurobio Dis. 120: 1-11). Additionally, increased expression of GPNMB was linked to SNP rs 199347, this risk SNP being located within the GPNMB gene (Murthy et al, Neurogenetics, 2017, 18: 121-133). [0177] Anti-MS4A4A antibodies of the present disclosure reduced GPNMB cell surface protein levels in human primary macrophages. In some aspects, an anti-MS4A4A antibody of the present disclosure decreases GPNMB cell surface protein levels in myeloid cells (e.g., macrophages, human macrophages, microglia) by at least 10%, by at least 20%, by at least 30%, by at least 40%, or by at least 50%. As increased GPNMB levels are associated with risk alleles for PD, a reduction in GPNMB following anti- MS4A4A antibody addition may provide a means for treatment of PD.

[0178] Provided herein are methods of using anti-MS4A4A antibodies, e.g., in combination with amyloid-beta targeting therapeutics. In some aspects, an anti-MS4A4A antibody can be the 4A-450 antibody. The 4A-450 antibody is described in WO 2021/022083, which is herein incorporated by reference in its entirety. The variable heavy chain and variable light chain sequences of the 4A-450 antibody are shown below in Table 1, with their corresponding CDRs (according to Kabat) underlined. The heavy chain CDR sequences are shown in Table 2, and the light chain CDR sequences are shown in Table 3. TABLE 1

TABLE 2

TABLE 3 [0179] In some aspects, provided herein are anti-MS4A4A antibodies comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NON; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:8; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NON, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NON.

[0180] In some aspects, provided herein are anti-MS4A4A antibodies comprising: (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:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NON; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0181] In some aspects, an anti-MS4A4A antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, an anti-MS4A4A antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 11. In some aspects, an anti-MS4A4A antibody comprises a VH and VL comprising the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In some aspects, an anti-MS4A4A antibody comprises the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences.

[0182] In another aspect, an anti-MS4A4A antibody comprises a full length heavy chain amino acid 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 NOs: 12-19. In certain aspects, a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to selected from the group consisting of SEQ ID NOs: 12-19 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MS4A4A antibody comprising that sequence retains the ability to bind to MS4A4A. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NOs: 12- 19. In certain aspects, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NOs: 12-19. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0183] In another aspect, an anti-MS4A4A antibody comprises a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In certain aspects, a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 17 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MS4A4A antibody comprising that sequence retains the ability to bind to MS4A4A. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 17. In certain aspects, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 17. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0184] In another aspect, an anti-MS4A4A antibody comprises a full length light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO:21. In certain aspects, a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence of SEQ ID NO:21 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MS4A4A antibody comprising that sequence retains the ability to bind to MS4A4A. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:21. In certain aspects, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:21. In certain aspects, substitutions, insertions, or deletions occur in regions outside the LVRs (i.e., in the FRs).

[0185] In some aspects, the anti-MS4A4A antibody comprises a full length heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19 and a full length light chain amino acid sequence of SEQ ID NO:21. In some aspects, the anti-MS4A4A antibody comprises a full length heavy chain amino acid sequence of SEQ ID NO: 17 and a full length light chain amino acid sequence of SEQ ID NO:21.

[0186] In some aspects, an anti-MS4A4A antibody comprises a heavy chain amino comprising the amino acid sequence of SEQ ID NO: 12. In some aspects, an anti-MS4A4A antibody comprises a heavy chain amino comprising the amino acid sequence of SEQ ID NO: 13. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14. In some aspects, an anti- MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16. In some aspects, an antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19.

[0187] In some aspects, an anti-MS4A4A antibody of the present disclosure competitively inhibits binding of the reference antibody 4A-450.

[0188] In some aspects, an anti-MS4A4A antibody of the present disclosure binds to an epitope of human MS4A4A that is the same as or overlaps with the MS4A4A epitope bound by reference antibody 4A-450. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0189] In some aspects, an anti-MS4A4A antibody of the present disclosure binds to extracellular domain 1 (ECL1) of MS4A4A. In some aspects, an anti-MS4A4A antibody of the present disclosure binds to extracellular domain 2 (ECL2) of MS4A4A. In some aspects, an anti-MS4A4A antibody of the present disclosure binds to one or more amino acids within the amino acid sequence SFHHPYCNYYGNSNNCHGTMS (amino acids 159-179) of SEQ ID NO: 1. In some aspects, an anti- MS4A4A antibody of the present disclosure binds to MS4A4A with the amino acid sequence of SEQ ID NO: 1 and binding to the MS4A4A protein is reduced by at least 20% when N166 of SEQ ID NO: 1 is mutated to A. In some aspects, an anti-MS4A4A antibody of the present disclosure binds to one or more amino acids within the amino acid sequence SFHHPYCNYYGNSNNCHGTMS (amino acids 159-179) of SEQ ID NO: 1 and binding to MS4A4A of SEQ ID NO: 1 is reduced by at least 20% when N166 of SEQ ID NO: 1 is mutated to A.

[0190] In some aspects, provided herein are anti-MS4A4A antibodies comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, or an amino acid with at least about 95% homology to the amino acidof SEQ ID NO:22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24 or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:27. [0191] In some aspects, provided herein are anti-MS4A4A antibodies comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22; (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:24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.

[0192] In some aspects, an anti-MS4A4A antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:28. In some aspects, an anti-MS4A4A antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:28. In some aspects, an anti-MS4A4A antibody comprises a VH and VL comprising the amino acid sequences of SEQ ID NO:28 and SEQ ID NO:29, respectively. In some aspects, an anti-MS4A4A antibody comprises the VH and VL sequences in SEQ ID NO:28 and SEQ ID NO:29, respectively, including post-translational modifications of those sequences. [0193] In another aspect, an anti-MS4A4A antibody comprises a full length heavy chain amino acid 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 NOs:30-37. In certain aspects, a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to selected from the group consisting of SEQ ID NOs:30-37 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MS4A4A antibody comprising that sequence retains the ability to bind to MS4A4A. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NOs:30- 37. In certain aspects, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NOs:30-37. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0194] In another aspect, an anti-MS4A4A antibody comprises a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:37. In certain aspects, a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:37 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MS4A4A antibody comprising that sequence retains the ability to bind to MS4A4A. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:37. In certain aspects, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:37. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0195] In another aspect, an anti-MS4A4A antibody comprises a full length light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO:38. In certain aspects, a full length heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence of SEQ ID NO:38 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MS4A4A antibody comprising that sequence retains the ability to bind to MS4A4A. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:38. In certain aspects, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:38. In certain aspects, substitutions, insertions, or deletions occur in regions outside the LVRs (i.e., in the FRs).

[0196] In some aspects, the anti-MS4A4A antibody comprises a full length heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs:30-37 and a full length light chain amino acid sequence of SEQ ID NO:38. In some aspects, the anti-MS4A4A antibody comprises a full length heavy chain amino acid sequence of SEQ ID NO:37 and a full length light chain amino acid sequence of SEQ ID NO:38. [0197] In some aspects, an anti-MS4A4A antibody comprises a heavy chain amino comprising the amino acid sequence of SEQ ID NO:30. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32. In some aspects, an anti- MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:33. In some aspects, an antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:35. In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:36. In some aspects, an anti-MS4A4A antibody comprises a heavy chain amino comprising the amino acid sequence of SEQ ID NO:37.

[0198] In some aspects, an anti-MS4A4A antibody of the present disclosure competitively inhibits binding of the reference antibody 4A-313.

[0199] In some aspects, an anti-MS4A4A antibody of the present disclosure binds to an epitope of human MS4A4A that is the same as or overlaps with the MS4A4A epitope bound by reference antibody 4A-313. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0200] In some aspects, provided herein are anti-MS4A4A antibodies comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid with at least about 95% homology to the amino acidof SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:39; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:40 or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:41, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NO:8; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NON, or an amino acid with at least about 95% homology to the amino acid of SEQ ID NON.

[0201] In some aspects, provided herein are anti-MS4A4A antibodies comprising: (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:39; (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:41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NON.

[0202] In some aspects, an anti-MS4A4A antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:42. In some aspects, an anti-MS4A4A antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:43. In some aspects, an anti-MS4A4A antibody comprises a VH and VL comprising the amino acid sequences of SEQ ID NO:42 and SEQ ID NO:43, respectively. In some aspects, an anti-MS4A4A antibody comprises the VH and VL sequences in SEQ ID NO:42 and SEQ ID NO:43, respectively, including post-translational modifications of those sequences.

[0203] In some aspects, an anti-MS4A4A antibody of the present disclosure competitively inhibits binding of the reference antibody 4A-21.

[0204] In some aspects, an anti-MS4A4A antibody of the present disclosure binds to an epitope of human MS4A4A that is the same as or overlaps with the MS4A4A epitope bound by reference antibody 4A-21. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0205] Any suitable competition assay or MS4A4A binding assay known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry, may be utilized to determine whether an anti-MS4A4A antibody competes with (or competitively inhibits the binding of) the reference antibody 4A-450 for binding to MS4A4A. In an exemplary competition assay, immobilized MS4A4A or cells expressing MS4A4A on the cell surface are incubated in a solution comprising a first labeled antibody (e.g., 4A-450) that binds to MS4A4A (e.g., human or non-human primate) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to MS4A4A. The second antibody may be present in a hybridoma supernatant. As a control, immobilized MS4A4A or cells expressing MS4A4A is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody.

After incubation under conditions permissive for binding of the first antibody to MS4A4A, excess unbound antibody is removed, and the amount of label associated with immobilized MS4A4A or cells expressing MS4A4A is measured. If the amount of label associated with immobilized MS4A4A or cells expressing MS4A4A is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to MS4A4A. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0206] Further provided herein are anti-MS4A4A antibodies which competitively inhibit binding of and/or compete for binding with an anti-MS4A4A antibody comprising (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:9. In some aspects, the antibody comprises the VH and VL sequences in SEQ ID NOTO and SEQ ID NO: 11, respectively.

[0207] Provided herein are anti-MS4A4A antibodies which bind to an epitope of human MS4A4A that is the same as or overlaps with the epitope bound by an anti-MS4A4A antibody comprising (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO:5, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NON. In some aspects, the antibody comprises the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In some aspects, the epitope of human MS4A4A is the same epitope as bound by the 4A-450 antibody.

[0208] Further provided herein are anti-MS4A4A antibodies which competitively inhibit binding of and/or compete for binding with an anti-MS4A4A antibody comprising (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27. In some aspects, the antibody comprises the VH and VL sequences in SEQ ID NO:28 and SEQ ID NO:29, respectively.

[0209] Provided herein are anti-MS4A4A antibodies which bind to an epitope of human MS4A4A that is the same as or overlaps with the epitope bound by an anti-MS4A4A antibody comprising (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO:23, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27. In some aspects, the antibody comprises the VH and VL sequences in SEQ ID NO:28 and SEQ ID NO:29, respectively. In some aspects, the epitope of human MS4A4A is the same epitope as bound by an the 4A-313 antibody.

[0210] Further provided herein are anti-MS4A4A antibodies which competitively inhibit binding of and/or compete for binding with an anti-MS4A4A antibody comprising (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:40, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:41, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NOV. In some aspects, the antibody comprises the VH and VL sequences in SEQ ID NO:42 and SEQ ID NO:43, respectively.

[0211] Provided herein are anti-MS4A4A antibodies which bind to an epitope of human MS4A4A that is the same as or overlaps with the epitope bound by an anti-MS4A4A antibody comprising (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO:39, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:40, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID N0:41, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:9. In some aspects, the antibody comprises the VH and VL sequences in SEQ ID NO:42 and SEQ ID NO:42, respectively. In some aspects, the epitope of human MS4A4A is the same epitope as bound by an the 4A-21 antibody.

[0212] In some aspects, the anti-MS4A4A antibody according to any of the above aspects is a monoclonal antibody, including a humanized and/or human antibody. In some aspects, the anti-MS4A4A antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In some aspects, the anti- MS4A4A antibody is a substantially full-length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein.

Multispecific Antibodies

[0213] Multispecific are antibodies that have binding specificities for at least two different epitopes, including those on the same or another polypeptide (e.g., one or more MS4A4A polypeptides of the present disclosure). In some aspects, the multispecific antibody can be a bispecific antibody. In some aspects, the multispecific antibody can be a trispecific antibody. In some aspects, the multispecific antibody can be a tetraspecific antibody. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies). In some aspects, the multispecific antibody comprises a first antigen binding region which binds to first site on MS4A4A and comprises a second antigen binding region which binds to a second site on MS4A4A. In some embodiment, the multispecific antibodies comprises a first antigen binding region which binds to MS4A4A and a second antigen binding region that binds to a second polypeptide.

[0214] Provided herein are multispecific antibodies comprises a first antigen binding region, wherein the first antigen binding region comprises the six HVRs of an antibody described herein, which binds to MS4A4A and a second antigen binding region that binds to a second polypeptide. In some aspects, the first antigen binding region comprises the VH or VL of an antibody described herein.

[0215] In some aspects of any of the multispecific antibodies, the second polypeptide is a) an antigen facilitating transport across the blood-brain-barrier; (b) an antigen facilitating transport across the bloodbrain-barrier 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), LRP8, diphtheria toxin receptor, CRM 197, a llama single domain antibody, TMEM 30(A), a protein transduction domain, TAT, Syn-B, penetratin, a poly -arginine peptide, an angiopep peptide, Basigin, Glucose Transporter Type 1 (Glutl), CD98, MfsD2a, and ANG1005; (c) a disease-causing protein selected from amyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursor protein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein Al, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non- ATG (RAN) translation products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine (PR) repeat peptides; (d) ligands and/or proteins expressed on immune cells, wherein the ligands and/or proteins selected from CD40, 0X40, 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) a protein, lipid, polysaccharide, or glycolipid expressed on one or more tumor cells and any combination thereof.

[0216] Numerous antigens are known in the art that facilitate transport across the blood-brain barrier (see, e.g., Gabathuler R. Neurobiol. Dis. 37:48-57 (2010)). Such second antigens include, without limitation, 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 penetratin, poly-arginine or generally positively charged peptides, Angiopep peptides such as ANG1005 (see, e.g., Gabathuler, 2010), and other cell surface proteins that are enriched on blood-brain barrier endothelial cells (see, e.g. , Daneman et al. PLoS One 5(10):el3741 (2010)).

[0217] The multivalent antibodies may recognize the MS4A4A antigen as well as without limitation additional antigens AP peptide, antigen or an oc-synuclein protein antigen or, Tau protein antigen or, TDP- 43 protein antigen or, prion protein antigen or, huntingtin protein antigen, or RAN, translation Products antigen, including the DiPeptide Repeats, (DPRs peptides) composed of glycine-alanine (GA), glycineproline (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 facilitate antibody transfer across the blood brain barrier. In some aspects, the second polypeptide is transferrin. In some aspects, the second polypeptide is Tau. In some aspects, the second polypeptide is A[3. In some aspects, the second polypeptide is TREM2. In some aspects, the second polypeptide is oc-synuclein.

[0218] The multivalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain or chains comprise two or more variable domains. For instance, the polypeptide chain or chains may comprise VDl-(Xl) _n -VD2-(X2) _n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. Similarly, the polypeptide chain or chains may comprise VH-CH1 -flexible linker-Vu-Cul-Fc region chain; or VH-CH1-VH-CH1-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 instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain. [0219] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain- light chain pairs having 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. Patent No. 5731168). See also WO 2013/026833 (CrossMab). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies (see, e.g., US Patent No. 4676980); using leucine; using "diabody" technology for making 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., in Tutt et al. J. Immunol. 147: 60 (1991).

[0220] Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g., US 2006/0025576). The antibody herein also includes a "Dual Acting FAb" or "DAF" comprising an antigen binding site that binds to multiple MS4A4A (see, US 2008/0069820, for example).

IV. A-Beta Targeting Therapeutics

[0221] Provided herein are also combinations of anti-MS4A4A antibodies and amyloid beta (A-beta) targeting therapeutics.

[0222] In some aspects, an anti-A-beta targeting therapeutic can prevent accumulation or reverse the deposition of A-beta (e.g., Api-42, Ap pyro 3-42, and/or AP4-42) within the brain and/or cerebrovasculature. In some aspects, an anti-A-beta targeting therapeutic can bind to and precipitate soluble Api-42 in blood plasma and/or in cerebrospinal fluid (CSF), thereby reducing the concentration of Api-42 in the serum and/or CSF, respectively.

[0223] In some aspects, an anti-A-beta targeting therapeutic binds to A-beta. In some aspects, an anti-A- beta targeting therapeutic binds to human A-beta. In some aspects, an anti-A-beta targeting therapeutic binds to human A-beta and murine A-beta. In some aspects, an anti-A-beta targeting therapeutic binds to amyloid oligomers and fibers, but not to not linearized amyloid species. In some aspects, an anti-A-beta targeting therapeutic binds to A-beta but does not significantly bind to amyloid precursor protein (APP). [0224] In some aspects, the A-beta targeting therapeutic can be one or more anti-A-beta antibodies. In some aspects, an anti-A-beta antibody is capable of binding fibrillary, oligomeric, and monomeric forms of A-beta. In some aspects, the binding of the anti-A-beta antibodies described herein can induce a conformational transition in amyloid beta. In some aspects, the anti-A-beta antibodies described herein capable of inhibiting, in vitro and in vivo, the aggregation of amyloidogenic monomeric peptides, specifically P-amyloid monomeric peptides such as, for example, Ap monomeric peptides 1-39, 1-40, 1-41, 1-42, or 1-43, into high molecular polymeric amyloid fibrils or filaments. In some aspects, one or more anti-A-beta antibodies can be administered with or combined in a therapy with an anti-MS4A4A antibody provided herein. Non-limiting examples of such anti-A-beta antibodies include lecanemab, donanemab, crenezumab, solanezumab, bapineuzumab, aducanumab, and gantenerumab. Additional A-beta antibodies have been disclosed in, e.g., US 2022/0098288, 2021/0009665, US 2020/3777582, US 2019/0262327, US 2019/0016791, US 2017/0369559, US 2015/0299299, US 2015/0320860, US 2013/0089537, US 2003/0073655, US 11,286,297, US 9,670,272, US 9,175,094, US 8,246,954, US 8,124,353, US 8,106,164, US 7,927,594, US 7,892,544, US 7,871,615, US 7,773,235, each of which is herein incorporated by reference in its entirety.

[0225] In some aspects, the A-beta targeting therapeutic can be one or more A-beta aggregation inhibitors. In some aspects, one or more A-beta aggregation inhibitors can be administered with or combined in a therapy with an anti-MS4A4A antibody provided herein. Non-limiting examples of A-beta aggregation inhibitors include EUND-005 (also referred to as AZD-103 or scyllo-inositol), tramiprosate, and PTI-80 (Exebryl-1®; ProteoTech). Additional A-beta aggregation inhibitors have been disclosed in, e.g., US 2015/0320860, which is herein incorporated by reference in its entirety.

[0226] Other A-beta targeting therapeutics include ACU103 (an anti-amyloid-beta oligomer monoclonal antibody; Acumen), CT1812 (small molecule; Cognition Therapeutics), ALZT-OP1 (AZ Therapeutics) and ANAVEX®2-73 (Blarcamesine).

V. Anti-MS4A4A Antibodies and A-Beta Targeting Antibodies

[0227] In some aspects, an anti-MS4A4A antibody and/or an A-beta targeting therapy that is an antibody according to any of the above aspects may incorporate any of the features, singly or in combination, as described in Sections 1-8 below:

(1) Antibody binding affinity

[0228] In some aspects of any of the antibodies provided herein, the antibody has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., IO’ ⁸ M or less, e.g., from 10" ⁸ M to 10" ¹³ M, e.g., from 10" ⁹ M to 10" ¹³ M). Dissociation constants may be determined through any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry (see, e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analyses. In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In some embodiment, an RIA is performed with the Fab version of an antibody of interest and its antigen, for example as described in Chen et al. J. Mol. Biol. 293:865-881(1999)). In some aspects, Kd is measured using a BIACORE surface plasmon resonance assay, for example, an assay using a BIACORE -2000 or a BIACORE -3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). In some aspects, the KD is determined using a monovalent antibody (e.g., a Fab) or a full-length antibody. In some aspects, the KD is determined using a full-length antibody in a monovalent form.

(2) Antibody fragments

[0229] In some aspects 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, e.g., WO 93/16185; and U.S. Patent Nos. 5571894 and 5587458. For 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.

[0230] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP404097; WO 1993/01161; Hudson et al. Nat. Med. 9: 129-134 (2003). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9: 129-134 (2003). Single-domain antibodies are antibody fragments comprising 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 aspects, a single-domain antibody is a human singledomain antibody (see, e.g., U.S. Patent No. 6248516).

[0231] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

(3) Chimeric and humanized antibodies

[0232] In some aspects of any of the antibodies provided herein, the antibody is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4816567. 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 a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. [0233] In some aspects of any of the antibodies provided herein, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In certain aspects, a humanized antibody is substantially non-immunogenic in humans. In certain aspects, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Pat. No. 5530101, 5693761; 5693762; and 5585089. In certain aspects, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen binding domain while reducing its immunogenicity are identified. See, e.g., U.S. Pat. Nos. 5766886 and 5869619. Generally, a humanized antibody comprises one or more variable domains in which HVRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0234] Humanized antibodies and methods of making them are reviewed, for example, in Almagro et al. Front. Biosci. 13: 161 9-1633 (2008), and are further described, e.g., in US Patent Nos. 5821337, 7527791, 6982321, and 7087409. Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best- fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g. , Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151 :2623 (1993)); 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)).

(4) Human Antibodies

[0235] In some aspects 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 described generally in van Dijk et al. Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg Curr. Opin. Immunol. 20:450-459 (2008).

[0236] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human MAbs with the desired specificity can be produced and selected. Certain exemplary methods are described in U.S. Pat. No. 5545807, EP 546073, and EP 546073. See also, for example, U.S. Patent Nos. 6075181 and 6150584 describing XENOMOUSE™ technology; U.S. Patent No. 5770429 describing HUMAB® technology; U.S. Patent No. 7041870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. [0237] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mousehuman heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol. 133:3001 (1984) and Boemer et al. J. Immunol. 147:86 (1991)). Human antibodies generated 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 those described, for example, in U.S. Patent No. 7189826 (describing 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 may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0238] In some aspects of any of the antibodies provided herein, the antibody is a human antibody isolated by in vitro methods and/or screening combinatorial libraries for antibodies with the desired activity or 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, repertoires of VH and VE genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. Am. Rev. Immunol. 12: 433- 455 (1994). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. 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): 1 19-132 (2004). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers comprising random sequence to encode the highly variable HVR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patent publications describing human antibody phage libraries include, for example: US Patent No. 5750373, and US Patent Publication Nos. 2007/0292936 and 2009/0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. (5) Constant Regions including Fc regions

[0239] In some aspects of any of the antibodies provided herein, the antibody comprises an Fc. In some aspects, the Fc is a human IgGl, IgG2, IgG3, and/or IgG4 isotype. In some aspects, the antibody is of the IgG class, the IgM class, or the IgA class.

[0240] In certain aspects of any of the antibodies provided herein, the antibody has an IgG2 isotype. In some aspects, the antibody contains a human IgG2 constant region. In some aspects, the human IgG2 constant region includes an Fc region. In some aspects, an anti-MS4A4A antibody induces the one or more MS4A4A activities or independently of binding to an Fc receptor.

[0241] In some aspects, the antibody binds an inhibitory Fc receptor. In certain aspects, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcyllB).

[0242] In certain aspects of any of the antibodies provided herein, the antibody has an IgGl isotype. In some aspects, the antibody contains a mouse IgGl constant region. In some aspects, the antibody contains a human IgGl constant region. In some aspects, the human IgGl constant region includes an Fc region. In some aspects, a human IgGl light chain constant region comprises the amino acid sequence of SEQ ID NO:20. In some aspects, the antibody binds an inhibitory Fc receptor. In certain aspects, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcyllB).

[0243] In certain aspects of any of the antibodies provided herein, the antibody has an IgG4 isotype. In some aspects, the antibody contains a human IgG4 constant region. In some aspects, the human IgG4 constant region includes an Fc region. In some aspects, the antibody binds an inhibitory Fc receptor. In certain aspects, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcyllB).

[0244] In certain aspects of any of the antibodies provided herein, the antibody has a hybrid IgG2/4 isotype. In some aspects, the antibody includes an amino acid sequence comprising amino acids 118 to 260 according to EU numbering of human IgG2 and amino acids 261-447 according to EU numbering of human IgG4 (WO 1997/11971; WO 2007/106585).

[0245] In some aspects, the Fc region increases clustering without activating complement as compared to a corresponding antibody comprising an Fc region that does not comprise the amino acid substitutions. In some aspects, the antibody induces one or more activities of a target specifically bound by the antibody.

[0246] It may also be desirable to modify an antibody of the present disclosure to modify effector function and/or to increase serum half-life of the antibody. For example, the Fc receptor binding site on the constant region may be modified or mutated to remove or reduce binding affinity to certain Fc receptors, such as FcyRI, FcyRII, and/or FcyRIII to reduce Antibody-dependent cell-mediated cytotoxicity. In some aspects, the effector function is impaired by removing N-glycosylation of the Fc region (e.g., in the CH2 domain of IgG) of the antibody. In some aspects, the effector function is impaired 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). [0247] In other aspects, it may also be desirable to modify an anti-MS4A4A antibody to modify effector function to increase finding selectivity toward the ITIM-containing FcyRIIb (CD32b) to increase clustering of MS4A4A antibodies on adjacent cells without activating humoral responses including Antibodydependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis.

[0248] To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example. As used herein, the term “salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgGz, IgGs, or IgG.0 that is responsible for increasing the in vivo serum half-life of the IgG molecule. Other amino acid sequence modifications.

(6) Antibody Variants

[0249] In some aspects 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 antibody.

(i) Substitution, Insertion, and Deletion Variants

[0250] In some aspects of any of the antibodies provided herein, antibody variants having one or more amino acid substitutions are provided. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody.

TABLE A: Amino Acid Substitutions

[0251] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as 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 groups based on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

[0252] For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class. Such substituted residues can be introduced, for example, into regions of a human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.

[0253] In making changes to the polypeptide or antibody described herein, according to certain aspects, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0254] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al. J. Mol. Biol., 157: 105-131 (1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain aspects, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain aspects, those which are within ±1 are included, and in certain aspects, those within ±0.5 are included.

[0255] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological aspects, as in the present case. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0256] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0+1); aspartate (+3.0+1); glutamate (+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). In making changes based upon similar hydrophilicity values, in certain aspects, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain aspects, those which are within ±1 are included, and in certain aspects, those within ±0.5 are included. One can also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions”.

[0257] In certain aspects, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g, conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen contacting residues in the HVRs. In certain aspects of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0258] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides comprising a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. , for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0259] Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment, such as an Fv fragment).

(ii) Glycosylation variants

[0260] In some aspects of any of the antibodies provided herein, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0261] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5- hydroxylysine may also be used.

[0262] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). [0263] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 according to Kabat numbering of the CH2 domain of the Fc region. The oligosaccharide may include various carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody of the disclosure may be made in order to create antibody variants with certain improved properties.

[0264] In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. See, e.g., US Patent Publication Nos. 2003/0157108 and 2004/0093621. Examples of publications related to "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 alpha- 1,6-fiicosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) and Kanda et a/. Biotechnol. Bioeng. 94(4):680-688 (2006)).

(Hi) Modified Constant regions

[0265] In some aspects of any of the antibodies provided herein, the antibody Fc is an antibody Fc isotypes and/or modification. In some aspects, the antibody Fc isotype and/or modification is capable of binding to Fc gamma receptor.

[0266] In some aspects of any of the antibodies provided herein, the modified antibody Fc is an IgGl modified Fc. In some aspects, the IgGl modified Fc comprises one or more modifications. For example, in some aspects, the IgGl modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some aspects, 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) R. J. Biol. Chem. 276, 6591-6604), 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), C226S, C229S, E233P, L234V, L234F, L235E (McEarchem 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, where the amino acid position is according to the EU numbering convention.

[0267] In some aspects of any of the IgGl modified Fc, the Fc comprises N297A mutation according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises D265A and N297A mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises N297A mutation according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises K322A mutation according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises N297A mutation according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises P33 IS mutation according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises D270A mutations according to EU numbering. In some aspects, the IgGl modified Fc comprises L234A and L235A mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises L234A and G237A mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises L234A, L235A and G237A mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises one or more (including all) of P238D, L328E, E233, G237D, H268D, P271G and A330R mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises one or more of S267E/L328F mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises the N325S and L328F mutations according to EU numbering (N325S/L328F). In some aspects of any of the IgGl modified Fc, the Fc comprises P238D, L328E, E233D, G237D, H268D, P271G and A33 OR mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises P238D, L328E, G237D, H268D, P271G and A33 OR mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises P238D, S267E, L328E, E233D, G237D, H268D, P271G and A330R mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises P238D, S267E, L328E, G237D, H268D, P271G and A330R mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises C226S, C229S, E233P, L234V, and L235A mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises L234F, L235E, and P331S mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises S267E and L328F mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises S267E mutations according to EU numbering. In some aspects of any of the IgGl modified Fc, the Fc comprises a substitute of the constant heavy 1 (CHI) and hinge region of IgGl with CHI and hinge region of IgG2 (amino acids 118-230 of IgG2 according to EU numbering) with a Kappa light chain. [0268] In some aspects of any of the IgGl modified Fc, the Fc includes two or more amino acid substitutions that increase antibody clustering without activating complement as compared to a corresponding antibody having an Fc region that does not include the two or more amino acid substitutions. Accordingly, in some aspects of any of the IgGl modified Fc, the IgGl modified Fc is an antibody comprising an Fc region, where the antibody comprises an amino acid substitution at position E430G and one or more amino acid substitutions in the Fc region at a residue position selected from: L234F, L235A, L235E, S267E, K322A, L328F, A330S, P33 IS, and any combination thereof according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G, L234A, L235A, and P331S according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions L234A, L235A, and P33 IS according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G and P33 IS according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G and K322A according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G, A330S, and P331S according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G, K322A, A330S, and P33 IS according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G, K322A, and A330S according to EU numbering. In some aspects, the IgGl modified Fc comprises an amino acid substitution at positions E430G, K322A, and P33 IS according to EU numbering.

[0269] In some aspects of any of the IgGl modified Fc, the IgGl modified Fc may further comprise herein may be combined with an A330L mutation (Lazar et al. Proc Natl Acad Sci USA, 103:4005-4010 (2006)), or one or more of L234F, L235E, and/or P331 S mutations (Sazinsky et al. Proc Natl Acad Sci USA, 105:20167-20172 (2008)), according to the EU numbering convention, to eliminate complement activation. In some aspects of any of the IgGl modified Fc, the IgGl modified Fc may further comprise one or more of A330L, A330S, L234F, L235E, and/or P331S according to EU numbering. In some aspects of any of the IgGl modified Fc, the IgGl modified Fc may further comprise one or more mutations to enhance the antibody half-life in human serum (e.g., one or more (including all) of M252Y, S254T, and T256E mutations according to the EU numbering convention). In some aspects of any of the IgGl modified Fc, the IgGl 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.

[0270] Other aspects of the present disclosure relate to antibodies having modified constant regions (i.e., Fc regions). An antibody dependent on binding to FcyR receptor to activate targeted receptors may lose its agonist activity if engineered to eliminate FcyR binding (see, e.g., Wilson et al. Cancer Cell 19: 101-113 (2011); Armour at al. Immunology 40:585-593 (2003); and White et al. Cancer Cell 27: 138-148 (2015)). As such, it is thought that an antibody with the correct epitope specificity can activate the target antigen, with minimal adverse effects, when the antibody has an Fc domain from a human IgG2 isotype (CHI and hinge region) or another type of Fc domain that is capable of preferentially binding the inhibitory FcyRIIB r receptors, or a variation thereof.

[0271] In some aspects of any of the antibodies provided herein, the modified antibody Fc is an IgG2 modified Fc. In some aspects, the IgG2 modified Fc comprises one or more modifications. For example, in some aspects, the IgG2 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some aspects of any of the IgG2 modified Fc, the one or more amino acid substitutions are selected from V234A (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 l(Suppl. l):27 (2000); Armour et al. The Haematology Journal l(Suppl.l):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 according to the EU numbering convention. In some aspects of any of the IgG2 modified Fc, the Fc comprises an amino acid substitution at positions V234A and G237A according to EU numbering. In some aspects of any of the IgG2 modified Fc, the Fc comprises an amino acid substitution at positions C219S or C220S according to EU numbering. In some aspects of any of the IgG2 modified Fc, the Fc comprises an amino acid substitution at positions A330S and P33 IS according to EU numbering. In some aspects of any of the IgG2 modified Fc, the Fc comprises an amino acid substitution at positions S267E and L328F according to EU numbering.

[0272] In some aspects of any of the IgG2 modified Fc, 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 aspects of any of the IgG2 modified Fc, the antibody has an IgG2 isotype with a Kappa light chain constant domain that comprises 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).

[0273] In some aspects of any of the IgG2 modified Fc, the Fc comprises a C220S amino acid substitution according to the EU numbering convention. In some aspects of any of the IgG2 modified Fc, the antibody has an IgG2 isotype with a Kappa light chain constant domain that comprises a C214S amino acid substitution according to the EU numbering convention.

[0274] In some aspects of any of the IgG2 modified Fc, the Fc comprises a C219S amino acid substitution according to the EU numbering convention. In some aspects of any of the IgG2 modified Fc, the antibody has an IgG2 isotype with a Kappa light chain constant domain that comprises a C214S amino acid substitution according to the EU numbering convention.

[0275] In some aspects of any of the IgG2 modified Fc, the Fc includes an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region (White et al. Cancer Cell 27: 138-148 (2015)). In certain aspects of any of the IgG2 modified Fc, the IgG2 isotype CHI and hinge region comprise the amino acid sequence of 118-230 according to EU numbering. In some aspects of any of the IgG2 modified Fc, the antibody Fc region comprises a S267E amino acid substitution, a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution according to the EU numbering convention.

[0276] In some aspects of any of the IgG2 modified Fc, the Fc further comprises one or more amino acid substitution at positions E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W according to EU numbering. In some aspects of any of the IgG2 modified Fc, the Fc may further comprise one or more mutations to enhance the antibody half-life in human serum (e.g., one or more (including all) of M252Y, S254T, and T256E mutations according to the EU numbering convention). In some aspects of any of the IgG2 modified Fc, the Fc may further comprise A330S and P33 IS.

[0277] In some aspects of any of the IgG2 modified Fc, the Fc is an IgG2/4 hybrid Fc. In some aspects, the IgG2/4 hybrid Fc comprises IgG2 aa 118 to 260 and IgG4 aa 261 to 447. In some aspects of any IgG2 modified Fc, the Fc comprises one or more amino acid substitutions at positions H268Q, V309L, A330S, and P33 IS according to EU numbering.

[0278] In some aspects of any of the IgGl 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.

[0279] In certain aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises one or more amino acid substitutions at a residue position selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P33 IS, E345R, E430G, S440Y, and any combination thereof according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G, L243A, L235A, and P33 IS according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G and P33 IS according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G and K322A according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G, A330S, and P33 IS according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G, K322A, A330S, and P33 IS according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G, K322A, and A330S according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E430G, K322A, and P33 IS according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions S267E and L328F according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some aspects of any of the IgGl and/or IgG2 modified Fc, the Fc comprises an amino acid substitution at positions E345R, E430G and S440Y according to EU numbering. [0280] In some aspects of any of the antibodies provided herein, the modified antibody Fc is an IgG4 modified Fc. In some aspects, the IgG4 modified Fc comprises one or more modifications. For example, in some aspects, the IgG4 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some aspects of any of the IgG4 modified Fc, the one or more amino acid substitutions 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 the EU numbering convention. In some aspects of any of the IgG4 modified Fc, the Fc may further comprise L235A, G237A, and E318A according to the EU numbering convention. In some aspects of any of the IgG4 modified Fc, the Fc may further comprise S228P and L235E according to the EU numbering convention. In some aspects of any of the IgG4 modified Fc, the IgG4 modified Fc may further comprise S267E and L328F according to the EU numbering convention.

[0281] In some aspects of any of the IgG4 modified Fc, the IgG4 modified Fc comprises may 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 stabilization.

[0282] In some aspects of any of the IgG4 modified Fc, the IgG4 modified Fc may further comprise one or more mutations to enhance the antibody half-life in human serum (e.g., one or more (including all) of M252Y, S254T, and T256E mutations according to the EU numbering convention).

[0283] In some aspects of any of the IgG4 modified Fc, the Fc comprises L235E according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises S228P mutation according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises S267E and L328F mutations according to EU numbering. In certain aspects of any of the IgG4 modified Fc, the Fc comprises one or more amino acid substitutions at a residue position selected from C127S, F234A, L235A, L235E, S267E, K322A, L328F, E345R, E430G, S440Y, and any combination thereof, according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at positions E430G, L243A, L235A, and P33 IS according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at positions E430G and P33 IS according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at positions E430G and K322A according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at position E430 according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc region comprises an amino acid substitution at positions E430G and K322A according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at positions S267E and L328F according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some aspects of any of the IgG4 modified Fc, the Fc comprises an amino acid substitution at positions E345R, E430G and S440Y according to EU numbering. [0284] In some aspects, an anti-MS4A4A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12.

[0285] In some aspects, an antibody has a human IgGl heavy chain without a C-terminal lysine. In some aspects, an antibody has a human IgGl heavy chain with a P33 IS mutation. In some aspects, an antibody has a human IgGl heavy chain with a P33 IS mutation and without a C-terminal lysine. In some aspects, an antibody has a human IgGl heavy chain with N325S and L328F mutations (N325S/L328F). In some aspects, an antibody has a human IgGl heavy chain with a N325S/L328F mutation and without a C- terminal lysine. In some aspects, an antibody has a human IgGl heavy chain with a K322A mutation. In some aspects, an antibody has a human IgGl heavy chain with a K322A mutation and without a C-terminal lysine. In the foregoing aspects, mutations are indicated according to EU numbering.

(7) Other antibody modifications

[0286] In some aspects of any of the antibodies, the antibody is a derivative. The term “derivative” refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain aspects, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain aspects, a chemically modified antigen binding protein can have a greater circulating half-life than an antigen binding protein that is not chemically modified. In certain aspects, a chemically modified antigen binding protein can have improved targeting capacity for desired cells, tissues, and/or organs. In some aspects, a derivative antigen binding protein is covalently modified to include one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat. Nos. 4640835, 4496689, 4301144, 4670417, 4791192 and 4179337. In certain aspects, a derivative antigen binding protein comprises one or more polymer, including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers.

[0287] In certain aspects, a derivative is covalently modified with polyethylene glycol (PEG) subunits. In certain aspects, one or more water-soluble polymer is bonded at one or more specific position, for example at the amino terminus, of a derivative. In certain aspects, one or more water-soluble polymer is randomly attached to one or more side chains of a derivative. In certain aspects, PEG is used to improve the therapeutic capacity for an antigen binding protein. In certain aspects, PEG is used to improve the therapeutic capacity for a humanized antibody. Certain such methods are discussed, for example, in U.S. Pat. No. 6133426, which is hereby incorporated by reference for any purpose. [0288] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “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 often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from: -CH2NH-, -CH2S-, - CH2-CH2-, -CH=CH-(cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used in certain aspects to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be generated 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 which cyclize the peptide.

[0289] Drug conjugation involves coupling of a biological active cytotoxic (anticancer) payload or drug to an antibody that specifically targets a certain tumor marker (e.g. a polypeptide that, ideally, is only to be found in or on tumor cells). Antibodies track these proteins down in the body 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 together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cancer. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other chemotherapeutic agents. Technics to conjugate antibodies are disclosed are known in the art (see, e.g., Jane de Lartigue OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al. Bioconjugate Chemistry 2t (1):5-13 (2010).

(8) Antibodies produced in host cells

[0290] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria (e.g., U.S. Patent Nos. 5648237, 5789199, and 5840523. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0291] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are also suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation patern (e.g., Gemgross Nat. Biotech. IT. 1409-1414 (2004); and Li et al. Nat. Biotech. 24:210-215 (2006)).

[0292] Suitable host cells for the expression of glycosylated antibody can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (e.g., U.S. Patent Nos. 5959177, 6040498, 6420548, 7125978, and 6417429, describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0293] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al. J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma 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, e.g., 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, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

VI. Pharmaceutical Compositions/Formulations

[0294] Provided herein are pharmaceutical compositions and/or pharmaceutical formulations comprising an anti-MS4A4A antibody of the present disclosure and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical compositions and/or pharmaceutical formulations described herein further comprise A-beta targeting therapeutics. In some aspects, pharmaceutical compositions and/or pharmaceutical formulations comprising an anti-MS4A4A antibody of the present disclosure are for administration in combination with A-beta targeting therapeutics. In some aspects, pharmaceutical compositions and/or pharmaceutical formulations comprising an A-beta targeting therapeutic and a pharmaceutically acceptable carrier are provided herein, wherein the pharmaceutical compositions and/or pharmaceutical formulations are for administration in combination with an anti-MS4A4A antibody.

[0295] In some aspects, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed. The anti-MS4A4A antibodies and A-beta targeting therapeutics described herein can be formulated into preparations in solid, semi-solid, liquid or gaseous forms. Examples of such formulations include, without limitation, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Pharmaceutically acceptable carriers can include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers of diluents, which are vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. In certain aspects, the pharmaceutical composition can comprise formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.

[0296] In some aspects of the pharmaceutical compositions described herein, an anti-MS4A4A antibody can be administered at the same time as an amyloid beta targeting therapeutic (e.g., as part of the same pharmaceutical composition or as part of different pharmaceutical compositions). In other aspects, an anti- MS4A4A antibody can be administered at different times than an amyloid beta targeting therapeutic. In additional aspects, an anti-MS4A4A antibody and an amyloid beta targeting therapeutic can be administered sequentially. In an aspect, an anti-MS4A4A antibody can be administered followed by the amyloid beta targeting therapeutic. In another aspect, an anti-MS4A4A antibody can be administered preceded by an amyloid beta targeting therapeutic.

VII. Articles of Manufacture

[0297] Provided herein are articles of manufacture (e.g., kits) comprising an anti-MS4A4A antibody and/or an A-beta therapeutic. Articles of manufacture can include one or more containers comprising an anti-MS4A4A antibody and/or an A-beta therapeutic described herein. Containers can be any suitable packaging including, but not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The containers can be unit doses, bulk packages (e.g., multi -dose packages), or sub-unit doses.

[0298] In some aspects, the kits can further comprise an additional agent. In some aspects, the additional agent is a pharmaceutically acceptable buffer or diluting agent including, but not limited to, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer's solution and dextrose solution.

[0299] In some aspects of any of the articles of manufacture provided herein, the article of manufacture further comprises instructions for use in accordance with the methods of this disclosure. The instructions can comprise information as to dosage, dosing schedule, and route of administration for the intended treatment. In some aspects, these instructions comprise a description of administration of the anti-MS4A4A antibody and/or A-beta therapeutic to prevent, reduce risk, or treat an individual having a disease, disorder, or injury selected from frontotemporal dementia, Alzheimer’s disease, late onset Alzheimer’s disease, early Alzheimer’s disease, cognitive decline or impairment, mild cognitive impairment, vascular dementia, vascular dementia, seizures, retinal dystrophy, atraumatic brain injury, a spinal cord injury, long-term depression, atherosclerotic vascular diseases, undesirable symptoms of normal aging, dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington’s disease, taupathy disease, 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, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatous disorders, Sarcoidosis, diseases of aging, age related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, inflammatory disorders, arthritis, multiple sclerosis, metabolic disorder, obesity, insulin resistance, type 2 diabetes, tissue or vascular damage, an injury, inflammatory cell debris or protein aggregates, abnormal circulating myeloid cells, unhealthy aging, age-related cognitive impairment, age-related brain atrophy, age-associated traits, including without limitation inflammation, neuronal loss, and cognitive deficits, such as cognitive deficits in the absence of known brain disease, including cognitive deficits of the frontal cerebral cortex of an older individual and, one or more undesirable symptoms of normal aging. Instructions for administration of an anti-MS4A4A antibody can comprise a description of administration in combination with an A-beta therapeutic. Instructions for administration of an A-beta therapeutic can comprise a description of administration in combination with an anti-MS4A4A antibody.

[0300] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.

EXAMPLES

EXAMPLE 1: Phagocytosis measurements in human iPSC-derived microglia treated with anti- MS4A4A antibody

[0301] Amyloid beta (A-beta) uptake in human induced pluripotent stem cell derived (iPSC)-derived microglia was tested. Cells were pre-treated for 48 hours with 1 pg/mL of an anti-MS4A4A antibody (4A- 450 IgGl antibody with NSLF Fc mutations, referred to herein as “4A-450 NSLF”), a control NSLF IgGl antibody, or with media only. Pre-treatment was followed by incubation with pHrodo™-labeled A-beta 1- 42 (A-beta peptide fragment 1-42) at 3 pM. Cells were imaged on Incucyte® Live-Cell Analysis Systems, and A-beta uptake was quantified as 1) an increase in the area of the pHrodo™ label normalized to cell number or 2) the integrated intensity (mean intensity/area) per cell number.

[0302] The 4A-450 NSLF antibody increased A-beta uptake in human iPSC-derived microglia compared to both NSLF IgGl antibody control or media only control. A two-fold increase in A-beta uptake was evident by two hours after A-beta was fed to cells and continued for the duration of the experiment (21 hours).

[0303] Fig. 1 shows the time course over 21 hours of A-beta uptake by iPSC microglia pre-treated with 4A-450 NSLF antibody, NSLF IgGl antibody, or with media only (measured as an increase in the area of the pHrodo™ label normalized to cell number or as the integrated intensity (mean intensity /area) per cell number). Fig. 2 shows bar graphs of the A-beta uptake quantified at the 2 hour time point (measured as an increase in the area of the pHrodo™ label normalized to cell number and the integrated intensity (mean intensity/area) per cell number for iPSC microglia pre-treated with 4A-450 NSLF antibody, NSLF IgGl antibody or with media only). Fig. 2 also shows the images of the cells after incubation with pHrodo™- labeled A-beta 1-42.

[0304] Thus, there was a time -dependent 2-fold increase in amyloid-beta uptake after treatment with 4A- 450 NSLF antibody compared to a NSLF IgG control in iPSC microglia. These results indicate that the 4A- 450 NSLF antibody can modulate phagocytosis of A-beta.

[0305] The phagocytosis process can be divided into 2 components: internalization of the phagocytosed substrate and digestion of the phagocytosed substrate by the lysosomal/autophagy system. Specifically, multiple findings in culture, animal models and human support the idea that internalization of the phagocytosed substrates such as amyloid beta might not be the only rate limiting factor in the removal of misfolded proteins and other debris. Instead, the evidence suggests that degradation of the phagocytosed material play a crucial role as well. Indeed, once the cargo is phagocytosed, microglia need to promptly degrade it to avoid the gorging and then choking on the phagocytosed material. Microglia that are not able to digest the phagocytosed substrates become dysfunctional and inflammatory.

[0306] The following literature discusses how degradation of phagocytosed A-beta is an additional rate limiting step. (Paresce DM, et al. Slow degradation of aggregates of the Alzheimer's disease amyloid betaprotein by microglial cells. J Biol Chem. 1997 Nov 14; 272(46):29390-7. doi: 10.1074/jbc.272.46.29390. PMID: 9361021. Chung H, et al. Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells. J Biol Chem. 1999 Nov 5;274(45): 32301-8. doi: 10.1074/jbc.274.45.32301. PMID: 10542270. Brazil MI et al. Effects of incorporation of immunoglobulin G and complement component C Iq on uptake and degradation of Alzheimer's disease amyloid fibrils by microglia. J Biol Chem. 2000 Jun 2; 275 (22): 16941-7. doi: 10.1074/jbc.M000937200. PMID: 10747968. Bard F, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 2000 Aug;6(8):916-9. doi: 10.1038/78682. PMID: 10932230; Majumdar A, et al. Degradation of fibrillar forms of Alzheimer's amyloid beta-peptide by macrophages. Neurobiol Aging. 2008 May;29(5):707-15. doi:

10.1016/j.neurobiolaging.2006.12.001. Epub 2007 Jan 11. PMID: 17222479; PMCID: PMC2424018.) [0307] Experiments were performed to demonstrate the impact of 4A-450 NSLF on both steps of this phagocytosis process.

[0308] A-beta uptake in human iPSC-derived microglia was tested in cells that were exposed to a low concentration of A-beta. Cells were pre-treated for 48 hours with 1 pg/mL of 4A-450 NSLF antibody, an NSLF IgGl antibody, or media only. Pre-treatment was followed by incubation with pHrodo™-labeled A- betal-42 at 0.3 pM (low concentration of A-beta). Cells were imaged on Incucyte® Live-Cell Analysis Systems, and A-beta uptake was quantified as 1) an increase in the area of the pHrodo™ label normalized to cell number or 2) as the integrated intensity (mean intensity/area) per cell number.

[0309] Fig. 3 A shows that pre-treatment with 4A-450 NSLF antibody increased the uptake of A-beta in cells compared with pre-treatment with NSLF IgGl antibody or media alone.

[0310] In a subsequent phagocytosis experiment, A-beta uptake in human iPSC-derived microglia was tested in cells that were stressed with a CSF1R inhibitor. It was hypothesized that cells would uptake more A-beta peptide under stressed conditions. Cells were pre-treated for 48 hours with 1 pg/mL of 4A-450 NSLF antibody, an NSLF IgGl antibody, or media only. This was followed by incubation with pHrodo™- labeled A-betal-42 at 0.3 pM (low A-beta concentration) and 10 pM BLZ-945 (a CSF1R inhibitor). Cells were imaged on Incucyte® Live-Cell Analysis Systems, and A-beta uptake was quantified as 1) an increase in the area of the pHrodo™ label normalized to cell number or 2) as the integrated intensity (mean intensity/area) per cell number.

[0311] Fig. 3B shows the time course of A-beta uptake over 21 hours by iPSC microglia treated and quantified as described above. Cells pre-treated with 4A-450 NSLF antibody and then treated with 0.3 pM A-betal-42 and 10 pM BLZ-945 showed the most uptake of A-beta.

[0312] In another experiment, A-beta uptake in human iPSC-derived microglia was tested in cells that were exposed to a high concentration of A-beta and exposed to a CSF1R inhibitor. Cells were pre-treated for 48 hours with 1 pg/mL of 4A-450 NSLF antibody, an NSLF IgGl antibody, or media only. This was followed by incubation with pHrodo™-labeled A-betal-42 at 3 pM (high A-beta concentration) and 10 pM BLZ-945. Cells were imaged on Incucyte® Live-Cell Analysis Systems, and A-beta uptake was quantified as 1) an increase in the area of the pHrodo™ label normalized to cell number or 2) as the integrated intensity (mean intensity/area) per cell number.

[0313] Fig. 3C shows the time course of A-beta uptake over 21 hours by iPSC microglia treated and quantified as described above. Cells pre-treated with 4A-450 NSLF antibody and then treated with 3 pM A-betal-42 and 10 pM BLZ-945 showed the most uptake of A-beta.

EXAMPLE 2: Treatment with 4A-450 NSLF Antibody Can Promote Lysosomal Activity in Macrophages

[0314] Certain lysosomal chaperones (e.g., progranulin, prosaposin, TMEM106B) are known to enhance the transport and activity of lysosomal enzymes, and various lysosomal enzymes (e.g., Cathepsin B & D) are known to degrade amyloid beta and tau (Suire CN, et al. Cathepsin D regulates cerebral AP42/40 ratios via differential degradation of Ap42 and Ap40. Alzheimers Res Ther. 2020 Jul 6; 12( 1): 80. doi: 10.1186/S13195-020-00649-8. PMID: 32631408; PMCID: PMC7339583). [0315] Experiments were performed to analyze the effect of 4A-450 NSLF antibody on the activity of lysosomal enzymes and the removal and elimination of misfolded proteins myelin debris and other substrates that require elimination by microglia.

[0316] Glucocerebrosidase (Gcase) is a lysosomal enzyme that helps break down glucocerebroside into a sugar (glucose) and a simpler fat molecule (ceramide). Gcase function is essential for brain and body health. GB1 is the gene encoding Gcase, and GBA1 loss of function mutations are the most prevalent cause of familial Parkinson’s disease (-10%) and the cause of Gaucher disease. Elevation of Gcase with enzyme replacement or gene therapy is believed to be beneficial.

[0317] In the GCase assay, macrophages from donors (n=6) were differentiated at Day -7. At Day 0, the macrophages were treated with 4A-450 NSLF antibody or NSLF IgGl antibody (1 pg/mL). At Day 2, 150 pM Gcase substrate was added to the cells, and the cells were imaged on an Operetta high content imager. [0318] Fig. 4 shows that treatment with 4A-450 NSLF antibody increased GCase activity as compared to control (NSLF IgGl antibody) as measured by quantifying the Gcase area in each field of view on fixed cells.

[0319] Fig. 5A shows images taken over a period of 120 minutes of unfixed macrophages treated with NSLF IgGl antibody (1 pg/mL) or 4A-450 NSLF antibody obtained from a representative donor. Fig. 5C quantifies the change in Gcase signal throughout the 120 minute time course as compared to a progranulin positive control that enhances Gcase activity and a conduritol-P-epoxide (CBE, ImM) negative control that blocks Gcase activity. The data shows that over the 120 minute time period, treatment with 4A-450 NSLF antibody increased GCase activity in human myeloid cells as compared to NSLF IgGl treated cells. Fig. 5B focuses in on the data at 1.5 hours. At 1.5 hours, treatment with 4A-450 NSLF antibody increased GCase activity similar to that of treatment with progranulin (5 pg/mL).

EXAMPLE 3: The 4/1-450 NSLF Antibody Promotes the Decrease of A-beta Plaque Density hi hiPSC Microglia

[0320] The effect of 4A-450 NSLF antibody on A-beta plaque density was studied in human iPSC microglia. iPSC-derived microglia were pre-treated with NSLF or 4A-450-NSLF for 48 hours or left untreated. 3 pM of A-beta 1-42 peptide were fed to cells for 4 days to allow for A-beta plaque formation. Cells were then fixed and stained with IBA-1 to stain microglia cytoplasm and methoxy-X04 to stain plaque conformations of A-beta. Images were taken on an Operetta and analyzed with ImageJ for integrated density of IBA1 and X04. X04 signal was divided by the IBA-1 signal to normalize for cell number. 13 images per well were taken with 7 wells per treatment. Image fields were averaged. Each well was plotted as a replicate. Integrated density measurements were background subtracted using the no treatment wells. Measurements were normalized to the A-beta only wells. [0321] Human iPSC microglia treated with the 4A-450 NSLF antibody displayed a reduced number and size of A-beta plaques. The 4A-450 NSLF antibody also reduced A-beta plaque area and density in three independent iPSC microglia lines (Carrie, Lupe, Line 20) exposed to A-beta peptide 42 (A-beta42). See Figure 6.

EXAMPLE 4: Treatment of mouse models of Alzheimer’s disease with combmations of A-beta and MS4A4A antibodies

[0322] The 5X FAD transgenic mouse model is a model of Alzheimer’s disease that overexpresses human amyloid precursor protein (APP) mutated with familial Alzheimer’s disease mutations (K670N, M671L, I716V), V7171) and human PSEN1 mutated with familial Alzheimer’s disease mutations (M146L and L286V). See Oakley H, et al. J Neurosci. 2006 Oct 4;26(40): 10129-40. Amyloid plaques, microgliosis, and neuroinflammation can be detected in these mice from two to four months of age with cognitive impairment beginning at six to nine months of age. Antibodies against A-beta can reduce plaque levels in this strain.

[0323] To test if anti-MS4A4A antibodies can enhance treatment with anti A-beta antibodies, 5X FAD mice are crossed to mice expressing a human MS4A4A transgene. Any of the anti-A-beta antibodies described above, such as aducanumab, may be used. Mice are treated at two months of age for 12 weeks with (i) 10 mg/kg anti-A-beta antibody (e.g., aducanumab) and 50 mg/kg of NSLF IgGl or (ii) 10 mg/kg aducanumab and 50 mg/kg 4A-450 NSLF IgGl antibody. Age-matched human-MS4A4A-5X FAD mice that are left untreated can be used as additional controls.

[0324] Microglia are analyzed for changes to gene signatures 48 hours after the final dose. To perform single cell RNA sequence analysis (scRNA-seq) of immune cells in brain, whole brains are dissected after transcardial perfusion with ice-cold PBS and dissociated. Immune cells are recovered after Percoll (GE) separation, as described (Mildner, A. et al. 2007. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat. Neurosci. 10: 1544-1553) and FACS sorted for CD45+ cells. scRNASeq is performed and analyzed using standard methods.

[0325] The results are analyzed to demonstrate that expression of disease-associated microglia (DAM) genes (Keren-Shaul H., et al, Cell. 169: 1276-1290.el7. 10.1016/j. cell.2017.05.018) that develop in 5X FAD mice are lower in the untreated animals compared to those treated with the combination of aducanumab and the NSLF IgGl antibody. The results are further analyzed to demonstrate that the combination of aducanumab and the 4A-450 NSLF IgGl antibody further reduces DAM gene expression compared to the combination of aducanumab and the NSLF IgGl antibody.

[0326] Immunofluorescence: The number, morphology, and distribution of cells are tested by immunofluorescence 48 hours after the final dose of the antibodies. Brain sections are blocked with PBS + 3% BSA and permeabilized with 0.25% Triton X-100 in blocking solution. Primary antibodies are added overnight at a dilution of 1:5,000 for Ibal (rabbit polyclonal, Wako), 1: 1,000 for CD68 (rat monoclonal, BioLegend), and 1:500 for Ap42 (rabbit recombinant monoclonal, Thermo Fisher Scientific) or 1: 1,000 for Api-16 (6E10, BioLegend), 1:500 for Sppl (goat polyclonal, R&D Systems), and 1: 1,000 for APP (22C11 mouse mAb, Millipore) at 4°C. Secondary antibodies are added as follows: anti-goat IgG Alexa Fluor 488 (donkey polyclonal, 1:2,000; Abeam), anti-rabbit IgG Alexa Fluor 555 (donkey polyclonal, 1: 1,000; Abeam), anti-rat IgG Alexa Fluor 555 (goat polyclonal, 1: 1,000, Invitrogen), anti-goat IgG Alexa Fluor 647 (donkey polyclonal, 1 : 1,000; Abeam), anti -rabbit IgG Alexa Fluor 647 (goat recombinant polyclonal, 1: 1,000; Invitrogen), anti-mouse IgG Alexa Fluor 647 (goat recombinant polyclonal, 1:2,000; Invitrogen) for 1.5 h at room temperature. Nuclei are counterstained with TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific), and methoxy-X04 (3 pg/ml; Tocris) is used to label A-beta plaques. Confocal pictures are taken on a Nikon AlRsi+ confocal laser-scanning microscope using a 20x 0.95-NA objective. z-Stacks with 1.1- pm steps in the z direction, 1,024 x 1,024-pixel resolution, are recorded. Three-dimensional reconstruction of microglia, plaques, A-beta, and dystrophic neurons and extraction of parameters are performed in Imaris v8.3 software (Bitplane), and further processing is analyzed using automated scripts in Matlab (MathWorks).

[0327] The results are analyzed to show that the combination of aducanumab and the NSLF IgGl antibody are partially reduce the coverage area of the MethoyXO4+ plaques compared to untreated animals, and to show that the effect is more profound when animals are treated with the combination of aducanumab and the 4A-450 NSLF IgGl antibody.

[0328] Image analyses: For measurement of distinct A-beta plaque morphology, 6E10 (primary antibody against A|31— 16), methoxy-X04, and merged z-stack images are exported from Imaris. The conformation of individual plaques in each treatment group of mice is determined as follows: filamentous, strong 6E10 staining without or less methoxy-X04 single; inert, only methoxy -X04+ or concomitant with little 6E10 staining; and compact, methoxy-X04+6E10+. The proportion of distinct plaque morphology is calculated by normalizing the total number of plaques from each z-stack image.

[0329] The results are analyzed to demonstrate that compared to untreated animals, the combination of aducanumab and the NSLF IgGl antibody partially reduce the coverage area of the filamentous plaques, and that this effect is more profound when animals are treated with the combination aducanumab and the 4A-450 NSLF IgGl antibody.

[0330] For measurement of neurite dystrophy around plaques, the volumes of dystrophic neurites and plaque, as well as their centers of mass, are determined on methoxy-X04 and APP image data.

[0331] The results are analyzed to demonstrate that compared to untreated animals, the combination of aducanumab and the NSLF IgGl antibody partially reduce the extent of dystrophic neurites, and that this effect is more profound when animals are treated with the combination of aducanumab and the 4A-450 NSLF IgGl antibody.

[0332] For measurement of soluble A-beta, cortical tissue and hippocampus are homogenized in PBS containing protease inhibitor (Roche) at 0.5x concentration, and remaining insoluble material is pelleted. Supernatant is collected as the PBS-soluble fraction, and the pellet is resuspended in 5.5 M guanidine and 50 mM Tris, pH 8.0, buffer, further homogenized, and incubated on a rotator for 3 hours at room temperature. The samples are centrifuged again to pellet insoluble material, and the supernatants are collected as the PBS-insoluble guanidine fraction. Api-40 and Api-42 are measured by MSD. The results are analyzed to demonstrate that compared to untreated animals, the combination of aducanumab and the NSLF IgGl antibody partially reduce the amount of soluble A-beta, and to show that this effect is more profound when animals are treated with the combination of aducanumab and the 4A-450 NSLF IgGl antibody.

Exemplary anti-MS4A4A antibody heavy and light chain amino acid sequences having wildtype huIgGl or Fc variants of wildtype huIgGl:

4A-450 (huIgGl heavy chain) (SEQ ID NO:12)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4A-450 (huIgGl heavy chain without C-terminal K) (SEQ ID NO:13)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

4A-450 (hlgGl heavy chain; P331S) (SEQ ID NO:14)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K VSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4A-450 (hlgGl heavy chain P331S without C-terminal K) (SEQ ID NO: 15)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K VSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

4A-450 (huIgGl heavy chain N325S/L328F) (SEQ ID NO:16)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K

VSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4A-450 (hlgGl heavy chain N325S/L328F without C-terminal K) (SEQ ID NO: 17)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K

VSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

4A-450 (huIgGl heavy chain K322A) (SEQ ID NO:18)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC A

VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4A-450 (huIgGl heavy chain K322A without C-terminal K) (SEQ ID NO: 19)

QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGV PTYAQ GFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC A

VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

4A-313 (huIgGl heavy chain constant region) (SEQ ID NO:30)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKS LSLSPGK

4A-313 (huIgGl heavy chain constant region without C-terminal K) (SEQ ID NO:31)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKS LSLSPG

4A-313 (hlgGl heavy chain constant region P331S) (SEQ ID NO:32)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKS LSLSPGK

4A-313 (hlgGl heavy chain constant region P331S without C-terminal K) (SEQ ID NO:33)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKS LSLSPG

4A-313 (huIgGl heavy chain constant region N325S/L328F) (SEQ ID NO:34)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSL SLSPGK

4A-313 (hlgGl heavy chain constant region N325S/L328F without C-terminal K) (SEQ ID NO:35)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSL SLSPG

4A-313 (huIgGl heavy chain constant region K322A) (SEQ ID NO:36)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD WLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKS LSLSPGK

4A-313 (huIgGl heavy chain constant region K322A without C-terminal K) (SEQ ID NO:37)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQD

WLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKS LSLSPG

Human IgGl Light Chain Constant Region (SEQ ID NO:20)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDST

YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

4A-450 - full length light chain (SEQ ID NO:21)

DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVS KLDSGVPDR

FSGSGSGTDFTLKISRVEAEDVGVYYCWOGIDFHQTFGGGTKVEIKRTVAAPSVFIF PPSDEOLKS

GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKV

YACEVTHQGLS SPVTKSFNRGEC

4A-313 - full length light chain (SEQ ID NO:38)

EIVLTQSPATLSLSPGERATLSCRASESVDNYGVSRMNWYQQKPGQAPRLLIYGASN OGSGIPAR

FSGSGSGTDFTLTISSLEPEDFAVYYCOOSKEVPPTFGGGTKVEIKRTVAAPSVFIF PPSDEOLKSG

TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVY

ACEVTHQGLSSPVTKSFNRGEC