Anti-pilra antibodies, uses thereof, and related methods and reagents

Abstract

Provided herein are anti-PILRA antibodies with the highly desirable selectivity: having comparable binding to cynomolgus and human PILRA proteins, but much weaker binding to human PILRB protein, as well as binding to both PILRA G78 and R78 variants. The binding and selectivity profiles of the antibodies described herein allow for them to be used in animal studies (e.g., monkeys) without the need to rely on a surrogate molecule and also when treating subjects with either PILRA variant. Further described herein, for the first time, are biological discoveries related to PILRA and the effects of reducing PILRA signaling in cells.

Description

ANTI-PILRA ANTIBODIES, USES THEREOF, AND RELATED METHODS AND REAGENTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 509,422, filed June 21, 2023, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Paired immunoglobulin-like type 2 receptor alpha (PILRA) is a transmembrane receptor that is expressed on various immune cells, such as microglia and is believed to function in inhibitory cell signaling pathways. A missense variant (G78R) of PILRA is associated with reduced risk of Alzheimer’s disease. The G78R variant alters the interaction of residues essential for sialic acid engagement, resulting in reduced binding for several PILRA ligands.

[0003] There remains a need for therapeutic agents that modulate PILRA activity.BRIEF SUMMARY

[0004] Described herein are antibodies that selectively bind to both cynomolgus monkey PILRA (cynoPILRA) and hPILRA, but may have comparatively lower binding to human PILRB (hPILRB). We have identified epitopes that allow for the desired selectivity. This selectivity profile is highly advantageous, but also very challenging, given the high homology between cynoPILRA and hPILRB. Having comparable binding between cyno and human proteins allows for conducting studies in monkeys without having to rely on a surrogate molecule. Binding to PILRB, by contrast, is not desired, because PILRB while having an extracellular domain highly similar to PILRA, has a different intracellular domain, which is expected to have different or even opposing activity. Further, the antibodies described herein also comprise a modified Fc polypeptide that can bind to a CD98 heavy chain (CD98hc) protein.

[0005] Furthermore, certain antibodies with this selectivity profile described herein also bind to, and have activity at, both PILRA variant forms (G78 and R78), thus ensuring thatthey can be used in a variety of populations, given that the frequency of each variant varies highly in different parts of the world.

[0006] In addition to developing highly useful antibodies, we have also made significant discoveries related to PILRA biology, including discovery of certain downstream effectors of PILRA signaling, and for the first time, have characterized effects of reducing signaling by the PILRA receptor in microglia. These insights allow, for the first time, linking PILRA ligand blocking to biological effects in cells, which provides novel approaches for both drug discovery as well as measuring biological impacts of known PILRA binders on cells and animals.

[0007] In one aspect, the disclosure features an isolated antibody comprising:(a) a variable region that specifically binds to a paired immunoglobulin-like type 2 receptor alpha (PILRA);(b) a first Fc polypeptide; and(c) a second Fc polypeptide, wherein the first Fc polypeptide is modified to specifically bind to a CD98 heavy chain (CD98hc) protein.

[0008] In some embodiments, the antibody specifically binds to both the G78 variant of the PILRA and the R78 variant of the PILRA. In some embodiments, the binding affinity for the G78 variant of the PILRA and the binding affinity for the R78 variant of the PILRA are within 50-fold (e.g., within 40-fold, 30-fold, 20-fold, 10-fold, 5-fold, or 2-fold) of each other.

[0009] In some embodiments, the PILRA is a cynomolgus monkey paired immunoglobulin-like type 2 receptor alpha (cynoPILRA). In some embodiments, the binding affinity for the cynoPILRA is at least 2-fold (e.g., at least 4-fold, 6-fold, 8-fold, 10-fold, 12- fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold) stronger than the binding affinity for a human paired immunoglobulin-like type 2 receptor beta (hPILRB).

[0010] In some embodiments, the PILRA is a human paired immunoglobulin-like type 2 receptor alpha (hPILRA).

[0011] In some embodiments, the variable region of the antibody comprises:(1) a CDR-H1 sequence comprising at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the sequence of GYTFTEYYMY(SEQ ID NO: 10), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 10;(2) a CDR-H2 sequence comprising at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the sequence of RIDPEDGGTD (SEQ ID NO: 11), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 11;(3) a CDR-H3 sequence comprising at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the sequence of TIRGTVFAF (SEQ ID NO: 12), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 12;(4) a CDR-L1 sequence comprising at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the sequence of RASEDIFNGLA (SEQ ID NO: 13), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 13;(5) a CDR-L2 sequence comprising at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the sequence of NAKTLHT (SEQ ID NO: 14), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 14; and(6) a CDR-L3 sequence comprising at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the sequence of QQYYDYPLT (SEQ ID NO: 15), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 15.

[0012] In some embodiments, the amino acid substitutions are conservative substitutions.

[0013] In some embodiments, the variable region of the antibody comprises: a CDR-H1 comprising the sequence of SEQ ID NO: 10 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 10; a CDR-H2 comprising the sequence of SEQ ID NO: 11 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 11; a CDR-H3 comprising the sequence of SEQ ID NO: 12 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 12; a CDR-L1 comprising the sequence of SEQ ID NO: 13 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 13; a CDR-L2 comprising the sequence of SEQ ID NO: 14 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 14; and a CDR-L3 comprising the sequence of SEQ ID NO: 15 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 15.

[0014] In some embodiments, the variable region of the antibody comprises: a CDR-H1 comprising the sequence of SEQ ID NO: 10; a CDR-H2 comprising the sequence of SEQ ID NO: 11; a CDR-H3 comprising the sequence of SEQ ID NO: 12; a CDR-L1 comprising the sequence of SEQ ID NO: 13; a CDR-L2 comprising the sequence of SEQ ID NO: 14; and a CDR-L3 comprising the sequence of SEQ ID NO: 15.

[0015] In some embodiments, the variable region of the antibody comprises a heavy chain variable region (VH) sequence that has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to SEQ ID NO: 16. In certain embodiments, the VH sequence comprises a sequence of SEQ ID NO: 16.

[0016] In some embodiments, the variable region of the antibody comprises a light chain variable region (VL) sequence that has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to SEQ ID NO: 17. In certain embodiments, the VL sequence comprises a sequence of SEQ ID NO: 17.

[0017] In some embodiments, the variable region of the antibody comprises: a VH sequence comprising SEQ ID NO: 16 and a VL sequence comprising SEQ ID NO: 17.

[0018] In some embodiments, the variable region of the antibody comprises a heavy chain variable region (VH) sequence that has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to SEQ ID NO:21. In certain embodiments, the VH sequence comprises a sequence of SEQ ID NO:21.

[0019] In some embodiments, the variable region of the antibody comprises a light chain variable region (VL) sequence that has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to SEQ ID NO:22. In certain embodiments, the VL sequence comprises a sequence of SEQ ID NO:22.

[0020] In some embodiments, the variable region comprises: a VH sequence comprising SEQ ID NO:21 and a VL sequence comprising SEQ ID NO:22.

[0021] In some embodiments, the CD98hc protein is a human CD98hc protein. In some embodiments, the CD98hc protein forms a complex with LAT1 (SLC7A5), LAT2 (SLC7A8), y+LATl (SLC7A7), y+LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11). In certain embodiments, the CD98hc protein forms a complex with LAT1 (SLC7A5).

[0022] In some embodiments of the antibodies described herein, the first Fc polypeptide comprises a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO:64. In certain embodiments, the first Fc polypeptide comprises: Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering.

[0023] In some embodiments of the antibodies described herein, the antibody has improved brain uptake compared to an antibody having a wild-type Fc dimer. In some embodiments, the antibody has at least two-fold (e.g., at least two, three, four, five, six, or seven-fold) improved brain uptake compared to the antibody having a wild-type Fc dimer. In particular embodiment, the antibody has between two-fold and seven-fold (e.g., between two-fold and six-fold, between two-fold and five-fold, between two-fold and four-fold, between two-fold and three-fold, between three-fold and seven-fold, between four-fold and seven-fold, between five-fold and seven-fold, or between six-fold and seven-fold; two-fold, three-fold, four-fold, five-fold, six-fold, or seven-fold) improved brain uptake compared to the antibody having a wild-type Fc dimer.

[0024] In some embodiments, the first Fc polypeptide has a T366W substitution and the second Fc polypeptide has T366S, L368A, and Y407V substitutions, according to EU numbering. In some embodiments, the first Fc polypeptide has T366S, L368A, and Y407V substitutions and the second Fc polypeptide has a T366W substitution, according to EU numbering.

[0025] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces effector function. In certain embodiments, the modification that reduces effector function comprises the substitutions of Ala at position 234 and Ala at position 235, according to EU numbering. In certain embodiments, the modification that reduces effector function comprises the substitution of Gly at position 329.

[0026] In some embodiments of the antibodies described herein, the antibody comprises:(i) a first heavy chain comprising: (1) a VH sequence having a CDR-H1, a CDR-H2, a CDR- H3 of SEQ ID NOS: 10-12, respectively, and (2) a first Fc polypeptide comprisingmodifications for CD98hc-binding, a knob mutation, and modifications that reduce or eliminate effector function;(ii) a second heavy chain comprising: (1) a VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and (2) a second Fc polypeptide comprising hole mutations, and modifications that reduce or eliminate effector function; and(iii) first and second light chains each comprising a VL sequence having a CDR-L1, a CDR- L2, a CDR-L3 of SEQ ID NOS: 13-15, respectively.

[0027] In some embodiments of the antibodies described herein, the antibody comprises:(i) the first heavy chain comprises: (1) the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and (2) the first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, Ala at position 442, Trp at position 366, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 66;(ii) the second heavy chain comprises: (1) the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and (2) the second Fc polypeptide comprising T366S, L368A, Y407V, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (c.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 63; and(iii) the first and second light chains each comprising the VL sequence having a CDR-L1, a CDR-L2, a CDR-L3 of SEQ ID NOS: 13-15, respectively. In some embodiments, the VH sequence in each of the first and second heavy chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 16. In some embodiments, the VL sequence in each of the first and second light chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 17. In some embodiments, the VH sequence in each of the first and second heavy chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 16 and the VL sequence in each of thefirst and second light chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 17.

[0028] In some embodiments of the antibodies described herein, the antibody comprises:(i) the first heavy chain comprises: (1) the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 16, and (2) the first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, Ala at position 442, Trp at position 366, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 66;(ii) the second heavy chain comprises: (1) the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 16, and (2) the second Fc polypeptide comprising T366S, L368A, Y407V, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 63; and(iii) the first and second light chains each comprising the VL sequence having a CDR-L1, a CDR-L2, a CDR-L3 of SEQ ID NOS: 13-15, respectively, and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO: 17.

[0029] In some embodiments of the antibodies described herein, the antibody comprises:(i) the first heavy chain comprises a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 18;(ii) the second heavy chain comprises a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 19; and(iii) the first and second light chains each comprising a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO:20.

[0030] In some embodiments of the antibodies described herein, the antibody comprises:(i) the first heavy chain comprises the VH sequence having a CDR-H1, a CDR-H2, a CORED of SEQ ID NOS: 10-12, respectively, and the first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, Ala at position 442, Trp at position 366, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 66;(ii) the second heavy chain comprises the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and the second Fc polypeptide comprising T366S, L368A, Y407V, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 63; and(iii) the first and second light chains each comprising the VL sequence having a CDR-L1, a CDR-L2, a CDR-L3 of SEQ ID NOS: 13-15. In some embodiments, the VH sequence in each of the first and second heavy chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:21. In some embodiments, the VL sequence in each of the first and second light chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:22. In some embodiments, the VH sequence in each of the first and second heavy chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:21 and the VL sequence in each of the first and second light chains comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:22.

[0031] In some embodiments of the antibodies described herein, the antibody comprises:(i) the first heavy chain comprises the VH sequence having a CDR-H1, a CDR-H2, a CDR- H3 of SEQ ID NOS: 10-12, respectively, and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:21, and the first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422,Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, Ala at position 442, Trp at position 366, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 66;(ii) the second heavy chain comprises the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:21, and the second Fc polypeptide comprising T366S, L368A, Y407V, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO: 63; and(iii) the first and second light chains each comprising the VL sequence having a CDR-L1, a CDR-L2, a CDR-L3 of SEQ ID NOS: 13-15, respectively, and at least 90% e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to SEQ ID NO:22.

[0032] In some embodiments, the antibody comprises:(i) the first heavy chain comprises a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO:23;(ii) the second heavy chain comprises a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO:24; and(iii) the first and second light chains each comprising a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0033] In some embodiments of the antibodies described herein, the antibody antagonizes hPILRA activity. In certain embodiments, the antibody blocks binding of a sialyated protein to hPILRA. Examples of sialyated proteins include, but are not limited to, a sialyated NPDC1, PANP, HSV-1 gB, COLECI 2, C4a, C4b, DAG1, and Clec4g.

[0034] In some embodiments, the antibody enhances phosphorylation of EGFR or STAT3, or decreases phosphorylation of STAT1. In certain embodiments, the antibody enhances cell migration. In certain embodiments, the antibody enhances microglia migration. In certainembodiments, the antibody enhances anti-inflammatory gene or protein expression. In particular embodiments, the antibody enhances IL1RN gene expression. In certain embodiments, the antibody reduces pro-inflammatory cytokine protein expression or secretion. In particular embodiments, the antibody reduces TNF, IL 6, and / or IP 10 expression. In some embodiments, the antibody increases cellular respiration. In some embodiments, the antibody increases mitochondrial respiration. In certain embodiments, the antibody increases ATP production. In certain embodiments, the antibody increases fatty acid metabolism. In certain embodiments, the antibody does not activate peripheral immune cells. In some embodiments, the antibody does not activate neutrophils and monocytes. In some embodiments, the antibody increases lipid storage or lipid levels in microglia.

[0035] In another aspect, the disclosure provides a pharmaceutical composition comprising the isolated antibody described herein and a pharmaceutically acceptable carrier.

[0036] In another aspect, the disclosure provides one or more polynucleotides comprising one or more nucleic acid sequences encoding the heavy chains and / or light chains of any one of the isolated antibodies described herien.

[0037] In another aspect, the disclosure provides one or more vectors comprising the one or more polynucleotides described herein.

[0038] In another aspect, the disclosure provides a host cell comprising the one or more polynucleotides described herein or the one or more vectors described herein.

[0039] In another aspect, the disclosure provides a method for producing an isolated antibody, comprising culturing the host cell described herein under conditions in which the isolated antibody described herein is expressed.

[0040] In another aspect, the disclosure provides a method of treating a neurodegenerative disease in a subject, comprising administering to the subject an isolated antibody described herein or the pharmaceutical composition described herein. In certain embodiments, the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis / parkinsonism- dementia complex of Guam (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillarytangles with calcification, Down’s syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadelopean PSP, Hallevorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington’s disease, inclusion-body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Parkinson’s disease, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle only dementia.

[0041] In another aspect, the disclosure provides a method for determining whether an antibody has activity at a PILRA protein, the method comprising:(a) contacting a cell that expresses the PILRA protein with the antibody;(b) either prior to, concurrently with, or following step (a), contacting a cell of the same type as in step (a) having lower or no PILRA expression with the antibody; and(c) measuring one of the following: phosphorylated STAT3 (pSTAT3) level and phosphorylated STAT1 (pSTATl) level, in both cells, wherein a change in the level of one of these measurements between the cells indicates that the antibody has activity at the PILRA protein of step (a), and wherein the antibody specifically binds to a CD98 heavy chain (CD98hc) protein.

[0042] In some embodiments of the method, step (c) measures pSTAT3 level. In certain embodiments, the cell of step (a) naturally expresses the PILRA protein.

[0043] In certain embodiments, the cell having lower PILRA expression has the PILRA protein knocked-out. In particular embodiments, the cell is a HEK cell. In particular embodiments, the cell is an iMicroglia. In particular embodiments, the cell is a PILRA LoF iMicroglia.

[0044] In certain embodiments, the cell of step (a) is engineered or modified to express or overexpress the PILRA protein. In some embodiments, the cell having lower PILRA expression naturally expresses the PILRA protein or is not engineered or modified to express the PILRA protein.

[0045] In some embodiments of the method, the antibody is from a library of antibodies. In some embodiments, the antibody is known to bind the PILRA protein. In some embodiments, it is unknown whether the antibody binds the PILRA protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIGS. 1A-1C show graphs of binding curves for anti-PILRA antibodies to parental HEK293 cells (FIG. 1A) or to hPILRA overexpressing HEK293 cells (FIGS. IB and 1C). Anti-PILRA antibodies bound to hPILRA expressed on HEK293 G78 PILRA OE cells and to HEK293 R78 PILRA OE cells in a dose-dependent manner. The data is expressed as median fluorescence intensity fluorescence obtained via FACS assay technology.

[0047] FIG. ID is a graph of a binding curve for anti-PILRA antibodies to hPILRB-DAP12 expressing HEK293 cells. No binding of anti-PILRA antibodies to HEK293 cells expressing hPILRB-DAP12.

[0048] FIGS. 2 A and 2B are graphs of binding curves for anti-PILRA antibodies to parental CHO KI cells (FIG. 2A) or cynoPILRA expressing CHO cells (FIG. 2B). Anti- PILRA antibodies bind to CHO cells expressing cynoPILRA. There was no binding to parental CHO-K1 cells.

[0049] FIG. 3A is a bar graph illustrating STAT3 Y705 expression in wild-type human iMicroglia and PILRA loss-of-function (LoF) iMicroglia. PILRA LoF iMicroglia had increased levels of phosphorylated STAT3 Y705 compared to wild-type human iMicroglia in serum-free media. Graphs show spot intensity expression above background as mean+ / - SEM. N=2 technical replicates. P>0.01, 2-way ANOVA.

[0050] FIGS. 3B and 3C are graphs of dose curves of anti-PILRA antibodies for phosphorylated STAT3 (Y705) expression in hPILRA expressing HEK293 cells. Dosedependent induction of pSTAT3 Y705 was observed in HEK293 cells expressing hPILRA G78 (FIG. 3B) or R78 (FIG. 3C). Anti-PILRA antibodies were dose titrated on HEK293 cells expressing hPILRA G78 or R78 and pSTAT3 Y705 induction was measured after 30 minutes. EC50 values showed nM potency for induction of pSTAT3 Y705 for each antibody. Data is presented as mean + / - SD fold expression over isotype control, n = 2 experiments.

[0051] FIG. 3D is a graph of dose curves of anti-PILRA antibodies for phosphorylated STAT3 (Y705) expression in HEK293 cells. Anti-PILRA antibodies did not induce pSTAT3 Y705 in parental HEK293 cells.

[0052] FIGS. 4A and 4B are bar graphs showing LPS-induced gene expression changes in wild type and PILRA LoF iMicroglia cells. PILRA LoF suppressed LPS-induced gene expression changes in TNF (FIG. 4 A) and CXCL10 (FIG. 4B) in PILRA LoF iMicrogliarelative to wild-type iMicroglia. Data is presented as mean + / - SEM, n=3 technical replicates.

[0053] FIGS. 4C and 4D are bar graphs showing LPS-induced cytokine expression changes in wild type and PILRA LoF iMicroglia cells. PILRA LoF suppressed LPS-induced cytokine expression changes in TNF alpha (FIG. 4C) and IP-10 (FIG. 4D) in PILRA LoF iMicroglia relative to wild-type iMicroglia. Data is presented as mean + / - SEM, n=3 technical replicates.

[0054] FIGS. 4E and 4F are bar graphs showing LPS-induced gene expression changes in wild type iMicroglia cells in the presence of anti-PILRA antibodies. Anti-PILRA antibodies attenuated LPS-induced IP-10 (FIG. 4E) and TNF alpha (FIG. 4F) cytokine secretion in wildtype iMicroglia, mimicking the phenotype observed in PILRA LoF iMicroglia. Data is presented as mean + / - SEM, n=3 technical replicates.

[0055] FIG. 5 shows a sequence alignment of the ECD and stalk region sequences of cynoPILRA, hPILRA, and hPILRB (positions are determined with reference to the sequence of SEQ ID NO: 1).

[0056] FIGS. 6A and 6B show systemic and brain pharmacokinetics profiles of Ab CL 2, which binds to CD98hc, compared to Ab CL 1, which does not bind to CD98hc.

[0057] FIGS. 7A and 7B show localization of anti-PILRA antibody (huIgG) on microglia, but not on neurons, as measured by huIgG signal on Ibal -positive microglia surface and NeuN-positive neuron surface. HuIgG signal in vehicle group indicates the background.

[0058] FIG. 8 shows PILRA LoF microglia exhibited increased lipid droplet accumulation compared to wild-type microglia (n=6 experiments). Complementation of PILRA in KO cells (PILRA KO + OE) reduced lipid droplets to wild-type levels (n=3 experiments).

[0059] FIGS. 9A and 9B show that anti-PILRA antibody Ab CL 2 significantly increased phospholipid synthesis-associated MG and DG lipids in microglia in vivo.

[0060] FIGS. 9C-9E show that plasmalogens (PE(P-18:0 / 18: l)), pyroglutamic acid, and phenylalanine, which are associated with anti-inflammation, were upregulated by Ab CL 2 in microglia in vivo.DETAILED DESCRIPTIONI. INTRODUCTION

[0061] PILRA is an inhibitory transmembrane receptor that is expressed on the cell surface of various immune cells, such as microglia, monocytes, macrophages, dendritic cells, and neutrophils. Without being bound to a particular theory, it is believed that upon ligand binding, PILRA acts as an inhibitory receptor by recruiting cytoplasmic phosphatases, such as PTPN6 / SHP-1 and PTPN11 / SHP-2, via their SH2 domains that block signal transduction through dephosphorylation of signaling molecules. A missense variant (G78R) of PILRA alters the sialic acid binding pocket of PILRA, leading to reduced binding of PILRA to several of its ligands, one of which is the sialyated herpes simplex virus type 1 glycoprotein B (HSV-1 gB). HSV-1 infection has been suggested to be present in some Alzheimer’s disease patients. The G78R variant of PILRA is proposed to protect individuals from Alzheimer’s disease by antagonizing or reducing PILRA signaling, thereby modifying microglial responses.

[0062] As detailed in the Examples section below, antibodies have been generated that specifically bind to human PILRA (hPILRA). Such antibodies are demonstrated to modulate one or more microglial functions regulated by PILRA. The antibodies disclosed herein have highly desirable characteristics. These include antibodies that selectively bind to both cynomolgus monkey (“cyno”) PILRA (cynoPILRA) and hPILRA, but may have comparatively lower binding to human PILRB (hPILRB). This is highly advantageous, but also very challenging, given the high homology between cynoPILRA and hPILRB. Having comparable binding between cyno and human PILRA allows for conducting studies in monkeys without having to employ a surrogate molecule. Binding to PILRB is not desired, because PILRB is thought to have different or opposing activity as compared to PILRA, given differences in their respective intracellular domains. Certain antibodies described herein can bind to cynoPILRA with a binding affinity that is within 100-fold (e.g., within 90- fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 5-fold, or 2-fold) relative to the binding affinity for hPILRA. The antibodies may also have a binding affinity for hPILRA that is at least 10-fold (e.g., at least 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold) stronger than its binding affinity for hPILRB.

[0063] The antibodies further comprise a modified Fc polypeptide containing mutations that permit binding of the Fc polypeptide to a CD98hc protein (CD98hc protein from, e.g., a human). In some aspects, the antibodies disclosed herein are able to bind, through the modified Fc polypeptide, to a CD98 heavy chain (CD98hc) protein (e.g., expressed on the surface of a brain endothelial cell (BECs)) and can thereby cross the blood-brain barrier (BBB) more effectively than antibodies lacking the CD98hc-binding Fc mutations.

[0064] CD98 is highly expressed on brain endothelial cells and therefore a promising target for receptor mediated transcytosis (RMT). CD98 is a heterodimer formed between CD98hc (4F2 heavy chain) and a CD98 light chain. To date six CD98 light chains have been identified, i.e., LAT1 (SLC7A5, 4F2 light chain), LAT2 (SLC7A8), y+LATl (SLC7A7), y+LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11). In complex, CD98 heavy chain transports the light chain to the cell surface where it functions as a large neutral amino acid transporter which preferentially transports branched-chain (valine, leucine, isoleucine) and aromatic (tryptophan, tyrosine, phenylalanine) amino acids. Leveraging the CD98 receptor- mediated transcytosis pathway, the modified Fc polypeptides containing a CD98hc-binding site described herein can be used to transport therapeutic agents across the BBB. This approach can substantially improve brain uptake of the therapeutic agents and is therefore highly useful for treating disorders and diseases where brain delivery is advantageous. In addition, this approach can be used to provide brain uptake and delivery to specific extracellular or neuro-oncology targets in the brain. For example, CD98hc-binding polypeptides provided herein may be used to target such extracellular targets or neuro- oncology targets while retaining wild-type effector function, if so desired. In addition, such CD98hc-binding polypeptides provided herein may be used to target such extracellular targets in cases where neuronal uptake is undesireable (e.g., the target is an antigen or plaque such as Abeta, Tau or alpha-synuclein). The CD98hc-binding polypeptides provided herein have distinct kinetic, biodistribution, and safety properties that may provide optimized and fit-for-purpose BBB transport platforms for protein-based therapeutics.

[0065] In certain embodiments, the antibodies disclosed herein further comprise mutations in an Fc polypeptide that reduce or eliminate effector function and mutations that increase in vivo half-life, e.g., by increasing binding of antibody Fc to Fc neonatal receptor (FcRn).

[0066] We have also discovered that antibodies binding to certain amino acid residues of the PILRA sequence can convey desirable properties. These include residues 63, 64, 78, 106,143, 116-118, and 182-186 of the PILRA sequence (e.g., SEQ ID NO: 1). In particular examples, we show that antibodies that bind to an epitope that includes (i) G78, K106, and E143 or (ii) T63 and A64 of hPILRA can also bind cynoPILRA but have reduced binding to hPILRB.II. DEFINITIONS

[0067] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0068] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0069] As used herein, the term “PILRA” refers to a paired immunoglobulin-like type 2 receptor alpha protein that is encoded by the gene PILRA. As used herein, a “PILRA” or “PILRA protein” refers to a native (i.e., wild-type) PILRA protein of any vertebrate, such as but not limited to human, non-human primates (e.g., cynomolgus monkey), rodents (e.g., mice, rat), and other mammals. In some embodiments, a PILRA protein is a human PILRA (hPILRA) protein having the sequence of SEQ ID NO: 1 :MGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYP WELATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNL QKQDQSVYFCRVELDTRSSGRQQWQSIEGTKLSITQAVTTTTQRPSSMTTTWRLSST TTTTGLRVTQGKRRSDSWHISLETAVGVAVAVTVLGIMILGLICLLRWRRRKGQQRT KATTPAREPFQNTEEPYENIRNEGQNTDPKLNPKDDGIVYASLALSSSTSPRAPPSHRP LKSPQNETLYSVLKA.

[0070] In some embodiments, a PILRA protein is a cynomolgus monkey PILRA (cynoPILRA) protein having the sequence of SEQ ID NO:2:MGRPLLLPLLLPLLPLLLPPAFLQPGGSAGSGPSGPYGVTQRKHLSAPMGGSVEIPFSF YHPWELAAAPNMKISWRRGNFHGEFFYRTRPAFIHEDYSNRLLLNWTEGQDRGLLR IWNLRKEDQSVYFCRVELDTRRSGRQRWQSIEGTKLTITQAVTTTTQRPSSMTTTRRP SSATTTAGLRVTQGKRHSDSWHLSLKTAVGVTVAVAVLGIMILGLICLLRWRRRKGQQRTKATTPAKEPFQNTEEPYENIRNEGQNTDPKPNPKDDGIVYASLALS S STSPRVP PSHHPLKSPQNETLYSVLKV.

[0071] As used herein, the term “PILRB” refers to a paired immunoglobulin-like type 2 receptor beta protein that is encoded by the gene PILRB. As used herein, a “PILRB” or “PILRB protein” refers to a native (i.e., wild-type) PILRB protein of any vertebrate, such as but not limited to human, non-human primates (e.g., cynomolgus monkey), rodents (e.g., mice, rat), and other mammals. In some embodiments, a PILRB protein is a human PILRB (hPILRB) protein having the sequence of SEQ ID NO:3:MGRPLLLPLLLLLQPPAFLQPGGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYP WELAIVPNVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQESGFLRISNLR KEDQSVYFCRVELDTRRSGRQQLQSIKGTKLTITQAVTTTTTWRPSSTTTIAGLRVTE SKGHSESWHLSLDTAIRVALAVAVLKTVILGLLCLLLLWWRRRKGSRAPSSDF.

[0072] As used herein, the term “CD98hc” or “CD98 heavy chain” refers to 4F2 cellsurface antigen heavy chain and is encoded by the SLC3A2 gene. CD98hc is also known as 4F2 heavy chain. The human CD98hc sequence is set forth in SEQ ID NO:5 and UNIPROT Accession No. P08195. CD98hc sequences from other species are also known (e.g., mouse, UNIPROT Accession No. P10852 and cynomolgus monkey, UNIPROT Accession No. G8F3Z0).

[0073] As used herein, the term “anti-PILRA antibody” refers to an antibody that specifically binds to a PILRA protein (e.g., human PILRA). In some embodiments, the anti- PILRA antibody comprises a modified Fc polypeptide that specifically binds to a CD98hc protein (e.g., hCD98hc).

[0074] As used herein, the term “antibody” refers to a protein with an immunoglobulin fold that specifically binds to an antigen via its variable regions. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, including full-length antibodies as well as single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The term “antibody,” as used herein, also includes antibody fragments that retain binding specificity via its variable regions, including but not limited to Fab, F(ab’)2, Fv, scFv, and bivalent scFv. Antibodies can contain light chains that are classified as either kappa or lambda. Antibodies can contain heavy chains that are classifiedas gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0075] As used herein, the term “full-length antibody” generally refers to an immunoglobulin molecule that has four polypeptide chains: two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain is composed of, from N- terminus to C-terminus, a heavy chain variable region (VH), a CHI constant domain, a hinge region, a CH2 constant domain, and a CH3 constant domain. Each light chain is composed of, from N-terminus to C-terminus, a light chain variable region (VL) and a CL constant domain. A Fab domain or fragment is formed from VH, CHI, VL, and CL domains. A full- length antibody can also be described as having two Fab domains and an Fc domain, where the Fc domain comprises two Fc polypeptides and each Fc polypeptide can include a CH2 domain, a CH3 domain, and may contain at least part of the hinge region of the antibody.

[0076] As used herein, the term “anti-PILRA antigen binding portion” refers to an antigen binding segment or entity that specifically binds to a PILRA protein (e.g., hPILRA and / or cynoPILRA). The terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably herein and refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen (e.g., a PILRA protein) via its variable region. Examples of antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains), F(ab’)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), single chain Fv (scFv), disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), a VL (light chain variable region), and a VH (heavy chain variable region).

[0077] The term “variable region” or “variable domain” refers to a domain in an antibody heavy chain or light chain that is derived from a germline Variable (V) gene, Diversity (D) gene, or Joining (J) gene (and not derived from a Constant (Cp and C5) gene segment), and that gives an antibody its specificity for binding to an antigen. Typically, an antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity determining regions.”

[0078] The term “complementarity determining region” or “CDR” refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are typically referred to asCDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which it is found, whereas a VL CDR1 or CDR-L1 is the CDR1 from the variable region of the light chain of the antibody in which it is found.

[0079] The “framework regions” or “FRs” of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences.

[0080] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), AbM, and observed antigen contacts (“Contact”). In some embodiments, CDRs are determined according to the Contact definition. See, MacCallum et al., J. Mol. BioL, 262:732-745 (1996). In some embodiments, CDRs are determined by a combination of Kabat, Chothia, and / or Contact CDR definitions.

[0081] The term “epitope” refers to the area or region of an antigen to which the CDRs of an antibody specifically binds and can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids. For example, where the target is a protein, the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope). In some embodiments, the epitope is phosphorylated at one amino acid (e.g., at a serine or threonine residue).

[0082] As used herein, the phrase “recognizes an epitope,” as used with reference to an anti-PILRA antibody, means that the antibody CDRs interact with or specifically bind to the antigen (i.e., the PILRA protein) at that epitope or a portion of the antigen containing that epitope.

[0083] A “monoclonal antibody” refers to antibodies produced by a single clone of cells or a single cell line and consisting of or consisting essentially of antibody molecules that are identical in their primary amino acid sequence.

[0084] A “polyclonal antibody” refers to an antibody obtained from a heterogeneous population of antibodies in which different antibodies in the population bind to different epitopes of an antigen.

[0085] A “chimeric antibody” refers to an antibody molecule in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen-binding site (i.e., variable region, CDR, or portion thereof) is linked to a constant region of a different or altered class, effector function and / or species, or in which the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity (e.g., CDR and framework regions from different species). In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human). Methods for producing chimeric antibodies are described in the art.

[0086] A “humanized antibody” is a chimeric immunoglobulin derived from a non-human source (e.g., murine) that contains minimal sequences derived from the non-human immunoglobulin outside the CDRs. In general, a humanized antibody will comprise at least one (e.g., two) antigen-binding variable domain(s), in which the CDR regions substantially correspond to those of the non-human immunoglobulin and the framework regions substantially correspond to those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin sequence. Methods of antibody humanization are known in the art.

[0087] A “human antibody” or a “fully human antibody” is an antibody having human heavy chain and light chain sequences, typically derived from human germline genes. In some embodiments, the antibody is produced by a human cell, by a non-human animal that utilizes human antibody repertoires (e.g., transgenic mice that are genetically engineered to express human antibody sequences), or by phage display platforms.

[0088] The term “specifically binds” refers to a molecule (e.g., an antibody or an antigenbinding portion thereof) that binds to an epitope or target with stronger affinity, stronger avidity, and / or greater duration to that epitope or target in a sample than it binds to anotherepitope or non-target compound (e.g., a structurally different antigen). In some embodiments, an antibody (or an antigen-binding portion thereof) that specifically binds to an epitope or target is an antibody (or an antigen-binding portion thereof) that binds to the epitope or target with at least 1.5-fold stronger affinity than other epitopes or non-target compounds, e.g., at least 1.5-fold, 2.5-fold, 5-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, or stronger affinity. The term “specific binding,” “specifically binds to,” or “is specific for” a particular epitope or target, as used herein, can be exhibited, for example, by a molecule having an equilibrium dissociation constant KD for the epitope or target to which it binds of, e.g., IO’4M or smaller, e.g., IO'5M, IO'6M, IO'7M, IO'8M, IO'9M, IO'10M, IO'11M, or IO'12M. It will be recognized by one of skill that an antibody that specifically binds to a target (e.g., a PILRA protein (e.g., a hPILRA and / or a cynoPILRA)) from one species may also specifically bind to orthologs of that target.

[0089] The term “binding affinity” is used herein to refer to the strength of a non-covalent interaction between two molecules, e.g., between an antibody (or an antigen-binding portion thereof) and an antigen. Thus, for example, the term may refer to 1 : 1 interactions between an antibody (or an antigen-binding portion thereof) and an antigen, unless otherwise indicated or clear from context. Binding affinity may be quantified by measuring an equilibrium dissociation constant (KD), which refers to the dissociation rate constant (kd, time'1) divided by the association rate constant (ka, time'1M'1). KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g., using Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, “binding affinity” includes not only formal binding affinities, such as those reflecting 1 : 1 interactions between an antibody (or an antigen-binding portion thereof) and an antigen, but also apparent affinities for which KD values are calculated that may reflect avid binding.

[0090] The term “cross-reacts,” as used herein, refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody was raised. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from another species than the antigen against which the antibody was raised. As a non-limiting example, an anti-PILRA antibody as described herein that is raised against a human PILRA peptide can exhibit cross-reactivity with a PILRA peptide or protein from a different species (e.g., cynomolgus monkey or mouse).

[0091] The term “modulate” refers to changing or altering one or more properties of a protein or a cell. Properties of a cell can be altered as a result of altering one or more properties of a protein (e.g., a PILRA protein) of the cell, i.e., by binding to the protein of the cell. Properties of a cell that can be modulated include, but are not limited to, cell growth, migration, survival, signaling, phagocytosis, and biomarker secretion. For example, a molecule that binds to a PILRA protein of a cell can cause one or more downstream signaling responses or activities of the cell as a result of PILRA-binding, thus, the molecule is said to modulate the signaling responses or activities of the cell. In some embodiments, the term “modulate” can refer to an increase or decrease in the signaling response or activity of the cell as a result of PILRA-binding, relative to the signaling response or activity of the cell without PILRA-binding. Examples of changes in signaling responses or activities of a cell as a result of PILRA-binding include, but are not limited to, changes phosphorylated STAT3 (pSTAT3) level, phosphorylated STAT1 (pSTATl) level, phosphorylated EGFR (pEGFR) level, cadherin expression, integrin expression, and cell (e.g., microglia) migration.

[0092] The terms “CH3 domain” and “CH2 domain” as used herein refer to immunoglobulin constant region domain polypeptides. In the context of IgG antibodies, a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme. CH2 and CH3 domain polypeptides may also be numbered by the IMGT (ImMunoGeneTics) numbering scheme in which the CH2 domain numbering is 1-110 and the CH3 domain numbering is 1-107, according to the IMGT Scientific chart numbering (IMGT website). CH2 and CH3 domains are part of the Fc region of an immunoglobulin. In the context of IgG antibodies, an Fc region refers to the segment of amino acids from about position 231 to about position 447 as numbered according to the EU numbering scheme. As used herein, the term “Fc region” may also include at least a part of a hinge region of an antibody. An exemplary partial hinge region sequence is DKTHTCPPCP (SEQ ID NO:59).

[0093] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a polypeptide “corresponds to” anamino acid in the SEQ ID NO: 1 when the residue aligns with the amino acid in SEQ ID NO: 1 when optimally aligned to SEQ ID NO: 1. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0094] As used herein, the term “Fc polypeptide” refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide that is characterized by an Ig fold as a structural domain. An Fc polypeptide contains constant region sequences including at least the CH2 domain and / or the CH3 domain and may contain at least part of the hinge region, but does not contain a variable region.

[0095] A “modified Fc polypeptide” refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion or insertion, as compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0096] The term “isolated,” as used with reference to a nucleic acid or protein (e.g., antibody), denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated nucleic acid or protein (e.g., antibody) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0097] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally occurring a-amino acids include, without limitation, D- alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D- phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D- lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline(D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D- tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. “Amino acid analogs” refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0098] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0099] The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.

[0100] The terms “polynucleotide” and “nucleic acid” interchangeably refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and doublestranded DNA, single- and double-stranded RNA, and hybrid molecules having mixtures of single- and double-stranded DNA and RNA.

[0101] The terms “conservative substitution” and “conservative mutation” refer to an alteration that results in the substitution of an amino acid with another amino acid that can becategorized as having a similar feature. Examples of categories of conservative amino acid groups defined in this manner can include: a “charged / polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gin (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R), and His (Histidine or H); an “aromatic group” including Phe (Phenylalanine or F), Tyr (Tyrosine or Y), Trp (Tryptophan or W), and (Histidine or H); and an “aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Vai (Valine or V), Leu (Leucine or L), He (Isoleucine or I), Met (Methionine or M), Ser (Serine or S), Thr (Threonine or T), and Cys (Cysteine or C). Within each group, subgroups can also be identified. For example, the group of charged or polar amino acids can be sub-divided into sub-groups including: a “positively-charged sub-group” comprising Lys, Arg and His; a “negatively-charged sub-group” comprising Glu and Asp; and a “polar sub-group” comprising Asn and Gin. In another example, the aromatic or cyclic group can be subdivided into sub-groups including: a “nitrogen ring sub-group” comprising Pro, His and Trp; and a “phenyl sub-group” comprising Phe and Tyr. In another further example, the aliphatic group can be sub-divided into sub-groups, e.g., an “aliphatic non-polar sub-group” comprising Vai, Leu, Gly, and Ala; and an “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys. Examples of categories of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained. In some embodiments, hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acid, e.g., in the active site, to preserve hydrophobicity.

[0102] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0103] For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment orusing publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0104] The terms “subject,” “individual,” and “patient,” as used interchangeably herein, refer to a mammal, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the subject, individual, or patient is a human.

[0105] The terms “treating,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of a neurodegenerative disease (e.g., Alzheimer’s disease or another neurodegenerative disease described herein), including any objective or subjective parameter such as abatement, remission, improvement in patient survival, increase in survival time or rate, diminishing of symptoms or making the disease more tolerable to the patient, slowing in the rate of degeneration or decline, or improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.

[0106] The term “pharmaceutically acceptable excipient” refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to a buffer, carrier, or preservative.

[0107] As used herein, a “therapeutic amount” or “therapeutically effective amount” of an agent (e.g., an antibody as described herein) is an amount of the agent that treats, alleviates, abates, or reduces the severity of symptoms of a disease in a subject. A “therapeutic amount” of an agent (e.g., an antibody as described herein) may improve patient survival, increase survival time or rate, diminish symptoms, make an injury, disease, or condition (e.g., a neurodegenerative disease) more tolerable, slow the rate of degeneration or decline, or improve a patient’s physical or mental well-being.

[0108] The term “administer” refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, an antibody as described herein is administered intravenously.

[0109] The term “control” or “control value” refers to a reference value or baseline value. Appropriate controls can be determined by one skilled in the art. In some instances, control values can be determined relative to a baseline within the same subject or experiment. In other instances, the control value can be determined relative to a control subject (e.g, a healthy control or a disease control) or an average value in a population of control subjects (e.g, healthy controls or disease controls, e.g., a population of 10, 20, 50, 100, 200, 500, 1000 control subjects or more).III. ANTI-PILRA ANTIBODIES

[0110] In one aspect, antibodies that specifically bind to a paired immunoglobulin-like type 2 receptor alpha (PILRA) protein (e.g., a hPILRA and / or a cynoPILRA protein) and comprise a modified Fc polypeptide that specifically binds to a CD98 heavy chain (CD98hc) protein are provided. In some embodiments, the antibody specifically binds to a hPILRA protein. In some embodiments, an anti-PILRA antibody is selective for PILRA over other PILR receptors (e.g., a paired immunoglobulin-like type 2 receptor beta (PILRB)).[OHl] In some embodiments, an anti-PILRA antibody is an antibody that comprises one or more complementarity determining region (CDR), heavy chain variable region, and / or light chain variable region sequences as disclosed herein. In some embodiments, an anti-PILRA antibody comprises one or more CDR, heavy chain variable region, and / or light chain variable region sequences as disclosed herein and further comprises one or more functional characteristics as disclosed herein, e.g., an antibody that antagonizes PILRA activity (e.g., blocks binding of a ligand to hPILRA, alters phosphorylation of downstream proteins (e.g., increase phosphorylation of STAT3), increases anti-inflammatory gene or protein expression, and / or reduces cytokine protein expression). In some embodiments, the anti-PILRA antibodies described herein also comprise a modified Fc polypeptide that specifically binds to a CD98hc protein (e.g., a hCD98hc).

[0112] In some embodiments, the anti-PILRA antibody is a fully human antibody. In some embodiments, the anti-PILRA antibody is a chimeric antibody. In some embodiments, the anti-PILRA antibody is a humanized and / or affinity matured antibody. In some embodiments, the anti-PILRA antibody is a fully human antibody.Anti-PILRA Antibody Sequences

[0113] In some embodiments, an anti-PILRA antibody comprises one or more CDRs selected from the group consisting of:(a) a heavy chain CDR1 (CDR-H1) sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of GYTFTEYYMY (SEQ ID NO: 10), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 10;(b) a heavy chain CDR2 (CDR-H2) sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of RIDPEDGGTD (SEQ ID NO: 11), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 11;(c) a heavy chain CDR3 (CDR-H3) sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of TIRGTVFAF (SEQ ID NO: 12), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12;(d) a light chain CDR1 (CDR-L1) sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of RASEDIFNGLA (SEQ ID NO: 13), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 13;(e) a light chain CDR2 (CDR-L2) sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of NAKTLHT (SEQ ID NO: 14), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 14; and(f) a light chain CDR3 (CDR-L3) sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of QQYYDYPLT (SEQ ID NO: 15), orhaving up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:15.

[0114] In some embodiments, an anti-PILRA antibody comprises one or more CDRs selected from the group consisting of:(a) a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 10;(b) a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 11;(c) a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 12;(d) a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 13;(e) a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 14; and(f) a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 15.

[0115] In some embodiments, an anti-PILRA antibody comprises one or more CDRs selected from the group consisting of:(a) a CDR-H1 sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of GYTFIGFYIH (SEQ ID NO:26), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:26;(b) a CDR-H2 sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of WINPESGDTT (SEQ ID NO:27), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:27;(c) a CDR-H3 sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of GNWNFPDTFDF (SEQ ID NO:28), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:28;(d) a CDR-L1 sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of RSSQSISIYLN (SEQ ID NO:29), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:29;(e) a CDR-L2 sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of VASSLQS (SEQ ID NO:30), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:30; and(f) a CDR-L3 sequence having at least 80% sequence identity (e.g., at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of QQSYSAPFT (SEQ ID NO:31), or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:31.

[0116] In some embodiments, an anti-PILRA antibody comprises one or more CDRs selected from the group consisting of:(a) a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:26;(b) a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:27;(c) a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:28;(d) a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:29;(e) a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:30; and(f) a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:31.

[0117] In some embodiments, an anti-PILRA antibody comprises two, three, four, five, or all six of (a)-(f). In some embodiments, an anti-PILRA antibody comprises the CDR-H1 of (a), the CDR-H2 of (b), and the CDR-H3 of (c). In some embodiments, an anti-PILRA antibody comprises the CDR-L1 of (d), the CDR-L2 of (e), and the CDR-L3 of (f). In some embodiments, a CDR having up to two amino acid substitutions has one amino acid substitution (e.g., one conservative substitution) relative to the reference sequence. In some embodiments, a CDR having up to two amino acid substitutions has two amino acid substitutions (e.g., two conservative substitutions) relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

[0118] In some embodiments, an anti-PILRA antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments, an anti-PILRA antibody comprises a VH sequence comprising the amino acid sequence of SEQ ID NO: 16.

[0119] In some embodiments, an anti-PILRA antibody comprises a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments, an anti-PILRA antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 17.

[0120] In some embodiments, an anti-PILRA antibody comprises: a heavy chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments, an anti-PILRA antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17.

[0121] In some embodiments, an anti-PILRA antibody comprises: a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively, and a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17.

[0122] In some embodiments, an anti-PILRA antibody comprises:(i) a first heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18;(ii) a second heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19; and(iii) first and second light chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20.

[0123] In some embodiments, an anti-PILRA antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:21. In some embodiments, an anti-PILRA antibody comprises a VH sequence comprising the amino acid sequence of SEQ ID NO:21.

[0124] In some embodiments, an anti-PILRA antibody comprises a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:22. Insome embodiments, an anti-PILRA antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO:22.

[0125] In some embodiments, an anti-PILRA antibody comprises: a heavy chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:22. In some embodiments, an anti-PILRA antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22.

[0126] In some embodiments, an anti-PILRA antibody comprises: a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively, and a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22.

[0127] In some embodiments, an anti-PILRA antibody comprises:(i) a first heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23;(ii) a second heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24; and(iii) first and second light chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25.

[0128] In some embodiments, an anti-PILRA antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:32. In some embodiments, an anti-PILRA antibody comprises a VH sequence comprising the amino acid sequence of SEQ ID NO: 32.

[0129] In some embodiments, an anti-PILRA antibody comprises a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:33. In some embodiments, an anti-PILRA antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO:33.

[0130] In some embodiments, an anti-PILRA antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:32, and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:33. In some embodiments, an anti-PILRA antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:32, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:33.

[0131] In some embodiments, an anti-PILRA antibody comprises: a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS:26-28, respectively, and a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS:29-31, respectively, and a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:33.

[0132] In some embodiments, an anti-PILRA antibody comprises:(i) a first heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:34;(ii) a second heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:35; and(iii) first and second light chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:36.

[0133] In some embodiments, a heavy chain sequence, or a portion thereof, and / or a light chain sequence, or a portion thereof, is derived from an anti-PILRA antibody described herein.Binding Characteristics of Anti-PILRA Antibodies

[0134] In some embodiments, an antibody as described herein that specifically binds to a PILRA protein (e.g., a hPILRA protein) binds to PILRA that is expressed on a cell (e.g., a cell line that endogenously expresses PILRA, such as immune cells, or a cell line that has been engineered to express PILRA, e.g., as described in the Examples section below). In some embodiments, an antibody that specifically binds to a PILRA protein as described herein binds to purified or recombinant PILRA protein of a portion thereof, or to a chimeric protein comprising PILRA or a portion thereof.

[0135] In some embodiments, an antibody that specifically binds to human PILRA protein exhibits cross-reactivity with one or more other PILRA proteins of another species. In some embodiments, an antibody that specifically binds to human PILRA protein exhibits crossreactivity with a cynomolgus monkey (“cyno”) PILRA protein (cynoPILRA).

[0136] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g, ELISA assay), immunoprecipitation, surface plasmon resonance (e.g, Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, CA)), and western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art, and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, kinetic exclusion assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0137] In some embodiments, an anti-PILRA antibody described herein specifically binds to a cynomolgus monkey paired immunoglobulin-like type 2 receptor alpha (cynoPILRA), wherein the binding affinity for the cynoPILRA is at least 2-fold (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold) stronger than the binding affinity for a human paired immunoglobulin-like type 2 receptor beta (hPILRB).

[0138] As described herein, in some embodiments, an anti-PILRA antibody described herein exhibits cross-reactivity with both hPILRA and cynoPILRA. In some embodiments, an anti-PILRA antibody described herein binds to both hPILRA and cynoPILRA.

[0139] In certain embodiments, the binding affinity of the anti-PILRA antibody for the cynoPILRA is within 100-fold (e.g., within 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50- fold, 40-fold, 30-fold, 20-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2- fold, or 1.5 fold) relative to the binding affinity for the hPILRA. In particular embodiments, the anti-PILRA antibody binds to the hPILRA with a binding affinity of between 0.1 nM and500 nM (e.g., between 0.1 nM and 400 nM, between 0.1 nM and 300 nM, between 0.1 nM and 200 nM, or between 0.1 nM and 100 nM). In particular embodiments, the anti-PILRA antibody binds to the hPILRA with a binding affinity of between 0.1 nM and 100 nM e.g., between 0.1 nM and 90 nM, between 0.1 nM and 80 nM, between 0.1 nM and 70 nM, between 0.1 nM and 60 nM, between 0.1 nM and 50 nM, between 0.1 nM and 40 nM, between 0.1 nM and 30 nM, between 0.1 nM and 20 nM, between 0.1 nM and 10 nM, between 0.1 nM and 5 nM, between 0.1 nM and 1 nM, between 1 nM and 100 nM, between 5 nM and 100 nM, between 10 nM and 100 nM, between 20 nM and 100 nM, between 30 nM and 100 nM, between 40 nM and 100 nM, between 50 nM and 100 nM, between 60 nM and 100 nM, between 70 nM and 100 nM, between 80 nM and 100 nM, or between 90 nM and 100 nM).

[0140] In some embodiments, an anti-PILRA antibody described herein selectively binds to hPILRA and / or cynoPILRA over hPILRB. In particular embodiments, the binding affinity of the antibody for the hPILRA is at least 10-fold (e.g., at least 10-fold, 20-fold, 40-fold, 60- fold, 80-fold, 100-fold, 120-fold, 140-fold, 160-fold, 180-fold, 200-fold, 220-fold, 240-fold, 260-fold, 280-fold, or 300-fold) stronger than the binding affinity for the hPILRB.Epitopes Recognized by Anti-PILRA Antibodies

[0141] In some embodiments, an anti-PILRA antibody recognizes an epitope of human PILRA that is the same or substantially the same as the epitope recognized by an antibody clone as described herein. As used herein, the term “substantially the same,” as used with reference to an epitope recognized by an antibody clone as described herein, means that the anti-PILRA antibody recognizes an epitope that is identical, within, or nearly identical to (e.g., has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or has one, two, or three amino acid substitutions,e.g., conservative substitutions, relative to), or has substantial overlap with (e.g., at least 50%, 60%, 70%, 80%, 90%, or 95% overlap with) the epitope recognized by the antibody clone as described herein.

[0142] In some embodiments, an anti-PILRA antibody recognizes an epitope of human PILRA that is the same or substantially the same as the epitope recognized by an antibody described herein (e.g., an antibody comprising a CDR-H1, a CDR-H2, and a CDR-H3 having the amino acid sequences of SEQ ID NOS: 10-12, respectively, and a CDR-L1, a CDR-L2, and a CDR-L3 having the amino acid sequences of SEQ ID NOS: 13-15, respectively) and variants of the same.

[0143] In some embodiments, an anti-PILRA antibody recognizes an epitope of human PILRA within the extracellular domain (ECD) of PILRA, e.g., the ECD comprising amino acids 20 to 143 of SEQ ID NO: 1. In some embodiments, an anti-PILRA antibody binds to human PILRA at an epitope within the stalk region of PILRA. In some embodiments, an anti-PILRA antibody is an antagonist that inhibits PILRA signaling.

[0144] In some embodiments, an anti-PILRA antibody binds to one or more amino acids at one or more of the following positions: 63, 64, 78, 106, 143, 116-118, and 182-186, wherein the positions are determined with reference to SEQ ID NO: 1. In particular embodiments, an anti-PILRA antibody described herein binds to one or more amino acids at one or more of the following positions in SEQ ID NO: 1 : 63, 64, 78, 106, 143, 116-118, and 182-186. FIG. 5 shows an alignment of the ECD and stalk region sequences of cynoPILRA, hPILRA, and hPILRB. In certain embodiments, the anti-PILRA antibody binds to one or more amino acids at one or more of the following positions: 78, 106, and 143 of hPILRA. In particular embodiments, the anti-PILRA antibody binds to G78, K106, and / or E143 of SEQ ID NO: 1. In some embodiments, the anti-PILRA antibody binds to G78 of SEQ ID NO:1. In some embodiments, the anti-PILRA antibody binds to R78 of SEQ ID NO:4. In some embodiments, the anti-PILRA antibody binds to KI 06 of SEQ ID NO:1. In some embodiments, the anti-PILRA antibody binds to E143 of SEQ ID NO: 1. In particular embodiments, the anti-PILRA antibody binds to G78, K106, and E143 of SEQ ID NO: 1. In particular embodiments, the anti-PILRA antibody binds to R78, K106, and E143 of SEQ ID NO:4.

[0145] In certain embodiments, the anti-PILRA antibody binds to one or more amino acids at one or more of the following positions: 63 and 64 of hPILRA. In particular embodiments,the anti-PILRA antibody binds to T63 and / or A64 of SEQ ID NO:1. In some embodiments, the anti-PILRA antibody binds to T63 of SEQ ID NO: 1. In some embodiments, the anti- PILRA antibody binds to A64 of SEQ ID NO:1. In particular embodiments, the anti-PILRA antibody binds to T63 and A64 of SEQ ID NO: 1.

[0146] In certain embodiments, the anti-PILRA antibody binds to one or more amino acids at one or more of the following positions: 106 and 116-118 of hPILRA. In some embodiments, the anti-PILRA antibody binds to QI 16, KI 17, and / or QI 18 of SEQ ID NO: 1 (e.g, QI 16, KI 17, and QI 18).

[0147] In some embodiments, an anti-PILRA antibody recognizes an epitope within stalk 2 region of hPILRA, e.g., QGKRR (SEQ ID NO:7) from positions 182-186 of SEQ ID NO: 1. In some embodiments, an anti-PILRA antibody recognizes an epitope comprising 1, 2, 3, or 4 amino acids within residues 182-186 of SEQ ID NO:1. In some embodiments, an anti- PILRA antibody recognizes an epitope comprising 2, 3, or 4 contiguous amino acids within residues 182-186 of SEQ ID NO: 1. In some embodiments, an anti-PILRA antibody recognizes an epitope comprising all five amino acids within residues 182-186 of SEQ ID NO: 1. In some embodiments, the anti-PILRA antibody binds to Q182, G183, K184, R185, and / or R186 of SEQ ID NO: 1 (e.g., Q182, G183, K184, R185, and R186).

[0148] In some embodiments, an anti-PILRA antibody recognizes an epitope within stalk 1 region of hPILRA, e.g., TTQRPSSM (SEQ ID NO:6) from positions 156-163 of SEQ ID NO: 1. In some embodiments, an anti-PILRA antibody recognizes an epitope comprising 1, 2, 3, 4, 5, 6, or 7 amino acids within residues 156-163 of SEQ ID NO: 1. In some embodiments, an anti-PILRA antibody recognizes an epitope comprising 2, 3, 4, 5, 6, or 7 contiguous amino acids within residues 156-163 of SEQ ID NO: 1. In some embodiments, an anti-PILRA antibody recognizes an epitope comprising all eight amino acids within residues 156-163 of SEQ ID NO: 1. In some embodiments, the anti-PILRA antibody binds to T156, T157, Q158, R159, P160, S161, S162, and / or M163 of SEQ ID NO: 1 (e.g., T156, T157, Q158, R159, P160, S 161, S162, and M163).Cross-Reactivity

[0149] In certain embodiments, an anti-PILRA antibody recognizes one or more epitopes that are conserved between hPILRA and cynoPILRA. In some embodiments, an anti-PILRA antibody binds to one or more amino acids at one or more of the following positions in hPILRA and / or in cynoPILRA: 64, 78, 139, 143, 156-163, and 182-185, wherein thepositions are determined with reference to SEQ ID NO: 1. In particular embodiments, an anti- PILRA antibody binds to one or more amino acids at one or more of the following positions in hPILRA and in cynoPILRA: 64, 78, 139, 143, 156-163, and 182-185, wherein the positions are determined with reference to SEQ ID NO: 1. In particular embodiments, the anti-PILRA antibody binds to A64, G78, W139, E143, T156, T157, Q158, R159, P160, S161, S162, M163, Q182, G183, K184, and / or R185 of hPILRA having the sequence of SEQ ID NO: 1 and A68, G82, W143, E147, T160, T161, Q162, R163, P164, S165, S166, M167, Q186, G187, K188, and / or R189 of cynoPILRA having the sequence of SEQ ID NO:2.

[0150] In particular embodiments, the anti-PILRA antibody binds to A64 of hPILRA having the sequence of SEQ ID NO: 1 and A68 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to G78 of hPILRA having the sequence of SEQ ID NO: 1 and G82 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to R78 of hPILRA having the sequence of SEQ ID NO:4 and G82 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to W139 of hPILRA having the sequence of SEQ ID NO: 1 and W143 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to E143 of hPILRA having the sequence of SEQ ID NO: 1 and E147 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to the same one or more amino acids within TTQRPSSM (SEQ ID NO:6) of both hPILRA (e.g., positions 156-163 of SEQ ID NO: 1) and cynoPILRA (e.g., positions 160 to 167 of SEQ ID NO:2). In particular embodiments, the anti-PILRA antibody binds to the same one or more amino acids within QGKR (SEQ ID NO:8) of both hPILRA (e.g., positions 182-185 of SEQ ID NO:1) and cynoPILRA (e.g., positions 186 to 189 of SEQ ID NO:2).

[0151] In particular embodiments, the anti-PILRA antibody binds to G78, K106, E143 of hPILRA having the sequence of SEQ ID NO:1 and G82, DUO, E147 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to R78, K106, E143 of hPILRA having the sequence of SEQ ID NO:4 and G82, DI 10, E147 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti- PILRA antibody binds to T63 and A64 of hPILRA having the sequence of SEQ ID NO: 1 and A67 and A68 of cynoPILRA having the sequence of SEQ ID NO:2. In particular embodiments, the anti-PILRA antibody binds to one or more positions within QGKRR (SEQ ID NO:7) of hPILRA (e.g., positions 182-186 of SEQ ID NO: 1) and the same correspondingpositions with QGKRH (SEQ ID NO: 9) of cynoPILRA (e.g., positions 186 to 190 of SEQ ID NO:2).Functional Characteristics of Anti-PILRA Antibodies

[0152] In some embodiments, an anti-PILRA antibody (e.g., an antibody having one or more CDR, heavy chain variable region, and / or light chain variable region sequences as disclosed) functions in one or more activities as disclosed herein. For example, in some embodiments, an anti-PILRA antibody antagonize or reduce PILRA activity, i.e., PILRA activity induced by a ligand.

[0153] In certain embodiments, an anti-PILRA antibody blocks the binding of a ligand to hPILRA. In particular embodiments, an anti-PILRA antibody blocks the binding of a sialyated protein to hPILRA, e.g., a sialyated form of any of the following proteins: neural proliferation differentiation and control protein 1 (NPDC1), PILRA-associated neural protein (PANP; PIANP), herpes simplex virus type 1 glycoprotein B (HSV-1 gB), collectin-12 (COLECI 2), complement component 4 A (C4a), complement component 4B (C4b), dystroglycan 1 (dystrophin-associated glycoprotein 1; DAG1), and c-type lectin domain family member G (Clec4g).

[0154] Further, in some embodiments, an anti-PILRA antibody alters phosphorylation of one or more downstream proteins, e.g., increases phosphorylation of EGFR or STAT3, or decreases phosphorylation of STAT1. In some embodiments, an anti-PILRA antibody induces or increases phosphorylation of one or more downstream proteins (e.g., EGFR or STAT3) if the level of downstream protein phosphorylation in a sample treated with the anti- PILRA antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value. In some embodiments, an anti-PILRA antibody induces phosphorylation of one or more downstream proteins (e.g., EGFR or STAT3) if the level of downstream protein phosphorylation in a sample treated with the anti-PILRA antibody is increased by at least 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to a control value. In some embodiments, an anti-PILRA antibody decreases phosphorylation of one or more downstream proteins (e.g., STAT1) if the level of downstream protein phosphorylation in a sample treated with the anti-PILRA antibody is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value. In some embodiments, an anti-PILRA antibody decreases phosphorylation of one or more downstream proteins (e.g., STAT1) if the level of downstream protein phosphorylation in a sample treated with the anti- PILRA antibody is decreased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to a control value.

[0155] In some embodiments, the control value is the level of downstream protein phosphorylation in an untreated sample (e.g., a sample comprising a PILRA-expressing cell that has not been treated with an anti -PILRA antibody, or a sample from a subject that has not been treated with an anti-PILRA antibody), or a sample that has been treated with a PILRA ligand but not an anti-PILRA antibody, or a sample treated with an appropriate non-PILRA- binding antibody.

[0156] For detecting and / or quantifying phosphorylation in a sample, in some embodiments, an immunoassay is used. In some embodiments, the immunoassay is an enzyme immunoassay (EIA), enzyme multiplied immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemiluminescence immunoassay (ECL). In some embodiments, phosphorylation is detected and / or quantified using an immunoassay that utilizes an amplified luminescent proximity homogenous assay (AlphaLISA®, PerkinElmer Inc.).

[0157] In some embodiments, phosphorylation is measured using a sample that comprises one or more cells, e.g., one or more PILRA-expressing cells (e.g., a cell line that endogenously expresses PILRA, such as human IPSC-derived microglia, or a cell line that has been engineered to express PILRA, e.g., as described in the Examples section below). In some embodiments, the sample comprises a fluid, e.g., blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises tissue (e.g., lung, brain, kidney, spleen, nervous tissue, or skeletal muscle) or cells from such tissue. In some embodiments, the sample comprises endogenous fluid, tissue, or cells (e.g., from a human or non-human subject).

[0158] Further, in some embodiments, an anti-PILRA antibody increases anti-inflammatory gene or protein expression. For example, an anti-PILRA antibody enhances IL1RN gene expression. In some embodiments, an anti-PILRA antibody enhances anti-inflammatory gene or protein expression if the level of anti-inflammatory gene or protein expression in a sampletreated with the anti-PILRA antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value. In other embodiments, an anti-PILRA antibody reduces pro- inflammatory cytokine protein expression or secretion. For example, an anti-PILRA antibody reduces TNF, IL-6, and / or IP- 10 expression. In some embodiments, an anti-PILRA antibody reduces cytokine protein expression if the level of cytokine protein expression in a sample treated with the anti-PILRA antibody is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value.

[0159] Further, in some embodiments, an anti-PILRA antibody enhances cell migration and / or cell function (e.g., for microglia, including IPSC-derived microglia and disease- associated microglia). Disease-associated microglia and methods of detecting disease- associated microglia are described in Keren-Shaul et al., Cell, 2017, 169: 1276-1290. In some embodiments, an anti-PILRA antibody enhances cell migration of one or more cell types (e.g., microglia, monocytes, or neutrophils). In some embodiments, an anti-PILRA antibody enhances cell function (e.g., ATP production, fatty acid metabolism, and / or cellular respiration) of one or more cell types (e.g., microglia, monocytes, or neutrophils). In some embodiments, an anti-PILRA antibody enhances the cell migration and / or cell function of microglia. In some embodiments, an anti-PILRA antibody enhances the cell migration and / or cell function of disease-associated microglia.

[0160] In some embodiments, an anti-PILRA antibody enhances cell migration and / or cell function if the level of activity in a sample treated with the anti-PILRA antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value. In some embodiments, an anti-PILRA antibody enhances cell migration and / or cell function if the level of activity in a sample treated with the anti-PILRA antibody is increased by at least 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to a control value. In some embodiments, the control value is the level of activity (e.g., migration or function) in an untreated sample (e.g., a sample that has not been treated with an anti- PILRA antibody), a sample that has been treated with a PILRA ligand but not an anti-PILRA antibody, or a sample treated with an appropriate non-PILRA-binding antibody.

[0161] In some embodiments, cell migration is measured using a chemotaxis assay. Chemotaxis assays are known in the art. In some embodiments, the cell migration assay (e.g., chemotaxis assay) is performed on a sample comprising cells that endogenously express PILRA, such as human IPSC-derived microglia. In some embodiments, the cell migration assay (e.g., chemotaxis assay) is performed on a sample comprising cells that have been engineered to express PILRA. In some embodiments, the cell migration assay is performed on a sample comprising cells in which PILRA has been deleted or rendered functionally inactive. In some embodiments, cell migration is measured using a chemotaxis assay as described in the Examples section below.

[0162] In some embodiments, cell function is measured using a functional assay that is appropriate for that cell. In some embodiments, an anti -PILRA antibody increases fatty acid metabolism (e.g., fatty acid oxidation). In some embodiments, an anti-PILRA antibody enhances cellular ATP production. In some embodiments, an anti-PILRA antibody enhances cellular respiration (e.g., mitochondrial or non-mitochondrial respiration). Changes in cellular ATP production and / or repiration can be evaluated using one or more assays, e.g., as described in the Examples section below.IV. FC POLYPEPTIDES AND MODIFICATIONS THEREOF

[0163] In some embodiments, the anti-PILRA antibodies described herein comprise a modified Fc polypeptide that specifically binds to a CD98hc protein. In some embodiments, the anti-PILRA antibodies comprise a first Fc polypeptide and a second Fc polypeptide, in which one of the first and second Fc polypeptides is modified to bind to a CD98hc protein. In some embodiments, one or both Fc polypeptides in the anti-PILRA antibody can further contain amino acid modifications that promote heterodimerization of the two Fc polypeptides in an Fc polypeptide dimer, modulate effector function, extend serum half-life, influence glycosylation, and / or reduce immunogenicity in humans. In some embodiments, the two Fc polypeptides present in the anti-PILRA antibody independently have an amino acid sequence identity of at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% to a corresponding wildtype Fc polypeptide (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc polypeptide) (e.g., SEQ ID NO:55). Examples and descriptions of modified Fc polypeptides (e.g., CD98hc-binding Fc polypeptides) can be found, e.g., in International Patent Publication No. PCT / US2022 / 053220, which is incorporated herein by reference in its entirety.

[0164] In some embodiments, only one Fc polypeptide of the Fc polypeptide dimer in an anti-PILRA antibody described herein has amino acid modifications that result in binding of the Fc polypeptide to a CD98hc. In some embodiments, both Fc polypeptides of the Fc polypeptide dimer in an anti-PILRA antibody described herein can have amino acid modifications that result in binding of the Fc polypeptides to a CD98hc.Fc Polypeptide Modifications for BBB Receptor Binding

[0165] Provided herein are anti-PILRA antibodies comprising an Fc polypeptide dimer that are capable of being transported across the BBB. Such an antibody comprises a modified Fc polypeptide that binds to a BBB receptor, e.g., a CD98hc. BBB receptors are expressed on BBB endothelia, as well as other cell and tissue types. In some embodiments, the BBB receptor is a CD98hc protein.

[0166] Amino acid residues designated in various Fc modifications, including those introduced in a modified Fc polypeptide that binds to a BBB receptor, e.g., CD98hc protein, are numbered herein using EU index numbering. Any Fc polypeptide, e.g., an IgGl, IgG2, IgG3, or IgG4 Fc polypeptide, may have modifications, e.g., amino acid substitutions, in one or more positions as described herein. In some embodiments, the domain that is modified for BBB (e.g., CD98hc) receptor-binding activity is a human Ig CH3 domain, such as an IgGl CH3 domain. The CH3 domain can be of any IgG subtype, i.e., from IgGl, IgG2, IgG3, or IgG4. In the context of IgGl antibodies, a CH3 domain refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme.

[0167] In some embodiments, a BBB (e.g., CD98hc) receptor-binding Fc polypeptide present in an anti-PILRA antibody described herein comprises at least eleven, twelve, thirteen, fourteen, or fifteen substitutions in a set of amino acid positions consisting of 378, 380, 382, 383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442. In some embodiments, the substitutions are selected from Ser, Vai, Asp, Glu, or Tyr at position 378, Leu, He, Met, Ala, Gin, Vai, or Lys at position 380, Asn, Ser, Leu, Met, Pro, Tyr, Lys, Ala, or Thr at position 382, Thr, Phe, Asn, Pro, Asp, Leu, His, or Gin at position 383, Lys, Arg, His, He, Leu, Phe, Tyr, Vai, or Gin at position 384, Phe or Tyr at position 385, Vai, Leu, Ala, He, Phe, Tyr, Ser, Thr, His, Arg, or Glu at position 386, Leu or He at position 387, Asp, Gin, Ala, Thr, His, or Vai at position 389, Thr, Vai, or Ala at position 391, Glu, Gin, or Ala at position 421, Leu, Met, lie, Thr, or Pro at position 422, Ala atposition 424, Asn at position 426, Leu, Thr, Pro, Tyr, Phe, He, Ala, Lys, His, or Trp at position 428, Ser at position 434, Leu, Vai, His, Phe, Pro, Arg or Trp at position 436, Phe or Trp at position 438, Leu, Pro, Glu, Asn, Vai, Ala, He, or Asp at position 440, Pro at position 441, and Ala, Vai, Met, Gin, Phe, Pro, Leu, Tyr, Lys, Arg, His, or Met at position 442.

[0168] In some embodiments, a BBB (e.g., CD98hc) receptor-binding Fc polypeptide present in an anti-PILRA antibody described herein comprises Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering.

[0169] In some embodiments, the BBB (e.g., CD98hc) receptor-binding Fc polypeptide has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 64 and in some embodiments has Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering. In particular embodiments, the BBB (e.g, CD98hc) receptor-binding Fc polypeptide has the sequence of SEQ ID NO:64. In some embodiments of the anti-PILRA antibodies described herein, one of the two Fc polypeptides in the Fc polypeptide dimer can be a BBB (e.g, CD98hc) receptor-binding Fc polypeptide having the sequence of SEQ ID NO: 64, while the other Fc polypeptide in the Fc polypeptide dimer can have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 55). In other embodiments of the anti-PILRA antibodies described herein, both Fc polypeptides in the Fc polypeptide dimer can be a BBB (e.g., CD98hc) receptor-binding Fc polypeptide having the sequence of SEQ ID NO:64.Fc Polypeptide Modifications for Heterodimerization

[0170] In some embodiments, the Fc polypeptides present in the anti-PILRA antibody include knob and hole mutations to promote heterodimer formation and hinder homodimer formation. Generally, the modifications introduce a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and thus hinder homodimer formation. Protuberances are constructedby replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). In some embodiments, such additional mutations are at a position in the Fc polypeptide that does not have a negative effect on binding of the polypeptide to a BBB receptor, e.g., CD98hc.

[0171] In one illustrative embodiment of a knob and hole approach for dimerization, position 366 (numbered according to the EU numbering scheme) of one of the Fc polypeptides present in the anti-PILRA antibody comprises a tryptophan in place of a native threonine. The other Fc polypeptide in the dimer has a valine at position 407 (numbered according to the EU numbering scheme) in place of the native tyrosine. The other Fc polypeptide may further comprise a substitution in which the native threonine at position 366 (numbered according to the EU numbering scheme) is substituted with a serine and a native leucine at position 368 (numbered according to the EU numbering scheme) is substituted with an alanine. Thus, one of the Fc polypeptides of an anti-PILRA antibody described herein has the T366W knob mutation and the other Fc polypeptide has the Y407V mutation, which is typically accompanied by the T366S and L368A hole mutations.

[0172] In some embodiments, one or both Fc polypeptides present in an anti-PILRA antibody described herein may also be engineered to contain other modifications for heterodimerization, e.g., electrostatic engineering of contact residues within a CH3-CH3 interface that are naturally charged or hydrophobic patch modifications.

[0173] For example, in some embodiments, an anti-PILRA antibody described herein can contain an Fc polypeptide dimer that has one Fc polypeptide having the T366W knob mutation and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:60 and the other Fc polypeptide having the T366S, L368A, and Y407V hole mutations and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:61. In certain embodiments, one or both Fc polypeptides in the Fc polypeptide dimer can be a CD98hc-binding Fc polypeptide. In particular embodiments, an anti-PILRA antibody described herein can contain an Fc polypeptide dimer that has (i) a first Fc polypeptide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:61, wherein thesequence includes Ser at position 366, Ala at position 368, and Vai at position 407, according to EU numbering; and (ii) a second Fc polypeptide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:65, wherein the sequence includes Trp at position 366 and in some embodiments has Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering.

[0174] In particular embodiments, an anti-PILRA antibody described herein can contain (i) a first Fc polypeptide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 60, wherein the sequence includes Trp at position 366, according to EU numbering, and (ii) a second Fc polypeptide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 67, wherein the sequence includes Ser at position 366, Ala at position 368, and Vai at position 407 and in some embodiments has Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering.Fc Polypeptide Modifications for Modulating Effector Function

[0175] In some embodiments, one or both Fc polypeptides present in an anti-PILRA antibody described herein may comprise modifications that reduce effector function, i.e., having a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an effector cell that mediates the effector function. Examples of antibody effector functions include, but are not limited to, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptor), and B-cell activation. Effector functions may vary with the antibody class. For example, native human IgGl and IgG3 antibodies can elicit ADCC and CDC activities upon binding to an appropriate Fc receptor present on an immune system cell; and native human IgGl, IgG2, IgG3, and IgG4 can elicit ADCP functions upon binding to the appropriate Fc receptor present on an immune cell.

[0176] In some embodiments, one or both Fc polypeptides in an Fc polypeptide dimer can comprise modifications that reduce or eliminate effector function. Illustrative Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 234 and 235 and / or at position 329, according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides comprise Ala residues at positions 234 and 235 (also referred to as “LALA” herein). In some embodiments, one or both Fc polypeptides comprise Gly residue at position 329 (also referred to as “P329G” or “PG” herein) or Ser residue at position 329 (also referred to as “P329S” or “PS” herein). In some embodiments, one or both Fc polypeptides comprise Ala residues at positions 234 and 235, and Gly residue at position 329 (also referred to as “LALA PG” herein). In some embodiments, one or both Fc polypeptides comprise Ala residues at positions 234 and 235, and Ser residue at position 329 (also referred to as “LALA PS” herein).

[0177] Additional Fc polypeptide mutations that modulate an effector function include, but are not limited to, the following: position 329 may have a mutation in which proline is substituted with a glycine or arginine or an amino acid residue large enough to destroy the Fc / Fcy receptor interface that is formed between proline 329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcyRIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, and P331S, according to the EU numbering scheme. Multiple substitutions may also be present, e.g., L234A and L235A of a human IgGl Fc region; L234A, L235A, and P329G of a human IgGl Fc region; L234A, L235A, and P329S of a human IgGl Fc region; S228P and L235E of a human IgG4 Fc region; L234A and G237A of a human IgGl Fc region; L234A, L235A, and G237A of a human IgGl Fc region; V234A and G237A of a human IgG2 Fc region; L235A, G237A, and E318A of a human IgG4 Fc region; and S228P and L236E of a human IgG4 Fc region, according to the EU numbering scheme. In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, e.g., substitutions at positions 298, 333, and / or 334, according to the EU numbering scheme.Fc Polypeptide Modifications for Extending Serum Half-Life

[0178] In some embodiments, modifications to enhance serum half-life can be introduced into any Fc polypeptides described herein. For example, in some embodiments, one or both Fc polypeptides in an Fc polypeptide dimer can comprise M428L and N434S substitutions (also referred to as LS substitutions), as numbered according to the EU numbering scheme.Alternatively, one or both Fc polypeptides in an Fc polypeptide dimer can have an N434S or N434A substitution. Alternatively, one or both Fc polypeptides in an Fc polypeptide dimer can have an M428L substitution. In other embodiments, one or both Fc polypeptides in an Fc polypeptide dimer can comprise M252Y, S254T, and T256E substitutions.

[0179] In some embodiments, one or both of the Fc polypeptides can have its C-terminal lysine removed (e.g, the Lys residue at position 447 of the Fc polypeptide, according to EU numbering). The C-terminal lysine residue is highly conserved in immunoglobulins across many species and may be fully or partially removed by the cellular machinery during protein production. In some embodiments, removal of the C-terminal lysines in the Fc polypeptides can improve the stability of the proteins.

[0180] In some embodiments, a hinge region (e.g., SEQ ID NO:58) or a portion thereof (e.g., SEQ ID NO:59) can be joined to an Fc polypeptide or a modified Fc polypeptide described herein. The hinge region can be from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgGl hinge region, e.g., human IgGl hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO:58) or a portion thereof (e.g, DKTHTCPPCP (SEQ ID NO:59)). In some embodiments, the hinge region is at the N-terminal region of the Fc polypeptide.V. ILLUSTRATIVE ANTI-PILRA ANTIBODIES

[0181] In some embodiments, an anti-PILRA antibody comprises:(i) a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and / or a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16 or 21, and a CDR- Ll, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively, and / or a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17 or 22; and(ii) a first Fc polypeptide; and(iii) a second Fc polypeptide, wherein one of the first and second Fc polypeptides is modified to specifically bind to a CD98hc protein. In some embodiments, the first Fc polypeptide is modified to specifically bind to the CD98hc protein.

[0182] In some embodiments, an anti-PILRA antibody comprises:(i) a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively; and(ii) a first Fc polypeptide; and(iii) a second Fc polypeptide, wherein one of the first and second Fc polypeptides is modified to specifically bind to a CD98hc protein. In some embodiments, the first Fc polypeptide is modified to specifically bind to the CD98hc protein.

[0183] In some embodiments, an anti-PILRA antibody comprises:(i) a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16 or 21, and a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17 or 22; and(ii) a first Fc polypeptide; and(iii) a second Fc polypeptide, wherein one of the first and second Fc polypeptides is modified to specifically bind to a CD98hc protein. In some embodiments, the first Fc polypeptide is modified to specifically bind to the CD98hc protein.

[0184] In some embodiments, the first Fc polypeptide comprises modifications for CD98hc-binding (e.g., Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering), a knob mutation (e.g., T366W), and modifications that reduce or eliminate effector function (e.g., L234A, L235A, and P329G). In certain embodiments, the first Fc polypeptide comprises a sequence having at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:66. In particular embodiments, the first Fc polypeptide comprises Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering, and a sequence having at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:66.

[0185] In some embodiments, the second Fc polypeptide comprises hole mutations (e.g., T366S, L368A, and Y407V) and modifications that reduce or eliminate effector function (e.g., L234A, L235A, and P329G). In certain embodiments, the second Fc polypeptide comprises a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 63. In particular embodiments, the second Fc polypeptide comprises Ser at position 366, Ala at position 368, Vai at position 407, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 63.

[0186] In particular embodiments, an anti-PILRA antibody comprises:(i) a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and / or a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively, and / or a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17; and(ii) a first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 66; and(iii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, Vai at position 407, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 63.

[0187] In particular embodiments, an anti-PILRA antibody comprises:(i) a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively; and(ii) a first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 66; and(iii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, Vai at position 407, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 63.

[0188] In particular embodiments, an anti-PILRA antibody comprises:(i) a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16, and a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17; and(ii) a first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 66; and(iii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, Vai at position 407, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO63.

[0189] In particular embodiments, an anti-PILRA antibody comprises: a first heavy chain having a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 18, a second heavy chain having a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 19, and first and second light chains each having a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:20.

[0190] In particular embodiments, an anti-PILRA antibody comprises:(i) a CDR-H1, a CDR-H2, a CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 10-12, respectively, and / or a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21, and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 13-15, respectively, and / or a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and(ii) a first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 66; and(iii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, Vai at position 407, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 63.

[0191] In particular embodiments, an anti-PILRA antibody comprises:(i) a VH sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21, and a VL sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and(ii) a first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 66; and(iii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, Vai at position 407, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 63.

[0192] In particular embodiments, an anti-PILRA antibody comprises: a first heavy chain having a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:23, a second heavy chain having a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:24, and first and second light chains each having a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:25.VI. CELL LINES AND METHODS OF ENGINEERING

[0193] Provided herein are also cells and cell lines that are homozygous for the gene encoding the G78 variant of the PILRA protein, homozygous for the gene encoding the R78 variant of the PILRA protein, or heterozygous for gene encoding the G78 variant and R78 variant of the PILRA protein. The disclosure provides an engineered human induced pluripotent stem cell (IPSC) or cell line that has been modified (i.e., genetically engineered) to express two copies of (i.e., homozygous for) the gene encoding R78 variant or the G78 variant of a PILRA protein. In some embodiments, the IPSC is modified at the endogenous genomic locus.

[0194] The disclosure also provides an engineered microglial cell or cell line that is derived from a human induced pluripotent stem cell (IPSC) that has been modified (i.e., genetically engineered) to express two copies of (i.e., homozygous for) the gene encoding the R78 variant or the G78 variant of a PILRA protein. An engineered microglial cell or cell line can also be derived from a human induced pluripotent stem cell (IPSC) that has been modified (i.e., genetically engineered) to express one copy of the gene encoding the R78 variant and one copy of the gene encoding the G78 variant of a PILRA protein (i.e., heterozygous for the gene encoding the R78 and G78 variants). In some embodiments, the IPSC is modified at the endogenous genomic locus. In some embodiments, the engineered microglial cell or cell line is derived by directed differentiation.

[0195] Also provided herein are two cell lines serving as a matched pair of cell lines (e.g., an IPSC line or microglia derived therefrom), in which one cell line expresses the G78 variant of the PILRA protein and the other cell line expresses the R78 variant of the PILRA protein. The disclosure provides a matched pair of cell lines, wherein: (a) the first cell line of the pair is homozygous for the gene encoding the R78 variant of a PILRA protein; and (b) thesecond cell line of the pair is homozygous for the gene encoding the G78 variant of a PILRA protein, in which both first and second cell lines of the pair are derived from the same parental cell line, and one or both cell lines have been engineered in the endogenous PILRA gene. In particular embodiments of the matched pair of cell lines, the parental cell line used to generate the matched pair of cell lines can be homozygous for the gene encoding the R78 variant of the PILRA protein, which means that only the cell line in the pair that is homozygous for the gene encoding the G78 variant of the PILRA protein needs to be generated from the parental cell line. In other embodiments of the matched pair of cell lines, the parental cell line used to generate the matched pair of cell lines can be homozygous for the gene encoding the G78 variant of the PILRA protein, which means that only the cell line in the pair that is homozygous for the gene encoding the R78 variant of the PILRA protein needs to be generated from the parental cell line. In yet other embodiments, the parental cell line is heterozygous for gene encoding the R78 variant and the G78 variant of the PILRA protein (z.e., one allele encoding the G78 variant and the other allele encoding the R78 variant). In this case, both cell lines in the matched pair need to be generated from the parental cell line.

[0196] In some embodiments of the matched pair of cell lines, a third cell line is included that is heterozygous for the gene encoding the G78 variant and the R78 variant of the PILRA protein. In some embodiments, the third cell line is derived from the parental cell line that is homozygous for the gene encoding the R78 variant or the G78 variant of the PILRA protein.

[0197] The disclosure also provides methods of generating a myeloid cell line, or a stem cell line capable of differentiating into a myeloid cell line (e.g., an IPSC line or microglia derived therefrom), with a modified PILRA gene, the method comprising: (a) determining whether an existing myeloid cell line, or an existing stem cell line, is homozygous for the gene encoding the R78 variant of a PILRA protein, homozygous for the gene encoding the G78 variant of a PILRA protein, or heterozygous for the gene encoding the R78 and G78 variants of a PILRA protein; and (b) engineering the cell line by modifying the gene encoding the PILRA protein to produce an engineered cell line that is homozygous for the gene encoding the R78 variant of the PILRA protein or the G78 variant of the PILRA protein, wherein the engineered cell line was not, prior to being engineered, homozygous for the gene that encodes the selected variant. In other words, depending on the existing cell line, the existing cell line may or may not need to be modified to generate the selected variant in the desired cell line.

[0198] The disclosure also provides methods of generating a matched pair of cell lines (e.g., an IPSC line or microglia derived therefrom), the method comprising: (a) determining whether an existing myeloid cell line, or an existing stem cell line capable of differentiating into a myeloid cell line, is homozygous for the gene encoding the R78 variant of a PILRA protein, homozygous for the gene encoding the G78 variant of a PILRA protein, or heterozygous for the gene encoding the R78 and G78 variants of a PILRA protein; and (b) engineering (i) a first cell line by modifying the gene encoding the PILRA protein to produce an engineered cell line that is homozygous for the gene encoding the R78 variant of the PILRA protein, and / or (ii) a second cell line by modifying the gene encoding the PILRA protein to produce an engineered cell line that is homozygous for the gene encoding the G78 variant of the PILRA protein. In some embodiments, the engineered cell line was not, prior to being engineered, homozygous for the gene that encodes the selected variant.

[0199] In particular embodiments, the existing cell line of step (a) is homozygous for the R78 variant of the PILRA protein, and the engineering of step (b) comprises modifying the existing cell line to produce an engineered cell line that is homozygous for the gene encoding the G78 variant of the PILRA protein. In particular embodiments, the existing cell line of step (a) is homozygous for the G78 variant of the PILRA protein, and the engineering of step (b) comprises modifying the existing cell line to produce an engineered cell line that is homozygous for the gene encoding the R78 variant of the PILRA protein. In other embodiments, the existing cell line of step (a) is heterozygous for the gene encoding the R78 and G78 variants of the PILRA protein, and the engineering of step (b) comprises modifying the existing cell line to produce an engineered cell line that is homozygous for the gene encoding the R78 variant of the PILRA protein, and an engineered cell line that is homozygous for the gene encoding the G78 variant of the PILRA protein.

[0200] Engineered cells or cell lines with modifications at an endogenous genomic locus (e.g., a PILRA gene locus) can be generated using a variety of methods and techniques, for example, the CRIPSR / Cas9 system, a zinc finger nuclease (ZFN), a Tale-effector domain nuclease (TALEN), and a transposon-mediated system. These methods typically comprise administering to the cell one or more polynucleotides encoding one or more nucleases such that the nuclease mediates modification of the endogenous gene by cleaving the DNA to create 5’ and 3’ cut ends in the DNA strand. In the presence of a donor sequence that is flanked by a left and a right homology arms that are substantially homologous to a sequence extending 5’ from the 5’ end and a sequence extending 3’ from the 3’ end, the donor isintegrated into the endogenous gene targeted by the nuclease via homology-directed repair (HDR). In some embodiments, the modification at the endogenous genomic locus is conducted using the CRISPR / Cas9 system. For example, a nucleic acid sequence encoding a heterologous gene encoding a PILRA with the desired variant is introduced into the endogenous PILRA genomic locus of the cell to be modified, which results in that the naturally occurring sequence that encodes the endogenous PILRA is replaced by the heterologous gene.CRISPR

[0201] In some embodiments, the introduction or knock-in of a heterologous gene encoding a PILRA with the desired variant is performed using the CRIPSR / Cas9 system. The CRISPR / Cas9 system includes a Cas9 protein and at least one to two ribonucleic acids that are capable of directing the Cas9 protein to and hybridizing to a target motif in the endogenous PILRA gene that is to be replaced. These ribonucleic acids are commonly referred to as the “single guide RNA” or “sgRNA.” The Cas9 protein then cleaves the target motif, which results in a double-strand break or a single-strand break. In the presence of a donor DNA that comprises the heterologous PILRA gene sequence flanked by two homology arms, the donor DNA is inserted into the target DNA, replacing the endogenous gene.

[0202] The Cas9 protein used in the disclosure can be a naturally occurring Cas9 protein or a functional derivative thereof. A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative of Cas9 to hydrolyze a DNA substrate into fragments. Suitable functional derivatives of a Cas9 polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas9 protein or a fragment thereof.

[0203] In some embodiments, the Cas9 protein is from Streptococcus pyogenes. Cas9 contains 2 endonuclease domains, including a RuvC-like domain which cleaves target DNA that is noncomplementary to the sgRNA, and an HNH nuclease domain which cleave target DNA complementary to sgRNA. The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence (2-5 nucleotides), known as a protospacer-associatedmotif (PAM), follows immediately 3’ of a target motif in the target sequence. In some embodiments, the PAM motif is an NGG motif. A donor DNA is introduced to the reaction. In one example, the donor DNA comprises the heterologous PILRA gene of the desired variant that is between a left homology arm and a right homology arm.

[0204] The sgRNAs can be selected depending on the particular CRISPR / Cas9 system employed and the sequence of the target polynucleotide. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas9 protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas9 protein, wherein the target motifs flank the genomic sequence to be replaced. Guide RNAs can be designed using software that is readily available, for example, at http: / / crispr.mit.edu.

[0205] In some embodiments, the donor DNA as disclosed herein comprises a nucleotide sequence that encodes the amino acid sequence of a hPILRA G78 variant. In some embodiments, the donor DNA as disclosed herein comprises a nucleotide sequence that encodes the amino acid sequence of a hPILRA R78 variant. The donor DNA as disclosed herein further comprises a left homology arm and a right homology arm that flank the nucleotide sequence and are designed to overlap the 5’ and 3’ exon sequences relative to the cleave site by the Cas9 protein. The homology arms may extend beyond the 5’ and 3’ exon sequences, and each of the homology arms may be at least 20, 30, 40, 50, 100, or 150 nucleotides in length. One of skilled in the art can readily determine the optimal length of the homology arm required for the experiment.

[0206] In some embodiments, the sgRNAs can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell to minimize off-target effects of the CRISPR / Cas9 system. Those skilled in the art will appreciate that a variety of techniques can be used to select suitable target motifs for minimizing off-target effects (e.g., bioinformatics analyses).Zinc finger nuclease (ZFN)

[0207] In some embodiments, the introduction or knock-in of a heterologous gene encoding a PILRA with the desired variant is performed using a ZFN. ZFNs are fusion proteins thatcomprise a non-specific cleavage domain (N) of FokI endonuclease and a zinc finger protein (ZFP). A pair of ZNFs are involved to recognize a specific locus in a target gene: one that recognizes the sequence upstream and the other that recognizes the sequence downstream of the site to be modified. The nuclease portion of the ZFN cuts at the specific locus. The donor DNA can then be inserted into the specific locus. Methods of using the ZFNs are well known, for example, as disclosed in US Pat. No. 9,045,763 and also in Durai et al., “Zinc Finger Nucleases: Custom-Designed Molecular Scissors for Genome Engineering of Plant and Mammalian cells,” Nucleic Acid Research, 33 (18):5978-5990 (2005), the disclosures of which are incorporated by reference in their entirety.Transcription activator-like effector nucleases (TALENs)

[0208] In some embodiments, the introduction or knock-in of a heterologous gene encoding a PILRA with the desired variant is performed using TALENs. TALENs are similar to ZFNs in that they bind as a pair around a genomic site and direct the same non-specific nuclease, FokI, to cleave the genome at a specific site, but instead of recognizing DNA triplets, each domain recognizes a single nucleotide. Methods of using the ZFNs are also well known, for example, as disclosed in US Pat. No. 9,005,973 and also Christian et al., “Targeting DNA Double-Strand Breaks with TAL Effector Nucleases,” Genetics, 186(2): 757-761 (2010), the disclosures of which are incorporated by reference in their entirety.

[0209] The disclosure also provides methods of generating a matched pair of cell lines from an existing myeloid cell line (e.g., an IPSC line or microglia derived therefrom), or an existing stem cell line capable of differentiating into a myeloid cell line, that is heterozygous for gene encoding the R78 and G78 variants of a PILRA protein, the method comprising: (a) engineering the existing cell line to produce a first engineered cell line that is homozygous for the gene encoding the R78 variant of the PILRA protein; and (b) engineering either the cell line generated in step (a) or the existing cell line to produce a second engineered cell line that is homozygous for the gene encoding the G78 variant of the PILRA protein. As described herein, the CRIPSR / Cas9 system can be used to generate the matched pair of cell lines using a donor DNA that comprises a nucleotide sequence that encodes the amino acid sequence of a hPILRA R78 variant or G78 variant.

[0210] In another aspect, the disclosure provides a method of generating a matched pair of cell lines from an existing myeloid cell line, or an existing stem cell line capable of differentiating into a myeloid cell (e.g., an IPSC line or microglia derived therefrom), that isheterozygous for gene encoding the R78 and G78 variants of a PILRA protein, the method comprising: (a) engineering the existing cell line to produce a first engineered cell line that is homozygous for the gene encoding the G78 variant of the PILRA protein; and (b) engineering either the cell line generated in step (a) or the existing cell line to produce a second engineered cell line that is homozygous for the gene encoding the R78 variant of the PILRA protein. As described herein, the CRIPSR / Cas9 system can be used to generate the matched pair of cell lines using a donor DNA that comprises a nucleotide sequence that encodes the amino acid sequence of a hPILRA R78 variant or G78 variant.

[0211] The disclosure also provides methods of generating a matched pair of cell lines from an existing myeloid cell line, or an existing stem cell line capable of differentiating into a myeloid cell line (e.g., an IPSC line or microglia derived therefrom), that is homozygous for the gene encoding the R78 variant of a PILRA protein, the method comprising: engineering the existing cell line to produce an engineered cell line that is homozygous for gene encoding the G78 variant of the PILRA protein. As described herein, the CRIPSR / Cas9 system can be used to generate the matched pair of cell lines using a donor DNA that comprises a nucleotide sequence that encodes the amino acid sequence of a hPILRA G78 variant.

[0212] The disclosure also provides methods of generating a matched pair of cell lines from an existing myeloid cell line, or an existing stem cell line capable of differentiating into a myeloid cell line (e.g., an IPSC line or microglia derived therefrom), that is homozygous for the gene encoding the G78 variant of a PILRA protein, the method comprising: engineering the existing cell line to produce an engineered cell line that is homozygous for gene encoding the R78 variant of the PILRA protein. As described herein, the CRIPSR / Cas9 system can be used to generate the matched pair of cell lines using a donor DNA that comprises a nucleotide sequence that encodes the amino acid sequence of a hPILRA R78 variant.

[0213] In some embodiments, the engineered cell, cell line, or cell model described herein is derived by directed differentiation.VII. METHODS OF SCREENING

[0214] The disclosure also provides methods for screening and identifying antibodies that that bind to and / or modulate expression or activity of a PILRA protein, i.e., antibodies that antagonize or reduce PILRA activity (i.e., antibodies that block binding of a ligand tohPILRA). In some embodiments, one or more downstream signaling responses that is related to PILRA binding and / or activation can be measured to identify PILRA-binding antibodies. For example, an antibody that binds to a PILRA protein of a cell can cause one or more downstream signaling responses or activities of the cell as a result of PILRA-binding. In some embodiments, an antibody that binds to a PILRA protein can cause an increase or decrease in the signaling response or activity of the cell as a result of PILRA-binding, relative to the signaling response or activity of the cell without PILRA-binding. Examples of changes in signaling responses or activities of a cell as a result of PILRA-binding include, but are not limited to, changes phosphorylated STAT3 (pSTAT3) level, phosphorylated STAT1 (pSTATl) level, phosphorylated EGFR (pEGFR) level, cadherin expression, integrin expression, and cell (e.g., microglia) migration. In particular embodiments, antibodies that bind to PILRA and antagonize or reduce PILRA activity can cause a downstream signaling response, such as an increase in pSTAT3 (e.g., pSTAT3 Y705, or pSTAT3 S727) level, an increase in pEGFR level, an increase in the expression level and / or cell secretion of a protein (e.g., cadherin, integrin), and / or an increase in cell (e.g., microglia) migration. Examples of other downstream signaling responses that can be caused by antibodies that bind to PILRA and antagonize or reduce PILRA activity can be, for example, elevated cellular respiration, elevated fatty acid metabolism (e.g., fatty acid oxidation), elevated ATP production, increased anti inflammatory gene or protein expression, and / or reduced cytokine protein expression.

[0215] Provided herein are methods for screening to determine whether an antibody has activity at a PILRA protein, the method comprising: (a) contacting a cell that expresses the PILRA protein with the antibody; (b) either prior to, concurrently with, or following step (a), contacting a cell of the same type as in step (a) having lower PILRA expression with the molecule; and (c) measuring one of the following: phosphorylated STAT3 (pSTAT3) level, phosphorylated STAT1 (pSTATl) level, phosphorylated EGFR (pEGFR) level, cadherin expression, integrin expression, and microglial migration in both cells. In some embodiments, a change in the level of one of these measurements between the cells indicates that the molecule has activity at the PILRA protein of step (a).

[0216] In certain embodiments of the methods for screening, the cell of step (a) naturally expresses the PILRA protein. In some embodiments, the cell having lower PILRA expression has the PILRA protein knocked-out or silenced. In particular embodiments, thecell can be a microglia. In certain embodiments, the cell is an iMicroglia (e.g., a PILRA LoF iMicroglia).

[0217] In certain embodiments of the methods for screening, the cell of step (a) is engineered or modified to express or overexpress the PILRA protein. In some embodiments, the cell having lower PILRA expression naturally expresses the PILRA protein or is not engineered or modified to express the PILRA protein.

[0218] In some embodiments, a library of antibodies can be screened using the methods described herein. In certain cases, the antibody is known to bind the PILRA protein. In other cases, it is unknown whether the antibody binds the PILRA protein.

[0219] Also provided herein are methods for determining whether an antibody that binds a PILRA protein modulates a signaling response or activity in a PILRA-expressing cell, the method comprising: (a) contacting the cell with the molecule; and (b) measuring one of the following: phosphorylated STAT3 (pSTAT3) level, phosphorylated STAT1 (pSTATl) level, phosphorylated EGFR (pEGFR) level, cadherin expression, integrin expression, and cell (e.g., microglia) migration. In some embodiments, a change in the level of one of the measurements indicates that the antibody modulates the signaling response or activity in the PILRA-expressing cell. In certain embodiments, the change is an increase or decrease in the level of one of the measurements when the antibody contacts the cell, relative to the level in the cell without the antibody, in particular, the changes described elsewhere in the application. In particular embodiments of these methods, the cell is in an in vitro assay. In other embodiments, the cell is in a mammal (i.e., in vivo methods).

[0220] In some embodiments, when the methods are used in vivo, and step (a) comprises administering the antibody to a mammal.

[0221] In some embodiments, a PILRA-expressing cell can be a microglia, a myeloid cell, a monocyte, or a neutrophil.Screening Assays

[0222] Screening assays to identify antibodies that that bind to and / or modulate expression or activity of a PILRA protein can be carried out by standard methods. The screening methods may involve high-throughput techniques. In addition, these screening techniques may be carried out in cultured cells or in organisms such as mice, worms, flies, or yeast.

[0223] Any number of methods is available for carrying out such screening assays. According to one approach, candidate antibodies are added at varying concentrations to the culture medium of PILRA-expressing cells. If downstream signaling such as phospho-STAT3 (pSTAT3) induction is used as a measurement of whether the antibody binds and / or modulate expression or activity of the PILRA protein, pSTAT3 levels can be measured in a cell that expresses the PILRA protein and compared to the pSTAT3 level in a corresponding cell that expresses a lower level of PILRA (e.g., a PILRA knockout). In other cases, the pSTAT3 level can be measured before and after adding the antibody to the cell. These levels of pSTAT3 can be compared.

[0224] In another approach, cellular secretions of proteins such as integrins and cadherins can also be measured to determine whether the antibody binds and / or modulate expression or activity of the PILRA protein, as it is demonstrated in the examples that anti-PILRA antibodies enhanced iMicroglial secretion of these proteins. Standard laboratory techniques can be used to isolate these proteins from the cell and detection of these proteins can be performed using, e.g., mass spectrometry, Western blot, and Proteome profiler kit; Human Soluble Receptor Array Kit - Non-Hematopoietic Panel (R&D ARY012).

[0225] In other embodiments, a candidate antibody that binds to a PILRA protein may be identified using a chromatography -based technique. For example, recombinant PILRA may be purified by standard techniques from cells engineered to express PILRA and may be immobilized on a column. A solution of candidate antibodies is then passed through the column, and an antibody specific for PILRA is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the antibody, the column is washed to remove non-specifically bound molecules, and the antibody of interest is then released from the column and collected. Antibodies isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography).Test Molecules

[0226] In general, potential antibodies can be identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the disclosure. Accordingly, virtually any number of chemicalextracts or molecules can be screened using the methods described herein. Examples of such extracts or molecules include, but are not limited to, plant-, fungal-, prokaryotic- or animalbased extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi -synthesis or total synthesis) of any number of molecules. Synthetic compound libraries are commercially available. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

[0227] When a crude extract is found to have an activity, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the characterization and identification of a chemical entity within the crude extract having the desired activity. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, molecules shown to be useful can be chemically modified according to methods known in the art.VIII. MEASURING ACTIVITY OF PILRA-BINDING ANTIBODIES IN A CELL OR ANIMAL

[0228] Also provided herein are methods for measuring the binding and / or activity of an antibody that binds to a PILRA protein (e.g., hPILRA G78 or R78). In some embodiments, the antibody antagonizes or reduces PILRA activity (i.e., antibodies that block binding of a ligand to hPILRA). Various measurements can be made to determine the binding and / or activity of a PILRA-binding antibody and its effects on the cell or animal. For example, it is demonstrated herein that the induction of phosphorylated STAT3 is a cellular downstream signaling response that is PILRA-dependent and happens when PILRA is antagonized.

[0229] In some embodiments, to measure the binding and / or activity of a PILRA-binding antibody, after cells were incubated with the PILRA-binding molecule, phosphorylated STAT3 (e.g., pSTAT3 Y705 and / or pSTAT3 S727) level can be measured using, for example, a Proteome Profiler Human Phospho-Kinase Array Kit (e.g., ARY003C, R&D Systems). In other embodiments, to measure phosphorylated protein levels, after the cellswere incubated with the PILRA-binding molecule, the cells can be fixed and the phosphorylated protein can be detected using standard immunocytochemistry protocol. Cells can then be imaged with a confocal microscope and images can be analyzed using a software to calculate mean fluorescent spot area and intensity per cell to determine phosphorylated protein level.

[0230] Other cellular responses that are dependent on PILRA binding (z.e., antagonizing) include, e.g., an increase in phosphorylated EGFR (e.g., pEGFR Y1086) level, which can also be measured using a phosphor-kinase array kit or immunocytochemistry, as mentioned above.

[0231] In some embodiments, measuring the phosphorylation level of STAT3 and / or EGFR upon PILRA-binding by the antibodies can be used to rank antagonistic effects of the molecules. For example, a PILRA-binding antibody whose binding resulted in the highest level of pSTAT3 can be determined to have the most antagonistic activity at the PILRA protein.

[0232] Other measurements that can be made to determine the binding and / or activity of a PILRA-binding antibody and its effects on the cell or animal include, for example, measuring cell migration, which is another cellular downstream signaling response that is PILRA- dependent and happens when PILRA is antagonized. Fro example, measurement and quantification of cell migration can be performed using a cell migration assay where a rubber stopper can be used to create a cell-free detection zone. The rubber stopper can then be removed upon addition of the PILRA-binding molecule, and a cell stain such as NucBlue or DAPI can be added. The cells can be imaged using microscopy and the images can be analyzed using a software to quantify nuclear labeling of the cells that migrated to the detection zone. Further, as PILRA-binding antibodies that antagonize PILRA also enhance the cell secretion of motile proteins, quantification of such motile proteins in the cell supernatant after addition of PILRA-binding molecule to the cells can also be performed. For example, soluble analytes in the supernatants can be analyzed with a proteome profiler kit, such as Human Soluble Receptor Array Kit - Non-Hematopoietic Panel (e.g., R&D ARY012). Examples of motile proteins that can be quantified in this manner include, but are not limited to, cadherins and integrins.

[0233] Measuring the binding and / or activity of a PILRA-binding antibody can be performed in a cell or an animal (e.g., mice, monkeys). For in vivo studies, an animal, e.g.,an animal expressing a PILRA protein (e.g., PILRA G78 or R78) can be administered a PILRA-binding antibody via any mode of administration available (e.g., IV, IP, oral, nasal, or transdermal administration). The appropriate cells, tissues, and / or fluid samples can be isolated from the animal to measure and quantify one or more of the PILRA-dependent downstream signaling responses described herein, such as pSTAT3 level, pEGFR level, amount of motile proteins (e.g., cadherins, integrins). In some embodiments, the cell or animal is homozygous for the gene encoding PILRA G78. In some embodiments, the cell or animal is homozygous for the gene encoding PILRA R78. In some embodiments, the cell or animal is heterozygous for the gene encoding PILRA G78 and R78 variants.IX. PREPARATION OF ANTIBODIES

[0234] In some embodiments, antibodies are prepared by immunizing an animal or animals (e.g., mice, rabbits, or rats) with an antigen or a mixture of antigens for the induction of an antibody response. In some embodiments, the antigen or mixture of antigens is administered in conjugation with an adjuvant (e.g., Freund’s adjuvant). After an initial immunization, one or more subsequent booster injections of the antigen or antigens may be administered to improve antibody production. Following immunization, antigen-specific B cells are harvested, e.g., from the spleen and / or lymphoid tissue. For generating monoclonal antibodies, the B cells are fused with myeloma cells, which are subsequently screened for antigen specificity. Methods of preparing antibodies are also described in the Examples section below.

[0235] The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Alternatively, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to selected antigens. Antibodies can also be made bispecific, i.e., able to recognize two different antigens. Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins.

[0236] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers. In embodiments inwhich an antibody comprises both a VH and VL region, the VH and VL regions may be expressed using a single vector, e.g., in a di-cistronic expression unit, or under the control of different promoters. In other embodiments, the VH and VL region may be expressed using separate vectors. A VH or VL region as described herein may optionally comprise a methionine at the N-terminus.

[0237] In some embodiments, the antibody is a chimeric antibody. Methods for making chimeric antibodies are known in the art. For example, chimeric antibodies can be made in which the antigen binding region (heavy chain variable region and light chain variable region) from one species, such as a mouse, is fused to the effector region (constant domain) of another species, such as a human. As another example, “class switched” chimeric antibodies can be made in which the effector region of an antibody is substituted with an effector region of a different immunoglobulin class or subclass.

[0238] In some embodiments, the antibody is a humanized antibody. Generally, a nonhuman antibody is humanized in order to reduce its immunogenicity. Humanized antibodies typically comprise one or more variable regions (e.g., CDRs) or portions thereof that are nonhuman (e.g., derived from a mouse variable region sequence), and possibly some framework regions or portions thereof that are non-human, and further comprise one or more constant regions that are derived from human antibody sequences. Methods for humanizing non- human antibodies are known in the art. Transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies. Other methods of humanizing antibodies include, for example, variable domain resurfacing, CDR grafting, grafting specificity-determining residues (SDR), guided selection, and framework shuffling.

[0239] As an alternative to humanization, fully human antibodies can be generated. As a non-limiting example, transgenic animals (e.g., mice) can be produced that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. As another example, human antibodies can be produced by hybridoma-based methods, such as by using primary human B cells for generating cell lines producing human monoclonal antibodies.

[0240] Human antibodies can also be produced using phage display or yeast display technology. In phage display, repertoires of variable heavy chain and variable light chain genes are amplified and expressed in phage display vectors. In some embodiments, the antibody library is a natural repertoire amplified from a human source. In some embodiments, the antibody library is a synthetic library made by cloning heavy chain and light chain sequences and recombining to generate a large pool of antibodies with different antigenic specificity. Phage typically display antibody fragments (e.g., Fab fragments or scFv fragments), which are then screened for binding to an antigen of interest.

[0241] In some embodiments, antibody fragments (such as a Fab, a Fab’, a F(ab’)2, a scFv, a VH, or a VHH) are generated. For example, an antibody fragment containing a VH sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 16 and / or a VL sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO: 17 can be generated. In another example, an antibody fragment containing a VH sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:21 and / or a VL sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity or 100% identity to the sequence of SEQ ID NO:22 can be generated. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab’)2 fragments. According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0242] In some embodiments, an antibody or an antibody fragment is conjugated to another molecule, e.g., polyethylene glycol (PEGylation) or serum albumin, to provide an extended half-life in vivo.X. NUCLEIC ACIDS, VECTORS, AND HOST CELLS

[0243] In some embodiments, the anti-PILRA antibodies as disclosed herein are prepared using recombinant methods. Accordingly, in some aspects, the disclosure provides isolatednucleic acids comprising a nucleic acid sequence encoding any of the anti-PILRA antibodies as described herein (e.g, any one or more of the CDRs, heavy chain variable regions, and light chain variable regions described herein); vectors comprising such nucleic acids; and host cells into which the nucleic acids are introduced that are used to replicate the antibodyencoding nucleic acids and / or to express the antibodies.

[0244] In some embodiments, a polynucleotide (e.g, an isolated polynucleotide) comprises a nucleotide sequence encoding an antibody as described herein. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding one or more amino acid sequences (e.g., CDR, heavy chain, or light chain sequences) disclosed in Table 1. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to a sequence (e.g., a CDR, heavy chain, or light chain sequence) disclosed in Table 1. In some embodiments, a polynucleotide as described herein is operably linked to a heterologous nucleic acid, e.g., a heterologous promoter.

[0245] Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof, include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in selecting clones containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0246] Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure. The expression vector may replicate in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno- associated viruses, retroviruses, and any other vector.

[0247] Suitable host cells for cloning or expressing a polynucleotide or vector as described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell isprokaryotic. In some embodiments, the host cell is eukaryotic, e.g., Chinese Hamster Ovary (CHO) cells or lymphoid cells. In some embodiments, the host cell is a human cell, e.g., a Human Embryonic Kidney (HEK) cell.

[0248] In another aspect, methods of making an anti-PILRA antibody as described herein are provided. In some embodiments, the method includes culturing a host cell as described herein (e.g., a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).XI. THERAPEUTIC METHODS USING ANTI-PILRA ANTIBODIES

[0249] In another aspect, therapeutic methods using an anti-PILRA antibody as disclosed herein (e.g., an anti-PILRA antibody as described in Sections III and V above) are provided. In some embodiments, methods of treating a neurodegenerative disease are provided. In some embodiments, methods of modulating one or more PILRA activities (e.g., in a subject having a neurodegenerative disease) are provided.

[0250] In some embodiments, methods of treating a neurodegenerative disease are provided. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis / parkinsonism-dementia complex of Guam (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadelopean PSP, Hallevorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington’s disease, inclusion-body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Parkinson’s disease, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle only dementia. In some embodiments, the neurodegenerative disease is Alzheimer’s disease. In some embodiments, the neurodegenerative disease is Nasu-Hakola disease. In some embodiments, theneurodegenerative disease is frontotemporal dementia. In some embodiments, the neurodegenerative disease is Parkinson’s disease. In some embodiments, the method comprises administering to the subject an isolated antibody that specifically binds to a hPILRA protein, e.g., an anti-PILRA antibody as described herein, or a pharmaceutical composition comprising an anti-PILRA antibody as described herein.

[0251] In some embodiments, an anti-PILRA antibody (or antigen-binding portion or pharmaceutical composition thereof) as described herein is used in treating a neurodegenerative disease that is characterized by PILRA activity. In some embodiments, the neurodegenerative disease that is characterized by PILRA activity is Alzheimer’s disease.

[0252] In some embodiments, methods of modulating one or more PILRA activities in a subject (e.g., a subject having a neurodegenerative disease) are provided. In some embodiments, the method comprises antagonizing or reducing PILRA activity, e.g., blocking binding of a ligand to hPILRA, altering phosphorylation of one or more downstream proteins (e.g., increases phosphorylation of EGFR or STAT3; decreases phosphorylation of STAT1), elevating cellular respiration, fatty acid metabolism (e.g., fatty acid oxidation), and ATP production, enhancing cell migration, increasing anti-inflammatory gene or protein expression, and / or reducing cytokine protein expression. Thus, in another aspect, methods of antagonizing PILRA activity, e.g., in a subject having a neurodegenerative disease, are provided. In some embodiments, the method of modulating one or more PILRA activities in a subject comprises administering to the subject an isolated antibody or an antigen-binding portion thereof that specifically binds to a hPILRA protein, e.g., an anti-PILRA antibody as describe herein, or a pharmaceutical composition comprising an anti-PILRA antibody as described herein.

[0253] In some embodiments, the subject to be treated is a human, e.g., a human adult or a human child.

[0254] In some embodiments, methods of reducing plaque accumulation in a subject having a neurodegenerative disease are provided. In some embodiments, the method comprises administering to the subject an antibody or pharmaceutical composition as described herein. In some embodiments, the subject has Alzheimer’s disease. In some embodiments, the subject is an animal model of a neurodegenerative disease (e.g., a 5XFAD or APP / PS1 mouse model). In some embodiments, plaque accumulation is measured by amyloid plaque imaging and / or Tau imaging, e.g., using positron emission tomography (PET)scanning. In some embodiments, administration of an anti-PILRA antibody reduces plaque accumulation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a baseline value (e.g., the level of plaque accumulation in the subject pirior to administration of the anti-PILRA antibody).

[0255] In some embodiments, an anti-PILRA antibody is administered to a subject at a therapeutically effective amount or dose. The dosages, however, may be varied according to several factors, including the chosen route of administration, the formulation of the composition, patient response, the severity of the condition, the subject’s weight, and the judgment of the prescribing physician. The dosage can be increased or decreased over time, as required by an individual patient. In certain instances, a patient initially is given a low dose, which is then increased to an efficacious dosage tolerable to the patient. Determination of an effective amount is well within the capability of those skilled in the art.

[0256] The route of administration of an anti-PILRA antibody as described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalational, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art. In some embodiments, the antibody is administered orally, intravenously, or intraperitoneally.

[0257] In some embodiments, the anti-PILRA antibody (and optionally another therapeutic agent) is administered to the subject over an extended period of time, e.g., for at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 days or longer.XII. PHARMACEUTICAL COMPOSITIONS AND KITS

[0258] In another aspect, pharmaceutical compositions and kits comprising an antibody that specifically binds to a hPILRA protein are provided. In some embodiments, the pharmaceutical compositions and kits are for use in treating a neurodegenerative disease. In some embodiments, the pharmaceutical compositions and kits are for use in modulating (e.g., enhancing or inhibiting) one or more PILRA activities, e.g., EGFR, STAT3, and / or STAT1 phosphorylation.Pharmaceutical Compositions

[0259] In some embodiments, pharmaceutical compositions comprising an anti-PILRA antibody are provided. In some embodiments, the anti-PILRA antibody is an antibody as described in Section III above.

[0260] In some embodiments, a pharmaceutical composition comprises an anti-PILRA antibody as described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that does not interfere with or otherwise inhibit the activity of the active agent. Various pharmaceutically acceptable excipients are well-known in the art.

[0261] In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are well- known in the art.

[0262] The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are merely exemplary and are in no way limiting.

[0263] For oral administration, an anti-PILRA antibody can be formulated by combining it with pharmaceutically acceptable carriers that are well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as a crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0264] An anti-PILRA antibody can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. For injection, the compound or compounds can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, compounds can be formulated in aqueous solutions, e.g., in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0265] Typically, a pharmaceutical composition for use in in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0266] Dosages and desired drug concentration of pharmaceutical compositions of the disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of one in the art. Suitable dosages are also described in above.Kits

[0267] In some embodiments, kits comprising an anti-PILRA antibody are provided. In some embodiments, the anti-PILRA antibody is an antibody as described in Sections III and V above.

[0268] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises an anti-PILRA antibody as described herein and further comprises one or more additional therapeutic agents for use in the treatment of a neurodegenerative disease, e.g., Alzheimer’s disease. In some embodiments, the therapeutic agent is an agent for use in treating a cognitive or behavioral symptom of a neurodegenerative disease (e.g., an antidepressant, a dopamine agonist, or ananti-psychotic). In some embodiments, the therapeutic agent is a neuroprotective agent (e.g., carbidopa / levodopa, an anticholinergic agent, a dopaminergic agent, a monoamine oxidase B (MAO-B) inhibitor, a catechol-O-methyl transferase (COMT) inhibitor, a glutamatergic agent, a histone deacetylase (HDAC) inhibitor, a cannabinoid, a caspase inhibitor, melatonin, an anti-inflammatory agent, a hormone (e.g., estrogen or progesterone), or a vitamin).

[0269] In some embodiments, the kit comprises an anti-PILRA antibody as described herein and further comprises one or more reagents for measuring anti-PILRA antibody induced activity (e.g., for measuring EGFR, STAT3, and / or STAT1 phosphorylation).

[0270] In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit for a therapeutic method as described above). While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.EXAMPLES

[0271] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner.EXAMPLE 1 - EVALUATION OF PILRA AND PILRB BINDING IN HEK293 CELLSPILRA Binding in HEK293 Cells

[0272] HEK293 cells expressing hPILRA G78 (also referred to as “HEK PILRA G78 cells” or “HEK PILRA G78 cells”) and HEK 293 cells expressing hPILRB DAP12 (“HEK hPILRB DAP12 cells”) were separately labeled with NucBlue Live ReadyProbes Reagent and rotated at 4°C for 30 minutes. After labelling, parental HEK293 cells were mixed with HEK G78 cells, while HEK 293 cells expressing hPILRA R78 (also referred to as “HEK PILRA R78 cells” or “HEK PILRA R78 cells”) were mixed separately with HEK hPILRB DAP12 cells. These cells were then incubated with various concentrations of anti-PILRA or isotype control antibodies for 30 minutes with rotation in 4°C using FACS diluent (PBS, 0.2% FBS, ImM EDTA). Cells were then washed twice with FACS diluent and incubatedwith Alexa Fluor 647-conjugated anti-human IgG for 30 minutes at 260 rpm in 4°C. Finally, the cells were washed once, and antibody binding was detected by FACS using BD FACS Canto II. Median fluorescence intensity (MFI) was derived from data analysis performed with the FLOJO software (FIGS. 1A-1C). Table 1 below lists each anti-PILRA antibody used in the Examples.Table 1

[0273] Binding of anti-PILRA antibodies to human PILRA G78 (common variant) and PILRA R78 (AD-protective variant) expressed on HEK293 cells (FIGS. IB and 1C) demonstrated cell surface target engagement of certain antibodies (e.g., Ab CLs 1, 2, and 4). The lack of binding of anti-PILRA antibodies (e.g., antibodies that do not have CD98hc- binding, e.g., Ab CLs 1 and 3) to parental HEK293 cells demonstrated specificity of binding (FIG. 1A). Finally, low binding of anti-PILRA antibodies with CD98hc-binding to parental HEK293 cells at high concentrations (FIG. 1 A) was not PILRA-driven, but driven by binding to CD98hc (e.g., Ab CLs 2 and 4, and CD98hc:Isotype Ctrl).PILRB Binding in HEK293 Cells

[0274] HEK hPILRB DAP12 cells were labeled with NucBlue Live ReadyProbes Reagent for 30 minutes with rotation at 4 °C. HEK hPILRA R78 and hPILRB DAP12 cells were mixed, washed, and incubated with various concentrations of anti-PILRA or isotype control antibodies for 30 minutes with rotation at 4 °C using FACS diluent (PBS, 0.2% FBS, and 1 mM EDTA). Cells were washed twice with FACS diluent, incubated with Alexa Fluor 647- conjugated anti-human IgG for 30 minutes with rotation at 4 °C, and washed once. Antibody binding to the cells was detected by FACS and median fluorescence intensity (MFI) was derived from data analysis performed with the FLOJO software (FIG. ID). Positive Ctrl Ab CL (see Table 1) was a PILRB-binding antibody used as a positive control. The results illustrate that the PILRA antibodies have relatively good specificity for PILRA binding and low off-target binding to PILRB.

[0275] There is high level of sequence similarity between hPILRA and hPILRB. Human PILRB receptor function is not fully understood. In contrast to PILRA, it is thought to be an activating receptor. Thus, human PILRB binding is a potential off-target safety risk. The sequence alignment in FIG. 5 highlights the limited epitopes on human PILRA conferring both human PILRA specificity and cyno PILRA cross-reactivity.PILRA Binding in CHO Cells

[0276] CHO-K1 and cynoPILRA expressing CHO cells were stained with NucBlue Live cell stain for 30 minutes with rotation at 4 °C. Cells were mixed, washed, and incubated with various concentrations of anti-PILRA or isotype control antibodies for 30 minutes with rotation at 4 °C using FACS diluent (PBS, 2% FBS, 1 mM EDTA). Cells were washed twice with FACS diluent and incubated with Alexa Fluor 647-conjugated anti-human IgG with rotation at 4 °C for 30 minutes. Antibody binding to cells was detected via BD FACS CantoII and median fluorescence intensity (MFI) was derived after analyzing results via FLOJO and PRISM software (FIGS. 2 A and 2B). Cell lines were run in duplicates to account for intra-variability. The results indicate that certain antibodies, including those with CD98hc binding capacity (e.g., Ab CLs 2 and 4) are able to specifically bind cynoPILRA expressed on CHO cells.

[0277] Overall, binding of anti-PILRA antibodies to cynoPILRA expressed on CHO-K1 cells demonstrated cell surface target engagement and cyno cross-reactivity. CynoPILRA cross-reactivity is a unique binding property which enables antibody safety assessment, TE / PK / PD studies in cyno.EXAMPLE 2 - CHARACTERIZATION OF ANTI-PILRA ANTIBODIESBinding Properties

[0278] The binding affinities of anti-PILRA antibodies to hPILRA, hPILRB, and cynoPILRA extracellular domain (ECD) were measured by SPR using a Biacore 8K instrument (Table 2). Antibodies were captured on Biacore™ Series S CM5 sensor chips immobilized with mouse anti-human Fab (human Fab capture kit from GE Healthcare) followed by injections of serial 3 -fold dilutions of recombinant ECD reagents at a flow rate of 30 pL / minute. Each sample was analyzed using a 3-minute association followed by a 10-minute dissociation. After each injection, the sensor chip was regenerated using a 50 mM glycine pH 2.0 regeneration buffer. A 1 : 1 Languir model of simultaneous fitting of konand koff was used for kinetics analysis.Epitope Mapping

[0279] PILRA binding epitopes of anti-PILRA antibodies were identified by SPR using a Biacore 8K instrument. Anti-PILRA antibodies were captured on Biacore™ Series S CM5 sensor chips immobilized with mouse anti-human Fab (human Fab capture kit from GE Healthcare) followed by injections of single point PILRA to PILRB mutant variants at 1 pM concentration.Ligand Blocking to Human PILRA

[0280] One possible mechanism of action to antagonize PILRA is to block binding of known sialylated ligands, including NPDC1, PANP, and HSV gB. Anti-PILRA antibodies that bind epitopes proximal to the ligand binding site G78 are likely to be more efficientlyblock (smaller) ligands than anti-PILRA antibodies that bind more distal epitopes. We have identified anti-PILRA antibodies that block ligand binding.

[0281] Ligand blocking characteristics of anti-PILRA antibodies were evaluated by SPR using a Biacore 8K instrument. Anti-PILRA antibodies were captured on Biacore™ Series S CM5 sensor chips immobilized with mouse anti-human Fab (human Fab capture kit from GE Healthcare) followed by injections of 300 nM recombinant hPILRA ECD. hPILRA-ligand interactions were monitored by subsequent injection of recombinant PILRA ligands: hNPDCl(35-181), hPANP(76-178), and HSV gB(23-279), which are known sialyated ligands of PILRA. Blockage of the ligands to bind to hPILRA demonstrated that the antibodies were able to antagonize hPILRA.

[0282] Table 2 below shows the binding affinities of anti-PILRA antibodies to hPILRA G78, hPILRA R78, hPILRB, cynoPILRA, and human CD98hc. The table also shows the binding epitope for each antibody, as well as the EC50 binding values measured in HEK293 or CHO cells expressing hPILRA G78, hPILRA R78, hPILRB, or cynoPILRA. The table also shows whether the antibodies blocked various tested ligands.Table 2EXAMPLE 3 - ANTI-PILRA ANTIBODY-INDUCED SIGNALING PATHWAYSEvaluation of Phosphokinase Protein Activity in Human iMicroglia and PILRA LoF iMicroglia - STAT3 Y705

[0283] Understanding PILRA downstream signaling is crucial to identifying antagonistic antibodies. Wild-type human iMicroglia and PILRA loss-of-function (LoF) iMicroglia were plated at DIV 72 in serum containing media. Media was changed after 24 hours to remove serum and cells were lysed after 72 hours. Phosphokinase levels pSTAT3 Y705 (FIG. 3A) were measured using Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R&D Systems) per manual instructions. The results demonstrate that PILRA loss of function enhances STAT3 signaling in iMicroglia.Evaluation of Anti-PILRA Antibody Downstream Signaling in HEK293 Cells Expressing hPILRA G78 or R78 - STAT3 Y705

[0284] HEK293 cells, human PILRA G78 expressing HEK293 cells, or human PILRA R78 expressing HEK293 cells were dose titrated with anti-PILRA antibodies (<200 nM) for 30 minutes in low serum condition (1% fetal bovine serum). Cells were fixed with 4% ice-cold paraformaldehyde and stained for phospho-STAT3 (Y705) (also referred to herein as “pSTAT3 (Y705)” or “pSTAT3 Y705”) using standard immunocytochemistry protocol (i.e., an automated robot for the staining) (FIG. 3B). Following a similar protocol, hPILRA R78 expressing HEK293 cells were also dose titrated with anti-PILRA antibodies and stained for pSTAT3 Y705 (FIG. 3C). Cells were imaged with a confocal microscope and images were analyzed in Harmony Software to calculate mean fluorescent spot area per cell. Data is presented as mean + / - SD fold expression over isotype control background, n=2 experiments. The results demonstrate that PILRA antibodies, including those with CD98hc binding capacity, induced phospho-STAT3 (Y705) signaling in both PILRA G78 expressing cells (FIG. 3B) and PILRA R78 expressing cells (FIG. 3C). FIG. 3D further shows that there was no induction of pSTAT3 Y705 by the same PILRA antibodies in parental HEK293 cells.

[0285] Table 3 lists EC50 values showing nM potency for induction of pSTAT3 Y705 (canonical) and pSTAT3 S727 (non-canonical) for each antibody in hPILRA G78-expressing or R78-expressing HEK293 cells. Evaluation of induction of phosphorylated STAT3 (S727) (also referred to herein as “pSTAT3 (S727)” or “pSTAT3 S727”) was carried out in a manner similar to that described above for pSTAT3 Y705. Induction of phosphorylated STAT3 Y705 and STAT3 S727 in hPILRA G78-expressing and hPILRA R78-expressing HEK293cells, but not in parental HEK293 cells, demonstrated specific PILRA-dependent downstream signaling. Further, the lack of signaling induction with isotype control antibody demonstrated PILRA selectivity and specificity.Table 3

[0286] In some embodiments, the dose-responsive induction of pSTAT3 (Y705) and / or pSTAT3 (S727) in hPILRA R78 or G78 expressing HEK293 cells can be used to rank order antagonistic antibodies based on potency and maximum effect.

[0287] Overall, as the results showed, basal changes in the phosphorylation states of STAT3 in anti-PILRA antibody dosed wild-type and PILRA LoF iMicroglia suggested that these pathways are downstream of PILRA, which is a significant discovery related to PILRA biology that has not been previously recognized. In some embodiments, an antagonistic anti- PILRA antibody modulates this pSTAT3 signaling effect in a manner similar to PILRA LoF.EXAMPLE 4 - EFFECT OF PILRA ON ANTI-INFLAMMATORY PHENOTYPE IN IMICROGLIA

[0288] Understanding PILRA-dependent functions in human iMicroglia in vitro could help to predict function in vivo. Modulation of inflammation state could be beneficial in neurodegenerative diseases. Anti-PILRA antibodies that phenocopy PILRA loss-of-function (LoF) iMicroglia functions are classified as functional antagonists. To assess PILRA- dependent transcriptional changes, wild-type iMicroglia and PILRA LoF iMicroglia were plated in serum containing media. Media was changed after 24 hours to remove serum. Lipopolysaccharide / endotoxin (LPS, 10 ng / ml) or vehicle was added 72 hours later tostimulate cytokine response. Cells were collected at 24 hours post-LPS treatment and RNA was isolated from 5 independent harvests (DIV 59, 63, 70, 73, 77) obtained from the same differentiation batch of wildtype iMicroglia or PILRA LoF iMicroglia. Two technical replicates were pooled for each condition per harvest.

[0289] To assess LPS-induced PILRA-dependent pro-inflammatory cytokine secretion, wild-type iMicroglia and PILRA LoF iMicroglia were plated in serum containing media at DIV53. Media was changed after 24 hours to remove serum and wild-type cells were dosed with antibodies (100 nM). LPS (10 ng / ml) was added 72 hours later to stimulate cytokine response. Supernatants were collected at 24 hours post-LPS treatment and run on commercial inflammatory panel kits (human pro-inflammatory (4-plex) MSD and human IP- 10 MSD, Meso Scale Discovery). Data is presented as mean + / - SEM, n=3 technical replicates.

[0290] For LPS-induced transcriptional changes, PILRA LoF suppressed LPS-induced TNF and CXCL10 gene expressions in PILRA LoF iMicroglia relative to wild-type iMicroglia (FIGS. 4A and 4B). For LPS-induced changes in cytokine secretion, PILRA LoF suppressed LPS-induced secretions of TNF alpha and IP-10 in PILRA LoF iMicroglia relative to wild-type iMicroglia (FIGS. 4C and 4D).

[0291] Moreover, anti-PILRA antibodies, including those with CD98hc binding capacity (e.g., Ab CL 2), were able to attenuate LPS-induced IP- 10 and TNF alpha cytokine secretion in wild-type iMicroglia, mimicking the phenotype observed in PILRA LoF iMicroglia (FIGS. 4E and 4F). Further, the lack of phenotype with isotype control antibody demonstrated specificity.

[0292] This example demonstrates that anti-PILRA antibodies stimulated IL IRA cytokine secretion in wild-type iMicroglia, mimicking the phenotype observed in PILRA LoF iMicroglia. Further, anti-PILRA antibodies attenuated the LPS-induced cytokine secretions in wild-type iMicroglia and IPSC-derived iMicroglia with endogenous level of hPILRA. Overall, the anti-PILRA antibodies, including those with CD98hc binding capacity (e.g., Ab CL 2), promoted anti-inflammatory phenotype.EXAMPLE 5 - PHARMACOKINETIC PROFILES OF ANTI-PILRA ANTIBODIES

[0293] To assess the pharmacokinetic profiles of anti-PILRA antibodies in vivo, BAC transgenic (BACtg) mice containing the human genomic sequence including the PILRA gene were generated. These mice were generated by microinjecting BAC clone CTD-2110B7 intoembryos from C57BL / 6J (JAX Stock# 000664) strain. This BAC clone contains the entire human PILRA (R78 version) and PILRB coding region and its regulatory elements, and expression of human PILRA in this model was confirmed.

[0294] Human PILRA BACtg mice were dosed once at 50 mg / kg of antibody Ab CL 1 or Ab CL 2 via intravenous injection (IV). Plasma and brain samples were obtained at the timepoints indicated. A MSD (Meso Scale Discovery)-based platform sandwich electrochemiluminescence immunoassay was used to quantify a total concentration of antibodies Ab CL 1 and Ab CL 2 in mouse plasma and brain lysate. As shown in FIG. 6A, differences in systemic exposure were observed between Ab CL 1 and Ab CL 2 as a result of differential binding with CD98hc in the periphery. As shown in FIG. 6B, the PK profiles demonstrate the ability of Ab CL 2, which binds to CD98hc, to access the brain relative to Ab CL 1, which does not bind to CD98hc. Ab CL 2 exhibited a more than seven-fold higher exposure in the brain compared to Ab CL 1.

[0295] This example demonstrates that Ab CL 2, an anti-PILRA antibody having CD98hc binding capacity, exhibited differences in systemic and brain exposure relative to Ab CL1, an anti-PILRA antibody lacking CD98hc binding capacity. In the brain, the higher drug exposure of Ab CL 2 was enabled or facilitated by CD98hc target engagement and delivery across the BBB.EXAMPLE 6 - LOCALIZATION OF ANTI-PILRA ANTIBODIES

[0296] Human PILRA BACtg mice expressing human PILRA (described above in Example 5) were intravenously administered a single dose of vehicle or 50 mg / kg Ab CL 2 (an anti-PILRA antibody with CD98hc binding capacity) and taken down at day 7 for tissue harvesting. Post PBS perfusion, the brain was dissected out and fixed in 4% PF A and then equilibrated in sucrose solution before sectioning at a thickness of 40 pm for Ibal, NeuN, and anti-huIgG staining. Brain images were obtained using a Leica SP8 Lightning confocal microscope, and huIgG signal on Ibal -positive and NeuN-positive cell surface were quantified. Data is presented as mean + / - SEM, n=4-5, mixed sex.

[0297] Measurement of huIgG signal on Ibal -positive microglia surface and NeuN-positive neuron surface in human PILRA BACtg brain showed localization of anti-PILRA antibody (huIgG) on microglia (FIG. 7A), but not on neuron (FIG. 7B), indicating CD98hc target engagement in vivo. HuIgG signal in the vehicle group indicates the background.EXAMPLE 7 - LIPID DROPLET FORMATION IN MICROGLIA IN VITRO

[0298] Wild-type, PILRA LoF and PILRA LoF+OE iMicroglia cells were plated at a density of 15k / well in serum-containing media on pre-coated 96-well plate. Media was changed after 24 hours to remove serum. Lipid droplet accumulation was assessed 92 hours later using BODIPYTM 493 / 503 neutral lipid assay from Thermo Fisher Scientific using manufacturers’ instructions. Data is presented as mean + / - SEM, n=3-6 experiments.

[0299] As shown in FIG. 8, PILRA LoF (PILRA KO) microglia exhibited increased lipid droplet accumulation compared to wild-type microglia (n=6 experiments). Further, complementation of PILRA in KO cells (PILRA KO + OE) reduced lipid droplets to wildtype levels (n=3 experiments).EXAMPLE 8 - EFFECTS OF ANTI-PILRA ANTIBODIES ON LIPID METABOLISM IN MICROGLIA IN VIVO

[0300] Human PILRA BACtg mice expressing human PILRA (described above in Example 5) were intravenously administered a single dose of vehicle or 50 mg / kg Ab CL 2 (an anti-PILRA antibody with CD98hc binding capacity) and taken down at day 7 for tissue harvesting. Brain tissue was dissociated into a single cell suspension using the Adult Brain Dissociation Kit (Miltenyi Biotec) followed by staining and FACS sorting. About 30,000 CD 11+ microglia were sorted directly into 400 pl of methanol containing surrogate internal standards (lipids and metabolites) for LCMS analysis. Data is presented as mean + / - SEM, n=6, mixed sex.

[0301] Anti-PILRA antibody (Ab CL 2) significantly increased phospholipid synthesis associated MG and DG lipids in microglia at day 7 post a single dose at 50 mg / kg, indicating the increased lipid storage by anti-PILRA antibody in vivo (FIGS. 9 A and 9B). Additionally, plasmalogens (PE(P-18:0 / 18: l)), pyroglutamic acid, and phenylalanine, which are associated with anti-inflammation, were also upregulated by Ab CL 2 in microglia in vivo (FIGS. 9C- 9E). The elevation of lipids and metabolites as a result of Ab CL 2 administration are consistent with the increase in lipid drop accumulation observed with PILRA LoF in microglia (Example 7, FIG. 8), demonstrating that a desirable pharmacodynamic response is induced by the anti-PILRA antibody in vivo.INFORMAL SEQUENCE LISTING

Claims

WHAT IS CLAIMED IS:

1. An isolated antibody comprising:(a) a variable region that specifically binds to a paired immunoglobulin-like type 2 receptor alpha (PILRA);(b) a first Fc polypeptide; and(c) a second Fc polypeptide, wherein the first Fc polypeptide is modified to specifically bind to a CD98 heavy chain (CD98hc) protein.

2. The isolated antibody of claim 1, wherein the antibody specifically binds to both the G78 variant of the PILRA and the R78 variant of the PILRA.

3. The isolated antibody of claim 2, wherein the binding affinity for the G78 variant of the PILRA and the binding affinity for the R78 variant of the PILRA are within 50- fold of each other.

4. The isolated antibody of any one of claims 1 to 3, wherein the PILRA is a cynomolgus monkey paired immunoglobulin-like type 2 receptor alpha (cynoPILRA).

5. The isolated antibody of claim 4, wherein the binding affinity for the cynoPILRA is at least 2-fold stronger than the binding affinity for a human paired immunoglobulin-like type 2 receptor beta (hPILRB).

6. The isolated antibody of any one of claims 1 to 3, wherein the PILRA is a human paired immunoglobulin-like type 2 receptor alpha (hPILRA).

7. The isolated antibody of any one of claims 1 to 6, wherein the variable region comprises:(1) a CDR-H1 sequence comprising at least 90% sequence identity to the sequence of GYTFTEYYMY (SEQ ID NO: 10), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 10;(2) a CDR-H2 sequence comprising at least 90% sequence identity to the sequence of RIDPEDGGTD (SEQ ID NO: 11), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 11 ;(3) a CDR-H3 sequence comprising at least 90% sequence identity to the sequence of TIRGTVFAF (SEQ ID NO: 12), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 12;(4) a CDR-L1 sequence comprising at least 90% sequence identity to the sequence of RASEDIFNGLA (SEQ ID NO: 13), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 13;(5) a CDR-L2 sequence comprising at least 90% sequence identity to the sequence of NAKTLHT (SEQ ID NO: 14), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 14; and(6) a CDR-L3 sequence comprising at least 90% sequence identity to the sequence of QQYYDYPLT (SEQ ID NO: 15), or having up to two amino acid substitutions relative to the sequence of SEQ ID NO: 15.

8. The isolated antibody of claim 7, wherein the amino acid substitutions are conservative substitutions.

9. The isolated antibody of any one of claims 1 to 8, wherein the variable region comprises: a CDR-H1 comprising the sequence of SEQ ID NO: 10 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 10; a CDR-H2 comprising the sequence of SEQ ID NO: 11 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 11; a CDR-H3 comprising the sequence of SEQ ID NO: 12 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 12; a CDR-L1 comprising the sequence of SEQ ID NO: 13 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 13; a CDR-L2 comprising the sequence of SEQ ID NO: 14 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 14; and a CDR-L3 comprising the sequence of SEQ ID NO: 15 or one or more conservative substitutions relative to the sequence of SEQ ID NO: 15.

10. The isolated antibody of any one of claims 1 to 9, wherein the variable region comprises: a CDR-H1 comprising the sequence of SEQ ID NO: 10; a CDR-H2 comprising the sequence of SEQ ID NO: 11; a CDR-H3 comprising the sequence of SEQ ID NO: 12; a CDR- L1 comprising the sequence of SEQ ID NO: 13; a CDR-L2 comprising the sequence of SEQ ID NO: 14; and a CDR-L3 comprising the sequence of SEQ ID NO: 15.

11. The isolated antibody of any one of claims 1 to 10, wherein the variable region comprises a heavy chain variable region (VH) sequence that has at least 85% sequence identity to SEQ ID NO: 16.

12. The isolated antibody of any one of claims 1 to 11, wherein the VH sequence comprises a sequence of SEQ ID NO: 16.

13. The isolated antibody of any one of claims 1 to 12, wherein the variable region comprises a light chain variable region (VL) sequence that has at least 85% sequence identity to SEQ ID NO: 17.

14. The isolated antibody of any one of claims 1 to 13, wherein the VL sequence comprises a sequence of SEQ ID NO: 17.

15. The isolated antibody of any one of claims 1 to 14, wherein the variable region comprises: a VH sequence comprising SEQ ID NO: 16 and a VL sequence comprising SEQ ID NO: 17.

16. The isolated antibody of any one of claims 1 to 10, wherein the variable region comprises a heavy chain variable region (VH) sequence that has at least 85% sequence identity to SEQ ID NO:21.

17. The isolated antibody of any one of claims 1 to 11, wherein the VH sequence comprises a sequence of SEQ ID NO:21.

18. The isolated antibody of any one of claims 1 to 12, wherein the variable region comprises a light chain variable region (VL) sequence that has at least 85% sequence identity to SEQ ID NO:22.

19. The isolated antibody of any one of claims 1 to 13, wherein the VL sequence comprises a sequence of SEQ ID NO:22.

20. The isolated antibody of any one of claims 1 to 14, wherein the variable region comprises: a VH sequence comprising SEQ ID NO:21 and a VL sequence comprising SEQ ID NO:22.

21. The isolated antibody of any one of claims 1 to 15, wherein the CD98hc protein is a human CD98hc protein.

22. The isolated antibody of any one of claims 1 to 21, wherein the CD98hc protein forms a complex with LAT1 (SLC7A5), LAT2 (SLC7A8), y+LATl (SLC7A7), y+LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11).

23. The isolated antibody of claim 22, wherein the CD98hc protein forms a complex with L ATI (SLC7A5).

24. The isolated antibody of any one of claims 1 to 23, wherein the first Fc polypeptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:64.

25. The isolated antibody of any one of claims 1 to 24, wherein the first Fc polypeptide comprises: Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, and Ala at position 442, according to EU numbering.

26. The isolated antibody of any one of claims 1 to 25, wherein the antibody has improved brain uptake compared to an antibody having a wild-type Fc dimer.

27. The isolated antibody of claim 26, wherein the antibody has at least two-fold improved brain uptake compared to the antibody having a wild-type Fc dimer.

28. The isolated antibody of claim 29, wherein the antibody has between two-fold and seven-fold improved brain uptake compared to the antibody having a wild-type Fc dimer.

29. The isolated antibody of any one of claims 1 to 28, wherein the first Fc polypeptide has a T366W substitution and the second Fc polypeptide has T366S, L368A, and Y407V substitutions, according to EU numbering.

30. The isolated antibody of any one of claims 1 to 26, wherein the first Fc polypeptide has T366S, L368A, and Y407V substitutions and the second Fc polypeptide has a T366W substitution, according to EU numbering.

31. The isolated antibody of any one of claims 1 to 30, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises a modification that reduces effector function.

32. The isolated antibody of claim 31, wherein the modification that reduces effector function comprises the substitutions of Ala at position 234 and Ala at position 235, according to EU numbering.

33. The isolated antibody of claim 31 or 32, wherein the modification that reduces effector function comprises the substitution of Gly at position 329.

34. The isolated antibody of any one of claims 1 to 33, comprising:(i) a first heavy chain comprising: (1) a VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and (2) a first Fc polypeptide comprising modifications for CD98hc-binding, a knob mutation, and modifications that reduce or eliminate effector function;(ii) a second heavy chain comprising: (1) a VH sequence having a CDR-H1, a CDR- H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and (2) a second Fc polypeptide comprising hole mutations, and modifications that reduce or eliminate effector function; and(iii) first and second light chains each comprising a VL sequence having a CDR-L1, a CDR-L2, a CDR-L3 of SEQ ID NOS:13-15, respectively.

35. The isolated antibody of claim 34, wherein:(i) the first heavy chain comprises: (1) the VH sequence having a CDR-H1, a CDR- H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% identity to SEQ ID NO: 16, and (2) the first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, Ala at position 442, Trp at position 366, Ala at position 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% identity to the sequence of SEQ ID NO: 66;(ii) the second heavy chain comprises: (1) the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% identity to SEQ ID NO: 16, and (2) the second Fc polypeptide comprising T366S, L368A, Y407V, Ala atposition 234, Ala at position 235, and Gly at position 329, according to EU numbering, and a sequence having at least 90% identity to the sequence of SEQ ID NO: 63; and(iii) the first and second light chains each comprising the VL sequence having a CDR- Ll, a CDR-L2, a CDR-L3 of SEQ ID NOS: 13-15, respectively, and at least 90% identity to SEQ ID NO: 17.

36. The isolated antibody of claim 35, wherein:(i) the first heavy chain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 18;(ii) the second heavy chain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 19; and(iii) the first and second light chains each comprising a sequence having at least 90% identity to the sequence of SEQ ID NO:20.

37. The isolated antibody of claim 34, wherein:(i) the first heavy chain comprises the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% identity to SEQ ID NO:21, and the first Fc polypeptide comprising Leu at position 380, Asn at position 382, Arg at position 384, Phe at position 385, Vai at position 386, Leu at position 387, Glu at position 421, He at position 422, Ala at position 424, Asn at position 426, Tyr at position 428, Phe at position 438, Asn at position 440, Ala at position 442, Trp at position 366, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% identity to the sequence of SEQ ID NO: 66;(ii) the second heavy chain comprises the VH sequence having a CDR-H1, a CDR-H2, a CDR-H3 of SEQ ID NOS: 10-12, respectively, and at least 90% identity to SEQ ID NO:21, and the second Fc polypeptide comprising T366S, L368A, Y407V, Ala at position 234, Ala at position 235, and Gly at position 329, numbering according to EU numbering, and a sequence having at least 90% identity to the sequence of SEQ ID NO: 63; and(iii) the first and second light chains each comprising the VL sequence having a CDR- Ll, a CDR-L2, a CDR-L3 of SEQ ID NOS: 13-15, respectively, and at least 90% identity to SEQ ID NO:22.

38. The isolated antibody of claim 35, wherein:(i) the first heavy chain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:23;(ii) the second heavy chain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:24; and(iii) the first and second light chains each comprising a sequence having at least 90% identity to the sequence of SEQ ID NO:25.

39. The isolated antibody of any one of claims 1 to 38, wherein the antibody antagonizes hPILRA activity.

40. The isolated antibody of any one of claims 1 to 39, wherein the antibody blocks binding of a sialyated protein to hPILRA.

41. The isolated antibody of claim 40, wherein the sialyated protein is a sialyated NPDC1, PANP, HSV-1 gB, COLEC12, C4a, C4b, DAG1, or Clec4g.

42. The isolated antibody of any one of claims 1 to 41, wherein the antibody enhances phosphorylation of EGFR or STAT3, or decreases phosphorylation of STATE43. The isolated antibody of any one of claims 1 to 42, wherein the antibody enhances cell migration.

44. The isolated antibody of claim 43, wherein the antibody enhances microglia migration.

45. The isolated antibody of any one of claims 1 to 44, wherein the antibody enhances anti-inflammatory gene or protein expression.

46. The isolated antibody of claim 45, wherein the antibody enhances IL1RN gene expression.

47. The isolated antibody of any one of claims 1 to 46, wherein the antibody reduces pro-inflammatory cytokine protein expression or secretion.

48. The isolated antibody of claim 47, wherein the antibody reduces TNF, IL-6, and / or IP- 10 expression.

49. The isolated antibody of any one of claims 1 to 48, wherein the antibody increases cellular respiration.

50. The isolated antibody of claim 49, wherein the antibody increases mitochondrial respiration.

51. The isolated antibody of any one of claims 1 to 50, wherein the antibody increases ATP production.

52. The isolated antibody of any one of claims 1 to 51, wherein the antibody increases fatty acid metabolism.

53. The isolated antibody of any one of claims 1 to 52, wherein the antibody does not activate peripheral immune cells.

54. The isolated antibody of claim 53, wherein the antibody does not activate neutrophils and monocytes.

55. The isolated antibody of any one of claims 1 to 54, wherein the antibody increases lipid storage or lipid levels in microglia.

56. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 55 and a pharmaceutically acceptable carrier.

57. One or more polynucleotides comprising one or more nucleic acid sequences encoding the heavy chains and / or light chains of the isolated antibody of any one of claims 1 to 55.

58. One or more vectors comprising the one or more polynucleotides of claim 57.

59. A host cell comprising the one or more polynucleotides of claim 57 or the one or more vectors of claim 58.

60. A method for producing an isolated antibody, comprising culturing the host cell of claim 59 under conditions in which the isolated antibody of any one of claims 1 to 55 is expressed.

61. A method of treating a neurodegenerative disease in a subject, comprising administering to the subject the isolated antibody of any one of claims 1 to 55 or the pharmaceutical composition of claim 56.

62. The method of claim 61, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis / parkinsonism-dementia complex of Guam (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler- Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadelopean PSP, Hallevorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington’s disease, inclusion-body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Parkinson’s disease, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle only dementia.

63. A method for determining whether an antibody has activity at a PILRA protein, the method comprising:(a) contacting a cell that expresses the PILRA protein with the antibody;(b) either prior to, concurrently with, or following step (a), contacting a cell of the same type as in step (a) having lower or no PILRA expression with the antibody; and(c) measuring one of the following: phosphorylated STAT3 (pSTAT3) level and phosphorylated STAT1 (pSTATl) level, in both cells, wherein a change in the level of one of these measurements between the cells indicates that the antibody has activity at the PILRA protein of step (a), and wherein the antibody specifically binds to a CD98 heavy chain (CD98hc) protein.

64. The method of claim 63, wherein step (c) measures pSTAT3 level.

65. The method of claim 63 or 64, wherein the cell of step (a) naturally expresses the PILRA protein.

66. The method of claim 63 to 65, wherein the cell having lower PILRA expression has the PILRA protein knocked-out.

67. The method of claim 66, wherein the cell is a HEK cell.

68. The method of claim 67, wherein the cell is an iMicroglia.

69. The method of claim 68, wherein the cell is a PILRA LoF iMicroglia.

70. The method of claim 63, wherein the cell of step (a) is engineered or modified to express or overexpress the PILRA protein.

71. The method of claim 70, wherein the cell having lower PILRA expression naturally expresses the PILRA protein or is not engineered or modified to express the PILRA protein.

72. The method of any one of claims 63 to 71, wherein the antibody is from a library of antibodies.

73. The method of any one of claims 63 to 72, wherein the antibody is known to bind the PILRA protein.

74. The method of any one of claims 63 to 72, wherein it is unknown whether the antibody binds the PILRA protein.

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