Anti-CD33 antibody

Abstract

This document describes isolated antibodies that specifically bind to the CD33 protein. Related nucleic acids, cells, and therapeutic uses are also described.

Description

[Technical Field]

[0001] Field of Invention This invention relates to antibodies capable of binding to CD33, and in particular, to novel therapeutic antibodies, but is not limited thereto. Methods for using anti-CD33 antibodies in the treatment of neurodegenerative diseases and cancer are also described. [Background technology]

[0002] background CD33 (Sigrec-3) is an inhibitory immune receptor and a type I transmembrane protein belonging to the family of sialic acid-binding immunoglobulin-like lectins (Sigrec). It is expressed on the cell surface of bone marrow cells, monocytes, macrophages, and microglia in the brain. In genome-wide association studies, CD33 was one of the top-ranked genes associated with the risk of developing Alzheimer's disease (AD). CD33 signaling in microglia is involved in AD pathology, and CD33 expression is elevated in AD patients, which is thought to regulate microglial activation and inhibit amyloid-beta clearance (Zhao et al., 2019).

[0003] Alzheimer's disease is the most common cause of dementia in older adults (Zhao et al., 2019). Novel therapies for Alzheimer's disease are being actively sought to alter the course of the disease. Current candidates targeting beta-amyloid, tau, and innate immunity in the brain have shown pharmacodynamic effects on pathological mechanisms in some cases in clinical trials, but have not yet demonstrated compelling disease modification in late-stage clinical trials (Golde et al., 2022).

[0004] Siglec, expressed on tumor-infiltrating immune cells, is suggested to influence anti-tumor immunity and may be a potential target for cancer immunotherapy (Stanczak & Laubli, 2023).

[0005] Antibodies capable of binding to CD33 have been developed. For example, U.S. Patent Application Publication 2022 / 0162309 describes humanized antibodies that bind to human CD33 and the use of these antibodies in Alzheimer's disease, dementia, frontotemporal dementia, vascular dementia, mixed dementia, tauopathic diseases, infections, and cancer. However, clinical trials for the lead antibody AL003 described in U.S. Patent Application Publication 2022 / 0162309 have been completed. Humanized anti-CD33 antibodies (lintuzumab) have been shown to have moderate activity in the treatment of acute myeloid leukemia (AML) (Jurcic, 2012). [Overview of the project] [Problems that the invention aims to solve]

[0006] Further antibodies capable of effectively binding to and regulating CD33 signaling are still needed. [Means for solving the problem]

[0007] Summary of the Invention This disclosure also includes combinations of the embodiments and preferred features described, unless such combinations are clearly unacceptable or expressly avoided.

[0008] According to a first aspect, the present invention provides an isolated antibody that specifically binds to the CD33 protein, and which increases the phagocytosis of cells expressing CD33 compared to a comparative antibody, and / or increases antibody binding to human CD33 protein containing mutations at positions 20, 21, 22 and 24 compared to antibody binding to human CD33 protein without such mutations.

[0009] The antibodies according to this embodiment may contain heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing any of the amino acid sequences of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005 CDRH2 containing any of the amino acid sequences 808, ATL_0005809, or ATL_0005810; CDRH3 containing any of the amino acid sequences 0ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810; or a CDR set in which each CDR contains 0, 1, or 2 amino acid substitutions compared to the above CDR set.

[0010] According to a second aspect, the present invention provides an isolated antibody that specifically binds to the CD33 protein and comprises a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising any of the amino acid sequences of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, antibodies ATL_0005802, ATL_0005853, ATL_0005854, A CDRH2 containing any of the amino acid sequences TL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810; CDRH3 containing any of the amino acid sequences 0ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810; or a CDR set in which each CDR contains 0, 1, or 2 amino acid substitutions compared to the above CDR set.

[0011] These CDR-containing antibodies were identified as CD33-binding antibodies in centenarians with superior cognitive abilities, suggesting a potential protective role in neurodegenerative diseases and good tolerability in treatment. These antibodies were found to be less affected by peripheral degradation than the comparative antibody, while simultaneously inducing phagocytosis in bone marrow and microglia cells to a similar or greater degree. These antibodies were also found to bind to different epitopes than the comparative antibody.

[0012] The antibody according to this embodiment may increase the phagocytosis of CD33-expressing cells compared to a comparative antibody, and / or antibody binding to human CD33 proteins containing mutations at positions 20, 21, 22, and 24 may be increased compared to antibody binding to human CD33 proteins without such mutations.

[0013] The antibody according to the first or second embodiment may have one or more of the following optional characteristics:

[0014] Antibody binding to human CD33 proteins containing mutations at positions 20, 21, 22, 24, and 132 may increase compared to antibody binding to human CD33 proteins without such mutations. Antibody binding to human CD33 proteins containing mutations at positions 47, 50, 51, and 52 may decrease compared to antibody binding to human CD33 proteins without such mutations. Antibodies cannot bind to human CD33 proteins containing mutations at positions 47, 50, 51, and 52. Antibody binding to human CD33 proteins containing mutations at positions 47, 50, 51, 52, and 122 may decrease compared to antibody binding to human CD33 proteins without such mutations. Antibodies cannot bind to human CD33 proteins containing mutations at positions 47, 50, 51, 52, and 122. Antibody binding to human CD33 proteins containing a mutation at position 83 may decrease compared to antibody binding to human CD33 proteins without such mutations.

[0015] The mutations may be selected from the following: N20R at position 20, F21V at position 21, W22R at position 22, Q24E at position 24, I47V at position 47, Y50H at position 50, D51T at position 51, K52R at position 52, Q83R at position 83, R122K at position 122, and P132T at position 132.

[0016] Binding may be measured using single-point ELISA. Human CD33 protein may contain residues 18–232 of human CD33. Unmutated human CD33 protein may be CD33M2_ECD_18-232_WT.

[0017] The antibody may have one or more or all of the following: (i) increased binding to the protein containing the sequence CD33M2_ECD_18-232_MutPos1[P1] compared to the protein containing the sequence CD33M2_ECD_18-232_WT, (ii) increased binding to the protein containing the sequence CD33M2_ECD_18-232_MutPos1_MutPos6[P6+1] compared to the protein containing the sequence CD33M2_ECD_18-232_WT, (iii) increased binding to the protein containing the sequence CD33M2_ECD_18-232_WT (iv) Compared to proteins containing the CD33M2_ECD_18-232_MutPos2[P2] sequence, there was a decrease in binding to proteins containing the CD33M2_ECD_18-232_MutPos2_MutPos5[P2+5] sequence, and (v) Compared to proteins containing the CD33M2_ECD_18-232_WT sequence, there was a decrease in binding to proteins containing the CD33M2_ECD_18-232_MutPos4[P4] sequence.

[0018] The antibodies of this disclosure may have lower peripheral clearance when administered to a subject compared to the comparative anti-CD33 antibody.

[0019] The antibodies of the present disclosure can increase the phagocytosis of Aβ plaques by microglial cells in vivo as compared to a control. The antibodies of the present disclosure can increase the phagocytosis of tau aggregates by microglia having an inflammatory phenotype (e.g., LPS-treated iPSC microglia) as compared to a control. The antibodies of the present disclosure can increase the phagocytosis of tau aggregates by microglia having an inflammatory phenotype to a greater extent than a comparative anti-CD33 antibody. The antibodies of the present disclosure cannot induce the release of one or more cytokines including IL-6 and / or MCP-1 by microglia in vitro and / or in vivo. The antibodies of the present disclosure can decrease the levels of IL-6 and / or MCP-1 released in vitro (e.g., LPS-treated human iPSC-derived microglia) and / or in vivo by microglia having an inflammatory phenotype. The antibodies of the present disclosure can decrease the inflammation-induced release of one or more markers of inflammation in a human neuronal cell culture assay and / or in the central nervous system of a subject. The one or more markers of inflammation can be selected from MCP-1, IP-10, GFAP, and IL-6.

[0020] The comparative antibody can be selected from an isotype control antibody, another CD33-binding antibody, and an antibody having the heavy chain variable sequence of ATL_5909 and the light chain variable sequence of ATL_5909. Phagocytosis can be evaluated by measuring a fluorescence signal associated with the uptake of labeled particles by imaging or flow cytometry. The cells can be monocytes or microglial cells. The cells can be human cells. The cells can be iPSC-derived microglial cells. The cells can be cells stimulated with an inflammatory signal (e.g., LPS) prior to exposure to the antibody.

[0021] The antibody can bind to a CD33 protein containing the V domain of CD33. The antibody cannot bind to a CD33 protein that does not contain the V domain of CD33. The antibody cannot bind to a CD33 protein having the sequence of protein CD33_human_ECD_C_domain_His_007.

[0022] The antibody can include a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising an amino acid sequence selected from the amino acid sequences of HCDR1_ATL_0005802, HCDR1_ATL_0005807, HCDR1_ATL_0005808: GYSFTSYW, HCDR1_ATL_0005853: GYKFNNNW, HCDR1_ATL_0005854: GYKFSNNW or an amino acid sequence having one or two mutations as compared with the above sequences. CDRH2 comprising an amino acid sequence selected from the amino acid sequences of HCDR2_ATL_0005802, HCDR2_ATL_0005807, HCDR2_ATL_0005808, HCDR2_ATL_0005853, HCDR2_ATL_0005854: IYPGDSDT or an amino acid sequence having one or two mutations as compared with the above sequences, and CDRH3 comprising an amino acid sequence selected from the amino acid sequences of HCDR3_ATL_0005802, ARPRGFGEYYFDY, HCDR3_ATL_0005853 ARHSGGLDGYTAAALDY, HCDR3_ATL_0005854 ATWGGSNWFVD or an amino acid sequence having one or two mutations as compared with the above sequences.

[0023] The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the sequence of HCDR1_ATL_0005802, CRH2 comprising the sequence of HCDR2_ATL_0005802 and CDRH3 comprising the sequence of HCDR3_ATL_0005802, or a set of CDRs comprising one or two mutations in CDRH1 and CDRH2 as compared with these sequences and / or one, two or three mutations in CDRH3 as compared with these sequences.

[0024] The antibody may contain heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005853, CRH2 containing the sequence HCDR2_ATL_0005853, and CDRH3 containing the sequence HCDR3_ATL_0005853, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences and / or one, two, or three mutations in CDRH3 compared to these sequences.

[0025] The antibody may contain heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005854, CRH2 containing the sequence HCDR2_ATL_0005854, and CDRH3 containing the sequence HCDR3_ATL_0005854, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences and / or one, two, or three mutations in CDRH3 compared to these sequences.

[0026] Antibodies may have a heavy chain variable domain (VH) with the following framework sequence: HFWR1 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, HFWR2 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810. HFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, and HFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or A framework array having 1 to 6 substitutions compared to the framework array above.

[0027] The antibody may have a heavy chain variable domain (VH) containing the framework sequence HFWR2 of ATL_0005802. The antibody may have a heavy chain variable domain (VH) containing a framework sequence with "A" at position 40 in standard IMGT numbering. Substitutions in the framework sequence of the heavy chain variable domain may be located at any position other than position 40 in standard IMGT numbering. The antibody may have a heavy chain variable domain (VH) containing CDRH1, CDRH2, and CDRH3 within the germline cell framework, provided that position 40 in standard IMGT numbering is A.

[0028] The antibody may have a heavy chain variable domain (VH) having the following framework sequence: HFWR1 of ATL_0005802, ATL_0005853 or ATL_0005854, HFWR2 of ATL_0005802, ATL_0005853 or ATL_0005854, HFWR3 of ATL_0005802, ATL_0005853 or ATL_0005854, and HFWR4 of ATL_0005802, ATL_0005853 or ATL_0005854.

[0029] The antibodies may have a heavy chain variable domain (VH) containing a sequence that has at least 95% sequence identity with a sequence selected from the VH sequences of the following antibodies: ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, ATL_0006040, ATL_0006041, ATL_0006042, ATL_0006043, ATL_0006044, ATL_0006045, ATL_0006046, ATL_0006047, and ATL_0006048. The antibody may have a heavy chain variable domain (VH) containing a sequence that has up to two mutations in each HCDR and up to three mutations in each framework region compared to a sequence selected from the above VH sequences. The antibody may have a heavy chain variable domain (VH) containing a sequence that has at least 95% sequence identity with a sequence selected from the VH sequences of antibodies ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence that has up to two mutations in each HCDR and up to three mutations in each framework region compared to a sequence selected from the above VH sequences.

[0030] The antibody can bind to human CD33. The antibody can bind to human CD33 with an EC50 of up to 2e-08M or up to 3e-09M, as evaluated by ELISA (e.g., binding to plated rhCD33).

[0031] The antibody may deplete less than 50% or less than 80% of CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody. The antibody may deplete less CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody than the same concentration of a control antibody.

[0032] The antibody may bind to CD33 more selectively than one or more other Siglecs. The antibody may bind to CD33 more selectively than one or more (or all) of the following: Siglec-6, Siglec-7, Siglec-8, and Siglec-9. The antibody may bind to human CD33 more selectively than one or more other homologs. The antibody may bind to human CD33 more selectively than mouse CD33 and cynomolgus monkey CD33.

[0033] The antibody may be an scFv antibody molecule, a nanobody, or a whole antibody. The antibody may contain an antibody constant region. The antibody may be a whole antibody. The antibody may be IgG1 or a variant thereof. The antibody may be the IgG1 variant L234A / L235A (LALA).

[0034] The antibody may contain a light chain variable domain (VL) having the following CDR: CDRL1 containing one of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, CDRL2 containing any of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and CDRL3 containing any of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or A set of CDRs in which each CDR contains 0, 1, or 2 amino acid substitutions, compared to the set of CDRs described above.

[0035] The VH domains CDRL1, CDRL2, and CDRL3 are located within the germline framework.

[0036] Antibodies may have a light chain variable domain (VL) with the following framework sequence: LFWR1 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, LFWR2 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, LFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, and LFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or A set of FWRs containing 1 to 6 amino acid substitutions, compared to the FWR set described above.

[0037] The antibody may have a heavy chain variable domain (VL) containing a selected sequence that has at least 95% sequence identity with a sequence selected from the VL sequences of the following antibodies: ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, ATL_0006040, ATL_0006041, ATL_0006042, ATL_0006043, ATL_0006044, ATL_0006045, ATL_0006046, ATL_0006047, and ATL_0006048. The antibody may have a light chain variable domain (VL) containing a sequence having up to two mutations in each LCDR and up to three mutations in each framework region compared to a sequence selected from the VL sequence. The antibody may have a light chain variable domain (VL) containing a sequence having at least 95% sequence identity with a sequence selected from the VL sequence of antibody ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence having up to two mutations in each LCDR and up to three mutations in each framework region compared to a sequence selected from the VL sequence.

[0038] According to a third aspect, an isolated VH domain of an antibody according to any embodiment of the first or second aspect is provided.

[0039] According to the fourth aspect, an isolated nucleic acid is provided which includes an antibody containing a VH or VL domain or a nucleotide sequence encoding a fragment thereof according to any embodiment of the first or second aspect.

[0040] This specification also describes a vector or set of vectors containing nucleic acids according to a fourth aspect, a host cell transformed in vitro with the nucleic acid, or a host cell containing the vector or set of vectors.

[0041] This specification also describes compositions comprising an antibody containing an antibody VH domain or an antibody VL domain according to any embodiment of the first or second aspect, and optionally, at least one additional component, including a pharmaceutically acceptable excipient, vehicle, or carrier.

[0042] This specification also describes antibodies in any embodiment of the first or second aspect for use in the treatment of a disease or disorder. This specification also describes a method for treating a disease or disorder in a subject in need thereof, comprising administering a therapeutically effective dose of an antibody in any embodiment of the first or second aspect. This specification also describes antibodies in any embodiment of the first or second aspect for use in the manufacture of a drug. The disease or disorder may be a disease related to microglial cell dysfunction. The disease or disorder may be a neurodegenerative disease or disorder. The disease or disorder may be a tauopathy. Neurodegenerative diseases include frontotemporal dementia (FTD), Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), HIV-induced encephalitis, chronic traumatic encephalopathy (CTE), vascular dementia, prion diseases, Lewy body dementia, spinal muscular atrophy (SMA), and motor neuron diseases (MND), such as amyotrophic lateral sclerosis (ALS) and progressive supranuclear palsy (PS). P) may be selected from spinocerebellar degeneration (SCA) types 1, 2, 6, 7, and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral-pallidoluysian atrophy (DRPLA), X-linked spinal muscular atrophy type 1 (SMAX1 / SBMA), Anderson Fabry (X-linked Fabry disease), and DNAJB6 myopathy, and optionally, neurodegenerative diseases may be selected from FTD, AD, HD, and PD. The disease or disorder may be cancer. The cancer may be selected from AML or cancer associated with hypersialylation of tumor cells and / or overexpression of CD33 by tumor cells. The disease or disorder may be a disease characterized by insufficient macrophage phagocytosis and / or macrophage dysfunction. For example, the disease may be COPD or IPF. The drug may be for the treatment of any of the above diseases or disorders.

[0043] Drawing Overview Herein, embodiments and experiments demonstrating the principles of this disclosure will be described with reference to the accompanying drawings. [Brief explanation of the drawing]

[0044] [Figure 1] The results of the process for identifying convergent BCR sequences in two individuals who were amyloid-positive but tau-negative and cognitively normal are schematically shown. The numbers shown are clonal type numbers. The two individuals shared 64 clonal types, and a representative sequence from one of the 64 shared clonal types was shown to bind to CD33 (see below). [Figure 2] The results of ELISA for the binding of ATL_0005082 (also referred to as ATL_5082 herein) to CD33 are shown. The isotype control is a commercially available human IgG1 isotype control (Absolute Antibody, Ab00102-10.0 Anti-Fluorescein), which does not show binding to CD33. Lysozyme = negative control antigen. CD33 = binding to recombinant human CD33. [Figure 3] The results of ELISA serum screening across a broad panel of individual donors are shown. The graphs show the level of autoreactivity to CD33 in multiple individuals by age. The Z-score is calculated as [raw signal - mean signal] / standard deviation (SD). [Figure 4A] This report presents the results of an ELISA for identifying CD33-reactive plasma from a cohort of individuals aged 110 years or older. Twelve plasma samples were tested by ELISA for binding to recombinant human CD33. The sample showing the highest signal for CD33 (arrow, SU_0000877) was selected for phage display library generation. [Figure 4B] The results of ELISA for identifying CD33-reactive plasma from a cohort of individuals aged 110 years or older are shown. Twelve plasma samples were tested by ELISA for binding to a lysozyme control. [Figure 5]The results of phage ELISA for identifying single-stranded variable fragment (scFv) clones that bind to the CD33 antigen are shown. Phage display scFv derived from phage display selection were tested for binding to recombinant CD33 (pink circles) and lysozyme control (black circles). [Figure 6] The results of ELISA for the binding of nine anti-CD33 antibodies and a comparative antibody (ATL_5503), identified by phage display, to the complete extracellular domain of CD33 (clear bar, "CD33_human_M17-H259_002_004") or the C2 domain (dark blue bar, "CD33_human_ECD_Cdomain_His_007") are shown. The results are presented as signal vs. isotype. [Figure 7A] This graph shows the quantification of CD33 levels on human monocytes after 5 hours of incubation with the indicated antibody at concentrations of 1 nM to 40 nM, as measured by flow cytometry. The graph shows the median MFI (fluorescence intensity) of CD14+CD33+ cells plotted against antibody concentration. The tested (ATL) antibody binds only to the V domain of the CD33 receptor, while the CD33 antibody used for detection by flow cytometry is specific to the C domain. The decrease in median fluorescence intensity may be due to the loss of the CD33 receptor from the cell membrane surface, as there is no interference between detection antibody binding and test antibody binding. ATL5909 shows complete CD33 depletion at all concentrations tested, while ATL5802 shows complete lack of CD33 depletion at all concentrations, comparable to the isotype control. ATL5810 has an intermediate profile, with a dose-dependent decrease in CD33 levels. [Figure 7B] This shows the quantification of CD33 levels on human monocytes after incubation for 5 hours with the indicated antibodies at concentrations of 1 nM to 40 nM, as measured by flow cytometry. The MFI of CD33 domain V in the CD14+CD16+ (bottom) or CD14+CD16- (top) of the indicated antibodies is shown. [Figure 7C]This shows the quantification of CD33 levels on human monocytes after incubation for 5 hours with the indicated antibodies at concentrations of 1 nM to 40 nM, as measured by flow cytometry. The MFI of CD33 domain C in the CD14+CD16+ (bottom) or CD14+CD16- (top) of the indicated antibodies is shown. It is noteworthy that since all test antibodies bind to the V domain, changes in domain V MFI can be due to both competition with the detected antibody and CD33 depletion. In contrast, assays using detection of domain C MFI more accurately represent CD33 depletion. [Figure 7D] This shows the quantification of CD33 levels on human monocytes after 5 hours of incubation with indicated antibody concentrations of 1 nM to 40 nM, as measured by flow cytometry. For CD14+CD16- and CD14+CD16+ cells, the area under the curve (AUC) of binding to the CD33 C and V domains as a function of concentration in the range of 40 to 1 nM (curves in the lower panels of B and C) is shown. The data are normalized to the AUC of the isotype control antibody set to 100. [Figure 7E] This shows the quantification of CD33 levels on human monocytes after 5 hours of incubation with indicated antibody concentrations of 1 nM to 40 nM, as measured by flow cytometry. The area under the curve (AUC) for binding to the CD33 C and V domains as a function of concentration in the range of 40 to 1 nM (curves in the upper panels of B and C). [Figure 8A] This paper outlines the setup and results of an in vitro phagocytic assay in microglia derived from inflammatory human iPSCs. The phagocytic assay is performed as follows: Microglia are incubated with LPS for 24 hours, followed by incubation with an anti-CD33 antibody (or isotype control) for 24 hours, and then the uptake of pHrodo-red labeled amyloid-beta is measured. In the low pH environment of cellular lysosomes after labeled amyloid-beta phagocytosis, only pHrodo-red fluoresces red. [Figure 8B]This document describes the setup and results of an in vitro phagocytic assay in inflammatory human iPSC-derived microglia. It also presents the results of a phagocytic assay testing the effects of ATL5802 and ATL5810 on microglial phagocytosis, comparing them to prior art antibody ATL5909, isotype control, and negative control (Aβ only), regarding the time course of the surface area of ​​red cells per well with antibody incubation. [Figure 8C] This document describes the setup and results of an in vitro phagocytic assay in inflammatory human iPSC-derived microglia. The results of the phagocytic assay in Panel B are shown, expressed as the area under the curve (μm²) of the surface area of ​​red cells per well over time (this represents the uptake of pHrodo red amyloid-beta into those cells by phagocytosis). [Figure 8D] This document describes the setup and results of in vitro phagocytic assays in inflammatory human iPSC-derived microglia. It also presents the results of phagocytic assays testing the effects of ATL5802, ATL5853, ATL5854, and ATL6044 on microglial phagocytosis compared to the prior art antibody ATL5909 and isotype controls. [Figure 8E] This paper describes the setup and results of in vitro phagocytic assays in microglia derived from inflammatory human iPSCs. It also shows the quantification of phagocytic assay results for panels D and F, expressed as the area under the curve (μm2) of the surface area of ​​red cells per well over time at 18 hours. The surface area of ​​red cells represents the uptake of pHrodol amyloid-beta into those cells and therefore indicates the level of phagocytosis. Statistical analysis was performed using one-way ANOVA against isotype controls. *=p<0.05, **=p<..., ***=p<..., ns=not significant. At least ATL_5854 showed a statistically significant difference when directly tested against ATL_5909 (one-way ANOVA). [Figure 8F]This document describes the setup and results of an in vitro phagocytic assay in microglia derived from inflammatory human iPSCs. It shows the results of experiments comparing the phagocytosis of labeled amyloid beta in iPSC-derived microglia treated with anti-TREM2 antibodies (ATL6166, ATL6167, and ATL6170) or ATL5802 (the upper and lower panels show repeats of the same experiment). [Figure 9A] The results of an ex vivo phagocytosis assay using human whole blood are shown. A flow cytometry plot of pHrodo red-labeled S. aureus bone marrow cells (pHrodo+ CD14 cells) is shown, which indicates pHrodo uptake in CD14+ cells. In the low pH environment of the lysosomes of cells after phagocytosis by labeled S. aureus, only pHrodo-red emits red fluorescence. [Figure 9B] The results of an ex vivo phagocytic assay using human whole blood are shown. pHrodo in CD14+ cells (which exhibits phagocytosis) is shown, measured as MFI of pHrodo in CD14+ myeloid cells incubated with the indicated antibodies (ATL5802, ATL5810, ATL5909) or isotype controls. Negative controls included wells without pHrodo-labeled S. aureus and untreated cells. [Figure 9C] The results of an ex vivo phagocytic assay using human whole blood are shown. Phagocytosis of pHrodo-labeled S. aureus in fresh, isolated CD14+ monocyte cells treated with 0.029 nM–30 nM ATL5802 (green), ATL5909 benchmark 1 (red), ATL4828 benchmark 2 (blue), and isotype control (yellow) is shown (number of red objects per well, corresponding to cells with pHrodo, normalized to the observed maximum). Latrantrin A was used as a control, expected to prevent intracellular actin polymerization and completely block phagocytosis. [Figure 10]This shows the CD33 levels in peripheral bone marrow cells in CD4+ NSG mice 24 hours after intraperitoneal (IP) injection of antibodies (ATL5802, 5810, 5909, or isotype control) at the indicated concentrations (10 mg / kg or 40 mg / kg). CD33 levels on the surface of human transplanted bone marrow cells (defined as huCD45+huCD14+ cells) were measured with antibodies specific to the C domain of CD33. The reported values ​​are the fluorescence intensity (MFI) of CD33 on human CD45+CD14+ cells. [Figure 11A] A schematic diagram of the mutation introduced into the CD33 V domain for epitope mapping of the antibodies described herein is shown, with the siaroglycan ligand indicated in gold in the center. Variants of the CD33 extracellular domain were prepared as recombinant proteins as shown. [Figure 11B] The corresponding results for mutations introduced into the CD33 V domain for epitope mapping of the antibodies described herein are shown. Nine IgG1 antibodies were tested by single-point ELISA against all of the wild-type CD33 and seven CD33 variant proteins, and binding was quantified compared to binding to the wild-type CD33 extracellular domain. Using the binding profiles of each antibody against various CD33 variants, the antibodies were grouped into four different epitope bins as shown. [Figure 12] This report shows CD33 levels on human CD11b+CD45+ cells in the brains of CD34+ NSG mice 24 hours after intraperitoneal (IP) injection of antibodies (ATL5802, 5810, 5909, or isotype control). Cells were isolated from brain homogenates using anti-CD11b magnetic beads and then stained with human and mouse anti-CD45. CD33 levels were assessed in human cells, and the median fluorescence intensity of CD33 staining is reported. [Figure 13]The results of a flow cytometry assay detecting the median fluorescence intensity of labeled S. aureus in human CD45+CD14+CD11b+ peripheral bone marrow cells from CD34+ NSG mice after 24-hour to 3-hour ex vivo incubation with pHrodo-labeled S. aureus following intraperitoneal injection of antibodies (ATL5802, 5810, 5909, or isotype control). [Figure 14] The SEC-HPLC chromatograms of ATL5802 under the specified forced degradation conditions (2 weeks at -80°C, 2 weeks at 40°C, overnight shaking, 3 freeze-thaw cycles (3×FT), and low pH maintenance) are shown. [Figure 15] The results of an ELISA test to examine the binding of recombinant human CD33 (rhCD33) to ATL5802 under the specified forced degradation conditions (2 weeks at -80°C, 2 weeks at 40°C, overnight shaking, 3 freeze-thaw cycles (3×FT), and low pH maintenance) are shown. [Figure 16A] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH FWR1. [Figure 16B] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH CDR1. [Figure 16C] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH FWR2. [Figure 16D] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH CDR2. [Figure 16E] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH FWR3. [Figure 16F] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH CDR3. [Figure 16G] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VH FWR4. [Figure 16H]The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VL FWR1. [Figure 16I] The alignment of the variable chain sequences of the selected anti-CD33 antibody is shown. The dashed line indicates the comparison antibody. VL CDR1. [Figure 16J] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VL FWR2. [Figure 16K] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody, VL CDR2. [Figure 16L] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VL FWR3. [Figure 16M] The alignment of the variable chain sequences of the selected anti-CD33 antibodies is shown. The dashed line indicates the comparison antibody. VL CDR3. [Figure 16N] The alignment of the variable chain sequences of the selected anti-CD33 antibody is shown. The dashed line indicates the comparison antibody, CL FWR4. [Figure 17A] The results of an in vivo pK test in C57BL / 6-Cd33tm1(CD33) mice are shown. ATL-5802 or ATL-5909 was administered intraperitoneally at a dose of 1 mg / kg to groups of mice (n=3 groups / group). Serum levels of hIgG1 were measured 4 hours, 24 hours, and on day 7 after a single antibody dose. A graph plotting the serum levels of ATL-5802 or ATL-5909 at the indicated time points is shown. [Figure 17B] The results of an in vivo pK test in C57BL / 6-Cd33tm1(CD33) mice are shown. ATL-5802 or ATL-5909 was administered intraperitoneally at a dose of 1 mg / kg to groups of mice (n=3 groups / group). Serum levels of hIgG1 were measured 4 hours, 24 hours, and on day 7 after a single antibody dose. The analysis of total antibody concentration over time (area under the curve, AUC) is shown. **p<0.01, unpaired t-test. [Figure 18]This report presents the results of an in vivo study in an animal model of Alzheimer's disease, testing the ability of ATL_5802 to alter the in vivo phagocytosis of amyloid-β in APPNL-GF mice xenografted with human microglia (hMG). After weekly administration of 40 mg / kg of either ATL_5802 (n=6) or the isotype control ATL_5338 (n=7) for 12 weeks, amyloid-β uptake by CD45+ human microglia was measured by flow cytometry using fluorescent amyloid-β labeling and methoxy-X04. Compared to mice treated with the control ATL_5338, mice treated with ATL_5802 showed a significantly increased percentage of methoxy-X04-positive human microglia cells, demonstrating that ATL_5802 increases in vivo amyloid-β phagocytosis by human microglia. *p<0.05, unpaired t-test. [Figure 19A] The results of a real-time live cell imaging study using LPS-stimulated microglia and pHrodo-labeled tau P301S aggregates are shown. Representative plots of the total red area (pHrodo.red) per well against elapsed time are shown for antibodies ATL_5338 (isotype control), ATL_5802, ATL_5909, ATL_6170, and LPS only. [Figure 19B] The results of a real-time live cell imaging study using LPS-stimulated microglia and pHrodo-labeled tau P301S aggregates are shown. The quantification of the total area under the curve (AUC) per well is shown in (A). [Figure 20A] The results of a multiplex immunoassay in iPSC microglia treated with ATL_5802 or ATL_5909 after 6 hours of pretreatment with LPS are shown. The concentration of IL-6 (pg / ml) in the supernatant of iPSC microglia treated with LPS and ATL5802 or isotype control antibody is shown. The data indicate that ATL_5802 reduces IL-6 release from microglia in response to LPS. Statistical analysis was performed using Student's t-test. *=p<0.05, **, p<0.01, ***, p<0.001, ns=not significant. [Figure 20B]The results of a multiplex immunoassay in iPSC microglia treated with ATL_5802 or ATL_5909 after 6 hours of pretreatment with LPS are shown. The concentration of MCP-1 (pg / ml) in the supernatant of iPSC microglia treated with LPS and ATL5802 or isotype control antibody is shown. The data indicate that ATL_5802 reduces MCP-1 release from microglia in response to LPS. Statistical analysis was performed using Student's t-test. *=p<0.05, **, p<0.01, ***, p<0.001, ns=not significant. [Figure 21] The figure shows the results of Western blotting of phosphorylated SYK (pSYK) in cultured microglia cells stimulated with LPS and treated with ATL_5802. The figure shows Western blotting of pSYK (Tyr525 / 526) and total SYK in microglia cells 20 minutes after treatment with isotype control antibody or ATL_5802. [Figure 22] The results of Western blotting of P2RY12 in LPS-stimulated cultured microglia cells 6 hours after treatment with ATL_5802 or an isotype control antibody are shown. The housekeeper molecule GAPDH was used as a control. [Figure 23A] The results of gene expression analysis after RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon-gamma (LI) or vehicle and treated with ATL_5802, ATL_5854, or ATL_5909 are shown. Principal component analysis (PCA) plots of RNAseq data showing principal component 1 (PC) vs. PC2 obtained from PCA calculated for the 25% most variant gene are also shown. [Figure 23B]This report presents the results of gene expression analysis after RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon-gamma (LI) or a vehicle and treated with ATL_5802, ATL_5854, or ATL_5909. A volcano plot of gene expression changes in iPSC-derived microglia treated with ATL_5802, compared to isotype controls, is shown under vehicle stimulation conditions. The volcano plot shows the expression ratio (x-axis) against statistical significance, expressed as -log10 (y-axis) of the adjusted p-value. Colored dots (to the left of the leftmost vertical line, to the right of the rightmost vertical line, and on the horizontal line) indicate statistically significant differences in expression according to the selected threshold: red (positive log2 expression ratio): upregulation with ATL_5802 treatment, blue (negative log2 expression ratio): downregulation. The horizontal line indicates the significance threshold, and the vertical line indicates the log2 expression ratio threshold. [Figure 23C] This document presents the results of gene expression analysis after RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon-gamma (LI) or a vehicle and treated with ATL_5802, ATL_5854, or ATL_5909. A volcano plot of gene expression changes in iPSC-derived microglia treated with ATL_5802, compared to isotype controls, under LPS / interferon-gamma stimulation conditions is shown. The volcano plot shows the expression ratio (x-axis) relative to statistical significance, expressed as -log10 (y-axis) of the adjusted p-value. Colored dots (to the left of the leftmost vertical line, to the right of the rightmost vertical line, and on the horizontal line) indicate statistically significant differences in expression according to the selected threshold: red (positive log2 expression ratio): upregulation with ATL_5802 treatment, blue (negative log2 expression ratio): downregulation. The horizontal line indicates the significance threshold, and the vertical line indicates the log2 expression ratio threshold. [Figure 23D]The results of gene expression analysis after RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon-gamma (LI) or vehicle and treated with ATL_5802, ATL_5854, or ATL_5909 are shown. For all stimulation and antibody treatments, the bar graphs (B) and (C) show the number of differentially expressed genes and the direction of their change (i.e., upregulation or downregulation). [Figure 23E] This shows the results of gene expression analysis after RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon-gamma (LI) or vehicle and treated with ATL_5802, ATL_5854, or ATL_5909. The bar graph shows the results of gene set enrichment analysis (GSEA) of oxidative phosphorylation and respiratory electron transport gene sets / pathways, including adjusted p-values ​​(x-axis) and normalized enrichment scores (NES, color intensity) for each comparison (y-axis) and pathway (vertical boxes 1-4). Red (top bar in each set of three horizontal bars): upper control; blue (middle and bottom bars in each set of three horizontal bars): lower control. NES is represented by color intensity. Vertical lines indicate significance thresholds. [Figure 23F] The results of gene expression analysis after RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon-gamma (LI) or vehicle and treated with ATL_5802, ATL_5854, or ATL_5909 are shown. Representative GSEA plots of the REACTOME pathway "respiratory electron transport" for all anti-CD33 antibodies compared to isotype controls when stimulated with LPS / interferon-gamma are shown. These plots indicate that this pathway is significantly enhanced (to a much greater extent than with the comparative antibody ATL_5909, though to a lesser extent with ATL_5854) when treated with ATL_5802. [Figure 24A] The results of the analysis of the effects of the antibodies disclosed herein in human central nervous system (CNS) co-culture experiments are shown. Fluorescence images of human CNS quad cell cultures containing glutamatergic / GABAergic neurons, astrocytes, and microglia derived from human iPSCs, as well as cells in such co-cultures, are shown. [Figure 24B] The results of the analysis of the effects of the antibody disclosed herein in human central nervous system (CNS) co-culture experiments are shown. Interleukin-6 (IL-6) levels are shown. Two-way ANOVA (n=2-3 wells) using Tukey's multiple comparison test, compared to the isotype-treated LPS / IFNγ group, showed *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The data demonstrate that ATL_5802 protects against inflammation in human iPSC-derived CNS quad cultures, as evidenced by the reduction in interleukin-6 (IL-6) and GFAP, a marker of astrogliosis. [Figure 24C] The results of the analysis of the effects of the antibody disclosed herein in human central nervous system (CNS) co-culture experiments are shown. GFAP levels are shown. Two-way ANOVA (n=2-3 wells) using Tukey's multiple comparison test, compared to isotype-treated LPS / IFNγ groups, showed *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. The data demonstrate that ATL_5802 protects against inflammation in human iPSC-derived CNS quad cultures, as evidenced by the reduction in interleukin-6 (IL-6) and GFAP, a marker of astrogliosis. [Figure 24D] The results of the analysis of the effects of the antibody disclosed herein in human central nervous system (CNS) co-culture experiments are shown. IP-10 levels are shown. Two-way ANOVA (n=2-3 wells) using Tukey's multiple comparison test, compared to the isotype-treated LPS / IFNγ group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The data show that ATL_5802 treatment under LPS / IFNγ conditions significantly reduces IP-10 (interferon-gamma-inducible protein 10) and MCP-1 levels. [Figure 24E]The results of the analysis of the effects of the antibody disclosed herein in human central nervous system (CNS) co-culture experiments are shown. MCP-1 levels are shown. Two-way ANOVA (n=2-3 wells) using Tukey's multiple comparison test showed *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared to the isotype-treated LPS / IFNγ group. The data indicate that ATL_5802 treatment under LPS / IFNγ conditions significantly reduces IP-10 (interferon-gamma-inducible protein 10) levels and MCP-1 levels. [Modes for carrying out the invention]

[0045] Detailed description of the invention Hereinafter, aspects and embodiments of the present invention will be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All references mentioned herein are incorporated herein by reference.

[0046] Antibodies and fragments thereof that can specifically bind to the CD33 protein or a fragment thereof are disclosed herein. As used herein, an antibody capable of "specific binding" to a target, or capable of "specifically binding" to a target, is an antibody that can bind through the association of an epitope recognition site with an epitope within the target. This is different from nonspecific binding, such as Fc-mediated binding, ionic and / or hydrophobic interactions. In other words, an antibody that specifically binds to a target generally recognizes and binds to a specific protein structure within the protein, rather than to the protein itself.

[0047] This disclosure refers to antibodies described herein using references designated as "ATL_000xxxx," "ATL_xxxx," or "xxxx," where "xxxx" is a four-digit reference number specific to the antibody described herein. All of the above notations are used interchangeably to refer to the same antibody or a part thereof (e.g., VH, VL, or a part thereof of the antibody). For example, antibody ATL_0005802 is interchangeably referred to herein as ATL_5802 and 5802. Furthermore, reference ATLX-1088 refers to ATL_5802.

[0048] CD33 is an inhibitory immune receptor belonging to the family of sialic acid-binding immunoglobulin-like lectins. CD33 is also known as sialic acid-binding immunoglobulin-like lectin 3 (Sigrec-3). The human CD33 gene (gene ID: 945) is located on human chromosome 19 (19q13.33) and consists of seven exons. The complete sequence of human CD33 is a 364-amino acid sequence available under the Uniprot identifier P20138. This consists of (i) an N-terminal signal peptide that induces CD33 into the secretory pathway (amino acids 1-17 of P20138), (ii) two extracellular domains consisting of an N-terminal Ig-like V-set domain and a C2-set domain for recognizing carbohydrate ligands (amino acids 18-259 of P20138), (iii) a transmembrane domain (amino acids 260-282 of P20138), and (iv) a cytoplasmic domain containing an immune receptor tyrosine inhibitory motif (ITIM) that mediates immune cell signaling (amino acids 283-364 of P20138) (Eskandari-Sedighi et al., 2023). CD33 undergoes alternative splicing to produce a long isoform designated hCD33M (M = "major") and a shorter isoform designated hCD33m (m = "minor") lacking a functional V domain, which is generated by exon 2 exclusion (amino acids 13-139 of hCD33M) (Hernandez-Caselles, et al., 2006). The major isoform of human CD33 (hCD33) is a 67 kDa transmembrane glycoprotein (canonical sequence, 364 amino acids) available under Uniprot identifier P20138-1 and is provided as SEQ ID NO: 176. The minor isoform is a 25 kDa protein available under Uniprot identifier P20138-3 (237 amino acids) and is provided as SEQ ID NO: 177. The antibodies described herein may or may not bind to hCD33M. CD33 is expressed on the cell surface of bone marrow cells, monocytes, macrophages, and microglia in the brain. CD33 is involved in the adhesion process of immune cells and mediates cell-cell interactions (Varki et al., 2006).

[0049] Genome-wide association studies have identified CD33 as a genetic regulator of susceptibility and pathology in late-onset Alzheimer's disease (Hollingworth et al. 2011). Upregulation of CD33 expression in microglia of AD patients is associated with more advanced cognitive decline or disease status (Siddiqui et al, 2017). This is thought to be due to altered activation of microglial toxic insoluble amyloid-β42 (Aβ42) species and inhibition of microglial phagocytosis. On the other hand, the AD protective allele of CD33 SNP rs3865444 reduces insoluble Aβ42 levels in AD brains, as well as reducing CD33 expression and increasing the proportion of cleaved non-signaling forms (Griciuc et al., 2013). Furthermore, CD33 has been suggested to interact with other microglial AD risk genes, such as TREM2, to influence the onset and pathogenesis of AD. In particular, it has been suggested that CD33 negatively regulates microglial activation via TREM2 / DAP12, reducing cellular functions such as microglial phagocytosis, and thereby decreasing the clearance of toxic Aβ42 species (Chan et al., 2015). Therefore, the antibodies described herein can increase phagocytosis in CD33-expressing cells, such as microglial phagocytosis, compared to the comparative antibody.

[0050] In addition to its role in inhibiting cellular processes such as phagocytosis, CD33 is involved in several other processes, including the regulation of cell adhesion and immune responses (Cao et al., 2010). CD33 is also highly expressed on leukemic blasts and myeloid leukemia initiation cells in acute myeloid leukemia (Bonnet et al., 1997; Vercauteren et al., 2007). Therefore, in some cases, the antibodies described herein may be useful in the treatment of AML by binding to CD33 in leukemic blast cells.

[0051] CD33 possesses endocytic properties (receptor internalization), which are utilized for targeting by antibody-drug conjugates (Laszlo et al., 2014). While this property can lead to a decrease in CD33 on the cell surface, it can also reduce the effectiveness of CD33-targeted therapies due to the internalization of anti-CD33 antibodies by CD33-expressing cells that are not the primary therapeutic target. For example, when targeting CD33-expressing microglia cells, CD33-expressing monocytes can act as a peripheral antibody sink, preventing the antibody from reaching a therapeutic dose in the brain with acceptable toxicity. In some cases, the antibodies described herein induce a reduction in CD33 endocytosis compared to comparator antibodies. CD33 internalization can be measured, for example, by determining cell surface levels of CD33 using flow cytometry in the presence of a candidate antibody. The cells may be human cells, such as microglia, bone marrow cells, monocytes, tumor cells, such as leukemic blast cells, and / or iPSC-derived microglia, and / or cells stimulated with an inflammatory signal (e.g., LPS) before exposure to the antibody. The comparative antibody may be selected from isotype control antibodies and / or antibodies having the heavy chain variable sequence of ATL_5909 and the light chain variable sequence of 5909.

[0052] Therapeutic strategies targeting TREM2, an anti-inflammatory receptor expressed in bone marrow cells, are being explored for the treatment of Alzheimer's disease (AD) with the aim of restoring normal microglial function. Indeed, loss of TREM2 function is known to reduce the microglial response to amyloid-beta plaques. Currently, several TREM2 agonists, such as Alector / Abbvie's AL002 (referred to herein as ATL6166, in Phase 2 clinical trials), Denali Therapeutic Inc.'s DNL919 (referred to herein as ATL6167, in Phase 1 clinical trials), and Vigil's VGL101 (referred to herein as ATL6170, in Phase 2 clinical trials), are in clinical trials as treatments for AD. Although CD33 is known to modulate TREM2 activity, to the best of our knowledge, no direct CD33 antagonist antibodies are currently in clinical development.

[0053] As used herein, the term “CD33” may refer to any protein having at least 80%, at least 90%, or at least 95% sequence identity with the CD33 sequence provided herein as Sequence ID No. 176, encompassing cleavage forms, derivatives, and variants of the CD33 sequence. The CD33 sequence may be a human CD33 sequence. The CD33 sequence may be a sequence containing at least a portion of the V domain of CD33 (also referred to as the “Ig-like V” domain) (amino acids 19-135 of Uniprot ID P20138-1 or its homolog). The CD33 sequence may further include one or more of the following: at least a portion of the C domain of CD33 (also referred to as the "Ig-like C2 type" domain) (amino acids 145-228 of Uniprot ID P20138-1 or its homolog), at least a portion of the transmembrane domain of CD33 (amino acids 260-282 of P20138-1 or its homolog), and at least a portion of the cytoplasmic domain of CD33 (amino acids 283-364 of P20138-1 or its homolog). The antibodies according to this disclosure may bind to CD33 proteins containing the V domain of human CD33 (amino acids 19-135 of Uniprot ID P20138 provided as SEQ ID NO: 176) or a sequence having at least 90%, 95%, 98%, or 99% sequence identity with the aforementioned sequence.

[0054] This disclosure relates primarily to antibody molecules, whether they are whole antibodies (e.g., IgG1, etc.) or antibody fragments (e.g., single-chain variable fragments (scFv), antibody fragments (Fab), or bivalent antibody fragments (F(ab')2), or single-domain antibodies (sdAb)). Antibody antigen-binding regions (also called "antigen-binding moieties") are provided, as well as antibody heavy chain variable (VH) domains and light chain variable (VL) domains. Complementarity-determining regions (CDRs) are provided within the VH and VL domains, and the CDRs may optionally be provided within different framework regions (FRs) to form the VH or VL domains. The antigen-binding sites may consist of antibody VH domains and / or VL domains.

[0055] The antibodies described herein may be provided in isolated forms. The term “antibody” includes its fragments or derivatives, or synthetic antibodies or synthetic antibody fragments. An antibody or its fragment may be a monoclonal antibody (mAb). An mAb is a homogeneous population of antibodies that specifically targets a single epitope of an antigen. Antibodies and methods for constructing and using them are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23 (9): 1126-1136 (2005). Considering the current technologies related to monoclonal antibody technology, antibodies can be prepared for most targets. Monoclonal and other antibody molecules can be taken out, and other antibodies or chimeric molecules that retain the specificity of the original antibody can be generated using recombinant DNA technology. Such techniques may include introducing the CDR or variable region of one antibody into different antibody molecules (see, for example, UK Patent Application Publication No. 2188638A and European Patent No. 0239400).

[0056] An "antigen-binding domain" represents a molecular portion that binds to all or part of a target antigen. Antibodies generally contain six complementarity-determining regions (CDRs): three within the VH region: HCDR1, HCDR2, and HCDR3, and three within the VL region: LCDR1, LCDR2, and LCDR3. Together, the six CDRs define a paratope of antigen-binding domains, which are part of the antigen-binding domain that binds to the target antigen. A monoclonal monospecific IgG antibody molecule contains two antigen-binding domains, each capable of binding to the same target (i.e., bivalent for a single target). A Fab fragment generally contains a VH domain, a CH1 domain, a VL domain, and a CL domain. A complete antibody may contain a pair of Fab fragments and an Fc fragment containing a pair of chains, each containing a CH2 domain and a CH3 domain. An Fv fragment contains a VH domain and a VL domain. The VH and VL regions contain framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDRs. From the N-terminus to the C-terminus, the VH region includes the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region includes the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0057] The term "ScFv molecule" refers to a molecule in which the VH and VL partner domains are covalently linked, for example, by a flexible oligopeptide. The Fab, Fv, ScFv, and dAb antibody fragments are all expressed in and can be secreted from E. coli, and therefore can be easily produced in large quantities. All antibodies and the F(ab')2 fragment are "bivalent." The term "bivalent" means that the antibody and F(ab')2 fragment have two antigen-binding sites. In contrast, the Fab, Fv, ScFv, and dAb fragments are monovalent and have only one antigen-binding site.

[0058] The antibodies according to this disclosure may be detectably labeled, or at least detectably labeled. For example, the antibody may be labeled with a radioactive atom, or a colored molecule, or a fluorescent molecule, or a molecule that can be readily detected by other means. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates, and radiolabels. The binding site (antibody or fragment thereof) may be directly or indirectly labeled with a detectable label. For example, the binding site may be an unlabeled antibody that can be detected by another antibody that is itself labeled. Alternatively, the first antibody may be indirectly labeled by a second antibody bound to biotin, with labeled streptavidin conjugating to the biotin.

[0059] Antibody "fragments" may contain any number of residues from the "parent" antibody while retaining target-binding ability. Fragments may lack effector function and may, for example, fail to bind completely to the Fc receptor compared to the parent, or may show reduced binding to the Fc receptor. Fragments are typically smaller than the parent antibody. Fragments may contain 50%, 60%, 70%, 80%, 90%, 95%, or more of the consecutive or discontinuous amino acids of the parent antibody. Fragments may contain 50, 100, 150, 200, 250, 300, or more consecutive or discontinuous amino acids of the parent antibody. Fragments may contain deletions within the Fc region or deletions within the Fc region. Fragments may retain the CDR and / or variable domains of the parent antibody without alteration. In some embodiments, the fragment is a Fab fragment or an F(ab')2 fragment. The CDR sequences are described herein using IMGT numbering (Lefranc, M.-P., Immunology Today, 18, 509 (1997)).

[0060] The antibodies according to this disclosure may have a VH having the following antibodies: ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, or a CDR set in which each CDR contains 0, 1, or 2 amino acid substitutions compared to the above CDR sets. Therefore, the isolated antibody may contain a heavy chain variable domain having the following CDRs: CDRH1 containing an amino acid sequence selected from (i) HCDR1_ATL_0005802, HCDR1_ATL_0005807, HCDR1_ATL_0005808:GYSFTSYW (SEQ ID NO: 44), (ii) HCDR1_ATL_0005809:GYSFNTYW (SEQ ID NO: 45), (iii) HCDR1_ATL_0005810:GYTFTSYY (SEQ ID NO: 46), (iv) HCDR1_ATL_0005853:GYKFNNNW (SEQ ID NO: 47), (v) HCDR1_ATL_0005854:GYKFSNNW (SEQ ID NO: 48), or (vi) an amino acid sequence having one or two mutations compared to the above amino acids. CDRH2 comprising (i) HCDR2_ATL_0005802, HCDR2_ATL_0005807, HCDR2_ATL_0005808, HCDR2_ATL_0005853, HCDR2_ATL_0005854:IYPGDSDT (SEQ ID NO: 52), (ii) HCDR2_ATL_0005809:IYPGDSET (SEQ ID NO: 53), (iii) HCDR2_ATL_0005810:INPSGGST (SEQ ID NO: 54), or (iv) an amino acid sequence selected from an amino acid sequence having one or two mutations compared to the above sequences, and (i) HCDR3_ATL_0005802:ARPRGFGEYYFDY (Sequence ID 59), (ii) HCDR3_ATL_0005807:ARQGAGPGGFDI (Sequence ID 60), (iii) HCDR3_ATL_0005808:ARGQGGGAGAFDI (Sequence ID 61), (iv) HCDR3_ATL_0005809:ARHRADAPSDAFDI (Sequence ID 62), (v) HCDR3_ATL_ CDRH3 containing an amino acid sequence selected from amino acid sequences having one or two mutations compared to the above sequences: (vi) HCDR3_ATL_0005853:ARHSGGLDGYTAAALDY (sequence number 64), (vii) HCDR3_ATL_0005854:ATWGGSNWFVD (sequence number 65), or (viii) an amino acid sequence having one or two mutations compared to the above sequences.

[0061] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005802, CRH2 containing the sequence HCDR2_ATL_0005802, CDRH3 containing the sequence HCDR3_ATL_0005802, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0062] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005853, CRH2 containing the sequence HCDR2_ATL_0005853, CDRH3 containing the sequence HCDR3_ATL_0005853, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0063] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005854, CRH2 containing the sequence HCDR2_ATL_0005854, CDRH3 containing the sequence HCDR3_ATL_0005854, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0064] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005810, CRH2 containing the sequence HCDR2_ATL_0005810, CDRH3 containing the sequence HCDR3_ATL_0005810, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0065] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 comprising the sequence HCDR1_ATL_0005807, CRH2 comprising the sequence HCDR2_ATL_0005807, CDRH3 comprising the sequence HCDR3_ATL_0005807, or a set of CDRs comprising one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0066] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005808, CRH2 containing the sequence HCDR2_ATL_0005808, CDRH3 containing the sequence HCDR3_ATL_0005808, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0067] The antibodies according to this disclosure may comprise heavy chain variable domains (VHs) having the following CDRs: CDRH1 containing the sequence HCDR1_ATL_0005809, CRH2 containing the sequence HCDR2_ATL_0005809, CDRH3 containing the sequence HCDR3_ATL_0005809, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRH3 compared to these sequences.

[0068] In the antibodies of this disclosure, at least one of the VH CDR1-3 sequences may differ. A variant may have one or two amino acid substitutions compared to the set of VH CDR1-3 described above. In embodiments, the antibodies of this disclosure include a CDR having a sequence that has one or two substitutions compared to the VH CDR sequence of any of the antibodies described above. For example, an antibody of this disclosure includes a CDR having the sequence of any of the antibodies described above, except that one or two of the CDRHs contain substitutions, and the total number of substitutions in the CDRH is two or less. In embodiments, a variant may have one, two, or three substitutions, preferably at most one or two, in each of the one or more of the VH CDR1-3 described above. Any CDRH1 region of the antibodies or fragments described herein may have an amino acid length of 8. Any CDRH2 region of the antibodies or fragments described herein may have an amino acid length of 8. Any CDRH3 region of the antibodies or fragments described herein may have an amino acid length of 13-21. In embodiments, the variant may have a VH CDR having at least 70%, at least 80%, or at least 90% sequence identity with any set of VH CDRs described herein.

[0069] The antibodies according to this disclosure may have a light chain variable domain (VL) CDR of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or a light chain variable domain (VL) having a CDR set in which each CDR contains 0, 1, or 2 amino acid substitutions compared to the above CDR set. Therefore, the isolated antibody may contain a light chain variable domain having the following CDRs: (i)LCDR1_ATL_0005802,LCDR1_ATL_0006040,LCDR1_ATL_0006041,LCDR1_ATL_0006042,LCDR1_ATL_0006043,LCDR1_ATL_0006044,LCDR1_ATL_0006045,LCDR1_ATL_0006046,LCDR1_ATL_0006047,LCDR1_ATL_0006048:SSNIGAGYD (Sequence ID 110), (ii)LCDR1_ATL_0005807:ALARQY (Sequence ID 111), (iii)LCDR1_ATL_0005808,LCDR1_ATL_0005854:SSDVGGYNY (SEQ ID NO: 112), (iv)LCDR1_ATL_0005809:SLRNYY (SEQ ID NO: 113), (v)LCDR1_ATL_0005810:SGSVSTSYY (SEQ ID NO: 114), (vi)LCDR1_ATL_0005853:QSLLHSDGYNY (SEQ ID NO: 115), or (vii)CDRL1 comprising an amino acid sequence selected from amino acid sequences having one or two mutations compared to the above sequences, (i) LCDR2_ATL_0005802, LCDR2_ATL_0006040, LCDR2_ATL_0006041, LCDR2_ATL_0006042, LCDR2_ATL_0006043, LCDR2_ATL_0006044, LCDR2_ATL_0006045, LCDR2_ATL_0006046, LCDR2_ATL_0006047, LCDR2_ATL_0006048: GNS (Sequence ID 119), (ii) LCDR2_ATL_0005854: DVS (Sequence ID 120), (ii i)LCDR2_ATL_0005807:KDS (SEQ ID NO: 121), (iv)LCDR2_ATL_0005808:DVT (SEQ ID NO: 123), (v)LCDR2_ATL_0005809:GKN (SEQ ID NO: 124), (vi)LCDR2_ATL_0005810:STN (SEQ ID NO: 125), (vii)LCDR2_ATL_0005853:VGS (SEQ ID NO: 126), or (viii)CDRL2 comprising an amino acid sequence selected from amino acid sequences having one or two mutations compared to the above sequences, and (i) LCDR3_ATL_0005802, LCDR3_ATL_0006040, LCDR3_ATL_0006041, LCDR3_ATL_0006042, LCDR3_ATL_0006043, LCDR3_ATL_0006044, LCDR3_ATL_0006045, LCDR3_ATL_0006046, LCDR3_ATL_0006047, LCDR3_ATL_0006048: QSYDSSLSGDV (Sequence ID 129), (ii) LCDR3_ATL_0005807: QSPDSSGTYPV (Sequence ID 130), (iii) LCDR3_AT CDRL3 containing an amino acid sequence selected from the following amino acid sequences having one or two mutations compared to the above sequences: (iv)LCDR3_ATL_0005809:NSRDSSGYHLGL(sequence number 132), (v)LCDR3_ATL_0005810:LLYMGSGIWM(sequence number 133), (vi)LCDR3_ATL_0005853:MQALQTPIT(sequence number 134), (vii)LCDR3_ATL_0005854:SSYTNSSTLEV(sequence number 236), or (viii) an amino acid sequence having one or two mutations compared to the above sequences.

[0070] The antibodies described herein may include a light chain variable domain having the following CDR: CDRL1 containing an amino acid sequence selected from LCDR1_ATL_0005802:SSNIGAGYD (SEQ ID NO: 110), LCDR1_ATL_0005854:SSDVGGYNY (SEQ ID NO: 112), LCDR1_ATL_0005853 QSLLHSDGYNY (SEQ ID NO: 115), or an amino acid sequence having one or two mutations compared to the above sequences, CDRL2 containing an amino acid sequence selected from LCDR2_ATL_0005802:GNS (SEQ ID NO: 119), LCDR2_ATL_0005854:DVS (SEQ ID NO: 120), LCDR2_ATL_0005853:VGS (SEQ ID NO: 126), or an amino acid sequence having one or two mutations compared to the above sequences, and CDRL3 containing an amino acid sequence selected from LCDR3_ATL_0005802:QSYDSSLSGDV (SEQ ID NO: 129), LCDR3_ATL_0005853:MQALQTPIT (SEQ ID NO: 134), LCDR3_ATL_0005854:SSYTNSSTLEV (SEQ ID NO: 236), or an amino acid sequence having one or two mutations compared to the above sequences.

[0071] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005802, CRL2 containing the sequence LCDR2_ATL_0005802, CDRH3 containing the sequence LCDR3_ATL_0005802, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0072] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005853, CRL2 containing the sequence LCDR2_ATL_0005853, CDRH3 containing the sequence LCDR3_ATL_0005853, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0073] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005854, CRL2 containing the sequence LCDR2_ATL_0005854, CDRH3 containing the sequence LCDR3_ATL_0005854, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0074] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005810, CRL2 containing the sequence LCDR2_ATL_0005810, CDRH3 containing the sequence LCDR3_ATL_0005810, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0075] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005807, CRL2 containing the sequence LCDR2_ATL_0005807, CDRH3 containing the sequence LCDR3_ATL_0005807, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0076] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005808, CRL2 containing the sequence LCDR2_ATL_0005808, CDRH3 containing the sequence LCDR3_ATL_0005808, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0077] The antibodies according to this disclosure may comprise light chain variable domains (VLs) having the following CDRs: CDRL1 containing the sequence LCDR1_ATL_0005809, CRL2 containing the sequence LCDR2_ATL_0005809, CDRH3 containing the sequence LCDR3_ATL_0005809, or a set of CDRs containing one or two mutations in CDRL1 and CDRL2 compared to these sequences (one or two mutations in each CDR or one or two mutations in both CDRs) and / or one, two or three mutations in CDRL3 compared to these sequences.

[0078] In the antibodies according to this disclosure, at least one of the VL CDR1-3 sequences may differ. A variant may have one, two, or three amino acid substitutions compared to the set of VL CDR1-3 described above. In embodiments, the antibodies according to this disclosure include a CDR having a sequence with one to three substitutions compared to the VL CDR sequences of the antibodies described herein. For example, the antibodies according to this disclosure may include substitutions where the total number of substitutions is two or less. In embodiments, a variant may have one, two, or three, preferably at most one or two substitutions in each of one or more of the VL CDR1-3 described above.

[0079] Any CDRL1 region of the antibody or fragment described herein may have a length of 6 to 11 amino acids. Any CDRL2 region of the antibody or fragment described herein may have a length of 3 amino acids. Any CDRL3 region of the antibody or fragment described herein may have a length of 9 to 11 amino acids. In embodiments, the variant may have a VL CDR having at least 70%, at least 80%, or at least 90% sequence identity with any set of VL CDRs described herein.

[0080] The VH CDR1-3 and, optionally, VL CDR1-3 of any of the above antibodies may be particularly useful in combination with several different framework regions. Therefore, light and / or heavy chains having the above CDR1-3 may have alternative framework regions. Preferred framework regions are known in the art and are described, for example, in M. Lefranc & G. Le Franc (2001) “The Immunoglobulin Facts Book”, Academic Press.

[0081] The antibodies disclosed herein may have a heavy chain variable domain (VH) having the following framework sequence: HFWR1 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or HFWR2 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810. , HFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 and HFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 or a framework sequence having 1 to 6 substitutions compared to the above framework sequence.

[0082] The antibodies described herein may have a heavy chain variable domain (VH) having the following framework sequence: HFWR1_ATL_0005807, HFWR1_ATL_0006041 QVQLVQSGAEVKKPGESLKISCKGS (Sequence ID 71), HFWR1_ATL_0005808 QVQLQQSGAEVKKPGESLKISCKGS (Sequence ID 72), HFWR1_ATL_0005809 QVQLQQSGGEVKKPGESLKISCKGS (Sequence ID 73), HFWR1_ATL_0005810 QVQLVQSGAEVKKPGASVKVSCKAS (Sequence ID 74), HFWR1_ATL_0005853 QVQLVQSGAEVKKTGESLRISCKAS (Sequence ID 235), HFWR1_ATL_0005854 QVQLVQSGAEVKKTGEYLRISCKAS (Sequence ID 75), HFWR1_ATL_0006040, HFWR1_ATL_0006044, HFWR1_ATL_0006045, HFWR1_ATL_0006046, HFWR1_ATL_0006047, HFWR1_ATL_0006048:EVQLVQSGAEVKKPGESLKISCKGS(Sequence ID 77), HFWR1_ATL_0006042:EVQLVESGAEVKKPGESLKISCKGS (Sequence ID 78), HFWR1_ATL_0006043:EVQLVQSGAEVKKPGESLRISCKGS(Sequence ID 79) HFWR1 containing an amino acid sequence selected from, HFWR2_ATL_0005802, HFWR2_ATL_0005853, HFWR2_ATL_0005854, HFWR2_ATL_0006044:IAWVRQMPGKGLEWMGI (Sequence ID 83), HFWR2_ATL_0005807, HFWR2_ATL_0005808, HFWR2_ATL_0006040, HFWR2_ATL_0006041, HFWR2_ATL_0006042, HFWR2_ATL_000 6043, HFWR2_ATL_0006045, HFWR2_ATL_0006046, HFWR2_ATL_0006047, HFWR2_ATL_0006048:IGWVRQMPGKGLEWMGI (Sequence number 84), HFWR2_ATL_0005809:IAWVRHAPGKGLEWMGI (Sequence ID 85), HFWR2_ATL_0005810:MHWVRQAPGQGLEWMGI(Sequence ID 86) HFWR2 containing an amino acid sequence selected from, HFWR3_ATL_0005802:RYSPSFEGQVTISADKSIGTAYLQWSSLKASDTAMYYC (Sequence ID 91), HFWR3_ATL_0005807:RYSPSFQGQVSISVDKSISTAFLQWSSLKSSDSAMYYC (Sequence ID 92), HFWR3_ATL_0005808, HFWR3_ATL_0006040, HFWR3_ATL_0006041, HFWR3_ATL_0006042, HFWR3_ATL_0006043, HFWR3_ATL_0006044, HFWR3_ATL_0006047, HFWR3_ATL_0006048:RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC (Sequence ID 93), HFWR3_ATL_0005809:RYSPSFQSQVTISADKSIDTAYLEWNTLEASDTAMYYC (Sequence ID 94), HFWR3_ATL_0005810:SYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC(Sequence ID 95), HFWR3_ATL_0005853:RYSPSFEGQVTISADKSSGIVYLQWTSLKASDTAIYYC (Sequence ID 96), HFWR3_ATL_0005854:RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAIYYC(Sequence ID 97) HFWR3 containing an amino acid sequence selected from, or A framework array having 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 substitutions compared to the framework arrays mentioned above.

[0083] The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005802 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005853 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005854 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005807 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005808 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005809 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) having framework sequences HFWR1, HFWR2, HFWR3, and HFWR4 of antibody ATL_0005810 or framework sequences having 1 to 6 substitutions compared to these framework sequences.

[0084] In the embodiment, substitutions in the framework sequence of the heavy chain variable domain may be located at any position other than position 40 in the standard IMGT numbering. In the embodiment, substitutions in the framework sequence do not include substitution at position 40 in the standard IMGT numbering. Substitutions in the heavy chain variable domain framework sequence may be located at any position in HFWR1 and / or HFRW3. Substitutions in the heavy chain variable domain framework sequence may be located at any position not in HFRW2. Substitutions in the heavy chain variable domain framework sequence may be selected from the following positions in the standard IMGT numbering: HFWR1: position 1, position 6, position 20; HFWR3: position 72, position 85. Substitutions in the heavy chain variable domain framework sequence may be selected from the following positions in the standard IMGT numbering: HFWR1: Q1E at position 1, E6Q at position 6, R20K at position 20; HFWR3: E72Q at position 72, G85S at position 85.

[0085] The antibodies of this disclosure may have a heavy chain variable domain (VH) having the framework sequence HFWR2 of ATL_0005802 (HFWR2_ATL_0005802, HFWR2_ATL_0005853, HFWR2_ATL_0005854). The isolated antibody may have a heavy chain variable domain (VH) having the framework sequence HFWR2 containing the sequence: IAWVRQMPGKGLEWMGI (SEQ ID NO: 83). The antibody may have a heavy chain variable domain (VH) having the framework sequence HFWR2 of ATL_0005802. The antibodies of this disclosure may have a heavy chain variable domain (VH) having a framework sequence containing A at position 40 in standard IMGT numbering.

[0086] The antibodies described herein may have a heavy chain variable domain (VH) having the following framework sequence: HFWR1(HFWR1_ATL_0005807, HFWR1_ATL_0006041, HFWR1_ATL_0005808, HFWR1_ATL_0005809, HFWR1_ATL_0005810, HFWR1_ATL_0005853, HFWR1_ATL_0005854, H FWR1_ATL_0006040, HFWR1_ATL_0006044, HFWR1_ATL_0006045, HFWR1_ATL_0006046, HFWR1_ATL_0006047, HFWR1_ATL_0006048, HFWR1_ATL_0006042, HFWR1_ATL_0006043), preferably HFWR1 of sequence numbers 70, 235, 75. HFWR2(HFWR2_ATL_0005802, HFWR2_ATL_0005853, HFWR2_ATL_0005854, HFWR2_ATL_0006044, HFWR2_ATL_0005807, HFWR2_ATL_0005808, HFWR2_ATL_0006040, HFWR2_ATL_000604) 1. HFWR2_ATL_0006042, HFWR2_ATL_0006043, HFWR2_ATL_0006045, HFWR2_ATL_0006046, HFWR2_ATL_0006047, HFWR2_ATL_0006048, HFWR2_ATL_0005809, HFWR2_ATL_0005810), preferably HFWR2 of sequence number 83, HFWR3(HFWR3_ATL_0005802HFWR3_ATL_0005807HFWR3_ATL_0005808HFWR3_ATL_0006040HFWR3_ATL_0006041HFWR3_ATL_0006042HFWR3_ATL_000604) 3, HFWR3_ATL_0006044, HFWR3_ATL_0006047, HFWR3_ATL_0006048, HFWR3_ATL_0005809, HFWR3_ATL_0005810, HFWR3_ATL_0005853, HFWR3_ATL_0005854), preferably HFWR3 of sequence numbers 91, 96, 97, and HFWR4 with array numbers 101, 104, 105 (HFWR4_ATL_0004828, HFWR4_ATL_0005802, HFWR4_ATL_0005853, HFWR4_ATL_0 005854, HFWR4_ATL_0006040, HFWR4_ATL_0006041, HFWR4_ATL_0006042, HFWR4_ATL_0006043, HFWR4_AT L_0006044, HFWR4_ATL_0006045, HFWR4_ATL_0006046, HFWR4_ATL_0006047, HFWR4_ATL_0006048, HFWR4_ATL_0005807, HFWR4_ATL_0005808, HFWR4_ATL_0005809, HFWR4_ATL_0005810), preferably HFWR4 of sequence number 101.

[0087] The antibodies of this disclosure may have VL domains CDRL1, CDRL2, and CDRL3 within their germline framework. The antibodies of this disclosure may have heavy chain variable domains (VH) containing CDRH1, CDRH2, and CDRH3 within their germline framework, provided that position 40 in the standard IMGT numbering is A.

[0088] The antibodies described herein may have a light chain variable domain (VL) having the following framework sequence: LFWR1_ATL_0005802:QAVLTQPSSVSGAPGQRVTISCTGS(Sequence ID 138), LFWR1_ATL_0005807:QSVLTQPPSVSVSPGQTARITCSGD (Sequence ID 139), LFWR1_ATL_0005808, LFWR1_ATL_0005854:QSALTQPASVSGSPGQSITISCTGT (Sequence ID 140), LFWR1_ATL_0005809:SSELTQDPAVSVAVGQTVRITCQGD(Sequence ID 141), LFWR1_ATL_0005810:QTVVTQEPSFSVSPGGTVTLTCGLS(Sequence ID 142), LFWR1_ATL_0005853:DVVMTQSPLSLPVNPGEPASISCRSS(Sequence ID 143), LFWR1_ATL_0006040, LFWR1_ATL_0006041, LFWR1_ATL_0006042, LFWR1_ATL_0006043, LFWR1_ATL_0006044, LFWR1_ATL_0006045, LFWR1_ATL_0006046:QSVLTQPPSVSGAPGQRVTISCTGS(Sequence ID 145), LFWR1_ATL_0006047 QAVLTQPPSVSGAPGQRVTISCTGS (Sequence ID 146), LFWR1_ATL_0006048 QSVLTQPSSVSGAPGQRVTISCTGS(Sequence ID 147) LFWR1 containing an amino acid sequence selected from, LFWR2_ATL_0005802:VHWYQQLPGTAPKLLIY(Sequence ID 151), LFWR2_ATL_0005807:AFWYQQKPGQAPVLVIY(Sequence ID 152), LFWR2_ATL_0005808, LFWR2_ATL_0005854:VSWYQQHPGKAPKLMIY (Sequence ID 153), LFWR2_ATL_0005809:ANWYQQKPGQAPVLVIY(Sequence ID 154), LFWR2_ATL_0005810:PSWYQQTPGQPPRTLIY(Sequence ID 155), LFWR2_ATL_0005853:LHWYLQKPGQSPQLLIY(Sequence ID 156) LFWR2 containing an amino acid sequence selected from, LFWR3_ATL_0005802:NRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC (Sequence ID 160), LFWR3_ATL_0005807:ERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYC (Sequence ID 161), LFWR3_ATL_0005808:NRPSGVSSRFSASKSGNTASLTISGLQAEDEADYYC (Sequence ID 162), LFWR3_ATL_0005809:NRPSGIPDRFSGSSSGNTASLTLTGAQAEDEADYYC (Sequence ID 163), LFWR3_ATL_0005810:TRSSGVPDRFSGSILGNKAALTITGAQADDESDYYC (Sequence ID 164), LFWR3_ATL_0005853:DRAPGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYC(Sequence ID 165), LFWR3_ATL_0005854:SRPSGVSYRFSGSKSGNTASLTISGLQAEDEADYYC(Sequence ID 166) LFWR3 containing an amino acid sequence selected from, and LFWR4_ATL_0005802:FGGGTKLTVL(Sequence ID 172), LFWR4_ATL_0005807, LFWR4_ATL_0005808, LFWR4_ATL_0005854:FGGGTKVTVL (Sequence ID 173), LFWR4_ATL_0005853:FGQGTRLEIK(Sequence ID 174) LFWR4 containing an amino acid sequence selected from, or A framework array having 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 substitutions compared to the framework arrays mentioned above.

[0089] The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005802 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005853 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005854 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005807 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005808 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005809 or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of this disclosure may have a light chain variable domain (VL) having framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody ATL_0005810 or framework sequences having 1 to 6 substitutions compared to these framework sequences.

[0090] Substitutions in the light chain variable domain framework sequence can be selected from the following positions in standard IMGT numbering: LFWR1: position 2, position 8. Substitutions in the light chain variable domain framework sequence can be selected from the following positions in standard IMGT numbering: LFWR1: A2S at position 2, S8P at position 8.

[0091] The antibody may have a light chain variable domain (VL) having the following framework sequence: LFWR1 of ATL_0005802, ATL_0005853 or ATL_0005854, LFWR2 of ATL_0005802, ATL_0005853 or ATL_0005854, LFWR3 of ATL_0005802, ATL_0005853 or ATL_0005854, and HFWR4 of ATL_0005802, ATL_0005853 or ATL_0005854.

[0092] In this specification, antibodies may have a VH (and optionally VL) region containing an amino acid sequence having a high percentage of sequence identity with the above-described VH and / or VL amino acid sequences. For example, an antibody according to the present invention may have a VH region that binds to CD33 and contains an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the VH region amino acid sequence of any of the above-described antibodies. Antibodies according to this disclosure may have a heavy chain variable domain (VH) containing a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NOs: 4-10 and 12-20. Antibodies according to this disclosure may have a heavy chain variable domain (VH) containing a sequence having at most two mutations in each HCDR and at most three mutations in each framework region compared to a sequence selected from SEQ ID NOs: 4-10 and 12-20.

[0093] The antibodies according to this disclosure may have a heavy chain variable domain (VH) containing a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NOs: 4, 9, and 10 (Antibodies ATL_0005802, ATL_0005853, ATL_0005854). The antibodies according to this disclosure may have a heavy chain variable domain (VH) containing a sequence having up to two mutations in each HCDR and up to three mutations in each framework region compared to a sequence selected from SEQ ID NOs: 4, 9, and 10 (Antibodies ATL_0005802, ATL_0005853, ATL_0005854).

[0094] Alternatively, or in addition, the antibodies of the present disclosure may have a VL region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the VL region amino acid sequence of any of the antibodies described above. For example, the antibodies of the present disclosure may have a VL region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the VL region amino acid sequence of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810.

[0095] The antibodies according to this disclosure may have a light chain variable domain (VL) containing a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NOs. The antibodies according to this disclosure may also have a light chain variable domain (VL) containing a sequence having up to two mutations in each LCDR and up to three mutations in each framework region compared to a sequence selected from SEQ ID NOs. 24-30 and 32-40.

[0096] The antibodies according to this disclosure may have a light chain variable domain (VL) containing a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NOs: 24, 29, and 30 (Antibodies ATL_0005802, ATL_0005853, ATL_0005854). The antibodies according to this disclosure may have a light chain variable domain (VL) containing a sequence having up to two mutations in each LCDR and up to three mutations in each framework region compared to a sequence selected from SEQ ID NOs: 24, 29, and 30 (Antibodies ATL_0005802, ATL_0005853, ATL_0005854).

[0097] The antibodies of this disclosure may have lambda (λ) or kappa (κ) light chains.

[0098] The overall identity percentage of the variable region or full-length heavy / light chain sequence can be combined with specific CDR sequences derived from the same antibody.

[0099] The percentage of sequence identity (%) is defined as the percentage of amino acid residues in a candidate sequence that are identical to residues in a comparison sequence, after aligning sequences to achieve maximum sequence identity and introducing gaps as necessary, without considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of each sequence. If the aligned sequences are of different lengths, the sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence, or if the comparison sequence is longer than the given sequence, the sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence. Sequence identity may be defined by referring to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximize the number of matches and minimize the number of gaps. Generally, default parameters of gap generation penalty = 12 and gap expansion penalty = 4 may be used. While the use of GAP is preferred, other algorithms can also be used, such as BLAST (Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method described in Pearson and Lipman (1988) PNAS USA 85: 2444-2448), SSEARCH (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), HMMER3 (Johnson LS et al BMC Bioinformatics. 2010 Aug 18; 11: 431), or Altschul et al. (1990) as listed above (generally using default parameters, e.g., Pearson Curr Prot Bioinformatics (2013) Chapt 3 Uniy 3.1 doi: 10.1002 / 0471250953). The TBLASTN program (see bi0301s42) can also be used.In particular, the psi-Blast algorithm can be used (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402). Sequence identity and similarity can also be determined using Genomequest® software (Gene-IT, Worcester MA USA). Sequence comparison is preferably performed over the entire length of the relevant sequences being compared.

[0100] The antibodies of this disclosure may include one or more substitutions within the framework of the VH region and / or VL region. As used herein, “substitution” refers to the exchange of one amino acid for another at a particular position in the same position of the baseline molecule. In some embodiments, the baseline molecule is an antibody illustrated herein, e.g., antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810.

[0101] In some embodiments, antibodies or fragments thereof according to this disclosure can cross the blood-brain barrier. In some embodiments, antibodies according to this disclosure may have one scFV chain, such as a transferrin receptor (Yu et al., 2014), and an scFv chain that binds to CD33 as described herein. In some embodiments, antibodies according to this disclosure include an antibody or fragment thereof that binds to CD33 as described herein (e.g., antibody, scFv, sdAb, etc.) and a further binding site that binds to another target. The other target may be a receptor in the brain, such as a signaling receptor. The further binding site may be an antibody, scFv, nanobody, or aptamer. The two binding sites of such a bispecific molecule may form a fusion protein.

[0102] Single-domain antibodies (sdAbs), also known as nanobodies, comprising the heavy chain CDR and / or VH sequence of any antibody described herein, are also described herein. Accordingly, antibodies or fusion molecules comprising a CD33-binding nanobody and a brain receptor-binding nanobody described herein are also described herein. Antibodies or fusion molecules comprising a CD33-binding scFV chain or nanobody and a brain receptor-binding aptamer are also described herein.

[0103] The antibodies described herein may increase the phagocytosis of CD33-expressing cells. The increase in phagocytosis may be measured in comparison to a comparative antibody. The comparative antibody may be an isotype control antibody. The comparative antibody may be another CD33-conjugating antibody. The comparative antibody may be an antibody having a heavy chain variable sequence and a light chain variable sequence of ATL_5909. The comparative antibody may be another CD33-conjugating antibody having a different epitope. Phagocytosis may be evaluated by measuring the fluorescence signal associated with the uptake of labeled particles by imaging or flow cytometry. Phagocytosis may be measured by detecting the fluorescence signal indicating phagocytosis of a substrate having pH-dependent fluorescence. The substrate may be selected from amyloid beta and S. aureus. The labeled substrate may be a substrate labeled with pHrodo red. CD33-expressing cells may be inflammatory human iPSC-derived microglia cells. Inflammatory cells may be cells stimulated with inflammatory signals. For example, cells may have been exposed to an inflammatory signal (e.g., LPS, IFN-γ) for at least 6, 12, or 24 hours, or for about 6, 12, or 24 hours, before being exposed to the antibody. CD33-expressing cells may be PBMCs. CD33-expressing cells may be cells isolated from human blood. CD33-expressing cells may be monocytes or microglia. CD33-expressing cells may be human cells. The cells may be iPSC-derived microglia. CD33-expressing cells may be cells stimulated with an inflammatory signal (e.g., LPS) before being exposed to the antibody.

[0104] The antibodies described herein can bind to human CD33. The antibodies described herein can bind to human CD33 with an EC50 of up to 2e-0.8M or up to 3e-0.9M, as evaluated by ELISA (e.g., binding to plated rhCD33). The antibodies disclosed herein can bind to human CD33 with an EC50 of up to 15 μg / ml, up to 12 μg / ml, up to 5 μg / ml, up to 3 μg / ml or up to 0.5 μg / ml. The antibodies disclosed herein can bind to human CD33 with an EC50 of up to 1e-7M, up to 8e-8M, up to 3e-8M, up to 2e-0.8M or up to 3e-0.9M. Binding to human CD33 can be evaluated by ELISA using plated rhCD33, such as Sino Biological's CD33_human_ECD, His_004_001, 12238-H08H.

[0105] The antibodies disclosed herein have a Koff of at least 1e-3s for binding to human CD33. -1 This is possible. The antibodies according to this disclosure have a Koff of 1e-3s for binding to human CD33. -1 ~5e-2s -1 It is possible that the antibodies relating to this disclosure may have a KD of up to 5e-7M for binding to human CD33. The antibodies relating to this disclosure may have a KD of 2e-8M to 5e-7M for binding to human CD33. Human CD33 may be rhCD33-his (e.g., R&D Systems, 10375-SL-050). KD and Koff may be measured by biolayer interferometry (BLI) with Octet-I. The antibodies relating to this disclosure may have a lower Koff for binding to human CD33 than the comparison antibodies. Preferred comparison antibodies are as described above.

[0106] The antibodies according to this disclosure may have a melting temperature Tm1 of at least 57 or 57-70. The antibodies according to this disclosure may have at least 95% SEC-HPLC purity monomer.

[0107] The antibodies described herein may reduce CD33 internalization in C33-expressing cells more effectively than comparative antibodies. The antibodies described herein may reduce the CD33 on the cell surface of human monocytes by less than 50% or less than 80% after 5 hours of incubation. The antibodies disclosed herein may reduce the CD33 on the cell surface of human monocytes to a lower degree than that of comparative antibodies at the same concentration after 5 hours of incubation. Preferred comparative antibodies are as described above.

[0108] The antibodies described herein may have increased binding to human CD33 proteins containing mutations at positions 20, 21, 22, and 24 compared to antibody binding to human CD33 proteins without such mutations. The antibodies described herein may have increased binding to human CD33 proteins containing mutations at positions 20, 21, 22, 24, and 132 compared to antibody binding to human CD33 proteins without such mutations. The antibodies described herein may have decreased binding to human CD33 proteins containing mutations at positions 47, 50, 51, and 52 compared to antibody binding to human CD33 proteins without such mutations. The antibodies described herein may not be able to bind to human CD33 proteins containing mutations at positions 47, 50, 51, and 52. The antibodies described herein may have decreased binding to human CD33 proteins containing mutations at positions 47, 50, 51, 52, and 122 compared to antibody binding to human CD33 proteins without such mutations. The antibodies described herein may not bind to human CD33 proteins containing mutations at positions 47, 50, 51, 52, and 122. The antibodies described herein may exhibit reduced binding to human CD33 proteins containing a mutation at position 83 compared to binding to human CD33 proteins without the aforementioned mutation. The mutations may be selected from: N20R at position 20, F21V at position 21, W22R at position 22, Q24E at position 24, I47V at position 47, Y50H at position 50, D51T at position 51, K52R at position 52, Q83R at position 83, R122K at position 122, and P132T at position 132. The positions may refer to locations in the full-length human CD33 sequence (CD33M (Uniprot ID: P20138-1)). Binding may be measured using single-point ELISA. The human CD33 protein may contain residues 18-232 of human CD33. The unmutated human CD33 protein may be CD33M2_ECD_18-232_WT (SEQ ID NO: 226). The antibodies described herein may increase binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos1[P1] (SEQ ID NO: 227) compared to proteins containing the sequence CD33M2_ECD_18-232_WT (SEQ ID NO: 226).The antibodies described herein may increase binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos1_MutPos6[P6+1](SEQ ID NO: 228) compared to proteins containing the sequence CD33M2_ECD_18-232_WT(SEQ ID NO: 226). The antibodies described herein may decrease binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos2[P2](SEQ ID NO: 229) compared to proteins containing the sequence CD33M2_ECD_18-232_WT(SEQ ID NO: 226). The antibodies described herein may reduce binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos2_MutPos5[P2+5](SEQ ID NO: 230) compared to proteins containing the sequence CD33M2_ECD_18-232_WT(SEQ ID NO: 226). The antibodies described herein may reduce binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos4[P4](SEQ ID NO: 232) compared to proteins containing the sequence CD33M2_ECD_18-232_WT(SEQ ID NO: 226). The antibodies described herein may interfere with sialic acid binding. The antibodies described herein may bind to an epitope such that antibody binding prevents sialic acid from binding to CD33.

[0109] The antibodies of this disclosure may bind to CD33 proteins containing the V domain of CD33. The antibodies of this disclosure may not bind to CD33 proteins that do not contain the V domain of CD33. The antibodies of this disclosure may not bind to CD33 proteins having the sequence of protein CD33_human_ECD_Cdomain_His_007 (SEQ ID NO: 175). The antibodies of this disclosure may bind to CD33 more selectively than one or more other Siglecs, and optionally, the antibodies may bind to CD33 more selectively than one or more (or all) of Siglecs-6, Siglecs-7, Siglecs-8, and Siglecs-9, and / or the antibodies may bind to human CD33 more selectively than one or more other homologs, and optionally, the antibodies may bind to human CD33 more selectively than mouse CD33 and cynomolgus monkey CD33.

[0110] The antibodies of this disclosure may reduce neuroinflammation (i.e., inflammation in the brain) compared to, for example, a comparative antibody (e.g., an isotype control antibody or ATL_5909). The level of neuroinflammation may be determined by measuring the level of pro-inflammatory cytokines in nerve cells, such as microglia (e.g., human Ipsc-derived microglia), using an immunoassay such as ELISA, as described herein. The pro-inflammatory cytokines may be one or more of monocyte chemotactic factor (MCP-1), IL-6, interferon-gamma-inducible protein 10 (IP10), and glial fibrillary acidic protein (GFAP).

[0111] The antibodies described herein may have lower peripheral clearance when administered to a subject compared to a comparative anti-CD33 antibody. The comparative anti-CD33 antibody may be ATL_5909. The subject may be an organism expressing the CD33 protein containing human CD33 exons 1-3 (e.g., a mouse). Administration may be parenteral. Administration may be intravenous or intraperitoneal.

[0112] The antibodies described herein can increase the phagocytosis of Aβ by microglial cells in vivo. This can be evaluated in vivo, for example, as described herein (see Example 10 and accompanying method). The effect of the antibodies on Aβ phagocytosis has been observed in Alzheimer's disease (AD) mice (e.g., APP). NL-G-F This can be evaluated using microglia derived from human microglia progenitor cells derived from iPSCs transplanted into the brains of knock-in mice.

[0113] The antibodies described herein can increase Aβ plaque clearance in subjects where it is needed. The antibodies can increase Aβ plaque clearance in subjects where it is needed by microglial phagocytosis.

[0114] The antibodies described herein can increase the phagocytosis of tau aggregates by microglia with an inflammatory phenotype (e.g., LPS-treated iPSC microglia). For example, the antibodies described herein can increase the phagocytosis of tau aggregates by microglia with an inflammatory phenotype to a greater extent than comparative anti-CD33 antibodies such as ATL_5909. This can be evaluated in vitro, as described herein, for example, in Example 11. The antibodies described herein can increase the phagocytosis of tau aggregates by microglia in subjects where it is needed.

[0115] The antibodies described herein may not induce significant release of any one or more cytokines by human iPSC-derived microglia, human PBMCs, and / or isolated human monocytes exposed to the antibodies in vitro, compared to a control (e.g., an isotype control antibody). The one or more cytokines may be selected from: CCL2 (MCP-1), CXCL8 (IL-8), IFN-γ, IL-10, IL-12p40, IL12p70, IL- 17A, IL-1β, IL-2, IL-23, IL-4, IL-6, TGF-β1, TNF-α, CCL17(TARC), CCL2(MIP-3α), CCL3(MIP-1α ), CCL4 (MIP-1β), CCL5 (RANTES), CXCL1 (Gro-α), CXCL5 (ENA-78), CXCL9 (MIG), CCL11 (eotaxin), CX3CL1 (fractalkine), IL-18, sRAGE, sTREM-1, sTREM-2, VEGF, VILIP-1, β-NGF, BDNF, CCL2 (MCP-1). The antibody may not induce the release of any of the above cytokines. The antibody may induce the release of one or more or all of the above cytokines by human iPSC-derived microglia, human PBMCs and / or isolated human monocytes exposed to the antibody in vitro at lower levels compared to the comparative anti-CD33 antibody (e.g., ATL_5909). The antibodies described herein may reduce the levels of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype in vitro (e.g., LPS-treated human iPSC-derived microglia) and / or in vivo (e.g., when administered to a subject requiring it). The antibodies described herein may reduce the levels of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype (e.g., LPS-treated human iPSC-derived microglia) in vitro (e.g., LPS-treated human iPSC-derived microglia) more significantly than the comparative anti-CD33 antibody (e.g., ATL_5909).

[0116] The antibodies described herein may increase the level of phosphorylated SYK protein in microglial cells treated with the antibodies in vitro compared to a control (e.g., an isotype control antibody). The microglial cells may be those exhibiting an inflammatory phenotype (e.g., microglial cells treated with LPS). SYK phosphorylation may increase without a significant increase in overall protein levels.

[0117] The antibodies described herein may increase the level of P2RY12 in microglia cells treated with the antibodies in vitro compared to a control (e.g., an isotype control antibody). The microglia cells may be those with an inflammatory phenotype (e.g., microglia cells treated with LPS).

[0118] The antibodies described herein may enhance TREM2 signaling in microglia cells treated with the antibodies in vitro compared to a control (e.g., an isotype control antibody). The microglia cells may be those exhibiting an inflammatory phenotype (e.g., microglia treated with LPS).

[0119] The antibodies described herein may alter the activity of the oxidative phosphorylation pathway in microglia compared to a control (e.g., treatment with an isotype control antibody). The activity of the oxidative phosphorylation pathway can be evaluated by RNA sequencing, followed optionally by gene set enrichment analysis, such as that described herein (see Example 14).

[0120] The antibodies described herein may reduce the pro-inflammatory release of one or more markers of inflammation in human neuronal cell culture assays compared to a control (e.g., exposure to an isotype control antibody). The human neuronal cell culture assay may be a co-culture assay including glutamatergic neurons, GABAergic neurons, microglia, and astrocytes. The pro-inflammatory release of one or more cytokines may be assessed by exposing cells to one or more pro-inflammatory signals, such as lipopolysaccharide (LPS) and / or interferon-gamma (INFγ). The one or more markers of inflammation may be selected from MCP-1, IP-10, GFAP, and IL-6. Therefore, the antibodies of this disclosure may result in a reduction in the expression of one or more markers of inflammation in the CNS, for subjects requiring such reduction. The one or more markers of inflammation may include one or more of MCP-1, IP-10, GFAP, and IL-6.

[0121] The antibodies described herein may selectively bind to CD33. The antibodies may not bind to any other human protein. Isolated nucleic acids encoding the antibodies, antigen-binding fragments, or polypeptides described herein are provided. Vectors containing the nucleic acids described herein and host cells containing the vectors are also provided. For example, the host cell may be a eukaryotic cell or a mammalian cell, such as a Chinese hamster ovary (CHO) cell, or a prokaryotic cell, such as Escherichia coli (E. coli). In some embodiments, the vector is a viral vector, such as a bacteriophage.

[0122] A method for producing an antibody or antibody fragment as described herein is further provided, comprising culturing host cells as described herein under conditions suitable for the expression of a vector encoding the antibody or antigen-binding fragment, and isolating and / or purifying the antibody or antibody fragment. The method further comprises incorporating the antibody or antibody fragment into a composition comprising at least one additional component.

[0123] Antibodies and their fragments may be used for therapeutic purposes.

[0124] The subject receiving treatment or diagnosis may be any animal or human. The subject is preferably a mammal, and more preferably a human. The subject may be male or female. The subject may be a patient. Therapeutic use may be in humans or animals (veterinary use). Unless otherwise specified, the subject is human.

[0125] Drugs and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by several routes, including (but not limited to) parenteral, intravenous, intra-arterial, intramuscular, oral, and nasal. Drugs and compositions may be formulated for injection.

[0126] Pharmaceutical compositions may be prepared using pharmaceutically acceptable “carriers” composed of materials considered safe and effective. “Pharmaceutically acceptable” means “generally considered safe,” e.g., physiologically tolerable, and typically does not cause allergic or similar adverse reactions, such as stomach upset, when administered to humans. In some embodiments, this term refers to molecular entities and compositions approved by a U.S. federal or state regulatory agency as GRAS-listed under Sections 5, 204(s) and 409 of the U.S. Food, Drug, and Cosmetic Act, which are subject to prior review and approval by the FDA or similar list, the United States Pharmacopeia, or another generally recognized pharmacopoeia for use in animals, more specifically in humans. The term “carrier” refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art will be familiar with such pharmaceutical carriers and methods of formulating pharmaceutical compositions using such carriers. Pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffers, isotonic agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., the anti-CFH antibody used in the composition. Therefore, those skilled in the art will understand that a composition without incompatibility among any of the components of the dosage form is provided. The excipients may be selected from the group consisting of buffers, solubilizers, isotonic agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0127] The dose is preferably a "therapeutic dose," which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, depends on the nature and severity of the disease being treated. Prescribing treatment, such as determining the dosage, is the responsibility of the general practitioner and other physicians, typically taking into account the disorder being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols described above can be found in Remington's Pharmaceutical Sciences, 20 thThis can be found in the 2000 edition published by Lippincott, Williams & Wilkins.

[0128] Conditions treatable according to the present invention include all those in which CD33 plays a role, including neurodegenerative disorders, particularly those characterized by an increase in toxic protein species such as Aβ42, which is characteristic of Alzheimer's disease (AD), and the phagocytosis of these toxic species is useful. The antibodies of this disclosure may be used to treat any neurological disease or disorder associated with the accumulation of toxic protein species. In fact, the antibodies of this disclosure have been shown to increase phagocytosis in microglia and therefore may be useful in treating any neurological disease or disorder in which phagocytosis of toxic protein species is impaired or insufficient. In addition, respiratory disorders characterized by impaired phagocytosis, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPD) (Donelly et al., 2012), may also be treated using the antibodies of this disclosure. Other conditions treatable according to the present invention include cancers in which sialyl acid can cause immunosuppression, such as AML and solid tumors, such as many solid tumors and cancers characterized by abnormal CD33 expression, such as acute myeloid leukemia (AML), including cancers in which tumors exhibit hypersialylation of their cell surface to evade recognition by immune cells (Stanczak & Laubli, 2023). The antibodies of this disclosure were originally identified by analysis of resilient AD patients. Subsequently, these antibodies were found in resilient centenarians and showed their relevance beyond AD. Therefore, antibodies of this disclosure for use as pharmaceuticals are also described herein. Antibodies for use in the treatment or prevention of neurodegenerative diseases, and / or respiratory diseases, and / or cancer are also described. Antibodies of this disclosure for use in the manufacture of pharmaceuticals, such as pharmaceuticals for the treatment or prevention of neurodegenerative diseases, or respiratory diseases, or cancer. This specification also describes a method for treating a subject diagnosed with or at risk of having a neurodegenerative disease, a respiratory disease, or cancer, which includes administering an effective dose of an antibody described herein to the subject.

[0129] Neurodegenerative diseases or disorders may be selected from the following: frontotemporal dementia (FTD), Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), human immunodeficiency virus (HIV)-induced encephalitis, chronic traumatic encephalopathy (CTE), vascular dementia, prion diseases, Lewy body dementia, spinal muscular atrophy (SMA), motor neuron diseases (MND), such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar degeneration (SCA) types 1, 2, 6, and 7. Types 1 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral-pallidoluysian atrophy (DRPLA), X-linked spinal muscular atrophy type 1 (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease), and DNAJB6 myopathy, multiple sclerosis (MS-CD33 single nucleotide polymorphisms are known to be associated with an increased risk of multiple sclerosis), and microgliosis, such as adult-onset leukoencephalopathy with axonal globulins and pigmentary glia (ALSP). For example, neurodegenerative diseases may be selected from FTD, AD, HD, and PD. Neurodevelopmental diseases or disorders may be tauopathies. Therefore, neurodegenerative disorders can include AD (Alzheimer's disease), CTE (chronic traumatic encephalopathy), PiD (Pick's disease), PSP (progressive supranuclear palsy), CBD (corticobasal degeneration), and AGD (argyrophilic grain dementia).

[0130] Respiratory diseases may include one or more of the following: COPD, cystic fibrosis, asthma, and idiopathic pulmonary fibrosis (IPD).

[0131] Cancers may be selected from cervical cancer, breast cancer, brain cancer, bladder cancer, colon adenocarcinoma, cervical cancer, fibrocarcinoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, non-small cell lung cancer, non-Hodgkin lymphoma, and hematological cancers, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma. In some embodiments, the cancer is AML. In embodiments, the cancer is selected from melanoma, hepatocellular carcinoma, pancreatic cancer, colon adenocarcinoma, cervical cancer, breast cancer, non-small cell lung cancer, head and neck cancer, and hematological cancers. All of these cancers are known to have tumor cells that suppress immune cells by hypersializing their cell surface, and therefore this suppression can be mitigated using the antibodies of this disclosure.

[0132] The antibodies of this disclosure may be used therapeutically in conjunction with one or more further therapeutic agents. As used herein, “further therapeutic agent” is an additional compound, protein, vector, antibody, cell, or entity having a therapeutic effect. The antibody may be administered concurrently with the further therapeutic agent. The antibody may be co-formulated with the further therapeutic agent. The antibody may be administered sequentially before or after the further therapeutic agent.

[0133] The antibodies described herein may be used as biomarkers to indicate that a subject is likely to respond to treatment with an antibody or antibody fragment as described herein. The Specified also describes a method for determining whether a subject is likely to respond to treatment with an antibody or antibody fragment as described herein, comprising: obtaining BCR sequence data from the subject; and using the sequence data, determining whether the subject's BCR repertoire contains one or more antibodies that are likely to bind to CD33 (e.g., antibodies as described herein, e.g., antibodies having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to any particular antibody or antibody fragment as described herein), wherein a subject whose BCR repertoire does not contain one or more antibodies that are likely to bind to CD33 is likely to respond to treatment with an antibody or antibody fragment as described herein.

[0134] Accordingly, this specification also describes a method for treating a subject diagnosed with or likely to have a disease in which CD33 plays a role (e.g., any disease in which the phagocytosis of CD33-expressing cells is impaired or insufficient, such as neurodegenerative disorders or cancer), comprising: obtaining BCR sequence data from the subject; using the sequence data, determining whether the subject's BCR repertoire contains one or more antibodies likely to bind to CD33; and administering a therapeutically effective amount of an antibody or antibody fragment described herein to a subject whose BCR repertoire does not contain one or more antibodies likely to bind to CD33.

[0135] Some of the methods of this disclosure involve a sample containing cells. The sample may be a culture of cells grown in vitro. For example, the culture may include a suspension of cells or cells cultured on a culture plate or in a culture dish. The methods of this disclosure may be carried out or products may be present in vitro, ex vivo, or in vivo.

[0136] According to some aspects of this disclosure, a parts kit comprising an antibody according to the present invention is provided. In some embodiments, the kit comprises the antibody according to the present invention and one or more reagents for use in immunochemistry, the antibody immobilized on a solid support, means for labeling the antibody, means for linking the antibody to a cytotoxic moiety, and further therapeutic agents.

[0137] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, are expressed in relation to means for performing the disclosed functions or methods or processes for obtaining the disclosed results, and may be used, separately or in any combination as needed, to realize the invention in its various forms.

[0138] Although the present invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art if this disclosure is given. Therefore, the exemplary embodiments of the present invention described above are illustrative and not limiting. Various modifications to the embodiments described can be made without departing from the spirit and scope of the invention.

[0139] To avoid any doubt, all theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0140] The headings used herein are for structural purposes only and should not be construed as limiting the subjects described.

[0141] Throughout this Specification, including the following claims, unless otherwise required by context, the words “comprise” and “include,” as well as variations such as “includes,” “includes,” and “includes,” shall be understood to mean that they include the integers or processes or groups of integers or processes described, but not any other integers or processes or groups of integers or processes.

[0142] When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “it” include multiple referents unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, another embodiment includes one particular value and / or another particular value. Similarly, where values ​​are expressed as approximations, the use of the antecedent “about” will be understood to mean that a particular value forms another embodiment. The term “about” with respect to numbers is optional and means, for example, + / - 10%.

[0143] [Table 1]

[0144] Table 2

[0145] Table 3

[0146] Table 4

[0147] Table 5

[0148] Table 6

[0149] Table 7

[0150] Table 8

[0151] Table 9

[0152] Table 10

[0153] Table 11

[0154] Table 12

[0155] Table 13

[0156] Table 14

[0157] Table 15

[0158] Throughout Table 0 above and this specification, antibodies may be referred to by the reference numbers shown in the above table (e.g., ATL_0005802), the same reference number without the leading "0" (e.g., ATL_5802), or the same reference with only the number (e.g., 5802). For example, antibody ATL_0005802 is interchangeably referred to as "ATL_0005802", "ATL_5802", and "5802". The notations "VH" and "VL", when added to an antibody reference (as either a prefix or suffix), refer to the heavy chain variable domain of the reference antibody. The notations HCDR1, HCDR2, and HCDR3, when added to an antibody reference (as either a prefix or suffix), refer to the heavy chain CDRs of the reference antibody (CDR1, CDR2, and CDR3, respectively). The notations LCDR1, LCDR2, and LCDR3, when added to an antibody reference (as either prefixes or suffixes), refer to the light chain CDRs of the reference antibody (CDR1, CDR2, and CDR3, respectively). The notations HFWR1, HFWR2, HFWR3, and HFWR4, when added to an antibody reference (as either prefixes or suffixes), refer to the heavy chain framework regions of the reference antibody (FWR1, FWR2, FWR3, and FWR4, respectively). The notations LFWR1, LFWR2, LFWR3, and LFWR4, when added to an antibody reference (as either prefixes or suffixes), refer to the light chain framework regions of the reference antibody (FWR1, FWR2, FWR3, and FWR4, respectively). [Examples]

[0159] Examples The following examples illustrate the identification of novel antibodies with therapeutic potential from a cohort of individuals with Alzheimer's disease or at risk of developing it, and from cognitively healthy centenarians aged 100 years or older who were serologically positive for CD33 antibodies (Example 1), as well as phage display analysis of the repertoire from CD33-reactive subjects (Example 2). Examples 3-15 show the results of functional screening and further characterization of some of the newly discovered antibodies.

[0160] Materials and methods Serum and plasma ELISA for identifying CD33-reactive subjects CD33 recombinant human antigen (R&D Systems, catalog number: 10375-SL-050) or negative control lysozyme (MP Biomedicals #195303) was directly absorbed into an ELISA plate at 3 μg / ml (50 μL per well) and incubated overnight at 4°C. The plate was washed with phosphate-buffered saline (PBS). The plate was blocked with 200 μl / well of blocking solution (1 w / v% bovine serum albumin (BSA) in PBS) at room temperature for 1 hour. Subsequently, the blocking solution was removed, and the serum and plasma samples to be evaluated were diluted 1 / 100 with the blocking solution (1 w / v% BSA in PBS) and applied to the plate. The plate was incubated at room temperature for 1 hour. The plate was washed with PBS / 0.1% Tween. Next, anti-human (Fab)'2-horseradish peroxidase (HRP) antibody (Jackson Immunoresearch #109-035-097, lot: 148466) was added to each well, and antibody binding was detected after incubation at room temperature for 1 hour. The plate was washed with PBS / 0.1% Tween, and 3,3',5,5-tetramethylbenzidine (TMB) solution (Life Technology, #002023) was added to each well. After incubation at room temperature for 5 minutes, a stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm using a Molecular Devices FilterMaxF5 plate reader.

[0161] Sequence analysis of scFv derived from phage ELISA and phage display. Each phagemid-supported TG1 clone from the glycerol stock was cultured at 37°C in 100 μL of 2TYAG (2TY medium supplemented with 100 μg / ml ampicillin and 2% glucose) with aeration until the optical density OD600 = 0.6. Subsequently, the cells were rescued for 1 hour with a helper phage (Invitrogen, catalog number: 18311-019) added at an MOI of 10. The medium was then replaced with 2TYAK (2TY medium supplemented with 100 μg / ml ampicillin and 50 mg / ml kanamycin) to prepare the phages. The cultures were then incubated overnight at 25°C with sufficient aeration. The following day, the phages were isolated from the bacteria by centrifugation at 3200 rpm for 10 minutes. The supernatant containing the phages was transferred to a new plate and blocked with 3 w / v% milk in PBS.

[0162] Recombinant human CD33 antigen (R&D Systems, catalog number: 10375-SL-050) or negative control lysozyme (MP Biomedicals #195303) was directly absorbed into ELISA plates at 3 μg / ml (50 μL per well) and incubated overnight at 4°C. Plates coated with each antigen were washed with PBS and blocked with 200 μL / well of blocking solution (3 w / v% milk in PBS) at room temperature for 1 hour. Subsequently, the blocking solution was removed and the blocked phage samples to be evaluated were applied to the plates. Plates were incubated at room temperature for 1 hour. Each plate was washed with PBS / 0.1% Tween and incubated with anti-M13 HRP (Sino Biological, #11973-MM05T-H) at room temperature for 1 hour to detect phage binding. Plates were washed with PBS / 0.1% Tween and TMB solution (Life Technology, #002023) was added. After incubating the plate at room temperature for 5 minutes, a stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm using a Molecular Devices FilterMaxF5 plate reader.

[0163] Single-point ELISA for binding to complete CD33 and CD33 C2 domains, as well as related CD33 proteins. Seven IgG1-converting antibodies were tested by single-point ELISA against the following antigen panel: human recombinant CD33 (Sino Biological catalog number: 12238-H08H), cynomolgus / rhesus monkey CD33 (Sino Biological, catalog number: 90303-C08H), human recombinant CD33 C2 domain (in-house produced), mouse CD33 (Sino Biological, catalog number: 50712-M08H), recombinant human siglec-6 / CD327 Fc chimera (R&D Systems, 2859-SL), recombinant human siglec-7 / CD328 Fc chimera (R&D Systems, catalog number: 1138-SL-050), recombinant human siglec-8 Fc chimera (R&D Systems, catalog number: 9045-SL), and recombinant human siglec-9 Fc chimera (R&D Systems, catalog number: 1139-SL). After single-point ELISA, EC50 values ​​were obtained by performing titration ELISA on each antibody and antigen pair that showed a positive signal.

[0164] Each recombinant protein antigen or negative control lysozyme (MP Biomedicals #195303) was directly absorbed into an ELISA plate at 3 μg / ml (50 μL per well) and incubated overnight at 4°C. Each plate was washed with PBS. The plates were blocked at room temperature for 1 hour with 200 μl / well of blocking solution (1 w / v% BSA in PBS). Subsequently, the blocking solution was removed, and the antibody to be evaluated was diluted to 100 μg / ml in blocking solution (1 w / v% BSA in PBS) and applied to the plate. The plates were incubated at room temperature for 1 hour. Each plate was washed with PBS / 0.1% Tween, and anti-human (Fab)'2-HRP (Jackson Immunoresearch #109-035-097, lot number: 148466) was added to the plate and incubated at room temperature for 1 hour to detect antibody binding. Each plate was washed with PBS / 0.1% Tween and TMB solution (Life Technology, #002023) was added. After incubating the plates at room temperature for 5 minutes, stop solution (0.5M sulfuric acid) was added. Absorbance was read at 450 nm using a Molecular Devices FilterMaxF5 plate reader.

[0165] Biolayer interferometry for characterizing CD33 binding reaction rates In biolayer interferometry (BLI) experiments, the binding of 11 IgG1-converting antibodies to human recombinant CD33 (R&D Systems, 10375-SL-050) was tested.

[0166] Each antibody was loaded into a column of eight anti-human IgG Fc capture biosensor chips (AHC chips, Sartorius Stedim part #18-5060), which were then equilibrated in kinetic buffer (Sartorius Stedim part #18-1105) and immersed in a dilution series of CD33 samples in kinetic buffer at 25°C. The binding reaction was measured. The chips were then immersed in kinetic buffer and the reaction was measured again. The binding and dissociation curves were fitted to the reaction measurements using the manufacturer's analysis software (Octet Data Analysis HT12.0), and kinetic parameters were calculated.

[0167] Phagocytosis in inflammatoryly stimulated human iPSC-derived microglia Induced pluripotent stem cell (iPSC)-derived microglia (FujiFilm (CDI) #R1131) were seeded at 20,000 cells / well. On day 3 of culture, 50% of the culture medium was changed, and the cells were rested for 3 days. On day 4, the cells were treated with 50 ng / ml LPS (tlrl-eklps Invivogen) for 24 hours, followed by treatment with 50 μg / ml test antibody for another 24 hours. Subsequently, the cells were treated with 0.5 μg / well pHrodo red-labeled amyloid-beta, and red fluorescence was monitored using Incucyte S3. (Beta-amyloid(1-42) aggregation kit, pHrodo Red-labeled rPeptide #1170-025, succinimidyl ester (ThermoFisher Scientific #P36600)).

[0168] Human whole blood phagocytic assay Blood was collected from an NHS Blood and Transplant apheresis cone and diluted 1:10 in RPMI medium (Invitrogen A4192301). 25 μL of diluted blood and 2 ml of 1× lysis buffer (ebioscience 00-4300-54 diluted 10× with water and then diluted to 1× immediately before use) were added to each well, and the wells were mixed by pipetting. The samples were incubated at room temperature for 15 minutes, then rotated at 400 g for 5 minutes to pellet residual leukocytes. The supernatant was discarded, and the cells were resuspended in 100 μL of RPMI medium containing 10 μg / ml S. aureus pHrodo-red and 10 μg / ml antibody. The samples were incubated at 37°C for 3 hours, then washed with 1 ml PBS / 2% FBS, pelleted by rotating at 400 g for 5 minutes, and then the supernatant was discarded. The samples were resuspended in 50 μl of 1 / 50 human Fc block (BD Biosciences 564219) diluted in PBS / 2% FBS and incubated at 2–8°C for 15 minutes. 50 μl of 1 / 100 diluted CD14 APC diluted in PBS / 2% FBS was added to the top of the Fc block and incubated at 2–8°C for a further 30 minutes. The samples were washed with 1 ml of PBS / 2% FBS as described above and then resuspended in 200 μL of PBS / 2% FBS / 1 / 10,000 DAPI for analysis using a BD FACSymphony flow cytometer. Live monocytes were gated (DAPI negative, CD14 positive), and pHrodo-red fluorescence of the live monocyte population was evaluated using the 561-586 / 15 channel.

[0169] Decrease in CD33 in human monocytes PBMC-isolated human monocyte cells, which had been frozen in advance, were thawed, washed, and rested overnight in RPMI medium (Invitrogen A4192301) + 10% fetal bovine serum (FBS) at 37°C and 5% CO2 (standard cell culture conditions). The following day, the rested monocyte cells were collected and washed once with RPMI + 10% FBS. Then, the cells were divided into 2 × 10⁻⁶ cells. 6 Resuspend at the final cell / ml concentration and add 50 μL to each well of a flat-bottomed 96-well plate (1 × 10⁶ cells). 5Cells / well (Corning, catalog number 3595) were added. Cells were allowed to rest in an incubator while reagents were prepared. Reagents (ATL_5802, ATL_5810, ATL5338, ATL_5909) were prepared at 60 μL / well in a round-bottom 96-well plate (Corning, catalog number 3799) at twice the experimental final concentration in RPMI + 10% FBS, at single concentrations (80 nM) or ranges (80, 60, 40, 30, 20, 2 nM). ATL5338 is an in-house prepared isotyped control based on FITC-conjugated antibody 4-4-20 described in Jung et al JMB 294, 163 (1999). Next, 50 μl / well of the reagent plate was transferred to a 96-well plate containing resting monocytes at 50 μl / well each, resulting in a final volume of 100 μl. The final concentrations were then set to 40, 30, 20, 10, and 1 nM. The plates were then incubated under standard cell culture conditions for 5 hours. After incubation, the plates were kept on ice to avoid any further antibody internalization. All subsequent steps were performed in the dark at 4°C. The cells were collected by centrifugation (300 g for 5 minutes), and the supernatant was removed. The cells were then resuspended in 50 μL of Live / Dead fixable violet dead cell stain (catalog number L34955) and incubated for 7 minutes. A washing step was performed by adding 150 μL of PBS / 2% FBS per well to the top of each sample, and the plates were centrifuged at 300 g for 5 minutes. The supernatant was removed, and the cells were resuspended in the following staining antibodies: anti-human CD33 domain V specific (Biolegend, catalog number 303428), anti-human CD33 domain C specific (Biolegend, catalog number 366620), anti-human CD45 (Biolegend, catalog number 304027), anti-human CD14 (Biolegend, catalog number 301806), anti-human CD16 (Biolegend, catalog number 302016), anti-human CD3 (Biolegend, catalog number 300412), and 1:50 human Fc block diluted in PBS-2% FBS (BD Biosciences 564219). Staining was performed at 4°C for 40 minutes.Next, the cells were washed twice as described above and resuspended in PBS-2% FBS at a final volume of 200 μl / well for analysis using a BD FACSymphony flow cytometer. Live monocytes were gated (negative for death staining, CD45 positive, CD3 negative, CD14 positive, CD16 positive / negative), and MFI released by two CD33 antibodies was recorded.

[0170] Regulation of phagocytosis by CD33 binding in freshly isolated human monocyte cells. Antibodies were prepared in RPMI + 10% FBS at a single concentration (60 nM) twice the final assay concentration, and as 1:4 serial dilutions starting from 60 nM, and distributed at 60 μl / well in round-bottom 96-well plates (Corning, catalog no. 3799). Latrantrin A was diluted 1:5000 for the wells to be used as negative controls. Phagocytic S. aureus (S. aureus) pHrodo bait (Thermo Fisher, catalog no. A10010) was prepared in RPMI + 10% FBS medium at 10 μg / ml twice the final concentration and distributed in flat-bottom 96-well plates (Corning, catalog no. 3595). Human monocytes were then isolated from fresh PBMCs according to the Miltenyi protocol (catalog no. 130-096-537). The resulting monocytes were 1 × 10⁶ cells. 6 The cells were resuspended at a concentration of cells / ml and 100 μl was distributed into each well of a round-bottom 96-well plate (Corning, catalog no. 3799). The cells were centrifuged at 300 g for 5 minutes, the supernatant was removed, and the cells were resuspended in the prepared antibody solution. The cells were then transferred to a plate containing pHrodo reagent in a final volume of 100 μl. The emitted fluorescence was recorded live using an Incucyte system (Sartorius).

[0171] Study Design: In vivo CD34+NSG mouse study There were 17 treatment groups. Each treatment group was labeled with a letter to conceal its identity from the observer. Mice were randomly assigned to each treatment group. This experiment consisted of the following 17 conditions (65 mice in total). 1. Control: Before and 24 hours after administration of 10 mg / kg 2. Test antibodies (ATL5802, ATL5810, and ATL5909) 10, 40 mg / kg, before administration and 24 hours prior to administration. 3. Control: Before administration of 10 mg / kg, 24 hours, and 7 days. 4. Test antibodies (ATL5802, ATL5810, and ATL5909): 10, 40 mg / kg, pre-administration, 24 hours, and 7 days. 5. Test antibodies (ATL5802, ATL5810, and ATL5909) 1 mg / kg, administered before, 4 hours, 24 hours, and for 7 days.

[0172] Groups 1 and 2 consisted of 3 mice each (n=3 biological replicates, total 21 mice), groups 3 and 4 consisted of 5 mice each (n=5 biological replicates, total 35 mice), and group 5 consisted of 3 mice each (n=3 biological replicates, total 9 mice). Blood volume was set according to UK Home Office guidelines (maximum 200 μl total per mouse): Groups 1 and 2 had 200 μl before administration, Groups 3 and 4 had 100 μl both before administration and at 24 hours, and Group 5 had 66 μl before administration, at 4 hours, and at 24 hours. The final time point for each group included end-stage blood samples. The mice were humanized from two separate donors (n=30 from donor 1 and n=35 from donor 2). Groups 1, 2, and 5 used animals from donor 1, and groups 3 and 4 used animals from donor 2.

[0173] The samples from each group are used as follows: • Groups 1 and 2 - Before administration (ELISA), 24 hours (ELISA and flow cytometry) • Groups 3 and 4 - Before administration (ELISA), 24 hours (flow cytometry), 7 days (ELISA and flow cytometry) • Group 5 - All time points (ELISA) The dosage for all treatments was 10 ml / kg.

[0174] Phagocytosis in peripheral blood monocytes from CD34+ mice Blood from CD34+NSG mice was processed as follows: 25 μL of mouse blood and 2 ml of 1× lysis buffer (ebioscience 00-4300-54 diluted to 10× with water and then diluted to 1× immediately before use) were added to each well, and the wells were mixed by pipetting. The samples were incubated at room temperature (18-22°C) for 15 minutes, then rotated at 400 g for 5 minutes to pellet residual leukocytes. The supernatant was discarded, and the cells were resuspended in 100 μL of RPMI medium containing 10 μg / ml S. aureus pHrodo. The samples were incubated at 37°C for 3 hours, then washed by adding 1 ml PBS / 2% FBS, pelleted by rotating at 400 g for 5 minutes, and then the supernatant was discarded. The samples were resuspended in 50 μl of PBS / 2% FBS containing 1 / 50 human Fc blocks (BD Biosciences 564219) and 1 / 100 mouse Fc blocks (Biolegend 156603), and incubated at 2–8°C for 15 minutes. Antibody cocktails were prepared containing the following antibodies in a 1:50 dilution in PBS / 2% FBS: anti-mouse CD45 AF488 (Biolegend 157608), anti-human CD45 APC (Biolegend 304012), and anti-human / mouse CD11b BV785 (Biolegend 101243). 50 μl of the antibody cocktail was added on top of the Fc blocks already present in the wells of each sample. The samples were incubated in the dark at room temperature for a further 30 minutes. The samples were washed with 1 ml of PBS / 2% FBS as described above, and then resuspended in 200 μL of PBS / 2% FBS / 1 / 10,000 DAPI for analysis using a BD FACSymphony flow cytometer. Live human monocytes were gated (DAPI negative, human CD45+, CD11b+ positive), and pHrodo-red fluorescence of the live monocyte population was evaluated using the 561-586 / 15 channel.

[0175] Size exclusion chromatography (SEC-HPLC) Antibody samples were diluted to 1 mg / ml in 20 mM histidine acetate, 150 mM NaCl, pH 5.5, and HPLC was performed using a Vanquish Flex (Thermo) on a Zorbax GF-250 SEC-HPLC column (Agilent). Samples were separated by size in a mobile phase of 20 mM sodium phosphate, 300 mM sodium sulfate, and 100 mM arginine at a flow rate of 0.75 ml / min, 25C, and 25 minutes per sample. Data were acquired at 280 nm, and chromatograms were integrated using Chromeleon software (Thermo Scientific).

[0176] Capillary isoelectric focusing (cIEF) Variant analysis was performed by preparing a master mix for diluting antibody samples for execution on a Maurice instrument (Protein Simple) cIEF cartridge. The final concentrations of the master mix were 0.35% methylcellulose (Protein Simple, 101876), 4% Pharmalite (pH 3-10) (Protein Simple, 17-0456-01), 10 mM arginine (Protein Simple, 042-691), and 0.01% pI markers 4.05 and 9.99 (Protein Simple, 046-029 and 046-034). Samples were diluted to 0.15-0.25 mg / ml in the master mix and run at 1500 volts for 1 minute, followed by 3000 volts for 4.5 minutes. A system compatibility standard (Protein Simples, 046-044) was also run at the start of the run. The stability data generated in weeks 2 and 4 were overlaid, and the charge species profiles were compared.

[0177] Thermal shift assay Protein thermal shift measurements were performed using Uncle (Unchained Labs). Antibodies were diluted to 1 mg / ml or 5 mg / ml in 20 mM histidine acetate, 150 mM NaCl, pH 5.5 buffer and passed through a temperature gradient of 25–95°C increasing at a rate of 0.5°C / min. The sample was tested three times, loading 8.8 μl into three different wells of the uni (Unchained Labs). The laser was set to achieve initial fluorescence in the 300–350 nm range with 10,000–50,000 counts. Melting temperatures (Tm1 / Tm2) and aggregation temperatures (Tagg / Tonset) were analyzed using Uncle Analysis software v6 (Unchained Labs). Tm measurements were calculated using the 350 / 330 nm ratio, while Tonset and Tagg were obtained from SLS readings at 266 nm.

[0178] Epitope Mapping Variants of the extracellular domain of CD33 were prepared as individual recombinant proteins. Since the mAb read panel was known to bind to human CD33 but not to cynomolgus monkey CD33, the variations were based on the differences between human CD33 and cynomolgus monkey CD33.

[0179] DNA sequences encoding human CD33 residues 21–232 were synthesized and cloned into pcDNA3.1(+) using Genscript. Insertions of native residues 18–20, single-residue and multi-residue cynomolgus monkey mutations were performed using the Q5 Site-Directed Mutagenesis Kit (NEB). The wild-type plasmid (residues 18–232) and seven mutant plasmids were individually transfected into 15 mL cultures of Expi293F cells (ThermoFisher) according to the manufacturer's instructions. The seven mutant plasmids were as follows: N20R F21V W22R Q24E[P1], I47V Y50H D51T K52R[P2], I67V R69L[P3], Q83R[P4], R122K[P5], P132T[P6], N20R F21V W22R Q24E P132T[P6+1], I47V Y50H D51T K52R R122K[P2+5]. These mutations are shown in Figure 11A. Five days after transfection, cells were removed from the culture by centrifugation, and proteins were purified from the AKTA pure (Cytiva) supernatant by Ni-affinity chromatography using a HisTrap excel column (Cytiva), followed by size exclusion chromatography using a Superdex Increase 10 / 300 GL column (Cytiva).

[0180] Eleven IgG1 antibodies were tested against wild-type CD33 and all seven CD33 mutant proteins using single-point ELISA. Each recombinant protein antigen or negative control lysozyme (MP Biomedicals #195303) was directly absorbed into an ELISA plate at 3 μg / ml (50 μL per well) and incubated overnight at 4°C. Each plate was washed with PBS. The plates were blocked at room temperature for 1 hour with 200 μl / well of blocking solution (1 w / v% BSA in PBS). Subsequently, the blocking solution was removed, and the antibody to be evaluated was diluted to 266 nM in blocking solution (1 w / v% BSA in PBS) and applied to the plate. The plates were incubated at room temperature for 1 hour. Plates were washed with PBS / 0.1% Tween, and anti-human (Fab)'2-HRP (Jackson Immunoresearch #109-035-097, lot number: 148466) was added to the plates. The plates were incubated at room temperature for 1 hour to detect antibody binding. Each plate was washed with PBS / 0.1% Tween and TMB solution (Life Technology, #002023) was added. After incubating the plates at room temperature for 5 minutes, a stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm using a Molecular Devices FilterMaxF5 plate reader. Absorbance values ​​were converted to percentage of wild-type CD33 binding (where wild-type = 100%).

[0181] [Table 16]

[0182] [Table 17]

[0183] [Table 18]

[0184] Mouse pK test For this study, male homozygous C57BL / 6-Cd33tm1(CD33) / Bcgen (common name: hCD33) were obtained from Biocytogen Technology Co., Ltd. and sent to Pharmaron (Ningbo, China) for this study. Mice (n = 3 / treatment group) were weighed at 6 - 8 weeks of age and administered the freshly prepared antibody solution at a dose of 5 ml / kg at single intraperitoneal (i.p.) doses of 1 mg / kg and / or 10 mg / kg. All mice had free access to food and water. Cage side observations were performed daily and clinical observations were performed before dosing and at each time point of sample collection.

[0185] Blood samples were collected from the orbital vein 4 hours, 24 hours, and 7 days (144 hours) after single i.p. administration of the antibody. Briefly, whole blood samples were left at room temperature for 30 minutes and then centrifuged at 3,500 × g for 15 minutes at 4°C to obtain the serum fraction. Serum samples were immediately transferred to cryovials and stored at -75°C until analysis.

[0186] Samples were processed and analyzed as follows. Serum samples were diluted with assay buffer (0.1% bovine serum albumin [BSA] - 0.05% Tween 20 - PBS), vortexed for 30 seconds, and then loaded into a microplate (Corning Incorporated 96 - well cell culture plate). The serum concentration of the test substance was determined using a custom - developed ELISA method (Pharmaron, Ningbo, China) using goat anti - human IgG Fc polyclonal primary antibody (5 μg / ml) and goat anti - human IgG monoclonal antibody, HRP (1:5000). Readings were obtained on a Molecular Devices SpectraMax ID3 and PK calculations were performed using WinNonlin (Phoenix™, version 8.3).

[0187] In vivo Alzheimer's disease model Male and female Rag2 - / - Il2rγ - / - hCSF1KIApp NL-G-F mice (hereinafter APP NL-G-FFor mice (referred to as mice), as described in Mancuso et al., 2022, iPSC-derived human microglial progenitor cells were transplanted on the 4th day after birth (P4). From about 4 months of age, mice were treated once a week for 12 weeks with 40 mg / Kg of ATL_5802 (n = 6) or an isotype control antibody ATL_5338 (n = 7). To evaluate phagocytosis in vivo, the fluorescent amyloid-β label, methoxy-X04 (MX04), was used, and the degree of amyloid-β uptake was evaluated by measuring the proportion of MX04-positive microglia using flow cytometry. At about 16 weeks of age, mice were injected i.p. with freshly prepared methoxy-X04 (ab142818, prepared in a 1:1 ratio of DMSO:HBSS) at 10 mg / kg. 24 hours after injection of MX04, mice were injected with the euthanasia agent Dolethal and perfused with ice-cold PBS. The harvested brain tissue was immediately placed in FACS buffer (1×PBS containing 2% FBS / FCS and 2 mM EDTA) for downstream processing by flow cytometry. For flow cytometry analysis, the Miltenyi neural tissue dissociation kit was used to dissociate the brain tissue according to the manufacturer's instructions. Samples were run on a MACSQUANT Analyzer 10 and gated for viability and human vs. mouse microglia using the following antibodies: fixable viability dye eFluor780 (1:2000, Invitrogen eBioscience catalog number 65-0865-14), APC mouse anti-human monoclonal CD45 antibody (1:50, BD Bioscience clone HI30 catalog number 555485), and PE-conjugated recombinant human anti-mouse CD11b antibody (1:100, Miltenyi Biotec catalog number 130-113-806). After removing non-viable cells, cells were gated using hCD45, the median fluorescence intensity (MFI) of the human microglial population was measured, and set as the threshold for each mouse. The percentage of MX04-positive microglia was calculated by quantifying the proportion of MX04+ microglial cells that were higher than this set threshold.

[0188] Tau phagocytosis in human iPSC-derived microglia iPSC-derived microglia (catalog number R1131) from FUJIFILM (CDI) were seeded at 20,000 cells / well, rested for 3 days, and 50% of the culture medium was changed on day 3. On day 4, the cells were treated with 50 ng / ml LPS (tlrl-eklps Invivogen) for 6 hours, followed by treatment with 50 μg / ml test antibody for a further 24 hours. The cells were then treated with 0.5 μg / well pHrodo.Red (ThermoFisher Scientific #P36600)-labeled TauP301S (Abcam #ab246003), which had been sonicated once for 5 minutes immediately before treatment. Red fluorescence was monitored using an Incucyte S3.

[0189] Quantification of cytokines in the culture medium of iPSC microglia, total PBMCs, and isolated monocytes. iCell microglia (Cellular Dynamics C1110) were seeded at 25,000 cells per well in iCell microglia medium in a flat-bottomed 96-well plate coated with poly-D-lysine (PDL). PBMCs were isolated from leukocyte apheresis cones (NHS BT) using Lymphopure (Biolegend 426201) and Leucosep tubes (Greiner 227290). After 24 hours, the medium was collected and cytokine levels were measured using a flow cytometry-based multiplex immunoassay (LEGENDplex® Biolegend 741081, 740930, 740502, 740796, or 741795). PBMCs were isolated from leukocyte apheresis cones (NHS BT) using Lymphopure (Biolegend 426201) and Leucosep tubes (Greiner 227290). Monocytes were isolated from PBMCs using a Pan monocyte isolation kit (Miltenyi 130-096-537) and MS column (Miltenyi #130-042-201). 100,000 PBMCs or monocytes per well were seeded in RPMI medium (Gibco A4192301) + 10% FBS in U-bottom plates (PBMCs) or flat-bottom plates (monocytes). Cells were treated with 50 μg / ml isotype control antibody (anti-fluorescein) or ATL_5802 antibody. Several assays involved treating cells with 50 ng / ml LPS (Invivogen, tlrl-eklps) for 6 hours before adding antibodies. After 24 hours, the medium was collected and cytokine levels were measured using a flow cytometry-based multiplex immunoassay (LEGENDplex® Biolegend 741081, 740930, 740502, 740796, or 741795).The clusters of CL(1:-C-2)(C1)(C) 、CXCL8(IL-8)、IFN-γ、IL-10、IL-12p40、IL12p70、IL -17A、IL-1β、IL-2、IL-23、IL-4、IL-6、TGF-β1、TNF-α、CCL17(TARC)、CCL2(MIP-3α)、CCL3(MIP-1α)、CCL4( MIP-1β)、CCL5(RANTES)、CXCL1(Gro-α)、CXCL5(ENA-78)、CXCL9(MIG)、CCL11(CX3CL1 )、IL-18、sRAGE、sTREM-1、sTREM-2、VEGF、VILIP-1、β -NGF, BDNF, CCL2(MCP-1) are also activated in the circulation. 、CCL17(TARC)、CCL2(MCP-1)、CCL5(RANTES)、CCL3( MIP-1α)、CXCL9(MIG)、CXCL5(ENA-78)、CCL20(MIP-3α)、CXCL1(GROα)、CCL4(MIP-1β) PBMC:IL-12p70、TNF-α(TNFSF2)、IL-6、IL-4、IL-10、IL-1β、CCL17(TARC)、IL-12p40、IL-23、IFN-γ PBMC-6:TNF-a、IL-6、IL-1 and IFN-γ iPSCミクログリア:CXCL8(IL-8)、CCL11(エオタキシン)、CCL17(TARC)、CCL2(MCP-1 )、CCL5(RANTES)、CCL3(MIP-1α)、CXCL9(MIG)、CXCL5(ENA-78)、CCL20( MIP-3α)、CXCL1(GROα)、CCL4(MIP-1β)VILIP-1、sTREM-2、BDNF、TGF-β1 VEGF、IL-6、sTREM-1、β-NGF、IL-18、TNF-α、sRAGE、CX3CL1( PBMC and monocytes: IL-4, IL-2, IL-1β, TNF-α (TNFSF2), CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN-γ, IL-12p70, TGF-β1 (Free Active), CXCL8 (IL-8). PBMC-4 donor: MCP-1, IL-1β, TNF-α, IL-6, IFN-γ, IL12p70, IL-4, IL-10, CCL17, IL12p40, IL-23, TARC Microglia: IL-4, IL-2, IL-1β, TNF-α (TNFSF2), CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN-γ, IL-12p70, TGF-β1 (Free Active), CXCL8 (IL-8).

[0190] Microglia cell culture Microglia cells (iCell microglia, FUJIFILM Cellular Dynamics, Inc., catalog number: R1131) were cultured according to the manufacturer's recommended protocol. Before seeding, the culture surface was prepared by coating with 0.1 mg / mL poly-D-lysine solution (Gibco) and incubated overnight to enhance cell adhesion. Subsequently, the cells were plated in 24-well plates at a density of 120,000 cells per well. After a 4-day rest period, the microglia cells underwent a stimulation protocol. This involved pretreatment of the cells with 50 ng / mL LPS (Sigma Aldrich, catalog number: L2637-10Mg) for 24 hours, followed by the addition of specific antibodies for 20 minutes, 6 hours, and 24 hours. Measuring phosphorylated protein at 20 minutes is generally considered ideal due to the transient nature of phosphorylation. As a result, changes in protein levels are observed using later time points such as 6 hours and 24 hours.

[0191] Western blotting For protein extraction, the inventors used RIPA cell yokai buffer consisting of 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 1% SDS, supplemented with Sigma's protease and phosphatase inhibitors. Automated Western blotting services were provided by RayBiotech, Inc. (Peachtree Corners, GA USA). A sample concentration of 0.1 mg / mL was loaded into an automated capillary electrophoresis system. For protein analysis, the following antibodies were used: Phospho Syk (Tyr525 / 526) (Cell Signaling, catalog no. 12710T) and total Syk (Cell Signaling, catalog no. 113198S). As a loading control, a gluceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody provided from RayBiotech's service library was used.

[0192] Culture of microglia cells for RNA sequencing Microglia cells (iCell microglia, FUJIFILM Cellular Dynamics, Inc., catalog number: R1131) were cultured according to the manufacturer's recommended protocol. Before seeding, the culture surface was prepared by coating with 0.1 mg / mL poly-D-lysine solution (Gibco) and incubated overnight to enhance cell adhesion. Subsequently, the cells were plated in 24-well plates at a density of 120,000 cells per well. After a 4-day resting period, the microglia cells were subjected to a stimulation protocol that mimicked an inflammatory state. This involved pre-treating cells with 10 ng / mL lipopolysaccharide (LPS, Sigma Aldrich, catalog number: L2637-10Mg) and 20 ng / mL interferon-gamma (IFN-γ, PeproTech, Inc., catalog number: 300-02-20μg) for 24 hours, followed by the addition of ATL_0005802, ATL_0005854, ATL_0005909, or ATL_0005338 (isotype) for a total of 48 hours of exposure to LPS / IFNγ(LI). This was compared to a state mimicking healthy cells pre-treated with the vehicle alone. After treatment, the cells were directly lysed in the wells by aspirating the cell culture medium and adding 400 μL of TRIzol reagent (Invitrogen, catalog number: 15596026). The TRIzol reagent ensured that the surface of each well was uniformly covered. The plate was gently agitated to promote complete mixing and effective cell lysis. The cell lysates, containing both the TRIzol reagent and cell contents, were then carefully transferred using a pipette into individually labeled tubes. These tubes were immediately placed on dry ice for rapid cooling, a crucial step in maintaining RNA integrity. Subsequently, the samples were stored at -80°C for long-term storage until RNA extraction was performed.

[0193] RNA extraction, library preparation, and sequencing. The collected samples were processed for next-generation sequencing as follows: Total RNA was extracted from TRIzol cryopreserved samples according to the manufacturer's recommendations. RNA integrity was assessed by Tapestation (Agilent Technologies, Palo Alto, CA, USA), and concentration was assessed by Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA, USA). Low-quality samples were excluded. Poly(A)mRNA was enriched with Oligod (T) beads according to the manufacturer's recommendations.

[0194] RNA libraries were prepared using the Twist RNA Library Prep kit and Twist UMI adapter system (Twist Bioscience) according to the manufacturer's recommendations, employing ERCC spike-in. Library size was assessed using a Tapestation, concentration using a Qubit 2.0 fluorometer, and final quantification was performed by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Multiplexed samples were sequenced using a single index with 2 × 150 bp reads on an Ilumina HiSeq4000 sequencer.

[0195] Bioinformatics analysis Quality control, sequence alignment, and quantification were performed as follows. Briefly, raw data quality was evaluated using FastQC (Andrews, 2010). Sequence reads were trimmed using fastp v.0.23.1 (Chen et al., 2018). UMI-based deduplication was performed using fastp v.0.23.1 (Chen et al., 2018). The trimmed and deduplication-free reads were mapped to Homo sapiens GRCh38.p7 containing the ERCC gene using STAR Aligner v.2.5.2b (Dobin et al., 2013). The number of unique gene hits was calculated using "featureCounts" from Subread v.1.5.2 (Liao et al., 2014).

[0196] Differential expression analysis was performed using R with DESeq2 v1.38.3 (Love et al. 2014). The Wald test was used to generate p-values ​​and log2 factor changes. Genes with a BH-adjusted p-value < 0.05 and an absolute log2 factor change > 1 were considered to be differentially expressed for each comparison of interest (i.e., vehicle + anti-CD33 antibody compared to vehicle + isotype and LI + anti-CD33 antibody compared to LI + isotype). Principal component analysis (PCA) was calculated using the DESeq2 "plotPCA" function (DESEq2 "varianceStabilizingTransformation" function) for variance-stabilizing counts, using the top 25% of the most variable genes. Volcano and PCA plots were plotted using the R library ggplot2 v.3.4.3 (cran.r-project.org / web / packages / ggplot2 / index.html).

[0197] Pathway Analysis: Gene sets were retrieved from the molecular signature database MSigDb (Broad Institute, www.gsea-msigdb.org), which includes curated Hallmark collections and REACTOME pathways, using the R package msigdbr v.7.5.1 (Dolgalev, 2022). Gene set enrichment analysis (Subramanian et al. 2005) was performed for each comparison of interest using fgsea v1.24.0 (Korotkevich, 2019) with corresponding gene lists ranked using Wald statistics. Gene sets / pathways with -log10 (adjusted p-value) > 10 were considered enriched in one condition compared to another. The direction of change was assessed using the normalized enrichment score (NE). The plots were created using fgsea, ggplot2, and the "plotEnrichment" function from ggpattern v.1.1.0-0 (cran.r-project.org / web / packages / ggpattern / readme / README.html).

[0198] Establishment of a quadruple culture system A quadruple culture system was established to reproduce the physiological state of the central nervous system using induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (Fujifilm, catalog number: R1061), GABAergic neurons (Fujifilm, catalog number: R1013), astrocytes (Fujifilm, catalog number: R1092), and microglia (Fujifilm, catalog number: R1131). These cells were cultured under standard conditions until they reached a confluence suitable for the experiment. Six days after the establishment of the quadruple culture system, the cells were pretreated with 10 ng / mL lipopolysaccharide (LPS, Sigma Aldrich, catalog number: L2637-10Mg) and 20 ng / mL interferon-gamma (IFNγ, PeproTech, Inc., catalog number: 300-02-20μg). The following day, the quadruple culture system was exposed to CD33 antibody, ATL_5802, or ATL_5338 (isotype control antibody). Each antibody was used at a concentration of 50 μg / mL. The cultures were incubated with these antibodies for a further 24 hours. The supernatant was collected on day 8 of the culture. Interleukin-6 (IL-6), GFAP, IP-10, and MCP-1 in the collected samples were quantified using an enzyme-linked immunosorbent assay (ELISA) service provided by RayBiotech, Inc. (Peachtree Corners, GA, USA). All ELISA assays were performed according to the manufacturer's protocol.

[0199] In vitro fused Retrogenix The screening was conducted by Charles River (UK) using their proprietary Retrogenix cell microarray technology (see www.criver.com / products-services / discovery-services / screening-and-profiling-assays / retrogenix-cell-microarray-technology?region=3696).

[0200] Example 1 - Convergence Analysis Disease-specific antibody sequences can be identified using convergent sequence clusters derived from the antibody repertoire of resilient populations. The inventors sought to identify candidate protective antibodies from individuals resilient to Alzheimer's disease (AD) in a prospective cohort (European Consortium for the Prevention of Alzheimer's Disease, ep-ad.org).

[0201] Pathophysiological changes in Alzheimer's disease can be detected in individuals decades before the onset of cognitive symptoms of dementia, i.e., in the preclinical stage of the disease. For example, the concentration of Aβ42 in cerebrospinal fluid (CSF) decreases with early disease progression compared to healthy controls, while the concentration of CSF phosphate tau increases. The "A / T / N" classification scheme (Jack et al., 2016) defines a convenient binary label for describing such biomarkers, where "A" refers to the value of a β-amyloid biomarker (amyloid positron emission tomography (PET) or CSF Aβ42), "T" refers to the value of a tau biomarker (CSF phosphate tau or tau PET), and "N" refers to a biomarker of neurodegeneration or nerve damage ([18F]-fluorodeoxyglucose-PET, structural MRI, or CSF total tau) (Jack et al. 2016). A level of less than 1,000 pg / mL is considered amyloid-positive, and a level greater than 27 pg / mL is considered pTau-positive (Amft et al., 2022; Blennow et al., 2019).

[0202] From a total cohort of 2,096 participants, 127, including 37 resilient AD subjects, were selected for convergent analysis based on data and sample availability (plasma and PBMC, as well as pTau and Aβ42 values in CSF and plasma), and resilience was defined by the absence of cognitive impairment using the A+T-CSF biomarker, as well as the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), Mini-Mental State Examination (MMSE), and Clinical Dementia Rating Sum of Boxes (CDR-SOB) (Duff et al., 2008; Balsis et al., 2015). Sequencing of the antibody repertoires of these resilient individuals revealed heavy chain variable (VH) sequences that converged between two resilient individuals, one of whom further carried the heterozygous ApoE3 / E4 genotype and had an additional predisposition to AD (Michaelson et al., 2014). Figure 1 shows the number of clone types, i.e., clusters of BCRs with high sequence similarity, identified in these two resilient individuals, indicating that 64 shared clone types existed between these two resilient individuals. A representative sequence from one of the 64 shared clone types was shown to bind to CD33.

[0203] Next, VH sequences identified in resilient individuals were paired with VL sequences using a transformer-based model that included an encoder-decoder model trained on a corpus of paired VH-VL sequences. Further details on how such models are trained and can be used are provided in International Publication No. 2022 / 223451 (which is incorporated herein by reference in its entirety). The trained model takes a VH sequence as input and produces a single complementary VL sequence as output. The resulting antibody, ATL_5082 (also referred herein as ATL_0005082), was included in an ELISA screening for binding to a panel of neurodegenerative linkage targets. The panel consisted of 14 targets, including BA-1-14 biotinylated peptide, BA-1-42 biotinylated peptide, tau-352, tau-441, NF-L (neurofilament light chain), Trem2, galectin-3, CD33 (Siglec 3), ApoE4, LRP8 (ApoE4 receptor), HTT exon 1 48Q (mutant HTT exon 1), transthyretin, baculovirus particles, and lysozyme. The antibody was shown to bind to CD33 (Figure 2). This indicates a correlation between CD33 reactivity and resilience to Alzheimer's disease (AD) in individuals resilient to AD in the EP-AD cohort.

[0204] Interestingly, serum and plasma autoreactivity to CD33 (defined as a z-score cutoff of 0.5 in serum ELISA) was observed in 21 subjects (25 samples), including one "person aged 110 or older" who was over 100 years old and had self-reported good cognitive function (a score of at least 28 out of 30 on the Mini-Mental State Examination, indicating a cognitively healthy individual (Holstege et al., 2018)) (Figure 3).

[0205] In addition, many cancers, particularly myeloid malignancies such as acute myeloid leukemia and lymphomas, are characterized by high levels of CD33 expression. Furthermore, it has been shown that the expression of CD33-related siglecs on tumor-associated macrophages supports cancer progression, and that inhibitory siglecs may inhibit immune cell activation, thereby suggesting siglec targeting as a possible immune checkpoint inhibitory therapeutic strategy (Stanczak et al., 2022). Therefore, the antibodies identified and described herein may be useful not only in the treatment and / or prevention of neurodegenerative diseases but also in cancer.

[0206] Example 2 - Deep mining by phage display of the repertoire from CD33-reactive targets Based on the results of Example 1, the inventors initiated screening serum and plasma libraries from "individuals aged 110 years or older" to identify potentially protective anti-CD33 antibodies. ELISA for CD33 was performed using plasma from a cohort of "individuals aged 110 years or older" (www.100plus.nl), a subset of the samples shown in Figure 3. All 12 plasma samples available from the cohort were analyzed, and the sample with the highest ELISA signal for CD33 (Figure 4A) and the lowest signal for the lysozyme control antigen (Figure 4B) was selected for phage library generation (subject SU_0000877 in Figure 4).

[0207] A phage library of scFv molecules presented on M13 phages was generated by cloning the heavy chain variable region (VH) repertoire of the target SU_0000877 into a sublibrary of phagemide vectors containing light chain variable region (VL) sequences from healthy donors. Size: 1.4 × 10⁻⁶ 8 A phage display library of clones was generated, and phages were generated for phage display selection.

[0208] To isolate antibodies that bind to the CD33 protein, 2–3 rounds of phage display panning selection were performed against the CD33 protein. Over 100 clones were analyzed by Sanger sequencing, and full-length sequences were selected for phage ELISA.

[0209] Next, full-length clones derived from phage display selection were analyzed by phage ELISA using CD33 antigen and lysozyme controls (Figure 5). Nine clones with a positive ELISA signal and unique sequences were selected for IgG conversion and further testing.

[0210] Nine antibodies derived from phage display panning selection were chosen for IgG conversion and generated as IgG1 with LALA mutations in the Fc region. IgG1 variants containing L234A / L235A substitutions reduced binding to IgG Fc receptors FcγRI, FcγRII, and FcγRIII, as well as complement component C1q, thereby reducing Fc-mediated toxicity (Lund et al. 1991). Next, these were tested by single-point ELISA against a panel of antigens including CD33 and related proteins (other sialic acid-binding immunoglobin-like lectins, siglecs): human recombinant CD33, cynomolgus monkey / rhesus monkey CD33, human recombinant CD33 C domain, mouse CD33, recombinant human siglec-6 / CD327 Fc chimera, recombinant human siglec-7 / CD328 Fc chimera, recombinant human siglec-8 Fc chimera, and recombinant human siglec-9 Fc chimera, together with the comparative antibodies ATL_5909 (also known as Alector's "AL003"), ATL_4828 (also known as gemtuzumab), and ATL_5503 (AL003 precursor).

[0211] Nine test antibodies (see data for ATL_5802, 5810, 5803, 5808, 5854, 5807, 5809, and 5853 in Table 2A) were found to bind to fully recombinant human CD33 (rhCD33-His) but not to the C2 domain (see also Figure 6, where single-point ELISA signals are shown normalized to isotype control = 100, and absorbance was measured at 450 nm). The antibodies further showed minimal species cross-reactivity in mouse (mCD33-His) and cynomolgus monkey (cCD33-His). Importantly, the tested antibodies showed good binding to CD33 but did not bind to the associated Siglec family proteins (Siglec-6-F, Siglec-7-F, Siglec-8-F, and Siglec-9-F), demonstrating selectivity for CD33. Dynamic data are shown in Table 2C. These data indicate that the comparative antibody has a higher affinity for CD33, which is primarily driven by a lower Koff (e.g., the on-rate of ATL5909 is about twice as high as that of ATL5802, but the off-rate is two orders of magnitude lower). This indicates that the antibodies of this disclosure dissociate more quickly from the target, which may be underpinned by a lower peripheral degradation rate (see Example 3 below). As shown in Examples 4 and 5, the antibodies described herein exhibit a very large effect on phagocytosis (even greater than the comparative antibody), demonstrating a complex relationship between the mechanism of action of the antibody and the dynamics of target binding.

[0212] [Table 19]

[0213] In conclusion, deep mining of the repertoire derived from CD33-reactive targets using phage display yielded a panel of eight selective CD33-binding antibodies, all of which bind to complete CD33 but not to the C2 domain.

[0214] Of these eight antibodies, two were selected for further investigation, antibody ATL5802 and antibody ATL5810, based on their CDR3H diversity, strong binding, epitope mapping (see Example 8), and different internalization behavior (see Example 3). These were then further investigated in Examples 3-7.

[0215] Epitope mapping was performed on these antibodies and the comparison antibodies as described in Example 8. This revealed that seven of these eight antibodies (5810, 5853, 5807, 5809, 5802, 5854, and 5805; see sequences in Table 2B) showed similar binding profiles indicating similar epitopes, and these respective binding profiles differed from those of comparison antibodies 4828 and 5909, as well as antibody 5803. Therefore, antibody 5803 was not investigated further.

[0216] [Table 20]

[0217] [Table 21]

[0218] Example 3 - Cell Binding and Internalization CD33 is known to be expressed at high levels on the cell membranes of peripheral blood cells such as monocytes. In addition to binding to recombinant CD33 as shown in Example 2, it was desirable to select a CD33 antibody that binds to endogenously expressed CD33 but minimizes the internalization of the antibody-CD33 complex by peripheral blood cells, and therefore avoids the acceleration of antibody clearance by this peripheral clearance mechanism. Accordingly, we tested the internalization of ATL5802 and ATL5810 by monocytes.

[0219] Figure 7A (see also Table 3) shows CD33 depletion in human monocytes 5 hours after the addition of ATL_5802 or ATL5810, compared to the comparative antibodies ATL5909 and ATL4828. ATL_5802 showed minimal CD33 depletion and therefore minimal internalization in monocytes, similar to the isotype control antibodies. ATL_5810 showed some CD33 depletion, but less than the prior art antibodies ATL5909 or ATL4828. Figures 7B and 7C show the depletion levels of the CD33 V domain and C domain (Figures 7B and 7C, respectively) in human CD14+CD16+ or CD14+CD16- cells (lower and upper panels in each figure) after the addition of specific antibodies in the range of 1 nM to 40 nM. Figures 7D and 7E show the areas under the curves in Figures 7B and 7C for CD14+CD16-monocytes (see also Table 3 below) and CD14+CD16+monocytes, respectively. In particular, all test antibodies in this disclosure showed less CD33 internalization than the comparative antibody 5909. ATL_5802 showed very little CD33 internalization (see Figure 7A), and ATL_5854 showed moderate CD33 internalization (see Figure 7B). It is worth noting that since all test antibodies bind to the V domain, the change in domain V to MFI can be due to both competition with the detection antibody and CD33 depletion. In contrast, assays using detection of domain C MFI faithfully represent CD33 depletion. We present separate data for both classical (CD14+CD16-) monocytes and non-classical / intermediate (CD14+CD16+) monocytes, demonstrating that the CD33 depletion behavior of the antibody is similar in both monocyte subsets.

[0220] [Table 22]

[0221] The comparative antibody ATL5909 has a non-linear PK profile (Ward et al., 2021), which is thought to be due to internalization into CD33-positive peripheral blood monocytes. In other words, ATL5909 rapidly internalizes by forming a complex with CD33, depleting CD33 on the cell surface. While this may be desirable at disease sites (e.g., brain tissue), this property leads to antibody precipitation in the periphery, where the antibody is internalized and degraded within monocytes. The fact that CD33 depletion in the periphery is not observed or is reduced with ATL5802 and ATL5810 (and all other test antibodies in this disclosure) suggests that these antibodies are not internalized by peripheral blood cells to the same extent as ATL5909, and therefore can be expected to have an improved PK profile compared to ATL5909, resulting in improved antibody availability in desired target regions such as the brain.

[0222] Example 4 - Phagocytosis Assay Microglia are endogenous innate immune cells in the central nervous system (CNS) and are essential for CNS health. Phagocytosis of toxic proteins such as amyloid-beta is a crucial function of microglia. The phagocytic pathway is suppressed by CD33 signaling, and CD33 is upregulated in microglia in AD patients, associated with cognitive decline. Therefore, it was interesting to test whether identified anti-CD33 antibodies could mitigate this CD33-mediated suppression of phagocytosis.

[0223] To investigate the effects of anti-CD33 antibodies ATL5802 and 5810, an in vitro assay was devised using induced pluripotent stem cell (iPSC)-derived microglia to measure amyloid-beta phagocytosis (see Methods). The effects of the anti-CD33 antibodies were not observed in basal quiescent iPSC microglia (data not shown). However, this was not considered representative of in vivo microglia, particularly microglia in neurodegenerative conditions. Therefore, induced pluripotent stem cell (iPSC)-derived microglia were first stimulated with LPS to induce an inflammatory response representing the in vivo pathological state of these cells in neurodegeneration. Following this step, they were incubated with a panel of anti-CD33 antibodies. Subsequently, cells were treated with pH-sensitive pHrodo red-labeled amyloid-beta, and phagocytosis was measured by monitoring the red fluorescence signal in each well. Increased intracellular levels of the red dye compared to isotype controls indicated increased phagocytosis (Figure 8A).

[0224] Figure 8B shows the results of assays using anti-CD33 antibodies ATL5802 and ATL5810. The graph in Figure 8B shows that incubation with ATL5802 increased phagocytosis in iPSC microglia compared to the prior art antibody ATL5909 and isotype controls. The results in Figure 8C support these results in repeated experiments with ATL5802, demonstrating that increased phagocytosis in inflammatory iPSC microglia in the presence of the antibody (ATL5802) of this disclosure is repeatable.

[0225] Next, the experiment was repeated using three other anti-CD33 antibodies, ATL_5853, ATL_5854, and ATL_6044 (germlined ATL_5802; see Example 7 below). The results of this experiment are shown in Figure 8D. The graph in Figure 8D shows that all of the test antibodies in this disclosure, namely antibodies ATL_5802, ATL_5854, and ATL_5853, increase phagocytosis compared to the prior art antibody ATL_5909 or the isotype control antibody.

[0226] Figures 8E and 8F further demonstrate that ATL_5802 induces greater phagocytosis compared to the agonist TREM2 antibody currently in clinical trials for the treatment of AD (ATL6166=Alector AL002, ATL6167=Denali DNL919, ATL6170=Vigil VGL101). TREM2 is a microglia-activating signaling receptor that supports microglial cell survival by promoting phagocytosis of Aβ plaques (McQuade et al., 2020).

[0227] To test the effect of anti-CD33 antibodies on phagocytic activity in human peripheral bone marrow cells (e.g., monocytes) known to express high levels of CD33, the above phagocytic assay was performed ex vivo using human peripheral blood mononuclear cells (PBMCs) derived from healthy donors. The ex vivo bone marrow assay measures the level of phagocytosis in bone marrow cells (CD14+) by flow cytometry using pH-staining dye (pHrodo)-labeled S. aureus (Figure 9A).

[0228] Figures 9B and 9C show that ATL5802 significantly increased phagocytosis in human bone marrow cells compared to the comparative antibodies ATL5909 (Alector) and ATL4828 (gemtuzumab). The results for ATL5810 suggested that the antibody likely induced phagocytosis, but the results were not statistically significant (compared to isotype controls) due to large variability in the cell assay.

[0229] In conclusion, these data suggest that the antibodies of this disclosure (as demonstrated particularly by antibodies ATL5802, ATL_5853, ATL_5854, and ATL_5810) are likely to enhance phagocytosis in a range of CD33-expressing human cells, including PBMCs and inflammatory iPSC-derived microglia. The data further demonstrate that the antibodies of this disclosure (as demonstrated particularly by antibodies ATL5802, ATL_5853, and ATL_5854) enhance phagocytosis in inflammatory human iPSC-derived microglia in vitro and in human bone marrow cells ex vivo.

[0230] Example 5 - In vivo testing of antibodies in CD34+ humanized mice The pharmacodynamic properties of ATL5802 and ATL5810 were modified by adding human hematopoietic stem cells that produce a humanized immune system (including a mixture of mouse and human monocytes) that can be targeted by an anti-CD33 antibody, resulting in a humanized mouse model called CD34. + Further in vivo testing was performed in the NSG(trademark) mouse model. It was necessary to test these antibodies in a humanized mouse model because substantial species differences exist between mice and humans due to the evolutionary divergence between human CD33 and non-primate CD33 in CD33 expression patterns and ligand recognition; therefore, the biology of mCD33 is not functionally related to the biology of hCD33 (Brinkman-Van der Linden, et al., 2003; Cao et al, 2009). In any case, the anti-CD33 antibodies described herein do not exhibit species cross-reactivity with mouse CD33, and therefore in vivo testing is only possible in humanized animal models.

[0231] First, the level of CD33 on humanized myeloid cells was tested as a way to assess in vivo internalization of CD33 (Figure 10). Figure 10 shows that injection of ATL5802 and ATL5810 minimized the decrease in CD33 on myeloid cells 24 hours after injection compared with the prior art antibody ATL5909. Figure 10 shows the mean fluorescence intensity (MFI) of CD45+CD14+CD33+ cells by flow cytometry. This indicates that peripheral drug loss is minimized with ATL5802 compared with ATL5909.

[0232] Figure 12 shows that 24 hours after intravenous injection of ATL5802, the expression level of CD33 in human CD45+ cells in the brain decreased in a dose-dependent manner, supporting the binding of ATL5802 to its target in the brain.

[0233] Furthermore, Figure 13 shows that intraperitoneal injection of ATL5802 in CD34+ mice compared to injection of ATL5909 in human peripheral bone marrow cells (CD45 + CD14 + CD11b + The study showed that pHrodo-labeled S. aureus cells were increased in phagocytosis, and this effect appears to be dose-dependent. The relatively large variability is due to the small number of human cells used in the experiment. Nevertheless, the trend observed in vivo supports the in vitro data shown above.

[0234] In summary, the ATL5802 is CD34 + These data indicate that ATL5802 exhibits minimal peripheral internalization in humanized mice, while showing increased target binding and phagocytosis in the brain. Therefore, ATL5802 was further characterized in terms of antibody expression and development potential.

[0235] Example 6 - Antibody Expression and Development Potential Feasibility studies were conducted to confirm that the identified antibodies were stable and therefore suitable for further optimization.

[0236] Initial baseline values ​​were calculated for all 10 antibodies selected in Example 2. These showed high monomer levels, as well as good thermal stability and cIEF values ​​(Table 4).

[0237] Protein aggregation during antibody storage must be kept to a minimum as it can trigger immunogenic reactions. Antibody purity and aggregation were evaluated using size exclusion chromatography (SEC-HPLC) after storage under a series of conditions including 2 weeks at -80°C, 2 weeks at 40°C, overnight shaking, 3 freeze-thaw cycles (3×FT), and low pH maintenance (Figure 14). Figure 14 shows that ATL5802 exhibits excellent stability, with very little change in monomer profile under these forced degradation conditions, which is further reflected in the 99% monomer values ​​in Table 4. Similar results were obtained for the other candidate antibodies identified in Example 2, all of which showed acceptable stability with respect to monomer percentage, with 5802 exhibiting particularly good thermal stability (Tm1).

[0238] [Table 23]

[0239] Furthermore, target binding of ATL5802 after storage under forced degradation conditions was tested using ELISA (Figure 15). ATL5802 showed consistent binding to CD33 under all conditions, indicating that the binding strength is not affected by temperature changes and freeze-thaw cycles.

[0240] In conclusion, all antibodies, particularly ATL5802, demonstrated excellent stability, and therefore ATL5802 was retained for further development.

[0241] Example 7 - Germlineization in the framework region The lead antibody ATL5802 has eight framework mutations from the corresponding germline sequence. Six are located in the VH region (IMGT positions 1, 6, 20, 40, 72, 85), and two are located in the VH region (IMGT positions 2 and 8). To test which of these could be reversed to the germline sequence without losing binding, nine variants of the original antibody were created and their binding to CD33 was tested by ELISA (Table 5). The following variants were tested: ATL6040, in which all mutations were reversed to the germline sequence; and ATL6041-ATL6048, in which all but one mutation were reversed and all but one were reversed. The variants were generated as IgG1 (LALA Fc mutation).

[0242] [Table 24]

[0243] All antibodies were generated and tested by titration ELISA using CD33 antigen and negative control antigen. Only one antibody, ATL6044, showed binding equivalent to the parent, suggesting that liability removal or a change to framework position G40A affects binding. This indicates that the A at position 40 in the VH of ATL5802 made a positive contribution to binding to the antigen, but all other positions can revert to germline. It should be noted that this does not indicate that strict adhesion to any framework sequence is necessary, as all antibodies still bind to the target regardless of the introduced germline mutation. Rather, this indicates that maintaining the A at position 40 of ATL5802 is particularly advantageous.

[0244] Example 8 - Epitope Mapping Figure 11B shows the results of the epitope mapping process schematically shown in Figure 11A. The antibodies were grouped into different epitope bins using the binding profiles of each antibody against various CD33 variants. Epitope bin 1 contained ATL4828 (comparative antibody gemtuzumab) and was characterized by complete loss of binding to variants P1 and P6+1, and approximately 50% loss of binding to variant P4. In contrast, epitope bin 2 contained ATL5909 (comparative antibody AL003) and was characterized by loss of binding to variants P2 and P2+5, but no effect on other variants. Bottles 3 and 4 were highly relevant and contained seven antibodies (5810, 5853, 5807, 5809, 5802, 5854, and 5808), which commonly exhibited loss of binding to variants P2 and P2+5, a 50% loss of binding to P4, and increased binding to P1 and P6+1. Bottle 5 contained antibody ATL5803 (which was not further characterized herein). This data suggests that other antibodies with the same epitopes as the variants that showed phagocytic enhancement activity (ATL5802, ATL5854, and ATL5853) are likely to exhibit therapeutic efficacy. The data further suggest that the different binding kinetics between the antibodies of this disclosure and the comparison antibodies shown in Example 2 are highlighted by different epitopes, and that the therapeutic efficacy of these antibodies may be improved by reduced peripheral depletion.

[0245] Finally, the epitope mapping data supports the existence of a different mechanism of action for the antibody in this disclosure compared to the prior art antibody, and is more consistent with the reduced internalization behavior observed in Example 3 compared to the comparative antibody. In particular, the epitope mapping data indicates that the antibody described herein can interfere with the binding of sialic acid to CD33.

[0246] In summary, these data indicate that the anti-CD33 antibodies described herein, particularly ATL5802, ATL5854, and ATL5853, may be therapeutic in the treatment and / or prevention of neurodegenerative diseases such as Alzheimer's disease (AD) and CD33-expressing cancers such as myeloid leukemia. This was further validated by extensive in vitro and in vivo studies described in later examples.

[0247] Figure 16 summarizes the sequences of the antibodies described herein, aligned using the IMGT antibody numbering rules. The sequences shown in Figure 16 are also summarized in Tables 5 (VH) and 6 (VL). In Tables 5 and 6, the antibodies of this disclosure are shown in bold. "Complete" refers to the complete sequence in VH (Table 5) or VL (Table 6).

[0248] [Table 25]

[0249] [Table 26]

[0250] Example 9 - Mouse pK test The antibodies described herein are specific to human CD33 and do not significantly cross-react with mouse CD33. Therefore, to evaluate the in vivo pharmacokinetics (PK) of the antibody ATL_5802, a transgenic mouse line was used in which exons 1-3 of the mouse Cd33 gene encoding the extracellular domain were replaced with human CD33 exons 1-3. Human CD33 expression was previously validated and characterized in this mouse model. Antibodies were administered intraperitoneally at a dose of 1 mg / kg, and serum human IgG1 levels were assessed at various time points after a single dose of human IgG1 antibody, ATL_5802, or ATL_5909.

[0251] The results of this pK test are shown in Figure 17. Figure 17A shows the serum levels of ATL_5802 and ATL_5909 at 4 hours, 24 hours, and 144 hours (7 days). Figure 17B shows the quantification of total antibody concentration over time (measured by area under the curve, AUC), revealing a significant increase in serum levels of ATL_5802 compared to ATL_5909.

[0252] These data indicate that ATL_5802 exhibits superior peripheral exposure compared to ATL_5909, suggesting that ATL_5802 has lower peripheral clearance than the comparison antibody.

[0253] In summary, these data indicate that the anti-CD33 antibodies described herein exhibit excellent pharmacokinetic properties, thus further supporting their potential for therapeutic use.

[0254] Example 10 - Alzheimer's disease model To test whether anti-CD33 antibodies have the ability to attenuate the pathology of Alzheimer's disease, the effect of ATL_5802 was tested in a xenograft mouse model of Alzheimer's disease. Here, human NL-GF mutation-carrying amyloid precursor protein (APP) mice (APP) NL-G-F Human microglia progenitor cells derived from iPSCs were xenotransplanted into mice (named "mice," Saito et al., 2014) as described in Fattorelli et al., 2021, Mancuso et al., 2019, and Mancuso et al., 2022. The transplanted cells matured through transcription to resemble ex vivo human primary microglia, and after 3 months, the mice exhibited signs of Alzheimer's disease pathology. Therefore, this mouse model can be used to investigate the ecology of human microglia in relation to the pathology of Alzheimer's disease.

[0255] Mice aged 4 months were administered either ATL_5802 or an isotype control antibody (ATL_5338) at a dose of 40 mg / kg via intravenous injection (IP) once a week for 3 months. Subsequently, methoxy-X04, a fluorescent dye that crosses the blood-brain barrier and binds to Aβ, was injected via IP. Microglia were then isolated using flow cytometry (cells were sorted using FAC and gated with hCD45+), and their phagocytic activity was evaluated by quantifying the percentage of methoxy-X04-positive human microglia.

[0256] The results of this experiment are shown in Figure 18. Mice treated with ATL_5802 showed a significant increase in the percentage of methoxy-X04+ microglia compared to control mice treated with the isotype, as measured by flow cytometry. These results indicate that ATL_5802 increases the phagocytosis of amyloid-β by human microglia in vivo in AppNL-GF mice xenografted with iPSC-derived human microglial progenitor cells.

[0257] In summary, these data indicate that ATL_5802 alleviates the pathology of Alzheimer's disease in vivo, at least by increasing the clearance of neurotoxic Aβ through microglial phagocytosis.

[0258] Example 11 - Tau phagocytosis by microglia Abnormal aggregation of pathological (mutant) tau protein into fibers, or so-called neurofibrillary entanglement, is characteristic of several neurodegenerative conditions. In humans and transgenic mice, mutations in P301 (e.g., P301S and P301L) in tau result in tau pathology with the diffusion of aggregated tau (Strang et al. 2018). Therefore, effectively clearing these aggregates from neurons is crucial for preventing and / or mitigating tau pathology. As described in Example 4, microglia are essential for the clearance of toxic extracellular proteins and for maintaining cellular health in the central nervous system.

[0259] To test whether the antibodies described herein can reduce the spread of mutant (P301S) aggregated tau, the uptake of these aggregates by microglia with an inflammatory phenotype was measured. Briefly, LPS-pretreated iPSC microglia were incubated with anti-CD33 antibody ATL5802, comparative anti-CD33 antibody ATL_5909, anti-TREM2 antibody ATL_6170, or isotype control antibody (ATL_5338), and the uptake of pHrodo-labeled mutant tau aggregates was subsequently measured using live-cell imaging.

[0260] The results of this experiment are shown in Figure 19. Figure 19A shows the increase in phagocytosis of labeled tau aggregates, measured as the total red area per well over time, after treatment with the anti-CD33 antibody ATL_5802 or the anti-TREM2 antibody ATL_6170, compared to isotype control antibodies using live cell imaging. In contrast, the comparative anti-CD33 antibody ATL_5909 tended to decrease tau uptake. Figure 19B shows the quantification of the total AUC area per well under each condition.

[0261] In summary, these results indicate that ATL_5802 increases phagocytosis of toxic tau aggregates by inflammatory microglia, thus contributing to the effective clearance of these aggregates. This is expected to reduce tau-related pathology and reduce and / or prevent (further) neurodegeneration.

[0262] Example 12 - Cytokine release from various cells CD33 is expressed not only on microglia but also on peripheral bone marrow cells such as monocytes. Modulation of these immune cells can lead to systemic release of cytokines, potentially resulting in undesirable consequences and safety concerns.

[0263] To test whether the anti-CD33 antibodies described herein induce undesirable cytokine release, iPSC microglia, PBMCs, and isolated monocytes from multiple donors were treated with isotype control antibodies or ATL5802, as shown in Table 7, and the cell media were assayed for a variety of cytokines, chemokines, and inflammatory mediators.

[0264] [Table 27]

[0265] [Table 28]

[0266] These results indicate that treatment with ATL5802 did not induce immune-mediated cytokine release. In particular, ATL5802 did not induce MCP-1 release by untreated microglia 24 hours after treatment at 100 mg / ml or 10 mg / ml (actually showing slightly lower levels than the isotype control), and this benefit was not shared by the comparative anti-CD33 antibody tested.

[0267] Treatment of iPSC microglia with LPS is known to induce the production of inflammatory cytokines MCP-1 and IL-6. To test whether the anti-CD33 antibody ATL_5802 can reduce the levels of these pro-inflammatory cytokines, microglia were treated with LPS and then with ATL_5802 or an isotype control antibody. After 24 hours, the culture medium was collected and cytokine levels were measured using a multiplex immunoassay.

[0268] The results are shown in Figure 20, demonstrating that treatment with ATL_5802 significantly reduces LPS-induced production of IL-6 (Figure 20A) and MCP-1 (Figure 20B) by iPSC microglia in response to LPS. This suggests that the anti-CD33 antibody described herein may have an anti-inflammatory effect in human iPSC microglia. This differs significantly from the isotype control antibody and the comparative anti-CD33 antibody ATL_5909.

[0269] MCP-1 recruits immune cells to inflammatory sites and is involved in the development of neuroinflammation and AD, autoimmune diseases (e.g., arthritis), and arteriosclerosis. Therefore, it is highly beneficial that the antibody of this disclosure does not induce cytokine release, including MCP-1, which is a beneficial effect not present in the comparative anti-CD33 antibody ATL_5909.

[0270] In summary, these data indicate that the anti-CD33 antibodies described herein do not induce any unwanted cytokine release, are safe for human use, and possess potent anti-inflammatory effects in vitro.

[0271] Example 13 - Changes in intracellular signaling in microglial cells after addition of ATL_5802 Spleen tyrosine kinase (SYK) is an important intracellular regulator in microglial activation and phagocytosis. It is particularly important in neurodegenerative diseases, promoting the clearance of toxic protein aggregates (Ennerfelt et al. 2022). In this example, we investigated changes in intracellular signaling after CD33 binding, focusing specifically on the phosphorylation state of Syk (pSYK) under inflammatory conditions.

[0272] Activation of ITAM receptors expressed on microglial cells, such as TREM2, leads to the recruitment and phosphorylation of ITAM-containing adapter molecules, followed by the recruitment of Syk. Upon activation, phosphorylation of SYK leads to upregulation of cytokine production, phagocytosis, and ROS production in microglia (Linnartz and Neumann, 2013). TREM2 is antagonistized by inhibitory signaling from ITAM receptors such as CD33. CD33 signaling via the ITAM domain recruits phosphatases that counter ITAM signaling by dephosphorylating the ITAM domain and ITAM-related kinases such as SYK (Huang et al., 2003).

[0273] Here, the inventors sought to investigate whether the anti-CD33 antibody described herein can regulate intracellular signaling after CD33 binding to ITAM, and in particular whether the antibody described herein can mitigate CD33-mediated suppression of ITAM receptor signaling.

[0274] To test this, the inventors measured the phosphorylation state of Syk (pSYK) under inflammatory conditions. Increased phosphorylation would indicate increased activation of the ITAM signaling pathway and therefore inhibition of the inhibitory effect of CD33.

[0275] Figure 21 shows the results of Western blotting of microglial cells treated with LPS and ATL_5802 or isotype control antibodies. This reveals a significant increase in SYK phosphorylation after ATL_5802 treatment, while the overall level of SYK protein remained stable. These findings suggest that ATL_5802 modulates signaling pathways, potentially leading to derepression of the TREM2 signaling axis and enhancement of TREM2 signaling, as evidenced by the increased levels of pSYK. This highlights the potential therapeutic value of targeting these specific pathways.

[0276] Next, the inventors investigated whether anti-CD33 antibodies described herein, such as ATL_5802, are effective against the purinergic receptor P2RY12. P2RY12 is a member of the P2 purinergic family of receptors and is a seven-transmembrane G protein-coupled receptor that responds to ADP / ATP and is involved in chemotaxis and phagocytosis. P2RY12 promotes microglial chemotaxis to sites characterized by necrotic or apoptotic cells, which is important for maintaining brain health and the response to injury or inflammation (Walker et al., 2020). The chemotactic response mediated by P2RY12 is a fundamental aspect of the microglial surveillance mechanism, enabling early detection and response to brain injury. Activation of P2RY12 enhances the phagocytic capacity of microglia and promotes the clearance of apoptotic cells, amyloid-beta plaques, and other debris associated with neurodegenerative diseases. Through its involvement in phagocytosis, P2RY12 contributes to the recovery from inflammation and the prevention of further tissue damage. By regulating P2RY12 activity, it may be possible to enhance beneficial microglial function while reducing harmful inflammation, thus protecting against neurodegeneration and supporting brain health.

[0277] To test whether ATL_5802 can enhance P2RY12 expression in microglia, Western blotting was performed using LPS-stimulated iPSC-induced microglia treated with ATL_5802 or an isotype control antibody.

[0278] Figure 22 shows the results of Western blotting of P2RY12 in microglia 6 hours after the addition of ATL_5802 or isotype control antibodies. ATL_5802 enhances the protein expression of the P2RY12 receptor, suggesting that the antibody may modulate intracellular signaling pathways in microglia that are crucial for responding to injury and inflammation. In particular, the data in Figure 22 show that the non-inflammatory control state has high P2RY12 expression, while the inflammatory control state has low P2RY12 expression. In contrast, after treatment with ATL_5802, the non-inflammatory state has higher P2RY12 expression compared to the isotype control (indicating microglia that have returned to a surveillance state characterized by high P2RY12 expression, in which the microglia do not actively participate in the inflammatory response but are ready to respond to new injury or threat), while the inflammatory state shows higher P2RY12 expression compared to the isotype control.

[0279] This indicates that ATL_5802 has the ability to enhance the protein expression of the P2RY12 receptor, which is indicative of inactivated microglia. This effect guides microglia to specific destinations, playing a crucial role in maintaining brain health and regulating responses to injury or inflammation. This alteration in signaling dynamics may result in a more effective immune response by microglia, a key factor in slowing the progression of neurodegenerative diseases.

[0280] In summary, these data indicate that the anti-CD33 antibodies described herein can drive intracellular signaling pathways in microglia that are crucial for their effective functions, such as phagocytosis, cytokine release, and chemotaxis, while inhibiting signaling pathways involved in microglial activation and functional suppression. This alteration in signaling dynamics may contribute to a more effective immune response, which is a key factor in slowing the progression of neurodegenerative diseases.

[0281] Example 14 - RNA sequencing of microglia derived from iPSCs To understand the transcriptional changes resulting from treatment with anti-CD33 antibodies in both healthy and inflammatory-mimicking conditions, genome-wide RNA sequencing was performed on iPSC-derived microglia. Briefly, iPSC-derived microglia were stimulated with a vehicle or LPS / interferon-gamma (LI) to mimic an inflammatory state, followed by treatment with ATL_5802, ATL_5909, ATL_5854, or isotype control antibodies, and then RNA sequencing was performed as described in the "Materials and Methods" section above.

[0282] The analysis results after RNA sequencing are shown in Figure 23. The PCA plot shown in Figure 23A shows that processing with ATL_5802 results in greater changes in gene expression than those driven by ATL_5854, ATL_5909, or ATL_5338 (isotype).

[0283] In LI or vehicle-stimulated cells, a comparison of expression profiles of samples treated with any anti-CD33 antibody and isotype control reveals several differentially expressed genes (DEGs) (Figures 23B-23C). As shown in Figures 23B and 23C, the largest number of differentially expressed genes were detected in iPSC-derived microglia treated with vehicle + ATL_5802 compared to those treated with vehicle + isotype control, demonstrating the effect of ATL_5802 in healthy cells. A total of 194 genes were upregulated with vehicle + ATL_5802 compared to vehicle + isotype control (see Figure 23D).

[0284] Interestingly, pathway analysis revealed statistically significant downregulation of several gene sets involved in oxidative phosphorylation (OXPHOS) in inflammatory microglia upon treatment with ATL_5802 compared to LI and isotype-treated samples (Figures 23E-23F). This result clarifies the differentiated mechanism of action of ATL_0005802, which suppresses the transcription of oxidative phosphorylation components and respiratory electron transport components.

[0285] Oxidative phosphorylation (OXPHOS) in microglia enables efficient ATP production under homeostatic conditions, while activation under inflammatory conditions, similar to the Warburg effect seen in tumors, relies on PI3K / mTOR / HIF1a-dependent aerobic glycolysis for faster ATP production (Laura et al., 2020). This, in turn, leads to microglia adopting a phagocytic phenotype. Microglia continuously monitor the brain parenchyma to detect neuronal damage and changes in homeostatic processes, and defects in cellular metabolism, including both glycolysis and OXPHOS, are associated with neurodegenerative diseases such as Alzheimer's disease (Baik et al., 2019). Enhancement of anaerobic glycolytic metabolism has been shown to restore phagocytic activity in microglia and improve cognitive impairment in a mouse model of AD (Baik et al., 2019). While these experiments only show a decrease in OXPHOS (and not a corresponding glycolytic boost), inhibition of CD33 inhibits SHIP1 / 2, negatively modulates PI3K, activates mTOR, and therefore boosts glycolysis, which is expected to have beneficial cognitive improvements.

[0286] Therefore, the result that ATL_5802 reduces the transcription of OXPHOS and respiratory electron transport components suggests that ATL_5802 may promote anaerobic glycolysis, thereby weakening the OXPHOS pathway. When OXPHOS is reduced, microglial cells that are subjected to CD33 antagonism by ATL_5802 have a beneficial reduction in the release of reactive oxygen species, a byproduct of OXPHOS.

[0287] In summary, these data suggest that ATL_5802 can modulate cellular metabolic pathways, ultimately leading to improved cognitive function in neurodegenerative diseases.

[0288] Example 15 - Quadruple culture using cortical neurons, astrocytes, and microglia Next, the inventors developed a human neuronal cell culture called the "CNS Quadruple Culture Platform" (Figure 24A). This platform integrates diverse cell types, particularly glutamatergic and GABAergic neurons, along with microglia and astrocytes, to accurately reproduce the complex intercellular dynamics of the human brain in a controlled in vitro environment. This in vitro approach allows for a deeper understanding of the interactions between various brain cell types and serves as an important experimental medium for human-related systems to evaluate the efficacy of ATL_5802 against markers of inflammation and astrocytes. To evaluate this, lipopolysaccharide (LPS) and interferon-gamma (INFγ) were introduced into the system to induce an inflammatory response and simulate the state of neuroinflammation, followed by analysis of inflammation, astrocytes, glial dysfunction, and microglial activation.

[0289] ATL_5802 significantly reduced LPS / INFγ-induced interleukin-6 (IL-6) levels compared to isotype controls (Figure 24B). Furthermore, ATL_5802 resulted in a significant reduction in GFAP, an astrocyte marker (Figure 24C), further suggesting the overall anti-inflammatory effect of ATL_5802.

[0290] Interferon-gamma-inducible protein 10 (IP-10) is a marker of microglial activation and mediates the initiation of neuroinflammatory processes. MCP-1 is a marker of glial dysfunction, and high CSF MCP-1 levels are associated with brain atrophy and cognitive impairment in AD. Therefore, these are important chemokines known to be elevated in AD. ATL_5802 significantly reduced both IP-10 (Figure 24D) and MCP-1 levels (Figure 24E) compared to isotype controls.

[0291] These results provide further evidence supporting the therapeutic potential of ATL_5802 in reducing neuroinflammation and thereby protecting against brain atrophy and cognitive impairment in neurodegenerative diseases.

[0292] Example 16 - Selectivity of ATL_5802 To evaluate the selectivity of ATL_5802, we screened its binding to fixed HEK293 cells expressing 6105 individual human proteins and an additional 400 human heterodimers. The screening library included human proteins linked to the cell membrane, secreted, and cell surface. The screening was performed without informing the operators of CD33, the target of ATL_5802. The test antibody ATL_5802 showed a single significant specific interaction with CD33 in both fixed and live cell microarrays (see Table 8). These data support that ATL_5802 is selective for CD33 and therefore unlikely to cross-react with other targets.

[0293] [Table 29]

[0294] References To provide a more detailed description and disclosure of the present invention and the art to which it pertains, several publications are referenced. A complete citation of these references is provided below. Each of these references in its entirety is incorporated herein. For standard molecular biology techniques, see Sambrook, J., Russel, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Amft, M., Ortner, M., Eichenlaub, U. Et al. The cerebrospinal fluid biomarker ratio Aβ42 / 40 identifies amyloid positron emission tomography positivity better than Aβ42 alone in a heterogeneous memory clinic cohort. Alz Res Therapy 14, 60 (2022). Andrews, S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data: www.bioinformatics.babraham.ac.uk / projects / fastqc / Baik, Sung Hoon et al.“A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer’s Disease.”Cell metabolism vol. 30, 3 (2019): 493-507. Balsis, Steve et al.“How Do Scores on the ADAS-Cog, MMSE, and CDR-SOB Correspond?.”The Clinical neuropsychologist vol. 29, 7 (2015): 1002-9. Blennow, K., Shaw, L.M., Stomrud, E. et al. Predicting clinical decline and conversion to Alzheimer’s disease or dementia using novel Elecsys Aβ(1-42), pTau and tTau CSF immunoassays. Sci Rep 9, 19024 (2019). Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. (1997) 3: 730. Brinkman-Van der Linden EC, Angata T, Reynolds SA, Powell LD, Hedrick SM, Varki A. CD33 / Siglec-3 binding specificity, expression pattern, and consequences of gene deletion in mice. Mol Cell Biol. 2003 Jun; 23 (12): 4199-206. Cao, H., de Bono, B., Belov, K. et al. Comparative genomics indicates the mammalian CD33rSiglec locus evolved by an ancient large-scale inverse duplication and suggests all Siglecs share a common ancestral region. Immunogenetics 61, 401-417 (2009). Cao, Huan, and Paul R Crocker.“Evolution of CD33-related siglecs: regulating host immune functions and escaping pathogen exploitation?.”Immunology vol. 132, 1 (2011): 18-26. Chan G, et al.: CD33 modulates TREM2: convergence of Alzheimer loci. Nat Neurosci 2015; 18: 1556-1558 Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884-i890, Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1; 29 (1): 15-21. Dolgalev I (2022). _msigdbr: MSigDB Gene Sets for Multiple Organisms in a Tidy Data Format_. R package version 7.5.1, <https: / / igordot.github.io / msigdbr / >. Donnelly, Louise E, and Peter J Barnes.“Defective phagocytosis in airways disease.”Chest vol. 141, 4 (2012): 1055-1062. Duff, Kevin et al.“Utility of the RBANS in detecting cognitive impairment associated with Alzheimer’s disease: sensitivity, specificity, and positive and negative predictive powers.”Archives of clinical neuropsychology: the official journal of the National Academy of Neuropsychologists vol. 23, 5 (2008): 603-12. Ennerfelt H, et al. SYK coordinates neuroprotective microglial responses in neurodegenerative disease. Cell. 2022 Oct 27; 185 (22): 4135-4152.e22. Eskandari-Sedighi, Ghazaleh et al.“CD33 isoforms in microglia and Alzheimer’s disease: Friend and foe.”Molecular aspects of medicine vol. 90 (2023): 101111. Fattorelli N, Martinez-Muriana A, Wolfs L, Geric I, De Strooper B, Mancuso R. Stem-cell-derived human microglia transplanted into mouse brain to study human disease. Nat Protoc. 2021 Feb; 16 (2): 1013-1033. Golde, Todd E.“Disease-Modifying Therapies for Alzheimer’s Disease: More Questions than Answers.”Neurotherapeutics: the journal of the American Society for Experimental NeuroTherapeutics vol. 19, 1 (2022): 209-227. Griciuc, Ana et al.“Alzheimer’s disease risk gene CD33 inhibits microglial uptake of amyloid beta.”Neuron vol. 78, 4 (2013): 631-43. Holstege H, Beker N, Dijkstra T, et al. The 100-plus Study of cognitively healthy centenarians: rationale, design and cohort description. Eur J Epidemiol. 2018; 33 (12): 1229-1249. Hollingworth, Paul et al.“Common variants at ABCA7, MS4A6A / MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer’s disease.”Nature genetics vol. 43, 5 (2011): 429-35. Huang, Zhen-Yu et al.“The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcgamma receptors FcgammaRIIB and FcgammaRIIA.”Journal of leukocyte biology vol. 73, 6 (2003): 823-9. Jack CR Jr, Bennett DA, Blennow K, et al. A / T / N: An unbiased descriptive classification scheme for Alzheimer disease biomarkers. Neurology. 2016; 87 (5): 539-547. Jung et al. Selection for improved protein stability by phage display. Journal Molecular Biology 294, 163-180 (1999). Jurcic, 2012. What Happened to Anti-CD33 Therapy for Acute Myeloid Leukemia? Curr Hematol Malig Rep (2012) 7: 65-73. Knopman, D.S., Amieva, H., Petersen, R.C. et al. Alzheimer disease. Nat Rev Dis Primers 7, 33 (2021). G. Korotkevich, V. Sukhov, A. Sergushichev. Fast gene set enrichment analysis. bioRxiv (2019), doi: 10.1101 / 060012 Lauro, Clotilde, and Cristina Limatola.“Metabolic Reprograming of Microglia in the Regulation of the Innate Inflammatory Response.”Frontiers in immunology vol. 11 493. 20 Mar. 2020. Laszlo et al. The past and future of CD33 as therapeutic target in acute myeloid leukemia. Blood Rev. 2014 Jul. Liao Y, Smyth GK and Shi W (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30 (7): 923-30. Linnartz, Bettina, and Harald Neumann.“Microglial activatory (immunoreceptor tyrosine-based activation motif)- and inhibitory (immunoreceptor tyrosine-based inhibition motif)-signaling receptors for recognition of the neuronal glycocalyx.”Glia vol. 61, 1 (2013): 37-46. Love, M.I., Huber, W., Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Genome Biology 15 (12): 550 (2014) Lund J, Winter G, Jones PT, Pound JD, Tanaka T, Walker MR et al. Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol. 1991; 147: 2657-2662 Mancuso R, et al. Stem-cell-derived human microglia transplanted in mouse brain to study human disease. Nat Neurosci. 2019 Dec; 22 (12): 2111-2116. Manscuso R., et al. A multi-pronged human microglia response to Alzheimer’s disease Ab pathology. Preprint at bioRxiv. 2022. doi: 10.1101 / 2022.07.07.499139. Michaelson DM. APOE ε4: the most prevalent yet understudied risk factor for Alzheimer’s disease. Alzheimers Dement. 2014; 10 (6): 861-868. McQuade, A., Kang, Y.J., Hasselmann, J. Et al. Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer’s disease. Nat Commun 11, 5370 (2020). Mancuso, Renzo et al.“Stem-cell-derived human microglia transplanted in mouse brain to study human disease.”Nature neuroscience vol. 22, 12 (2019) Siddiqui SS, Springer SA, Verhagen A, Sundaramurthy V, Alisson-Silva F, Jiang W, Ghosh P, Varki A: The Alzheimer’s disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool. J Biol Chem 2017; 292: 15312-15320. Stanczak MA et al. Targeting cancer glycosylation repolarizes tumor-associated macrophages allowing effective immune checkpoint blockade. Science Translational Medicine 14, eabj1270 (2022) 2 November 2022. Stanczak, Michal A, and Heinz Laeubli.“Siglec receptors as new immune checkpoints in cancer.”Molecular aspects of medicine vol. 90 (2023): 101112. Strang KH, et al. Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies. J Biol Chem. 2018 Feb 16; 293 (7): 2408-2421. doi: 10.1074 / jbc.M117.815357. Epub 2017 Dec 19. Erratum in: J Biol Chem. 2018 Mar 23; 293 (12): 4579. Trinidad Hernandez-Caselles, et al., A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing, Journal of Leukocyte Biology, Volume 79, Issue 1, Jan 2006, Pages 46-58 Walker, D.G.; et al. Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer’s Disease Brains. Int. J. Mol. Sci. 2020, 21, 678. Ward et al., 2021. A phase I study of AL003 in Healthy Volunteers and Participants with Alzheimer’s Disease. Abstract P45 in“Abstract: Symposia, Conferences, Oral communications: 14 th Clinical Trials on Alzheimer’s Disease (CTAD) November 9-12, 2021.”The journal of prevention of Alzheimer’s disease vol. 8,S1 (2021): S2-S72. Varki A, Angata T: Siglecs - the major subfamily of I-type lectins. Glycobiology 2006; 16: 1R-27R. Vercauteren S, Zapf R, Sutherland H. Primitive AML progenitors from most CD34+ patients lack CD33 expression but progenitors from many CD34- AML patients express CD33. Cytotherapy (2007) 9: 194-204. Yu YJ, Atwal JK, Zhang Y, et al. Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci Transl Med. 2014; 6 (261): 261ra154. Zhao, Lingzhi.“CD33 in Alzheimer’s Disease - Biology, Pathogenesis, and Therapeutics: A Mini-Review.”Gerontology vol. 65, 4 (2019): 323-331.

Claims

1. An isolated antibody that specifically binds to the CD33 protein, wherein, compared to a control antibody, it increases the phagocytosis of cells expressing CD33, and / or antibody binding to human CD33 protein containing mutations at positions 20, 21, 22, and 24 is increased compared to antibody binding to human CD33 protein without such mutations.

2. Antibody binding to human CD33 proteins containing mutations at positions 20, 21, 22, 24, and 132 is increased compared to antibody binding to human CD33 proteins without such mutations, and / or Antibody binding to human CD33 proteins containing mutations at positions 47, 50, 51, and 52 is reduced compared to antibody binding to human CD33 proteins without such mutations, and / or The antibody does not bind to human CD33 protein containing mutations at positions 47, 50, 51, and 52, and / or Antibody binding to human CD33 proteins containing mutations at positions 47, 50, 51, 52, and 122 is reduced compared to antibody binding to human CD33 proteins without such mutations, and / or The antibody does not bind to human CD33 protein containing mutations at positions 47, 50, 51, 52 and 122, and / or The isolated antibody according to claim 1, wherein antibody binding to a human CD33 protein containing a mutation at position 83 is reduced compared to antibody binding to a human CD33 protein without the mutation.

3. The aforementioned mutation is a. N20R in 20th place, b. F21V in 21st place, c. W22R in 22nd place, d. Q24E in 24th place, e. I47V in 47th place, f. Y50H in 50th place, g. D51T in 51st place, h. K52R in 52nd place, i. Q83R in 83rd place, j. R122K in 122nd place, and k. P132T in 132nd place An isolated antibody according to claim 1 or 2, selected from the above.

4. The isolation antibody according to any one of claims 1 to 3, wherein the binding is measured using single-point ELISA, and / or the human CD33 protein comprises residues 18 to 232 of human CD33, and / or the human CD33 protein is CD33M2_ECD_18-232_WT.

5. a. Increased binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos1[P1] compared to proteins containing the sequence CD33M2_ECD_18-232_WT. b. Increased binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos1_MutPos6[P6+1] compared to proteins containing the sequence CD33M2_ECD_18-232_WT, c. Reduced binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos2[P2] compared to proteins containing the sequence CD33M2_ECD_18-232_WT. d. Compared to proteins containing the sequence CD33M2_ECD_18-232_WT, there is a decrease in binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos2_MutPos5[P2+5], and e. Reduced binding to proteins containing the sequence CD33M2_ECD_18-232_MutPos4[P4] compared to proteins containing the sequence CD33M2_ECD_18-232_WT. An isolated antibody according to any one of claims 1 to 4, comprising one or more or all of the above.

6. The isolated antibody according to any one of claims 1 to 5, wherein the comparative antibody is selected from an isotype control antibody, another CD33-conjugated antibody, and an antibody having a heavy chain variable sequence and a light chain variable sequence of ATL_5909, and / or the phagocytosis is evaluated by measuring a fluorescence signal associated with the uptake of labeled particles by imaging or flow cytometry.

7. The isolated antibody according to any one of claims 1 to 6, wherein the cells are monocytes or microglia, and optionally, the cells are human cells and / or iPSC-derived microglia, and / or cells stimulated with an inflammatory signal (e.g., LPS) prior to exposure to the antibody.

8. An isolated antibody according to any one of claims 1 to 7, which binds to a CD33 protein containing the V domain of CD33, and / or does not bind to a CD33 protein that does not contain the V domain of CD33, and / or does not bind to a CD33 protein having the sequence of protein CD33_human_ECD_C domain_His_007.

9. The following CD-R: CDRH1 containing one of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, CDRH2 containing one of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, CDRH3 containing any of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or A set of CDRs in which each CDR contains 0, 1, or 2 amino acid substitutions, compared to the set of CDRs described above. An isolated antibody according to any one of claims 1 to 8, comprising a heavy chain variable domain (VH) having a heavy chain variable domain.

10. The following CD-R: HCDR1_ATL_0005802, HCDR1_ATL_0005807, HCDR1_ATL_0005808: GYSFTSYW (Sequence ID 44), HCDR1_ATL_0005853:GYKFNNNW (Sequence ID 47), HCDR1_ATL_0005854:GYKFSNNW (Sequence ID 48), or Amino acid sequences having one or two mutations compared to the above sequences CDRH1 containing an amino acid sequence selected from, HCDR2_ATL_0005802, HCDR2_ATL_0005807, HCDR2_ATL_0005808, HCDR2_ATL_0005853, HCDR2_ATL_0005854, IYPGDSDT (Sequence ID 52), or Amino acid sequences having one or two mutations compared to the above sequences CDRH2 containing an amino acid sequence selected from, and HCDR3_ATL_0005802, ARPRGGEYYFDY (Sequence ID 59), HCDR3_ATL_0005853 ARHSGGLDGYTAAAALDY (Sequence ID 64), HCDR3_ATL_0005854 ATWGGSNWFVD (Sequence ID 65), or Amino acid sequences having one or two mutations compared to the above sequences CDRH3 containing an amino acid sequence selected from The isolated antibody according to claim 9, comprising a heavy chain variable domain (VH) having the above.

11. The following CD-R: A set of CDRs containing the sequence HCDR1_ATL_0005802, CRH2 containing the sequence HCDR2_ATL_0005802, and CDRH3 containing the sequence HCDR3_ATL_0005802, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to the sequence and / or one, two, or three mutations in CDRH3 compared to the sequence, or A set of CDRs containing the sequence HCDR1_ATL_0005853, CRH2 containing the sequence HCDR2_ATL_0005853, and CDRH3 containing the sequence HCDR3_ATL_0005853, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to the sequence and / or one, two, or three mutations in CDRH3 compared to the sequence, or A set of CDRs containing the sequence HCDR1_ATL_0005854, CRH2 containing the sequence HCDR2_ATL_0005854, and CDRH3 containing the sequence HCDR3_ATL_0005854, or a set of CDRs containing one or two mutations in CDRH1 and CDRH2 compared to the sequences and / or one, two, or three mutations in CDRH3 compared to the sequences. An isolated antibody according to claim 9 or 10, comprising a heavy chain variable domain (VH) having the above.

12. The following framework array: HFWR1 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, HFWR2 antibody ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 HFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and HFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or Framework arrays having 1 to 6 substitutions compared to the framework arrays above. An isolated antibody according to any one of claims 1 to 11, having a heavy chain variable domain (VH) having a heavy chain variable domain (VH).

13. An isolated antibody according to any one of claims 1 to 12, having a heavy chain variable domain (VH) having the framework sequence HFWR2 of ATL_0005802, and / or having a heavy chain variable domain (VH) having a framework sequence containing A at position 40 in standard IMGT numbering.

14. The isolated antibody according to claim 12 or 13, wherein the substitution in the framework sequence of the heavy chain variable domain is located at any position other than position 40 in standard IMGT numbering.

15. An isolated antibody according to any one of claims 1 to 14, having a heavy chain variable domain (VH) having the following framework sequence: HFWR1 of ATL_0005802, ATL_0005853 or ATL_0005854, HFWR2 of ATL_0005802, ATL_0005853 or ATL_0005854, HFWR3 of ATL_0005802, ATL_0005853 or ATL_0005854 and HFWR4 of ATL_0005802, ATL_0005853 or ATL_0005854.

16. Antibodies: Having a heavy chain variable domain (VH) containing a sequence that has at least 95% sequence identity with a sequence selected from the VH sequences of ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, ATL_0006040, ATL_0006041, ATL_0006042, ATL_0006043, ATL_0006044, ATL_0006045, ATL_0006046, ATL_0006047 and ATL_0006048, or a sequence selected from the VH sequences In comparison, the isolated antibody according to any one of claims 1 to 15 has a heavy chain variable domain (VH) containing a sequence having up to two mutations in each HCDR and up to three mutations in each framework region, and optionally, a sequence having at least 95% sequence identity with a sequence selected from the VH sequences of antibody ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence having up to two mutations in each HCDR and up to three mutations in each framework region compared to a sequence selected from the VH sequences.

17. (i) having a heavy chain variable domain (VH) containing CDRH1, CDRH2 and CDRH3 within the germline framework, provided that the 40th position in the standard IMGT numbering is A, and / or (ii) being an scFv antibody molecule, nanobody or whole antibody, and / or comprising an antibody constant region, and / or being a whole antibody, and / or being IgG1 or a variant thereof, optionally being an IgG1 variant L234A / L235A (LALA), according to any one of claims 1 to 16.

18. Binds to human CD33, optionally, as evaluated by ELISA, with an EC50 of up to 2e-08M or up to 3e-09M (e.g., binding of plated rhCD33), and / or An isolated antibody according to any one of claims 1 to 17, which binds more selectively to CD33 than one or more other Siglecs, optionally binds more selectively to CD33 than one or more (or all) of Siglecs-6, Siglecs-7, Siglecs-8, and Siglecs-9, and / or binds more selectively to human CD33 than one or more other homologs, optionally binds more selectively to human CD33 than mouse CD33 and cynomolgus monkey CD33.

19. An isolated antibody according to any one of claims 1 to 18, wherein, after 5 hours of incubation with the antibody, the CD33 on the cell surface of human monocytes is depleted by less than 50% or less than 80%, and / or, after 5 hours of incubation with the antibody, the CD33 on the cell surface of human monocytes is depleted to a lower degree than that of a comparative antibody of the same concentration.

20. The following CD-R: CDRL1 containing one of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, A CDRL2 containing any of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and A CDRL3 containing any of the amino acid sequences ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or A set of CDRs in which each CDR contains 0, 1, or 2 amino acid substitutions, compared to the set of CDRs described above. An isolated antibody according to any one of claims 1 to 19, comprising a light chain variable domain (VL) having a light chain variable domain.

21. The CDRL1, CDRL2, and CDRL3 of the VL domain are located within the germline framework, and / or the antibody is in the following framework sequence: LFWR1 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, LFWR2 antibody ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 LFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and LFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810, or A set of FWRs containing 1 to 6 amino acid substitutions, compared to the set of FWRs described above. The isolated antibody according to claim 20, having a light chain variable domain (VL) having the following characteristics.

22. Antibodies: Having a light chain variable domain (VL) containing a selected sequence that has at least 95% sequence identity with a sequence selected from the VL sequences of ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, ATL_0006040, ATL_0006041, ATL_0006042, ATL_0006043, ATL_0006044, ATL_0006045, ATL_0006046, ATL_0006047 and ATL_0006048, or selected from the VL sequences The isolated antibody according to any one of claims 1 to 21, having a light chain variable domain (VL) containing a sequence having up to two mutations in each LCDR and up to three mutations in each framework region compared to the sequence, and optionally having a sequence having at least 95% sequence identity with a sequence selected from the VL sequences of antibody ATL_0005802, ATL_0005853, or ATL_0005854, or a light chain variable domain (VL) containing a sequence having up to two mutations in each LCDR and up to three mutations in each framework region compared to a sequence selected from the VL sequences.

23. When administered to the target population, it exhibits lower peripheral clearance compared to the comparative anti-CD33 antibody and / or increases Aβ phagocytosis by microglial cells in vivo compared to the control and / or increases tau aggregate phagocytosis by microglia with an inflammatory phenotype (e.g., LPS-treated iPSC microglia) and / or increases tau aggregate phagocytosis by microglia with an inflammatory phenotype to a greater extent than the comparative anti-CD33 antibody and / or increases IL-6 and / or MCP-1 phagocytosis by microglia in vitro and / or in vivo. An isolated antibody according to any one of claims 1 to 22, which does not induce the release of one or more cytokines including and / or reduces the levels of IL-6 and / or MCP-1 released in vitro (e.g., LPS-treated human iPSC-derived microglia) and / or in vivo by microglia having an inflammatory phenotype, and / or reduces the inflammation-inducible release of one or more markers of inflammation in a human neuronal cell culture assay and / or in the central nervous system of a subject, wherein the one or more markers of inflammation are optionally selected from MCP-1, IP-10, GFAP and IL-6.

24. An isolated nucleic acid, vector, or set of vectors comprising a nucleotide sequence encoding an antibody or fragment thereof containing a VH or VL domain as described in any one of claims 1 to 23.

25. A host cell containing the vector described in claim 24, or a host cell transformed in vitro using the nucleic acid described in claim 24.

26. (i) Diseases related to microglial cell dysfunction, (ii) Optionally, tauopathy or frontotemporal dementia (FTD), Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), HIV-induced encephalitis, chronic traumatic encephalopathy (CTE), vascular dementia, prion diseases, Lewy body dementia, spinal muscular atrophy (SMA), motor neuron diseases (MND), such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar degeneration (SCA) types 1, 2, 6, 7 and 17, Machado Joseph disease (MJD / SCA3), dentatorubral-pallidoluysian atrophy (DRPLA), X-linked spinal muscular atrophy type 1 (SM An antibody according to any one of claims 1 to 23 for use in the treatment of a neurodegenerative disease or disorder selected from AX1 / SBMA), Anderson Fabry (X-linked Fabry disease), and DNAJB6 myopathy, wherein the neurodegenerative disease is optionally selected from FTD, AD, HD, and PD; (iii) optionally selected from cancer, which is associated with hypersialylation of AML or tumor cells and / or overexpression of CD33 by tumor cells; and (iv) an antibody according to any one of claims 1 to 23 for use in the treatment of a disease or disorder selected from a disease characterized by insufficient macrophage phagocytosis and / or macrophage dysfunction, which is optionally selected from COPD or IPF.

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