Anti-CD33 antibodies

Abstract

Isolated antibodies that specifically bind to a CD33 protein are described. Related nucleic acids, cells and therapeutic uses are also described.

Description

TECHNICAL FIELD

[0001] The present application relates to antibodies capable of binding to CD33, and in particular, but not exclusively, to novel therapeutic antibodies. Methods of using the anti-CD33 antibodies to treat neurodegenerative diseases and cancer are also described. BACKGROUND

[0002] CD33 (Siglec-3) is an inhibitory immune receptor and is a type I transmembrane protein belonging to the sialic acid-binding immunoglobulin-like lectin (Siglec) family, which is expressed on the cell surface of myeloid cells, monocytes, macrophages and microglia cells in the brain. CD33 is one of the top-ranking genes associated with risk of developing Alzheimer’s disease (AD) in genome-wide association studies. CD33 signalling in microglia is associated with AD pathology and CD33 expression is elevated in AD patients, which is thought to modulate microglial activation and inhibit clearance of beta amyloid (Zhao et al., 2019).

[0003] Alzheimer’s disease is the most common cause of dementia in the elderly (Zhao et al., 2019). New treatments for Alzheimer’s disease are being actively sought to change the course of the disease. Current candidates targeting beta-amyloid, Tau and innate immunity in the brain show pharmacodynamic effects on pathological mechanisms in some cases in clinical trials, but have so far not shown convincing disease modification in later-stage clinical trials (Golde et al., 2022).

[0004] Siglecs expressed on tumour infiltrating immune cells have been shown to influence anti-tumour immunity and represent potential targets for cancer immunotherapy (Stanczak & Laubli, 2023).

[0005] Antibodies capable of binding to CD33 have been developed. For example, US 2022 / 0162309 describes humanised antibodies that bind to human CD33, and the use of these antibodies in the treatment of Alzheimer’s disease, dementia, frontotemporal dementia, vascular dementia, mixed dementia, tauopathies, infections and cancer. However, clinical trials of the lead antibody AL003 described in US 2022 / 0162309 have been terminated. Humanised anti-CD33 antibodies (lintuzumab) have been shown to have modest activity in the treatment of acute myeloid leukemia (AML) (Jurcic, 2012).

[0006] There remains a need for additional antibodies that are capable of binding CD33 and modulating CD33 signaling in an effective manner. SUMMARY

[0007] The present application includes combinations of the described aspects and preferred features, except where such combinations are obviously not permitted or explicitly avoided.

[0008] According to a first aspect, the present application provides an isolated antibody that specifically binds to a CD33 protein, wherein the antibody increases phagocytosis of a cell expressing CD33 compared to a comparative antibody, and / or wherein the antibody increases binding to a human CD33 protein comprising a mutation at positions 20, 21, 22 and 24 compared to binding of the antibody to a human CD33 protein that does not have the mutation.

[0009] According to the present aspect, the antibody can comprise a heavy chain variable domain (VH) having CDRs comprising the amino acid sequence of CDRH1 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; CDRH2 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; CDRH3 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a CDR set comprising zero, one or two amino acid substitutions in each CDR compared to the above CDR sets.

[0010] According to a second aspect, the present application provides an isolated antibody that specifically binds to a CD33 protein, wherein the antibody comprises a heavy chain variable domain (VH) having a CDRH1 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; a CDRH2 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; a CDRH3 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a CDR set comprising zero, one or two amino acid substitutions in each CDR as compared to the above CDR sets.

[0011] In centenarians with superior cognitive abilities, antibodies having these CDRs were identified as CD33 binders, suggesting that they can have a protective effect in neurodegenerative diseases and can have a good therapeutic tolerance. These antibodies were identified as being less susceptible to peripheral degradation as compared to comparative antibodies, while being able to induce phagocytosis of myeloid and microglial cells to the same or higher extent as compared to comparative antibodies. These antibodies were identified as binding to a different epitope as compared to comparative antibodies.

[0012] According to the present aspect, the antibody can improve phagocytosis of CD33-expressing cells as compared to comparative antibodies, and / or the antibody can improve binding to a human CD33 protein comprising a mutation at positions 20, 21, 22 and 24 as compared to binding of the antibody to a human CD33 protein not having said mutation.

[0013] According to the first or second aspect, the antibody can have any one or more of the following optional features.

[0014] The antibody can bind to a human CD33 protein comprising a mutation at position 20, 21, 22, 24, and 132. The antibody can bind to a human CD33 protein comprising a mutation at position 47, 50, 51, and 52. The antibody can not bind to a human CD33 protein comprising a mutation at position 47, 50, 51, and 52. The antibody can bind to a human CD33 protein comprising a mutation at position 47, 50, 51, 52, and 122. The antibody can not bind to a human CD33 protein comprising a mutation at position 47, 50, 51, 52, and 122. The antibody can bind to a human CD33 protein comprising a mutation at position 83. The antibody can not bind to a human CD33 protein comprising a mutation at position 83.

[0015] The mutation can be selected from the group consisting of: 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] The binding can be measured as using a single point ELISA. The human CD33 protein can comprise residues 18 to 232 of human CD33. The human CD33 protein without the mutation can be CD33M2_ECD_18-232_WT.

[0017] The antibody can have one or more or all of: (i) increased binding to a protein comprising the sequence of CD33M2_ECD_18-232_MutPosl [Pl] as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, (ii) increased binding to a protein comprising the sequence of CD33M2_ECD_18-232_MutPosl_MutPos6 [P6+1] as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, (iii) decreased binding to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos2 [P2] as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, (iv) decreased binding to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos2_MutPos5 [P2+5] as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, and (v) decreased binding to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos4 [P4] as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT.

[0018] The antibody of the present disclosure can have a lower peripheral clearance when administered to a subject as compared to a comparative anti-CD33 antibody.

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

[0020] The antibody can be selected from the group consisting of: 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 assessed by measuring the fluorescence signal associated with uptake of labeled particles by imaging or flow cytometry. The cells can be monocytes or microglia cells. The cells can be human cells. The cells can be iPSC-derived microglia cells. The cells can be cells that have been stimulated with an inflammatory signal (e.g., LPS) prior to exposure to the antibody.

[0021] The antibody can bind to a CD33 protein comprising a V domain of CD33. The antibody can not bind to a CD33 protein that does not comprise a V domain of CD33. The antibody can not bind to a CD33 protein having the sequence of protein CD33_human_ECD_Cdomain_His_007.

[0022] The antibody can comprise a heavy chain variable domain (VH) having CDRs comprising:

[0023] a CDRH1 comprising an amino acid sequence selected from the group consisting of: ; or an amino acid sequence having 1 or 2 mutations compared to the above sequences;

[0024] a CDRH2 comprising an amino acid sequence selected from the group consisting of: or an amino acid sequence having 1 or 2 mutations compared to the above sequences; and

[0025] a CDRH3 comprising an amino acid sequence selected from the group consisting of:

[0026] or an amino acid sequence having 1 or 2 mutations compared to the above sequences.

[0027] The antibody can comprise a heavy chain variable domain (VH) having CDRs comprising: a CDRH1 comprising the sequence of HCDR1_ATL_0005802, a CRH2 comprising the sequence of HCDR2_ATL_0005802, and a CDRH3 comprising the sequence of HCDR3_ATL_0005802; or a CDR set comprising 1 or 2 mutations in the CDRH1 and CDRH2 compared to these sequences and / or comprising 1, 2, or 3 mutations in the CDRH3 compared to these sequences.

[0028] The antibody can comprise a heavy chain variable domain (VH) with CDRs comprising a CDRH1 comprising the sequence of HCDR1_ATL_0005853, a CRH2 comprising the sequence of HCDR2_ATL_0005853, and a CDRH3 comprising the sequence of HCDR3_ATL_0005853; or a CDR set comprising 1 or 2 mutations in the CDRH1 and CRH2 compared to these sequences and / or comprising 1, 2, or 3 mutations in the CDRH3 compared to these sequences.

[0029] The antibody can comprise a heavy chain variable domain (VH) with CDRs comprising a CDRH1 comprising the sequence of HCDR1_ATL_0005854, a CRH2 comprising the sequence of HCDR2_ATL_0005854, and a CDRH3 comprising the sequence of HCDR3_ATL_0005854; or a CDR set comprising 1 or 2 mutations in the CDRH1 and CRH2 compared to these sequences and / or comprising 1, 2, or 3 mutations in the CDRH3 compared to these sequences.

[0030] The antibody can have a heavy chain variable domain (VH) with a framework sequence of:

[0031] HFWR1 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810,

[0032] HFWR2 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810,

[0033] HFWR3 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and

[0034] HFWR4 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, or

[0035] a framework sequence having one to six substitutions compared to the above framework sequences.

[0036] The antibody can have a heavy chain variable domain (VH) with the framework sequence HFWR2 of ATL_0005802. The antibody can have a heavy chain variable domain (VH) with a framework sequence comprising 'A' at position 40 in standard IMGT numbering. The substitution in the framework sequence of the heavy chain variable domain can be at any position in the framework sequence other than position 40 in standard IMGT numbering. The antibody can have a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3 within a germline framework, provided that position 40 in standard IMGT numbering is A.

[0037] The antibody can have a heavy chain variable domain (VH) with the 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.

[0038] The antibody can have a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of 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. The antibody can have a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of the VH sequences described above. The antibody can have a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of the VH sequences of antibodies ATL_0005802, ATL_0005853 or ATL_0005854, or a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of the VH sequences.

[0039] The antibody can bind to human CD33. The antibody can bind to human CD33 with an EC50 of at most 2e-08 M or at most 3e-09 M, as assessed by ELISA (e.g., binding to plated rhCD33).

[0040] The antibody can deplete CD33 on the cell surface of human monocytes by less than 50% or less than 80% after 5 hours of incubation with the antibody. The antibody can deplete CD33 on the cell surface of human monocytes to a lesser extent compared to a comparator antibody at the same concentration after 5 hours of incubation with the antibody.

[0041] The antibody can selectively bind to CD33 relative to one or more other siglecs. The antibody can selectively bind to CD33 relative to one or more (or all) of siglec-6, siglec-7, siglec-8, and siglec-9. The antibody can selectively bind to human CD33 relative to other one or more homologs. The antibody can selectively bind to human CD33 relative to mouse CD33 and cyno CD33.

[0042] The antibody can be an scFv antibody molecule, a nanobody, or a whole antibody. The antibody can comprise an antibody constant region. The antibody can be a whole antibody. The antibody can be an IgG1 or a variant thereof. The antibody can be an IgG1 variant L234A / L235A (LALA).

[0043] The antibody can comprise a light chain variable domain (VL) having the CDRs:

[0044] CDRL1 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810,

[0045] CDRL2 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and

[0046] CDRL3 comprising the amino acid sequence of any one of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or

[0047] a CDR set comprising zero, one, or two amino acid substitutions in each CDR as compared to the CDR sets described above.

[0048] CDRL1, CDRL2, and CDRL3 of the VL domain can be within a germline framework.

[0049] The antibody can have a light chain variable domain (VL) with the following framework sequences:

[0050] LFWR1 of any one of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810;

[0051] LFWR2 of any one of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810;

[0052] LFWR3 of any one of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; and

[0053] LFWR4 of any one of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or

[0054] a FWR set containing one to six amino acid substitutions as compared to the FWR sets described above.

[0055] The antibody can have a light chain variable domain (VL) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of VL sequences of 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 can have a light chain variable domain (VL) comprising a sequence having at most 2 mutations in each LCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of the VL sequences. The antibody can have a light chain variable domain (VL) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of VL sequences of antibodies ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence having at most 2 mutations in each LCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of the VL sequences.

[0056] According to a third aspect, there is provided an isolated VH domain of an antibody according to any embodiment of the first or second aspect.

[0057] According to a fourth aspect, there is provided an isolated nucleic acid comprising a nucleotide sequence encoding an antibody according to any embodiment of the first or second aspect, including a VH or VL domain, or a fragment thereof.

[0058] Also described herein are vectors or sets of vectors comprising a nucleic acid according to the fourth aspect, host cells transformed in vitro with the nucleic acid, or host cells comprising the vectors or sets of vectors.

[0059] Also described herein are compositions comprising an antibody according to any embodiment of the first or second aspect (including an antibody VH domain or an antibody VL domain) and at least one additional component, which optionally comprises a pharmaceutically acceptable excipient, carrier, or vehicle.

[0060] Also described herein is an antibody according to any embodiment of the first or second aspect for use in the treatment of a disease or disorder. Also described herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antibody according to any embodiment of the first or second aspect. Also described herein is an antibody according to any embodiment of the first or second aspect for use in the manufacture of a medicament. The disease or disorder can be a disease associated with microglial dysfunction. The disease or disorder can be a neurodegenerative disease or disorder. The disease or disorder can be a tauopathy. The neurodegenerative disorder can be selected from the group consisting of frontotemporal dementia (FTD), Alzheimer’s disease (AD), Huntington’s Disease (HD), Parkinson’s disease (PD), amyotrophic laterals sclerosis (ALS), human immunodeficiency virus (HIV)-induced encephalitis, Chronic traumatic encephalopathy (CTE), vascular dementia, prion disease, Lewy body disease, Spinal muscular atrophy (SMA), Motor Neuron Disease (MND) such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxia (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral pallidoluysian atrophy (DRPLA), spinal bulbar muscular atrophy X-linked type 1 (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease), and DNAJB6 myopathy, optionally wherein the neurodegenerative disease is selected from the group consisting of FTD, AD, HD and PD. The disease or disorder can be a cancer.The cancer can be selected from AML or a cancer associated with tumor cell hypersyalilation and / or overexpression of CD33 by tumor cells. The disease or disorder can be a disease characterized by insufficient macrophage phagocytosis and / or macrophage dysfunction. For example, the disease can be COPD or IPF. The medicament can be for treating any of the above diseases or disorders. BRIEF DESCRIPTION OF DRAWINGS

[0061] Some embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings, in which:

[0062] Figure 1 Results of the process of identifying convergent BCR sequences in two individuals positive for amyloid but negative for tau and cognitively normal are shown schematically. The numbers shown are the number of clonotypes. These two individuals share 64 clonotypes and a representative sequence of one of the 64 shared clonotypes is shown to bind to CD33 (see below).

[0063] Figure 2 ELISA results showing that ATL_0005082 (also referred to herein as ATL_5082) binds to CD33 are shown. The isotype control is a commercially available human IgGl isotype control (Absolute Antibody; Ab00102-10.0 Anti-Fluorescein) and shows no binding to CD33. Lysozyme = negative control antigen. CD33 = binding to recombinant human CD33.

[0064] Figure 3 Results of a serum screen of a broad panel of individual donors by ELISA are shown. The graph shows the level of auto-reactivity to CD33 by multiple individuals by age. Z-scores were calculated as [raw signal - average signal] / standard deviation (SD).

[0065] Figure 4 shows the results of an ELISA to identify CD33 reactive plasma from a cohort of supercentenarians. Twelve plasma samples were tested for binding to recombinant human CD33 (A) and lysozyme control (B) by ELISA. The subject with the highest signal for CD33 (arrow, SU_0000877) was selected for phage display library generation.

[0066] Figure 5Results of a phage ELISA to identify single-chain variable fragment (scFv) clones that bind to the CD33 antigen are shown. Phage-displayed scFv derived from phage display selection were tested for binding to recombinant CD33 (pink circles) and a lysozyme control (black circles).

[0067] Figure 6 Results of an ELISA for binding of nine anti-CD33 antibodies identified by phage display and a comparative antibody (ATL_5503) to the full extracellular region of CD33 (transparent bar, "CD33 Human M17-H259_002_004") or the C2 domain (dark blue bar, "CD33 Human ECD C Domain His 007") are shown. Results are shown as signal relative to isotype.

[0068] Figure 7 shows quantification of CD33 levels on human monocytes 5 hours after incubation with the indicated antibodies at concentrations ranging from 1 nM to 40 nM, measured by flow cytometry. The plots in (A) show MFI (Median fluorescence intensity) on CD14+CD33+ cells plotted against antibody concentration. The (ATL) antibodies tested bind only the V domain of the CD33 receptor, whereas the CD33 antibodies used for flow cytometry detection are specific for the C domain. Reduction in median fluorescence intensity can be attributed to loss of cell membrane surface CD33 receptor, as there is no interference between detection antibody binding and test antibody binding. ATL5909 shows complete depletion of CD33 at all concentrations tested, whereas ATL5802 shows complete non-depletion of CD33 comparable to isotype control at all concentrations. ATL5810 shows an intermediate profile, with CD33 levels decreasing in a dose-dependent manner. (B) shows MFI of CD33 domain V on CD14+CD16+ (bottom) or CD14+CD16- (top) for the indicated antibodies. (C) shows MFI of CD33 domain C on CD14+CD16+ (bottom) or CD14+CD16- (top) for the indicated antibodies. It is noted that as all test antibodies bind in the V domain, changes in MFI of domain V can be attributed to both competition with detection antibodies and depletion of CD33. In contrast, using an assay that detects MFI in the C domain of CD33 truly represents CD33 depletion. (D) Area under the curve (AUC) of binding to CD33 C domain and V domain as a function of concentration (40 to 1 nM) (curves on bottom panels in B and C) for CD14+CD16- cells and CD14+CD16+ cells. Data are normalized to AUC of isotype control antibody (set to 100). (E) Area under the curve of binding to CD33 C domain and V domain as a function of concentration (40 to 1 nM) (curves in top panels in B and C).

[0069] Figure 8 shows the setup and results of an in vitro phagocytosis assay of microglia derived from inflammatory human iPSCs. (A) Schematically illustrates how the phagocytosis assay was performed. Microglia were incubated with LPS for 24 hours, followed by incubation with anti-CD33 antibody (or isotype control) for 24 hours, and then the uptake of pHrodo red-labeled β-amyloid protein was measured. pHrodo red only fluoresces red in the low pH environment of the cell lysosomes after phagocytosis of the labeled β-amyloid protein. (B) Shows the results of the phagocytosis assay that tested the effect of ATL5802 and ATL5810 on microglia phagocytosis compared to prior art antibody ATL5909, isotype control, and negative control (Aβ only), directly as the surface area / well of red cells over time under antibody incubation. (C) Shows the results of the phagocytosis assay in Figure B, expressed as the surface area (µm²) of red cells over time. 2 (Area under the curve) / well represents the uptake of pHrodo red-β amyloid protein by these cells via phagocytosis. (D) shows the results of a phagocytosis assay that tested the effects of ATL5802, ATL_5853, ATL_5854, and ATL_6044 on microglia phagocytosis compared to prior art antibody ATL5909 and isotype control. (E) shows the quantification of the phagocytosis assay results in Figures D and F, expressed as red cell surface area (µm²) over time after 18 hours. 2 Area under the curve (AUC) / well. The surface area of ​​red cells represents the uptake of pHrodo red β-amyloid in these cells and thus indicates the level of phagocytosis. Statistical analysis was performed using univariate Anova relative to isotype controls. * = p < 0.05; ** = p < …; *** = p < .., ns = not significant. At least ATL_5854 showed statistically significant differences in direct testing relative to ATL_5909 (univariate Anova). (F) shows the results of experiments comparing the phagocytosis of labeled β-amyloid in iPSC-derived microglia treated with anti-TREM2 antibodies (ATL6166; ATL 6167; and ATL6170) or ATL5802 (top and bottom plots show replicates of the same experiments).

[0070] Figure 9 shows the results of an in vitro phagocytosis assay using human whole blood. (A) shows myeloid cells of Staphylococcus aureus (S. aureus) labeled with pHrodo red. +Flow cytometry plots of CD14 cells indicating uptake of pHrodo in CD14+ cells. PHrodo Red only emits red fluorescence in the low pH environment of the lysosome after phagocytosis of labeled S. aureus has occurred. (B) shows pHrodo in CD14 + cells (indicative of phagocytosis) measured as MFI of pHrodo within CD14+ myeloid cells incubated with the indicated antibodies (ATL5802; ATL5810; ATL5909) or isotype control. Negative controls include wells with no pHrodo-labeled S. aureus and untreated cells. (C) shows phagocytosis of pHrodo-labeled S. aureus on freshly isolated CD14+ monocytes treated with 0.029 nM to 30 nM of ATL5802 (green shading), ATL5909 benchmark 1 (red), ATL4828 benchmark 2 (blue), and isotype control (yellow) (red object counts per well, corresponding to cells with pHrodo normalized to the maximum observed). Latrunculin A, which prevents actin polymerization in cells, was used as a control and was expected to completely block phagocytosis.

[0071] Figure 10 shows CD33 levels on peripheral myeloid cells in CD34+ 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 engrafted myeloid cells (defined as huCD45+ huCD14+ cells) were measured with an antibody specific for the C domain of CD33. Values reported are human CD45 + CD14 + fluorescence intensity (MFI) of CD33 on CD14

[0072] Figure 11 shows a schematic of mutations introduced to the CD33 V domain for epitope mapping of the antibodies described herein (A) and the corresponding results (B). As shown in A, variants of the CD33 extracellular domain were prepared as individual recombinant proteins. Nine IgGl antibodies were tested against wild type CD33 and all 7 CD33 variant proteins in a single point ELISA and binding was quantified relative to binding to the wt CD33 extracellular domain. Utilizing the binding profile of each antibody to the different CD33 variants, antibodies were divided into 4 different epitope bins as shown in B.

[0073] Figure 12 CD34+ cells are shown 24 hours after intraperitoneal (ip) injection of antibody (ATL5802; 5810; 5909 or isotype control). CD34 + CD33 levels on human CD11b+CD45+ cells in the brain of NSG mice. Cells were isolated from brain homogenate using anti-CD11b magnetic beads and subsequently stained for human and mouse anti-CD45. CD33 levels on human cells were assessed and median fluorescence intensity of CD33 staining is reported.

[0074] Figure 13 Flow cytometry results are shown for detection of CD34+ cells 24 hours after intraperitoneal injection of antibody (ATL5802; 5810; 5909 or isotype control) (incubated with labeled S. aureus for 3 hours ex vivo). + Human CD45+CD14 + CD11b + Median fluorescence intensity of pHrodo-labeled S. aureus in peripheral myeloid cells.

[0075] Figure 14 SEC-HPLC chromatograms of ATL5802 under specified forced degradation conditions (2 weeks at -80°C, 2 weeks at 40°C, shaking overnight, 3 freeze-thaw cycles (3x FT); and low pH maintenance) are shown.

[0076] Figure 15 Results of ELISA testing ATL5802 binding to recombinant human CD33 (rhCD33) under specified forced degradation conditions (2 weeks at -80°C, 2 weeks at 40°C, shaking overnight, 3 freeze-thaw cycles (3x FT); and low pH maintenance) are shown.

[0077] Figure 16 shows an alignment of variable chain sequences of selected anti-CD33 antibodies. Dashed lines indicate comparison antibodies.

[0078]

[0079] Figure 17 shows the results of an in vivo pK study in C57BL / 6-Cd33tm1 (CD33) mice. Groups of mice (n=3 / group) were administered 1 mg / kg of ATL-5802 or ATL-5909 intraperitoneally, and serum levels of hlgGl were measured at 4 hours, 24 hours, and 7 days after the single antibody administration. (A) shows a plot of serum levels of ATL_5802 or ATL_5909 at the indicated time points. (B) analysis of total antibody concentration over time (area under the curve; AUC). **p<0.01, unpaired t-test.

[0080] Figure 18 Figure 16 shows the results of an in vivo study in an animal model of Alzheimer’s disease testing ATL_5802 to alter the phagocytosis of amyloid-beta by human microglia (hMG) xenografted into the brains of mice. Mice were treated with 40 mg / kg of ATL_5802 (n=6) or isotype control ATL_5338 (n=7) weekly for 12 weeks. The percentage of hMG that were positive for Methoxy-X04, a fluorescent amyloid-beta tag, was measured by flow cytometry. The percentage of hMG positive for Methoxy-X04 was significantly higher in mice treated with ATL_5802 compared to mice treated with control ATL_5338, indicating that ATL_5802 increased the phagocytosis of amyloid-beta by hMG in vivo. *p<0.05, unpaired t-test. NL-G-F Figure 16 shows the results of an in vivo study in an animal model of Alzheimer’s disease testing ATL_5802 to alter the phagocytosis of amyloid-beta by human microglia (hMG) xenografted into the brains of mice. Mice were treated with 40 mg / kg of ATL_5802 (n=6) or isotype control ATL_5338 (n=7) weekly for 12 weeks. The percentage of hMG that were positive for Methoxy-X04, a fluorescent amyloid-beta tag, was measured by flow cytometry. The percentage of hMG positive for Methoxy-X04 was significantly higher in mice treated with ATL_5802 compared to mice treated with control ATL_5338, indicating that ATL_5802 increased the phagocytosis of amyloid-beta by hMG in vivo. *p<0.05, unpaired t-test.

[0081] Figure 19 shows the results of a real-time live cell imaging study using LPS-primed microglia and pHrodo-labeled Tau P301S aggregates. (A) Representative plots of total red (pHrodo. red) area per well versus elapsed time for antibodies ATL_5338 (isotype control); ATL_5802; ATL_5909; ATL_6170; and LPS alone. (B) Quantification of the area under the curve (AUC) for total red per well shown in (A).

[0082] Figure 20 shows the results of multiplex immunoassay in iPSC microglia treated with ATL_5802 or ATL_5909 after 6 hours of pre-treatment with LPS. (A) shows the concentration of IL-6 (pg / ml) in the supernatant of iPSC microglia treated with LPS and ATL 5802 or isotype control antibody. The data indicate that ATL_5802 reduces microglial IL-6 release in response to LPS. (B) shows the concentration of MCP-1 (pg / ml) in the supernatant of iPSC microglia treated with LPS and ATL 5802 or isotype control antibody. The data indicate that ATL_5802 reduces microglial MCP-1 release in response to LPS. Statistical analysis was performed using Student t-test, * = p<0.05; ** = p<0.01; *** = p<0.001, ns = not significant.

[0083] Figure 21 Figure 21 shows the results of western blot for phosphorylated SYK (pSYK) in cultured microglia stimulated with LPS and treated with ATL_5802. The figure shows western blots for pSYK (Tyr525 / 526) and total SYK in microglia 20 minutes after treatment with isotype control antibody or ATL_5802.

[0084] Figure 22 Figure 22 shows the results of western blot for P2RY12 in cultured microglia stimulated with LPS 6 hours after treatment with ATL_5802 or isotype control antibody. The housekeeper molecule GAPDH was used as a control.

[0085] Figure 23 shows results of gene expression analysis following RNA sequencing of iPSC-derived microglia stimulated with LPS / interferon gamma (LI) or vehicle and treated with ATL_5802, ATL_5854 or ATL_5909. (A) shows a principal component analysis (PCA) plot of RNAseq data showing principal component 1 (PC) vs. PC2, PCA calculated on 25% most variable genes. (B-C) show volcano plots of gene expression changes in iPSC-derived microglia treated with ATL_5802 relative to isotype control, under conditions of vehicle stimulation (B) or LPS / interferon gamma stimulation (C). Volcano plots show fold change (x-axis) vs. statistical significance, expressed as -loglO of adjusted p-value (y-axis). Colored points (points to the left of the leftmost vertical line, to the right of the rightmost vertical line and above the horizontal line) represent statistically significant differential expression according to selected thresholds; red (positive log2 fold change): upregulated upon treatment with ATL_5802, blue (negative log2 fold change): downregulated. Horizontal lines mark the significance threshold, vertical lines mark the log2 fold change threshold. (D) shows a bar plot with the number of differentially expressed genes and their direction of change (i.e. up- or down-regulation) as shown in (B) and (C) for all stimulations and antibody treatments. (E) bar plot shows Gene Set Enrichment analysis (GSEA) results for oxidative phosphorylation and respiratory electron transport gene set / pathway, with adjusted p-value (x-axis) and normalised enrichment score (NES) (colour intensity) (y-axis) and pathways (vertical boxes 1-4) for each comparison. Red (top bar in each set of 3 horizontal bars): upregulated, blue (middle and bottom bars in each set of 3 horizontal bars): downregulated. NES is represented by intensity of colour. Vertical lines mark the significance threshold. (F) representative GSEA plot for REACTOME pathway “respiratory electron transport” for all anti-CD33 antibodies compared to isotype control when stimulated with LPS / interferon gamma. These plots show that this pathway is significantly more enriched upon treatment with ATL_5802 (and to a lesser extent ATL_5854) compared to the comparison antibody ATL_5909.

[0086] Figure 24 shows the results of an analysis of the effect of antibodies of the present disclosure in human central nervous system (CNS) co-culture experiments. (A) shows a cartoon schematic explaining a human CNS quad culture comprising human iPSC-derived glutamatergic / GABAergic neurons, astrocytes and microglia, and fluorescent images showing cells in such a co-culture. (B-E) Levels of interleukin-6 (IL-6) (B); GFAP (C), IP-10 (D); and MCP-1 (E). Two-way ANOVA with Tukey’s multiple comparison test (n = 2-3 wells), *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001 compared to the isotype-treated LPS / IFNy group. The data in B-C demonstrate that ATL_5802 has an anti-inflammatory protective effect in human iPSC-derived CNS quad cultures, as evidenced by a decrease in interleukin-6 (IL-6) and a reduction in the astrocyte proliferation marker GFAP. The data in D-E demonstrate that treatment with ATL_5802 significantly reduces IP-10 (Interferon gamma-induced protein 10) and MCP-1 levels under LPS / IFNy conditions. Specific embodiments

[0087] Some aspects and embodiments of the present application will now be discussed with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are hereby incorporated by reference.

[0088] Disclosed herein are antibodies and fragments thereof that are capable of specifically binding to a CD33 protein or fragment thereof. As used herein, an antibody that is capable of "specifically binding" or "specifically binds" to a target is one that binds via an epitope recognition site to an epitope within the target. This is distinguished from non-specific binding, such as Fc-mediated binding, ionic and / or hydrophobic interactions. In other words, an antibody that specifically binds to a target recognizes and binds to a specific protein structure within the target, rather than recognizing and binding to protein in general.

[0089] The present disclosure relates to the antibodies described herein using the reference designation "ATL_000xxxx", "ATL_xxxx", or "xxxx", where "xxxx" is a four-digit reference number specific to the antibody described herein. All of the above designations can be used interchangeably to refer to the same antibody or portion thereof (e.g., the VH, VL, or portion thereof of the antibody). For example, antibody ATL_0005802 can be referred to interchangeably herein as ATL_5802 and 5802. In addition, reference number ATLX-1088 refers to ATL_5802.

[0090] CD33 is an inhibitory immune receptor belonging to the sialic acid-binding immunoglobulin-like lectin family. CD33 is also known as sialic acid-binding immunoglobulin-like lectin 3 (Siglec-3). The human CD33 gene (Gene ID: 945) is located on human chromosome 19 (19ql3.33) and consists of seven exons. The complete sequence of human CD33 is a 364 amino acid long sequence, available under Uniprot identifier P20138. It consists of (i) an N-terminal signal peptide (amino acids 1-17 of P20138) that targets CD33 to the secretory pathway; (ii) two extracellular domains consisting of an N-terminal Ig-like V-set domain and a C2-set domain for recognition of carbohydrate ligands (amino acids 18-259 of P20138); (iii) a transmembrane domain (amino acids 260-282 of P20138); and (iv) a cytoplasmic domain with an immunoreceptor tyrosine-based inhibitory motif (ITIM) that mediates immune cell signaling (amino acids 283-364 of P20138) (Eskandari-Sedighi et al., 2023). CD33 undergoes alternative splicing to generate a long isoform, denoted hCD33M (M ='major'), and a short isoform, denoted hCD33m (m ='minor'), which is generated by exclusion of exon 2 (amino acids 13-139 of hCD33M) and lacks a functional V domain (Hernandez-Caselles, et al., 2006). The major isoform of human CD33 (hCD33) is a 67 kDa transmembrane glycoprotein, available under Uniprot identifier P20138-1 (canonical sequence, 364 amino acids), provided as SEQ ID NO: 176. The minor isoform is a 25 kDa protein, available under Uniprot identifier P20138-3 (237 amino acids), provided as SEQ ID NO: 177. The antibodies described herein can bind to hCD33M, but not to hCD33m. CD33 is expressed on the cell surface of myeloid cells, monocytes, macrophages, and microglial cells in the brain. CD33 is involved in adhesion processes of immune cells and mediates cell-cell interactions (Varki et al., 2006).

[0091] 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 on microglia in AD patients has been associated with later stage cognitive decline or disease state (Siddiqui et al., 2017). This is thought to be due to altered microglial activation and inhibition of microglia-mediated phagocytosis of toxic insoluble amyloid beta 42 (Aβ42) species. On the other hand, the AD protective allele of CD33 SNP rs3865444 results in lower CD33 expression and a higher proportion of a truncated, non-signaling form, and a reduction in insoluble Aβ42 levels in the AD brain (Griciuc et al., 2013). In addition, CD33 has been shown to interact with other microglia AD risk genes, such as TREM2, to influence AD onset and pathogenesis. In particular, CD33 has been shown to negatively regulate TREM2 / DAP12-mediated microglial activation, resulting in decreased cellular function, such as microglial phagocytosis, which in turn results in decreased clearance of toxic Aβ42 species (Chan et al., 2015). Thus, the antibodies described herein are capable of increasing phagocytosis of cells expressing CD33, such as microglial phagocytosis, compared to comparator antibodies.

[0092] In addition to its role in inhibiting cellular processes, such as phagocytosis, CD33 is involved in several additional processes, including cell adhesion and modulation of immune responses (Cao et al., 2010). CD33 is also highly expressed in leukemic blasts of acute myeloid leukemia as well as myeloid leukemia initiating cells (Bonnet et al., 1997; Vercauteren et al., 2007). Thus, in some cases, the antibodies described herein can be used to treat AML by binding to CD33 in leukemic blasts.

[0093] CD33 has endocytic properties (receptor internalization) that have been exploited for antibody-drug conjugate targeting (Laszlo et al., 2014). While this property can lead to a reduction of CD33 at the cell surface, it can also reduce the efficacy of CD33-targeted therapies, as anti-CD33 antibodies are internalized by CD33-expressing cells that are not the primary therapeutic target. For example, when targeting CD33-expressing microglia, CD33-expressing monocytes can represent a peripheral sink that can prevent the antibody from reaching a therapeutic dose with acceptable toxicity in the brain. In some cases, the antibodies described herein induce reduced CD33 endocytosis compared to a comparator antibody. For example, CD33 internalization can be measured by determining cell surface levels of CD33 using flow cytometry in the presence of a candidate antibody. The cells can be human cells, such as microglia, myeloid cells, monocytes, tumor cells (e.g., leukemic blasts), and / or iPSC-derived microglia and / or cells that have been stimulated with an inflammatory signal (e.g., LPS) prior to exposure to the antibody. The comparator antibody can be selected from the group consisting of: an isotype control antibody, and / or an antibody having the heavy chain variable sequence of ATL_5909 and the light chain variable sequence of ATL_5909.

[0094] Therapeutic strategies against TREM2, an anti-inflammatory receptor expressed in myeloid cells, have been explored for the treatment of AD, aiming to restore normal microglial function. Indeed, loss of TREM2 function is known to reduce microglial response to beta-amyloid plaques. For example, several TREM2 agonists are currently in clinical studies for AD treatment, including AL002 by Alector / Abbvie (referred to herein as ATL6166, in a phase 2 clinical trial), DNL919 by Denali Therapeutics (referred to herein as ATL6167, in a phase 1 clinical trial), and VGL101 by Vigil (referred to herein as ATL6170, in a phase 2 clinical trial). While CD33 is known to modulate TREM2 activity, to the best of the inventors’ knowledge, no direct CD33 antagonist antibody is currently in clinical development.

[0095] The term "CD33" as used herein encompasses truncations, derivatives, and variants of the CD33 sequence provided herein as SEQ ID NO: 176 or homologs thereof, and can refer to any protein having at least 80%, at least 90%, or at least 95% sequence identity to the sequence. The CD33 sequence can be a human CD33 sequence. The CD33 sequence can be a sequence comprising at least a portion of the V domain of CD33 (also known as an "Ig-like V-type" domain) (amino acids 19-135 of Uniprot ID P20138-1 or homologs thereof). The CD33 sequence can further comprise one or more of at least a portion of the C domain of CD33 (also known as an "Ig-like C2-type" domain) (amino acids 145-228 of Uniprot ID P20138-1 or homologs thereof), at least a portion of the transmembrane domain of CD33 (amino acids 260-282 of P20138-1 or homologs thereof), and at least a portion of the cytoplasmic domain of CD33 (amino acids 283-364 of P20138-1 or homologs thereof). Antibodies according to the present disclosure can bind to a CD33 protein comprising the V domain of human CD33 (amino acids 19-135 of Uniprot ID P20138-1 provided as SEQ ID NO: 176) or a sequence having at least 90%, 95%, 98%, or 99% sequence identity to the sequence.

[0096] The present disclosure relates primarily to antibody molecules, whether intact antibodies (e.g., IgG, e.g., IgG1) or antibody fragments (e.g., single chain variable fragments (scFv), antibody fragments (Fab) or divalent antibody fragments (F(ab')2), single-domain antibodies (sdAb). Antibody antigen binding regions (also referred to as "antigen binding portions") are provided, as are antibody heavy chain variable (VH) domains and light chain variable (VL) domains. Within the VH and VL domains, complementarity determining regions (CDRs) are provided, which can be provided within different framework regions (FRs) to form the VH or VL domain as the case can be. The antigen binding site can be composed of antibody VH domains and / or VL domains.

[0097] Antibodies according to the present disclosure can be provided in isolated form. The term "antibody" encompasses fragments or derivatives thereof, or synthetic antibodies or antibody fragments. The antibody or fragment thereof can be a monoclonal antibody (mAb). mAbs are a homogeneous population of antibodies that specifically target a single epitope on an antigen. Antibodies and methods of their construction and use are well known in the art and are described in, for example, Holliger & Hudson, Nature Biotechnology 23(9): 1126-1136 (2005). Given today's technology associated with monoclonal antibody technology, antibodies can be made against most targets. Monoclonal antibodies and other antibody molecules can be utilized and recombinant DNA technology used to produce additional antibodies or chimeric molecules that retain the specificity of the original antibody. Such technology can involve the introduction of the CDRs or variable regions of one antibody into a different antibody molecule (see, for example, GB 2188638A and EP 0239400).

[0098] An "antigen binding domain" describes the portion of a molecule that binds to all or part of a target antigen. Antibodies typically comprise six complementarity determining regions (CDRs); three in the VH region: HCDR1, HCDR2, and HCDR3, and three in the VL region: LCDR1, LCDR2, and LCDR3. The six CDRs together define the paratope, which is the portion of the antigen binding domain that binds to the target antigen. Monoclonal monospecific IgG antibody molecules comprise two antigen binding domains, each of which is capable of binding the same target (i.e., it is bivalent for a single target). Fab fragments typically comprise a VH domain, a CH1 domain, a VL domain, and a CL domain. A complete antibody can comprise a pair of Fab fragments and a Fc fragment comprising a pair of chains, each chain comprising a CH2 domain and a CH3 domain. Fv fragments comprise a VH domain and a VL domain. The VH and VL regions comprise framework regions (FRs) flanking each CDR, which provide a scaffold for the CDRs. From N- to C-terminus, the VH region comprises the following structure: N-terminal C-terminal; and the VL region comprises the following structure: N-terminal C-terminal.

[0099] 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. Fab, Fv, ScFv, and sdAb antibody fragments can all be expressed in and secreted from E. coli, making it easy to produce large quantities of the fragments. Complete antibodies and F(ab')2 fragments are "bivalent." The term "bivalent" means that the antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv, and dAb fragments are monovalent, having only one antigen combining site.

[0100] Antibodies according to the present disclosure can be detectably labeled or at least capable of detection. For example, the antibodies can be labeled with radioactive atoms or colored or fluorescent molecules or can be readily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates, and radioactive labels. The binding moiety (antibody or fragment thereof) can be directly labeled with a detectable label, or it can be indirectly labeled. For example, the binding moiety can be an unlabeled antibody that can be detected by another antibody that is labeled itself. Alternatively, the second antibody can bind to biotin, and the binding of labeled streptavidin to the biotin is used to indirectly label the first antibody.

[0101] A "fragment" of an antibody can comprise any number of residues of the "parent" antibody while retaining target binding ability. Fragments can lack effector function, e.g., a fragment can be completely incapable of binding an Fc receptor or show diminished binding to an Fc receptor relative to the parent. Fragments are typically smaller than the parent antibody. A fragment can comprise 50%, 60%, 70%, 80%, 90%, 95% or more contiguous or non-contiguous amino acids of the parent antibody. A fragment can comprise 50, 100, 150, 200, 250, 300 or more contiguous or non-contiguous amino acids of the parent antibody. A fragment can comprise a deletion in the Fc region, or a deletion of the Fc region. Fragments can retain unaltered CDRs and / or variable domains of the parent antibody. In some embodiments, the fragment is a Fab fragment or a F(ab')2 fragment. CDR sequences are described herein using IMGT numbering (Lefranc, M.-P., Immunology Today, 18, 509 (1997)).

[0102] Antibodies according to the present disclosure can have a VH having the VH-CDRs of the following antibody: or a CDR set comprising zero, one or two amino acid substitutions in each CDR as compared to the above CDR sets. Thus, an isolated antibody can comprise a heavy chain variable domain having the CDRs:

[0103] CDRH1 comprising an amino acid sequence selected from the group consisting of:

[0104]

[0105] or (vi) an amino acid sequence having 1 or 2 mutations compared to the above sequences;

[0106] CDRH2 comprising an amino acid sequence selected from the group consisting of:

[0107]

[0108] or (iv) an amino acid sequence having 1 or 2 mutations compared to the above sequences; and

[0109] CDRH3 comprising an amino acid sequence selected from the group consisting of:

[0110]

[0111] or (viii) an amino acid sequence having 1 or 2 mutations compared to the above sequences.

[0112] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising a CDRH1 comprising a sequence of a CDRH2 comprising a sequence of and a CDRH3 comprising a sequence of or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[0113] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising a CDRH1 comprising a sequence of a CDRH2 comprising a sequence of and a CDRH3 comprising a sequence of or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[0114] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising a CDRH1 comprising a sequence of a CDRH2 comprising a sequence of and a CDRH3 comprising a sequence of CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences.

[0115] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising CDRH1 of the sequences, CDRH2 of the sequences, and CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences. An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising CDRH1 of the sequences, CDRH2 of the sequences, and CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences.

[0116] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising CDRH1 of the sequences, CDRH2 of the sequences, and CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences. An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising

[0117] CDRH1 of the sequences, CDRH2 of the sequences, and CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences. An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising CDRH1 of the sequences, CDRH2 of the sequences, and CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences.

[0118] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) having CDRs comprising CDRH1 of the sequences, CDRH2 of the sequences, and CDRH3 of the sequences, or a CDR set comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences. ​​​CDR set comprising CDRH3 of the sequences of any of SEQ ID NOs: 1-3, or comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2, or 3 mutations in CDRH3 compared to these sequences.

[0119] In antibodies according to the disclosure, at least one of the VH CDR 1-3 sequences can be varied. Variants can have one or two amino acid substitutions compared to the sets of VH CDR 1-3 described above. In some embodiments, antibodies according to the disclosure comprise a CDR having a sequence with one or two substitutions compared to the VH CDR sequences of any of the antibodies described herein. For example, an antibody according to the disclosure can comprise VH CDRs having the sequences of any of the antibodies above, except that one or two of the CDRH sequences comprise a substitution, wherein the total number of substitutions in the CDRH sequences is not more than two. In some embodiments, variants can have one, two, or three substitutions in each of one or more of the VH CDR 1-3 described above, preferably with at most one or two substitutions. The CDRH1 region of any of the antibodies or fragments described herein can be 8 amino acids in length. The CDRH2 region of any of the antibodies or fragments described herein can be 8 amino acids in length. The CDRH3 region of any of the antibodies or fragments described herein can be 13 to 21 amino acids in length. In some embodiments, variants can have VH CDRs with at least 70%, at least 80%, or at least 90% sequence identity to any of the VH CDR sets described herein.

[0120] Antibodies according to the disclosure can have a light chain variable domain (VL) having an antibody the CDRs of the light chain variable domain (VL) of any of the antibodies described above, or a CDR set comprising zero, one, or two amino acid substitutions in each CDR compared to the CDR sets above. Thus, an isolated antibody can comprise a light chain variable domain having CDRs:

[0121] CDRL1 comprising an amino acid sequence selected from the group consisting of:

[0122]

[0123] or (vii) an amino acid sequence having 1 or 2 mutations compared to the sequences above;

[0124] CDRL2 comprising an amino acid sequence selected from the group consisting of:

[0125]

[0126] or (viii) an amino acid sequence having 1 or 2 mutations compared to the above sequences; and

[0127] CDRL3 comprising an amino acid sequence selected from the group consisting of:

[0128]

[0129] or (viii) an amino acid sequence having 1 or 2 mutations compared to the above sequences.

[0130] An antibody according to the present disclosure can comprise a light chain variable domain (VL) having CDRs comprising a CDRL1 comprising a sequence of:

[0131] CDRL1 comprising an amino acid sequence selected from the group consisting of:

[0132]

[0133] or an amino acid sequence having 1 or 2 mutations compared to the above sequences;

[0134] CDRL2 comprising an amino acid sequence selected from the group consisting of:

[0135]

[0136] or an amino acid sequence having 1 or 2 mutations compared to the above sequences; and

[0137] CDRL3 comprising an amino acid sequence selected from the group consisting of:

[0138]

[0139] or an amino acid sequence having 1 or 2 mutations compared to the above sequences.

[0140] An antibody according to the present disclosure can comprise a light chain variable domain (VL) having CDRs comprising a CDRL1 comprising a sequence of:

[0141] An antibody according to the present disclosure can comprise a light chain variable domain (VL) having CDRs comprising a CDRL1 comprising a sequence of: ​​​​​The CDRH3 sequence, or the CDRL1 and CDRL2 sequences containing 1 or 2 mutations (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs) compared to these sequences, and / or the CDR group containing 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[0142] Antibodies according to this disclosure may comprise a light chain variable domain (VL) having the following CDRs: The CDRL1 sequence contains The sequence of CRL2 and containing The CDRH3 sequence, or the CDRL1 and CDRL2 sequences containing 1 or 2 mutations (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs) compared to these sequences, and / or the CDR group containing 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[0143] Antibodies according to this disclosure may comprise a light chain variable domain (VL) having the following CDRs: The CDRL1 sequence contains The sequence of CRL2 and containing The CDRH3 sequence, or the CDRL1 and CDRL2 sequences containing 1 or 2 mutations (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs) compared to these sequences, and / or the CDR group containing 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[0144] Antibodies according to this disclosure may comprise a light chain variable domain (VL) having the following CDRs: The CDRL1 sequence contains The sequence of CRL2 and containing The CDRH3 sequence, or the CDRL1 and CDRL2 sequences containing 1 or 2 mutations (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs) compared to these sequences, and / or the CDR group containing 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[0145] Antibodies according to this disclosure may comprise a light chain variable domain (VL) having the following CDRs: The CDRL1 sequence contains The sequence of CRL2 and containing CDR set comprising a CDRH1 comprising the sequence of

[0146] An antibody according to the present disclosure can comprise a heavy chain variable domain (VH) comprising a CDRH1 comprising the sequence of a CDRH2 comprising the sequence of a CDRH3 comprising the sequence of or comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences (1 or 2 mutations in each CDR, or 1 or 2 mutations in both CDRs), and / or comprising 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[0147] In an antibody according to the present disclosure, at least one of the VL CDR1 to 3 sequences can be varied. A variant can have 1, 2 or 3 amino acid substitutions compared to the set of VL CDR1 to 3 described above. In some embodiments, an antibody according to the present disclosure comprises a CDR having a sequence with 1 to 3 substitutions compared to the VL CDR sequences of the antibodies described herein. For example, an antibody according to the present disclosure can comprise substitutions, wherein the total number of substitutions is not more than 3. In some embodiments, a variant can have one, two or three substitutions in each of one or more of the VL CDR1 to 3 described above, preferably at most one or two substitutions.

[0148] The length of the CDRL1 region of any of the antibodies or fragments described herein can be 6 to 11 amino acids. The length of the CDRL2 region of any of the antibodies or fragments described herein can be 3 amino acids. The length of the CDRL3 region of any of the antibodies or fragments described herein can be 9 to 11 amino acids. In some embodiments, a variant can have a VL CDR having at least 70%, at least 80% or at least 90% sequence identity to any of the VL CDR sets described herein.

[0149] The VH CDR 1 to 3 and optionally VL CDR 1 to 3 of any of the antibodies described above can also particularly be used in combination with a number of different framework regions. Thus, a light chain and / or heavy chain having CDR 1 to 3 as described above can have an alternative framework region. Suitable 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.

[0150] An antibody of the present disclosure can have a heavy chain variable domain (VH) having the framework sequence of the antibody HFWR1 of any one of the antibodies, an antibody HFWR2 of any one of the antibodies, an antibody HFWR3 of any one of the antibodies, and an antibody HFWR4 of any one of the antibodies, or a framework sequence having one to six substitutions compared to the above framework sequences.

[0151] An antibody according to the present disclosure can have a heavy chain variable domain (VH) having the framework sequence of:

[0152] HFWR1 comprising an amino acid sequence selected from the group consisting of:

[0153]

[0154] HFWR2 comprising an amino acid sequence selected from the group consisting of:

[0155]

[0156] HFWR3 comprising an amino acid sequence selected from the group consisting of:

[0157]

[0158] or a framework sequence having one to six, one to five, one to four, one to three, or one to two substitutions compared to the above framework sequences.

[0159] An antibody of the present disclosure can have a heavy chain variable domain (VH) having the framework sequence HFWR1, HFWR2, HFWR3, and HFWR4 of the antibody , or a framework sequence having one to six substitutions compared to these framework sequences. An antibody of the present disclosure can have a heavy chain variable domain (VH) having the framework sequence HFWR1, HFWR2, HFWR3, and HFWR4 of the antibody , or a framework sequence having one to six substitutions compared to these framework sequences. An antibody of the present disclosure can have a heavy chain variable domain (VH) having the framework sequence HFWR1, HFWR2, HFWR3, and HFWR4 of the antibody The frame sequences HFWR1, HFWR2, HFWR3, and HFWR4, or having 1 to 6 alternative frame sequences compared to these frame sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) that has an antibody The frame sequences HFWR1, HFWR2, HFWR3, and HFWR4, or having 1 to 6 alternative frame sequences compared to these frame sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) that has an antibody The frame sequences HFWR1, HFWR2, HFWR3, and HFWR4, or having 1 to 6 alternative frame sequences compared to these frame sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) that has an antibody The frame sequences HFWR1, HFWR2, HFWR3, and HFWR4, or having 1 to 6 alternative frame sequences compared to these frame sequences. The antibodies of this disclosure may have a heavy chain variable domain (VH) that has an antibody The frame sequences HFWR1, HFWR2, HFWR3 and HFWR4, or the frame sequences with 1 to 6 alternatives compared to these frame sequences.

[0160] In some implementations, substitutions in the frame sequence of the heavy-chain variable domain can be located anywhere in the standard IMGT number except for bit 40. In some implementations, substitutions in the frame sequence do not include substitution at bit 40 of the standard IMGT number. Substitutions in the frame sequence of the heavy-chain variable domain can be located anywhere in HFWR1 and / or HFRW3. Substitutions in the frame sequence of the heavy-chain variable domain can be located anywhere outside HFRW2. Substitutions in the frame sequence of the heavy-chain variable domain can be selected from the following positions in the standard IMGT number: HFWR1: bits 1, 6, and 20; HFWR3: bits 72 and 85. Substitutions in the frame sequence of the heavy-chain variable domain can be selected from the following positions in the standard IMGT number: HFWR1: bit 1: Q1E, bit 6: E6Q, bit 20: R20K; HFWR3: bit 72: E72Q, bit 85: G85S.

[0161] The antibody disclosed herein may have a heavy chain variable domain (VH) having The frame sequence HFWR2 ( The isolated antibody may have a heavy chain variable domain (VH) containing a sequence. the framework sequence HFWR2. An antibody of the present disclosure can have a heavy chain variable domain (VH) having the framework sequence HFWR2. the framework sequence HFWR2. An antibody of the present disclosure can have a heavy chain variable domain (VH) having the framework sequence comprising A at position 40 in standard IMGT numbering.

[0162] An antibody according to the present disclosure can have a heavy chain variable domain (VH) having the following framework sequence:

[0163] HFWR1 of SEQ ID NO: 70, 71, 72, 73, 74, 235, 75, 77, 78, or 79

[0164]

[0165] preferably the HFWR1 of SEQ ID NO: 70, 235, 75;

[0166] HFWR2 of SEQ ID NO: 83, 84, 85, 86

[0167]

[0168]

[0169] preferably the HFWR2 of SEQ ID NO: 83;

[0170] HFWR3 of SEQ ID NO: 91, 92, 93, 94, 95, 96, 97

[0171]

[0172] preferably the HFWR2 of SEQ ID NO: 91, 96, 97, and

[0173] HFWR4 of SEQ ID NO: 101, 104, 105

[0174]

[0175] preferably the HFWR4 of SEQ ID NO: 101.

[0176] An antibody of the present disclosure can have CDRL1, CDRL2, and CDRL3 of a VL domain within a germline framework. An antibody of the present disclosure can have a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3 within a germline framework, provided that position 40 in the standard IMGT numbering is A.

[0177] An antibody according to the present disclosure can have a light chain variable domain (VL) having the following framework sequences:

[0178] LFWR1 comprising an amino acid sequence selected from the group consisting of:

[0179]

[0180] LFWR2 comprising an amino acid sequence selected from the group consisting of:

[0181]

[0182] LFWR3 comprising an amino acid sequence selected from the group consisting of:

[0183]

[0184]

[0185] LFWR4 comprising an amino acid sequence selected from the group consisting of:

[0186]

[0187] or a framework sequence having 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 substitutions compared to the above framework sequences.

[0188] An antibody of the present disclosure can have a light chain variable domain (VL) having the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody or a framework sequence having 1 to 6 substitutions compared to these framework sequences. An antibody of the present disclosure can have a light chain variable domain (VL) having the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody or a framework sequence having 1 to 6 substitutions compared to these framework sequences. An antibody of the present disclosure can have a light chain variable domain (VL) having the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody 1, or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of the present disclosure can have a light chain variable domain (VL) having the framework sequence of antibody the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody 1, or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of the present disclosure can have a light chain variable domain (VL) having the framework sequence of antibody the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody 1, or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of the present disclosure can have a light chain variable domain (VL) having the framework sequence of antibody the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody 1, or framework sequences having 1 to 6 substitutions compared to these framework sequences. The antibodies of the present disclosure can have a light chain variable domain (VL) having the framework sequence of antibody the framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of antibody 1, or framework sequences having 1 to 6 substitutions compared to these framework sequences.

[0189] The 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. The substitutions in the light chain variable domain framework sequence can be selected from the following positions in standard IMGT numbering: LFWR1: position 2: A2S, position 8: S8P.

[0190] The antibody can have a light chain variable domain (VL) having the following framework sequence:

[0191] LFWR1 of antibody 1; LFWR2 of antibody 1; LFWR3 of antibody 1; and LFWR4 of antibody 1.

[0192] In the present specification, an antibody can have a VH (and optionally a VL) region comprising an amino acid sequence having a high percentage of sequence identity to a VH and / or VL amino acid sequence described above. For example, an antibody according to the present application includes an antibody that binds CD33 and has a VH region comprising an amino acid sequence having at least one of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH region amino acid sequence of any of the antibodies described herein. An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-10 and 12-20. An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of SEQ ID NOs: 4-10 and 12-20.

[0193] An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 9, and 10 (antibodies 1, 3, and 4, respectively). An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of SEQ ID NOs: 4, 9, and 10 (antibodies 1, 3, and 4, respectively). An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of SEQ ID NOs: 4, 9, and 10 (antibodies 1, 3, and 4, respectively).

[0194] Alternatively or additionally, an antibody of the present disclosure can have a VL region comprising an amino acid sequence having at least one of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL region amino acid sequence of any of the antibodies described herein. For example, an antibody of the present disclosure can have a VL region comprising an amino acid sequence having at least one of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL region amino acid sequence of any of the antibodies described herein. Alternatively or additionally, an antibody of the present disclosure can have a VL region comprising an amino acid sequence having at least one of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL region amino acid sequence of any of the antibodies described herein. For example, an antibody of the present disclosure can have a VL region comprising an amino acid sequence having at least one of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL region amino acid sequence of any of the antibodies described herein.

[0195] An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-23. An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-23.

[0196] An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (antibodies 1-23, respectively). An antibody according to the present disclosure can have a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (antibodies 1-23, respectively).

[0197] An antibody of the present disclosure can have a lambda (l) or kappa (K) light chain.

[0198] The overall percent identity of the variable region or full length heavy chain / light chain sequence can be combined with the particular CDR sequences from the same antibody.

[0199] ​Percent (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the residues in the comparison sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences. When the sequences to be compared have different lengths, the sequence identity of the shorter comparison sequence can be determined over the entire length of the longer given sequence, or when the comparison sequence is longer than the given sequence, the sequence identity of the comparison sequence can be determined over the entire length of the shorter given sequence. Sequence identity can be defined with reference 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 maximizes the number of matches and minimizes the number of gaps. In general, the default parameters can be used, where the gap creation penalty = 12 and the gap extension penalty = 4. The use of GAP can be preferred, but other algorithms can also be used, such as BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of 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 the TBLASTN program of Altschul et al. (1990) supra, typically with the default parameters (see e.g. Pearson Curr Prot Bioinformatics (2013) Chapt 3 Uniy 3.1 doi:10.1002 / 0471250953.bi0301s42). In particular, the psi-Blast algorithm (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402) can be used. Sequence identity and similarity can also be determined using Genomequest™ software (Gene-IT, Worcester MA USA). Sequence comparison is preferably performed over the full length of the relevant sequences to be compared.

[0200] Antibodies of the present disclosure can comprise one or more substitutions within the framework of the VH and / or VL region. As used herein, a "substitution" refers to the replacement of one amino acid with another amino acid at a particular position relative to the same position in a baseline molecule. In some embodiments, the baseline molecule is an antibody exemplified herein, e.g., an antibody .

[0201] In some embodiments, an antibody or fragment thereof according to the present disclosure is capable of crossing the blood brain barrier. In some embodiments, an antibody of the present disclosure can have one scFV chain, e.g., a transferrin receptor (Yu et al., 2014), and an scFv chain that binds CD33 as described herein. In some embodiments, an antibody according to the present disclosure comprises an antibody or fragment thereof that binds CD33 as described herein (e.g., as an antibody, scFv, sdAb, etc.), and an additional binding moiety that binds to another target. The other target can be a receptor in the brain, e.g., a transferrin receptor. The additional binding moiety can be an antibody, scFv, nanobody, or aptamer. The two binding moieties of such a bispecific molecule can form a fusion protein.

[0202] Also described herein are single domain antibodies (sdAbs), also known as nanobodies, comprising the heavy chain CDR and / or VH sequence of any of the antibodies described herein. Thus, also described herein are antibodies or fusion molecules comprising a nanobody that binds CD33 as described herein, and a nanobody that binds a receptor in the brain. Also described herein are fusion molecule antibodies comprising an scFV chain or nanobody that binds CD33 as described herein, and an aptamer that binds a receptor in the brain.

[0203] An antibody described herein can increase phagocytosis of a CD33-expressing cell. The increase in phagocytosis can be measured in comparison to a comparative antibody. The comparative antibody can be an isotype control antibody. The comparative antibody can be another CD33-binding antibody. The comparative antibody can be an antibody having the heavy chain variable sequence of ATL_5909 and the light chain variable sequence of ATL_5909. The comparative antibody can be another CD33-binding antibody having a different epitope. Phagocytosis can be assessed by measuring fluorescence signal associated with uptake of labeled particles by imaging or flow cytometry. Phagocytosis can be measured by detecting fluorescence signal indicative of phagocytosis of a substrate having pH-dependent fluorescence. The substrate can be selected from the group consisting of beta amyloid and S. aureus. The labeled substrate can be a substrate labeled with pHrodo Red. The CD33-expressing cell can be an inflammatory human iPSC-derived microglia. The inflammatory cell can be a cell that has been stimulated with an inflammatory signal. For example, the cell can have been exposed to an inflammatory signal (e.g., LPS, IFN-g) for at least 6, 12, or 24 hours, or about 6, 12, or 24 hours, prior to exposure to the antibody. The CD33-expressing cell can be a PBMC. The CD33-expressing cell can be a cell isolated from human blood. The CD33-expressing cell can be a monocyte or microglia. The CD33-expressing cell can be a human cell. The CD33-expressing cell can be an iPSC-derived microglia. The CD33-expressing cell can be a cell that has been stimulated with an inflammatory signal (e.g., LPS) prior to exposure to the antibody.

[0204] An antibody described herein can bind human CD33. An antibody described herein can bind human CD33 with an EC50 of at most 2e-08 M or at most 3e-09 M, as assessed by ELISA (e.g., binding of plated rhCD33). An antibody of the present disclosure can bind human CD33 with an EC50 of at most 15 pg / ml, at most 12 pg / ml, at most 5 pg / ml, at most 3 pg / ml, or at most 0.5 pg / ml. An antibody of the present disclosure can bind human CD33 with an EC50 of at most 1e-7 M, at most 8e-8 M, at most 3e-8 M, at most 2e-08 M, or at most 3e-09 M. Binding to human CD33 can be assessed by ELISA using plated rhCD33 (e.g., CD33_Human_ECD, His_004_001 from SinoBiological, 12238-H08H).

[0205] An antibody according to the present disclosure can have a Koff of at least 1e-3 s -1 for binding to human CD33. An antibody according to the present disclosure can have a Koff of 1e-3 s -1 to 5e-2 s -1Koff for binding to human CD33. An antibody according to the disclosure can have a KD for binding to human CD33 of at most 5e-7 M. An antibody according to the disclosure can have a KD for binding to human CD33 of 2e-8 M to 5e-7 M. The human CD33 can be rhCD33-his (e.g., R&D Systems, 10375-SL-050). The KD and Koff can be as measured by biolayer interferometry (BLI) on octet-I. An antibody according to the disclosure can have a lower Koff for binding to human CD33 than a comparison antibody. Suitable comparison antibodies are described above.

[0206] An antibody according to the disclosure can have a melting temperature Tm1 of at least 57, or 57 to 70. An antibody according to the disclosure can have a purity SEC-HPLC % monomer of at least 95%.

[0207] An antibody described herein can result in lower CD33 internalization on C33 expressing cells compared to a comparison antibody. The antibody can result in less than 50%, or less than 80% depletion of CD33 on the surface of human monocyte cells after 5 hours of incubation with the antibody. The antibody can result in a lower degree of depletion of CD33 on the surface of human monocyte cells after 5 hours of incubation with the antibody compared to a comparison antibody at the same concentration. Suitable comparison antibodies are described above.

[0208] The antibody described herein can have reduced binding to a human CD33 protein comprising a mutation at position 47, 50, 51, 52, and 122 compared to the binding of the antibody to a human CD33 protein that does not comprise the mutation. The antibody described herein can not bind to a human CD33 protein comprising a mutation at position 47, 50, 51, 52, and 122. The antibody described herein can have reduced binding to a human CD33 protein comprising a mutation at position 83 compared to the binding of the antibody to a human CD33 protein that does not comprise the mutation. The mutation can be selected from the group consisting of: at position 20: N20R, at position 21: F21V, at position 22: W22R, at position 24: Q24E, at position 47: I47V, at position 50: Y50H, at position 51: D51T, at position 52: K52R, at position 83: Q83R, at position 122: R122K, and at position 132: P132T. The positions can refer to positions in the full-length human CD33 sequence (CD33M (Uniprot ID: P20138-1)). Binding can be as determined using a single point ELISA. The human CD33 protein can comprise residues 18 to 232 of human CD33. The human CD33 protein that does not comprise the mutation can be SEQ ID NO: 226. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 226 compared to a protein comprising the sequence of SEQ ID NO: 226. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 226 compared to a protein comprising the sequence of SEQ ID NO: 227. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 227 compared to a protein comprising the sequence of SEQ ID NO: 226. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 226 compared to a protein comprising the sequence of SEQ ID NO: 228. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 228 compared to a protein comprising the sequence of SEQ ID NO: 226. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 226 compared to a protein comprising the sequence of SEQ ID NO: 229. The antibody described herein can have reduced binding to a protein comprising the sequence of SEQ ID NO: 229 compared to a protein comprising the sequence of Compared to proteins containing the sequence (SEQ ID NO: 226), antibodies as described herein may have reduced [specific properties / effects]. The binding of proteins containing the sequence (SEQ ID NO: 230). Compared to proteins containing the sequence (SEQ ID NO: 226), antibodies as described herein may have reduced [specific properties / effects]. The binding of proteins with the sequence (SEQ ID NO: 232). The antibody described herein interferes with the binding of sialic acid. The antibody described herein binds to an epitope, thereby interfering with the binding of sialic acid to CD33.

[0209] The antibody disclosed herein can bind to CD33 proteins containing the V domain of CD33. The antibody disclosed herein may not bind to CD33 proteins that do not contain the V domain of CD33. The antibody disclosed herein may not bind to proteins containing the V domain of CD33. The antibody of this disclosure binds to the CD33 protein of the sequence (SEQ ID NO: 175). The antibody may selectively bind to CD3 relative to one or more other siglecs, optionally, wherein the antibody selectively binds to CD33 relative to one or more (or all) of siglec-6, siglec-7, siglec-8, and siglec-9, and / or wherein the antibody selectively binds to human CD33 relative to one or more other homologs, optionally, wherein the antibody selectively binds to human CD33 relative to mouse CD33 and cynomolgus monkey CD33.

[0210] The antibodies disclosed herein can reduce neuroinflammation (i.e., inflammation in the brain), for example, compared to comparative antibodies (e.g., allotype antibodies or ATL_5909). The level of neuroinflammation can be determined by measuring the levels of pro-inflammatory cytokines in nerve cells (e.g., microglia as described herein, such as microglia derived from human Ipsc) (e.g., using an immunoassay, such as ELISA). Pro-inflammatory cytokines can be one or more of the following: monocyte chemoattractant protein-1 (MCP-1), IL-6, interferon-gamma-induced protein 10 (IP10), and glial fibrillary acidic protein (GFAP).

[0211] The antibodies described herein can have lower peripheral clearance when administered to a subject compared to a comparative anti-CD33 antibody. The comparative anti-CD33 antibody can be ATL_5909. The subject can be a subject (e.g., a mouse) expressing a CD33 protein comprising human CD33 exons 1-3. The administration can be parenteral. The administration can be intravenous or intraperitoneal.

[0212] The antibodies described herein can increase phagocytosis of Aβ by microglia in vivo. This can be assessed in vivo, e.g., as described herein (see Example 10 and accompanying methods). The effect of the antibody on Aβ phagocytosis can be assessed on microglia derived from human microglial progenitor cells from iPSCs grafted into the brain of an Alzheimer’s disease (AD) mouse (e.g., an APP NL-G-F knock-in mouse).

[0213] The antibodies described herein can increase clearance of Aβ plaques in a subject in need thereof. The antibody can increase clearance of Aβ plaques by microglial phagocytosis in a subject in need thereof.

[0214] The antibodies described herein can increase phagocytosis of tau aggregates by microglia having an inflammatory phenotype (e.g., iPSC microglia treated with LPS). The antibodies described herein can increase phagocytosis of tau aggregates by microglia having an inflammatory phenotype to a greater extent compared to a comparative anti-CD33 antibody (e.g., as ATL_5909). This can be assessed in vitro as described herein, e.g., as in Example 11. The antibodies described herein can increase phagocytosis of tau aggregates by microglia in a subject in need thereof.

[0215] The antibodies described herein can not induce significant release of any one or more cytokines from human iPSC-derived microglia, human PBMCs, and / or human isolated monocytes exposed to the antibody in vitro compared to a control (e.g., an isotype control antibody). The one or more cytokines can be selected from the group consisting of:

[0216]

[0217] The antibody can not induce release of any of the above cytokines. The antibody can induce lower levels of release of any one or more or all of the above cytokines from human iPSC-derived microglia, human PBMCs, and / or human isolated monocytes exposed to the antibody in vitro compared to a comparative anti-CD33 antibody (e.g., ATL_5909). The antibody as described herein can reduce the level 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 in need thereof). The antibody as described herein can more reduce the level of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype in vitro (e.g., LPS-treated human iPSC-derived microglia) compared to a comparative anti-CD33 antibody (e.g., ATL_5909).

[0218] The antibody as described herein can increase the level of phosphorylated SYK protein in microglia treated with the antibody in vitro compared to a control (e.g., an isotype control antibody). The microglia can be microglia with an inflammatory phenotype (e.g., microglia treated with LPS). SYK phosphorylation can be increased without a significant increase in total protein levels.

[0219] The antibody as described herein can increase the level of P2RY12 in microglia treated with the antibody in vitro compared to a control (e.g., an isotype control antibody). The microglia can be microglia with an inflammatory phenotype (e.g., microglia treated with LPS).

[0220] The antibody as described herein can enhance TREM2 signaling in microglia treated with the antibody in vitro compared to a control (e.g., treatment with an isotype control antibody). The microglia can be microglia with an inflammatory phenotype (e.g., microglia treated with LPS).

[0221] The antibody as described herein can 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 assessed by RNA sequencing, optionally followed by gene set enrichment analysis, e.g., as described herein (see Example 14).

[0222] The antibodies described herein can reduce the release of one or more inflammatory markers induced by inflammation in a human neural cell culture assay compared to a control (e.g., exposure to an isotype control antibody). The human neural cell culture assay can be a co-culture assay comprising glutamatergic and GABAergic neurons, microglia, and astrocytes. The release of one or more cytokines induced by inflammation can be assessed by exposing the cells to one or more pro-inflammatory signals (e.g., such as lipopolysaccharide (LPS) and / or Interferon-gamma (INFy). The one or more inflammatory markers can be selected from the group consisting of MCP-1, IP-10, GFAP, and IL-6. Accordingly, the antibodies of the present disclosure can reduce the expression of one or more inflammatory markers in the CNS of a subject in need thereof. The one or more inflammatory markers can comprise one or more of MCP-1, IP-10, GFAP, and IL-6.

[0223] The antibodies described herein can selectively bind to CD33. The antibodies can not bind to any other human protein. Isolated nucleic acids encoding an antibody, antigen binding fragment, or polypeptide as described herein are provided. Vectors comprising the nucleic acids described herein and host cells comprising the vectors are also provided. For example, the host cell can be a eukaryotic cell or a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, or can be a prokaryotic cell, such as E. coli. In some embodiments, the vector is a viral vector, such as a bacteriophage.

[0224] Methods for making an antibody or antibody fragment as described herein are also provided, the methods comprising culturing a host cell as described herein under conditions suitable for expression of a vector encoding the antibody or antibody fragment, and isolating and / or purifying the antibody or antibody fragment. The methods further comprise formulating the antibody or antibody fragment into a composition comprising at least one additional component.

[0225] The antibodies and fragments thereof can be used in therapy.

[0226] The subject to be treated or diagnosed can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be male or female. The subject can be a patient. The therapeutic use can be in humans or animals (veterinary use). Unless otherwise indicated, the subject is a human.

[0227] The medicaments and pharmaceutical compositions according to some aspects of the present application can be formulated for administration by a variety of routes including, but not limited to, parenteral, intravenous, intra-arterial, intramuscular, oral, and nasal. The medicaments and compositions can be formulated for injection.

[0228] Pharmaceutical compositions can be prepared using a pharmaceutically acceptable "carrier" consisting of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., as are physiologically tolerable, and do not typically produce an allergic or similar untoward reaction, such as gastric upset or the like, when administered to an animal, and more particularly to a human. In some embodiments, the term refers to molecular entities and compositions approved by a regulatory agency of the Federal or a state government of the United States, such as the GRAS list under the Federal Food, Drug and Cosmetic Act 5 204(s) and 409, which undergo pre-market review and approval by the FDA or similar listing, the U.S. Pharmacopeia, or another generally recognized pharmacopeia, for use in animals, and more particularly in humans. The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those of skill in the art are familiar with such pharmaceutical carriers and methods of using such carriers to compound pharmaceutical compositions. The pharmaceutical compositions provided herein can include one or more excipients, such as a solvent, a solubility enhancer, a suspending agent, a buffer, an isotonicity agent, an antioxidant, or an antimicrobial preservative. When used, the excipients in the composition will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., the anti-CFH antibody used in the composition. Thus, the skilled artisan will appreciate that compositions are provided in which there is no incompatibility between the components of the dosage form. Excipients can be selected from buffers, solubilizers, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0229] It is preferred to administer in a "therapeutically effective amount" sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage, etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, method of administration, and other factors known to medical practitioners. Some examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Ed., 2000, pub. Lippincott, Williams & Wilkins.

[0230] Disorders treatable according to the present application include any disorder in which CD33 plays a role, including neurodegenerative diseases, and in particular those characterized by increased toxic protein species, such as the Ab42 characteristic of Alzheimer’s disease (AD), in which phagocytosis of these toxic species is useful. The antibodies of the present disclosure can be used to treat any neurological disease or disorder associated with accumulation of toxic protein species. Indeed, the antibodies of the present disclosure have been shown to increase phagocytosis by microglia and can therefore be useful in treating any neurological disease or disorder in which phagocytosis of toxic protein species is impaired or deficient. In addition, respiratory diseases characterized by phagocytosis defects, including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPD) (Donelly et al., 2012), can also be treated using the antibodies of the present disclosure. Other disorders treatable according to the present application include cancers, such as solid tumors in which sialic acid can cause immunosuppression and AML, such as cancers in which tumors display high sialylation on their cell surface to avoid recognition by immune cells (Stanczak & Laubli, 2023), including many solid tumors, and cancers characterized by abnormal expression of CD33, such as acute myeloid leukemia (AML). The antibodies of the present disclosure were initially identified by analysis of resilient AD individuals. These antibodies were subsequently found in resilient centenarians, suggesting their relevance beyond AD. Accordingly, it is also described herein that the antibodies of the present disclosure are for use as a medicament. It is also described that the antibodies are for use in the treatment or prevention of a neurodegenerative disease, and / or a respiratory disease, and / or a cancer. It is also described herein that the antibodies of the present disclosure are for use in the manufacture of a medicament, such as a medicament for the treatment or prevention of a neurodegenerative disease, or a respiratory disease, or a cancer. It is also described herein a method of treating a subject diagnosed with, or at risk of, a neurodegenerative disease, or a respiratory disease, or a cancer, the method comprising administering to the subject an antibody as described herein in a therapeutically effective amount.

[0231] The neurodegenerative disease or disorder can be selected from: 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 disease, Lewy body disease, spinal muscular atrophy (SMA), motor neuron disease (MND) such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxia (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral pallidoluysian atrophy (DRPLA), X-linked type 1 spinal and bulbar muscular atrophy (SMA X1 / SBMA), anderson-fabry (X-linked fabry disease) and DNAJB6 myopathy, multiple sclerosis (MS - a single nucleotide polymorphism in CD33 is known to be associated with an increased risk of multiple sclerosis) and microglia disease such as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). For example, the neurodegenerative disease can be selected from FTD, AD, HD and PD. The neurodegenerative disease or disorder can be a tauopathy. Thus, the neurodegenerative disease can be AD (Alzheimer’s disease), CTE (chronic traumatic encephalopathy), PiD (Pick’s disease), PSP (progressive supranuclear palsy), CBD (corticobasal degeneration), AGD (argyrophilic grain disease).

[0232] The respiratory disease can comprise one or more of: COPD, cystic fibrosis, asthma and idiopathic pulmonary fibrosis (IPD).

[0233] The cancer can be selected from: cervical cancer, breast cancer, brain cancer, bladder cancer, colon adenocarcinoma, cervical cancer, fibrosarcoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, non-small cell lung cancer, non-Hodgkin’s lymphoma and hematologic malignancies 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 some 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 hematologic malignancies. All of these cancers are known to have tumor cells that sialylate their cell surface to suppress immune cells, and thus the antibodies of the disclosure can be used to alleviate this suppression.

[0234] The antibodies of the present disclosure can be used in therapy with one or more additional therapeutic agents. As used herein, an "additional therapeutic agent" is an additional compound, protein, vector, antibody, cell, or entity that has a therapeutic effect. The antibodies described herein can be co-administered with an additional therapeutic agent. The antibodies can be co-formulated with an additional therapeutic agent. The antibodies can be administered sequentially before or after an additional therapeutic agent.

[0235] The antibodies described herein can be used as a biomarker to indicate that a subject is likely to respond to treatment using an antibody or antibody fragment as described herein. Also described herein are methods of determining whether a subject is likely to respond to treatment with an antibody or antibody fragment as described herein, the method comprising obtaining BCR sequence data from the subject, and using the sequence data to determine whether the subject's BCR repertoire comprises one or more antibodies likely to bind to CD33 (e.g., an antibody as described herein, e.g., an antibody that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to any particular antibody or antibody fragment described herein), wherein a subject whose BCR repertoire does not comprise one or more antibodies likely to bind to CD33 as described herein is likely to respond to treatment with an antibody or antibody fragment as described herein.

[0236] Accordingly, also described herein are methods of treating a subject diagnosed with or likely to have a disease in which CD33 plays a role (e.g., any disease in which phagocytosis of CD33-expressing cells is impaired or deficient, e.g., a neurodegenerative disease or a cancer), the method comprising: obtaining BCR sequence data from the subject; using the sequence data to determine whether the subject's BCR repertoire comprises one or more antibodies likely to bind to CD33; and administering to a subject whose BCR repertoire does not comprise one or more antibodies likely to bind to CD33 a therapeutically effective amount of an antibody or antibody fragment as described herein.

[0237] Some methods of the present disclosure involve a sample comprising cells. The sample can be a cell culture grown in vitro. For example, the culture can comprise cells or a suspension of cells grown in a culture plate or dish. Methods according to the present disclosure can be performed in vitro, ex vivo, or in vivo, or the product can be present in vitro, ex vivo, or in vivo.

[0238] According to some aspects of the present disclosure, there is provided a kit of parts comprising an antibody according to the present application. In some embodiments, the kit comprises an antibody according to the present application and one or more of the following: reagents for immunochemistry; antibodies immobilized to a solid support; means for labeling antibodies; means for linking antibodies to cytotoxic moieties; and additional therapeutic agents.

[0239] ***

[0240] Features disclosed in the foregoing description, or in the appended claims, or in the drawings, in their particular form or in terms of the manner in which the disclosed function is performed, or in terms of the method or process for obtaining the disclosed result, may suitably be used alone or in any combination of such features to implement the invention in its various forms.

[0241] Although the invention has been described in conjunction with the foregoing exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the invention set forth above are to be considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

[0242] To avoid any doubt, any theoretical explanations provided herein are offered for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0243] Any section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter described.

[0244] Throughout this specification (including the appended claims), unless the context otherwise requires, the words “comprising” and “including” and variations thereof shall be understood to imply the inclusion of the indicated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0245] It is important to note that, unless the context clearly indicates otherwise, nouns without quantifiers as used in the specification and appended claims include plural pronouns. A range herein may be expressed as “about” a particular value, and / or “about” another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to numerical values ​​is optional and means, for example, + / - 10%.

[0246] sequence

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] Table 0. Sequences used in this specification.

[0253] In the above Table 0 and throughout this specification, antibodies can be referred to by their reference number as shown in the above table (e.g. ATL_0005802), or by the same reference number without the initial string “0” (e.g. ATL_5802), or by the same reference with only the number (e.g. 5802). For example, antibody ATL_0005802 can be referred to interchangeably as “ATL_0005802”, “ATL_5802”, and “5802”. The symbols “VH” and “VL”, when appended to an antibody reference (whether as a prefix or a suffix), refer to the heavy chain variable domain of the reference antibody. The symbols HCDR1, HCDR2, and HCDR3, when appended to an antibody reference (whether as a prefix or a suffix), refer to the heavy chain CDRs (CDR1, CDR2, and CDR3, respectively) of the reference antibody. The symbols LCDR1, LCDR2, and LCDR3, when appended to an antibody reference (whether as a prefix or a suffix), refer to the light chain CDRs (CDR1, CDR2, and CDR3, respectively) of the reference antibody. The symbols HFWR1, HFWR2, HFWR3, and HFWR4, when appended to an antibody reference (whether as a prefix or a suffix), refer to the heavy chain framework regions (FWR1, FWR2, FWR3, and FWR4, respectively) of the reference antibody. The symbols LFWR1, LFWR2, LFWR3, and LFWR4, when appended to an antibody reference (whether as a prefix or a suffix), refer to the light chain framework regions (FWR1, FWR2, FWR3, and FWR4, respectively) of the reference antibody.

[0254] Examples

[0255] The following examples demonstrate the identification of new antibodies with therapeutic potential from a cohort of subjects with Alzheimer's disease or considered at risk of developing Alzheimer's disease, as well as cognitively healthy centenarians that are seropositive for CD33 antibodies (Example 1), as well as phage display analysis from libraries 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.

[0256] Materials and Methods

[0257] Serum and plasma ELISA for identifying subjects with CD33 reactivity

[0258] CD33 recombinant human antigen (R&D Systems, cat: 10375-SL-050) or negative control lysozyme (MP Biomedicals #195303) were directly adsorbed to ELISA plates at 3 pg / ml (50 pl per well) and incubated overnight at 4°C. Plates were washed with phosphate-buffered saline (PBS). Plates were blocked with 200 pl / well of blocking solution (1% bovine serum albumin (BSA) w / v in PBS) for 1 hour at room temperature. After this, the blocking solution was removed and serum and plasma samples to be evaluated were diluted 1 / 100 in blocking solution (1% BSA w / v in PBS) and applied to the plates. Plates were incubated for 1 hour at room temperature. Plates were 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 incubated for 1 hour at room temperature to detect antibody binding. Plates were washed with PBS / 0.1% Tween and 3,3',5,5;-tetramethylbenzidine (TMB) solution (Life Technology; #002023) was added to each well. Plates were incubated for 5 minutes at room temperature and then a stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm on a Molecular Devices FilterMax F5 plate reader.

[0259] Phage ELISA and sequence analysis of scFv derived from phage display

[0260] Phages were prepared by growing each phagemid-bearing TG1 clone from a glycerol stock in 100 μΐ of 2TY AG (2TY medium supplemented with 100 μg / ml ampicillin and 2% glucose) at 37°C and aeration to an optical density OD600=0.6, followed by rescue with helper phage (Invitrogen, cat: 18311-019) added at MOI 10 for 1 hour and medium change to 2TYAK (2TY medium supplemented with 100 μg / ml ampicillin and 50 mg / ml kanamycin). Cultures were then incubated overnight at 25°C and good aeration, and on the next day phages were isolated from bacteria by centrifugation at 3200 rpm for 10 minutes. Phage-containing supernatant was transferred to a new plate and blocked with 3% milk w / v in PBS.

[0261] CD33 recombinant human antigen (R&D Systems, cat: 10375-SL-050) or negative control lysozyme (MP Biomedicals #195303) were directly adsorbed to ELISA plates at 3 μg / ml (50 μΐ per well) and incubated overnight at 4°C. Each antigen-coated plate was washed with PBS and blocked with 200 ul / well of blocking solution (3% milk w / v in PBS) for 1 hour at room temperature. After this, the blocking solution was removed and the blocked phage samples to be evaluated were applied to the plates. Plates were incubated for 1 hour at room temperature. Each plate was washed with PBS / 0.1% Tween and incubated with anti-M13 HRP (Sino Biological, #11973-MM05T-H) for 1 hour at room temperature to detect phage binding. Plates were washed with PBS / 0.1% Tween and TMB solution (Life Technology; #002023) was added. Plates were incubated for 5 minutes at room temperature and then a stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm on a Molecular Devices FilterMax F5 plate reader.

[0262] Single point ELISA binding to full CD33 and CD33 C2 domain and related CD33 proteins

[0263] Seven IgGl switcher antibodies were tested in single point ELISA against a panel of antigens: human recombinant CD33 (SinoBiological cat: 12238-H08H), Cynomologus / Rhesus CD33 (SinoBiological, cat: 90303-C08H), human recombinant CD33 C2 domain (produced in house), mouse CD33 (SinoBiological, cat: 50712-M08H), recombinant human Siglec-6 / CD327 Fc chimera (R&D Systems, 2859-SL), recombinant human Siglec-7 / CD328 Fc chimera (R&D Systems, cat: 1138-SL-050), recombinant human Siglec-8 Fc chimera (R&D Systems, cat: 9045-SL), recombinant human Siglec-9 Fc chimera (R&D Systems, cat: 1139-SL). Following the single point ELISA, for each antibody and antigen pair for which a positive signal was detected, a titration ELISA was performed to obtain EC50 values.

[0264] Each recombinant protein antigen or negative control lysozyme (MP Biomedicals #195303) was directly adsorbed to ELISA plates at 3 pg / ml (50 mΐ per well) and incubated overnight at 4°C. Each plate was washed with PBS. The plates were blocked with 200 ul / well of blocking solution (1% BSA w / v in PBS) for 1 hour at room temperature. After this, the blocking solution was removed and the antibodies to be evaluated were diluted to 100 pg / ml in blocking solution (1% BSA w / v in PBS) and applied to the plates. The plates were incubated for 1 hour at room temperature. Each plate was washed with PBS / 0.1% Tween and anti-human (Fab)’2-HRP (Jackson Immunoresearch #109-035-097; Lot: 148466) was added to the plates and incubated for 1 hour at room temperature to detect antibody binding. Each plate was washed with PBS / 0.1% Tween and TMB solution (Life Technology; #002023) was added. The plates were incubated for 5 minutes at room temperature and then a stop solution (0.5 M sulfuric acid) was added. The absorbance was read at 450 nm on a Molecular Devices FilterMax F5 plate reader.

[0265] Biolayer interferometry for characterization of CD33 binding kinetics

[0266] Eleven IgGl switch-type antibodies were tested for binding to human recombinant CD33 (R&D Systems, 10375-SL-050) in a BioLayer Interferometry (BLI) experiment.

[0267] Each antibody was loaded onto a row of eight anti-human IgG Fc capture biosensor tips (AHC tips, Sartorius Stedim part #18-5060) and the row was then equilibrated in kinetics buffer (Sartorius Stedim part #18-1105) before being dipped into a dilution series of CD33 analyte in kinetics buffer at 25 °C. Binding responses were measured. The tips were then dipped into kinetics buffer and responses were measured. Association and dissociation curves were fitted to the response metrics using the manufacturer’s analysis software (Octet Data Analysis HT12.0) and kinetic parameters were calculated.

[0268] Phagocytosis of inflammatory stimulated human iPSC-derived microglia

[0269] Induced pluripotent stem cell (iPSC) derived microglia (FujiFilm (CDI) # R1131) were seeded at 20000 cells / well and rested for 3 days with a 50% media change on day 3 of culture. On day 4, cells were treated with 50 ng / ml LPS (tlrl-eklps Invivogen) for 24 hours and then treated with 50 μg / ml test antibody for a further 24 hours. Cells were then treated with 0.5 μg / well of pHrodo red labelled beta amyloid and red fluorescence was monitored using Incucyte S3. (Beta Amyloid (1-42) Aggregation Kit, rPeptide #A-1170-025 labelled with pHrodo Red, Succinimidyl Ester (ThermoFisher Scientific #P36600)).

[0270] Human whole blood phagocytosis assay

[0271] Blood was derived from NHS Blood and Transplant Apheresis Cone and diluted 1 :10 in RPMI medium (Invitrogen A4192301). 25 mI diluted blood and 2 ml 1 x lysis buffer (ebioscience 00-4300-54, 10x in water diluted to 1 x just before use) were added to each well and mixed thoroughly by pipetting. Samples were incubated for 15 minutes at room temperature and then spun at 400 g for 5 minutes to sediment the remaining white blood cells. The supernatant was discarded to waste and the cells were resuspended in 100 mI of RPMI medium containing 10 pg / ml S. aureus pHrodo Red and 10 pg / ml antibody. Samples were incubated for 3 hours at 37°C and then washed by adding 1 ml PBS / 2% FBS, spinning at 400 g for 5 minutes to sediment and then discarding the supernatant to waste. Samples were resuspended in 50 ul of 1 / 50 human Fc Block diluted in PBS / 2% FBS (BD Biosciences 564219) and incubated for 15 minutes at 2 to 8°C. On top of the Fc Block, 50 mI of anti-human CD14 APC diluted 1 / 100 in PBS / 2% FBS was added and incubated for another 30 minutes at 2 to 8°C. Samples were washed as above with 1 ml PBS / 2% FBS and then resuspended in 200 ul PBS / 2% FBS containing 1 :10,000 DAPI for analysis on a BD FACSymphony flow cytometer. Live monocytes were gated (DAPI negative, CD14 positive) and the pHrodo Red fluorescence within the live monocyte population was assessed using the 561-586 / 15 channel.

[0272] Depletion of CD33 on human monocytes

[0273] Previously frozen PBMC isolated human monocytes were thawed, washed and left O / N at 37°C, 5% C02 (standard cell culture conditions) in RPMI medium (Invitrogen A4192301) + 10% fetal bovine serum (FBS). The next day, the settled monocytes were collected and washed once in RPMI + 10% FBS. Cells were then resuspended at a final concentration of 2 x 10 6 cells / ml and 50 mI were plated in each well of a flat bottom 96 well plate, i.e. 1 x 10 5Cells were plated at 50,000 cells / well (Corning, Cat. 3595). Cells were rested in the incubator while reagents were prepared. Reagents (ATL_5802, ATL_5810, ATL5338, ATL_5909) were prepared in round bottom 96 well plates (Corning, cat. 3799) at twice the final concentration in RPMI + 10% FBS, either at a single concentration (80 nM) or a series (80, 60, 40, 30, 20, 2 nM) in 60 μl / well. ATL5338 is an isotype control produced in-house based on the FITC conjugated antibody 4-4-20 described in Jung et al JMB 294, 163 (1999). 50 μl / well was then transferred from the reagent plate to the 96 well plate containing 50 μl / well of rested mononuclear cells, for a final volume of 100 μl, and subsequent final concentrations of 40, 30, 20, 10, 1 nM. The plate was then incubated for 5 hours under standard cell culture conditions. After incubation, the plate was maintained on ice to avoid any further internalization of the antibody. All subsequent steps were performed at 4°C in the dark. Cells were collected by centrifugation (at 300 g for 5 minutes) and the supernatant was removed. Cells were then resuspended in 50 μl Live / Dead fixable violet dead cell stain (Cat. L34955) and incubated for 7 minutes. A washing step was performed by adding 150 μl PBS / 2% FBS per well on top of each sample and the plate was 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, cat. 303428), anti-human CD33 domain C specific (Biolegend, cat. 366620), anti-human CD45 (Biolegend, cat. 304027), anti-human CD14 (Biolegend, cat. 301806), anti-human CD16 (Biolegend, cat. 302016), anti-human CD3 (Biolegend, cat. 300412) plus 1:50 human Fc block (BD Biosciences 564219) diluted in PBS-2% FBS. Staining was performed at 4°C for 40 minutes. Cells were then washed twice as above and resuspended in a final volume of 200° μl / well PBS-2% FBS for analysis on a BD FACSymphony flow cytometer. Live mononuclear cells were gated (dead stain negative, CD45 positive, CD3 negative, CD14 positive, CD16 positive / negative) and the MFI emitted by the two CD33 antibodies was recorded.

[0274] Phagocytosis modulation by CD33 engagement with freshly isolated human monocytes

[0275] Antibodies were prepared in RPMI + 10% FBS at twice the final assay concentration in a single concentration (60 nM) and 1:4 serial dilutions starting from 60 nM and distributed in round bottom 96 well plates (Corning, cat. 3799) 60 ul / well. For the wells used as negative controls, Latrunculin A was diluted 1:5000. S. aureus pHrodo baits for phagocytosis (Thermo Fisher, cat. A10010) were prepared in RPMI + 10% FBS medium at double the final concentration of 10 pg / ml and distributed in flat bottom 96 well plates (Corning, Cat. 3595). Then, human monocytes were isolated from fresh PBMCs following the Miltenyi protocol (Cat. 130-096-537). The obtained monocytes were resuspended at 1 x 10 6

[0276] Study design for in vivo CD34+ NSG mouse studies

[0277] There were 17 treatment groups. Each treatment group was given a letter so that the observer was unaware of the identity of the treatment group. Mice were randomly assigned to treatment groups.

[0278] This experiment consisted of 17 conditions (total of 65 animals):

[0279] 1. Control 10 mg / kg pre-dose & 24 hours

[0280] 2. Test antibodies (ATL5802, ATL5810 and ATL5909) 10, 40 mg / kg; pre-dose & 24 hours

[0281] 3. Control, 10 mg / kg pre-dose, 24 hours, 7 days

[0282] 4. Test antibodies (ATL5802, ATL5810 and ATL5909) 10, 40 mg / kg; pre-dose, 24 hours, 7 days

[0283] ​5. Test antibody (ATL5802, ATL5810 and ATL5909) 1 mg / kg; pre-dose, 4 hours, 24 hours, 7 days

[0284] Group 1 and 2 consisted of 3 animals per group (n = 3 biological replicates, total of 21 animals), Group 3 and 4 consisted of 5 animals per group (n = 5 biological replicates, total of 35 animals), and Group 5 consisted of 3 animals per group (n = 3 biological replicates, total of 9 animals). Blood volumes were guided by UK Home Office guidelines (maximum of 200 μΐ per mouse in total), therefore Group 1 and 2 were 200 μΐ pre-dose, Group 3 and 4 were 100 μΐ pre-dose and 24 hours, and Group 5 were 66 μΐ pre-dose, 4 hours and 24 hours. The final time point for each group included a terminal blood sample. Mice were humanised from two separate donors (n = 30 from the first donor and n = 35 from the second donor). Groups 1, 2 and 5 will use animals from donor 1, with Groups 3 and 4 from donor 2.

[0285] Samples for each group were used as follows:

[0286] • Groups 1 and 2 - pre-dose (ELISA), 24 hours (ELISA & flow cytometry)

[0287] • Groups 3 and 4 - pre-dose (ELISA), 24 hours (flow cytometry), 7 days (ELISA & flow cytometry)

[0288] • Group 5 - all time points (ELISA) Dose volumes for all treatments were 10 ml / kg.

[0289] Phagocytosis of peripheral monocytes from CD34+ mice

[0290] Blood from CD34+ NSG mice was processed as follows. 25 mΐ of mouse blood and 2 ml of lx lysis buffer (ebioscience 00-4300-54, 10x in water diluted to lx just before use) were added to each well and mixed thoroughly by pipetting. The samples were incubated for 15 minutes at room temperature (18 to 22 °C) and then spun at 400 g for 5 minutes to pellet the remaining white blood cells. The supernatant was discarded and the cells were resuspended in 100 mΐ of RPMI medium containing 10 pg / ml of S. aureus pHrodo. The samples were incubated for 3 hours at 37 °C and then washed by adding 1 ml of PBS / 2% FBS, spinning at 400 g for 5 minutes to pellet, and then discarding the supernatant to waste. The samples were resuspended in 50 ul of PBS / 2% FBS containing 1 / 50 Human Fc Block (BD Biosciences 564219) and 1 / 100 Mouse Fc Block (Biolegend 156603) and incubated for 15 minutes at 2 to 8 °C. An antibody mix containing the following antibodies was prepared at a dilution of 1 / 50 in PBS / 2% FBS: Anti-mouse CD45 AF488 (Biolegend 157608), Anti-human CD45 APC (Biolegend 304012), Anti-human / mouse CD11b BV785 (Biolegend 101243). To each sample, 50 mΐ of antibody mix was added on top of the Fc block in the well. The samples were incubated for another 30 minutes at room temperature in the dark. The samples were washed as described above with 1 ml of PBS / 2% FBS and then resuspended in 200 mΐ of PBS / 2% FBS containing 1:10,000 DAPI for analysis on a BD FACSymphony flow cytometer. Live human monocytes were gated (DAPI negative, human CD45+, CD11b+ positive) and the pHrodo fluorescence within the live human myeloid population was assessed using the 561-586 / 15 channel.

[0291] Size exclusion chromatography (SEC-HPLC)

[0292] Antibody samples were diluted to 1 mg / ml in 20 mM Histidine acetate, 150 mM NaCl pH 5.5 for running on a Vanquish Flex (Thermo Scientific) on a Zorbax GF-250 SEC-HPLC column (Agilent). The 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 at +25C for 25 minutes per sample. Data were collected at 280 nm and the chromatograms were integrated using Chromeleon software (Thermo Scientific).

[0293] Capillary isoelectric focusing (cIEF)

[0294] Charge variant analysis was performed by preparing a master mix to dilute the antibody samples for running on a Maurice instrument (Protein Simple) on a cIEF cartridge. The master mix had the following final concentrations: methylcellulose 0.35% (Protein Simple, 101876), pharmalyte pH 3 to 10 4% (Protein Simple, 17-0456-01), 10 mM arginine (Protein Simple, 042-691), pi markers 4.05 and 9.99 0.01% (Protein Simple, 046-029 and 046-034). The samples were diluted in the master mix at 0.15 to 0.25 mg / ml and run at 1500 volts for 1 minute followed by 3000 volts for 4.5 minutes. A system suitability standard (Protein Simple, 046-044) was also run at the start of the run. Stability data generated at 2 weeks and 4 weeks were overlaid to compare the charge species profile.

[0295] Thermal shift assay

[0296] Protein thermal shift measurements were performed on Uncle (Unchained labs). Antibodies were diluted to 1 mg / ml or 5 mg / ml in a buffer of 20 mM Histidine acetate, 150 mM NaCl, pH 5.5 and run through a temperature ramp of 25°C to 95°C at a rate of 0.5°C / min. Samples were run in triplicate with 8.8 μl loaded in 3 different wells of the uni (Unchained Lab). Laser settings were set to achieve an initial fluorescence in the range of 300 to 350 nm of 10000 to 50000 counts. The melting temperature (Tml / Tm2) and the aggregation temperature (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.

[0297] Epitope mapping

[0298] Variants of the CD33 extracellular domain were prepared as individual recombinant proteins. The variations were based on differences between human CD33 and cynomolgus CD33, as the lead sequence of the mAb is known to bind to human but not to cynomolgus CD33.

[0299] A DNA sequence encoding human CD33 residues 21 to 232 was synthesized and cloned into pcDNA3.1 (+) by Genscript. Insertion of natural residues 18 to 20, single residue and multiple residue cynomolgus mutations were performed using the Q5 site-directed mutagenesis kit (NEB). Wild type plasmid (residues 18 to 232) and seven mutant plasmids were transfected into 15 mL Expi293F cell (Thermofisher) cultures according to the manufacturer’s instructions. The seven mutant plasmids were: These mutations were in Figure 11AFigure 6. ELISA of the eleven IgGl antibodies against wild type CD33 and all seven CD33 variant proteins. The supernatant was purified by Ni-affinity on an AKTA pure (Cytiva) using a HisTrap excel column (Cytiva) followed by size exclusion chromatography using a Superdex Increase 10 / 300 GL column (Cytiva) 5 days after transfection. Eleven IgGl antibodies were tested in a single point ELISA against wild type CD33 and all seven CD33 variant proteins. Each recombinant protein antigen or negative control lysozyme (MP Biomedicals #195303) was directly adsorbed to an ELISA plate at 3 pg / ml (50 mΐ per well) and incubated overnight at 4°C. Each plate was washed with PBS. The plates were blocked with 200 ul / well of blocking solution (1% BSA w / v in PBS) for 1 hour at room temperature. After this, the blocking solution was removed and the antibodies to be evaluated were diluted to 266 nM in blocking solution (1% BSA w / v in PBS) and applied to the plates. The plates were incubated for 1 hour at room temperature. Each plate was washed with PBS / 0.1% Tween and anti-human (Fab)’2-HRP (Jackson Immunoresearch #109-035-097; Lot: 148466) was added to the plates and incubated for 1 hour at room temperature to detect antibody binding. Each plate was washed with PBS / 0.1% Tween and TMB solution (Life Technology; #002023) was added. The plates were incubated for 5 minutes at room temperature before a stop solution (0.5 M sulfuric acid) was added. The absorbance was read at 450 nm on a Molecular Devices FilterMax F5 plate reader. The absorbance values were converted to % of WT CD33 binding, where WT = 100%.

[0300] Antigen constructs

[0301] The following Table 1 summarizes the antigen constructs used in these examples.

[0302] Experiments Antigen effects Antigen constructs ( / lots) used and construct description Supplier and catalog number Phage display Selection antigens CD33_human_M17-H259_002_004 CHO-derived human Siglec-3 / CD33 protein extracellular domain Met17-His259 with C-terminal 6-His tag R&D Systems, 10375-SL-050 ELISA Binding characterization (C domain only) CD33_human_ECD_Cdomain_His_007 HEK-derived human Siglec-3 / CD33 protein C domain Asp140-Thr232 with C-terminal 6-His tag Internal SEQ ID NO: 175 ELISA Binding characterization CD33_human_ECD, His_004_001 HEK-derived human Siglec-3 / CD33 protein extracellular domain Met1-His259 with C-terminal polyhistidine tag Sino Biological, 12238-H08H BLI Binding characterization (kinetics) CD33_human_M17-H259_002_006 CHO-derived human Siglec-3 / CD33 protein extracellular domain Met17-His259 with C-terminal 6-His tag R&D Systems, 10375-SL-050 ELISA Cross-reactivity check (mouse) CD33_mouse_His_002_001 HEK-derived mouse Siglec-3 / CD33 protein extracellular domain Met1-Glu240 with C-terminal polyhistidine tag Sino Biological, 50712-M08H ELISA Cross-reactivity check (cynomolgus monkey) CD33_cyno_His_001_001 HEK-derived cynomolgus monkey Siglec-3 / CD33 protein extracellular domain Met1-Gly248 with C-terminal polyhistidine tag Sino Biological, 90303-C08H ELISA Off-target binding check Table 1: Summary of antigen constructs used in experiments SIGL6_human_Fc chimera_001_001 CHO-expressed human Siglec-6 extracellular domain Gln27-Val331 with C-terminal human IgGl Fc fusion domain (Pro100-Lys330) R&D Systems, 2859-SL ELISA Off-target binding check SIGL7_human_Gln19-Gly357_001_002 NS0-expressed human Siglec-7 extracellular domain Gln19-Gly357 with C-terminal human IgGl Fc fusion domain (Pro100-Lys330) R&D Systems, 1138-SL ELISA Off-target binding check SIGL8_human_Fc chimera_001 NS0-expressed human Siglec-8 extracellular domain Met17-Ala363 with C-terminal human IgGl Fc fusion domain (Pro100-Lys330) R&D Systems, 9045-SL ELISA Off-target binding check SIGL9_human_Fc chimera_001 NS0-expressed human Siglec-9 extracellular domain Gln18-Gly348 with C-terminal human IgGl Fc fusion domain (Pro100-Lys330) R&D Systems, 1139-SL ELISA Epitope mapping CD33M2_ECD_18-232_WTHEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag (wild-type sequence) (sequence provided in Table 0 without terminal 6xH tag) Internal SEQ ID NO: 226 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos1 [P1] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutations N20R F21V W22R Q24E (sequence provided in Table 0 without terminal 6xH tag) Internal SEQ ID NO: 227 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos1_MutPos6 [P6+1] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutations N20R F21V W22R Q24E P132T (sequence provided in Table 0 without terminal 6xH tag) Internal SEQ ID NO: 228 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos2 [P2] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutations I47V Y50H D51T K52R (sequence provided in Table 0 without terminal 6xH tag) Internal SEQ ID NO: 229 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos2_MutPos5 [P2+5] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutations I47V Y50H D51T K52R R122K (sequence provided in Table 0 without terminal 6xH tag) internal SEQ ID NO: 230 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos3 [P3] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutation I67V R69L (sequence provided in Table 0 without terminal 6xH tag) internal SEQ ID NO: 231 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos4 [P4] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutation Q83R (sequence provided in Table 0 without terminal 6xH tag) internal SEQ ID NO: 232 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos6 [P6] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutation P132T (sequence provided in Table 0 without terminal 6xH tag) internal SEQ ID NO: 233 ELISA Epitope mapping CD33M2_ECD_18-232_MutPos5 [P5] HEK-derived human Siglec-3 / CD33 protein residues 18 to 232 with C-terminal 6-His tag and mutation R122K (sequence provided in Table 0 without terminal 6xH tag) internal SEQ ID NO: 234

[0303] Table 1. Overview of antigen constructs used in examples.

[0304] Mouse pK study

[0305] Male homozygous C57BL / 6-Cd33tm1 (CD33) / Bcgen (common name: hCD33) were obtained from Biocytogen Technology Co., Ltd and shipped to Pharmaron (Ningbo, China) for this study. At 6 to 8 weeks of age, mice (n = 3 per treatment group) were weighed and administered a single intraperitoneal (i.p.) dose of freshly prepared antibody solution at 1 mg / kg and / or 10 mg / kg in a dosing volume of 5 ml / kg. All mice had free access to food and water. Cage-side observations were performed daily and clinical observations were performed prior to dosing and at each sample collection time point.

[0306] Blood samples were collected from the orbital vein 4 hours, 24 hours, and 7 days (144 hours) after single i.p. administration of antibody. Briefly, whole blood samples were allowed to stand at room temperature for 30 minutes and then centrifuged at 3,500 x g for 15 minutes at 4 °C to obtain the serum fraction. Serum samples were immediately transferred to cryogenic vials and stored at -75 °C until analysis.

[0307] Samples were treated and analyzed as follows. Serum samples were diluted in assay buffer (0.1% bovine serum albumin [BSA] - 0.05% Tween 20 - PBS) and vortexed for 30 seconds before loading into microplates (Corning Incorporated 96-well cell culture plates). Serum concentrations of test articles were determined using a custom-developed ELISA method (Pharmaron, Ningbo, China) using goat anti-human IgG Fc polyclonal primary antibody (5 ug / ml) and goat anti-human IgG monoclonal antibody, HRP (1:5000). Readouts were acquired on a Molecular Devices SpectraMax ID3 and PK calculations were performed using WinNonlin (Phoenix™, version 8.3).

[0308] Alzheimer’s disease model in vivo

[0309] On postnatal day 4 (P4), male and female Rag2- / - Il2rg- / - hCSF1KI App NL-G-F Mice (hereafter referred to as APP NL-G-FMancuso et al., 2022). Starting at approximately 4 months of age, mice were treated once weekly for 12 weeks with 40 mg / kg of ATL_5802 (n=6) or isotype control antibody ATL-5338 (n=7). To assess phagocytosis in vivo, the fluorescent beta amyloid label, methoxy-X04 (MX04) was used and the extent of beta amyloid uptake was assessed by measuring the percentage of MX04 positive microglia using flow cytometry. At approximately 16 weeks of age, mice were injected i.p. with freshly prepared methoxy-X04 (ab142818, prepared in DMSO:HBSS at a 1:1 ratio) at 10 mg / kg. 24 hours after injection of MX04, mice were injected with the euthanasia agent Dolethal and perfused with ice cold PBS. Harvested brain tissue was immediately placed in FACS buffer (1x PBS containing 2% FBS / FCS and 2 mM EDTA) for downstream processing by flow cytometry. For flow cytometry analysis, brain tissue was dissociated using the Miltenyi neural tissue dissociation kit according to 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 Cat#65-0865-14), APC mouse anti-human monoclonal CD45 antibody (1:50, BD Bioscience clone HI30 Cat#555485) and PE conjugated recombinant human anti-mouse CD11b antibody (1:100, Miltenyi Biotec Cat#130-113-806). After exclusion of non-viable cells, cells were gated using hCD45 and the median florescent intensity (MFI) of the human microglia population was measured and set as a threshold value for each mouse. The percentage of MX04 positive microglia was calculated by quantifying the proportion of MX04+ microglia above this set threshold.

[0310] Tau phagocytosis by human iPSC-derived microglia

[0311] iPSC-derived microglia from FujiFilm (CDI) (Cat. No. R1131) were seeded at 20,000 cells / well and rested for 3 days, 50% of the medium was changed on day 3 of culture. On day 4, cells were treated with 50 ng / ml LPS (tlrl-eklps, Invivogen) for 6 hours and subsequently treated with 50 pg / ml test antibody for 24 hours. Cells were then treated with 0.5 pg / well of Tau P301S (Abeam #ab246003) labelled with pHrodo. Red (ThermoFisher Scientific #P36600), which was directly sonicated once for 5 minutes prior to treatment. Red fluorescence was monitored using Incucyte S3.

[0312] Quantification of cytokines in culture medium of iPSC microglia, total PBMC and isolated monocytes

[0313] iCell microglia (Cellular Dynamics C1110) were seeded at 25,000 cells / well in iCell microglia media in flat bottom poly-D-lysine (PDL) coated 96 well plates. PBMCs were isolated from leukapheresis cones (NHS BT) using Lymphopure (Biolegend 426201) and Leucosep tubes (Greiner 227290). After 24 hours, the media was harvested and cytokine levels were measured using a flow cytometry based multiplex immunoassay (LEGENDplex TM Biolegend 741081, 740930, 740502, 740796 or 741795).

[0314] PBMCs were isolated from leukapheresis cones (NHS BT) using Lymphopure (Biolegend 426201) and Leucosep tubes (Greiner 227290).

[0315] Monocytes were isolated from PBMCs using a Pan Monocyte Isolation kit (Miltenyi 130-096-537) and MS columns (Miltenyi #130-042-201).

[0316] PBMCs or monocytes were plated at 100,000 cells per well in RPMI media (Gibco A4192301) + 10% FBS in U-bottom plates (PBMCs) or flat bottom plates (monocytes). Cells were treated with 50 µg / ml of isotype control antibody (anti-fluorescein) or ATL_5802 antibody. Some assays included treating cells with 50 ng / ml LPS (Invivogen, tlrl-eklps) for 6 hours prior to antibody addition. After 24 hours, media was harvested and cytokine levels were measured using flow cytometry-based multiplex immunoassays (LEGENDplex TM Biolegend 741081, 740930, 740502, 740796, or 741795).

[0317] Cytokines tested in these immunoassays include:

[0318]

[0319] (CXCL8 (IL-8), CCL11 (eotaxin), CCL17 (TARC), CCL2 (MCP-1), CCL5 (RANTES), CCL3

[0320] Monocytes: CXCL8 (IL-8), CCL11 (eotaxin), CCL17 (TARC), CCL2 (MCP-1), CCL5 (RANTES), CCL3

[0321]

[0322] iPSC microglia: CXCL8 (IL-8), CCL11 (eotaxin), CCL17 (TARC), CCL2 (MCP-1), CCL5 (RANTES), CCL3

[0323]

[0324] Microglia culture

[0325] Microglia cells were cultured according to the manufacturer’s recommended protocol (iCell Microglia, FUJIFILM Cellular Dynamics, Inc., Cat. No: R1131). Prior to cell seeding, the culture surface was prepared by coating with a 0.1 mg / ml solution of poly-D-lysine (Gibco) and incubated overnight to enhance cell adhesion. Subsequently, cells were plated in 24-well plates at a density of 120,000 cells / well. After a 4-day resting period, microglia cells were subjected to the stimulation protocol. This included pre-treatment of cells with 50 ng / ml LPS (Sigma Aldrich, Cat. No: L2637-10Mg) for 24 hours, followed by addition of specific antibodies for 20 minutes, 6 hours, and 24 hours. Due to the transient nature of phosphorylation, measuring phosphorylated proteins at the 20-minute mark is generally considered ideal. Therefore, later time points, such as 6 and 24 hours, were used to observe changes in protein levels.

[0326] Western blot

[0327] For protein extraction, we employed RIPA cell lysis buffer, which consists of 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% deoxycholic acid sodium, and 1% SDS, supplemented with protease and phosphatase inhibitors from Sigma. Automated Western testing services were provided by RayBiotech, Inc. (Peachtree Corners, GA USA). Samples were loaded into the automated capillary electrophoresis instrument at a concentration of 0.1 mg / mL. The following antibodies were employed for protein analysis: Phospho-SYK (Tyr525 / 526) (Cell Signaling; Cat. No 12710T) and total SYK (Cell Signaling; Cat. No 113198S). As a loading control, we used a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, which was provided by RayBiotech from their service library.

[0328] Microglia culture for RNA sequencing

[0329] Microglia cells were cultured according to the manufacturer’s recommended protocol (iCell Microglia, FUJIFILM Cellular Dynamics, Inc., Cat. No: R1131). Prior to cell seeding, the culture surface was prepared by coating with a 0.1 mg / ml solution of poly-D-lysine (Gibco) and incubated overnight to enhance cell adhesion. Subsequently, cells were plated in 24-well plates at a density of 120,000 cells / well. After a 4-day resting period, microglia cells were subjected to a stimulation protocol mimicking inflammatory conditions. This included pre-treatment of cells (i.e. prior to antibody treatment) with 10 ng / ml lipopolysaccharide (LPS, Sigma Aldrich, Cat. No: L2637-10Mg) and 20 ng / ml interferon-gamma (IFN-gamma, PeproTech, Inc., Cat. No: 300-02-20ug) for 24 hours, followed by addition of ATL_0005802, ATL_0005854, ATL_0005909 or ATL_0005338 (isotype) for another 24 hours, resulting in a total exposure time of 48 hours for LPS / IFN gamma (LI). This was compared to conditions mimicking healthy conditions, where cells were pre-treated with vehicle only. After treatment, cell culture medium was aspirated and cells were lysed directly in the wells by addition of 400 pL TRIzol reagent (Invitrogen, Cat. No: 15596026). Ensure that the TRIzol reagent covers the surface of each well evenly. Gently rock the plate to facilitate thorough mixing and efficient cell lysis. The cell lysate, containing both TRIzol reagent and cell contents, was then carefully transferred to a separately labeled tube using a pipette. These tubes were immediately placed on dry ice for rapid cooling, a critical step to maintain RNA integrity. Subsequently, samples were stored at -80 °C for long-term storage until RNA extraction.

[0330] RNA extraction, library preparation and sequencing

[0331] Collected samples were processed for next generation sequencing as follows. Total RNA was extracted from TRIzol frozen stored samples following the manufacturer’s recommendations. RNA integrity was assessed by Tapestation (Agilent Technologies, Palo Alto, CA, USA) and concentration by Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA, USA). Low quality samples were excluded. Poly(A) mRNA was enriched with Oligod(T) beads following the manufacturer’s recommendations.

[0332] Libraries were prepared from RNA with ERCC spike-in using Twist RNA Library Preparation Kit and Twist UMI Adapter system (Twist Bioscience) following the manufacturer recommendations. Library size was assessed on Tapestation, concentration was assessed by Qubit 2.0 fluorometer and final quantification was assessed by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Multiplexed samples were sequenced on an Illumina HiSeq4000 sequencer using 2x150bp reads with single index.

[0333] Bioinformatic analysis

[0334] Quality control, sequence alignment and quantification were performed as follows. Briefly, raw data quality was evaluated with 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). Trimmed and deduplicated reads were mapped to Homo sapiens GRCh38.p7 with ERCC genes using STAR aligner v.2.5.2b (Dobin et al., 2013). Unique gene hit counts were computed by using “featureCounts” of Subread v.1.5.2 (Liao et al., 2014).

[0335] Differential expression analysis was performed with R using DESeq2 v1.38.3 (Love et al. 2014). Wald test was used to generate p-values and log2 fold changes. For each comparison of interest (i.e. vehicle + anti-CD33 antibody vs. vehicle + isotype, and LI + anti-CD33 antibody vs. LI + isotype), genes with BH adjusted p-value < 0.05 and absolute log2 fold change > 1 were considered as differentially expressed genes. Principal Component Analysis (PCA) was computed based on variance stabilized counts using the “plotPCA” function from DESeq2 (DESEq2 ‘varianceStabilizingTransformation’ function) and using the top 25% of genes with highest variability. Volcano and PCA plots were plotted using R library ggplot2 v.3.4.3 (Wickham, 2016).

[0336] ​Pathway analysis: Gene sets were retrieved from the molecular signature database MSigDb (Broad Institute, www.gsea-msigdb.org) using the R package msigdbr v.7.5.1 (Dolgalev, 2022) including curated HALLMARK sets and REACTOME pathways. For each comparison of interest, gene set enrichment analysis was performed by fgsea v1.24.0 (Korotkevich, 2019) using the corresponding gene list ordered based on Wald statistics (Subramanian et al. 2005). Gene sets / pathways with -log10(adjusted p-value) > 10 were considered enriched in one condition compared to the other. The directionality of changes was assessed using the normalised enrichment score (NES). Plots were generated using the “plotEnrichment” function of fgsea, ggplot2 and ggpatern v.1.1.0-0 (Wilk, 2021).

[0337] Establishment of quadruple culture system

[0338] ​Induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (Fujifilm; Cat. No: R1061), GABAergic neurons (Fujifilm; Cat. No: R1013), astrocytes (Fujifilm; Cat. No: R1092) and microglia (Fujifilm; Cat. No: R1131) were used to establish a quad-culture system to recreate physiological conditions of the central nervous system. These cells were cultured under standard conditions until they reached the appropriate confluence for the experiments. On day 6, after establishing the quad-culture system, cells were pre-treated with 10 ng / ml lipopolysaccharide (LPS, Sigma Aldrich, Cat. No: L2637-10Mg) and 20 ng / ml interferon-gamma (IFNy, PeproTech, Inc., Cat. No: 300-02-20ug). The next day, the quad-culture system was exposed to CD33 antibody, ATL_5802 or ATL_5338 (isotype control antibody). Each antibody was used at a concentration of 50 pg / mL. The cultures were incubated with these antibodies for a further 24 hours. On day 8 of the culture, supernatants were collected. Quantification of interleukin-6 (IL-6), GFAP, IP-10 and MCP-1 in the collected samples was performed 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.

[0339] In vitro binding Retrogenix

[0340] The screen was performed by Charles River (UK) using their proprietary Retrogenix cell microarray technology (see )

[0341] Example 1 - Convergence analysis

[0342] Convergent sequence clusters from the antibody repertoire of resistant individuals can be used to identify disease-specific antibody sequences. The inventors sought to identify candidate protective antibodies from Alzheimer’s disease (AD) resistant individuals in a prospective cohort (European Alzheimer’s Disease Consortium for Dementia Prevention, ep-ad.org).

[0343] The pathophysiological changes of Alzheimer’s disease can be detected in individuals decades before the cognitive symptoms of dementia appear (i.e., the preclinical stage of the disease). For example, the concentration of Aβ42 in cerebrospinal fluid (CSF) is reduced, while the concentration of phospho-tau in CSF is elevated in early disease progression relative to healthy controls. The “A / T / N” classification scheme (Jack et al., 2016) defines a convenient binary label to describe such biomarkers: where “A” refers to the value of a beta-amyloid biomarker (amyloid positron emission tomography (PET) or CSF Aβ42); “T” refers to the value of a tau biomarker (CSF phospho-tau or tau PET); and “N” refers to a biomarker of neurodegeneration or neuronal injury ([18F]-fluorodeoxyglucose-PET, structural MRI, or CSF total tau) (Jack et al. 2016). Below 1000 pg / mL is considered amyloid positive, and above 27 pg / mL is considered pTau positive (Amft et al., 2022; Blennow et al., 2019).

[0344] From the total cohort of 2096 participants, 127 participants were selected for convergence analysis based on data and sample availability (plasma, PBMCs, and pTau and Aβ42 values from CSF and plasma), including 37 AD-resistant subjects, where resistance was defined by A+T- CSF biomarkers, and no cognitive impairment was found using Alzheimer’s disease Assessment scale—Cognitive subscale (ADAS-Cog), Mini-Mental State Examination (MMSE), and Clinical Dementia Rating Scale Sum of Boxes (CDR-SOB) (Duff et al., 2008; Balsis et al., 2015). Sequencing of the antibody repertoire of these resistant individuals revealed convergent heavy chain variable (VH) sequences between two resistant individuals, one of whom also carried the heterozygous Apo E3 / E4 genotype, further predisposing them to AD (Michaelson et al., 2014). Figure 1 The number of clonotypes (i.e., clusters of BCRs with high sequence similarity) identified in these two resistant individuals is shown, indicating 64 shared clonotypes between the two resistant individuals. Representative sequences from one of the 64 shared clonotypes are shown to bind to CD33.

[0345] Subsequently, the VH sequences identified in the resistant individuals were paired with VL using a converter-based model, which comprises an encoder-decoder model trained on a library of paired VH-VL sequences. More details on how such a model can be trained and used are provided in WO 2022 / 223451 (which is incorporated herein by reference in its entirety). The trained model takes a VH sequence as input and generates a single complementary VL sequence as output. The resulting antibody ATL_5082 (also referred to herein as ATL_0005082) was included in an ELISA screening for binding to a panel of targets associated with neurodegeneration. 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 148Q (mutant HTT exon 1), transthyretin, baculovirus particles, lysozyme. The antibody was shown to bind to CD33 (Figure 1) (p<0.05, one-way ANOVA with Dunnett’s post-hoc test, n=21, 25 samples). Figure 2 This indicates that there is an association between CD33 reactivity and AD resistance in Alzheimer’s disease (AD) resistant individuals in the EP-AD cohort.

[0346] Interestingly, serum and plasma auto-reactivity to CD33 (defined as z-score cut-off of 0.5 in serum ELISA) was observed in 21 subjects (25 samples), including one “supercentenarian” - i.e. an individual aged over 100 years with self-reported good cognitive function (Mini Mental State Examination score of at least 28 out of 30, a cognitively healthy individual (Holstege et al., 2018) (Figure 2). Figure 3

[0347] In addition, many cancers are characterized by high CD33 expression levels, in particular myeloid malignancies, such as acute myeloid leukemia and lymphoma. Furthermore, it has been shown that expression of CD33-related siglecs on tumor-associated macrophages supports cancer progression, and inhibitory siglecs can inhibit immune cell activation, suggesting that targeting siglecs is a possible immune checkpoint inhibition treatment strategy (Stanczak et al., 2022). Thus, the antibodies identified and described herein are not only useful for the treatment and / or prevention of neurodegenerative diseases, but also for cancer.

[0348] Example 2 - Deep mining of libraries from CD33-reactive subjects by phage display

[0349] ​Based on the results in Example 1, the inventors set out to screen a serum and plasma library from "supercentenarians" to identify possible protective anti-CD33 antibodies. ELISAs against CD33 were performed using plasma from a cohort of "supercentenarians" (www.100plus.nl) Figure 3 A subset of samples in Example 1. All twelve plasma samples available from this cohort were analyzed and the sample with the highest CD33 ELISA signal Figure 4A ) and low lysozyme control antigen signal Figure 4B ) was selected for phage library generation (subject SU_0000877 in Figure 4).

[0350] A phage library of scFv molecules displayed on M13 phage was generated by cloning the heavy chain variable region (VH) repertoire of subject SU_0000877 into a phagemid vector sub-library with light chain variable region (VL) sequences from healthy donors. A phage display library of 1.4 x 10 8 clones was generated and phage for phage display screening was produced.

[0351] To isolate antibodies that bind to CD33 protein, two to three rounds of phage display panning were performed on CD33 protein. Over 100 clones were analyzed by Sanger sequencing and full length sequences were selected for phage ELISA.

[0352] Subsequently, full length clones derived from the phage display selection were analyzed by phage ELISA using CD33 antigen and lysozyme control Figure 5 ). Nine clones with ELISA positive signal and unique sequences were selected for IgG conversion and further testing.

[0353] Nine antibodies derived from phage display panning were selected for IgG conversion and were made as IgGl with LALA mutations in the Fc region, IgGl variants containing L234A / L235A substitutions that reduce binding to IgG Fc receptors FcyRI, FcyRII, and FcyRIII, and to complement component Clq, reducing Fc-mediated toxicity (Lund et al. 1991). Next, these antibodies were tested in a single point ELISA against a panel of antigens including CD33 and related proteins (other sialic acid-binding immunoglobulin-like lectins, siglecs): human recombinant CD33, cynomolgus / rhesus 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, recombinant human Siglec-9 Fc chimera, along with comparative antibodies ATL_5909 (also known as “AL003” from Alector), ATL_4828 (also known as gemtuzumab), and ATL_5503 (AL003 precursor). Following the single point ELISA, a titration ELISA was performed for each antibody-antigen pair that detected a positive signal to obtain EC50 values for binding to CD33 (see Table 1).

[0354] Nine of the test antibodies (see data for ATL_5802, 5810, 5803, 5808, 5854, 5807, 5809, 5853 in Table 2A) were found to bind to full-length recombinant human CD33 (rhCD33-His) but not to the C2 domain (see also Table 2B). Figure 6where single point ELISA signal was normalized to isotype control = 100 and absorbance was measured at 450 nm). Antibodies further showed minimal species cross-reactivity in mice (mCD33-His) and cynomolgus monkey (cCD33-His). Importantly, while the test antibodies showed good binding to CD33, they did not bind to related Siglec family proteins (Siglec-6-F; Siglec-7-F; Siglec-8-F; Siglec-9-F), indicating selectivity for CD33. Kinetic data are shown in Table 2C. These data indicate that while the comparative antibodies have higher affinity for CD33, this is primarily due to lower Koff (e.g., the on-rate of ATL5909 is about 2-fold higher than that of ATL5802, but the off-rate is 2 orders of magnitude lower). This indicates that the antibodies of the present disclosure dissociate more quickly from the target, which can be the reason for their lower peripheral degradation rates (see Example 3 below). As demonstrated in Examples 4 and 5, the antibodies described herein show very significant effects on phagocytosis (even higher than the comparative antibodies), indicating a complex relationship between the mode of action of the antibodies and the target binding kinetics.

[0355]

[0356] Table 2A shows the binding signal of each antibody measured by ELISA to the following: human recombinant CD33 (rhCD33-His); human recombinant CD33 C domain (hCD33(C domain)-His); mouse CD33 (mCD33-His); cynomolgus / rhesus CD33 (cCD33-His); recombinant human siglec-6 / CD327 Fc chimera (Siglec-6 Fc); recombinant human Siglec-7 / CD328 Fc chimera (Siglec-7 Fc); recombinant human Siglec-8 Fc chimera (Siglec-8 Fc); recombinant human Siglec-9 Fc chimera (Siglec-9 Fc); and negative control (lysozyme). Data shown are raw absorbance values measured at 450 nm, normalized to isotype control set to a value of 100. ATL_0005909 is a comparative antibody (Alector AL003); ATL_0004828 is a comparative antibody (gemtuzumab). ATL_0005503 is a comparative antibody (2F5 of Alector, murine precursor to AL003 described in US11136390B2).

[0357] In summary, a set of 8 selective CD33 binding antibodies were obtained by deep mining of libraries from CD33 reactive subjects by phage display, all of which bind to full-length CD33 but not to the C2 domain.

[0358] Of these eight antibodies, two were selected for further study based on CDR3H diversity, strong binding, epitope mapping (see Example 8), and different internalization behavior (see Example 3), including antibody ATL5802 and antibody ATL5810. These antibodies were further studied in Examples 3 to 7.

[0359] Epitope mapping of these antibodies and comparative antibodies was performed as described in Example 8. The results showed that seven of the eight antibodies (5810, 5853, 5807, 5809, 5802, 5854, and 5805, see SEQ ID NO: 2B) exhibited similar binding profiles, indicating similar epitopes, and these respective binding profiles were different from the binding profiles of comparative antibodies 4828 and 5909 and antibody 5803. Therefore, antibody 5803 was not further studied.

[0360]

[0361] Table 1B shows the CDR3-H and CDR3-L sequences of the eight CD33 antibodies identified by phage display, as well as comparative antibodies ATL_0005909, ATL_0004828, ATL_0005503 (indicated by *).

[0362]

[0363]

[0364] Table 2C shows kinetic data for the sequences of the anti-CD33 antibodies identified by phage display, as well as comparative antibodies (indicated by *).

[0365] Example 3 - Cell binding and internalization

[0366] CD33 is known to be expressed at high levels in the cytoplasmic membrane of peripheral blood cells, such as monocytes. It is desirable to select a CD33 antibody that, in addition to binding to recombinant CD33 as shown in Example 2, also binds to endogenously expressed CD33, but minimizes internalization of the antibody-CD33 complex by peripheral blood cells, and thereby avoids accelerated clearance of the administered antibody by this peripheral clearance mechanism. Therefore, monocytes were tested for internalization of ATL5802 and ATL5810.

[0367] Figure 7A(see also Table 3) shows CD33 depletion on human monocytes 5 hours after addition of ATL_5802 or ATL5810 compared to the comparator antibodies ATL5909 and ATL4828. ATL_5802 shows minimal CD33 depletion on monocytes, and thus minimal internalization, similar to the isotype control antibody. ATL_5810 shows some CD33 depletion, but less than the prior art antibodies ATL5909 or ATL4828. Figure 7B and 7C shows the level of CD33 V-domain and C-domain depletion on human CD14+CD16+or CD14+CD16- cells (lower and upper panels of each graph, respectively) after addition of the indicated antibodies at a concentration of 1 nM to 40 nM. Figure 7B and Figure 7C ). Figure 7D and Figure 7E the area under the curve of CD14+CD16- monocytes (see also Table 3) and CD14+CD16+ monocytes, respectively. Figure 7B , 7C . Notably, all of the test antibodies in the present disclosure show lower CD33 internalization than the comparator antibody 5909. ATL_5802 shows very low CD33 internalization (see Figure 7A ), while ATL_5854 shows intermediate CD33 internalization (see Figure 7B . Notably, since all of the test antibodies bind to the V-domain, the change in MFI for domain V can be due to both competition with the detection antibody and depletion of CD33. In contrast, using the assay for MFI in domain C will truly reflect CD33 depletion. Data are shown for classical (CD14+CD16-) and non-classical / intermediate (CD14+CD16+) monocytes, respectively, to show that the CD33 depletion behavior of the antibodies is similar in both monocyte subpopulations.

[0368]

[0369]

[0370] Table 3. Results of flow cytometry experiments to measure cell binding and internalization. * indicates comparator antibodies.

[0371] The comparator antibody ATL5909 has a non-linear PK profile (Ward et al., 2021), and this is likely to be caused by its internalization into CD33-positive peripheral monocytes. In other words, ATL5909 is rapidly internalized in complex with CD33, and depletes CD33 on the cell surface. While this can be desirable at the site of disease (e.g. brain tissue), this property would lead to peripheral deposition of the antibody, where the antibody is internalized and degraded in monocytes. The fact that no or reduced CD33 depletion is observed in the periphery with ATL5802 and ATL5810 (as well as all other test antibodies in this disclosure) indicates that these antibodies are internalized to a different extent by peripheral blood cells than ATL5909, and can therefore be expected to have an improved PK profile compared to ATL5909, and improved availability of the antibody in the desired target region (e.g. the brain).

[0372] Example 4 - Phagocytosis assay

[0373] Microglia are brain-resident innate immune cells residing in the central nervous system and are essential for CNS health. Phagocytosis of toxic proteins such as beta amyloid is an important function of microglia. The phagocytosis pathway is inhibited by CD33 signaling, and notably CD33 is upregulated in microglia of AD patients and correlates with cognitive decline. It is therefore of interest to test whether the identified anti-CD33 antibodies can alleviate this CD33-mediated phagocytosis inhibition.

[0374] To investigate the effect of the anti-CD33 antibodies ATL5802 and ATL5810, an in vitro assay was designed using induced pluripotent stem cell (iPSC)-derived microglia to measure phagocytosis of beta amyloid (see Methods). No effect of the anti-CD33 antibodies was observed in iPSC microglia in basal, resting state (data not shown). However, this cannot be considered representative of microglia in vivo, and especially microglia in the context of neurodegeneration. Therefore, the induced pluripotent stem cell (iPSC)-derived microglia were first stimulated with LPS to induce an inflammatory response, which represents the in vivo pathological state of these cells in neurodegeneration. This step was followed by incubation with a panel of anti-CD33 antibodies. Cells were subsequently treated with pH-sensitive pHrodo red-labeled beta amyloid, and phagocytosis was measured by monitoring the red fluorescent signal in each well, with an increase in intracellular red dye level compared to isotype control indicating enhanced phagocytosis Figure 8A .

[0375] Figure 8B Results of the assay using the anti-CD33 antibodies ATL5802 and ATL5810 are shown.Figure 8B The figure shows that incubation with ATL5802 enhanced phagocytosis in iPSC microglia compared to existing technology antibody ATL5909 and isotype control. Figure 8C The results were confirmed in repeat experiments against ATL_5802, showing that phagocytosis in inflammatory iPSC microglia was repeatedly enhanced in the presence of the antibody (ATL_5802) of this disclosure, while no such enhancement was observed in the comparative antibody ATL_5909.

[0376] Next, the experiment was repeated using three other anti-CD33 antibodies: ATL_5853, ATL_5854, and ATL_6044 (strain ATL5802, see Example 7 below). The results of this experiment are shown in... Figure 8D middle. Figure 8D The figures show that all the antibodies tested in this disclosure (i.e., antibodies ATL_5802, ATL_5854, and ATL_5853) enhanced phagocytosis compared to the prior art antibody ATL_5909 or the isotype control antibody.

[0377] Figure 8E and 8F Further studies showed that ATL_5802 induced a greater degree of enhanced phagocytosis compared to TREM2 antibodies (ATL6166=Alector AL002; ATL6167=Denali DNL919; ATL6170=Vigil VGL101) currently used in clinical trials to treat AD. TREM2 is a microglia-activating signaling receptor and supports microglia survival by promoting phagocytosis of Aβ plaques (McQuade et al., 2020).

[0378] To test the effect of anti-CD33 antibodies on phagocytic activity in human peripheral myeloid cells (e.g., on monocytes) known to express high levels of CD33, the aforementioned phagocytosis assay was performed in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. This in vitro myeloid assay used pH-dependent dye (pHrodo)-labeled Staphylococcus aureus to measure the level of phagocytosis in myeloid cells (CD14+) by flow cytometry. Figure 9A ).

[0379] Figure 9B and 9CIt is shown that ATL5802 significantly enhances phagocytosis of human myeloid cells compared to the comparator antibodies ATL5909 (Alector) and ATL4828 (gemtuzumab). Results for ATL5810 do indeed suggest that this antibody can induce phagocytosis, but the results did not reach significance due to high variability in the cellular assay (compared to isotype control).

[0380] In summary, these data show that the antibodies of the disclosure can enhance phagocytosis of a range of human cells expressing CD33, including PBMCs and inflammatory iPSC-derived microglia (in particular as shown for antibodies ATL5802, ATL_5853, ATL_5854 and ATL_5810). The data further show that the antibodies of the disclosure (in particular as shown for antibodies ATL5802, ATL_5853, ATL_5854) improve phagocytosis of inflammatory human iPSC-derived microglia in vitro and of human myeloid cells ex vivo.

[0381] Example 5 - In vivo testing of antibodies in CD34+ humanized mice

[0382] In CD34 + NSG TM The pharmacodynamic properties of ATL5802 and ATL5810 were further tested in vivo in a mouse model, a humanized mouse model modified by the addition of human hematopoietic stem cells to generate a humanized immune system (a mixture of mouse and human monocytes) that can be targeted by anti-CD33 antibodies. Due to evolutionary divergence between human CD33 and non-primate CD33, there are significant species differences between mice and humans in CD33 expression patterns and ligand recognition, and thus the biology of mCD33 is not functionally relevant to the biology of hCD33 (Brinkman-Van der Linden, et al., 2003; Cao et al, 2009), and it was therefore necessary to test these antibodies in a humanized mouse model. In any case, none of the anti-CD33 antibodies described herein showed species cross-reactivity with mouse CD33, and thus in vivo testing was only performed in humanized animal models.

[0383] First, CD33 levels on humanized myeloid cells were tested as a way to assess in vivo internalization of CD33 Figure 10 ). Figure 10 It is shown that injection of ATL5802 and ATL5810 resulted in minimal depletion of CD33 on myeloid cells 24 hours after injection compared to the prior art antibody ATL5909. Figure 10The mean fluorescence intensity (MFI) of CD45+CD14+CD33+ cells obtained by flow cytometry is shown. This indicates that the peripheral drug loss of ATL5802 is minimal compared to ATL5909.

[0384] Figure 12 The study showed that 24 hours after intraperitoneal injection of ATL5802, the expression level of CD33 in human CD45+ cells in the brain decreased in a dose-dependent manner, which supports the target binding of ATL5802 in the brain.

[0385] in addition, Figure 13 This indicates that, compared to ATL5909 injection, intraperitoneal injection of ATL5802 in CD34+ mice resulted in increased incidence of human peripheral myeloid cells (CD45+). + CD14 + CD11b + The cells exhibited enhanced phagocytosis of pHrodo-labeled Staphylococcus aureus, and this effect showed a dose-dependent effect. The relatively large variability was due to the small number of human cells used in the experiments. Nevertheless, the trends observed in vivo confirmed the in vitro data presented above.

[0386] In summary, these data indicate that the ATL5802 in CD34 + In humanized mice, ATL5802 exhibited minimal peripheral internalization, along with target binding and enhanced phagocytosis in the brain. Therefore, further characterization of ATL5802 was conducted regarding antibody expression and development potential.

[0387] Example 6 - Antibody expression and developability

[0388] To ensure that the identified antibodies are stable and therefore suitable for further optimization, a developmental study was conducted.

[0389] Initial baseline values ​​were calculated for all 10 antibodies selected in Example 2, which exhibited high monomer levels as well as good thermal stability and cIEF values ​​(Table 4).

[0390] Protein aggregation during antibody storage must be kept to a minimum as it can induce immunogenic responses. Size exclusion chromatography (SEC-HPLC) was used to evaluate antibody purity and aggregation after storage under a range of conditions, including -80°C for 2 weeks, 40°C for 2 weeks, overnight shaking, 3 freeze-thaw cycles (3×FT), and low pH maintenance. Figure 14 ). Figure 14It is shown that under these forced degradation conditions, the monomer profile of ATL5802 showed little change, indicating excellent stability, and the 99% monomer values in Table 4 further reflect this. Similar results were obtained for the other candidate antibodies identified in Example 2, all of which showed acceptable stability in terms of monomer, while 5802 also showed particularly good thermal stability (Tm1).

[0391]

[0392] Table 4 shows the results of the thermal stability assay (melting temperature (Tm1) / aggregation (Tagg); cIEF SEC-HPLC (% monomer).

[0393] In addition, the 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 potency was not affected by temperature changes and freeze-thaw cycles.

[0394] In summary, all antibodies, but particularly ATL5802, showed excellent stability, and therefore ATL5802 was retained for further development.

[0395] Example 7 - Germlining in the framework regions

[0396] The lead antibody ATL5802 has 8 framework mutations relative to the corresponding germline sequence: 6 in the VH (IMGT positions 1, 6, 20, 40, 72, 85) and 2 in the VL region (IMGT positions 2 and 8). To test which of these mutations can be reversed to the germline sequence without losing binding, 9 variants of the original antibody were prepared and tested for their binding to CD33 by ELISA (Table 5). The following variants were tested: ATL6040, which has all mutations reversed to the germline sequence; and ATL6041 to ATL6048, which have all but one mutation reversed. The variants were produced as IgGl (LALA Fc mutation).

[0397]

[0398] Table 5 shows the germline mutations introduced in the respective variants of ATL_5802 and their EC50 values obtained by ELISA.

[0399] All antibodies have been made and subjected to titration ELISA testing using CD33 antigen and negative control antigen. Only one antibody, ATL6044, exhibited comparable binding to the parent, suggesting that the change or liability at framework position G40A removes an influence on binding. This suggests that the A at position 40 in the ATL5802 VH makes a positive contribution to binding to the antigen, but all other positions can be reversed back to germline. It is important to note that this does not mean that any framework sequence must be followed strictly, as all antibodies are still able to bind to the target regardless of the germlinisation mutations introduced. Rather, it suggests that it is particularly advantageous to retain the A at position 40 of ATL5802.

[0400] Example 8 - Epitope mapping

[0401] Figure 11B Results of the epitope mapping process are shown Figure 11A The results of the epitope mapping process are shown schematically in Figure 6. The binding profiles of each antibody to the different CD33 variants were used to group the antibodies into different epitope bins. Epitope bin 1 contains ATL4828 (comparator antibody Gemtuzumab), and is characterised by complete loss of binding to variants PI and P6+1, and approximately 50% loss of binding to variant P4. In contrast, epitope bin 2 contains ATL5909 (comparator antibody AL003), and is characterised by loss of binding to variants P2 and P2+5, but no effect on any other variants. Bins 3 and 4 are highly related groups, and contain seven antibodies (5810, 5853, 5807, 5809, 5802, 5854, 5808) that share the commonality of loss of binding to variants P2 and P2+5, 50% loss of binding to P4, and increased binding to PI and P6+1. Bin 5 contains antibody ATL5803 (not further characterised herein). These data suggest that other antibodies with the same epitope as the variants with proven enhanced phagocytosis activity (ATL5802, ATL5854 and ATL5853) can exhibit therapeutic efficacy. The data further suggest that the different binding kinetics of the antibodies of the disclosure shown in Example 2 compared to the comparator antibodies can be caused by different epitopes, which can support the improved therapeutic efficacy of these antibodies by reducing peripheral depletion.

[0402] Finally, the epitope mapping data also support a different mode of action of the antibodies of the disclosure compared to prior art antibodies, and further align with the reduced internalisation behaviour observed in Example 3 compared to the comparator antibodies. In particular, the epitope mapping data suggest that the antibodies described herein can interfere with sialic acid binding to CD33.

[0403] Taken together, these data indicate that the anti-CD33 antibodies described herein, and in particular ATL5802, ATL5854 and ATL5853, can show therapeutic efficacy in the treatment and / or prevention of neurodegenerative diseases (e.g. AD) as well as CD33-expressing cancers (e.g. myeloid leukemia). This is further validated by the large number of in vitro and in vivo studies described in the subsequent examples.

[0404] 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 Table 5 (VH) and Table 6 (VL). In Table 5 and Table 6, the antibodies of the present disclosure are shown in bold. “Complete” refers to the complete sequence of the VH (Table 5) or VL (Table 6).

[0405] Reference Annotation FWR1 CDR1 FWR2 CDR2 FWR3 CDR3 FWR4 Complete ATL_0004828 Gatixmab 67 41 80 49 88 56 101 1 ATL_0005338 Isotype control 68 42 81 50 89 57 102 2 ATL_0005503 2F5, AL-003 murine precursor 69 43 82 51 90 58 103 3 ATL_0005909 AL003, as described in sequence AB-64.1.2 in US2022 / 0162309 43 87 55 98 66 106 11 ATL_0005802 Lead 70 44 83 52 91 59 101 4 ATL_0005807 Back up 71 44 84 52 92 60 104 5 ATL_0005808 Back up 72 44 84 52 93 61 104 6 ATL_0005809 Back up 73 45 85 53 94 62 104 7 ATL_0005810 Back up 74 46 86 54 95 63 105 8 ATL_0005853 Lead 235 47 83 52 96 64 101 9 ATL_0005854 Lead 75 48 83 52 97 65 101 10 ATL_0006040 Full germline (FG; x8 reverted) 77 44 84 52 93 59 101 12 ATL_0006041 FG minus VH E1Q 71 44 84 52 93 59 101 13 ATL_0006042 FG minus VH Q6E 78 44 84 52 93 59 101 14 ATL_0006043 FG minus VH K20R 79 44 84 52 93 59 101 15 ATL_0006044 FG minus VH G40A 77 44 83 52 93 59 101 16 ATL_0006045 FG minus VH Q72E 77 44 84 52 99 59 101 17 ATL_0006046 FG minus VH S85G 77 44 84 52 100 59 101 18 ATL_0006047 FG minus VL S2A 77 44 84 52 93 59 101 19 ATL_0006048 FG minus VL P8S 77 44 84 52 93 59 101 20

[0406] Table 5. Summary of VH sequences. All numbers are SEQ ID Nos. mentioned in Table 0.

[0407] Reference Annotation FWR1 CDR1 FWR2 CDR2 FWR3 CDR3 FWR4 Complete ATL_0004828 Gatixmab 135 107 148 116 157 126 168 21 ATL_0005338 Isotype control 136 108 149 117 158 127 170 22 ATL_0005503 2F5, AL-003 murine precursor 137 109 150 118 159 128 171 23 ATL_0005909 AL003, as described in sequence AB-64.1.2 in US2022 / 0162309 144 109 150 118 167 128 169 31 ATL_0005802 Lead 138 110 151 119 160 129 172 24 ATL_0005807 Back up 139 111 152 121 161 130 173 25 ATL_0005808 Back up 140 112 153 122 162 131 173 26 ATL_0005809 Back up 141 113 154 123 163 132 172 27 ATL_0005810 Back up 142 114 155 124 164 133 172 28 ATL_0005853 Lead 143 115 156 125 165 134 174 29 ATL_0005854 Lead 140 112 153 120 166 236 173 30 ATL_0006040 Full germline (FG; x8 reverted) 145 110 151 119 160 129 172 32 ATL_0006041 FG minus VH E1Q 145 110 151 119 160 129 172 33 ATL_0006042 FG minus VH Q6E 145 110 151 119 160 129 172 34 ATL_0006043 FG minus VH K20R 145 110 151 119 160 129 172 35 ATL_0006044 FG minus VH G40A 145 110 151 119 160 129 172 36 ATL_0006045 FG minus VH Q72E 145 110 151 119 160 129 172 37 ATL_0006046 FG minus VH S85G 145 110 151 119 160 129 172 38 ATL_0006047 FG minus VL S2A 146 110 151 119 160 129 172 39 ATL_0006048 FG minus VL P8S 147 110 151 119 160 129 172 40

[0408] Table 6. Summary of VL sequences. All numbers are SEQ ID Nos. mentioned in Table 0.

[0409] Example 9 - Mouse pK study

[0410] The antibodies described herein are specific for human CD33 and do not show significant cross-reactivity with mouse CD33. Therefore, to assess the in vivo pharmacokinetics (PK) of antibody ATL_5802, a transgenic mouse line was used in which exons 1-3 encoding the extracellular domain of the mouse Cd33 gene were replaced by human CD33 exons 1-3. Expression of human CD33 has been validated and characterized in this mouse model before. Antibodies were administered intraperitoneally at 1 mg / kg and human IgGl levels in serum were assessed at different time points after a single administration of human IgGl antibodies ATL_5802 or ATL_5909.

[0411] The results of this pK study are shown in Figure 17. Figure 17A Serum levels of ATL_5802 and ATL_5909 are shown at 4h, 24h and 144h (7d). Figure 17B The quantification of total antibody concentration over time (measured as area under the curve (AUC)) is shown and shows a significant increase in serum levels of ATL_5802 compared to ATL-5909.

[0412] These data indicate superior peripheral exposure of ATL_5802 compared to ATL_5909, which indicates a lower peripheral clearance of ATL_5802 compared to the comparator antibody.

[0413] In summary, these data demonstrate that the anti-CD33 antibodies described herein show superior pharmacokinetic properties, further supporting their potential use in therapy.

[0414] Example 10 - Alzheimer's disease model

[0415] To test whether anti-CD33 antibodies can alleviate Alzheimer’s disease pathology, the effect of ATL_5802 was tested in a mouse model of Alzheimer’s disease xenograft. Here, mice carrying a human NL-G-F mutated amyloid precursor protein (APP) mice (named AppNL-G-F mice) were xenotransplanted with iPSC-derived human microglial progenitors as described in Fattorelli et al., 2021, Mancuso et al., 2019 and Mancuso et al., 2022. Here, the transplanted cells were matured ex vivo to resemble human primary microglia at the transcriptional level and, after three months, the mice developed signs of Alzheimer’s disease pathology. Therefore, this mouse model can be used to study human microglia biology in the context of Alzheimer’s disease pathology. NL-G-F Mice were treated i.p. once a week with 40 mg / kg of ATL_5802 or isotype control antibody (ATL_5338) starting at 4 months of age for three months. After this, mice were i.p. injected with methoxy-X04, a fluorescent dye able to cross the blood brain barrier and bind to Ab. After this, microglia were isolated using flow cytometry (FAC sorted cells and gated on hCD45+) and their phagocytic capacity was assessed by quantifying the percentage of methoxy-X04 positive human microglia.

[0416] In summary, these data demonstrate that ATL_5802 alleviates AD pathology in vivo at least by increasing the clearance of neurotoxic Ab via microglial phagocytosis.

[0417] The results of this experiment are shown in Figure 18 Compared to isotype treated control mice, mice treated with ATL_5802 showed a significant increase in the percentage of methoxy-X04+ microglia as measured by flow cytometry. These results indicate that ATL_5802 enhances the phagocytosis of Ab amyloid by human microglia in vivo in AppNL-G-F mice xenotransplanted with iPSC-derived human microglial progenitors.

[0418] In summary, these data demonstrate that ATL_5802 alleviates AD pathology in vivo at least by increasing the clearance of neurotoxic Ab via microglial phagocytosis.

[0419] Example 11 - Microglial phagocytosis of Tau

[0420] Pathological (mutant) tau protein aggregates into abnormal filaments, or so-called neurofibrillary tangles, and is a hallmark of multiple neurodegenerative disorders. Mutations in tau, such as P301S and P301L, cause tau pathology with spreading of aggregated tau in humans and transgenic mice (Strang et al. 2018). Therefore, efficient clearance of these aggregates from neurons is crucial to prevent and / or reduce tau pathology. Microglia are essential for the clearance of toxic extracellular proteins and maintenance of cellular health in the central nervous system, as described in Example 4.

[0421] To test whether the antibodies described herein are able to reduce the spreading of mutant (P301S) aggregated tau, the uptake of these aggregates by microglia with an inflammatory phenotype was measured. Briefly, iPSC microglia pre-treated with LPS 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 subsequently live cell imaging was used to measure the uptake of pHrodo-labelled mutant Tau aggregates.

[0422] The results of this experiment are shown in Figure 19. Figure 19A It is shown that, using live cell imaging, phagocytosis of labelled tau aggregates is increased after treatment with anti-CD33 antibody ATL_5802 or anti-TREM2 antibody ATL_6170 relative to isotype control antibody, as measured as total area in red per well over time. In contrast, comparative anti-CD33 antibody ATL_5909 tends to decrease uptake of tau. Figure 19B Quantification of the AUC of total area per well under each condition is shown.

[0423] In summary, these results show that ATL_5802 enhances phagocytosis of toxic tau aggregates by inflammatory microglia, thereby contributing to efficient clearance of these aggregates. This is expected to reduce tau-related pathology and to mitigate and / or prevent (further) neurodegeneration.

[0424] Example 12 - Cytokine release from different cells

[0425] CD33 is expressed on microglia, but also on peripheral myeloid cells, such as monocytes. Modulation of these immune cells can lead to systemic release of cytokines, which can have undesirable consequences and safety concerns.

[0426] To test whether the anti-CD33 antibodies described herein would cause an undesirable release of cytokines, iPSC microglia, PBMCs, and isolated monocytes from multiple donors were treated with isotype control antibody or ATL5802, and various different cytokines, chemokines, and inflammatory mediators were measured in the cell culture media, as shown in Table 7.

[0427]

[0428] Table 7. Cytokine levels were measured in supernatants of iPSC microglia, monocytes, and PBMCs treated with isotype control antibody or ATL_5802. NS = no significant difference between the anti-fluorescein isotype control antibody and cells treated with ATL5802 (t-test). NA = not applicable, as the test was not performed for that cell type.

[0429] These results show that treatment with ATL5802 does not cause immune-mediated cytokine release. In particular, ATL_5802 did not cause untreated microglia to release MCP-1 after treatment at 100 μg / ml or 10 μg / ml for 24 hours (in fact showed slightly lower levels than the isotype control), whereas the comparative anti-CD33 antibodies tested did not show this advantage.

[0430] LPS treatment of iPSC microglia is known to cause production of the inflammatory cytokines MCP-1 and IL-6. To test whether the anti-CD33 antibody ATL-5802 could reduce the levels of these pro-inflammatory cytokines, microglia were treated with LPS, followed by treatment with ATL-5802 or isotype control antibody. After 24 hours, the media was collected, and cytokine levels were measured using a multiplex immunoassay.

[0431] The results are shown in Figure 20, and show that treatment with ATL_5802 significantly reduced the production of IL-6 ( Figure 20A ) and MCP-1 ( Figure 20B ) by LPS-induced iPSC microglia in response to LPS. This shows that the anti-CD33 antibodies described herein can have an anti-inflammatory effect in human iPSC microglia. This is significantly different from the isotype control antibody and the comparative anti-CD33 antibody ATL_5909.

[0432] MCP-1 recruits immune cells to sites of inflammation, and is involved in the pathogenesis of neuroinflammation and AD, autoimmune diseases (e.g., arthritis), and atherosclerosis. Thus, it is highly beneficial that the antibodies of the present disclosure do not cause release of cytokines including MCP-1, whereas the comparative anti-CD33 antibody ATL_5909 does not have this advantage.

[0433] In summary, these data show that the anti-CD33 antibodies described herein do not cause any unwanted cytokine release, indicating that they are safe for human use, and have potent anti-inflammatory effects in vitro.

[0434] Example 13 - Changes in intracellular signaling in microglia after addition of ATL_5802

[0435] Spleen tyrosine kinase (SYK) is a key intracellular regulator of microglial activation and phagocytosis. It is particularly important in neurodegenerative diseases because it contributes to the clearance of toxic protein aggregates (Ennerfelt et al. 2022). In this example, the inventors investigated changes in intracellular signaling following CD33 binding, with a particular focus on the phosphorylation state of Syk (pSYK) under inflammatory conditions.

[0436] Activation of ITAM-containing receptors expressed on microglia, such as TREM2, leads to the recruitment and phosphorylation of ITAM-containing adaptor molecules, which in turn recruit SYK. Following activation, phosphorylation of SYK leads to the upregulation of cytokine production, phagocytosis and ROS production in microglia (Linnartz and Neumann, 2013). TREM2 is antagonized by inhibitory signaling from ITIM receptors, such as CD33. CD33 signaling through ITIM domains recruits phosphatases that counteract ITAM signaling through dephosphorylation of ITAM domains and ITAM-associated kinases, such as SYK (Huang et al., 2003).

[0437] Here, the inventors sought to investigate whether the anti-CD33 antibodies described herein are able to modulate intracellular signaling following CD33 engagement with ITIMs, and in particular whether the antibodies described herein are able to alleviate CD33-mediated inhibition of ITAM receptor signaling. To test this, the inventors measured the phosphorylation state of Syk (pSYK) under inflammatory conditions. An increase in phosphorylation indicates an increase in activation of the ITAM signaling pathway and thereby inhibition of CD33.

[0438] Figure 21 Western blot results are shown for microglia treated with LPS and ATL_5802 or isotype control antibody. This shows that SYK phosphorylation is significantly increased following ATL_5802 treatment, while the overall level of SYK protein remains stable. These findings indicate that ATL_5802 modulates the signaling pathway, likely leading to de-inhibition of the TREM2 signaling axis, and to an increase in TREM2 signaling, as evidenced by the increase in pSYK levels. This highlights the potential therapeutic value of targeting these particular pathways.

[0439] Next, the inventors investigated whether the anti-CD33 antibodies described herein (e.g., ATL_5802) have an effect on the purinergic receptor P2RY12. P2RY12 is a member of the P2 purinergic receptor family (which is a family of seven-transmembrane G protein-coupled receptors that respond to ADP / ATP) and is associated with chemotaxis and phagocytosis. P2RY12 promotes microglial cell chemotaxis to sites characterized by necrotic or apoptotic cells, which is critical for maintaining brain health and responding to injury or inflammation (Walker et al., 2020). P2RY12-mediated chemotactic responses are an essential aspect of the surveillance mechanism of microglial cells, enabling early detection and response to brain injury. Activation of P2RY12 enhances the phagocytic capacity of microglial cells, promoting clearance of apoptotic cells, beta-amyloid plaques, and other debris associated with neurodegenerative diseases. Through its involvement in phagocytosis, P2RY12 helps to resolve inflammation and prevent further tissue damage. By modulating P2RY12 activity, it can enhance beneficial microglial cell functions while mitigating detrimental inflammation, thereby protecting against neurodegeneration and supporting brain health.

[0440] To test whether ATL_5802 can enhance P2RY12 expression in microglial cells, western blots were performed using LPS-stimulated iPSC-induced microglial cells treated with ATL_5802 or isotype control antibody.

[0441] Figure 22 Western blot results of P2RY12 in microglial cells 6 hours after addition of ATL_5802 or isotype control antibody are shown. ATL_5802 enhanced protein expression of the P2RY12 receptor, indicating that the antibody is able to modulate intracellular signaling pathways important for the response to injury and inflammation in microglial cells. Specifically, Figure 22 The data in FIG. 6 show that the non-inflammatory control condition has high P2RY12 expression, whereas the inflammatory control condition has low P2RY12 expression. In contrast, after treatment with ATL_5802, the non-inflammatory condition has higher P2RY12 expression than the isotype control (indicating that the microglial cells have returned to a surveillance state characterized by high P2RY12 expression. In this state, microglial cells are not actively engaged in an inflammatory response, but are ready to respond to new injury or threat), and the inflammatory condition shows higher P2RY12 expression than the isotype control.

[0442] This indicates that ATL_5802 is able to enhance protein expression of P2RY12 receptor, indicating that microglia are in a non-activated state. This effect primes microglia to specific targets, playing a crucial role in maintaining brain health and coordinating responses to injury or inflammation. This change in signaling kinetics can lead to a more effective immune response from microglia, a key factor in slowing progression of neurodegenerative diseases.

[0443] In summary, these data indicate that the anti-CD33 antibodies described herein are able to drive intracellular signaling pathways in microglia that are critical for their effective function (e.g., phagocytosis, cytokine release, and chemotaxis), while simultaneously inhibiting signaling pathways involved in suppressing microglial activation and function. This change in signaling kinetics can contribute to a more effective immune response, a key factor in slowing progression of neurodegenerative diseases.

[0444] Example 14 - RNAseq of iPSC-derived microglia

[0445] To understand the transcriptional changes induced by treatment with anti-CD33 antibodies in both healthy and inflammation mimicking conditions, iPSC-derived microglia were subjected to whole genome RNA sequencing studies. Briefly, iPSC-derived microglia were treated with vehicle or stimulated with LPS / interferon gamma (LI) to mimic inflammatory conditions, followed by treatment with ATL_5802, ATL_5909, ATL_5854, or isotype control antibody, followed by RNA sequencing, as described in the “Materials and Methods” section above.

[0446] The results of the analysis following RNA sequencing are shown in Figure 23. Figure 23A The PCA plot shown in Figure 23 indicates that the changes in gene expression induced by treatment with ATL_5802 were greater than those driven by ATL_5854, ATL_5909, or ATL_5338 (isotype).

[0447] Comparison of the expression profiles of samples treated with any of the anti-CD33 antibodies and isotype control in LI- or vehicle-stimulated cells revealed a number of differentially expressed genes (DEGs) Figure 23B to 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, as shown in Figure 23B and 23C , indicating the effect of ATL_5802 in healthy cells. A total of 194 genes were upregulated in vehicle + ATL_5802 compared to vehicle + isotype control (see Figure 23D ).

[0448] Interestingly, pathway analysis revealed a high statistical significance downregulation of several gene sets involved in oxidative phosphorylation (OXPHOS) in inflammatory microglia after treatment with ATL_5802 compared to LI and isotype treated samples Figure 23E to 23F This result reveals the differential mechanism of action of ATL_0005802, inhibiting the transcription of oxidative phosphorylation and respiratory electron transport components.

[0449] Oxidative phosphorylation (OXPHOS) in microglia enables efficient ATP production under steady-state conditions, whereas activation under inflammatory conditions relies on PI3K / mTOR / HIF1a-dependent aerobic glycolysis to produce ATP more rapidly, similar to the Warburg effect observed in tumors (Laura et al., 2020). This in turn causes microglia to assume a phagocytic phenotype. Microglia continuously monitor the brain parenchyma to detect neuronal damage and changes in homeostatic processes, and cellular metabolic defects involving both glycolysis and OXPHOS have been shown to be associated with neurodegenerative diseases such as Alzheimer’s disease (Baik et al., 2019). It has been shown that the enhancement of anaerobic glycolytic metabolism restored phagocytic activity of microglia and improved cognitive impairment in a mouse AD model (Baik et al., 2019). While these experiments only showed a decrease in OXPHOS (not a corresponding enhancement of glycolysis), inhibiting CD33 would inhibit SHIP1 / 2, negatively regulate PI3K, activate mTOR, and thereby enhance glycolysis, which is expected to be beneficial for cognitive improvement.

[0450] The result of ATL_5802 decreasing the transcription of OXPHOS and respiratory electron transport components thus suggests that ATL_5802 can enhance anaerobic glycolysis and thereby inhibit the OXPHOS pathway. With the decrease in OXPHOS, microglia undergoing CD33 antagonism by ATL_5802 would beneficially reduce the release of reactive oxygen species (a byproduct of OXPHOS).

[0451] In summary, these data show that ATL_5802 is able to modulate cellular metabolic pathways, ultimately leading to cognitive function improvement in neurodegenerative diseases.

[0452] Example 15 - Quadruple culture of cortical neurons, astrocytes and microglia

[0453] Next, the inventors developed a human neural cell culture called “CNS quadruple culture platform” (CNS QCP) that recapitulates the cellular and molecular features of the CNS in vitro (Fig. 1). Figure 24A). This platform integrates multiple cell types co-cultured - specifically, glutamatergic and GABAergic neurons, as well as microglia and astrocytes - to precisely replicate the complex intercellular dynamics of the human brain in a controlled in vitro environment. This in vitro approach enables a deeper understanding of the interactions between different brain cell types and serves as a key experimental intermediary for assessing the effectiveness of ATL_5802 on inflammatory and astrogliosis markers in a human-relevant system. To assess this, lipopolysaccharide (LPS) and interferon-gamma (INFy) were introduced into the system to induce an inflammatory response, mimicking a neuroinflammatory environment, and subsequently analyzed for inflammation, astrogliosis, glial dysfunction, and microglial activation.

[0454] ATL_5802 significantly reduced LPS / INFy-induced interleukin-6 (IL-6) levels relative to isotype control ( Figure 24B ). In addition, ATL_5802 resulted in a significant reduction in the astrogliosis marker GFAP ( Figure 24C ), further demonstrating the overall anti-inflammatory effects of ATL_5802.

[0455] Interferon gamma-induced 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 key 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 control.

[0456] These results provide further evidence to support the therapeutic potential of ATL_5802 in reducing neuroinflammation, thereby preventing brain atrophy and cognitive impairment in neurodegenerative diseases.

[0457] Example 16 - Selectivity of ATL_5802

[0458] To assess the selectivity of ATL_5802, a screen was performed with fixed HEK293 cells expressing 6105 individual human proteins and an additional 400 human heterodimers bound to the screen. The screen library contains plasma membrane, secreted, and cell surface-tethered human proteins. The screen was performed by operators blinded to the target of ATL_5802, CD33. Antibody ATL_5802 showed a single, significant specific interaction with CD33 on both fixed cell and live cell microarrays (see Table 8). These data confirm that ATL_5802 is selective for CD33 and therefore is unlikely to cross-react with other targets.

[0459]

[0460] Table 8. Microarray binding screen results for ATL5802 confirming selective binding to CD33.

[0461] References

[0462] To more fully describe and disclose the application and the state of the art to which the application pertains, a number of publications are cited herein. Full citations for these references are provided below. The entire contents of each of these references are incorporated herein. For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0463]

[0464]

[0465]

Claims

1. An isolated antibody that specifically binds to a CD33 protein, wherein the antibody increases phagocytosis of a cell expressing CD33 as compared to a comparative antibody, and / or wherein the antibody binds to a human CD33 protein comprising a mutation at positions 20, 21, 22, and 24 that is increased as compared to binding of the antibody to a human CD33 protein that does not have the mutation.

2. The isolated antibody of claim 1, wherein the antibody binds to a human CD33 protein comprising a mutation at positions 20, 21, 22, 24, and 132 that is increased as compared to binding of the antibody to a human CD33 protein that does not have the mutation, and / or wherein the antibody binds to a human CD33 protein comprising a mutation at positions 47, 50, 51, and 52 that is decreased as compared to binding of the antibody to a human CD33 protein that does not have the mutation, and / or wherein the antibody does not bind to a human CD33 protein comprising a mutation at positions 47, 50, 51, and 52, and / or wherein the antibody binds to a human CD33 protein comprising a mutation at positions 47, 50, 51, 52, and 122 that is decreased as compared to binding of the antibody to a human CD33 protein that does not have the mutation, and / or wherein the antibody does not bind to a human CD33 protein comprising a mutation at positions 47, 50, 51, 52, and 122, and / or wherein the antibody binds to a human CD33 protein comprising a mutation at position 83 that is decreased as compared to binding of the antibody to a human CD33 protein that does not have the mutation.

3. The isolated antibody of claim 1 or claim 2, wherein the mutation is selected from the group consisting of: a. at position 20: N20R, b. at position 21: F21V, c. at position 22: W22R, d. at position 24: Q24E, e. at position 47: I47V, f. at position 50: Y50H, g. at position 51: D51T, h. at position 52: K52R, i. at position 83: Q83R, j. at position 122: R122K, and k. at position 132: P132T.

4. The isolated antibody of any preceding claim, wherein the binding is measured using a 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. The isolated antibody of any preceding claim, wherein the antibody has one or more or all of: a. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPosl [P1] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, b. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos2 [P2] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, c. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos3 [P3] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, d. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos4 [P4] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, e. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos5 [P5] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, f. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos6 [P6] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, g. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos7 [P7] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, h. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos8 [P8] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, i. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPos9 [P9] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, j. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPoslO [P10] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, k. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPosl l [Pll] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT, and / or l. binds to a protein comprising the sequence of CD33M2_ECD_18-232_MutPosl2 [P12] that is increased as compared to binding to a protein comprising the sequence of CD33M2_ECD_18-232_WT. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ b. increased binding to a protein comprising the CD33M2_ECD_18-232_MutPosl_MutPos6 [P6+1] sequence as compared to binding to a protein comprising the CD33M2_ECD_18-232_WT sequence, c. decreased binding to a protein comprising the CD33M2_ECD_18-232_MutPos2 [P2] sequence as compared to binding to a protein comprising the CD33M2_ECD_18-232_WT sequence, d. decreased binding to a protein comprising the CD33M2_ECD_18-232_MutPos2_MutPos5 [P2+5] sequence as compared to binding to a protein comprising the CD33M2_ECD_18-232_WT sequence, e. decreased binding to a protein comprising the CD33M2_ECD_18-232_MutPos4 [P4] sequence as compared to binding to a protein comprising the CD33M2_ECD_18-232_WT sequence.

6. The isolated antibody of any preceding claim, wherein the comparison antibody is selected from the group consisting of: 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, and / or wherein the phagocytosis is assessed by measuring a fluorescence signal associated with uptake of labeled particles by imaging or flow cytometry.

7. The isolated antibody of any preceding claim, wherein the cell is a monocyte or microglial cell, optionally wherein the cell is a human cell and / or a microglial cell derived from iPSCs and / or a cell that has been stimulated with an inflammatory signal (e.g. LPS) prior to exposure to the antibody.

8. The isolated antibody of any preceding claim, wherein the antibody binds to a CD33 protein comprising the V domain of CD33, and / or wherein the antibody does not bind to a CD33 protein that does not comprise the V domain of CD33, and / or wherein the antibody does not bind to a CD33 protein having the sequence of protein CD33_human_ECD_Cdomain_His_007.

9. The isolated antibody of any preceding claim, wherein the antibody comprises a heavy chain variable domain (VH) having: a CDRH1 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; a CDRH2 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; and a CDRH3 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810. CDRH3 containing the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, or ATL_0005810; or The CDR group, compared to the CDR group mentioned above, contains zero, one, or two amino acid substitutions in each CDR.

10. The isolated antibody according to claim 9, wherein the antibody comprises a heavy chain variable domain (VH) having the following CDR: CDRH1 contains an amino acid sequence selected from the following: Or an amino acid sequence with one or two mutations compared to the above sequence; CDRH2 contains an amino acid sequence selected from the following: Or an amino acid sequence with one or two mutations compared to the above sequence; and CDRH3 contains an amino acid sequence selected from the following: Or an amino acid sequence with one or two mutations compared to the above sequence.

11. The isolated antibody according to claim 9 or claim 10, wherein the antibody comprises a heavy chain variable domain (VH) having the following CDR: CDRH1 containing the sequence HCDR1_ATL_0005802, CRH2 containing the sequence HCDR2_ATL_0005802, and CDRH3 containing the sequence HCDR3_ATL_0005802; or a CDR group that contains one or two mutations in CDRH1 and CDRH2 compared to these sequences and / or contains one, two, or three mutations in CDRH3 compared to these sequences; or CDRH1 containing the sequence HCDR1_ATL_0005853, CRH2 containing the sequence HCDR2_ATL_0005853, and CDRH3 containing the sequence HCDR3_ATL_0005853; or a CDR group that contains one or two mutations in CDRH1 and CDRH2 compared to these sequences and / or contains one, two, or three mutations in CDRH3 compared to these sequences; or CDRH1 containing the sequence HCDR1_ATL_0005854, CRH2 containing the sequence HCDR2_ATL_0005854, and CDRH3 containing the sequence HCDR3_ATL_0005854; or a group of CDRs that contains 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.

12. The isolated antibody according to any of the preceding claims, wherein the antibody has a heavy chain variable domain (VH) having the following framework sequence: HFWR1 of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, and ATL_0005810. HFWR2 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR3 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and HFWR4 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, or a framework sequence having one to six substitutions compared to the framework sequences described above.

13. The isolated antibody of any preceding claim, wherein the antibody has a heavy chain variable domain (VH) with framework sequence HFWR2 of ATL_0005802, and / or wherein the antibody has a heavy chain variable domain (VH) with a framework sequence comprising A at position 40 in standard IMGT numbering.

14. The isolated antibody of claim 12 or claim 13, wherein the substitutions in the framework sequence of the heavy chain variable domain are at any position in standard IMGT numbering except position 40.

15. The isolated antibody of any preceding claim, wherein the antibody has a heavy chain variable domain (VH) with 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. The isolated antibody of any preceding claim, wherein the antibody has a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of VH sequences of 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, or wherein the antibody has a heavy chain variable domain (VH) comprising a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of the VH sequences; optionally wherein the antibody has a heavy chain variable domain (VH) comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of VH sequences of antibodies ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence having at most 2 mutations in each HCDR and at most 3 mutations in each framework region compared to a sequence selected from the group consisting of the VH sequences.

17. The isolated antibody of any preceding claim, wherein the antibody: (i) has a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3 within a germline framework, with the proviso that position 40 is A in standard IMGT numbering; and / or (ii) is a scFv antibody molecule, a nanobody, or a whole antibody, and / or wherein the antibody comprises an antibody constant region, and / or wherein the antibody is a whole antibody, and / or wherein the antibody is an IgG1 or a variant thereof, optionally wherein the antibody is an IgG1 variant L234A / L235A (LALA).

18. The isolated antibody of any preceding claim, wherein the antibody binds to human CD33, optionally wherein the antibody binds to human CD33 with an EC50 of at most 2e-08 M or at most 3e-09 M, as assessed by ELISA (e.g. binding to rhCD33 plated), and / or wherein the antibody selectively binds to CD33 relative to other one or more siglecs; optionally wherein the antibody selectively binds to CD33 relative to one or more (or all) of siglec-6, siglec-7, siglec-8, and siglec-9; and / or wherein the antibody selectively binds to human CD33 relative to other one or more homologs; optionally wherein the antibody selectively binds to human CD33 relative to mouse CD33 and cynomolgus CD33.

19. The isolated antibody of any preceding claim, wherein the antibody causes less than 50% or less than 80% depletion of CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody, and / or wherein the antibody causes a lower degree of depletion of CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody compared to the same concentration of a comparator antibody.

20. The isolated antibody according to any of the preceding claims, wherein the antibody comprises a light chain variable domain (VL) having: a CDR L1 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, a CDR L2 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, and a CDR L3 comprising the amino acid sequence of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a CDR set comprising zero, one or two amino acid substitutions in each CDR compared to the CDR sets described above.

21. The isolated antibody according to claim 20, wherein the CDR L1, CDR L2 and CDR L3 of the VL domain are within the germline framework, and / or wherein the antibody has a light chain variable domain (VL) with the following framework sequence: LFWR1 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; LFWR2 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; LFWR3 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; and LFWR4 of any of antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a FWR set that contains one to six amino acid substitutions compared to the FWR sets described above.

22. The isolated antibody of any preceding claim, wherein the antibody has a light chain variable domain (VL) comprising a selected sequence having at least 95% sequence identity to a sequence selected from the VL sequences of 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, or wherein the antibody has a light chain variable domain (VL) comprising a sequence having at most 2 mutations in each LCDR and at most 3 mutations in each framework region compared to a sequence selected from the VL sequences; optionally wherein the antibody has a light chain variable domain (VL) comprising a sequence having at least 95% sequence identity to a sequence selected from the VL sequences of antibodies ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence having at most 2 mutations in each LCDR and at most 3 mutations in each framework region compared to a sequence selected from the VL sequences.

23. The isolated antibody of any preceding claim, wherein the antibody has lower peripheral clearance when administered to a subject compared to a comparative anti-CD33 antibody, and / or wherein the antibody increases phagocytosis of Ab by microglia in vivo compared to a control, and / or increases phagocytosis of tau aggregates by microglia having an inflammatory phenotype (e.g. LPS-treated iPSC microglia) compared to a control, and / or increases phagocytosis of tau aggregates by microglia having an inflammatory phenotype to a greater extent compared to a comparative anti-CD33 antibody, and / or does not induce release of one or more cytokines including IL-6 and / or MCP-1 by microglia in vitro and / or in vivo, and / or reduces the level of IL-6 and / or MCP-1 released by microglia having an inflammatory phenotype in vitro (e.g. LPS-treated human iPSC-derived microglia) and / or in vivo, and / or reduces release of one or more markers of inflammation induced by inflammation in a human neural cell culture assay and / or in the central nervous system of a subject, optionally wherein the one or more markers of inflammation are selected from the group consisting of: MCP-1, IP-10, GFAP and IL-6.

24. An isolated nucleic acid, vector or vector set comprising a nucleotide sequence encoding an antibody according to any preceding claim, including a VH or VL domain, or fragment thereof.

25. A host cell comprising the vector of claim 24, or a host cell transformed in vitro with the nucleic acid of claim 24.

26. The antibody of any one of claims 1 to 23 for use in the treatment of a disease or disorder selected from the group consisting of: (i) a disease associated with microglial dysfunction; (ii) a neurodegenerative disease or disorder, optionally wherein the neurodegenerative disease or disorder is a tauopathy or a disease or disorder selected from the group consisting of 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 disease, Lewy body disease, spinal muscular atrophy (SMA), motor neuron disease (MND) such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxia (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral pallidoluysian atrophy (DRPLA), X-linked type 1 spinal and bulbar muscular atrophy (SMAX1 / SBMA), anderson-fabry (X-linked fabry disease) and DNAJB6 myopathy, optionally wherein the neurodegenerative disease is selected from the group consisting of FTD, AD, HD and PD; (iii) a cancer, optionally wherein the cancer is AML or a cancer associated with high tumor cell sialylation and / or tumor cell overexpression of CD33; and (iv) a disease characterized by insufficient macrophage phagocytosis and / or macrophage dysfunction, optionally wherein the disease is COPD or IPF.

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