Anti-CD33 antibodies

Description

"ANTI-CD33 ANTIBODIES" FIELD OF THE INVENTION

[001] The present invention relates to antibodies capable of binding to CD33 and, particularly, though not exclusively, to novel therapeutic antibodies. Methods for the use of anti-CD33 antibodies in the treatment of neurodegenerative diseases and cancer are also described. FUNDAMENTALS

[002] CD33 (Siglec-3) is an inhibitory immune receptor and 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 in the brain. CD33 has been one of the highest-ranked genes associated with the risk of developing Alzheimer's disease (AD) in genome-wide association studies. CD33 signaling in microglia has been implicated in AD pathology, and CD33 expression is elevated in AD patients, where it is believed to modulate microglial activation and inhibit β-amyloid clearance (Zhao et al., 2019).

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

[004] It has been suggested that siglecs expressed on immune cells that infiltrate tumors influence antitumor immunity and represent a potential target for cancer immunotherapy (Stanczak & Laubli, 2023). Petition 870250089689, dated 02 / 10 / 2025, page 15 / 221 2 / 144

[005] Antibodies capable of binding to CD33 have been developed. For example, US patent 2022 / 0162309 describes humanized 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, taupatia disease, infection, and cancer. However, clinical trials of the lead antibody AL003, described in US patent 2022 / 0162309, have been terminated. A humanized anti-CD33 antibody (lintuzumab) has demonstrated modest activity in the treatment of acute myeloid leukemia (AML) (Jurcic, 2012).

[006] There is still a need for more antibodies that are able to bind to and modulate CD33 signaling effectively. SUMMARY OF THE INVENTION

[007] The invention includes the combination of the described preferred aspects and features, except where such combination is clearly inadmissible or expressly avoided.

[008] According to a first aspect, the invention provides an isolated antibody that specifically binds to a CD33 protein, wherein the antibody increases the phagocytosis of a cell expressing CD33 compared to a comparator antibody and / or wherein the binding of the antibody to a human CD33 protein comprising mutations at positions 20, 21, 22 and 24 is increased compared to the binding of the antibody to a human CD33 protein without said mutations.

[009] The antibody, according to the present aspect, may comprise a variable heavy chain (VH) domain with the following CDRs: CDRH1 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; CDRH2 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, Petition 870250089689, dated 02 / 10 / 2025, p. 16 / 221 3 / 144 ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; CDRH3 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a set of CDRs containing zero, one, or two amino acid substitutions in each CDR compared to the set of CDRs above.

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

[011] Antibodies with these CDRs have been identified as ligands for CD33 in centenarians with exceptional cognitive ability, indicating a probable protective role in neurodegenerative diseases, as well as probable good therapeutic tolerability. These antibodies were identified as undergoing less peripheral degradation than the comparator antibodies, being able to induce phagocytosis in myeloid and microglial cells to the same or greater extent than the comparator antibodies. These antibodies were identified as binding to a different epitope than the comparator antibodies. Petition 870250089689, dated 02 / 10 / 2025, page 17 / 221 4 / 144

[012] The antibody, according to the present aspect, can increase the phagocytosis of a cell expressing CD33 compared to a comparator antibody, and / or the binding of the antibody to a human CD33 protein comprising mutations at positions 20, 21, 22 and 24 can be increased compared to the binding of the antibody to a human CD33 protein without said mutations.

[013] The antibody, according to the first or second aspect, may have one or more of the following optional characteristics.

[014] Antibody binding to a human CD33 protein comprising mutations at positions 20, 21, 22, 24, and 132 may be increased compared to antibody binding to a human CD33 protein without said mutations. Antibody binding to a human CD33 protein comprising mutations at positions 47, 50, 51, and 52 may be reduced compared to antibody binding to a human CD33 protein without said mutations. The antibody may not bind to a human CD33 protein comprising mutations at positions 47, 50, 51, and 52. Antibody binding to a human CD33 protein comprising mutations at positions 47, 50, 51, 52, and 122 may be reduced compared to antibody binding to a human CD33 protein without said mutations. The antibody may not bind to a human CD33 protein comprising mutations at positions 47, 50, 51, 52, and 122.The binding of the antibody to a human CD33 protein that includes mutations at positions 83 may be reduced compared to the binding of the antibody to a human CD33 protein without said mutations.

[015] Mutations can be selected from: position 20: N20R, position 21: F21V, position 22: W22R, position 24: Q24E, position 47: I47V, position 50: Y50H, position 51: D51T, position 52: K52R, position 83: Q83R, position 122: R122K and position 132: P132T. Petition 870250089689, dated 02 / 10 / 2025, page 18 / 221 5 / 144

[016] The linkage can be measured by means of a single-point ELISA. The human CD33 protein may comprise residues 18-232 of human CD33. The human CD33 protein without mutations may be CD33M2_ECD_18-232_WT.

[017] The antibody may have one or more, or all of the following: (i) increased binding to a protein comprising the sequence CD33M2_ECD_18232_MutPos1[P1] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, (ii) increased binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos1_MutPos6 [P6+1] compared to a protein comprising the sequence CD33M2_ECD_18232_WT, (iii) reduced binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos2 [P2] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, (iv) reduced binding to a protein comprising the sequence CD33M2_ECD_18232_MutPos2_MutPos5 [P2+5] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, and (v) reduced binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos4 [P4] compared to a protein comprising the sequence CD33M2_ECD_18232_WT.

[018] A dissemination antibody may have lower peripheral clearance when administered to an individual, compared to a comparator anti-CD33 antibody.

[019] A disclosure antibody can increase the phagocytosis of Iβ plaques by microglia cells in vivo compared to a control. A disclosure antibody can increase the phagocytosis of tau aggregates by microglia with an inflammatory phenotype (e.g., LPS-treated iPSC microglia) compared to a control. A disclosure antibody can increase the phagocytosis of tau aggregates by microglia with an inflammatory phenotype to a greater extent than Petition 870250089689, dated 02 / 10 / 2025, page 19 / 221 6 / 144 a comparative anti-CD33 antibody. A disclosure antibody may not induce the release of one or more cytokines, including IL-6 and / or MCP-1, by microglia in vitro and / or in vivo. A disclosure antibody may reduce the levels of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype in vitro (e.g., LPS-treated human iPSC-derived microglia) and / or in vivo. A disclosure antibody may reduce the inflammation-induced release of one or more inflammation markers in a human neural cell culture assay and / or in the central nervous system of an individual. The inflammation marker(s) may be selected from: MCP-1, IP-10, GFAP, and IL-6.

[020] A comparative antibody can be selected from: an isotype control antibody, another CD33-binding antibody, and an antibody with the variable heavy chain sequence of ATL_5909 and the variable light chain sequence of ATL_5909. Phagocytosis can be assessed by measuring a fluorescence signal associated with the uptake of a labeled particle by imaging or flow cytometry. The cell can be a monocyte or a microglial cell. The cell can be a human cell. The cell can be an iPSC-derived microglial cell. The cell can be a cell that has been stimulated with an inflammatory signal (e.g., LPS) prior to antibody exposure.

[021] The antibody can bind to a CD33 protein that includes the CD33 V domain. The antibody may not bind to a CD33 protein that does not include the CD33 V domain. The antibody may not bind to a CD33 protein that has the CD33_HUMAN_ECD_Cdomain_His_007 protein sequence.

[022] The antibody may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising an amino acid sequence selected from: HCDR1_ATL_0005802; HCDR1_ATL_0005807; HCDR1_ATL_0005808: GYSFTSYW; HCDR1_ATL_0005853:GYKFNNNW; Petition 870250089689, dated 02 / 10 / 2025, page 20 / 221 7 / 144 HCDR1_ATL_0005854:GYKFSNNW; or an amino acid sequence with 1 or 2 mutations compared to the sequences above; a CDRH2 comprising an amino acid sequence selected from: HCDR2_ATL_0005802; HCDR2_ATL_0005807; HCDR2_ATL_0005808; HCDR2_ATL_0005853; HCDR2_ATL_0005854; IYPGDSDT; or an amino acid sequence with 1 or 2 mutations compared to the sequences above; and a CDRH3 comprising an amino acid sequence selected from: HCDR3_ATL_0005802; ARPRFGEYYFDY HCDR3_ATL_0005853 ARHSGGLDGYTAAALDY; HCDR3_ATL_0005854 ATWGGSNWFVD; or an amino acid sequence with 1 or 2 mutations compared to the sequences above.

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

[024] The antibody may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005853, a CDRH2 comprising the sequence HCDR2_ATL_0005853 and a CDRH3 comprising the sequence HCDR3_ATL_0005853, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[025] The antibody may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence of Petition 870250089689, dated 02 / 10 / 2025, page 21 / 221 8 / 144 HCDR1_ATL_0005854, a CRH2 comprising the sequence HCDR2_ATL_0005854 and a CDRH3 comprising the sequence HCDR3_ATL_0005854, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[026] The antibody may have a variable heavy chain (VH) domain with the following structural sequences: HFWR1 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR2 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 and HFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 or structural sequences with one to six substitutions compared to the structural sequences above.

[027] The antibody may have a variable heavy chain (VH) domain with an HFWR2 structural sequence of ATL_0005802. The antibody may have a variable heavy chain (VH) domain with a structural sequence comprising an “A” at position 40 in the standard IMGT numbering. Substitutions in the variable heavy chain domain structural sequences may be located at any position other than position 40 in the standard IMGT numbering. The antibody may have a variable heavy chain (VH) domain comprising CDRH1, CDRH2, and CDRH3 within a germline structure, provided that position 40 in the standard IMGT numbering is A. Petition 870250089689, dated 02 / 10 / 2025, page 22 / 221 9 / 144

[028] The antibody may have a variable heavy chain (VH) domain with the following structural sequences: HFWR1 from ATL_0005802, ATL_0005853 or ATL_0005854; HFWR2 from ATL_0005802, ATL_0005853 or ATL_0005854; HFWR3 from ATL_0005802, ATL_0005853 or ATL_0005854; and HFWR4 from ATL_0005802, ATL_0005853 or ATL_0005854.

[029] The antibody may have a variable heavy chain (VH) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from the VH sequence of the antibodies: ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, ATL_0006040, ATL_0006041, ATL_0006042, ATL_0006043, ATL_0006044, ATL_0006045, ATL_0006046, ATL_0006047 and ATL_0006048. The antibody may have a variable heavy chain (VH) domain comprising a sequence that exhibits a maximum of 2 mutations in each HCR and a maximum of 3 mutations in each structural region, compared to a sequence selected from the VH sequences above.The antibody may have a variable heavy chain (VH) domain comprising a sequence that exhibits at least 95% sequence identity with a sequence selected from the VH sequence of antibodies ATL_0005802, ATL_0005853, or ATL_0005854, or a sequence that exhibits at most 2 mutations in each HCR and at most 3 mutations in each structural region, compared to a sequence selected from the aforementioned VH sequences.

[030] 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 (such as plated rhCD33 binding).

[031] The antibody can deplete CD33 on the cell surface of human monocytes by less than 50%, or less than 80% after 5 hours of Petition 870250089689, dated 02 / 10 / 2025, p. 23 / 221 10 / 144 incubation with the antibody. The antibody can deplete CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody to a lesser extent than a comparator antibody at the same concentration.

[032] The antibody can selectively bind to CD33 with respect to one or more siglecs. The antibody can selectively bind to CD33 with respect to one or more (or all): siglec-6, siglec-7, siglec-8 and siglec-9. The antibody can selectively bind to human CD33 with respect to one or more homologs. The antibody can selectively bind to human CD33 with respect to mouse CD33 and canine CD33.

[033] The antibody may be an scFv antibody molecule, a nanobody, or a complete antibody. The antibody may comprise a constant region of the antibody. The antibody may be a complete antibody. The antibody may be an IgG1 or a variant thereof. The antibody may be an L234A / L235A variant of IgG1 (LALA).

[034] The antibody may comprise a variable light chain (VL) domain with the following CDRs: CDRL1 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, CDRL2 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 and CDRL3 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 or a set of CDRs containing zero, one or two amino acid substitutions in each CDR, compared to the set of CDRs above. Petition 870250089689, dated 02 / 10 / 2025, page 24 / 221 11 / 144

[035] The CDRL1, CDRL2 and CDRL3 of the VL domain may be within a germline structure.

[036] The antibody may have a variable light chain (VL) domain with the following structural sequences: LFWR1 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; LFWR2 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; LFWR3 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; and LFWR4 of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a set of FWRs containing one to six amino acid substitutions compared to the FWR set above.

[037] The antibody may have a variable light chain (VL) domain comprising a selected sequence that has at least 95% sequence identity with a sequence selected from the VL sequence of the antibodies: ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, ATL_0006040, ATL_0006041, ATL_0006042, ATL_0006043, ATL_0006044, ATL_0006045, ATL_0006046, ATL_0006047 and ATL_0006048. The antibody may have a variable light chain (VL) domain comprising a sequence that exhibits a maximum of 2 mutations in each LCDR and a maximum of 3 mutations in each structural region, compared to a sequence selected from said VL sequences. The antibody may have a variable light chain (VL) domain comprising a sequence that exhibits at least 95% sequence identity with a sequence selected from the VL sequence of antibodies ATL_0005802, ATL_0005853. Petition 870250089689, dated 02 / 10 / 2025, p. 25 / 221 12 / 144 or ATL_0005854, or a sequence that exhibits a maximum of 2 mutations in each LCDR and a maximum of 3 mutations in each structural region, compared to a sequence selected from the aforementioned VL sequences.

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

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

[040] Also described in this report is a vector or set of vectors comprising the nucleic acid, according to the fourth aspect, a host cell transformed in vitro with said nucleic acid or a host cell comprising said vector or set of vectors.

[041] Also described in this report is a composition comprising an antibody, including an antibody VH domain or an antibody VL domain, according to any embodiment of the first or second aspect, and at least one additional component, optionally comprising a pharmaceutically acceptable excipient, vehicle or carrier.

[042] Also described in this report 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 in this report is a method for treating a disease or disorder in an individual in need thereof, comprising administering a therapeutically effective amount of an antibody, according to any embodiment of the first or second aspect. Also described in this report is an antibody, according to any embodiment of the first or second aspect, for use in Petition 870250089689, dated 02 / 10 / 2025, page 26 / 221 13 / 144 manufacturing of a drug. The disease or disorder may be a disease associated with microglia cell dysfunction. The disease or disorder may be a neurodegenerative disease or disorder. The disease or disorder may be a tauopathy.Neurodegenerative disorders 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 diseases, Lewy body disease, spinal muscular atrophy (SMA), motor neuron disease (MND), such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxias (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubra pallidal atrophy (DRPLA), X-linked spinal bulbar muscular atrophy type 1 (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease). and DNAJB6 myopathies, optionally, in which the neurodegenerative disease is selected from FTD, AD, HD, and PD. The disease or disorder may be cancer.The cancer may be selected based on AML or a cancer associated with hypersalicylation of tumor cells and / or overexpression of CD33 by tumor cells. The disease or disorder may be characterized by insufficient phagocytosis of macrophages and / or macrophage dysfunction. For example, the disease may be COPD or IPF. The medication may be for the treatment of any of the above diseases or disorders. SUMMARY OF FIGURES

[043] The methods of implementation and experiments that illustrate the principles of disclosure will now be discussed with reference to the attached figures in which:

[044] Figure 1 schematically illustrates the results of a process to identify convergent BCR sequences in two individuals who are amyloid-positive, tau-negative, and cognitively normal. The numbers indicated Petition 870250089689, dated 02 / 10 / 2025, page 27 / 221 14 / 144 are the clonotype numbers. The two individuals shared 64 clonotypes, and a representative sequence from one of the 64 shared clonotypes was shown to bind to CD33 (see below).

[045] Figure 2 shows the results of an ELISA for the binding of ATL_0005082 (also referred to in this report as ATL_5082) to CD33. The isotype control is a commercially available human IgG1 isotype control (Absolute Antibody; Ab00102-10.0 Anti-Fluorescein) and does not bind to CD33. Lysozyme = negative control antigen. CD33 = binding to recombinant human CD33.

[046] Figure 3 shows the results of serum screening by ELISA in a large panel of individual donors. The graph shows the level of autoreactivity to CD33 in multiple individuals by age. The Z-score is calculated as [raw signal - mean signal] / standard deviation (SD).

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

[048] Figure 5 shows the results of a phage ELISA for identification of single-strand variable fragment (scFv) clones that bind to the CD33 antigen. The scFvs displayed on phages, derived from phage display selections, were tested for binding to recombinant CD33 (pink circles) and to the lysozyme control (black circles).

[049] Figure 6 shows the ELISA result for the binding of nine anti-CD33 antibodies identified by phage display and a comparative antibody (ATL_5503) to the complete extracellular region of CD33 (light bars, “CD33 HUMAN_M17-H259_002_004”) or to the C2 domain (dark blue bars, Petition 870250089689, dated 02 / 10 / 2025, page 28 / 221 15 / 144 “CD33_HUMAN_ECD_Cdomain_His_007”). The results are presented as sign versus isotype.

[050] Figure 7 shows the quantification of CD33 levels in human monocytes 5 hours after incubation with the indicated antibodies at a concentration between 1 nM and 40 nM, measured by flow cytometry. The graph in (A) shows MFI (mean fluorescence intensity) in CD14+CD33+ cells plotted against antibody concentration. The tested antibodies (ATL) bind exclusively to the V domain of the CD33 receptor, while the CD33 antibody used for detection by flow cytometry is specific for the C domain. The reduction in mean fluorescence intensity can be attributed to the loss of the CD33 receptor from the cell membrane surface, since there is no interference between the binding of the detection antibody and the binding of the test antibody. ATL5909 shows complete CD33 depletion at all tested concentrations, while ATL5802 shows a complete absence of CD33 depletion, comparable to the isotype control, at all concentrations.ATL5810 has an intermediate profile, with a dose-dependent decrease in CD33 levels. (B) shows MFI of the CD33 V domain in CD14+CD16+ (below) or CD14+CD16 (above) of the indicated antibodies. (C) shows MFI of the CD33 C domain in CD14+CD16+ (below) or CD14+CD16- (above) of the indicated antibodies. It is worth noting that, since all test antibodies bind to the V domain, changes in the V domain MFI can be attributed to either competition with the detection antibody or CD33 depletion. In contrast, the assay using MFI detection in the C domain will be a true representation of CD33 depletion. (D) Area under the curve (AUC) of binding to the C domain of CD33 and to the V domain as a function of concentration ranging from 40 to 1 nM for CD14+CD16- cells and CD14+CD16+ cells (curves in the lower panels in B and C). Data are normalized to the AUC of the isotype control antibody, which was defined as. Petition 870250089689, dated 02 / 10 / 2025, p. 29 / 221 16 / 144 100. (E) Area under the binding curve to the C domain of CD33 and to the V domain as a function of concentration varying from 40 to 1 nM (curves in the upper panels in B and C).

[051] Figure 8 shows the setup and results of an in vitro phagocytosis assay in microglia derived from human inflammatory iPSCs. (A) schematically shows how the phagocytosis assay is performed. Microglia are incubated with LPS for 24 hours, followed by incubation with an anti-CD33 antibody (or isotype control) for 24 hours, followed by measurement of uptake of pHrodo red-labeled beta-amyloid. pHrodo red fluoresces red only in the low pH environment of the cell lysosome after phagocytosis of the labeled beta-amyloid. (B) shows the results of a phagocytosis assay testing the effect of ATL5802 and ATL5810 on phagocytosis in microglia, compared to the previous technique antibody ATL5909, an isotype control and a negative control (Aβ only), in terms of red blood cell surface area per well over time with antibody incubation.(C) shows the results of the phagocytosis assay for panel B represented as the area under the curve of the surface area (μm2) of red blood cells per well over time, which represents the uptake of amyloid-red beta pHrodo in these cells by phagocytosis. (D) shows the results of a phagocytosis assay testing the effect of ATL5802, ATL_5853, ATL_5854, and ATL_6044 on phagocytosis in microglia, compared to the antibody from the previous technique ATL5909 and the isotype control. (E) shows the quantification of the phagocytosis assay results in panels D and F, represented as the area under the curve of the surface area (μm2) of red blood cells per well over time, after 18 hours. The surface area of ​​the red blood cells represents the uptake of amyloid-red beta pHrodo in these cells and therefore indicates the level of phagocytosis. Statistical analysis was performed using one-way ANOVA versus isotype control. * = p < 0.05; ** = p < ...; *** = p < .., ns = not significant.At least ATL_5854 showed one. Petition 870250089689, dated 02 / 10 / 2025, page 30 / 221 17 / 144 statistically significant difference when tested directly vs ATL_5909 (one-way ANOVA) (F) shows the results of experiments comparing phagocytosis of labeled beta amyloid in iPSC-derived microglia cells treated with anti-TREM2 antibodies (ATL6166; ATL 6167; and ATL6170) or with ATL5802 (the top and bottom panels show replicates of the same experiments).

[052] Figure 9 shows the results of an ex vivo phagocytosis assay using human whole blood. (A) shows a flow cytometry graph of S. aureus myeloid cells labeled with pHrodo red (pHrodo+CD14 cells), indicating uptake of pHrodo into CD14+ cells. pHrodo-red only fluoresces red in the low pH environment of the cell lysosome after phagocytosis of labeled S. aureus has occurred. (B) shows pHrodo in CD14+ cells (indicating phagocytosis) measured as MFI of pHrodo within CD14+ myeloid cells incubated with the indicated antibodies (ATL5802; ATL5810; ATL5909) or isotype control. Negative controls included wells without pHrodo-labeled S. aureus and untreated cells. (C) shows phagocytosis of S.pHrodo-labeled aureus cells in newly isolated CD14+ monocyte cells (red object count per well, corresponding to pHrodo cells, normalized to the maximum observed value) treated with ATL5802 from 0.029 nM to 30 nM (shades of green), ATL5909 benchmark 1 (red), ATL4828 benchmark 2 (blue), and isotype control (yellow). Latrunculin A prevents actin polymerization in cells and was used as a control, which is expected to completely block phagocytosis.

[053] Figure 10 shows CD33 levels in peripheral myeloid cells in NSG CD34+ mice 24 hours after intraperitoneal (ip) injection of antibody (ATL5802; 5810; 5909 or isotype control) at the indicated concentrations (10 mg / kg or 40 mg / kg). The CD33 level on the surface of grafted human myeloid cells (defined as huCD45+huCD14+ cells) was measured with Petition 870250089689, dated 02 / 10 / 2025, page 31 / 221 18 / 144 is a specific antibody for the C domain of CD33. The reported values ​​are the fluorescence intensity (MFI) of CD33 in human CD45+CD14+ cells.

[054] Figure 11 shows a schematic illustration of the mutations introduced into the V domain of CD33, with the sialoglycan ligand shown centrally in gold, for epitope mapping of the antibodies described in this report (A) and corresponding results (B). Variants of the CD33 extracellular domain were prepared as individual recombinant proteins, as indicated in A. Nine IgG1 antibodies were tested in a single-point ELISA against wild-type CD33 and all 7 variant CD33 proteins, and the binding relative to the wild-type CD33 extracellular domain was quantified. The binding profile of each antibody to the different CD33 variants was used to group the antibodies into 4 different epitope compartments, as shown in B.

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

[056] Figure 13 shows the results of a flow cytometry assay that detects the mean fluorescence intensity of pHrodo-labeled S. aureus in human CD45+CD14+CD11b+ peripheral myeloid cells in CD34+ NSG mice 24 hours after intraperitoneal antibody injection (ATL5802; 5810; 5909 or isotype control) - 3 hours of ex vivo incubation with labeled S. aureus. Petition 870250089689, dated 02 / 10 / 2025, page 32 / 221 19 / 144

[057] Figure 14 shows the SEC-HPLC chromatograms for ATL5802 under the indicated forced degradation conditions (2 weeks at -80 °C, 2 weeks at 40 °C, overnight agitation, 3 freeze-thaw cycles (3x FT); and maintenance at low pH).

[058] Figure 15 shows the results of an ELISA test of ATL5802 binding to recombinant human CD33 (rhCD33) under the indicated forced degradation conditions (2 weeks at -80 °C, 2 weeks at 40 °C, overnight agitation, 3 freeze-thaw cycles (3x FT); and maintenance of low pH).

[059] Figure 16 shows a variable chain sequence alignment for selected anti-CD33 antibodies. Dashed lines indicate comparative antibodies. A. VH FWR1. B. VH CDR1. C. VH FWR2. D. VH CDR2. E. VH FWR3. F. VH CDR3. G. VH FWR4. H. VL FWR1. I. VL CDR1. J. VL FWR2. K. VL CDR2. L. VL FWR3. M. VL CDR3. N. CL FWR4.

[060] Figure 17 shows the results of an in vivo pK study in C57BL / 6-Cd33tm1 (CD33) mice. Groups of mice (n = 3 / group) received 1 mg / kg of ATL-5802 or ATL-5909 intraperitoneally, and serum hIgG1 levels were measured 4 hours, 24 hours, and 7 days after a single antibody administration. (A) shows a graph plotting 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.

[061] Figure 18 shows the results of an in vivo study in an animal model of Alzheimer's disease, testing the ability of ATL_5802 to alter in vivo phagocytosis of amyloid-β in APPNL-GF mice xenotransplanted with human microglia (hMG). After 12 weeks of weekly treatment with 40 mg / kg of ATL_5802 (n = 6) or isotype control, ATL_5338 (n = 7), the uptake of Petition 870250089689, dated 02 / 10 / 2025, page 33 / 221 The phagocytosis of 20 / 144 amyloid-β by human CD45+ microglia was measured by flow cytometry using the fluorescent amyloid-β marker, Methoxy-X04. There was a significant increase in the percentage of Methoxy-X04-positive human microglia cells in mice treated with ATL_5802 compared to mice treated with the control, ATL_5338, indicating that ATL_5802 increased amyloid-β phagocytosis by human microglia in vivo. *p<0.05, unpaired t-test.

[062] Figure 19 shows the results of a real-time live-cell imaging study using LPS-prepared microglia and pHrodo-labeled Tau P301S aggregates. (A) Representative graph of total red area (pHrodo.red) 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) of total red per well is shown in (A).

[063] Figure 20 shows the results of a multiplex immunoassay in iPSC microglia pre-treated with LPS for 6 hours, followed by treatment with ATL_5802 or ATL_5909. (A) Shows the concentration (pg / ml) of IL-6 in the supernatants of iPSC microglia treated with LPS and ATL5802 or an isotype control antibody. The data show that ATL_5802 decreases microglial IL-6 release in response to LPS. (B) Shows the concentration of MCP-1 (pg / ml) in the supernatants of iPSC microglia treated with LPS and ATL5802 or an isotype control antibody. The data show that ATL_5802 decreases microglial MCP-1 release in response to LPS. Statistical analysis was performed using a Student's t-test, * = p<0.05; ** = p<0.01; *** = p < 0.001, ns = not significant.

[064] Figure 21 shows the results of a Western blot for phosphorylated SYK (pSYK) in cultured microglia cells, stimulated with LPS and treated with ATL_5802. The figure shows a Western blot for pSYK (Tyr525 / 526) and total SYK in microglia cells 20 minutes after treatment with isotype control antibody or ATL_5802. Petition 870250089689, dated 02 / 10 / 2025, page 34 / 221 21 / 144

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

[066] Figure 23 shows the results of a gene expression analysis after RNA sequencing in 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) versus PC2, from PCA calculated for 25% of the most variable genes. (BC) shows volcano plots of gene expression changes in iPSC-derived microglia treated with ATL_5802 versus isotype control in stimulated vehicle (B) or LPS / interferon gamma stimulated conditions (C). The volcano plot shows fold changes (x-axis) versus statistical significance, expressed as -log10 of the adjusted p-value (y-axis).Colored dots (dots to the left of the leftmost vertical line, to the right of the rightmost vertical line, and above the horizontal line) indicate differentially expressed genes with statistical significance, according to the selected thresholds; red (positive log2FoldChange): upregulated after treatment with ATL_5802, blue (negative log2FoldChange): downregulated. The horizontal line marks the threshold for significance, the vertical lines mark the thresholds for log2FoldChange. (D) shows a bar graph with the number of differentially expressed genes, as shown in (B) and (C), and their direction of change (i.e., upregulated or downregulated), for all stimuli and antibody treatments.(E) Bar chart showing the results of a gene set enrichment analysis (GSEA) for oxidative phosphorylation and respiratory electron transport gene sets / pathways with fitted p-values ​​(x-axis) and normalized enrichment scores (NES, intensity. Petition 870250089689, dated 02 / 10 / 2025, p. 35 / 221 22 / 144 of the color) by comparison (y-axis) and pathway (vertical boxes 1 to 4). 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 color intensity. The vertical line marks the significance limit. (F) Representative GSEA plot for the REACTOME “Respiratory Electron Transport” pathway for all anti-CD33 antibodies compared to the isotype control when stimulated with LPS / interferon gamma. These plots show that the pathway is significantly more enriched after treatment with ATL_5802 (and to a lesser extent with ATL_5854) compared to the comparator antibody ATL_5909.

[067] Figure 24 shows the results of the antibody disclosure effect analysis in a human central nervous system (CNS) co-culture experiment. (A) Shows an animated drawing illustrating human CNS quadruple cell cultures comprising human iPSC-derived glutamatergic / GABAergic neurons, astrocytes, and microglia, and a fluorescent image showing cells in such co-culture. (B - E) Interleukin-6 (IL-6) levels (B); GFAP (C), IP-10 (D); and MCP-1 (E). Two-way ANOVA with Tukey's multiple comparisons test (n = 2 to 3 wells), *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001 compared to the isotype-treated LPS / IFNy group. Data from BC show that ATL_5802 protects against inflammation in Quad-CNS cultures derived from human iPSCs, as evidenced by a reduction in interleukin-6 (IL-6) and a decrease in GFAP, a marker of astrogliosis.Data in DE show a significant reduction in IP-10 (interferon-gamma-induced protein 10) and MCP-1 levels with ATL_5802 treatment under LPS / IFNy conditions. Petition 870250089689, dated 02 / 10 / 2025, page 36 / 221 23 / 144 DETAILED DESCRIPTION OF THE INVENTION

[068] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Other aspects and embodiments will be evident to those skilled in the art. All documents mentioned in this text are incorporated into this report by reference.

[069] Antibodies and their fragments capable of specifically binding to the CD33 protein or a fragment thereof are described in this report. As used in this report, an antibody capable of “specific binding” or “target-specific binding” is one capable of binding through the association of the epitope recognition site with an epitope within the target. This is different from non-specific binding, for example, 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 it, rather than to proteins in general.

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

[071] CD33 is an inhibitory immune receptor belonging to the family of sialic acid-binding immunoglobulin-like lectins. CD33 is also known as sialic acid-binding immunoglobulin-like lectin 3 (Siglec-3). The human CD33 gene (gene ID: 945) is located on chromosome 19 (19q13.33) in humans and consists of seven exons. The complete sequence of Petition 870250089689, dated 02 / 10 / 2025, p. 37 / 221 24 / 144 Human CD33 is a 364-amino acid sequence available under the Uniprot identifier P20138. It consists of (i) an N-terminal signal peptide that directs CD33 to the secretory pathway (amino acids 1 to 17 of P20138); (ii) two extracellular domains, consisting of the N-terminal Ig-like V-set domain for carbohydrate ligand recognition and a C2-set domain (amino acids 18 to 259 of P20138); (iii) a transmembrane domain (amino acids 260 to 282 of P20138); and (iv) a cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs (ITIM) that mediate immune cell signaling (amino acids 283 to 364 of P20138) (Eskandari-Sedighi et al., 2023). CD33 undergoes alternative splicing to generate a long isoform, denoted as hCD33M (M = 'longer'), and a short isoform denoted as hCD33m (m = 'shorter'), which is generated by the deletion of exon 2 (amino acids 13 to 139 of hCD33M) and does not have a functional V domain (Hernández-Caselles et al., 2006).The major isoform of human CD33 (hCD33) is a 67 kDa transmembrane glycoprotein, available under the 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 the Uniprot identifier P20138-3 (237 amino acids), provided as SEQ ID NO: 177. The antibodies described in this report can bind to hCD33M and not hCD33m. CD33 is expressed on the cell surface of myeloid cells, monocytes, macrophages, and microglia in the brain. CD33 participates in immune cell adhesion processes and mediates cell-cell interaction (Varki et al., 2006).

[072] Genome-wide association studies have identified CD33 as a genetic modulator of susceptibility and pathology in late-onset Alzheimer's disease (Hollingworth et al., 2011). Increased CD33 expression in the microglia of AD patients is associated with cognitive decline or more advanced disease status (Siddiqui et al., 2017). This is believed to be due to altered microglial activation and inhibition of microglia-mediated phagocytosis. Petition 870250089689, dated 02 / 10 / 2025, p. 38 / 221 25 / 144 toxic insoluble beta-amyloid 42 (Αβ42) species. On the other hand, a protective allele of the CD33 SNP rs3865444 results in lower expression of CD33 and a higher proportion of a truncated, non-signaling form, along with reduced levels of insoluble Άβ42 in the AD brain (Griciuc et al., 2013). Furthermore, it is suggested that CD33 interacts with other AD risk genes in microglia, such as TREM2, influencing the onset and pathogenesis of the disease. In particular, it is suggested that CD33 downregulates TREM2 / DAP12-mediated microglial activation, resulting in reduced cellular function, such as microglial phagocytosis, which, in turn, leads to reduced clearance of toxic Ae42 species (Chan et al., 2015). Consequently, the antibodies described in this report are able to increase the phagocytosis of cells expressing CD33, such as microglia phagocytosis, compared to a comparator antibody.

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

[074] CD33 possesses endocytic properties (receptor internalization), a property that has been exploited for targeting by antibody-drug conjugates (Laszlo et al., 2014). Although this property may result in a decrease in CD33 on the cell surface, it may also reduce the effectiveness of CD33-targeted therapies due to the internalization of anti-CD33 antibodies by CD33-expressing cells that are not the primary therapeutic targets. For example, when targeting microglia cells that express CD33, monocytes that Petition 870250089689, dated 02 / 10 / 2025, page 39 / 221 26 / 144 expressing CD33 may represent a peripheral reservoir of antibodies, which may prevent the antibody from reaching therapeutic doses in the brain with acceptable toxicity. In some cases, the antibodies described in this report induce reduced CD33 endocytosis compared to a comparator antibody. CD33 internalization can be measured, for example, by determining CD33 levels on the cell surface using flow cytometry in the presence of a candidate antibody. The cell may be a human cell, such as a microglial cell, myeloid cells, a monocyte, a tumor cell, such as a leukemic blast cell, and / or an iPSC-derived microglial cell and / or a cell that has been stimulated with an inflammatory signal (e.g., LPS) prior to antibody exposure.A comparative antibody can be selected from: an isotype control antibody and / or an antibody with the variable heavy chain sequence of ATL_5909 and the variable light chain sequence of ATL_5909.

[075] Therapeutic strategies targeting TREM2, an anti-inflammatory receptor expressed on 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 the microglial response to beta-amyloid plaque. For example, several TREM2 agonists are currently under clinical investigation for the treatment of AD, including AL002 from Alector / Abbvie (referred to in this document as ATL6166, in a phase 2 clinical trial), DNL919 from Denali Therapeutic Inc (referred to in this document as ATL6167, in a phase 1 clinical trial), and VGL101 from Vigil (referred to in this document as ATL6170; in a phase 2 clinical trial). Although CD33 is known to modulate TREM2 activity, to our knowledge, no direct CD33 antagonist antibodies are currently in clinical development.

[076] As used in this report, the term “CD33” encompasses truncations, derivatives, and variants of the CD33 sequence provided in this report. Petition 870250089689, dated 02 / 10 / 2025, p. 40 / 221 27 / 144 as SEQ ID NO: 176 or a homolog thereof, and may refer to any protein with at least 80%, at least 90%, or at least 95% sequence identity with said sequence. The CD33 sequence may be a human CD33 sequence. The CD33 sequence may be a sequence comprising at least part of the V domain (also called the “Ig-like V-type” domain) of CD33 (amino acids 19 to 135 of Uniprot ID P20138-1 or a homolog thereof). The CD33 sequence may also comprise one or more of the following: at least part of the C domain (also called the “Ig-like C2 type” domain) of CD33 (amino acids 145 to 228 of the Uniprot ID P20138-1 or a homolog thereof), at least part of the transmembrane domain of CD33 (amino acids 260 to 282 of P20138-1 or a homolog thereof), and at least part of the cytoplasmic domain of CD33 (amino acids 283 to 364 of P20138-1 or a homolog thereof).The antibodies, according to this disclosure, can bind to CD33 proteins comprising the human CD33 domain V (amino acids 19 to 135 of Uniprot ID P20138-1, provided as SEQ ID NO:176) or to a sequence that has at least 90%, 95%, 98% or 99% sequence identity with said sequence.

[077] This disclosure relates primarily to antibody molecules, whether whole antibodies (e.g., IgG, such as IgG1) or antibody fragments (e.g., single-chain variable fragment (scFv), antibody fragments (Fab) or bivalent antibody fragments (F(ab')2), single-domain antibody (sdAb)). Antigen-binding regions of the antibody (also called “antigen-binding portions”) are provided, as well as variable heavy chain (VH) and variable light chain (VL) domains of the antibody. Within the VH and VL domains, complementarity-determining regions (CDRs) are provided, which may be provided within different structure regions (FRs). Petition 870250089689, dated 02 / 10 / 2025, page 41 / 221 28 / 144 to form VH or VL domains, as appropriate. An antigen-binding site may consist of a VH domain and / or a VL domain of the antibody.

[078] Antibodies, according to the present disclosure, can be provided in isolated form. The term “antibody” encompasses a fragment or derivative thereof, or a synthetic antibody or antibody fragment. An antibody or fragment thereof can be a monoclonal antibody (mAb). mAbs are homogeneous populations of antibodies that specifically target a single epitope on an antigen. Antibodies and methods for their construction and use are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 (2005). Considering current techniques regarding monoclonal antibody technology, antibodies can be prepared for most targets. It is possible to use monoclonal antibody molecules and others, and to use recombinant DNA technology techniques to produce other antibodies or chimeric molecules that retain the specificity of the original antibody.Such techniques may involve the introduction of CDRs or variable regions of one antibody into a different antibody molecule (see, for example, GB 2188638A and EP0239400).

[079] An “antigen-binding domain” describes the part of a molecule that binds to all or part of the target antigen. An antibody generally comprises 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 of the antigen-binding domain, which is the part of the antigen-binding domain that binds to the target antigen. A monospecific monoclonal IgG antibody molecule contains two antigen-binding domains, each of which is capable of binding to the same target (i.e., it is bivalent for a single target). A Fab fragment generally comprises a VH domain, a CH1 domain, a VL domain, and a CL domain. An antibody Petition 870250089689, dated 02 / 10 / 2025, page 42 / 221 A complete 29 / 144 may comprise a pair of Fab fragments and an Fc fragment comprising a pair of chains, each comprising a CH2 domain and a CH3 domain. An Fv fragment comprises a VH domain and a VL domain. The VH and VL regions comprise structure regions (FRs) on each side of each CDR, which provide a structure for the CDRs. From the N-terminal to the C-terminal, the VH regions comprise the following structure: N-terminal - [HFR1] - [HCDR1] [HFR2] - [HCDR2] - [HFR3] - [HCDR3] - [HFR4] - C-terminal; and the VL regions comprise the following structure: N-terminal - [LFR1] - [LCDR1] - [LFR2] [LCDR2] - [LFR3] - [LCDR3] - [LFR4] - C-terminal.

[080] The term “ScFv molecules” refers to molecules 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 be expressed and secreted by E. coli, thus allowing for the easy production of large quantities of said fragments. Whole antibodies and F(ab')2 fragments are “bivalent.” The term “bivalent” means that said antibodies and F(ab')2 fragments possess two antigen-binding sites. In contrast, Fab, Fv, ScFv, and dAb fragments are monovalent, possessing only one antigen-binding site.

[081] Antibodies, according to this disclosure, may be labeled in a detectable manner or at least capable of detection. For example, the antibody may be labeled with a radioactive atom, a colored molecule, a fluorescent molecule, or a molecule that can be easily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radiolabels. The binding fraction (antibody or fragment thereof) may be labeled directly with a detectable marker or may be labeled indirectly. For example, the binding fraction may be an unlabeled antibody that can be detected by another Petition 870250089689, dated 02 / 10 / 2025, p. 43 / 221 30 / 144 antibody that is also labeled. Alternatively, the second antibody may have biotin bound to it, and the binding of the labeled streptavidin to biotin is used to indirectly label the first antibody.

[082] An antibody “fragment” may comprise any number of residues from a “parental” antibody, while retaining the ability to bind to the target. A fragment may have no effector function, for example, it may be completely unable to bind or exhibit reduced binding to the Fc receptor, relative to the parental antibody. A fragment is typically smaller than the parental antibody. A fragment may comprise 50%, 60%, 70%, 80%, 90%, 95% or more of the contiguous or non-contiguous amino acids of the parental antibody. A fragment may comprise 50, 100, 150, 200, 250, 300 or more contiguous or non-contiguous amino acids of the parental antibody. A fragment may comprise deletions in the Fc region or in the Fc region. A fragment may retain the CDRs and / or variable domains of the parental antibody unchanged. In some embodiments, a fragment is either a Fab fragment or an F(ab')2 fragment. CDR sequences are described in this report using IMGT numbering (Lefranc, M.-P., Immunology Today, 18, 509 (1997)).

[083] Antibodies, according to the present disclosure, may have a VH with the VH CDRs of the following antibodies: ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, or a set of CDRs containing zero, one, or two amino acid substitutions in each CDR, compared to the set of CDRs above. Thus, the isolated antibody may comprise a variable heavy chain domain with the following CDRs: a CDRH1 comprising an amino acid sequence selected from: (i) HCDR1_ATL_0005802; HCDR1_ATL_0005807; HCDR1_ATL_0005808: GYSFTSYW (SEQ ID NO:44); (ii) HCDR1_ATL_0005809: GYSFNTYW (SEQ ID Petition 870250089689, dated 02 / 10 / 2025, p. 44 / 221 31 / 144 NO:45); (iii) HCDR1_ATL_0005810: GYTFTSYY (SEQ ID NO:46); (iv) HCDR1_ATL_0005853: GYKFNNNW (SEQ ID NO:47); (v) HCDR1_ATL_0005854: GYKFSNNW (SEQ ID NO:48); or (vi) an amino acid sequence with 1 or 2 mutations compared to the sequences above; a CDRH2 comprising an amino acid sequence selected from: (i) HCDR2_ATL_0005802; HCDR2_ATL_0005807; HCDR2_ATL_0005808; HCDR2_ATL_0005853; HCDR2_ATL_0005854: IYPGDSDT (SEQ ID NO: 52); (ii) HCDR2_ATL_0005809: IYPGDSET (SEQ ID NO: 53); (iii) HCDR2_ATL_0005810: INPSGGST (SEQ ID NO: 54); or (iv) an amino acid sequence with 1 or 2 mutations compared to the sequences above; and a CDRH3 comprising an amino acid sequence selected from from: (i) HCDR3_ATL_0005802: ARRPRGFGEYYFDY (SEQ ID NO: 59); (ii) HCDR3_ ATL_ 0005807: FILEGAGPGGFDI (SEQ ID NO:60); (iii) HCDR3_ ATL_ 0005808: ARGQGGGAGAFDI (SEQ ID NO:61); (iv) HCDR3_ ATL_ 0005809: ARHRADAPSDAFDI (SEQ ID NO:62); (v) HCDR3_ ATL_ 0005810: : ARDWHNTPQFDTDYYYYGMDV (SEQ ID NO:63); (vi) HCDR3_ ATL_ 0005853: ARHSGGLDGYTAAALDY (SEQ ID NO:64); (vii) HCDR3_ATL_0005854: ATWGGSNWFVD (SEQ ID NO:65); or (viii) an amino acid sequence with 1 or 2 mutations compared to the sequences above.

[084] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005802, a CDRH2 comprising the sequence HCDR2_ATL_0005802 and a CDRH3 comprising the sequence HCDR3_ATL_0005802, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences. Petition 870250089689, dated 02 / 10 / 2025, page 45 / 221 32 / 144

[085] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005853, a CDRH2 comprising the sequence HCDR2_ATL_0005853 and a CDRH3 comprising the sequence HCDR3_ATL_0005853, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[086] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005854, a CDRH2 comprising the sequence HCDR2_ATL_0005854 and a CDRH3 comprising the sequence HCDR3_ATL_0005854, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[087] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005810, a CDRH2 comprising the sequence HCDR2_ATL_0005810 and a CDRH3 comprising the sequence HCDR3_ATL_0005810, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[088] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005807, a CRH2 Petition 870250089689, dated 02 / 10 / 2025, page 46 / 221 33 / 144 comprising the sequence HCDR2_ATL_0005807 and a CDRH3 comprising the sequence HCDR3_ATL_0005807, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[089] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005808, a CDRH2 comprising the sequence HCDR2_ATL_0005808 and a CDRH3 comprising the sequence HCDR3_ATL_0005808, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[090] An antibody, according to the present disclosure, may comprise a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005809, a CDRH2 comprising the sequence HCDR2_ATL_0005809 and a CDRH3 comprising the sequence HCDR3_ATL_0005809, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

[091] In an antibody, according to the present disclosure, at least one of the HV CDR 1-3 sequences may vary. A variant may have one or two amino acid substitutions compared to the HV CDR 1-3 set described above. In some embodiments, an antibody, according to the present disclosure, comprises CDRs with sequences that have one or two substitutions compared to the HV CDR sequences of any antibody. Petition 870250089689, dated 02 / 10 / 2025, p. 47 / 221 34 / 144 described in this report. For example, an antibody, according to the disclosure, may comprise HV CDRs with the sequences of any of the above antibodies, except that one or two of the HV CDRs comprise a substitution, wherein the total number of substitutions among the HV CDRs does not exceed two. In some embodiments, a variant may have one, two, or three substitutions, preferably at most one or two substitutions in each or more of the HV CDR1-3 described above. The CDRH1 regions of any antibodies or fragments described in this report may have a length of 8 amino acids. The CDRH2 regions of any antibodies or fragments described in this report may have a length of 8 amino acids. The CDRH3 regions of any antibodies or fragments described in this report may have a length of 13 to 21 amino acids.In some embodiments, a variant may have VH CDRs that have at least 70%, at least 80%, or at least 90% sequence identity with any set of VH CDRs described in this report.

[092] Antibodies, according to the present disclosure, may have a variable light chain (VL) domain with the variable light chain (VL) domain CDRs of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, or a set of CDRs containing zero, one, or two amino acid substitutions in each CDR, compared to the set of CDRs above. Thus, the isolated antibody may comprise a variable light chain domain with the following CDRs: a CDRL1 comprising an amino acid sequence selected from: (i) LCDR1_ATL_0005802; LCDR1_ATL_0006040; LCDR1_ATL_0006041; LCDR1_ATL_0006042; LCDR1_ATL_0006043; LCDR1_ATL_0006044; LCDR1_ATL_0006045; LCDR1_ATL_0006046; LCDR1_ATL_0006047; Petition 870250089689, dated 02 / 10 / 2025, page 48 / 221 35 / 144 LCDR1_ATL_0006048: SSNIGAGYD (SEQ ID NO: 110); (ii) LCDR1_ATL_0005807:ALARQY (SEQ ID NO: 111); (iii) LCDR1_ATL_0005808; LCDR1_ATL_0005854:SSDVGGYNY (SEQ ID NO: 112); (iv) LCDR1_ATL_0005809:SLRNYY (SEQ ID NO: 113); (v) LCDR1_ATL_0005810:SGSVSTSYY (SEQ ID NO: 114); (vi) LCDR1_ATL_0005853:QSLLHSDGYNY (SEQ ID NO: 115); or (vii) an amino acid sequence with 1 or 2 mutations compared to the sequences above; a CDRL2 comprising an amino acid sequence selected from: (i) LCDR2_ATL_0005802; LCDR2_ATL_0006040; LCDR2_ATL_0006041; LCDR2_ATL_0006042; LCDR2_ATL_0006043; LCDR2_ATL_0006044; LCDR2_ATL_0006045; LCDR2_ATL_0006046; LCDR2_ATL_0006047; LCDR2_ATL_0006048: GNS (SEQ ID NO:119); (ii) LCDR2_ATL_0005854: DVS (SEQ ID NO:120); (iii) LCDR2_ATL_0005807:KDS (SEQ ID NO:121); (iv) LCDR2_ATL_0005808:DVT (SEQ ID NO:123); (v) LCDR2_ATL_0005809:GKN (SEQ ID NO:124); (vi) LCDR2_ATL_0005810:STN (SEQ ID NO:125); (vii) LCDR2_ATL_0005853: VGS (SEQ ID NO:126); or (viii) an amino acid sequence with 1 or 2 mutations compared to the sequences above; and a CDRL3 comprising an amino acid sequence selected from: (i) LCDR3_ATL_0005802; LCDR3_ATL_0006040; LCDR3_ATL_0006041; LCDR3_ATL_0006042; LCDR3_ATL_0006043; LCDR3_ATL_0006044; LCDR3_ATL_0006045; LCDR3_ATL_0006046; LCDR3_ATL_0006047; LCDR3_ATL_0006048: QSYDSSLSGDV (SEQ ID NO: 129); (ii) LCDR3_ATL_0005807: QSPDSSGTYPV (SEQ ID NO: 130); (iii) LCDR3_ATL_0005808:SSYTSSSTLEV (SEQ ID NO: 131); (iv) LCDR3_ATL_0005809:NSRDSSGYHLGL (SEQ ID NO: 132); (v) LCDR3_ATL_0005810: LLYMGSGIWM (SEQ ID NO: 133); (vi) LCDR3_ATL_0005853:MQALQTPIT (SEQ ID NO: 134); (vii) Petition 870250089689, dated 02 / 10 / 2025, page 49 / 221 36 / 144 LCDR3_ATL_0005854:SSYTNSSTLEV (SEQ ID NO: 236); or (viii) an amino acid sequence with 1 or 2 mutations compared to the sequences above.

[093] An antibody, according to the present disclosure, may comprise a variable light chain domain with the following CDRs: a CDRL1 comprising an amino acid sequence selected from: LCDR1_ATL_0005802: SSNIGAGYD (SEQ ID NO: 110); LCDR1_ATL_0005854:SSDVGGYNY (SEQ ID NO: 112); LCDR1_ATL_0005853 QSLLHSDGYNY (SEQ ID NO: 115); or an amino acid sequence with 1 or 2 mutations compared to the sequences above; a CDRL2 comprising an amino acid sequence selected from: LCDR2_ATL_0005802: GNS (SEQ ID NO:119); LCDR2_ATL_0005854: DVS (SEQ ID NO:120); LCDR2_ATL_0005853: VGS (SEQ ID NO:126); or an amino acid sequence with 1 or 2 mutations compared to the sequences above; and a CDRL3 comprising an amino acid sequence selected from: LCDR3_ATL_0005802: QSYDSSLSGDV (SEQ ID NO: 129); LCDR3_ATL_0005853: MQALQTPIT (SEQ ID NO: 134); LCDR3_ATL_0005854: SSYTNSSTLEV (SEQ ID NO: 236); or an amino acid sequence with 1 or 2 mutations compared to the sequences above.

[094] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005802, a CRL2 comprising the sequence LCDR2_ATL_0005802 and a CDRH3 comprising the sequence LCDR3_ATL_0005802, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences. Petition 870250089689, dated 02 / 10 / 2025, page 50 / 221 37 / 144

[095] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005853, a CRL2 comprising the sequence LCDR2_ATL_0005853 and a CDRH3 comprising the sequence LCDR3_ATL_0005853, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[096] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005854, a CRL2 comprising the sequence LCDR2_ATL_0005854 and a CDRH3 comprising the sequence LCDR3_ATL_0005854, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[097] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005810, a CRL2 comprising the sequence LCDR2_ATL_0005810 and a CDRH3 comprising the sequence LCDR3_ATL_0005810, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[098] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005807, a CRL2 Petition 870250089689, dated 02 / 10 / 2025, page 51 / 221 38 / 144 comprising the sequence LCDR2_ATL_0005807 and a CDRH3 comprising the sequence LCDR3_ATL_0005807, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[099] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005808, a CRL2 comprising the sequence LCDR2_ATL_0005808 and a CDRH3 comprising the sequence LCDR3_ATL_0005808, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[0100] An antibody, according to the present disclosure, may comprise a variable light chain (VL) domain with the following CDRs: a CDRL1 comprising the sequence LCDR1_ATL_0005809, a CRL2 comprising the sequence LCDR2_ATL_0005809 and a CDRH3 comprising the sequence LCDR3_ATL_0005809, or a set of CDRs comprising 1 or 2 mutations in CDRL1 and CDRL2 (1 or 2 mutations in each CDR or 1 or 2 mutations in both CDRs) compared to these sequences and / or 1, 2 or 3 mutations in CDRL3 compared to these sequences.

[0101] In an antibody, according to the present disclosure, at least one of the VL CDR1-3 sequences may vary. A variant may have 1, 2, or 3 amino acid substitutions compared to the VL CDR1-3 set described above. In some embodiments, an antibody, according to the disclosure, comprises CDRs with sequences that have between 1 and 3 substitutions compared to the VL CDR sequences of an antibody described herein. Petition 870250089689, dated 02 / 10 / 2025, p. 52 / 221 39 / 144 report. For example, an antibody, according to the disclosure, may comprise one substitution, wherein the total number of substitutions does not exceed 3. In some embodiments, a variant may have one, two, or three, preferably at most one or two substitutions in each or more of the VL CDR1-3s described above.

[0102] The CDRL1 regions of any antibodies or fragments described in this report may have a length between 6 and 11 amino acids. The CDRL2 regions of any antibodies or fragments described in this report may have a length of 3 amino acids. The CDRL3 regions of any antibodies or fragments described in this report may have a length of 9 to 11 amino acids. In some embodiments, a variant may have VL CDRs that have at least 70%, at least 80%, or at least 90% sequence identity with any set of VL CDRs described in this report.

[0103] The VH CDRs 1-3 and, optionally, the VL CDRs 1-3 of any of the antibodies described above may also be particularly useful in conjunction with several different structural regions. Consequently, light and / or heavy chains with CDRs 1-3, as described above, may possess an alternative structural region. Suitable structural regions are known in the art and described, for example, in M. Lefranc & G. Le Franc (2001) “The Immunoglobulin Facts Book”, Academic Press.

[0104] A disclosure antibody may have a variable heavy chain (VH) domain with the following structure sequences: HFWR1 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR2 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR3 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, Petition 870250089689, dated 02 / 10 / 2025, page 53 / 221 40 / 144 ATL_0005808, ATL_0005809, ATL_0005810 and HFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 or structure sequences with one to six substitutions compared to the structure sequences above.

[0105] An antibody, according to the disclosure, may have a variable heavy chain (VH) domain with the following structural sequences: an HFWR1 comprising an amino acid sequence selected from: H FWR1_ATL_0005807; H FWR1_ATL_0006041 QVQLVQSGAEVKKPGESLKISCKGS (SEQ ID NO: 71) HFWR1_ATL_0005808 QVQLQQSGAEVKKPGESLKISCKGS (SEQ ID NO:72) HFWR1_ATL_0005809 QVQLQQSGGEVKKPGESLKISCKGS (SEQID NO:73) HFWR1_ATL_0005810 QVQLVQSGAEVKKPGASVKVSCKAS (SEQID NO:74) HFWR1_ATL_0005853 QVQLVQSGAEVKKTGESLRISCKAS (SEQID NO:235) HFWR1_ATL_0005854 QVQLVQSGAEVKKTGEYLRISCKAS (SEQID NO:75) HFWR1_ATL_0006040; HFWR1_ATL_0006044; HFWR1_ATL_0006045; HFWR1_ATL_0006046; HFWR1_ATL_0006047; HFWR1_ATL_0006048: EVQLVQSGAEVKKPGESLKISCKGS (SEQ ID NO: 77) HFWR1_ATL_0006042: EVQLVESGAEVKKPGESLKISCKGS (SEQ ID NO:78) HFWR1_ATL_0006043: EVQLVQSGAEVKKPGESLRISCKGS (SEQ ID NO:79) Petition 870250089689, dated 02 / 10 / 2025, page 54 / 221 41 / 144 an HFWR2 comprising an amino acid sequence selected from: HFWR2_ATL_0005802; HFWR2_ATL_0005853; HFWR2_ATL_0005854; HFWR2_ATL_0006044: IAWVRQMPGKGLEWMGI (SEQ ID NO: 83) HFWR2_ATL_0005807; HFWR2_ATL_0005808; HFWR2_ATL_0006040; HFWR2_ATL_0006041; HFWR2_ATL_0006042; HFWR2_ATL_0006043; HFWR2_ATL_0006045; HFWR2_ATL_0006046; HFWR2_ATL_0006047; HFWR2_ATL_0006048: IGWVRQMPGKGLEWMGI (SEQ ID: 84) HFWR2_ATL_0005809: IAWVRHAPGKGLEWMGI (SEQ ID NO: 85) HFWR2_ATL_0005810: MHWVRQAPGQGLEWMGI (SEQ ID NO: 86) an HFWR3 comprising an amino acid sequence selected from: HFWR3_ATL_0005802: RYSPFEGQVTISADKSIGTAYLQWSSLKASDTAMYYC (SEQ ID NO:91) HFWR3_ATL_0005807: RYSPSFQGQVSISVDKSISTAFLQWSSLKSDSAMYYC (SEQ ID NO:92) HFWR3_ATL_0005808; HFWR3_ATL_0006040; HFWR3_ATL_0006041; HFWR3_ATL_0006042; HFWR3_ATL_0006043; HFWR3_ATL_0006044; HFWR3_ATL_0006047; HFWR3_ATL_0006048: RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC (SEQ ID NO:93) HFWR3_ATL_0005809: RYSPSFQSQVTISADKSIDTAYLEWNTLEASDTAMYYC (SEQ ID NO: 94) HFWR3_ATL_0005810: SYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC (SEQ ID NO: 95) HFWR3_ATL_0005853: RYSPFEGQVTISADKSSGIVYLQWTSLKASDTAIYYC (SEQ ID NO: 96) Petition 870250089689, dated 02 / 10 / 2025, p. 55 / 221 42 / 144 HFWR3_ATL_0005854: RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAIYYC (SEQ ID NO: 97) or structure sequences with one to six, one to five, one to four, one to three, or one to two substitutions compared to the structure sequences above.

[0106] A disclosure antibody may have a variable heavy chain (VH) domain with the HFWR1, HFWR2, HFWR3, and HFWR4 structure sequences of antibody ATL_0005802, or structure sequences with one to six substitutions compared to these structure sequences. A disclosure antibody may have a variable heavy chain (VH) domain with the HFWR1, HFWR2, HFWR3, and HFWR4 structure sequences of antibody ATL_0005853, or structure sequences with one to six substitutions compared to these structure sequences. A disclosure antibody may have a variable heavy chain (VH) domain with the HFWR1, HFWR2, HFWR3, and HFWR4 structure sequences of antibody ATL_0005854, or structure sequences with one to six substitutions compared to these structure sequences.An antibody for disclosure may have a variable heavy chain (VH) domain with the HFWR1, HFWR2, HFWR3, and HFWR4 structural sequences of antibody ATL_0005807, or structural sequences with one to six substitutions compared to these structural sequences. An antibody for disclosure may have a variable heavy chain (VH) domain with the HFWR1, HFWR2, HFWR3, and HFWR4 structural sequences of antibody ATL_0005808, or structural sequences with one to six substitutions compared to these structural sequences. An antibody for disclosure may have a variable heavy chain (VH) domain with the HFWR1, HFWR2, HFWR3, and HFWR4 structural sequences of antibody ATL_0005809, or structural sequences with one to six substitutions compared to these structural sequences. An antibody for dissemination may have a variable heavy chain (VH) domain with the sequences. Petition 870250089689, dated 02 / 10 / 2025, page 56 / 221 43 / 144 of the HFWR1, HFWR2, HFWR3 and HFWR4 structures of the ATL_0005810 antibody, or structure sequences with one to six substitutions compared to these structure sequences.

[0107] In some embodiments, substitutions in the variable domain structure sequences of the heavy chain may be located at any position other than position 40 in the standard IMGT numbering. In some embodiments, the substitutions in the structure sequences do not include a substitution at position 40 in the standard IMGT numbering. Substitutions in the variable domain structure sequences of the heavy chain may be located at any position in HFWR1 and / or HFRW3. Substitutions in the variable domain structure sequences of the heavy chain may be located at any position other than in HFRW2. Substitutions in the variable domain structure sequences of the heavy chain may be selected from the following positions in the standard IMGT numbering: HFWR1: positions 1, 6, 20; HFWR3: positions 72, 85.Substitutions in the variable domain structure sequences of the heavy chain can be selected from the following positions in the standard IMGT numbering: HFWR1: at position 1: Q1E, at position 6: E6Q, at position 20: R20K; HFWR3: at position 72: E72Q, at position 85: G85S.

[0108] A disclosure antibody may have a variable heavy chain (VH) domain with an HFWR2 structural sequence of ATL_0005802 (HFWR2_ATL_0005802; HFWR2_ATL_0005853; HFWR2_ATL_0005854). The isolated antibody may have a variable heavy chain (VH) domain with an HFWR2 structural sequence comprising the sequence: IAWVRQMPGKGLEWMGI (SEQ ID NO: 83). A disclosure antibody may have a variable heavy chain (VH) domain with an HFWR2 structural sequence of ATL_0005802. A disclosure antibody may have a variable domain Petition 870250089689, dated 02 / 10 / 2025, page 57 / 221 44 / 144 heavy chain (VH) with a structural sequence comprising an A at position 40 in the standard IMGT numbering.

[0109] An antibody, according to the disclosure, may have a variable heavy chain (VH) domain with the following structural sequences: HFWR1 of SEQ ID NOs: 70, 71, 72, 73, 74, 235, 75, 77, 78 or 79 (HFWR1_ATL_0005807; HFWR1_ATL_0006041; HFWR1_ATL_0005808; HFWR1_ATL_0005809; HFWR1_ATL_0005810; HFWR1_ATL_0005853; HFWR1_ATL_0005854; HFWR1_ATL_0006040; HFWR1_ATL_0006044; HFWR1_ATL_0006045; HFWR1_ATL_0006046;HFWR1_ATL_0006047; HFWR1_ATL_0006048; HFWR1_ATL_0006042; HFWR1_ATL_0006043), preferably SEQ ID NOS: 70, 235, 75; HFWR2 of SEQ ID NOs; 83, 84, 85, 86 (HFWR2_ATL_0005802; HFWR2_ATL_0005853; HFWR2_ATL_0005854; HFWR2_ATL_0006044; HFWR2_ATL_0005807; HFWR2_ATL_0005808; HFWR2_ATL_0006040; HFWR2_ATL_0006041; HFWR2_ATL_0006042; HFWR2_ATL_0006043; HFWR2_ATL_0006045; HFWR2_ATL_0006046; HFWR2_ATL_0006047; HFWR2_ATL_0006048; HFWR2_ATL_0005809; HFWR2_ATL_0005810), preferably SEQ ID NO:83; HFWR3 from SEQ ID NOs: 91, 92, 93, 94, 95, 96, 97 (HFWR3_ATL_0005802 HFWR3_ATL_0005807; HFWR3_ATL_0005808; HFWR3_ATL_0006040; HFWR3_ATL_0006041; HFWR3_ATL_0006042; HFWR3_ATL_0006043; HFWR3_ATL_0006044; HFWR3_ATL_0006047; HFWR3_ATL_0006048; HFWR3_ATL_0005809; HFWR3_ATL_0005810 HFWR3_ATL_0005853; HFWR3_ATL_0005854), preferably SEQ ID Nos: 91, 96, 97 and HFWR4 of SEQ ID NOs: 101, 104, 105 (HFWR4_ATL_0004828; HFWR4_ATL_0005802; HFWR4_ATL_0005853; HFWR4_ATL_0005854; Petition 870250089689, dated 02 / 10 / 2025, page 58 / 221 45 / 144 HFWR4_ATL_0006040; HFWR4_ATL_0006043; HFWR4_ATL_0006046; HFWR4_ATL_0005807; HFWR4_ATL_0006041; HFWR4_ATL_0006044; HFWR4_ATL_0006047; HFWR4_ATL_0005808; HFWR4_ATL_0006042 HFWR4_ATL_0006045 HFWR4_ATL_0006048 HFWR4_ATL_0005809 HFWR4_ATL_0005810), preferably SEQ ID NO: 101.

[0110] A disclosure antibody may have the CDRL1, CDRL2, and CDRL3 of the VL domain within a germline structure. A disclosure antibody may have a variable heavy chain (VH) domain comprising CDRH1, CDRH2, and CDRH3 within a germline structure, provided that position 40 in the standard IMGT numbering is A.

[0111] An antibody, according to the disclosure, may have a variable light chain (VL) domain with the following structural sequences: an LFWR1 comprising an amino acid sequence selected from: LFWR1_ATL_0005802: QAVLTQPSSVSGAPGQRVTISCTGS (SEQ ID NO: 138) LFWR1_ATL_0005807: QSVLTQPPSVSVSPGQTARITCSGD (SEQ ID NO:139) LFWR1_ATL_0005808; LFWR1_ATL_0005854: QSALTQPASVSGSPGQSITISCTGT (SEQ ID NO:140) LFWR1_ATL_0005809: SSELTQDPAVSVAVGQTVRITCQGD (SEQ ID NO:141) LFWR1_ATL_0005810: QTVVTQEPSFSVSPGGTVTLTCGLS (SEQ ID NO: 142) LFWR1_ATL_0005853: DVVMTQSPLSLPVNPGEPASISCRSS (SEQ ID NO: 143) Petition: 870250089689, on 10 / 02 / 2025, page. 59 / 221 46 / 144 LFWR1_ATL_0006040; LFWR1_ATL_0006041; LFWR1_ATL_0006042; LFWR1_ATL_0006043; LFWR1_ATL_0006044; LFWR1_ATL_0006045; LFWR1_ATL_0006046: QSVLTQPPSVSGAPGQRVTISCTGS (ID SEQ N°: 145) LFWR1_ATL_0006047 QAVLTQPPSVSGAPGQRVTISCTGS (SEQ ID NO: 146) LFWR1_ATL_0006048 QSVLTQPSSVSGAPGQRVTISCTGS (SEQ ID NO: 147) uma LFWR2 compreendendo uma sequência de aminoácidos seleciónada a partir de: LFWR2_ATL_0005802: VHWYQQLPGTAPKLLIY (SEQ ID NO: 151) LFWR2_ATL_0005807: AFWYQQKPGQAPVLVIY (SEQ ID NO: 152) LFWR2_ATL_0005808; LFWR2_ATL_0005854: VSWYQQHPGKAPKLMIY (SEQ ID NO:153) LFWR2_ATL_0005809: ANWYQQKPGQAPVLVIY (SEQ ID NO:154) LFWR2_ATL_0005810: PSWYQQTPGQPPRTLIY (SEQ ID NO: 155) LFWR2_ATL_0005853: LHWYLQKPGQSPQLLIY (SEQ ID NO:156) uma LFWR3 compreendendo uma sequência de aminoácidos seleciónada a partir de: LFWR3_ATL_0005802: NRPSGVPDRFSGSSKSGTSASLAITGLQAEDEADYYC (SEQ ID NO:160) LFWR3_ATL_0005807: ERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYC (SEQ ID NO:161) LFWR3_ATL_0005808: NRPSGVSSRFSASKSGNTASLTISGLQAEDEADYYC (SEQ ID NO:162) LFWR3_ATL_0005809: NRPSGIPDRFSGSSSGNTASLTLTGAQAEDEADYYC (SEQ ID NO:163) Petition: 870250089689, on 10 / 02 / 2025, page. 60 / 221 47 / 144 LFWR3_ATL_0005810: TRSSGVPDRFSGSILGNKAALTITGAQADDESDYYC (SEQ ID NO:164) LFWR3_ATL_0005853: DRAPGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYC (SEQ ID NO:165) LFWR3_ATL_0005854: SRPSGVSYRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO:166); and an LFWR4 comprising an amino acid sequence selected from: LFWR4_ATL_0005802: FGGGTKLTVL (SEQ ID NO:172) LFWR4_ATL_0005807; LFWR4_ATL_0005808; LFWR4_ATL_0005854: FGGGTKVTVL (SEQ ID NO:173) LFWR4_ATL_0005853: FGQGTRLEIK (SEQ ID NO:174) or structure sequences with one to six, one to five, one to four, one to three, or one to two substitutions compared to the structure sequences above.

[0112] A disclosure antibody may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005802, or structure sequences with one to six substitutions compared to these structure sequences. A disclosure antibody may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005853, or structure sequences with one to six substitutions compared to these structure sequences. A disclosure antibody may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005854, or structure sequences with one to six substitutions compared to these structure sequences. An antibody from the disclosure may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005807, or sequences of Petition 870250089689, dated 02 / 10 / 2025, page 61 / 221 48 / 144 structure with one to six substitutions compared to these structure sequences. A disclosure antibody may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005808, or structure sequences with one to six substitutions compared to these structure sequences. A disclosure antibody may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005809, or structure sequences with one to six substitutions compared to these structure sequences. An antibody from the disclosure may have a variable light chain (VL) domain with the LFWR1, LFWR2, LFWR3, and LFWR4 structure sequences of antibody ATL_0005810, or structure sequences with one to six substitutions compared to these structure sequences.

[0113] Substitutions in the variable domain structure sequences of the light chain can be selected at the following positions in the standard IMGT numbering: LFWR1: positions 2, 8. Substitutions in the variable domain structure sequences of the light chain can be selected at the following positions in the standard IMGT numbering: LFWR1: at position 2: A2S, at position 8: S8P.

[0114] The antibody may have a variable light chain (VL) domain with the following structural sequences: LFWR1 of ATL_0005802, ATL_0005853 or ATL_0005854; LFWR2 of ATL_0005802, ATL_0005853 or ATL_0005854; LFWR3 of ATL_0005802, ATL_0005853 or ATL_0005854; and LFWR4 of ATL_0005802, ATL_0005853, or ATL_0005854.

[0115] In this descriptive report, antibodies may have VH (and optionally VL) regions comprising an amino acid sequence with a high percentage of sequence identity with the VH and / or VL amino acid sequences described above. For example, antibodies according to the present Petition 870250089689, dated 02 / 10 / 2025, page 62 / 221 49 / 144 invention, include antibodies that bind to CD33 and have a VH region comprising an amino acid sequence with at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the VH region of any antibody described in this report. An antibody, according to the disclosure, may have a variable heavy chain (VH) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from SEQ ID NOs: 4 to 10 and 12 to 20. An antibody, according to the disclosure, may have a variable heavy chain (VH) domain comprising a sequence that has at most 2 mutations in each HCDR and at most 3 mutations in each structure region compared to a sequence selected from SEQ ID NOs: 4 to 10 and 12 to 20.

[0116] An antibody, according to the disclosure, may have a variable heavy chain (VH) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from SEQ ID NOs: 4, 9 and 10 (antibodies ATL_0005802, ATL_0005853, ATL_0005854). An antibody, according to the disclosure, may have a variable heavy chain (VH) domain comprising a sequence that has at most 2 mutations in each HCDR and at most 3 mutations in each structure region compared to a sequence selected from SEQ ID NOs: 4, 9 and 10 (antibodies ATL_0005802, ATL_0005853, ATL_0005854).

[0117] Alternatively or additionally, disclosure antibodies may have a VL region comprising an amino acid sequence with at least 70%, more preferably, one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the VL region of any antibody described in this report. For example, disclosure antibodies may have a VL region comprising Petition 870250089689, dated 02 / 10 / 2025, p. 63 / 221 50 / 144 an amino acid sequence with at least 70%, more preferably, one with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the VL region of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810

[0118] An antibody, according to the disclosure, may have a variable light chain (VL) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from SEQ ID NOs: 24 to 30 and 32 to 40. An antibody, according to the disclosure, may have a variable light chain (VL) domain comprising a sequence that has at most 2 mutations in each LCDR and at most 3 mutations in each structure region compared to a sequence selected from SEQ ID NOs: 24 to 30 and 32 to 40.

[0119] An antibody, according to the disclosure, may have a variable light chain (VL) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from SEQ IDs NOs: 24, 29, 30 (antibodies ATL_0005802, ATL_0005853, ATL_0005854). An antibody, according to the disclosure, may have a variable light chain (VL) domain comprising a sequence that has at most 2 mutations in each LCDR and at most 3 mutations in each structure region compared to a sequence selected from SEQ IDs NOs: 24, 29, 30 (antibodies ATL_0005802, ATL_0005853, ATL_0005854).

[0120] Dissemination antibodies can have a lambda (λ) or kappa (κ) light chain.

[0121] The overall percent identity of a variable region or complete light / heavy chain sequence can be combined with specific CDR sequences of the same antibody. Petition 870250089689, dated 02 / 10 / 2025, page 64 / 221 51 / 144

[0122] Sequence identity percent (%) is defined as the percentage of amino acid residues in a candidate sequence that are identical to the residues in a comparison sequence after sequence alignment and the introduction of gaps, if necessary, to achieve maximum sequence identity, without considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated along the entire length of the respective sequences. When the aligned sequences have different lengths, the sequence identity of the shorter comparison sequence can be determined along the entire length of the longer sequence provided, or, when the comparison sequence is longer than the provided sequence, the sequence identity of the comparison sequence can be determined along the entire length of the shorter sequence provided.Sequence identity can be defined with reference to the GAP algorithm (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, standard parameters can be used, with a gap creation penalty = 12 and a gap extension penalty = 4. The use of GAP may be preferred, but other algorithms can be used, for example, 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. August 18, 2010; 11: 431) or the TBLASTN program, by Altschul et al. (1990) above, generally employing standard parameters (see, for example, Pearson Curr Prot Bioinformatics (2013) Chapt 3 Uniy 3.1 doi:10.1002 / 0471250953.bi0301s42). In particular, the psi-Blast algorithm can be used (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402). The identity and a. Petition 870250089689, dated 02 / 10 / 2025, p. 65 / 221 52 / 144 Sequence similarity can also be determined using Genomequest™ software (Gene-IT, Worcester, MA, USA). Sequence comparisons are preferably made over the entire length of the relevant sequences to be compared.

[0123] The antibodies of this disclosure may comprise one or more substitutions within the structure of the VH and / or VL region. As used in this report, “substitution” refers to the exchange of one amino acid for another at a specific position, relative to the same position in a base molecule. In some embodiments, the base molecules are antibodies exemplified in this report, for example, antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810.

[0124] In some embodiments, antibodies or fragments thereof, according to the present disclosure, are capable of crossing the blood-brain barrier. In some embodiments, the antibodies of the present disclosure may have an scFV chain, such as the transferrin receptor (Yu et al., 2014), as well as an scFv chain that binds to CD33, as described in this report. In some embodiments, the antibodies, according to the disclosure, comprise an antibody or fragment thereof that binds to CD33, as described in this report (such as, for example, an antibody, scFv, sdAb, etc.) and an additional binding fragment that binds to another target. The other target may be a receptor in the brain, such as the transferrin receptor. The additional binding fragment may be an antibody, an scFv, a nanobody, or an aptamer. The two binding fragments of such a bispecific molecule may form a fusion protein.

[0125] Single-domain antibodies (sdAbs), also known as nanobodies, comprising the heavy chain CDRs and / or the VH sequence of any antibody described in this report, are also described in this report. Petition 870250089689, dated 02 / 10 / 2025, p. 66 / 221 53 / 144 Thus, antibodies or fusion molecules comprising a nanobody that binds to CD33, as described in this report, and a nanobody that binds to a receptor in the brain are also described in this report. Also described in this report are antibodies of fusion molecules comprising an scFV chain or nanobody that binds to CD33, as described in this report, and an aptamer that binds to a receptor in the brain.

[0126] The antibodies described in this report can increase the phagocytosis of a cell expressing CD33. The increase in phagocytosis can be measured in comparison to a comparator antibody. A comparator antibody can be an isotype control antibody. A comparator antibody can be another CD33-binding antibody. A comparator antibody can be an antibody with the variable heavy chain sequence of ATL_5909 and the variable light chain sequence of ATL_5909. A comparator antibody can be another CD33-binding antibody with a different epitope. Phagocytosis can be assessed by measuring a fluorescence signal associated with the uptake of a labeled particle by imaging or flow cytometry. Phagocytosis can be measured by detecting a fluorescence signal indicative of phagocytosis of a substrate that has pH-dependent fluorescence. The substrate can be selected from beta-amyloid and S. aureus.The labeled substrate may be a pH-red labeled substrate. The cell expressing CD33 may be an inflammatory human iPSC-derived microglial cell. An inflammatory cell may be a cell that has been stimulated with an inflammatory signal. For example, the cell may have been exposed to an inflammatory signal (e.g., LPS, IFN-γ) for at least 6, 12, or 24 hours, or approximately 6, 12, or 24 hours before antibody exposure. The cell expressing CD33 may be a PBMC. The cell expressing CD33 may be a cell isolated from human blood. The cell expressing CD33 may be a monocyte or a microglial cell. The cell expressing CD33 may be a human cell. The cell that... Petition 870250089689, dated 02 / 10 / 2025, page 67 / 221 54 / 144 expressing CD33 may be a microglial cell derived from an iPSC. The cell expressing CD33 may be a cell that was stimulated with an inflammatory signal (e.g., LPS) prior to antibody exposure.

[0127] The antibodies described in this report can bind to human CD33. The antibodies described in this report can bind to human CD33 with an EC50 of at most 2e-08 M, or at most 3e-09 M, as assessed by ELISA (such as rhCD33 plated binding). A disclosure antibody can bind to human CD33 with an EC50 of at most 15 μg / ml, at most 12 μg / ml, at most 5 μg / ml, at most 3 μg / ml or at most 0.5 μg / ml. An antibody from the dissemination can bind to human CD33 with an EC50 of at most 1e-7M, at most 8e-8M, at most 3e-8M, at most 2e-8M, or at most 3e-9M. Binding to human CD33 can be assessed by ELISA using rhCD33-plated cells, such as CD33_HUMAN_ECD, His_004_001 from Sino Biological, 12238-H08H.

[0128] An antibody, according to the disclosure, may have a Koff for binding to human CD33 of at least 1e-3 s-1. An antibody, according to the disclosure, may have a Koff for binding to human CD33 between 1e-3 s-1 and 5e-2 s-1. An antibody, according to the disclosure, may have a KD for binding to human CD33 of at most 5e-7 M. An antibody, according to the disclosure, may have a KD for binding to human CD33 between 2e-8 M and 5e-7 M. A human CD33 may be a rhCD33-his (e.g., R&D Systems, 10375-SL-050). KD and Koff may be measured by biolayer interferometry (BLI) in the octet-I. An antibody, according to the disclosure, may have a Koff for binding to human CD33 lower than that of a comparator antibody. Suitable comparative antibodies are those described above.

[0129] An antibody, according to the disclosure, can have a melting point Tm1 of at least 57, or between 57 and 70. An antibody, according to the Petition 870250089689, dated 02 / 10 / 2025, page 68 / 221 55 / 144 present disclosure, may have a SEC-HPLC monomer purity of at least 95%.

[0130] The antibodies described in this report may result in less internalization of CD33 in C33-expressing cells compared to a comparator antibody. An antibody described in this report may 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. A disclosure antibody may deplete CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody to a lesser extent than a comparator antibody at the same concentration. Suitable comparator antibodies are those described above.

[0131] The antibodies described in this report may exhibit increased binding to a human CD33 protein comprising mutations at positions 20, 21, 22, and 24, compared to antibody binding to a human CD33 protein without said mutations. The antibodies described in this report may exhibit increased binding to a human CD33 protein comprising mutations at positions 20, 21, 22, 24, and 132, compared to antibody binding to a human CD33 protein without said mutations. The antibodies described in this report may exhibit reduced binding to a human CD33 protein comprising mutations at positions 47, 50, 51, and 52, compared to antibody binding to a human CD33 protein without said mutations. The antibodies described in this report may not bind to a human CD33 protein comprising mutations at positions 47, 50, 51, and 52.The antibodies described in this report may have reduced binding to a human CD33 protein comprising mutations at positions 47, 50, 51, 52, and 122, compared to antibody binding to a human CD33 protein without these mutations. The antibodies described in this report may not bind to a human CD33 protein comprising mutations at positions 47, 50, 51, 52, and 122. Petition 870250089689, dated 02 / 10 / 2025, p. 69 / 221 56 / 144 antibodies described in this report may have reduced binding to a human CD33 protein comprising mutations at positions 83, compared to antibody binding to a human CD33 protein without said mutations. The mutations may be selected from: 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 may refer to the position in the complete human CD33 sequence (CD33M (Uniprot ID: P20138-1)). Binding can be measured using a single-point ELISA. The human CD33 protein may comprise residues 18-232 of human CD33. The human CD33 protein without the aforementioned mutations may be CD33M2_ECD_18-232_WT (SEQ ID NO: 226).An antibody, as described in this report, may have greater binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos1[P1] (SEQ ID NO: 227) compared to a protein comprising the sequence CD33M2_ECD_18-232_WT (SEQ ID NO: 226). An antibody, as described in this report, may have greater binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos1_MutPos6 [P6+1] (SEQ ID NO: 228) compared to a protein comprising the sequence CD33M2_ECD_18232_WT (SEQ ID NO: 226). An antibody, as described in this report, may have reduced binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos2 [P2] (SEQ ID NO: 229) compared to a protein comprising the sequence CD33M2_ECD_18-232_WT (SEQ ID NO: 226).An antibody, as described in this report, may have reduced binding to a protein comprising the sequence CD33M2_ECD_18232_MutPos2_MutPos5 [P2+5] (SEQ ID NO: 230) compared to a protein comprising the sequence CD33M2_ECD_18-232_WT (SEQ ID NO: 226). An antibody, as described in this report, may have reduced binding to a... Petition 870250089689, dated 02 / 10 / 2025, p. 70 / 221 57 / 144 protein comprising the sequence CD33M2_ECD_18-232_MutPos4 [P4] (SEQ ID NO: 232) compared to a protein comprising the sequence CD33M2_ECD_18-232_WT (SEQ ID NO: 226). The antibodies described in this report may interfere with sialic acid binding. The antibodies described in this report may bind to an epitope in such a way that antibody binding interferes with sialic acid binding to CD33.

[0132] A disclosure antibody may bind to a CD33 protein comprising the CD33 V domain. A disclosure antibody may not bind to a CD33 protein that does not comprise the CD33 V domain. A disclosure antibody may not bind to a CD33 protein possessing the CD33_HUMAN_ECD_Cdomain_His_007 protein sequence (SEQ ID NO: 175). A disclosure antibody may selectively bind to CD33 with respect to one or more siglecs, optionally wherein the antibody selectively binds to CD33 with respect to one or more (or all): siglec-6, siglec-7, siglec-8 and siglec-9, and / or wherein the antibody selectively binds to human CD33 with respect to one or more homologs, optionally wherein the antibody selectively binds to human CD33 with respect to mouse CD33 and canine CD33.

[0133] An antibody from this disclosure can reduce neuroinflammation (i.e., inflammation in the brain), for example, compared to a comparator antibody (e.g., an isotype control antibody or ATL_5909). The level of neuroinflammation can be determined by measuring the levels of pro-inflammatory cytokines in neural cells, such as microglia (e.g., human Ipsc-derived microglia), as described in this report (e.g., using an immunoassay, such as an ELISA). The pro-inflammatory cytokines may 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). Petition 870250089689, dated 02 / 10 / 2025, page 71 / 221 58 / 144

[0134] An antibody described in this report may exhibit lower peripheral clearance when administered to an individual compared to a comparator anti-CD33 antibody. The comparator anti-CD33 antibody may be ATL_5909. The individual may be an individual (e.g., a mouse) expressing a CD33 protein comprising exons 1-3 of human CD33. Administration may be parenteral. Administration may be intravenous or intraperitoneal.

[0135] An antibody described in this report can enhance AP phagocytosis by microglia cells in vivo. This can be evaluated in vivo, for example, as described in this report (see Example 10 and attached methods). The effect of the antibody on Aβ phagocytosis can be evaluated in microglia from human microglia progenitors derived from iPSCs transplanted into the brains of mice with Alzheimer's disease (AD) (e.g., mice with knock-in for APPNL'GF).

[0136] An antibody described in this report can increase the clearance of AP plaques in an individual in need of it. The antibody can increase the clearance of AP plaques in an individual in need of it through microglial phagocytosis.

[0137] An antibody described in this report can increase the phagocytosis of tau aggregates by microglia with an inflammatory phenotype (e.g., LPS-treated iPSC microglia). An antibody described in this report can increase the phagocytosis of tau aggregates by microglia with an inflammatory phenotype to a greater extent than a comparative anti-CD33 antibody, such as, for example, ATL_5909. This can be evaluated in vitro, as described in this report, such as, for example, in Example 11. An antibody described in this report can increase the phagocytosis of tau aggregates by microglia in an individual in need. Petition 870250089689, dated 02 / 10 / 2025, page 72 / 221 59 / 144

[0138] An antibody described in this report may not induce significant release of one or more cytokines by human iPSC-derived microglia, human PBMCs, and / or isolated human monocytes exposed to the antibody in vitro, compared to a control (e.g., isotype control antibody). One or more cytokines may be selected from: CCL2 (MCP-1); CXCL8 (IL-8); IFN-γ; IL-10; IL-12p40; IL12p70; IL-17A; IL-1β; IL-2; IL-23; IL-4; IL-6; TGF-β1; TNF-α; CCL17 (TARC); CCL2 (MIP-3α); CCL3 (MIP-1α); CCL4 (MIP-1β); CCL5 (RANTES); CXCL1 (Gro-α); CXCL5 (ENA-78); CXCL9 (MIG); CCL11 (Eotaxin); CX3CL1 (Fractalcin); IL-18; sRAGE; sTREM-1; sTREM-2; VEGF; VILIP-1; β-NGF; BDNF; CCL2 (MCP-1). The antibody may not induce the release of any of the above cytokines.The antibody can induce lower release levels of one, more, or all of the above cytokines by human iPSC-derived microglia, human PBMCs, and / or isolated human monocytes exposed to the antibody in vitro, compared to a comparative anti-CD33 antibody (e.g., ATL_5909). An antibody, as described in this report, can reduce the levels of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype in vitro (e.g., human iPSC-derived microglia treated with LPS) and / or in vivo (e.g., when administered to an individual in need). An antibody as described in this report can reduce the levels of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype in vitro (e.g., human iPSC-derived microglia treated with LPS) more than a comparative anti-CD33 antibody (e.g., ATL_5909).

[0139] An antibody described in this report can increase the levels of phosphorylated SYK protein in antibody-treated microglia cells in vitro compared to a control (e.g., antibody isotype control). The microglia cells may be microglia cells with inflammatory phenotypes (e.g., LPS-treated microglia cells). SYK phosphorylation may increase without a significant increase in overall protein levels. Petition 870250089689, dated 02 / 10 / 2025, page 73 / 221 60 / 144

[0140] An antibody described in this report can increase P2RY12 levels in microglia cells treated with the antibody in vitro compared to a control (e.g., antibody isotype control). The microglia cells may be microglia cells with inflammatory phenotypes (e.g., LPS-treated microglia cells).

[0141] An antibody described in this report can increase TREM2 signaling in microglia cells treated with the antibody in vitro compared to a control (e.g., antibody isotype control). The microglia cells may be microglia cells with inflammatory phenotypes (e.g., LPS-treated microglia cells).

[0142] An antibody described in this report 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, for example, as described in this report (see Example 14).

[0143] An antibody described in this report can reduce the inflammation-induced release of one or more inflammatory markers 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 inflammation-induced release of one or more cytokines can be assessed by exposing the cells to one or more pro-inflammatory signals, such as, for example, lipopolysaccharide (LPS) and / or interferon-gamma (INFγ). The inflammatory marker(s) can be selected from: MCP-1, IP-10, GFAP, and IL-6. Thus, the antibodies of the present invention can reduce the expression of one or more markers of Petition 870250089689, dated 02 / 10 / 2025, page 74 / 221 61 / 144 inflammation in the CNS of an individual in need of the same. The inflammation marker(s) may include one or more of MCP-1, IP-10, GFAP, and IL-6.

[0144] An antibody described in this report may selectively bind to CD33. The antibody may not bind to any other human protein. Isolated nucleic acids encoding an antibody, antigen-binding fragment, or polypeptide as described in this report are provided. Also provided is a vector comprising a nucleic acid described in this report and a host cell comprising the vector. For example, the host cell may be a eukaryotic or mammalian cell, for example, a Chinese hamster ovary (CHO) cell, or it may be a prokaryotic cell, for example, E. coli. In some embodiments, the vector is a viral vector, for example, a bacteriophage.

[0145] Methods are also provided for the production of an antibody, or antibody fragment, as described in this report. The method comprises cultivating a host cell, as described in this report, under conditions suitable for the expression of a vector encoding the antibody, or antibody fragment, and isolating and / or purifying the antibody, or antibody fragment. The method further comprises formulating the antibody, or antibody fragment, into a composition that includes at least one additional component.

[0146] Antibodies and their fragments can be used in therapy.

[0147] The subject to be treated or diagnosed may be any animal or human. The subject is preferably a mammal, more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic uses may be in humans or animals (veterinary use). Unless otherwise specified, the subject is a human.

[0148] Medicines and pharmaceutical compositions, according to aspects of the present invention, can be formulated for administration by Petition 870250089689, dated 02 / 10 / 2025, page 75 / 221 62 / 144 various routes, including, but not limited to, parenteral, intravenous, intra-arterial, intramuscular, oral and nasal. Medications and compositions may be formulated for injection.

[0149] Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier,” composed of materials considered safe and effective. “Pharmaceutically acceptable” refers to entities and molecular compositions that are “generally considered safe,” for example, that are physiologically tolerable and that do not normally produce an allergic or similar reaction, such as gastric discomfort and the like, when administered to a human being. In some embodiments, this term refers to entities and molecular compositions approved by a U.S. federal or state government regulatory agency, as listed under Section 5204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, which are subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia, or another generally recognized pharmacopoeia for use in animals and, more particularly, in humans.The term “carrier” refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and methods for composing pharmaceutical compositions using such carriers. The pharmaceutical compositions provided in this report may include one or more excipients, for example, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants, or antimicrobial preservatives. When used, the excipients in the compositions will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredients, i.e., the anti-CFH antibodies used in the composition. Thus, those skilled in the art will understand that compositions are provided in which there is no incompatibility between any of the components of the formulation. Petition 870250089689, dated 02 / 10 / 2025, page 76 / 221 63 / 144 pharmaceutical. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents and preservatives.

[0150] Administration is preferably in a “therapeutically effective amount,” which is sufficient to demonstrate benefit to the individual. The amount actually administered, the rate and time of administration will depend on the nature and severity of the disease being treated. Prescribing treatment, for example, decisions about dosage, etc., is the responsibility of general practitioners and other physicians, and normally takes into account the disorder being treated, the patient's condition, the site of administration, the method of administration, and other factors known to physicians. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, published by Lippincott, Williams & Wilkins.

[0151] Treatable conditions according to the present invention include any in which CD33 plays a role, including neurodegenerative disorders and, in particular, those characterized by increased toxic protein species, such as Aβ42, characteristic of Alzheimer's Disease (AD), where phagocytosis of these toxic species is useful. The antibodies of the present disclosure can be used in the treatment of any neurological disease or disorder associated with the accumulation of toxic protein species. In fact, the antibodies of the present disclosure have been shown to increase phagocytosis in microglia and are therefore likely to be useful in the treatment of any neurological disease or disorder in which phagocytosis of toxic protein species is impaired or insufficient. Furthermore, respiratory disorders characterized by defective phagocytosis, including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPD) (Donelly et al., 2012), can also be treated. Petition 870250089689, dated 02 / 10 / 2025, page 77 / 221 64 / 144 with antibodies from the present disclosure. Other treatable conditions according to the present invention include cancers such as AML and solid tumors in which sialic acid may be causing immunosuppression, such as cancers in which the tumors exhibit hypersialylation of their cell surface, so as to avoid recognition by immune cells (Stanczak & Laubli, 2023), including many solid tumors, as well as cancers characterized by aberrant CD33 expression, such as acute myeloid leukemia (AML). The antibodies of the present invention were originally identified through analysis of resilient individuals with AD. These antibodies were subsequently found in resilient centenarians, indicating their relevance beyond AD. Thus, the antibodies of the present invention for use as a medicament are also described in this report.This report also describes antibodies for use in the treatment or prevention of a neurodegenerative disease, and / or respiratory disease, and / or cancer. It also describes antibodies of the present invention for use in the manufacture of a medicament, such as a medicament for the treatment or prevention of a neurodegenerative disease, or respiratory disease, or cancer. This report also describes a method for treating an individual who has been diagnosed as having or at risk of having a neurodegenerative disease, or respiratory disease, or cancer, the method comprising administering an antibody, as described in this report, in a therapeutically effective quantity to the subject.

[0152] A neurodegenerative disease or disorder may 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 diseases, Lewy body disease, spinal muscular atrophy (SMA), motor neuron disease (MND), etc. Petition 870250089689, dated 02 / 10 / 2025, page 78 / 221 65 / 144 such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxias (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubra pallidal-Luisian atrophy (DRPLA), X-linked spinal bulbar muscular atrophy type 1 (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease) and DNAJB6 myopathies, multiple sclerosis (MS, a single nucleotide polymorphism in CD33, is known to be associated with an increased risk of multiple sclerosis) and microgliopathies, such as adult-onset leukoencephalopathy with axonal spheroids and glia pigment (ALSP). For example, neurodegenerative disease can be selected from FTD, AD, HD and PD. A neurodegenerative disease or disorder can be a tauopathy.Thus, neurodegenerative disorders can include AD (Alzheimer's disease), CTE (chronic traumatic encephalopathy), PiD (Pick's disease), PSP (progressive supranuclear palsy), CBD (corticobasal degeneration), and AGD (argyrophilic grain disease).

[0153] A respiratory disease may comprise one or more of the following: COPD, cystic fibrosis, asthma, and idiopathic pulmonary fibrosis (IPD).

[0154] A cancer may be selected from: cervical cancer, breast cancer, brain cancer, bladder cancer, colon adenocarcinoma, cervical cancer, fibrocarcinoma, head and neck cancer, hepatocellular carcinoma, renal cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, non-small cell lung cancer, non-Hodgkin 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 other 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 malignancies. Petition 870250089689, dated 02 / 10 / 2025, page 79 / 221 66 / 144 hematological. It is known that all these cancers have tumor cells that hypersialize their cell surfaces to suppress immune cells, and the antibodies in this disclosure could therefore be used to alleviate this suppression.

[0155] The antibodies of the present invention can be used in therapy with one or more additional therapeutic agents. As used in this report, an “additional therapeutic agent” is an additional compound, protein, vector, antibody, cell, or entity with a therapeutic effect. The antibodies described in this report 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.

[0156] The antibodies described in this report can be used as biomarkers that indicate whether an individual is likely to respond to therapy with an antibody or antibody fragment, as described in this report. A method for determining whether an individual is likely to respond to treatment with an antibody or antibody fragment, as described in this report, is also described in this report.The method comprises obtaining BCR sequence data from the individual and determining, using said sequence data, whether the individual's BCR repertoire comprises one or more antibodies likely to bind to CD33 (e.g., antibodies, as described in this report, such as, for example, antibodies with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with any specific antibody or antibody fragment described in this report), wherein an individual whose BCR repertoire does not comprise one or more antibodies likely to bind to CD33, as described in this report, is likely to respond to treatment. Petition 870250089689, dated 02 / 10 / 2025, page 80 / 221 67 / 144 with an antibody or antibody fragment thereof, as described in this report.

[0157] Thus, this report also describes a method for treating an individual who has been diagnosed as having or is likely to have a disease in which CD33 plays a role (for example, any disease in which cells expressing CD33 have impaired or insufficient phagocytosis, such as, for example, a neurodegenerative disorder or cancer), the method comprising: obtaining BCR sequence data from the individual; determining, using the sequence data, whether the individual'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 thereof, as described in this report.

[0158] Some methods in this disclosure involve a sample containing cells. The sample may be a culture of cells grown in vitro. For example, the culture may comprise a cell suspension or cells grown in a culture plate or vessel. The methods, according to this disclosure, may be performed, or the products may be present, in vitro, ex vivo, or in vivo.

[0159] According to some aspects of the present disclosure, a kit of parts comprising an antibody according to the present invention is provided. In some embodiments, the kit comprises an antibody according to the present invention and one or more of the following: reagents for use in immunochemistry; antibodies immobilized on a solid support; means for labeling the antibodies; means for linking the antibodies to a cytotoxic fraction; and an additional therapeutic agent. Petition 870250089689, dated 02 / 10 / 2025, page 81 / 221 68 / 144

[0160] The features disclosed in the preceding description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, can, separately or in any combination of such features, be used to carry out the invention in various forms thereof.

[0161] Although the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art upon reading this description. Consequently, the exemplary embodiments of the invention presented above are considered illustrative and not limiting. Various alterations to the described embodiments may be made without departing from the spirit and scope of the invention.

[0162] To avoid any doubt, any theoretical explanations provided in this report are given for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0163] All section headings used in this report are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0164] Throughout this descriptive report, including the claims that follow, unless the context requires otherwise, the words “comprise” and “include”, and variations such as “comprises”, “comprising” and “including” will be understood as implying the inclusion of a stated whole number or step or group of whole numbers or steps, but not the exclusion of any other whole number or step or group of whole numbers or steps.

[0165] It should be noted that, as used in the descriptive report and the attached claims, the singular forms “a”, “an” and “the” include Petition 870250089689, dated 02 / 10 / 2025, page 82 / 221 69 / 144 references in the plural, unless the context clearly indicates otherwise. Ranges may be expressed in this report as “from about” a specific value and / or up to “about” another specific value. When such a range is expressed, another realization includes from a specific value and / or up to another specific value. Similarly, when values ​​are expressed as approximations, by using the antecedent “about”, it is understood that the specific value forms another realization. The term “about” in relation to a numerical value is optional and means, for example, + / - 10%. Sequences S EQ ID NO Descrição Sequência 1 ATL_0004828 VH EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIH WVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRA TLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPW LAYWGQGTLVTVSS 2 ATL_0005338 VH EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYW MNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSV KGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTG SYYGMDYWGQGTSVTVSS 3 ATL_0005503 VH EVQLQQSGPELVKPGASVKISCKASGYTFTDYNL HWVKLSHGKSLEWIGFIYPSNGITGYNQKFKNKAT LTVDNSSSTAYMELRSLTSEDSAVYYCARSTVDY FDYWGQGTTLTVSS 4 ATL_0005802 VH QVQLVESGAEVKKPGESLRISCKGSGYSFTSYWI AWVRQMPGKGLEWMGIIYPGDSDTRYSPSFEGQ VTISADKSIGTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVSS 5 ATL_0005807 VH QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VSISVDKSISTAFLQWSSLKSSDSAMYYCARQGA GPGGFDIWGQGTMVTVSS 6 ATL_0005808 VH QVQLQQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKAS DTAMYYCARGQGGGAGAFDIWGQGTMVTVSS 7 ATL_0005809 VH QVQLQQSGGEVKKPGESLKISCKGSGYSFNTYWI AWVRHAPGKGLEWMGIIYPGDSETRYSPSFQSQ VTISADKSIDTAYLEWNTLEASDTAMYYCARHRADATL_0005810 Petition 870250089689, dated 02 / 10 / 2025, page 83 / 221 70 / 144 9 ATL_0005853 VH QVQLVQSGAEVKKTGESLRISCKASGYKFNNNWI AWVRQMPGKGLEWMGIIYPGDSDTRYSPSFEGQ VTISADKSSGIVYLQWTSLKASDTAIYYCARHSGG LDGYTAAALDYWGQGTLVTVSS 10 ATL_0005854 VH QVQLVQSGAEVKKTGEYLRISCKASGYKFSNNWI AWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAIYYCATWGGS NWFVDWGQGTLVTVSS 11 ATL_0005909 VH QVQLVQSGAEVKKPGASVKISCKASGYTFTDYNL HWVRQAPGQGLEWIGFIYPSNRITGYAQKFQGRA TLTVDNSTSTAYMELSSLRSEDTAVYYCARSDVD YFDYWGQGTLLTVSS 12 ATL_0006040 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSD TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYCARPRGFGEYYFDYWGQGTLVTVSS 13 ATL_0006041 VH QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVSS 14 ATL_0006042 VH EVQLVESGAEVKKPGESLKISCKGSGYSFTSYWIG WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQV TISADKSISTAYLQWSSLKASDTAMYYCARPRGFG EYYFDYWGQGTLVTVSS 15 ATL_0006043 VH EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVSS 16ATL_0006044 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIA WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQV TISADKSISTAYLQWSSLKASDTAMYYCARPRGFG EYYFDYWGQGTLVTVSS 17 ATL_0006045 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFEGQ VTISADKSISTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVS S 18 ATL_0006046 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSIGTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVSS 19 ATL_0006047 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVSS 20 ATL_0006048 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYYCARPRGF GEYYFDYWGQGTLVTVSS 21 ATL_0004828 VL DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRF LTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGS GSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFG QGTKVEVK Petição 870250089689, de 02 / 10 / 2025, pág. 84 / 221 71 / 144 22 ATL_0005338 VL DVVMTQTPLSLPVSLGDQASISCRSSQSLVNSNG NTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFS GSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWT FGGGTKLEIK 23 ATL_0005503 VL DIVLTQSPASLAVSLGQRATMSCRASQSVSTSTYS YMHWYQQKPGQPPKLLIKYASNLESGVPARFSGS GSGTDFTLNIHPVEEEDTAT YYCQHSWEIPLTFGAGTKLELK 24 ATL_0005802 VL QAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 25 ATL_0005807 VL QSVLTQPPSVSVSPGQTARITCSGDALARQYAFW YQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTT VTLTISGVQAEDEADYYCQSPDSSGTYPVFGGGT KVTVL 26 ATL_0005808 VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIYDVTNRPSGVSSRFSASK SGNTASLTISGLQAEDEADYYCSSYTSSSTLEVFG GGTKVTVL 27 ATL_0005809 VL SSELTQDPAVSVAVGQTVRITCQGDSLRNYYANW YQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGN TASLTLTGAQAEDEADYYCNSRDSSGYHLGLFGG GTKLTVL 28 ATL_0005810 VL QTVVTQEPS FSVS PGGTVTLTCGLSSGSVSTSYY PSWYQQTPGQPPRTLIYSTNTRSSGVPDRFSGSI LGNKAALTITGAQADDESDYYCLLYMGSGIWMFG GGTKLTVL 29 ATL_0005853 VL DVVMTQSPLSLPVNPGEPASISCRSSQSLLHSDGYNYLHWYLQKPGQSPQLLIYVGSDRAPGVPDRFS GSGSGTDFTLKINRVEAEDVGVYYCMQALQTPITF GQGTRLEIK 30 ATL_0005854 VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIYDVSSRP SGVSYRFSGSKSGNTASLTISGLQAEDEADYYCS SYTNSSTLEVFGGGTKVTVL 31 ATL_0005909 VL DIVLTQSPDSLAVSLGERATINCRASQSVSTSTYS YMHWYQQKPGQPPKLLIKYASNLESGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCQHSWEIPLTFGQ GTKLEIK 32 ATL_0006040 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 33 ATL_0006041 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 34 ATL_0006042 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL Petição 870250089689, de 02 / 10 / 2025, pág. 85 / 221 72 / 144 35 ATL_0006043 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 36 ATL_0006044 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 3 7 ATL_0006045 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 38 ATL_0006046 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 39 ATL_0006047 VL QAVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 40 ATL_0006048 VL QSVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDEADYYCQSYDSSLSGDVF GGGTKLTVL 41 HCDR1_ATL_0004828 GYTITDSN 42 HCDR1_ATL_0005338 GFTFSDYW 43 HCDR1_ATL_0005503; HCDR1_ATL_0005909 GYTFTDYN 44 HCDR1_ATL_0005802; HCDR1_ATL_0005807; HCDR1_ATL_0005808; HCDR1_ATL_0006040;HCDR1_ATL_0006041; HCDR1_ATL_0006042; HCDR1_ATL_0006043; HCDR1_ATL_0006044; HCDR1_ATL_0006045; HCDR1_ATL_0006046; HCDR1_ATL_0006047; HCDR1 ATL 0006048 GYSFTSYW 45 HCDR1_ATL_0005809 GYSFNTYW 46 HCDR1_ATL_0005810 GYTFTSYY 47 HCDR1_ATL_0005853 GYKFNNNW 48 HCDR1_ATL_0005854 GYKFSNNW 49 HCDR2_ATL_0004828 IYPYNGGT 50 HCDR2_ATL_0005338 IRNKPYNYET 51 HCDR2_ATL_0005503 IYPSNGIT; Petição 870250089689, de 02 / 10 / 2025, pág. 86 / 221 73 / 144 52 HCDR2_ATL_0005802; HCDR2_ATL_0005807; HCDR2_ATL_0005808; HCDR2_ATL_0005853; HCDR2_ATL_0005854; HCDR2_ATL_0006040; HCDR2_ATL_0006041; HCDR2_ATL_0006042; HCDR2_ATL_0006043; HCDR2_ATL_0006044; HCDR2_ATL_0006045; HCDR2_ATL_0006046; HCDR2_ATL_0006047; HCDR2 ATL 0006048 IYPGDSDT 53 HCDR2_ATL_0005809 IYPGDSET 54 HCDR2_ATL_0005810 INPSGGST 55 HCDR2_ATL_0005909 IYPSNRIT 56 HCDR3_ATL_0004828 VNGNPWLAY 57 HCDR3_ATL_0005338 TGSYYGMDY 58 HCDR3_ATL_0005503 ARSTVDYFDY 59 HCDR3_ATL_0005802; HCDR3_ATL_0006040; HCDR3_ATL_0006041; HCDR3_ATL_0006042; HCDR3_ATL_0006043; HCDR3_ATL_0006044; HCDR3_ATL_0006045; HCDR3_ATL_0006046; HCDR3_ATL_0006047;HCDR3 ATL 0006048 ARPRGFGEYYFDY 60 HCDR3_ATL_0005807 ARQGAGPGGFDI 61 HCDR3_ATL_0005808 ARGQGGGAGAFDI 62 HCDR3_ATL_0005809 ARHRADAPSDAFDI 6 3 HCDR3_ATL_0005810 ARDWHNTPQFDTDYYYYGMDV 64 HCDR3_ATL_0005853 ARHSGGLDGYTAAALDY 65 HCDR3_ATL_0005854 ATWGGSNWFVD 66 HCDR3_ATL_0005909 ARSDVDYFDY 67 HFWR1_ATL_0004828 EVQLVQSGAEVKKPGSSVKVSCKAS 68 HFWR1_ATL_0005338 EVKLDETGGGLVQPGRPMKLSCVAS 69 HFWR1_ATL_0005503 EVQLQQSGPELVKPGASVKISCKAS 70 HFWR1_ATL_0005802 QVQLVESGAEVKKPGESLRISCKGS 71 HFWR1_ATL_0005807; HFWR1_ATL_0006041 QVQLVQSGAEVKKPGESLKISCKGS 72 HFWR1_ATL_0005808 HFWR1_ATL_0005810 QVQLVQSGAEVKKPGASVKVSCKAS; Petition 870250089689, dated 02 / 10 / 2025, page 87 / 221 74 / 144 235 HFWR1_ATL_0005853 QVQLVQSGAEVKKTGESLRISCKAS 75 HFWR1_ATL_0005854 QVQLVQSGAEVKKTGEYLRISCKAS 76 HFWR1_ATL_0005909 QVQLVQSGAEVKKPGASVKISCKAS 77 HFWR1_ATL_0006040; HFWR1_ATL_0006044; HFWR1_ATL_0006045; HFWR1_ATL_0006046 ;HFWR1_ATL_0006047; HFWR1 ATL 0006048 EVQLVQSGAEVKKPGESLKISCKGS 78 HFWR1_ATL_0006042 EVQLVESGAEVKKPGESLKISCKGS 79 HFWR1_ATL_0006043 EVQLVQSGAEVKKPGESLRISCKGS 80 HFWR2_ATL_0004828 IHWVRQAPGQSLEWIGY 81 HFWR2_ATL_0005338 MNWVRQSPEKGLEWVAQ 82 HFWR2_ATL_0005503 LHWVKLSHGKSLEWIGF 83 HFWR2_ATL_0005802; HFWR2_ATL_0005853; HFWR2_ATL_0005854; HFWR2 ATL 0006044 IAWVRQMPGKGLEWMGI 84 HFWR2_ATL_0005807; HFWR2_ATL_0005808; HFWR2_ATL_0006040; HFWR2_ATL_0006041; HFWR2_ATL_0006042; HFWR2_ATL_0006043; HFWR2_ATL_0006045; HFWR2_ATL_0006046; HFWR2_ATL_0006047;HFWR2 ATL 0006048 IGWVRQMPGKGLEWMGI 85 HFWR2_ATL_0005809 IAWVRHAPGKGLEWMGI 86 HFWR2_ATL_0005810 MHWVRQAPGQGLEWMGI 87 HFWR2_ATL_0005909 LHWVRQAPGQGLEWIGF 88 HFWR3_ATL_0004828 DYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTA FYYC 89 HFWR3_ATL_0005338 YYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMG IYYC 90 HFWR3_ATL_0005503 GYNQKFKNKATLTVDNSSSTAYMELRSLTSEDSA VYYC 91 HFWR3_ATL_0005802 RYSPSFEGQVTISADKSIGTAYLQWSSLKASDTAM YYC 92 HFWR3_ATL_0005807 RYSPSFQGQVSISVDKSISTAFLQWSSLKSDSAMY YC 93 HFWR3_ATL_0005808; HFWR3_ATL_0006040; HFWR3_ATL_0006041; HFWR3_ATL_ 0006042; HFWR3_ATL_0006043; HFWR3_ATL_0006044; HFWR3_ATL_0006047; HFWR3_ATL_0006048 RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYC; Petition 870250089689, dated 02 / 10 / 2025, page 88 / 221 75 / 144 94 HFWR3_ATL_0005809 RYSPSFQSQVTISADKSIDTAYLEWNTLEASDTAM YYC 95 HFWR3_ATL_0005810 SYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYC 96 HFWR3_ATL_0005853 RYSPSFEGQVTISADKSSGIVYLQWTSLKASDTAI YYC 97 HFWR3_ATL_0005854 RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAI YYC 98 HFWR3_ATL_0005909 GYAQKFQGRATLTVDNSTSTAYMELSSLRSEDTA VYYC 99 HFWR3_ATL_0006045 RYSPSFEGQVTISADKSISTAYLQWSSLKASDTAM YYC 100 HFWR3_ATL_0006046 RYSPSFQGQVTISADKSIGTAYLQWSSLKASDTA MYYC 101 HFWR4_ATL_0004828; HFWR4_ATL_0005802; HFWR4_ATL_0005853; HFWR4_ATL_0005854; HFWR4_ATL_0006040; HFWR4_ATL_0006041; HFWR4_ATL_0006042; HFWR4_ATL_0006043; HFWR4_ATL_0006044; HFWR4_ATL_0006045; HFWR4_ATL_0006046; HFWR4_ATL_0006047; HFWR4 ATL 0006048 WGQGTLVTVSS 102 HFWR4_ATL_0005338 WGQGTSVTVSS 103 HFWR4_ATL_0005503 WGQGTTLTVSS 1 04 HFWR4_ATL_0005807; HFWR4_ATL_0005808; HFWR4 ATL 0005809 WGQGTMVTVSS 105 HFWR4_ATL_0005810 WGQGTTVTVSS 106 HFWR4_ATL_0005909 WGQGTLLTVSS 107 LCDR1_ATL_0004828 ESLDNYGIRF 108 LCDR1_ATL_0005338 QSLVNSNGNTY 109 LCDR1_ATL_0005503;LCDR1_ATL_0005909 QSVSTSTYSY 110 LCDR1_ATL_0005802; LCDR1_ATL_0006040; LCDR1_ATL_0006041; LCDR1_ATL_0006042; LCDR1_ATL_0006043; LCDR1_ATL_0006044; LCDR1_ATL_0006045; LCDR1_ATL_0006046; LCDR1_ATL_0006047; LCDR1 ATL 0006048 SSNIGAGYD 111 LCDR1_ATL_0005807 ALARQY; Petition 870250089689, dated 02 / 10 / 2025, page 89 / 221 76 / 144 112 LCDR1_ATL_0005808; LCDR1_ATL_0005854 SSDVGGYNY 113 LCDR1_ATL_0005809 SLRNYY 114 LCDR1_ATL_0005810 SGSVSTSYY 115 LCDR1_ATL_0005853 QSLLHSDGYNY 116 LCDR2_ATL_0004828 AAS 117 LCDR2_ATL_0005338 KVS 118 LCDR2_ATL_0005503; LCDR2_ATL_0005909 YAS 119 LCDR2_ATL_0005802; LCDR2_ATL_0006040; LCDR2_ATL_0006041; LCDR2_ATL_0006042; LCDR2_ATL_0006043; LCDR2_ATL_0006044; LCDR2_ATL_0006045; LCDR2_ATL_0006046; LCDR2_ATL_0006047; LCDR2 ATL 0006048 GNS 120 LCDR2_ATL_0005854 DVS 121 LCDR2_ATL_0005807 KDS 122 LCDR2_ATL_0005808 TVP 123 LCDR2_ATL_0005809 GKN 124 LCDR2_ATL_0005810 STN 125 LCDR2_ATL_0005853 VGS 126 LCDR3_ATL_0004828 QQTKEVPWS 127 LCDR3_ATL_0005338 SQSTHVPWT 128 LCDR3_ATL_0005503; LCDR3_ATL_0005909 QHSWEIPLT 129 LCDR3_ATL_0005802; LCDR3_ATL_0006040; LCDR3_ATL_0006041; LCDR3_ATL_0006042; LCDR3_ATL_0006043; LCDR3_ATL_ 0006044; LCDR3_ATL_0006045; LCDR3_ATL_0006046; LCDR3_ATL_0006047;LCDR3 ATL 0006048 QSYDSSLSGDV 130 LCDR3_ATL_0005807 QSPDSSGTYPV 131 LCDR3_ATL_0005808 SSYTSSSTLEV 132 LCDR3_ATL_0005809 NSRDSSGYHLGL 133 LCDR3_ATL_0005810 LLYMGSGIWM 134 LCDR3_ATL_0005853 MQALQTPIT; Petition 870250089689, dated 02 / 10 / 2025, pp. 90 / 221 77 / 144 236 LCDR3_ATL_0005854 SSYTNSSTLEV 135 LFWR1_ATL_0004828 DIQLTQSPSTLSASVGDRVTITCRAS 136 LFWR1_ATL_0005338 DVVMTQTPLSLPVSLGDQASISCRSS 137 LFWR1_ATL_0005503 DIVLTQSPASLAVSLGQRATMSCRAS 138 LFWR1_ATL_0005802 QAVLTQPSSVSGAPGQRVTISCTGS 139 LFWR1_ATL_0005807 QSVLTQPPSVSVSPGQTARITCSGD 140 LFWR1_ATL_0005808; LFWR1_ATL_0005854 QSALTQPASVSGSPGQSITISCTGT 141 LFWR1_ATL_0005809 SSELTQDPAVSVAVGQTVRITCQGD 142 LFWR1_ATL_0005810 QTVVTQEPSFSVSPGGTVTLTCGLS 143 LFWR1_ATL_0005853 DVVMTQSPLSLPVNPGEPASISCRSS 144 LFWR1_ATL_0005909 DIVLTQSPDSLAVSLGERATINCRAS 145 LFWR1_ATL_0006040; LFWR1_ATL_0006041; LFWR1_ATL_0006042; LFWR1_ATL_0006043; LFWR1_ATL_0006044; LFWR1_ATL_ 0006045; LFWR1 ATL 0006046 QSVLTQPPSVSGAPGQRVTISCTGS 146 LFWR1_ATL_0006047 QAVLTQPPSVSGAPGQRVTISCTGS 147 LFWR1_ATL_0006048 QSVLTQPSSVSGAPGQRVTISCTGS 148 LFWR2_ATL_0004828 LTWFQQKPGKAPKLLMY 149 LFWR2_ATL_0005338 LRWYLQKPGQSPKVLIY 150 LFWR2_ATL_0005503; LFWR2_ATL_0005909 MHWYQQKPGQPPKLLIK 151 LFWR2_ATL_0005802; LFWR2_ATL_0006040; LFWR2_ATL_0006041;LFWR2_ATL_0006042; LFWR2_ATL_0006043; LFWR2_ATL_0006044; LFWR2_ATL_0006045; LFWR2_ATL_0006046; LFWR2_ATL_0006047; LFWR2 ATL 0006048 VHWYQQLPGTAPKLLIY 152 LFWR2_ATL_0005807 AFWYQQKPGQAPVLVIY 153 LFWR2_ATL_0005808; LFWR2_ATL_0005854 VSWYQQHPGKAPKLMIY 154 LFWR2_ATL_0005809 ANWYQQKPGQAPVLVIY 155 LFWR2_ATL_0005810 PSWYQQTPGQPPRTLIY 156 LFWR2_ATL_0005853 LHWYLQKPGQSPQLLIY 157 LFWR3_ATL_0004828 NQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATY YC 158 LFWR3_ATL_0005338 NRVSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY FC; Petition: 870250089689, on 10 / 02 / 2025, page. 91 / 221 78 / 144 159 LFWR3_ATL_0005503 NLESGVPARFSGSGSGTDFTLNIHPVEEEEDTATY YC 160 LFWR3_ATL_0005802; LFWR3_ATL_0006040; LFWR3_ATL_0006041; LFWR3_ATL_0006042; LFWR3_ATL_0006043; LFWR3_ATL_0006044; LFWR3_ATL_0006045; LFWR3_ATL_0006046; LFWR3_ATL_0006047; LFWR3 ATL 0006048 NRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY YC 161 LFWR3_ATL_0005807 ERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYY C 162 LFWR3_ATL_0005808 NRPSGVSSRFSASKSGNTASLTISGLQAEDEADY YC 163 LFWR3_ATL_0005809 NRPSGIPDRFSGSSSGNTASLTLTGAQAEDEADY YC 164 LFWR3_ATL_0005810 TRSSGVPDRFSGSILGNKAALTITGAQADDESDYY C 165 LFWR3_ATL_0005853 DRAPGVPDRFSGSGSGTDFTLKINRVEAEDVGVY YC 166 LFWR3_ATL_0005854 SRPSGVSYRFSGSKSGNTASLTISGLQAEDEADY YC 167 LFWR3_ATL_0005909 NLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY C 168 LFWR4_ATL_0004828 FGQGTKVEVK 169 LFWR4_ATL_0005909 FGQGTKLEIK 170 LFWR4_ATL_0005338 FGGGTKLEIK 171 LFWR4_ATL_0005503 FGAGTKLELK 172 LFWR4_ATL_0005802; LFWR4_ATL_0005809; LFWR4_ATL_0005810; LFWR4_ATL_0006040; LFWR4_ATL_0006041; LFWR4_ATL_0006042; LFWR4_ATL_0006043; LFWR4_ATL_0006044;LFWR4_ATL_0006045; LFWR4_ATL_0006046; LFWR4_ATL_0006047; LFWR4 ATL 0006048 FGGGTKLTVL 173 LFWR4_ATL_0005807; LFWR4_ATL_0005808; LFWR4 ATL 0005854 FGGGTKVTVL 174 LFWR4_ATL_0005853 FGQGTRLEIK 175 CD33_HUMAN_ECD_C domain_His_007, domínio de proteína C ETGDLTHRPKILIPGTLEPGHSKNLTCSVSWASEQ GTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHG TN LTCQVKFAGAGVTTE RTIQLNVTGTKH HHHHH; Petition: 870250089689, on 10 / 02 / 2025, page. 92 / 221 79 / 144 Siglec-3 / CD33 humana derivado de HEK Asp140-Thr232 com uma etiqueta 6-His Cterminal 176 CD33M (ID Uniprot: P20138-1 ) MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEG LCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISR DSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSL SIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVH VTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQG TPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGT NLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIF PGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLI FFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQK KSKLHGPTETSSCSGAAPTVEMDEELHYASLNFH GMNPSKDTSTEYSEVRTQ 177 CD33m (Uniprot ID: P20138-3 ) MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTC SVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSSVLI ATL_0006166_VH (Alector AL002) QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSHW MNWVRQAPGQGLEWMGRIYPGGGDTNYAQKFR GRVTITADESTSTAYMELSSLRSEDTAVYYCARLL RNQPGASYAMDYWGQGTLVTVSS 179 ATL_0006166_VL (Alector AL002)DVVMTQSPDSLAVSLGERATINCRSSQSLVHSNR YTYLHWYQQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTRVPYT FGQGTKLEIK 180 ATL_0006166_HCDR1 GYAFSSHW 181 ATL_0006166_HCDR2 IYPGGGDT 182 ATL_0006166_HCDR3 ARLLRNQPGASYAMDY 183 ATL_0006166_HFWR1 QVQLVQSGAEVKKPGSSVKVSCKAS 184 ATL_0006166_HFWR2 MNWVRQAPGQGLEWMGR 185 ATL_0006166_HFWR3 NYAQKFRGRVTITADESTSTAYMELSSLRSEDTAV YYC 186 ATL_0006166_HFWR4 WGQGTLVTVSS 187 ATL_0006166_LCDR1 QSLVHSNRYTY 188 ATL_0006166_LCDR2 KVS 189 ATL_0006166_LCDR2 SQSTRVPYT 190 ATL_0006166_LFWR1 DVVMTQSPDSLAVSLGERATINCRSS 191 ATL_0006166_LFWR2 LHWYQQKPGQSPKLLIY 1 92 ATL_0006166_LFWR3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YC 193 ATL_0006166_LFWR4 FGQGTKLEIK 194 ATL_0006167_VH (Denali DNL919) QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIH WVRQAPGQGLEWMGWIDPEQGESKYAPKFQGR VTITADTSTSTAYMELSSLRSEDTAVYYCHADHGN Petição 870250089689, de 02 / 10 / 2025, pág. 93 / 221 80 / 144 196 ATL_0006167_VL (Denali DNL919) ATL_0006167_HCDR1 GFSIEDFY 197 ATL_0006167_HCDR2 IDPEQGES 198 ATL_0006167_HCDR3 HADHGNYGSTMDY 199 ATL_0006167_HFWR1 QVQLVQSGAEVKKPGASVKVSCKAS 200 ATL_0006167_HFWR2 IHWVRQAPGQGLEWMGW 201 ATL_0006167_HFWR3 KYAPKFQGRVTITADTSTSTAYMELSSLRSEDTAV YYC 202 ATL_0006167_HFWR4 WGQGTLVTVSS 2 03 ATL_0006167_LCDR1 QHINVW 204 ATL_0006167_LCDR2 KAS 205 ATL_0006167_LCDR2 QQGQTYPRT 206 ATL_0006167_LFWR1 DIQMTQSPSSLSASVGDRVTITCHAS 207 ATL_0006167_LFWR2 LSWYQQKPGKAPKLLIY 208 ATL_0006167_LFWR3 NLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYY C 209 ATL_0006167_LFWR4 FGQGTKVEIK 210 ATL_0006170_VH (Vigil VGL101) EVQLVQSGAEVKIKPGESLKISCKGSGYSFTSYWI GWVRQMPGKGLEWMGIIYPGDADARYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYFCARRRQG IFGDALDFWGQGTLVTVSS 211 ATL_0006170_VL (Vigil VGL101) EIVMTQSPATLSVSPGERATLSCRASQSVSSNLA WFQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQPEDFAVYYCLQDNNFPPTFGQGTK VDIKRT 212 ATL_0006170_HCDR1 GYSFTSYW 213 ATL_0006170_HCDR2 IYPGDADA 2 14 ATL_0006170_HCDR3 ARRRQGIFGDALDF 215 ATL_0006170_HFWR1 EVQLVQSGAEVKIKPGESLKISCKGS 216 ATL_0006170_HFWR2 IGWVRQMPGKGLEWMGI 217 ATL_0006170_HFWR3 RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YFC 218 ATL_0006170_HFWR4 WGQGTLVTVSS 219 ATL_0006170_LCDR1 QSVSSN 220 ATL_0006170_LCDR2 GÁS 221 ATL_0006170_LCDR2 LQDNNFPPT 222 ATL_0006170_LFWR1 EIVMTQS PATLSVSPG E RATLSCRAS 223 ATL_0006170_LFWR2 LAWFQQKPGQAPRLLIY 224 ATL_0006170_LFWR3 TRATGIPARFSGSGSGTEFTLTISSLQPEDFAVYY C 225 ATL_0006170_LFWR4 FGQGTKVDIKRT 226 CD33M2_ECD_18232_WT DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYD KNSPVHGYWFREGAISRDSPVATNKLDQEVQEE TQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFR Petition: 870250089689, on 10 / 02 / 2025, page. 94 / 221 81 / 144 M E RGSTKYSYKSPQLSV HVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQ GTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHG TNLTCQVKFAGAGVTTERTIQLNVT 227 CD33M2_ECD_18232_MutPos1[P1] DPRVRLEVQESVTVQEGLCVLVPCTFFHPIPYYDK NSPVHGYWFREGAIISRDSPVATNKLDQEVQEET QGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRM ERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEP GHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGP RTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTE RTIQLNVT 228 CD33M2_ECD_18232_MutPos1_MutPos6 [P6+1] DPRVRLEVQESVTVQEGLCVLVPCTFFHPIPYYDK NSPVHGYWFREGAIISRDSPVATNKLDQEVQEET QGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRM ERGSTKYSYKSTQLSVHVTDLTHRPKILIPGTLEPG HSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPR TTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTER TIQLNVT 229 CD33M2_ECD_18232_MutPos2 [P2] DPNFWLQVQESVTVQEGLCVLVPCTFFHPVPYHT RNSPVHGYWFREGAIISRDSPVATNKLDQEVQEE TQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFR MERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLE PGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLG PRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTT ERTIQLNVT 230 CD33M2_ECD_18232_MutPos2_MutPos 5 [P2+5] DPNFWLQVQESVTVQEGLCVLVPCTFFHPVPYHTRNSPVHGYWFREGAIISRDSPVATNKLDQEVQEE TQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFR MEKGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLE PGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLG PRTTHSSVLIITPRQDHGTNLTCQVKFAGAGVTT ERTIQLNVT 231 CD33M2_ECD_18232_MutPos3 [P3] DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYD KNSPVHGYWFREGAIISRDSPVATNKLDQEVQEE TQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFR MERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLE PGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLG PRTTHSSVLIITPRQDHGTNLTCQVKFAGAGVTT ERTIQLNVT 232 CD33M2_ECD_18232_MutPos4 [P4] 233 CD33M2_ECD_18232_MutPos6 [P6] DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYD KNSPVHGYWFREGAIISRSPVATNKLDQEVQEE TQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFR MERGSTKYSYKSTQLSVHVTDLTHRPKILIPGTLE PGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLG PRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTT Petition 870250089689, dated 02 / 10 / 2025, page 95 / 221 82 / 144 ERTIQLNVT 234 CD33M2_ECD_18232_MutPos5 [P5] DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYD KNSPVHGYWFREGAIISRDSPVAT NKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDAR RRDNGSYFFRMEKGSTKYSYKSPQLSVHVTDLTH RPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFS WLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQ VKFAGAGVTTERTIQLNVT 235 HFWR1_ATL_0005853 QVQLVQSGAEVKKTGESLRISCKAS 236 LCDR3_ATL_0005854 SSYTNSSTLEV Table 0. Sequences used in this descriptive report.

[0166] In Table 0 above and throughout this descriptive report, antibodies may be referenced by their reference numbers, as indicated in the table above (e.g., ATL_0005802), or by the same reference number without the initial sequence '0' (e.g., ATL_5802), or by the same reference with only the number (e.g., 5802). For example, the antibody ATL_0005802 may be referenced interchangeably as “ATL_0005802”, “ATL_5802”, and “5802”. The notations “VH” and “VL”, when appended to an antibody reference (either as a prefix or suffix), refer to the variable domain of the referenced antibody heavy chain. The notations HCDR1, HCDR2, and HCDR3, when attached to an antibody reference (either as a prefix or suffix), refer to the heavy chain CDRs (CDR1, CDR2, and CDR3, respectively) of the referenced antibody.The notations LCDR1, LCDR2, and LCDR3, when attached to an antibody reference (either as a prefix or suffix), refer to the light chain CDRs (CDR1, CDR2, and CDR3, respectively) of the referenced antibody. The notations HFWR1, HFWR2, HFWR3, and HFWR4, when attached to an antibody reference (either as a prefix or suffix), refer to the heavy chain structure regions (FWR1, FWR2, FWR3, and FWR4, respectively) of the referenced antibody. The notations LFWR1, LFWR2, LFWR3, and LFWR4, when attached to an antibody reference (either as a prefix or suffix), refer to the light chain structure regions (FWR1, FWR2, FWR3, and FWR4, respectively) of the referenced antibody. Petition 870250089689, dated 02 / 10 / 2025, p. 96 / 221 83 / 144 Examples

[0167] The examples below demonstrate the identification of novel antibodies with therapeutic potential from a cohort of individuals who have or are considered at risk of developing Alzheimer's disease, as well as from cognitively healthy centenarians who are seropositive for CD33 antibodies (Example 1), as well as the analysis of phage repertoires from CD33-reactive individuals (Example 2). Examples 3 to 15 show the results of functional screening and further characterization of some of the newly discovered antibodies. Materials and methods Serum and plasma ELISA to identify individuals with CD33 reactivity

[0168] Recombinant human CD33 antigen (R&D Systems, cat: 10375-SL050) or negative control lysozyme (MP Biomedicals #195303) were absorbed directly onto ELISA plates at 3 μg / ml (50 μl per well) and incubated overnight at 4 °C. Plates were washed with phosphate-buffered saline (PBS). Plates were blocked with 200 μl / well of blocking solution (1% bovine serum albumin (BSA) w / v in PBS) for 1 hour at room temperature. Then, the blocking solution was removed and the 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. The plates were incubated at room temperature for 1 hour. The plates were washed with PBS / 0.1% Tween.Next, the anti-human (Fab)'2-horseradish peroxidase (HRP) antibody (Jackson Immunoresearch No. 109-035-097; Lot: 148466) was added to each well and incubated for 1 hour at room temperature to detect antibody binding. The plates were washed with PBS / 0.1% Tween and a solution of 3,3',5,5'-tetramethylbenzidine (TMB) (Life Technology; No. 002023) was added to each well. The plates were incubated for 5 minutes at room temperature before the addition. Petition 870250089689, dated 02 / 10 / 2025, page 97 / 221 84 / 144 of the stopping solution (0.5 M sulfuric acid). Absorbance was read on a Molecular Devices FilterMaxF5 plate reader at 450 nm. Phage ELISA and sequence analysis in scFv derived from phage display

[0169] Phages were prepared by culturing each TG1 clone containing the phagemid from a glycerol stock in 100 μl of 2TYAG (2TY medium supplemented with 100 μg / ml ampicillin and 2% glucose) at 37 °C with aeration until optical density OD600 = 0.6, followed by rescue with helper phage added at MOI 10 (Invitrogen, cat: 18311-019) for 1 hour and changing the medium to 2TYAK (2TY medium supplemented with 100 μg / ml ampicillin and 50 mg / ml kanamycin). The cultures were then incubated overnight at 25 °C with good aeration and, the following day, the phages were separated from the bacteria by centrifugation for 10 minutes at 3200 rpm. The supernatant containing the phage was transferred to a new plate and blocked with 3% w / v milk in PBS.

[0170] Recombinant human antigen CD33 (R&D Systems, cat: 10375-SL050) or negative control Lysozyme (MP Biomedicals No 195303) were absorbed directly onto the ELISA plate at 3 μg / ml (50 μl per well) and incubated overnight at 4 °C. Each antigen-coated plate was washed with PBS and blocked with 200 μl / well of blocking solution (3% milk w / v in PBS) for 1 hour at room temperature. Then, the blocking solution was removed and samples of blocked phages to be evaluated were applied to the plates. The plates were incubated at room temperature for 1 hour. Each plate was washed with PBS / 0.1% Tween and incubated with HRP Anti-M13 (Sino Biological, No. 11973-MM05T-H) for 1 hour at room temperature to detect phage binding. The plates were washed with 0.1% PBS / Tween solution and TMB (Life Technology; No. 002023). The plates were incubated for 5 minutes at room temperature before... Petition 870250089689, dated 02 / 10 / 2025, pp. 98 / 221 85 / 144 of the addition of the stopping solution (0.5 M sulfuric acid). Absorbance was read on a Molecular Devices FilterMaxF5 plate reader at 450 nm. Single-point ELISA for binding to the full CD33 domain and CD33 C2 and CD33-related proteins.

[0171] Seven converted IgG1 antibodies were tested in a single-point ELISA against a panel of antigens: recombinant human CD33 (Sino Biological cat: 12238-H08H), Cynomolgus / Rhesus CD33 (Sino Biological, cat: 90303-C08H), recombinant human CD33 C2 domain (in-house production), mouse CD33 (Sino Biological, cat: 50712-M08H), recombinant human Fc Siglec-6 / CD327 chimera (R&D Systems, 2859-SL), recombinant human Fc Siglec-7 / CD328 chimera (R&D Systems cat: 1138-SL-050), recombinant human Fc Siglec-8 chimera (R&D Systems, cat: 9045-SL), recombinant human Fc Siglec-9 chimera (R&D Systems, cat: 1139-SL). After the single-point ELISA, for each antibody-antigen pair for which a positive signal was detected, the titration ELISA was performed to obtain the EC50 values.

[0172] Each recombinant protein antigen or negative control lysozyme (MP Biomedicals #195303) was absorbed directly onto the ELISA plate at 3 μg / ml (50 μl per well) and incubated overnight at 4 °C. Each plate was washed with PBS. The plates were blocked with 200 μl / well of blocking solution (1% w / v BSA in PBS) for 1 hour at room temperature. Then, the blocking solution was removed and the antibodies to be evaluated were diluted to 100 μg / ml in blocking solution (1% w / v BSA in PBS) and applied to the plate. The plates were incubated at room temperature for 1 hour. Each plate was washed with 0.1% PBS / Tween and with (Fab)'2-HRP anti-human (Jackson Immunoresearch No. 109-035-097; Lot: 148466) added to the plate and incubated for 1 hour at room temperature to detect antibody binding. Each plate was washed with 0.1% PBS / Tween and TMB solution (Life Technology; No.) added. Petition 870250089689, dated 02 / 10 / 2025, pp. 99 / 221 86 / 144 (002023). The plates were incubated for 5 minutes at room temperature before the addition of the stopping solution (0.5 M sulfuric acid). Absorbance was read using a Molecular Devices FilterMaxF5 plate reader at 450 nm. Biolayer interferometry for characterization of CD33 binding kinetics

[0173] Eleven converted IgG1 antibodies were tested in a biolayer interferometry (BLI) experiment for binding to recombinant human CD33 (R&D Systems, 10375-SL-050).

[0174] Each antibody was loaded onto a row of eight anti-human Fc IgG capture biosensor tips (AHC tips, Sartorius Stedim, part No. 18-5060), and the row was then equilibrated with kinetic buffer (Sartorius Stedim, part No. 18-1105) before immersion in a series of CD33 analyte dilutions in kinetic buffer at 25 °C. The binding response was measured. The tips were then immersed in kinetic buffer and the response was measured. Association and dissociation curves were fitted to the response measurements using the manufacturer's analysis software (Octet Data Analysis HT12.0) and kinetic parameters were calculated. Phagocytosis in microglia derived from human iPSCs stimulated by inflammation.

[0175] Microglia derived from induced pluripotent stem cells (iPSCs) (FujiFilm (CDI) # R1131) were seeded at 20,000 cells / well and left to stand for 3 days, with a 50% medium change on day 3 of culture. On day 4, cells were treated with 50 ng / ml of LPS (tlrl-eklps Invivogen) for 24 hours and then with 50 μg / ml of test antibody for another 24 hours. Cells were then treated with 0.5 μg / well of pHrodo red-labeled β-amyloid and red fluorescence was monitored with the Incucyte S3 (Beta Aggregation Kit). Petition 870250089689, dated 02 / 10 / 2025, pp. 100 / 221 87 / 144 Amyloid(1-42), rPeptide #A-1170-025 labeled with pHrodo Red, Succinimidyl Ester (ThermoFisher Scientific #P36600). Phagocytosis assay in human whole blood

[0176] Blood was obtained from an apheresis cone from NHS Blood and Transplant and diluted 1:10 in RPMI medium (Invitrogen A4192301). 25 μL of diluted blood and 2 ml of 1x lysis buffer (ebioscience 00-4300-54 diluted 10x in water to 1x immediately before use) were added to each well and thoroughly mixed by pipetting. Samples were incubated at room temperature for 15 minutes and then centrifuged at 400g for 5 minutes to pellet any remaining leukocytes. The supernatant was discarded and the cells resuspended in 100 μL of RPMI medium containing 10 μg / ml of S. aureus pHrodo-red and 10 μg / ml of antibody. The samples were incubated at 37 °C for 3 hours and then washed by adding 1 ml of PBS / 2% FBS, centrifuging at 400 g for 5 minutes to sediment, and then discarding the supernatant.Samples were resuspended in 50 μl of a 1 / 50 human Fc block diluted in PBS / 2% FBS (BD Biosciences 564219) and incubated for 15 minutes at 2–8 °C. 50 μl of anti-human CD14 APC diluted 1 / 100 in PBS / 2% FBS was added to the Fc block and incubated for a further 30 minutes at 2–8 °C. Samples were washed as above with 1 ml of PBS / 2% FBS and then resuspended in 200 μl of PBS / 2% FBS / 1 in 10,000 DAPI for analysis on a BD FACSymphony flow cytometer. Live monocytes were selected (DAPI negative, CD14 positive) and the 561-586 / 15 channel was used to evaluate pHrodo-red fluorescence within the live monocyte population. CD33 depletion in human monocytes

[0177] Human monocyte cells isolated from previously frozen PBMCs were thawed, washed, and maintained at rest O / N in RPMI medium (Invitrogen A4192301) + 10% Fetal Bovine Serum (FBS) at 37°C, 5% of Petition 870250089689, dated 02 / 10 / 2025, pp. 101 / 221 88 / 144 CO2 (standard cell culture condition). The following day, resting monocyte cells were collected and washed once in RPMI medium + 10% FBS. The cells were then resuspended at a final concentration of 2 x 10⁶ cells / ml, and 50 μl were seeded into each well of a 96-well flat-bottom plate, resulting in 1 x 10⁵ cells / well (Corning, Cat. 3595). The cells remained at rest in the incubator while the reagents were prepared. The reagents (ATL_5802, ATL_5810, ATL5338, ATL_5909) were prepared at concentrations twice the final concentrations of the experiment in RPMI + 10% FBS, at a single concentration (80 nM) or in a range of concentrations (80, 60, 40, 30, 20, 2 nM) in 60 μl / well in a 96-well round-bottom plate (Corning, cat. 3799). ATL5338 is an internally produced isotype control based on the FITC 4-4-20 binding antibody described in Jung et al. JMB 294, 163 (1999).Next, 50 μl / well of the reagent plate was transferred to the 96-well plate of 50 μl / well containing resting monocytes to a final volume of 100 μl and then to a final concentration of 40, 30, 20, 10, 1 nM. The plate was subsequently incubated for 5 hours under standard cell culture conditions. After incubation, the plate was kept on ice to prevent any further internalization of antibodies. All subsequent steps were performed at 4 °C in the dark. Cells were collected by centrifugation (5 minutes at 300 g) and the supernatant was removed. Then, the cells were resuspended in 50 μl of Live / Dead fixable violet dead cell dye (Cat. L34955) and incubated for 7 minutes. A washing step was performed by adding 150 μl of PBS / 2% FBS per well on top of each sample, and the plate was centrifuged at 300 g for 5 minutes.The supernatants were removed and the cells resuspended in stained antibodies: V domain specific for anti-human CD33 (Biolegend, cat. 303428), C domain specific for anti-human CD33 (Biolegend, cat. 366620), anti-human CD45 (Biolegend, cat. 304027), anti-CD14. Petition 870250089689, dated 02 / 10 / 2025, pp. 102 / 221 89 / 144 human (Biolegend, cat. 301806), anti-human CD16 (Biolegend, cat. 302016), anti-human CD3 (Biolegend, cat. 300412) plus human Fc block 1:50 (BD Biosciences 564219) diluted in PBS-2% FBS. Staining was performed at 4°C for 40 minutes. Then, the cells were washed twice, as described above, and resuspended in a final volume of 200 μl / well of PBS-2% FBS for analysis on a BD FACSymphony flow cytometer. Live monocytes were labeled (negative for dead staining, positive for CD45, negative for CD3, positive for CD14, positive / negative for CD16) and the MFI emitted by the two antibodies against CD33 was recorded. Modulation of phagocytosis via CD33 involvement in newly isolated human monocyte cells

[0178] Antibodies were prepared at twice the final assay concentration, as a single concentration (60 nM) and as a 1:4 serial dilution, starting with 60 nM in RPMI + 10% FBS, and distributed in a 96-well round-bottom plate (Corning, cat. 3799), 60 μl / well. Latrunculin A was diluted 1:5000 for the wells used as negative control. S. aureus pHrodo bait (Thermo Fisher, cat. A10010) for phagocytosis was prepared in RPMI + 10% FBS medium at twice the final concentration of 10 μg / ml and distributed in a 96-well flat-bottom plate (Corning, cat. 3595). Next, human monocyte cells were isolated from fresh PBMCs following the Miltenyi protocol (cat. 130-096-537). The obtained monocyte cells were resuspended at 1x10⁶ cells / ml, with 100 μl distributed into each well of a 96-well round-bottom plate (Corning, cat. 3799).The cells were centrifuged at 300 g for 5 minutes, the supernatant was removed, and the cells were resuspended in the prepared antibody solutions before transfer to a plate containing the pHrodo reagent to a final volume of 100 μl. The emitted fluorescence was recorded live using the Incucyte system (Sartorius). Petition 870250089689, dated 02 / 10 / 2025, pp. 103 / 221 90 / 144 Study design in CD34+ NSG mice in vivo

[0179] There were 17 treatment groups. Each treatment group was assigned a letter so that the observer would not know the identity of the treatment groups. The mice were randomly assigned to the treatment groups. This experiment consisted of 17 conditions (65 animals in total): 1. Control dose: 10 mg / kg pre-dose and 24 hours 2. Antibodies tested (ATL5802, ATL5810 and ATL5909) 10, 40 mg / kg; Predose and 24 hours 3. Control: 10 mg / kg Pre-dose, 24 hours, 7 days 4. Antibodies tested (ATL5802, ATL5810 and ATL5909) 10, 40 mg / kg; Predose, 24 hours, 7 days 5. Antibodies tested (ATL5802, ATL5810 and ATL5909) 1 mg / kg; Pre-dose, 4 hours, 24 hours, 7 days

[0180] Groups 1 and 2 consisted of 3 animals per group (n = 3 biological replicates, 21 animals in total), groups 3 and 4 consisted of 5 animals per group (n = 5 biological replicates, 35 animals in total), and group 5 consisted of 3 animals per group (n = 3 biological replicates, 9 animals in total). Blood volumes were guided by UK Home Office guidelines (maximum 200 μl per mouse in total), so groups 1 and 2 received 200 μl pre-dose, groups 3 and 4 received 100 μl pre-dose and at 24 hours, and group 5 received 66 μl pre-dose, at 4 and 24 hours. The final time point for each group involved a terminal blood sample. Mice were humanized from two separate donors (n = 30 from one and n = 35 from the second). Groups 1, 2, and 5 used animals from donor 1, and groups 3 and 4 used animals from donor 2.

[0181] Samples from each group are used as follows: • Groups 1 and 2 - Pre-dose (ELISA), 24 hours (ELISA and Flow Cytometry) Petition 870250089689, dated 02 / 10 / 2025, pp. 104 / 221 91 / 144 • Group 3 and 4 - Pre-dose (ELISA), 24 hours (Flow cytometry), 7 days (ELISA and Flow cytometry) • Group 5 - All time points (ELISA) The dose volume for all treatments was 10 ml / kg. Phagocytosis in peripheral monocytes of CD34+ mice

[0182] Blood from CD34+ NSG mice was processed as follows: 25 μl of mouse blood and 2 ml of 1x lysis buffer (ebioscience 00-4300-54 diluted 10 times in water to 1x immediately before use) were added to each well and thoroughly mixed by pipetting. Samples were incubated at room temperature (18 to 22 °C) for 15 minutes and then centrifuged at 400 g for 5 minutes to pellet any remaining leukocytes. The supernatant was discarded and the cells resuspended in 100 μl of RPMI medium containing 10 μg / ml of S. aureus pHrodo. The samples were incubated at 37°C for 3 hours and then washed by adding 1 ml of PBS / 2% FBS, centrifuging at 400 g for 5 minutes to form pellets, and then discarding the supernatant in the waste. The samples were resuspended in 50 μl of PBS / 2% FBS containing 1 in 50 blocks of human Fc (BD Biosciences 564219) and 1 / 100 blocks of mouse Fc (Biolegend 156603) and incubated for 15 minutes between 2 and 8 °C.An antibody cocktail was prepared containing the following antibodies at a 1:50 dilution in PBS / 2% FBS: CD45 AF488 anti-mouse (Biolegend 157608), CD45 APC anti-human (Biolegend 304012), and CD11b BV785 anti-human / mouse (Biolegend 101243). 50 μl of the antibody cocktail were added to each sample, over the Fc block already present in the well. Samples were incubated for a further 30 minutes at room temperature in the dark. Samples were washed, as described above, with 1 ml of PBS / 2% FBS and then resuspended in 200 μl of PBS / 2% FBS / 1 in 10,000 DAPI for analysis on a BD FACSymphony flow cytometer. Live human monocytes. Petition 870250089689, dated 02 / 10 / 2025, pp. 105 / 221 92 / 144 were subjected to group selection (DAPI negative, human CD45+, CD11b+ positive) and the 561-586 / 15 channel was used to evaluate pHrodo fluorescence in the live human myeloid population. Size exclusion chromatography (SEC-HPLC)

[0183] Antibody samples were diluted to 1 mg / ml in 20 mM histidine acetate, 150 mM NaCl, pH 5.5, to be processed on a Zorbax GF-250 SEC-HPLC column (Agilent) in a Vanquish Flex (Thermo Scientific). Samples were separated by size in a mobile phase containing 20 mM sodium phosphate, 300 mM sodium sulfate, and 100 mM arginine, at a flow rate of 0.75 ml / minute at 25 °C, for 25 minutes per sample. Data acquisition at 280 nm, using Chromeleon software (Thermo Scientific), was used to integrate the chromatograms. Capillary isoelectric focusing (cIEF)

[0184] Charge variant analysis was performed by preparing a master mix to dilute antibody samples to be run on a cIEF cartridge in a Maurice instrument (Protein Simple). The master mix contained a final methylcellulose concentration of 0.35% (Protein Simple, 101876), pharmalyte pH of 3 to 10 4% (Protein Simple, 17-0456-01), 10 mM arginine (Protein Simple, 042-691), pI markers of 4.05 and 9.99 0.01% (Protein Simple, 046-029 and 046-034). Samples were diluted to 0.15 to 0.25 mg / ml in the master mix and run for 1 minute at 1500 volts, followed by 4.5 minutes at 3000 volts. A system suitability standard (Protein Simple, 046-044) was also run at the start of the run. Stability data generated at 2 and 4 weeks were overlaid to compare the loading species profile. Thermal displacement test

[0185] Protein thermal displacement measurements were performed on an Uncle (Unchained Labs). Antibodies were diluted to 1 mg / ml or 5 mg / ml Petition 870250089689, dated 02 / 10 / 2025, pp. 106 / 221 93 / 144 samples were stored in 20 mM histidine acetate buffer, 150 mM NaCl, pH 5.5, and subjected to a temperature ramp from 25 to 95 °C, increasing at a rate of 0.5 °C / minute. Samples were processed in triplicate, loading 8.8 μI into 3 different wells of a uni (Unchained Labs). Laser settings were adjusted to achieve initial fluorescence in the 300 to 350 nm range from 10,000 to 50,000 counts. The melting temperature (Tm1 / Tm2) and aggregation temperature (Tagg / Tonset) were analyzed using Uncle Analysis v6 software (Unchained Labs). The Tm measurement was calculated using the 350 / 330 nm ratio, while Tonset and Tagg were obtained from SLS readings at 266 nm. Epitope mapping

[0186] Variants of the extracellular domain of CD33 were prepared as individual recombinant proteins. The variations were based on the differences between human CD33 and cynomolgus monkey CD33, since the main panel of mAbs was known to bind to human CD33 but not to cynomolgus monkey CD33.

[0187] The DNA sequence encoding residues 21 to 232 of human CD33 was synthesized and cloned into pcDNA3.1(+) by Genscript. Insertion of native residues 18 to 20 and single- and multi-residue cynomolgus mutations was conducted using the Q5 Site-Directed Mutagenesis (NEB) kit. The wild-type plasmid (residues 18 to 232) and seven mutant plasmids were individually transfected into 15 mL cultures of Expi293F (Thermofisher) cells, according to the manufacturer's instructions. The seven mutant plasmids were: N20R F21V W22R Q24E [P1]; I47V Y50H D51T K52R [P2]; I67V R69L [P3]; Q83R [P4]; R122K [P5]; P132T [P6]; N20R F21V W22R Q24E P132T [P6+1]; I47V Y50H D51T K52R R122K [P2+5]. These mutations are illustrated in Figure 11A. Cells were removed from cultures by centrifugation 5 days after transfection, and proteins were purified from the supernatant in a pure AKTA (Cytiva) by Petition 870250089689, dated 02 / 10 / 2025, pp. 107 / 221 94 / 144 affinity with Ni, using a HisTrap Excel column (Cytiva), followed by size exclusion chromatography, using a Superdex Increase 10 / 300 GL column (Cytiva).

[0188] Eleven IgG1 antibodies were tested in a single-point ELISA against wild-type CD33 and all 7 variant CD33 proteins. Each recombinant protein antigen or negative control Lysozyme (MP Biomedicals #195303) was absorbed directly onto an ELISA plate at 3 μg / ml (50 μl per well) and incubated overnight at 4 °C. Each plate was washed with PBS. The plates were blocked with 200 μl / well of blocking solution (1% w / v BSA in PBS) for 1 hour at room temperature. Then, the blocking solution was removed and the antibodies to be evaluated were diluted to 266 nM in blocking solution (1% w / v BSA in PBS) and applied to the plate. The plates were incubated at room temperature for 1 hour. Each plate was washed with 0.1% PBS / Tween and (Fab)'2-HRP anti-human (Jackson Immunoresearch No. 109035-097; Lot: 148466) was added to the plate and incubated for 1 hour at room temperature to detect antibody binding.Each plate was washed with 0.1% PBS / Tween and TMB solution (Life Technology; No. 002023). The plates were incubated for 5 minutes at room temperature before the addition of the stopping solution (0.5 M sulfuric acid). Absorbance was read on a Molecular Devices FilterMaxF5 plate reader at 450 nm. Absorbance values ​​were converted to % wild-type CD33 binding (WT), where WT = 100%. Antigen constructs

[0189] Table 1 below summarizes the antigen constructs used in these examples. Experiment: Role of the antigen; Antigen construct ( / lot) used; Construct description; Provided catalog number; Phage display; Selection antigen CD33_HUMAN_M17-H259_002_004; Extracellular domain Met17-His259 from R&D Systems. Petition 870250089689, dated 02 / 10 / 2025, pp. 108 / 221 95 / 144 Human Siglec-3 / CD33 protein derived from CHO with a C-terminal 6His tag 10375-SL-050 ELISA Binding characterization (C domain only) CD33_HUMAN_ECD_Cdomain_His_007 C domain of human Siglec-3 / CD33 protein derived from HEK Asp140Thr232 with a C-terminal 6His tag In-house SEQ ID NO: 175 ELISA Binding characterization CD33_HUMAN_ECD, His_004_001 Extracellular Met1-His259 domain of human Siglec-3 / CD33 protein derived from HEK with a C-terminal polyhistidine tag Sino Biological, 12238H08H BLI Binding characterization (kinetics) CD33_HUMAN_M17-H259_002_006 Extracellular domain Met17-His259 of human Siglec-3 / CD33 protein derived from CHO with a C-terminal 6His tag R&D Systems, 10375-SL050 ELISA Cross-reactivity check (mouse) CD33_MOUSE_His_002_001 Extracellular domain Met1-Glu240 of mouse Siglec-3 / CD33 protein derived from HEK with a C-terminal polyhistidine tag Sino Biological,50712M08H ELISA Cross-reactivity check (cyno) CD33_CYNO_His_001_001 Extracellular domain Met1-Gly248 of monkey cynomolgus Siglec-3 / CD33 protein derived from HEK with a C-terminal polyhistidine tag Sino Biological, 90303C08H ELISA Off-target binding check SIGL6_HUMAN_Fc Chimera_001_001 Extracellular domain Gln27-Val331 of human Siglec-6 expressed by CHO with a C-terminal human IgG1 Fc fusion domain (Pro100-Lys330) R&D Systems, 2859-SL ELISA Off-target binding check SIGL7_HUMAN_Gln19-Gly357_001_002 Extracellular domain Gln19-Gly357 of Human Siglec-7 expressed in NS0 with a C-terminal human IgG1 Fc fusion domain (Pro100-Lys330) R&D Systems, 1138-SL ELISA Off-target binding verification SIGL8_HUMAN_Fc Chimera_001 Extracellular domain of human Siglec-8 expressed by NS0 Met17Ala363 with a C-terminal human IgG1 Fc fusion domain (Pro100Lys330) R&D Systems,9045-SL ELISA Linkage verification outside of SIGL9_HUMAN_Fc Chimera_001 Extracellular domain Gln18-Gly348 of R&D Systems, Petition 870250089689, dated 02 / 10 / 2025, pp. 109 / 221 96 / 144 Human Siglec-9 target expressed by NS0 with a C-terminal human IgG1 Fc fusion domain (Pro100-Lys330) 1139-SL ELISA CD33M2_ECD_18-232_WT epitope mapping Human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag (wild type) (sequence provided in Table 0 without terminal 6xH tag) In-house SEQ ID NO: 226 ELISA CD33M2_ECD_18-232_MutPos1[P1] epitope mapping Human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag and N20R F21V W22R Q24E mutations (sequence provided in Table 0) (without 6xH terminal label) In-house SEQ ID NO: 227 ELISA Epitope Mapping CD33M2_ECD_18232_MutPos1_MutPos6 [P6+1] Human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a 6-His C-terminal label and N20R F21V W22R Q24E P132T mutations (sequence provided in Table 0 without 6xH terminal label) In-house SEQ ID NO: 228 ELISA Epitope Mapping CD33M2_ECD_18-232_MutPos2 [P2]Human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag and I47V Y50H D51T K52R mutations (sequence provided in Table 0 without terminal 6xH tag) In-house SEQ ID NO: 229 ELISA Epitope Mapping CD33M2_ECD_18232_MutPos2_MutPos5 [P2 + 5] Human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag and I47V Y50H D51T K52R R122K mutations (sequence provided in Table 0 without terminal 6xH tag) In-house SEQ ID NO: 230 ELISA Epitope Mapping CD33M2_ECD_18-232_MutPos3 [P3] Human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag and I67V R69L mutations (sequence provided in Table 0 without terminal 6xH tag) In-house SEQ ID NO: 231 ELISA Mapping CD33M2 ECD 18-232 _MutPos4 [P4] Internal Petition 870250089689, dated 02 / 10 / 2025, pp. 110 / 221 97 / 144 Epitope Mapping of human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag and Q83R mutation (sequence provided in Table 0 without terminal 6xH tag) SEQ ID NO: 232 ELISA Epitope Mapping CD33M2_ECD_18-232_MutPos6 [P6] Epitope Mapping of human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a C-terminal 6-His tag and P132T mutation (sequence provided in Table 0 without terminal 6xH tag) In-house SEQ ID NO: 233 ELISA Epitope Mapping of human Siglec-3 / CD33 protein residues derived from HEK 18-232 with a 6-His C-terminal label and R122K mutation (sequence provided in Table 0 without 6xH terminal label) In-house SEQ ID NO: 234 Table 1. Summary of antigen constructs used in the examples. Study of pK in mice

[0190] Homozygous male C57BL / 6-Cd33tm1(CD33) / Bcgen mice (common name: hCD33) were obtained from Biocytogen Technology Co., Ltd. and shipped to Pharmaron (Ningbo, China) for this study. At 6–8 weeks of age, mice (n = 3 / treatment group) were weighed and received a single intraperitoneal (ip) dose of freshly prepared antibody solutions at 1 mg / kg and / or 10 mg / kg, with a dosing volume of 5 ml / kg. All mice had free access to food and water. Cageside observations were conducted daily, and clinical observations were made before administration and at each sample collection time.

[0191] Blood samples were collected from the orbital vein 4 hours, 24 hours, and 7 days (144 hours) after a single intraperitoneal (ip) antibody administration. Briefly, whole blood samples were left at room temperature for 30 minutes and then centrifuged at 3,500 xg 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. Petition 870250089689, dated 02 / 10 / 2025, pp. 111 / 221 98 / 144

[0192] Samples were processed 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 microwell plates (96-well cell culture plates from Corning Incorporated). Serum concentrations of the test subjects were determined using a custom-developed ELISA method (Pharmaron, Ningbo, China), using a goat anti-human Fc polyclonal IgG primary antibody (5 μg / ml) and a goat anti-human IgG monoclonal antibody, HRP (1:5000). Readings were obtained on a Molecular Devices SpectraMax ID3 and pharmacokinetic calculations were performed using WinNonlin (Phoenix™, version 8.3). In vivo Alzheimer's disease model

[0193] Male and female Rag2- / - Il2ry- / - hCSFIKI AppNL-GF mice (hereinafter referred to as APPNL-GF mice) were transplanted with human microglia progenitors derived from iPSCs on postnatal day 4 (P4), as described in Mancuso et al., 2022. From 4 months of age, mice were treated once a week for 12 weeks with 40 mg / kg of ATL_5802 (n = 6) or with the isotype control antibody, ATL-5338 (n = 7). To assess in vivo phagocytosis, the fluorescent amyloid-β marker, Methoxy-X04 (MX04), was used, and the extent of amyloid-β uptake was evaluated by measuring the percentage of MX04-positive microglia by flow cytometry. At approximately 16 weeks of age, mice were injected with freshly prepared Methoxy-X04 (ab142818, prepared at a 1:1 ratio with DMSO:HBSS) at 10 mg / kg, i.p. Twenty-four hours after the MX04 injection, the mice were injected with the euthanasia agent, Dolethal, and perfused with ice-cold PBS.The collected brain tissue was immediately placed in FACS buffer (1X PBS with 2% FBS / SFB and 2 mM EDTA) for processing. Petition 870250089689, dated 02 / 10 / 2025, pp. 112 / 221 99 / 144 posterior by flow cytometry. For flow cytometry analysis, brain tissue was dissociated using the Miltenyi neural tissue dissociation kit, according to the manufacturer's instructions. Samples were run on the MACSQUANT Analyzer 10 and controlled for viability and human versus mouse microglia using the following antibodies: Fixable Viability Dye eFluor780 (1:2000, Invitrogen eBioscience Cat#65-0865-14), mouse anti-human CD45 monoclonal antibody (1:50, clone BD Bioscience HI30 Cat#555485), and PE-conjugated recombinant human anti-mouse CD11b antibody (1:100, Miltenyi Biotec Cat No 130-113-806). After excluding non-viable cells, the cells were controlled using hCD45, and the median fluorescent intensity (MFI) of the human microglia population was measured and set as a threshold for each mouse.The percentage of MX04-positive microglia was calculated by quantifying the proportion of MX04+ microglia cells that were greater than this defined limit. Phagocytosis of Tau in microglia derived from human iPSCs

[0194] FujiFilm iPSC-derived microglia (CDI) (Catalog Code R1131) were seeded at 20,000 cells / well and held at rest for 3 days, with a 50% medium change on day 3 of culture. On day 4, cells were treated with 50 ng / ml LPS (tlrl-eklps, Invivogen) for 6 hours and then with 50 μg / ml test antibody for 24 hours. Cells were then treated with 0.5 μg / well of pHrodo.Red-labeled TauP301S (ThermoFisher Scientific #P36600) (Abcam #ab246003) and sonicated once for 5 minutes immediately before treatment. Red fluorescence was monitored using Incucyte S3. Quantification of cytokines in iPSC microglia, total PBMCs, and isolated monocytes.

[0195] iCell Microglia cells (Cellular Dynamics C1110) were seeded at 25,000 cells per well in iCell Microglia medium in a 96-well plate. Petition 870250089689, dated 02 / 10 / 2025, pp. 113 / 221 100 / 144 flat-bottomed poly-D-lysine (PDL) coated tubes. PBMCs were isolated from a leukapheresis cone (NHS BT) using Lymphopure (Biolegend 426201) and Leucosep (Greiner 227290) tubes. After 24 hours, the medium was collected and cytokine levels were measured using a flow cytometry-based multiplex immunoassay (LEGENDplex™ Biolegend 741081, 740930, 740502, 740796 or 741795).

[0196] PBMCs were isolated from a leukapheresis cone (NHS BT) using Lymphopure (Biolegend 426201) and Leucosep tubes (Greiner 227290).

[0197] Monocytes were isolated from PBMCs using a Pan Monocyte Isolation kit (Miltenyi 130-096-537) and MS columns (Miltenyi #130-042-201).

[0198] 100,000 PBMCs or monocytes per well were seeded in RPMI medium (Gibco A4192301) + 10% FBS in a U-bottom plate (PBMC) or flat-bottom plate (monocytes). Cells were treated with 50 μg / ml of isotype control antibody (antifluorescein) or ATL_5802 antibody. Some assays involved treating cells with 50 ng / ml of LPS (Invivogen, tlrleklps) for 6 hours before antibody addition. After 24 hours, the medium was harvested and cytokine levels were measured using a flow cytometry-based multiplex immunoassay (LEGENDplex™ Biolegend 741081, 740930, 740502, 740796, or 741795).

[0199] The cytokines tested in these immunoassays include: CCL2 (MCP-1); CXCL8 (IL-8); IFN-γ; IL-10; IL-12p40; IL12p70; IL-17A; IL-13; IL-2; IL-23; IL-4; IL-6; TGF-βΤ TNF-α; CCL17 (TARC); CCL2 (MIP-3a); CCL3 (MIP-1a); CCL4 (MIP-13); CCL5 (RANTES); CXCL1 (Gro-α); CXCL5 (ENA-78); CXCL9 (MIG); CCL11 (Eotaxin); CX3CL1 (Fractalcin); IL-18; sRAGE; stream-1; sTREM-2; VEGF; VILIP-1; β-NGF; BDNF; CCL2 (MCP-1). Details of the tested cytokines are provided below: Petition 870250089689, dated 02 / 10 / 2025, p. 114 / 221 101 / 144 Monocytes: CXCL8 (IL-8), CCL11 (Eotaxin), CCL17 (TARC), CCL2 (MCP1), CCL5 (RANTES), CCL3 (MIP-1a), CXCL9 (MIG), CXCL5 (ENA-78), CCL20 (MIP3α), CXCL1(GROMaa), CXCL1(GROMaa) PBMC: IL-12p70, TNF-a (TNFSF2), IL-6, IL-4, IL-10, IL-1β(CCL17 (TARC), IL-12p40, IL-23, IFN-γ PBMC - 6 donors: TNF-a, IL-6, IL-1beta, IFN-y iPSC Microglia: CXCL8 (IL-8), CCL11 (Eotaxin), CCL17 (TARC), CCL2 (MCP-1), CCL5 (RANTES), CCL3 (MIP-18MIG (CCLA), CXCL9 (MIP-3a), CXCL1 (GROa), CCL4 (MIP-1p) VILIP-1, sTREM-2, BDNF, TGF-e1, VEGF, IL-6, sTREM-1, β-NGF, IL-18, TNF-a, sRAGE, CX3CL1 (Fractalkine) PBMC and Monocytes: IL-4, IL-2, IL-1e, TNF-α (TNFSF2), CCL2 (MCP-1), IL17A, IL-6, IL-10, IFN-γ, IL-12p70, TGF-e1 (Ativo Livre), CXCL8 (IL-8). PBMC - 4 donors: MCP-1, IL-1 β , TNF- a , IL-6, IFN-y, IL12p70, IL-4, IL10, CCL17, IL12p40, IL-23, TARC Microglia: IL-4, IL-2, IL-1 β , TNF -a (TNFSF2), CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN- γ , IL-12p70, TGF- β 1 (Ativo Livre), CXCL8 (IL-8). Microglia Cell Culture

[0200] Microglia cells (iCell Microglia, FUJIFILM Cellular Dynamics, Inc., Catalog No: R1131) were cultured according to manufacturer-recommended protocols. Prior to cell seeding, culture surfaces were prepared by coating with 0.1 mg / mL Poly-D-Lysine solution (Gibco) and incubated overnight to enhance cell adhesion. Subsequently, cells were seeded into 24-well plates at a density of 120,000 cells per well. After a four-day resting period, microglia cells were subjected to a stimulation protocol. This involved pretreating the cells with 50 ng / mL LPS (Sigma Aldrich, Catalog No: L2637-10Mg) for 24 hours, followed by the addition of antibodies. Petition 870250089689, dated 02 / 10 / 2025, pp. 115 / 221 102 / 144 specific times for 20 minutes, 6 hours, and 24 hours. Measuring phosphorylated proteins at the 20-minute mark is generally considered ideal due to the transient nature of phosphorylation. Consequently, later time points, such as 6 and 24 hours, are used to observe changes in protein levels. Western blotting

[0201] For protein extraction, we used RIPA cell lysis buffer, composed of 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, and 1% SDS, supplemented with protease and phosphatase inhibitors from Sigma. Auto Western Testing Service was provided by RayBiotech, Inc. (Peachtree Corners, GA, USA). A sample concentration of 0.1 mg / mL was loaded into the automated capillary electrophoresis machine. The following antibodies were employed for protein analysis: SYK phospho (Tyr525 / 526) (Cell Signaling; Catalog No. 12710T) and total SYK (Cell Signaling; Catalog No. 113198S). As a charge control, we used the antibody Gluceraldehyde-3-phosphate dehydrogenase (GAPDH), provided by RayBiotech from their service library. Microglia cell culture for RNA sequencing

[0202] Microglia cells (iCell Microglia, FUJIFILM Cellular Dynamics, Inc., Catalog No: R1131) were cultured according to manufacturer-recommended protocols. Prior to cell seeding, culture surfaces were prepared by coating with 0.1 mg / mL Poly-D-Lysine solution (Gibco) and incubated overnight to enhance cell adhesion. Subsequently, cells were seeded into 24-well plates at a density of 120,000 cells per well. After a four-day resting period, microglia cells were subjected to a stimulation protocol to simulate inflammatory conditions. This involved pretreating the cells (i.e., prior to antibody treatment) with 10 ng / mL of lipopolysaccharide (LPS, Petition 870250089689, dated 02 / 10 / 2025, pp. 116 / 221 103 / 144 Sigma Aldrich, Catalog No: L2637-10Mg) and 20 ng / mL of interferon-gamma (IFNgamma, PeproTech, Inc., Catalog No: 300-02-20ug) for 24 hours, followed by the addition of ATL_0005802, ATL_0005854, ATL_0005909, or ATL_0005338 (isotype) for another 24 hours, resulting in a total exposure period of 48 hours to LPS / IFNy (LI). This is compared to a condition mimicking healthy conditions, in which cells are pre-treated with vehicle only. After treatment, the cell culture medium was aspirated and the cells were lysed directly into the wells, adding 400 μL of TRIzol reagent (Invitrogen, catalog number: 15596026). The TRIzol reagent was ensured to uniformly cover the surface of each well. The plates were gently agitated to facilitate thorough mixing and effective cell lysis. The cell lysate, composed of both the TRIzol reagent and the cell contents, was then carefully transferred to individually labeled tubes using a pipette.These tubes were immediately placed on dry ice for rapid cooling, a critical step to preserve the integrity of the RNA. Subsequently, the samples were stored at -80 °C for long-term preservation until RNA extraction. RNA extraction, library preparation and sequencing

[0203] The collected samples were processed for next-generation sequencing as follows. Total RNA was extracted from cryopreserved TRIzol samples, following the manufacturer's recommendations. RNA integrity was assessed using Tapestation (Agilent Technologies, Palo Alto, CA, USA) and concentration using Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA, USA). Samples with low quality were excluded. Poly(A) mRNA was enriched with Oligod(T) beads, following the manufacturer's recommendations.

[0204] Libraries were prepared from RNA with added ERCC using the Twist RNA Library Prep Kit and the Twist UMI Adapter System (Twist Bioscience), following the manufacturer's recommendations. The library size was Petition 870250089689, dated 02 / 10 / 2025, pp. 117 / 221 104 / 144 evaluated on Tapestation, concentration measured using a Qubit 2.0 fluorometer, and final quantification performed by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Multiplexed samples were sequenced with a single index using 2x150 bp reads on an Illumina HiSeq4000 sequencer. Bioinformatic analysis

[0205] Quality control, sequence alignment, and quantification were performed as follows. Briefly, the quality of the raw data was assessed using FastQC (Andrews, 2010). Sequence reads were trimmed using fastp v.0.23.1 (Chen et al., 2018). UMI-based deduplication was performed using fastp v.0.23.1 (Chen et al., 2018). Trimmed and deduplicated reads were mapped to Homo sapiens GRCh38.p7 with ERCC genes using the STAR aligner v.2.5.2b (Dobin et al., 2013). Single gene hit counts were calculated using 'featureCounts' from Subread v.1.5.2 (Liao et al., 2014).

[0206] Differential expression analysis was performed with R using DESeq2 v1.38.3 (Love et al. 2014). Wald's test was used to generate p-values ​​for log2 times changes. Genes with an adjusted BH p-value < 0.05 and an absolute log2 times change > 1 were considered differentially expressed genes for each comparison of interest (i.e., vehicle + anti-CD33 antibodies compared to vehicle + isotype and LI + anti-CD33 antibodies compared to LI + isotype). Principal component analysis (PCA) was calculated using the DESeq2 'plotPCA' function on variance-stabilized counts (DESEq2 'varianceStabilizingTransformation' function) and using the 25% most variable genes. The volcano and PCA plots were plotted using the R library ggplot2 v.3.4.3 (cran.r-project.org / web / packages / ggplot2 / index.html). Pathway analysis: The R package msigdbr v.7.5.1 (Dolgalev, 2022) was used to retrieve gene sets from the Molecular Signature Database. Petition 870250089689, dated 02 / 10 / 2025, pp. 118 / 221 105 / 144 MSigDb (Broad Institute, www.gsea-msigdb.org), including the curated HALLMARK collection and REACTOME pathways. Gene set enrichment analysis (Subramanian et al. 2005) was performed with fgsea v1.24.0 (Korotkevich, 2019) for each comparison of interest, using the corresponding gene list classified using Wald statistics. Gene sets / pathways with a -log10 (adjusted p-value) > 10 were considered enriched under one condition compared to another. The directionality of change was assessed using the normalized enrichment score (NES). Plots were generated using the 'plotEnrichment' function from fgsea, ggplot2, and ggpattern v.1.1.0-0 (cran.rproject.org / web / packages / ggpattern / readme / README.html). Establishment of the Quad-culture system

[0207] Glutamatergic neurons derived from induced pluripotent stem cells (iPSCs) (Fujifilm; Catalog No: R1061), GABAergic neurons (Fujifilm; Catalog No: R1013), astrocytes (Fujifilm; Catalog No: R1092), and microglia (Fujifilm; Catalog No: R1131) were used to establish a quad-culture system to recapitulate the physiological conditions of the central nervous system. These cells were cultured under standard conditions until they reached the appropriate confluence for experimentation. On the sixth day after establishing the quad-culture system, the cells were pre-treated with 10 ng / mL of lipopolysaccharide (LPS, Sigma Aldrich, Catalog No: L2637-10Mg) and 20 ng / mL of interferon-gamma (IFNy, PeproTech, Inc., Catalog No: 300-02-20ug). The following day, the quad-culture system was exposed to the antibody CD33, ATL_5802, or ATL_5338 (isotype control antibody). Each antibody was used at a concentration of 50 μg / mL.The cultures were incubated with these antibodies for an additional 24 hours. On the eighth day of culture, the supernatant was 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) provided by RayBiotech, Inc. (Peachtree Corners, GA). Petition 870250089689, dated 02 / 10 / 2025, pp. 119 / 221 106 / 144 (USA). All ELISA assays were conducted in accordance with the manufacturer's protocols. Retrogenix in vitro binding

[0208] The screening was carried out by Charles River (United Kingdom) using its patented Retrogenix Cell Microarray technology (see, www.criver.com / productsservices / discovery-services / screening-and-profiling-assays / retrogenix-cellmicroarray-technology?region=3696). Example 1 - Convergence Analysis

[0209] Convergent sequence clusters derived from the antibody repertoire of resilient individuals can be used to identify disease-specific antibody sequences. The present inventors sought to identify candidate protective antibodies from individuals resilient to Alzheimer's Disease (AD) in a prospective cohort (European Consortium for the Prevention of Alzheimer's Dementia, ep-ad.org).

[0210] Pathophysiological changes in Alzheimer's disease can be detected in individuals decades before the onset of cognitive symptoms of dementia, i.e., in the preclinical phase of the disease. For example, the concentration of A042 in cerebrospinal fluid (CSF) is reduced in the early progression of the disease compared to healthy controls, while the concentration of phosphotau in CSF is increased. The ‘A / T / N’ classification scheme (Jack et al., 2016) defines convenient binary labels to describe such biomarkers, where “A” refers to the value of a β-amyloid biomarker (amyloid positron emission tomography (PET) or Ap42 in CSF); “T”, the value of a tau biomarker (phosphotau in CSF or tau PET); and “N”, biomarkers of neurodegeneration or neuronal injury ([18F]fluorodeoxyglucose-PET, structural magnetic resonance imaging or total tau in CSF) (Jack et al., 2016). Below 1,000 pg / mL is considered positive for amyloid, and above Petition 870250089689, dated 02 / 10 / 2025, pages 120 / 221 107 / 144 of 27 pg / mL is considered positive for pTau (Amft et al., 2022; Blennow et al., 2019).

[0211] From the total cohort of 2,096 participants, 127 were selected for convergence analysis based on data and sample availability (plasma and PBMC, and pTau and Aβ42 values ​​from CSF and plasma), including 37 AD-resilient individuals, where resilience is defined by A+TLCR biomarkers and no cognitive impairment using the Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-Cog), the Mini-Mental State Examination (MMSE), and the Clinical Dementia Assessment Box Sum Scale (CDR-SOB) (Duff et al., 2008; Balsis et al., 2015). Sequencing of the antibody repertoires of these resilient individuals revealed a convergent variable heavy chain (VH) sequence between two resilient individuals, one of whom also carries a heterozygous Apo E3 / E4 genotype, further predisposing them to AD (Michaelson et al., 2014).Figure 1 shows the number of clonotypes, that is, clusters of BCRs with high sequence similarity, identified in these two resilient individuals, showing that there were 64 shared clonotypes between them. A representative sequence from one of the 64 shared clonotypes was shown to bind to CD33.

[0212] The VH sequence identified in resilient individuals was subsequently paired with a VL using a transformer-based model, comprising an encoder-decoder model trained on a corpus of paired VH-VL sequences. Further details on how such a model can be trained and used are provided in WO 2022 / 223451 (the full text of which is incorporated herein by reference). The trained model receives a VH sequence as input and generates a single complementary VL sequence as output. The resulting antibody, ATL_5082 (also referred to in this report as ATL_0005082), was included in an ELISA screening for binding to a panel of targets. Petition 870250089689, dated 02 / 10 / 2025, pages 121 / 221 108 / 144 linked to neurodegeneration. The panel consisted of 14 targets, including biotinylated peptide BA-1-14, biotinylated peptide BA-1-42, Tau-352, Tau-441, NF-L (neurofilament light chain), Trem2, Galectin-3, CD33 (Siglec 3), ApoE4, LRP8 (ApoE4 Receptor), HTT exon 1 48Q (mutant HTT exon 1), transthyretin, baculovirus particles, and lysozyme. The antibody was shown to bind to CD33 (Figure 2). This indicated a link between CD33 reactivity and AD resilience in Alzheimer's Disease (AD) resilient individuals in the EP-AD cohort.

[0213] Interestingly, serum and plasma autoreactivity to CD33 (defined as a z-score cutoff point of 0.5 on serum ELISA) was observed in 21 individuals (25 samples), including a “supercentenarian,” that is, an individual over 100 years old with self-reported good cognitive function (score of at least 28 out of 30 on a mini-mental state examination, cognitively healthy individuals (Holstege et al., 2018)) (Figure 3).

[0214] Furthermore, many cancers are characterized by high levels of CD33 expression, particularly myeloid neoplasms such as acute myeloid leukemia and lymphoma. In addition, it has been shown that the expression of CD33-related siglecs in tumor-associated macrophages favors cancer progression and that inhibitory siglecs can inhibit the activation of immune cells, thus suggesting the targeting of siglecs as a possible therapeutic strategy for inhibiting the immune checkpoint (Stanczak et al., 2022). Therefore, the antibodies identified and described in this report may be useful not only in the treatment and / or prevention of neurodegenerative diseases, but also in cancer. Example 2 - deep mining by displaying phages from repertoires of individuals reactive to CD33

[0215] Based on the results of Example 1, the inventors set out to screen serum and plasma libraries of “supercentenarians” to identify Petition 870250089689, dated 02 / 10 / 2025, pages 122 / 221 109 / 144 possible protective anti-CD33 antibodies. An ELISA against CD33 was performed using plasma from a cohort of “supercentenarians” (www.100plus.nl), a subset of the samples in Figure 3. All twelve available plasma samples from the cohort were analyzed, and the sample with the highest ELISA signal for CD33 (Figure 4A) and the lowest signal for the Lysozyme control antigen (Figure 4B) was chosen for phage library generation (Individual SU_0000877 in Figure 4).

[0216] A phage library of scFv molecules displayed on an M13 phage was generated by cloning the variable region (VH) heavy chain repertoire of individual SU_0000877 into a phagemid vector sub-library with variable region (VL) light chain sequences from healthy donors. A phage display library with a size of 1.4 x 108 clones was generated and phages were produced for the phage display selections.

[0217] To isolate antibodies that bind to the CD33 protein, two to three rounds of panning selection of phages displaying the CD33 protein were performed. More than 100 clones were analyzed by Sanger sequencing and complete sequences were selected for phage ELISA.

[0218] Complete clones derived from phage display selections were subsequently analyzed by phage ELISA using CD33 antigen and lysozyme control (Figure 5). Nine clones with a positive ELISA signal and unique sequence were selected for IgG conversion and further testing.

[0219] The nine antibodies derived from phage display panning selections were selected for IgG conversion and produced as LALA-mutated IgG1 variants of the Fc region, comprising L234A / L235A substitutions, reducing binding to Fc receptors of IgG FcyRI, FcyRII, and FcyRIII, as well as to the complement component C1q, thus reducing Fc-mediated toxicity (Lund et al., 1991). Subsequently, these, along with the antibodies Petition 870250089689, dated 02 / 10 / 2025, pages 123 / 221 110 / 144 comparative antigens ATL_5909 (also known as “AL003” from Alector), ATL_4828 (also known as Gemtuzumab); and ATL_5503 (a precursor of AL003), were tested by single-point ELISA against a panel of antigens comprising CD33 and related proteins (other sialic acid-binding immunoglobulin-like lectins, siglecs): recombinant human CD33, Cynologous / Rhesus CD33, recombinant human CD33 C domain, mouse CD33, recombinant human Fc siglec-6 / CD327 chimera, recombinant human Fc siglec-7 / CD328 chimera, recombinant human Fc siglec-8 chimera, recombinant human Fc siglec-9 chimera. After the single-point ELISA, for each antibody-antigen pair for which a positive signal was detected, the titration ELISA was performed to obtain the EC50 value for CD33 binding (see Table 1).

[0220] The nine antibodies tested (see data for ATL_5802; 5810; 5803; 5808; 5854; 5807; 5809; 5853 in Table 2A) were found to bind to the complete recombinant human CD33 (rhCD33-His), but not to the C2 domain (see also Figure 6, where the single-point ELISA signal is shown normalized to the control isotype = 100 and absorbance was measured at 450 nm). The antibodies also showed minimal species cross-reactivity in mice (mCD33-His) and cynomolgus monkey (cCD33-His). Importantly, while the tested antibodies showed good binding to CD33, they did not bind to related proteins of the Siglec family (Siglec-6-F; Siglec-7-F; Siglec-8-F; Siglec-9F), indicating selectivity for CD33. Kinetic data are presented in Table 2C.These data demonstrate that, although the comparative antibodies exhibit a higher affinity for CD33, this is mainly due to a lower Koff (for example, the activation rate for ATL5909 versus ATL5802 is approximately twice as high, but the deactivation rate is two orders of magnitude lower). This indicates that the antibodies in this publication dissociate more rapidly. Petition 870250089689, dated 02 / 10 / 2025, pp. 124 / 221 111 / 144 of the target, which may be behind its lower rate of peripheral degradation (see Example 3 below). As demonstrated in Examples 4 and 5, the antibodies currently described exhibit very significant effects on phagocytosis (even greater than the comparative antibodies), indicating a complex relationship between the antibody's mode of action and the kinetics of engagement with the target. Anticorpo rhCD33-His EC50 (μg / ml) ligação ao CD33-His rh hCD33(domínio C)His mCD33-His cCD33-His Siglec-6-Fc Siglec-7-Fc Siglec-8-Fc Siglec-9-Fc Lisozima ATL_0005802 4536 0,21 100 91 5 137 52 63 62 71 ATL_0005810 3561 2,57 144 104 6 154 98 125 166 113 ATL_0005803 3363 0,41 104 90 30 191 85 103 124 67 ATL_0005808 4229 3,15 104 90 10 200 114 171 244 86 ATL_0005854 3595 10,74 106 104 50 98 91 100 71 110 ATL_0005807 3852 3,00 105 95 6 181 90 109 161 97 ATL_0005809 3620 3,01 100 92 6 150 59 73 97 67 ATL_0005853 4116 2,31 101 104 35 94 89 97 67 114 ATL_0005909 3905 0,17 102 96 52 83 81 87 83 99 ATL_0004828 4958 0,07 102 97 9 131 53 106 64 81 ATL_0005503 4651 0,14 140 95 8 128 51 70 54 76 Table 2A shows the binding signal, measured by ELISA, of each antibody for binding to recombinant human CD33 (rhCD33-His); recombinant human 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). The data shown are raw absorbance values ​​measured at 450 nm, normalized to a control. Petition 870250089689, dated 02 / 10 / 2025, pp. 125 / 221 112 / 144 isotype defined at value 100. ATL_0005909 is a comparator antibody (Alector AL003). ATL_0004828 is a comparator antibody (Gemtuzumab). ATL_0005503 is a comparator antibody (2F5 from Alector, a murine precursor of AL003 described in US 11136390B2).

[0221] In conclusion, deep mining by displaying phages from repertoires of CD33-reactive individuals resulted in a panel of 8 selective CD33-binding antibodies that bind fully to CD33 but not to the C2 domain.

[0222] Among these 8 antibodies, two were selected for further investigation based on CDR3H diversity, strong binding, epitope mapping (see Example 8) and different internalization behaviors (see Example 3), including antibody ATL5802 and antibody ATL5810. These were then investigated in more detail in Examples 3 to 7.

[0223] As explained in Example 8, epitope mapping was performed for these antibodies and comparator antibodies. This revealed that 7 of these 8 antibodies (5810, 5853, 5807, 5809, 5802, 5854, and 5805, see Sequence Table 2B) showed similar binding profiles, indicating similar epitopes, and these respective binding profiles were different from those of comparator antibodies 4828 and 5909, and antibody 5803. Therefore, antibody 5803 was not investigated in further detail.______________________________________________ Anticorpo HCR3 LCDR3 ATL_0005909* CARSDVDYFDYW (SEQ ID NO: 237) CQHSWEIPLTF (SEQ ID NO: 247) ATL_0005802 CARPRGFGEYYFDYW (SEQ ID NO: 238) CQSYDSSLSGDVF(SEQ ID NO: 248) ATL_0005810 CARDWHNTPQFDTDYYYYGMDVW (SEQ ID NO: 239) CLLYMGSGIWMF (SEQ ID NO: 249) ATL_0005808 CARGQGGGAGAFDIW (SEQ ID NO: 240) CSSYTSSSTLEVF(SEQ ID NO: 250) ATL_0005854 CATWGGSNWFVDW (SEQ ID NO: 241) CSSYTNSSTLEVF(SEQ ID NO: 251) ATL_0005807 CARQGAGPGGFDIW (SEQ ID NO: 242) CQSPDSSGTYPVF (SEQ ID NO: 252) Petition: 870250089689, on 10 / 02 / 2025, page. 126 / 221 113 / 144 ATL_0005809 CARHRADAPSDAFDIW (SEQ ID NO: 243) CNSRDSSGYHLGLF (SEQ ID NO: 253) ATL_0005853 CARHSGGLDGYTAAALDYW (SEQ ID NO: 244) CMQALQTPITF (SEQ ID NO: 254) ATL_0004828* CVNGNPWLAYW (SEQ ID NO: 245) CQQTKEVPWSF (SEQ ID NO: 255) ATL_0005503* CARSTVDYFDYW (SEQ ID NO: 246) CQHSWEIPLTF (SEQ ID NO: 247) Table 1B shows the CDR3-H and CDR3-L sequences of eight CD33 antibodies identified by phage display, as well as comparative antibodies ATL_0005909, ATL_0004828; ATL_0005503 (indicated by *). Antibody KD M (Octet / BLI) R&D rhCD33-his Kon 1 / Ms (Octet / BLI) R&D rhCD33-his Koff 1 / s (Octet / BLI) R&D rhCD33-his ATL_0005909* 1.11E-10 7.96E+05 8.86E-05 ATL_0005802 2.42E-08 3.24E+05 7.83E-03 ATL_0005810 3.18E-08 2.15E+05 6.82E-03 ATL_0005803 2.41E-08 1.28E+05 3.09E-03 ATL_0005808 6.05E-08 1.85E+05 1.12E-02 ATL_0005854 1.62E-07 1.74E+05 2.82E-02 ATL_0005807 1.25E-07 1.19E+05 1.48E-02 ATL_0005809 3.79E-07 4.53E+04 1.72E-02 ATL_0005853 3.49E-08 1.84E+05 6.43E-03 ATL_0004828* 4.20E-10 4.99E+05 2.10E-04 ATL_0005503* 1.89E-10 7.12E+05 1.34E-04 Table 2C shows kinetic data for anti-CD33 antibody sequences identified by phage display, as well as comparative antibodies (*). Example 3 - Cellular connection and internalization

[0224] CD33 is known to be expressed at high levels on the plasma membrane of peripheral blood cells, such as monocytes. In addition to binding to recombinant CD33, as demonstrated in Example 2, it was desirable to select an antibody against CD33 that would bind to the expressed CD33. Petition 870250089689, dated 02 / 10 / 2025, pp. 127 / 221 114 / 144 endogenously, but resulted in minimal internalization of the antibody-CD33 complex by peripheral blood cells and, therefore, avoided accelerated clearance of the administered antibodies through this peripheral clearance mechanism. Therefore, the internalization of ATL5802 and ATL5810 by monocytes was tested.

[0225] Figure 7A (see also Table 3) shows CD33 depletion in human monocytes 5 hours after the addition of ATL_5802 or ATL5810 compared to the comparator antibodies ATL5909 and ATL4828. ATL_5802 showed minimal CD33 depletion and therefore minimal internalization in monocytes, similar to the isotype control antibody. ATL_5810 showed some CD33 depletion, but less than the prior technique antibodies ATL5909 or ATL4828. Figures 7B and 7C show the depletion levels of the V domain and C domain of CD33 (respectively for Figures 7B and 7C) in human CD14+CD16+ or CD14+CD16- cells (lower and upper panels, respectively, in each figure) after the addition of the specified antibodies at a concentration ranging from 1 nM to 40 nM. Figures 7D and 7E show the area under the curves of Figures 7B and 7C for CD14+CD16- monocytes (see also Table 3 below) and CD14+CD16+ monocytes, respectively.Notably, all tested antibodies from the disclosure showed lower CD33 internalization than the comparator antibody 5909. Antibody ATL_5802 shows very little CD33 internalization (see Figure 7A), and antibody ATL_5854 shows moderate CD33 internalization (see Figure 7B). It is worth highlighting that, since all test antibodies bind to the V domain, changes in the V domain MFI can be attributed to either competition with the detection antibody or CD33 depletion. In contrast, the assay using MFI detection in the C domain will be a faithful representation of CD33 depletion. Separate data are shown for classical monocytes (CD14+CD16-) and... Petition 870250089689, dated 02 / 10 / 2025, pp. 128 / 221 115 / 144 non-classical / intermediate monocytes (CD14+CD16+) to show that the CD33 antibody depletion behavior is similar in both monocyte subsets. Antibody Change in CHO-CD33 cell binding relative to the isotype mean n = 3 Remaining CD33 monocytes, AUC 5 hours, CD14+CD16-, normalized to isotype ATL 0005909* ND 8.7 ATL 0005802 8.6 100.0 ATL 0005810 20.4 38.8 ATL 0005808 10.7 23.8 ATL 0005854 8.7 56.6 ATL 0005807 4.2 40.6 ATL 0005809 3.2 87.7 ATL 0005853 2.3 72.6 ATL 0004828* 4.0 ND ATL 0005503* 4.1 ND Table 3. Results of flow cytometry experiments measuring cell binding and internalization. * denotes comparative antibodies.

[0226] The comparator antibody ATL5909 has a non-linear pharmacokinetic profile (Ward et al., 2021), likely caused by internalization into peripheral CD33-positive monocytes. In other words, ATL5909 rapidly internalizes in complex with CD33 and depletes CD33 on the cell surface. While this may be desirable at the site of disease (e.g., brain tissue), this property causes antibody deposition in the periphery, where the antibody is internalized and degraded within monocytes. The fact that no CD33 depletion or reduced depletion was observed in the periphery with ATL5802 and ATL5810 (and all other antibodies tested in the disclosure) is an indication that these antibodies are not internalized by peripheral blood cells to the same extent as ATL5909 and, therefore, an improved pharmacokinetic profile compared to ATL5909 can be expected, as well as greater antibody availability in desired target areas, such as the brain. Petition 870250089689, dated 02 / 10 / 2025, pp. 129 / 221 116 / 144 Example 4 - Phagocytosis assay

[0227] Microglia are innate immune cells residing in the brain, located in the central nervous system, essential for CNS health. Phagocytosis of toxic proteins, such as β-amyloid, is an important function of microglia. The phagocytosis pathway is suppressed by CD33 signaling, and notably, CD33 is upregulated in the microglia of AD patients and is associated with cognitive decline. Therefore, it was interesting to test whether the identified anti-CD33 antibodies are capable of alleviating this CD33-mediated suppression of phagocytosis.

[0228] To investigate the effect of the anti-CD33 antibodies ATL5802 and 5810, an in vitro assay using microglia derived from induced pluripotent stem cells (iPSCs) was developed to measure beta-amyloid phagocytosis (see methods). No effect of the anti-CD33 antibodies was observed in iPSC microglia in a resting basal state (data not shown). However, this was not considered representative of microglia in vivo, and especially of microglia in a neurodegenerative context. Microglia derived from induced pluripotent stem cells (iPSCs) were therefore first stimulated with LPS to induce an inflammatory response representative of the in vivo pathological state of these cells in neurodegeneration. This step was followed by incubation with the anti-CD33 antibody panel.The cells were subsequently treated with a pH-sensitive pHrodo red labeled β-amyloid, and phagocytosis was measured by monitoring the red fluorescence signal in each well, with increased intracellular levels of red dye compared to the isotype control indicating increased phagocytosis (Figure 8A).

[0229] Figure 8B shows the results of the assay with the antiCD33 antibodies ATL5802 and ATL5810. The graph in Figure 8B shows that incubation with ATL5802 results in increased phagocytosis in iPSC microglia compared to the ATL5909 antibody from the previous technique and an isotype control. The results in Petition 870250089689, dated 02 / 10 / 2025, pp. 130 / 221 117 / 144 Figure 8C confirms these results in a repeated experiment for ATL_5802, indicating a replicable increase in phagocytosis in iPSC inflammatory microglia in the presence of the disclosure antibody (ATL_5802), but not the comparator antibody ATL_5909.

[0230] Next, the experiment was repeated with three other anti-CD33 antibodies, ATL_5853; ATL_5854; and ATL_6044 (ATL5802 sprouted, see Example 7 below). The results of this experiment are shown in Figure 8D. The graph in Figure 8D shows that all tested antibodies from the disclosure, namely antibodies ATL_5802, ATL_5854, and ATL_5853, increase phagocytosis compared to antibody ATL_5909 from the previous technique or an isotype control antibody.

[0231] Figures 8E and F further show that ATL_5802 induces a greater extent of increased phagocytosis compared to TREM2 agonist antibodies currently in clinical trials for the treatment of AD (ATL6166 = Alector AL002; ATL6167 = Denali DNL919; ATL6170 = Vigil VGL101). TREM2 is a microglia activating signaling receptor and assists in the survival of microglia cells by promoting phagocytosis of Aβ plaques (McQuade et al., 2020).

[0232] To test the effect of anti-CD33 antibodies on phagocytic activity in human peripheral myeloid cells, which are known to express high levels of CD33 (e.g., in monocytes), the phagocytosis assay described above was performed ex vivo using human peripheral blood mononuclear cells (PBMCs) from healthy donors. The ex vivo myeloid assay uses pH-dependent dye-labeled S. aureus (pHrodo) to measure the level of phagocytosis in myeloid cells (CD14+) by flow cytometry (Figure 9A).

[0233] Figures 9B and 9C show that ATL5802 significantly increases phagocytosis in human myeloid cells compared to the comparator antibodies ATL5909 (Alector) and ATL4828 (Gemtuzumab). The results for the Petition 870250089689, dated 02 / 10 / 2025, pp. 131 / 221 118 / 144 ATL5810 indicated that the antibody likely induced phagocytosis, but the results did not reach significance (compared to the isotype control) due to high variability in the cell assay.

[0234] In conclusion, these data demonstrate that disclosure antibodies likely enhance phagocytosis in a variety of human cells expressing CD33, including PBMCs and microglia derived from inflammatory iPSCs (as demonstrated, in particular, by antibodies ATL5802, ATL_5853, ATL_5854, and ATL_5810). The data further demonstrate that disclosure antibodies (as demonstrated, in particular, by antibodies ATL5802, ATL_5853, and ATL_5854) enhance phagocytosis in microglia derived from inflammatory human iPSCs in vitro, as well as in human myeloid cells ex vivo. Example 5 - In vivo antibody test in humanized CD34+ mice

[0235] The pharmacodynamic properties of ATL5802 and ATL5810 were tested in vivo in a murine CD34+ NSG™ model, a humanized murine model modified by the addition of human hematopoietic stem cells to produce a humanized immune system (comprising a mixture of murine and human monocytes) that can be targeted by anti-CD33 antibodies. Testing these antibodies in a humanized murine model was necessary because there are substantial differences between murine and human species in CD33 expression patterns and ligand recognition, due to evolutionary divergence between human CD33 and non-primate CD33, and the biology of mCD33 is therefore not functionally relevant to the biology of hCD33 (Brinkman-Van der Linden et al., 2003; Cao et al., 2009). In any case, the anti-CD33 antibodies described in this report do not show cross-reactivity between species with murine CD33 and, therefore, in vivo testing is only possible in humanized animal models. Petition 870250089689, dated 02 / 10 / 2025, pp. 132 / 221 119 / 144

[0236] First, CD33 levels in humanized myeloid cells were tested to assess in vivo CD33 internalization (Figure 10). Figure 10 shows that injection of ATL5802 and ATL5810 results in minimal CD33 depletion in myeloid cells 24 hours after injection, compared to the ATL5909 antibody from the previous technique. Figure 10 shows the mean fluorescence intensity (MFI) of CD45+CD14+CD33+ cells by flow cytometry. This indicates minimal peripheral drug loss from ATL5802 compared to ATL5909.

[0237] Figure 12 shows that there is a dose-dependent decrease in CD33 expression levels in human CD45+ cells in the brain 24 hours after ip injection of ATL5802, supporting the targeted engagement of ATL5802 in the brain.

[0238] Furthermore, Figure 13 demonstrates that intraperitoneal injection of ATL5802 in CD34+ mice results in increased phagocytosis of pHrodo-labeled S. aureus by human peripheral myeloid cells (CD45+CD14+CD11b+ cells) compared to ATL5909 injection, and this effect appears to be dose-dependent. The relatively large variability is due to the small number of human cells in the experiment. However, the trends observed in vivo confirm the in vitro data shown above.

[0239] Taken together, these data demonstrate that ATL5802 exhibits minimal peripheral internalization in humanized CD34+ mice, in addition to exhibiting target involvement in the brain and increased phagocytosis. Therefore, ATL5802 was further characterized in terms of antibody expression and development. Example 6 - Antibody expression and development

[0240] To ensure that the identified antibodies are stable and therefore suitable for further optimization, a development study was carried out. Petition 870250089689, dated 02 / 10 / 2025, pp. 133 / 221 120 / 144

[0241] Baseline values ​​were calculated for all 10 antibodies selected in Example 2, which demonstrated high monomer levels and good thermostability and cIEF values ​​(Table 4).

[0242] Protein aggregation during antibody storage should be kept to a minimum, as it can cause immunogenic reactions. Size exclusion chromatography (SEC-HPLC) was used to evaluate the purity and aggregation of antibodies after storage under a variety of conditions, including 2 weeks at -80 °C, 2 weeks at 40 °C, overnight shaking, 3 freeze-thaw cycles (3x FT); and low pH maintenance (Figure 14). Figure 14 shows that ATL5802 shows very little change in the monomer profile under these forced degradation conditions, indicating excellent stability, and this is further reflected by the 99% monomer value in Table 4. Similar results were obtained for the other candidate antibodies identified in Example 2, all of which showed acceptable stability in terms of % monomer, and 5802 additionally showed particularly good thermostability (Tm1)._____________________________________ Antibody Thermostability - Tm1 Thermostability - Tagg cIEF - pl of the main species Purity: SECHPLC % monomer ATL_0005802 67.5 59.4 7.76 99 ATL_0004828 66.9 71.4 8.67 100 ATL_0005503 62.8 74.4 7.9 90 ATL_0005909 65.0 71.1 7.66 99 ATL_0005810 59.2 56.8 8.21 98 ATL_0005808 67.1 67.7 8.72 98 ATL_0005854 59.6 58.8 8.77 97 ATL_0005807 65.2 64.4 8.48 99 ATL_0005809 57.7 56.3 6.83 96 ATL_0005853 61.9 60 7.97 98 Petition 870250089689, dated 02 / 10 / 2025, pp. 134 / 221 121 / 144 Table 4 shows the results of a thermostability test (melting temperature (Tm1) / aggregation (Tagg); cIEF SEC-HPLC (% monomer).

[0243] Furthermore, the target binding of ATL5802 was tested after storage under forced degradation conditions using ELISA (Figure 15). ATL5802 showed consistent binding to CD33 under all conditions, indicating that binding strength is not affected by temperature changes and freeze-thaw cycles.

[0244] In conclusion, all antibodies, but in particular ATL5802, showed excellent stability and therefore ATL5802 was retained for further development. Example 7 - Germline in structural regions

[0245] The main antibody ATL5802 has 8 structural mutations in the corresponding germline sequence: 6 located in the VH region (positions 1, 6, 20, 40, 72, and 85 of IMGT) and 2 in the VL region (positions 2 and 8 of IMGT). To test which of these can be reverted to the germline sequence without loss of binding, 9 variants of the original antibody were produced and tested for CD33 binding by ELISA (Table 5). The following variants were tested: ATL6040, which had all mutations reverted to the germline sequence, and ATL6041-ATL6048, which had all mutations, except one, reverted. The variants were produced as IgG1 (LALA Fc mutation). Germline (IMGT numbering) ELISA EC50 Lysozyme Binding 1 ATL_0005802 Original 2.14e-10 No 2 ATL_0006040 Complete germline (FG; reversals x8) 1.79e-08 No 3 ATL 0006041 FG less VH E1Q 1.93e-08 No 4 ATL 0006042 FG less VH Q6E 3.31e-08 No 5 ATL 0006043 FG less VH K20R 1.97e-08 No 6 ATL 0006044 FG less VH G40A 4.77e-10 No 7 ATL 0006045 FG less VH Q72E 2.01e-08 No 8 ATL 0006046 FG less VH S85G 2.00e-08 No Petition 870250089689, dated 02 / 10 / 2025, pp. 135 / 221 122 / 144 9 ATL 0006047 FG less VL S2A 2.14e-08 No 10 ATL 0006048 FG less VL P8S 2.14e-08 No Table 5 shows the germline mutations introduced into each variant of ATL_5802, as well as their EC50 values ​​obtained by ELISA.

[0246] All antibodies were produced and tested by titration ELISA with CD33 antigen and negative control antigen. Only one antibody, ATL6044, showed comparable binding to the original antibody, suggesting that removing deficiencies or alterations at the G40A position of the structure impacts binding. This demonstrated that the A at position 40 of the VH of ATL5802 contributed positively to antigen binding, but all other positions could be reverted to germline. Note that this does not indicate that strict adherence to any sequence of the structure is necessary, as all antibodies still bind to the target regardless of the germline mutation introduced. Instead, this indicates that maintaining A at position 40 of ATL5802 is particularly advantageous. Example 8 - Epitope Mapping

[0247] Figure 11B shows the results of the epitope mapping process, schematically illustrated in Figure 11A. The binding profile of each antibody to the different CD33 variants was used to group the antibodies into different epitope compartments. Epitope compartment 1 contained ATL4828 (comparative antibody Gemtuzumab) and was characterized by a complete loss of binding to variants P1 and P6+1 and a loss of approximately 50% to variant P4. In contrast, epitope compartment 2 contained ATL5909 (comparative antibody AL003) and was characterized by loss of binding to variants P2 and P2+5, but no effect on any of the other variants. Groups 3 and 4 were highly related groups and contained seven antibodies (5810, 5853, 5807, 5809, 5802, 5854, and 5808), which had in common a loss of Petition 870250089689, dated 02 / 10 / 2025, pp. 136 / 221 123 / 144 binding for variants P2 and P2+5, a 50% loss of binding for P4, and an increase in binding for P1 and P6+1. Group 5 comprised antibody ATL5803 (which was not characterized in more detail in this report). These data suggest that other antibodies with the same epitopes as the variants with confirmed phagocytosis-enhancing activity (ATL5802, ATL5854, and ATL5853) are likely to exhibit therapeutic effects. The data further suggest that the engagement kinetics shown in Example 2, which differs for the disclosure antibodies compared to the comparator antibodies, may be underscored by different epitopes, potentially corroborating enhanced therapeutic effects for these antibodies by reducing peripheral depletion.

[0248] Finally, epitope mapping data also corroborate a different mode of action of the antibodies in the present disclosure compared to the antibodies in the prior art, being even more consistent with the reduced internalization behavior observed in Example 3 compared to the comparator antibodies. In particular, epitope mapping data indicate that the antibodies described in this report may interfere with the binding of sialic acid to CD33.

[0249] Taken together, these data demonstrate that the anti-CD33 antibodies described in this study, in particular antibodies ATL5802, ATL5854 and ATL5853, likely have therapeutic effects in the treatment and / or prevention of neurodegenerative diseases, such as AD, as well as cancers that express CD33, such as myeloid leukemia. This was subsequently validated through extensive in vitro and in vivo studies, described in the following examples.

[0250] Figure 16 summarizes the antibody sequences described in this report, aligned using the IMGT antibody numbering convention. The sequences shown in Figure 16 are also summarized in Table 5 (VH) and in Petition 870250089689, dated 02 / 10 / 2025, pp. 137 / 221 124 / 144 Table 6 (VL). In Tables 5 and 6, the antibodies from the disclosure are in bold. “Complete” refers to the complete sequence of VH (Table 5) or VL (Table 6). Reference Notes FW R1 CD R1 FW R2 CD R2 FW R3 CD R3 FW R4 Complete ATL_0004 828 Gemtuzumab 67 41 80 49 88 56 101 1 ATL_0005 338 Isotype control 68 42 81 50 89 57 102 2 ATL_0005 503 2F5, murine precursor AL003 69 43 82 51 90 58 103 3 ATL_0005 909 AL003, described as sequence AB-64.1.2 in US2022 / 016 2309 43 87 55 98 66 106 11 ATL_0005 802 Lead 70 44 83 52 91 59 101 4 ATL_0005 807 Backup 71 44 84 52 92 60 104 5 ATL_0005 808 Backup 72 44 84 52 93 61 104 6 ATL_0005 809 Backup 73 45 85 53 94 62 104 7 ATL_0005 810 Backup 74 46 86 54 95 63 105 8 ATL_0005 853 Lead 235 47 83 52 96 64 101 9 ATL_0005 854 Lead 75 48 83 52 97 65 101 10 ATL_0006 040 Complete germline (fg; reversals x8) 77 44 84 52 93 59 101 12 ATL_0006 041 FG minus VH E1Q 71 44 84 52 93 59 101 13 ATL_0006 042 FG minus VH Q6E 78 44 84 52 93 59 101 14 ATL_0006 043 FG minus VH K20R 79 44 84 52 93 59 101 15 ATL 0006 FG minus 77 44 83 52 93 59 101 16 Petition 870250089689, dated 02 / 10 / 2025, pp. 138 / 221 125 / 144 044 VH G40A ATL 045 _0006 FG less VH Q72E 77 44 84 52 99 59 101 17 ATL 046 _0006 FG less VH S85G 77 44 84 52 100 59 101 18 ATL 047 _0006 FG less VL S2A 77 44 84 52 93 59 101 19 ATL 048 _0006 FG less VL P8S 77 44 84 52 93 59 101 20 Table 5. Summary of VH sequences. All numbers are SEQ ID Nos referring to Table 0. Reference Notes FW R1 CD R1 FW R2 CD R2 FW R3 CD R3 FW R4 Complete ATL_0004 828 Gemtuzumab 135 107 148 116 157 126 168 21 ATL_0005 338 Isotype control 136 108 149 117 158 127 170 22 ATL_0005 503 2F5, murine precursor AL003 137 109 150 118 159 128 171 23 ATL_0005 909 AL003, described as sequence AB-64.1.2 in US2022 / 016 2309 144 109 150 118 167 128 169 31 ATL_0005 802 Lead 138 110 151 119 160 129 172 24 ATL_0005 807 Backup 139 111 152 121 161 130 173 25 ATL_0005 808 Backup 140 112 153 122 162 131 173 26 ATL_0005 809 Backup 141 113 154 123 163 132 172 27 ATL_0005 810 Backup 142 114 155 124 164 133 172 28 ATL_0005 853 Lead 143 115 156 125 165 134 174 29 ATL_0005 854 Lead 140 112 153 120 166 236 173 30 ATL 0006 Lineage 145 110 151 119 160 129 172 32 Petition 870250089689, dated 02 / 10 / 2025, pp. 139 / 221 126 / 144 040 Complete germination (FG; reversals x8) ATL_0006 041 FG less VH E1Q 145 110 151 119 160 129 172 33 ATL_0006 042 FG less VH Q6E 145 110 151 119 160 129 172 34 ATL_0006 043 FG less VH K20R 145 110 151 119 160 129 172 35 ATL_0006 044 FG less VH G40A 145 110 151 119 160 129 172 36 ATL_0006 045 FG less VH Q72E 145 110 151 119 160 129 172 37 ATL_0006 046 FG minus VH S85G 145 110 151 119 160 129 172 38 ATL_0006 047 FG minus VL S2A 146 110 151 119 160 129 172 39 ATL_0006 048 FG minus VL P8S 147 110 151 119 160 129 172 40 Table 6. Summary of VL sequences. All numbers are SEQ ID Nos referring to Table 0. Example 9 - Study of pK in mice

[0251] The antibodies described in this report are specific for human CD33 and do not exhibit significant cross-reactivity with murine CD33. Therefore, to evaluate the in vivo pharmacokinetics (PK) of the ATL_5802 antibody, a transgenic murine strain was used in which exons 1 to 3 of the murine CD33 gene, which encode the extracellular domain, were replaced by exons 1 to 3 of human CD33. The expression of human CD33 has been previously validated and characterized in this murine model. The antibodies were administered intraperitoneally at 1 mg / kg and serum levels of human IgG1 were evaluated at different times after a single administration of human IgG1 antibodies, ATL_5802 or ATL_5909.

[0252] The results of this pK study are shown in Figure 17. Figure 17A shows the serum levels of ATL_5802 and ATL_5909 at 4 hours, 24 hours Petition 870250089689, dated 02 / 10 / 2025, pp. 140 / 221 127 / 144 and 144 hours (7 days). Figure 17B shows the quantification of total antibody concentration over time (measured by the area under the curve; AUC) and reveals significantly increased serum levels of ATL_5802 compared to ATL-5909.

[0253] These data demonstrate superior peripheral exposure of ATL_5802 compared to ATL_5909, suggesting that the peripheral clearance of ATL_5802 is lower than that of the comparator antibody.

[0254] Together, these data demonstrate that the anti-CD33 antibodies described in this report exhibit excellent pharmacokinetic properties, further reinforcing their potential use in therapy. Example 10 - Alzheimer's disease model

[0255] To test whether anti-CD33 antibodies are capable of attenuating Alzheimer's disease pathology, the effect of ATL_5802 was tested in a murine xenotransplantation model of Alzheimer's disease. In this report, mice with amyloid precursor protein (APP) carrying human NLGF mutations (named APPNL'GF mice, Saito et al., 2014) were xenotransplanted with human microglial progenitors derived from iPSCs, as described in Fattorelli et al., 2021, Mancuso et al., 2019, and Mancuso et al., 2022. In this report, the transplanted cells mature to transcriptionally resemble human primary microglia ex vivo, and after three months, the mice show signs of Alzheimer's disease pathology. Thus, this murine model can be used to investigate human microglia biology in the context of Alzheimer's disease pathology.

[0256] Starting at 4 months of age, mice were treated with 40 mg / kg of ATL_5802 or isotype control antibody (ATL_5338) intraperitoneally (IP) once a week for three months. Subsequently, an intraperitoneal injection of Methoxy-X04, a fluorescent dye that crosses the blood-brain barrier and binds to Aβ, was performed. Later, the microglia Petition 870250089689, dated 02 / 10 / 2025, pp. 141 / 221 128 / 144 was isolated by flow cytometry (the cells were separated by FACs and subjected to hCD45+ activation) and its phagocytic capacity was evaluated by quantifying the percentage of human microglia positive for Methoxy-X04.

[0257] The results of this experiment are shown in Figure 18. Mice treated with ATL_5802 showed a significant increase in the percentage of Methoxy-X04+ microglia compared to control mice treated with the isotype, as measured by flow cytometry. These results indicate that ATL_5802 increases the in vivo phagocytosis of β-amyloid by human microglia in AppNL-GF mice xenotransplanted with iPSC-derived human microglia progenitors.

[0258] Together, these data demonstrate that ATL_5802 alleviates AD pathology in vivo, at least by increasing the clearance of neurotoxic Aβ via microglial phagocytosis. Example 11 - Phagocytosis of Tau by microglia

[0259] The aggregation of pathological (mutant) tau protein into abnormal filaments, or so-called neurofibrillary tangles, is a feature of multiple neurodegenerative conditions. In humans and transgenic mice, P301 mutations (e.g., P301S and P301L) in the tau protein lead to tau protein pathology with dissemination of aggregated tau protein (Strang et al., 2018). Effective elimination of these aggregates from neurons is therefore of great importance for preventing and / or reducing tau protein pathology. As described in Example 4, microglia are essential for the elimination of toxic extracellular proteins and for maintaining cellular health in the central nervous system.

[0260] To test whether the antibodies described in this report are capable of reducing the spread of mutant tau aggregates (P301S), the uptake of these aggregates by microglia with an inflammatory phenotype was measured. In short, iPSC Petition 870250089689, dated 02 / 10 / 2025, pp. 142 / 221 129 / 144 microglia pretreated with LPS were incubated with the anti-CD33 antibody ATL5802, the comparative anti-CD33 antibody ATL_5909; the anti-TREM2 antibody ATL_6170; or an isotype control antibody (ATL_5338), and the uptake of pHrodo-labeled mutant Tau aggregates was subsequently measured using live cell imaging.

[0261] The results of this experiment are shown in Figure 19. Figure 19A shows an increase in phagocytosis of labeled tau aggregates, measured as the total red area per well over time, after treatment with the anti-CD33 antibody ATL_5802 or the anti-TREM2 antibody ATL_6170 compared to an isotype control antibody using live cell imaging. In contrast, the comparative anti-CD33 antibody ATL_5909 tended to decrease tau uptake. Figure 19B shows the AUC quantification of the total area per well for each condition.

[0262] Taken together, these results indicate that ATL_5802 increases the phagocytosis of toxic tau aggregates by inflammatory microglia, thus contributing to the effective elimination of these aggregates. This is expected to reduce tau-associated pathology and reduce and / or prevent (subsequently) neurodegeneration. Example 12 - Cytokine release from different cells

[0263] CD33 is expressed in microglia, but also in peripheral myeloid cells, such as monocytes. Modulation of these immune cells can cause systemic release of cytokines, with potentially undesirable consequences and safety concerns.

[0264] To test whether the anti-CD33 antibodies described in this report cause unwanted cytokine release, iPSC microglia, PBMCs, and monocytes isolated from various donors were treated with isotype control antibody or Petition 870250089689, dated 02 / 10 / 2025, pp. 143 / 221 130 / 144 ATL5802 eo cell meio foi analyzed for several cytokines, chemokines and different inflammatory mediators, as set out in Table 7. iPSC Microglia Isolated monocytes Total PBMC CCL2 (MCP-1) NS NS NS NS CXCL8 (IL-8) NS NS NS IFN-γ NS NS NS IL-10 NS NS NS IL-12p40 NS NS NS IL12p70 NS NS NS IL-17A NS NS NS IL-W NS IL-2 IL-23 IL-4 IL-6 TGF-P1 TNF-α CCL17 (TARC) CCL2 (MIP-3α) CCL3 (MIP-1α) CCL4 (MIP-1P) NS NS AND CCL5(RANTES) NS NS NS AND CXCL1 (Gro-α) NS NS NS AND CXCL5 (ENA-78) NS NS NS AND CXCL9 (MIG) NS NS NS AND CCL11 (Eotaxina) NS NS NS AND CX3CL1 (Fractalina) NS NA NA IL-18 NS NA NA sRAGE NS NA NA sTREM-1 NS AND sTREM-2 NS AND VEGF NS AND VILIP-1 NS AND β-NGF NS AND BDNF NS AND CCL2 (MCP-1) NS AND AND Table 7. Cytokine levels were measured in the supernatants of iPSC microglia, monocytes, and PBMCs treated with isotype control antibody or ATL_5802. NS = no significant difference between the antifluorescein isotype control antibody and cells treated with ATL5802 (t-test). NA = Not applicable, as it was not tested for this cell type. Petition 870250089689, dated 02 / 10 / 2025, pp. 144 / 221 131 / 144

[0265] These results indicate that treatment with ATL5802 did not cause the release of immune-mediated cytokines. In particular, ATL_5802 did not cause the release of MCP-1 by untreated microglia after 24 hours of treatment with 100 μg / ml or 10 μg / ml (showing levels slightly below the isotype control), a benefit that was not shared by the comparative anti-CD33 antibody tested.

[0266] It is known that LPS treatment of iPSC microglia leads to the production of the inflammatory cytokines MCP-1 and IL-6. To test whether the anti-CD33 antibody ATL-5802 was able to reduce the levels of these pro-inflammatory cytokines, microglia were treated with LPS, followed by treatment with ATL-5802 or an isotype control antibody. After 24 hours, the medium was collected and cytokine levels were measured using a multiplex immunoassay.

[0267] The results are shown in Figure 20 and indicate that treatment with ATL_5802 significantly reduces LPS-induced IL-6 (Figure 20A) and MCP-1 (Figure 20B) production by iPSC microglia in response to LPS. This suggests that the anti-CD33 antibodies described in this report are capable of exerting an anti-inflammatory effect on human iPSC microglia. This is significantly different from the isotype control antibody and the comparative anti-CD33 antibody ATL_5909.

[0268] MCP-1 recruits immune cells to the site of inflammation and is implicated in the pathogenesis of neuroinflammation and Alzheimer's disease, autoimmune diseases (e.g., arthritis), and atherosclerosis. Therefore, it is very beneficial that the antibodies in this disclosure do not lead to the release of cytokines, including MCP-1, a benefit that was not present in the comparative anti-CD33 antibody ATL_5909. Petition 870250089689, dated 02 / 10 / 2025, pp. 145 / 221 132 / 144

[0269] Taken together, these data show that the anti-CD33 antibodies described in this report do not cause any unwanted cytokine release, suggesting that they are safe for human use and have potent anti-inflammatory effects in vitro. Example 13 - Changes in intracellular signaling in microglia cells after the addition of ATL 5802

[0270] Splenic tyrosine kinase (SYK) is a key intracellular regulator of microglial activation and phagocytosis. It is particularly significant in neurodegenerative diseases, where it assists in the elimination of toxic protein aggregates (Ennerfelt et al., 2022). In this example, the inventors investigated changes in intracellular signaling after CD33 involvement, with a specific focus on the phosphorylation state of Syk (pSYK) under inflammatory conditions.

[0271] Activation of ITAM receptors expressed on microglia cells, such as TREM2, results in the recruitment and phosphorylation of ITAM-containing adaptor molecules, which, in turn, recruit SYK. After activation, SYK phosphorylation results in 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 neutralize ITAM signaling by dephosphorylating ITAM domains, as well as ITAM-associated kinases such as SYK (Huang et al., 2003).

[0272] In this study, the inventors sought to investigate whether the anti-CD33 antibodies described in this report are capable of modulating intracellular signaling after the interaction of CD33 with ITIM and, in particular, whether the antibodies described in this report are capable of alleviating the repression of ITAM receptor signaling mediated by CD33. To test this, the inventors measured the phosphorylation state of Syk (pSYK) under inflammatory conditions. The increase in Petition 870250089689, dated 02 / 10 / 2025, pp. 146 / 221 133 / 144 phosphorylation would indicate increased activation of the ITAM signaling pathway and, therefore, inhibition of the suppressive effect of CD33.

[0273] Figure 21 shows the results of a Western blot of microglia cells treated with LPS and ATL_5802 or an isotype control antibody. This revealed a notable increase in SYK phosphorylation after treatment with ATL_5802, while overall SYK protein levels remained stable. These findings suggest that ATL_5802 modulates signaling pathways, possibly resulting in the derepression of the TREM2 signaling axis and leading to increased TREM2 signaling, as evidenced by the increased pSYK levels. This underscores the potential therapeutic value of targeting these specific pathways.

[0274] Next, the inventors investigated whether the anti-CD33 antibodies described in this report, such as ATL_5802, have an effect on the purinergic receptor P2RY12. P2RY12 is a member of the P2 purinergic receptor family, a G protein-coupled receptor spanning seven transmembrane membranes and responding to ADP / ATP, being associated with chemotaxis and phagocytosis. P2RY12 promotes microglial chemotaxis towards sites characterized by necrotic or apoptotic cells, which is important for maintaining brain health and responses to injury or inflammation (Walker et al., 2020). The P2RY12-mediated chemotactic response is a fundamental aspect of the microglial surveillance mechanism, allowing for early detection and response to brain damage. Activation of P2RY12 increases the phagocytic capacity of microglia, promoting the elimination of apoptotic cells, beta-amyloid plaques, and other debris associated with neurodegenerative diseases.Through its involvement in phagocytosis, P2RY12 contributes to the resolution of inflammation and the prevention of further tissue damage. By modulating the activity of P2RY12, it may be possible to enhance beneficial microglial functions while simultaneously mitigating [inflammation]. Petition 870250089689, dated 02 / 10 / 2025, pp. 147 / 221 134 / 144 reduces harmful inflammation, thus protecting against neurodegeneration and contributing to brain health.

[0275] To test whether ATL_5802 is capable of increasing P2RY12 expression in microglia, a western blot was performed using LPS-stimulated iPSC-induced microglia treated with ATL_5802 or isotype control antibody.

[0276] Figure 22 shows the results of a western blot for P2RY12 in microglia 6 hours after the addition of ATL_5802 or isotype control antibody. ATL_5802 increases the protein expression of the P2RY12 receptor, suggesting that the antibody is able to modulate intracellular signaling pathways in microglia important for responding to injury and inflammation. In particular, the data in Figure 22 show that non-inflamed control conditions exhibit high P2RY12 expression, while inflamed control conditions exhibit low P2RY12 expression. In contrast, after treatment with ATL_5802, non-inflamed conditions show higher P2RY12 expression compared to the isotype control (indicative that the microglia returned to a state of surveillance, characterized by high P2RY12 expression).In this state, microglia are not actively involved in inflammatory responses, but are ready to respond to new damage or threats, and inflamed conditions show higher expression of P2RY12 compared to the isotype control.

[0277] This demonstrates that ATL_5802 has the ability to increase the protein expression of the P2RY12 receptor, which indicates the absence of microglial activation. This effect guides microglia to specific destinations, playing a vital role in maintaining brain health and orchestrating responses to injury or inflammation. This change in signaling dynamics may result in a more effective microglial immune response, a crucial factor in slowing the progression of neurodegenerative diseases. Petition 870250089689, dated 02 / 10 / 2025, pp. 148 / 221 135 / 144

[0278] Taken together, these data indicate that the anti-CD33 antibodies described in this report are capable of activating intracellular signaling pathways in microglia that are important for their effective function, such as phagocytosis, cytokine release, and chemotaxis, while simultaneously inhibiting signaling pathways involved in suppressing microglial activation and function. This alteration in signaling dynamics may contribute to a more effective immune response, a crucial factor in slowing the progression of neurodegenerative diseases. Example 14 - RNAseq in microglia derived from iPSCs

[0279] To understand the transcriptional changes resulting from treatment with anti-CD33 antibodies, both in healthy conditions and in conditions that simulate inflammation, a genome-wide RNA sequencing study of iPSC-derived microglia was performed. Briefly, iPSC-derived microglia were stimulated with vehicle or with LPS / interferon gamma (LI) to simulate 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.

[0280] The results of the analysis after RNA sequencing are shown in Figure 23. The PCA plot shown in Figure 23A indicates that treatment with ATL_5802 leads to greater changes in gene expression than those caused by ATL_5854, ATL_5909, or ATL_5338 (isotype).

[0281] Comparison of the expression profiles of samples treated with any anti-CD33 antibody and the isotype control in LI- or vehicle-stimulated cells shows a number of differentially expressed genes (DEGs) (Figure 23 BC). The highest number of differentially expressed genes was detected in microglia derived from iPSCs treated with vehicle + ATL_5802 compared to those treated with vehicle + isotype control, as shown in Figures 23B and C, indicating an effect of ATL_5802 on healthy cells. A total of 194 Petition 870250089689, dated 02 / 10 / 2025, pp. 149 / 221 136 / 144 genes were upregulated in vehicle + ATL_5802 compared to vehicle + isotype control (see Figure 23D).

[0282] Interestingly, pathway analysis revealed a statistically significant downregulation of several gene sets involved in oxidative phosphorylation (OXPHOS) after treatment with ATL_5802 in inflammatory microglia, compared to samples treated with LI and isotype (Figure 23E to F). This result reveals a differentiating mechanism of action for ATL_0005802, whereby the transcription of components of oxidative phosphorylation and respiratory electron transport is attenuated.

[0283] Oxidative phosphorylation (OXPHOS) in microglia allows for efficient ATP production under homeostatic conditions, while 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 adopt a phagocytic phenotype. Microglia continuously monitor the brain parenchyma to detect neuronal damage and alterations in homeostatic processes, and defects in cellular metabolism involving glycolysis and OXPHOS have been implicated in neurodegenerative diseases such as Alzheimer's disease (Baik et al., 2019). Increased anaerobic glycolytic metabolism has been shown to restore microglial phagocytic activity and improve cognitive impairment in a murine model of AD (Baik et al., 2019).Although these experiments only show a decrease in OXPHOS (but not the corresponding glycolytic increase), CD33 inhibition would inhibit SHIP1 / 2, downregulate PI3K, activate mTOR, and therefore initiate glycolysis, which would be expected to have a beneficial cognitive improvement.

[0284] The result that ATL_5802 reduces the transcription of OXPHOS and components of respiratory electron transport therefore suggests that ATL_5802 may stimulate anaerobic glycolysis and thus attenuate the OXPHOS pathway. With the Petition 870250089689, dated 02 / 10 / 2025, pp. 150 / 221 137 / 144 decrease in OXPHOS, microglia cells subjected to CD33 antagonism by ATL_5802 will have a beneficial reduction in the release of reactive oxygen species, a byproduct of OXPHOS.

[0285] Together, these data suggest that ATL_5802 is capable of modulating cellular metabolic pathways, ultimately leading to improved cognitive function in neurodegenerative diseases. Example 15 - Quad-culture with Cortical Neurons, Astrocytes, and Microglia

[0286] Next, the inventors developed a human neural cell culture called the “CNS quad-culture platform” (Figure 24A). This platform integrates a diverse set of cell types—specifically, glutamatergic and GABAergic neurons, along with microglia and astrocytes—co-cultured to accurately replicate the complex intercellular dynamics of the human brain in a controlled in vitro environment. This in vitro approach allows for a deeper understanding of the interactions between various brain cell types and acts as a critical experimental intermediary for a human-relevant system to assess the efficacy of ATL_5802 on markers of inflammation and astrogliosis. To evaluate this, lipopolysaccharide (LPS) and interferon-gamma (INFy) were introduced into the system to induce an inflammatory response, simulating neuroinflammatory conditions, followed by analysis of inflammation, astrogliosis, glial dysfunction, and microglial activation.

[0287] ATL_5802 significantly reduced LPS / INFy-induced interleukin-6 (IL6) levels compared to the isotype control (Figure 24B). Furthermore, ATL_5802 led to a significant reduction in the astrogliosis marker, GFAP (Figure 24C), further suggesting a general anti-inflammatory effect of ATL_5802.

[0288] 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 elevated levels of MCP-1 in CSF are Petition 870250089689, dated 02 / 10 / 2025, pp. 151 / 221 138 / 144 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 IP-10 (Figure 24D) and MCP-1 (Figure 24E) levels compared to the isotype control.

[0289] These results provide further evidence supporting the therapeutic potential of ATL_5802 in reducing neuroinflammation, thereby protecting against brain atrophy and cognitive impairment in neurodegenerative diseases. Example 16 - Selectivity of ATL 5802

[0290] To evaluate the selectivity of ATL_5802, a screening was performed for binding to fixed HEK293 cells expressing 6105 individual human proteins, as well as another 400 human heterodimers. The screening library includes secreted, plasma membrane, and cell surface-anchored human proteins. Screening was performed with operators blinded to the ATL_5802 target, CD33. The ATL_5802 test antibody showed a single significant specific interaction with CD33 in both fixed and live cell microarrays (see Table 8). These data confirm that ATL_5802 is selective for CD33 and is therefore unlikely to cross-react with other targets. Sample ID Gene ID Unique Clone Number Protein Name Uniprot ID Library Screen (fixed cells) Confirmation Screen (fixed cells) ATL_5802 CD33 0583 Myeloid cell surface antigen CD33 P20138-1 Rep1: medium / strong Rep2: medium / strong Rep1: medium / strong Rep2: medium / strong Sample ID Gene ID Confirmation Screen (live cells) Comments ATL5802 CD33 strong 364aa, CD33M isoform, single-pass type I membrane protein Petition 870250089689, dated 02 / 10 / 2025, pp. 152 / 221 139 / 144 Table 8. Results of a microarray binding screening for ATL5802 confirming selective binding to CD33. REFERENCES

[0291] Several publications are cited above to describe and disclose more fully the invention and the state of the art to which it relates. Full citations of these references are provided below. The full text of each of these references is incorporated into this report. For standard molecular biology techniques, see Sambrook, J., Russel, DW Molecular Cloning, A Laboratory Manual. 3rd ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Amft, M., Ortner, M., Eichenlaub, U. Et al. The cerebrospinal fluid biomarker ratio Αβ42 / 40 identifies amyloid positron emission tomography positivity better than Αβ42 alone in a heterogeneous memory clinical cohort. Alz Res Therapy 14, 60 (2022). Andrews, S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data: www.bioinformatics.babraham.ac.uk / projects / fastqc / Baik, Sung Hoon et al. “A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease.” Cell metabolism vol. 30,3 (2019): 493507. Balsis, Steve et al. “How Do Scores on the ADAS-Cog, MMSE, and CDRSOB Correspond?.” The Clinical neuropsychologist vol. 29,7 (2015): 1002-9. Blennow, K., Shaw, L.M., Stomrud, E. et al. Predicting clinical decline and conversion to Alzheimer’s disease or dementia using novel Elecsys Ae(1-42), pTau and tTau CSF immunoassays. Sci Rep 9, 19024 (2019). Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. (1997) 3:730. Petição 870250089689, de 02 / 10 / 2025, pág. 153 / 221 140 / 144 Brinkman-Van der Linden EC, Angata T, Reynolds SA, Powell LD, Hedrick SM, Varki A. CD33 / Siglec-3 binding specificity, expression pattern, and consequences of gene deletion in mice. Mol Cell Biol. 2003 Jun;23(12):4199-206. Cao, H., de Bono, B., Belov, K. et al. Comparative genomics indicates the mammalian CD33rSiglec locus evolved by an ancient large-scale inverse duplication and suggests all Siglecs share a common ancestral region. Immunogenetics 61, 401-417 (2009). Cao, Huan, and Paul R Crocker. “Evolution of CD33-related siglecs: regulating host immune functions and escaping pathogen exploitation?.” Immunology vol. 132,1 (2011): 18-26. Chan G, et al.: CD33 modulates TREM2: convergence of Alzheimer loci. Nat Neurosci 2015; 18: 1556-1558 Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884-i890, Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1;29(1):15-21. Dolgalev I (2022). _msigdbr: MSigDB Gene Sets for Multiple Organisms in a Tidy Data Format_. R package version 7.5.1, <https: / / igordot.github.io / msigdbr / >. Donnelly, Louise E, and Peter J Barnes. “Defective phagocytosis in airways disease.” Chest vol. 141,4 (2012): 1055-1062. Duff, Kevin et al. “Utility of the RBANS in detecting cognitive impairment associated with Alzheimer’s disease: sensitivity, specificity, and positive and negative predictive powers.” Archives of clinical neuropsychology : the official journal of the National Academy of Neuropsychologists vol. 23,5 (2008): 603-12. Petição 870250089689, de 02 / 10 / 2025, pág. 154 / 221 141 / 144 Ennerfelt H, et al. SYK coordinates neuroprotective microglial responses in neurodegenerative disease. Cell. 2022 Oct 27;185(22):4135-4152.e22. Eskandari-Sedighi, Ghazaleh et al. “CD33 isoforms in microglia and Alzheimer’s disease: Friend and foe.” Molecular aspects of medicine vol. 90 (2023): 101111. Fattorelli N, Martinez-Muriana A, Wolfs L, Geric I, De Strooper B, Mancuso R. Stem-cell-derived human microglia transplanted into mouse brain to study human disease. Nat Protoc. 2021 Feb;16(2):1013-1033. Golde, Todd E. “Disease-Modifying Therapies for Alzheimer’s Disease: More Questions than Answers.” Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics vol. 19,1 (2022): 209-227. Griciuc, Ana et al. “Alzheimer’s disease risk gene CD33 inhibits microglial uptake of amyloid beta.” Neuron vol. 78,4 (2013): 631-43. Holstege H, Beker N, Dijkstra T, et al. The 100-plus Study of cognitively healthy centenarians: rationale, design and cohort description. Eur J Epidemiol. 2018;33(12):1229-1249. Hollingworth, Paul et al. “Common variants at ABCA7, MS4A6A / MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer’s disease.” Nature genetics vol. 43,5 (2011): 429-35. Huang, Zhen-Yu et al. “The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcgamma receptors FcgammaRIIB and FcgammaRIIA.” Journal of leukocyte biology vol. 73,6 (2003): 823-9. Jack CR Jr, Bennett DA, Blennow K, et al. A / T / N: An unbiased descriptive classification scheme for Alzheimer disease biomarkers. Neurology. 2016;87(5):539547. Jung et al. Selection for improved protein stability by phage display. Journal Molecular Biology 294, 163-180 (1999). Petição 870250089689, de 02 / 10 / 2025, pág. 155 / 221 142 / 144 Jurcic, 2012. What Happened to Anti-CD33 Therapy for Acute Myeloid Leukemia? Curr Hematol Malig Rep (2012) 7:65-73. Knopman, D.S., Amieva, H., Petersen, R.C. et al. Alzheimer disease. Nat Rev Dis Primers 7, 33 (2021). G. Korotkevich, V. Sukhov, A. Sergushichev. Fast gene set enrichment analysis. bioRxiv (2019), doi:10.1101 / 060012 Lauro, Clotilde, and Cristina Limatola. “Metabolic Reprograming of Microglia in the Regulation of the Innate Inflammatory Response.” Frontiers in immunology vol. 11 493. 20 Mar. 2020. Laszló et al. The past and future of CD33 as therapeutic target in acute myeloid leukemia. Blood Rev. 2014 Jul. Liao Y, Smyth GK and Shi W (2014). featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics,30(7):923-30. Linnartz, Bettina, and Harald Neumann. “Microglial activatory (immunoreceptor tyrosine-based activation motif)- and inhibitory (immunoreceptor tyrosine-based inhibition motif)-signaling receptors for recognition of the neuronal glycocalyx.” Glia vol. 61,1 (2013): 37-46. Love, M.I., Huber, W., Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Genome Biology 15(12):550 (2014) Lund J, Winter G, Jones PT, Pound JD, Tanaka T, Walker MR et al. Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol. 1991; 147:2657-2662 Mancuso R, et al. Stem-cell-derived human microglia transplanted in mouse brain to study human disease. Nat Neurosci. 2019 Dec;22(12):2111-2116. Petição 870250089689, de 02 / 10 / 2025, pág. 156 / 221 143 / 144 Manscuso R., et al. A multi-pronged human microglia response to Alzheimer’s disease Ab pathology. Preprint at bioRxiv. 2022. doi: 10.1101 / 2022.07.07.499139. Michaelson DM. APOE ε4: the most prevalent yet understudied risk factor for Alzheimer’s disease. Alzheimers Dement. 2014;10(6):861-868. McQuade, A., Kang, Y.J., Hasselmann, J. Et al. Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer’s disease. Nat Commun 11, 5370 (2020). Mancuso, Renzo et al. “Stem-cell-derived human microglia transplanted in mouse brain to study human disease.” Nature neuroscience vol. 22,12 (2019) Siddiqui SS, Springer SA, Verhagen A, Sundaramurthy V, Alisson-Silva F, Jiang W, Ghosh P, Varki A: The Alzheimer’s disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool. J Biol Chern 2017; 292: 15312-15320. Stanczak MA et al. Targeting cancer glycosylation repolarizes tumorassociated macrophages allowing effective immune checkpoint blockade. Science Translational Medicine 14, eabj1270 (2022) 2 November 2022. Stanczak, Michal A, and Heinz Lãubli. “Siglec receptors as new immune checkpoints in cancer.” Molecular aspects of medicine vol. 90 (2023): 101112. Strang KH, et al. Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies. J Biol Chern. 2018 Feb 16;293(7):2408-2421. doi: 10.1074 / jbc.M117.815357. Epub 2017 Dec 19. Erratum in: J Biol Chern. 2018 Mar23;293(12 ):4579. Trinidad Hernández-Caselles, et al., A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing, Journal of Leukocyte Biology, Volume 79, Issue 1, Jan 2006, Pages 46-58 Petição 870250089689, de 02 / 10 / 2025, pág. 157 / 221 144 / 144 Walker, D.G.; et al. Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer’s Disease Brains. Int. J. Mol. Sci. 2020, 21,678. Ward et al., 2021. A phase I study of AL003 in Healthy Volunteers and Participants with Alzheimer’s Disease. Abstract P45 in “Abstract: Symposia, Conferences, Oral communications: 14thClinical Trials on Alzheimer’s Disease (CTAD) November 9-12, 2021.” The journal of prevention of Alzheimer’s disease vol. 8,S1 (2021): S2-S72. Varki A, Angata T: Siglecs - the major subfamily of I-type lectins. Glycobiology 2006; 16: 1R-27R. Vercauteren S, Zapf R, Sutherland H. Primitive AML progenitors from most CD34+ patients lack CD33 expression but progenitors from many CD34- AML patients express CD33. Cytotherapy (2007) 9:194-204. Yu YJ, Atwal JK, Zhang Y, et al. Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci Transl Med. 2014;6(261):261ra154. Zhao, Lingzhi. “CD33 in Alzheimer’s Disease - Biology, Pathogenesis, and Therapeutics: A Mini-Review.” Gerontology vol. 65,4 (2019): 323-331.

Claims

CLAIMS 1. Isolated antibody that specifically binds to a CD33 protein, CHARACTERIZED in that the antibody increases the phagocytosis of a cell expressing CD33 compared to a comparator antibody and / or in that the binding of the antibody to a human CD33 protein comprising mutations at positions 20, 21, 22 and 24 is increased compared to the binding of the antibody to a human CD33 protein without said mutations.

2. Isolated antibody, according to claim 1, CHARACTERIZED in that the binding of the antibody to a human CD33 protein comprising mutations at positions 20, 21, 22, 24 and 132 is increased compared to the binding of the antibody to a human CD33 protein without said mutations, and / or in that the binding of the antibody to a human CD33 protein comprising mutations at positions 47, 50, 51 and 52 is reduced compared to the binding of the antibody to a human CD33 protein without said mutations, and / or in that the antibody does not bind to a human CD33 protein comprising mutations at positions 47, 50, 51 and 52, and / or in that the binding of the antibody to a human CD33 protein comprising mutations at positions 47, 50, 51, 52 and 122 is reduced compared to the binding of the antibody to a human CD33 protein without said mutations, and / or where the antibody does not bind to a human CD33 protein comprising mutations at positions 47, 50, 51, 52 and 122. It binds to a human CD33 protein comprising mutations at positions 47, 50, 51, 52, and 122.and / or where antibody binding to a human CD33 protein comprising mutations at positions 83 is reduced compared to antibody binding to a human CD33 protein without said mutations. Petition 870250089689, dated 02 / 10 / 2025, p. 159 / 221 2 / 12, 3. Isolated antibody, according to claim 1 or 2, CHARACTERIZED in that the mutations are selected from: 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. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that 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. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody has one or more, or all of the following: a. increased binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos1[P1] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, b. increased binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos1_MutPos6[P6+1] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, Petition 870250089689, 10 / 02 / 2025, p. 160 / 221 3 / 12 c. reduced binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos2 [P2] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, d. reduced binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos2_MutPos5 [P2+5] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT, and e.Reduced binding to a protein comprising the sequence CD33M2_ECD_18-232_MutPos4 [P4] compared to a protein comprising the sequence CD33M2_ECD_18-232_WT.

6. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the comparative antibody is selected from: an isotype control antibody, another CD33-binding antibody, and an antibody with the variable ATL_5909 heavy chain sequence and the variable ATL_5909 light chain sequence, and / or in which phagocytosis is assessed by measuring a fluorescence signal associated with the uptake of a labeled particle by imaging or flow cytometry.

7. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the cell is a monocyte or a microglial cell, optionally, wherein the cell is a human cell and / or a microglial cell derived from iPSC and / or a cell that has been stimulated with an inflammatory signal (e.g., LPS) prior to exposure to the antibody.

8. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody binds to a CD33 protein comprising the CD33 V domain, and / or in that the antibody does not bind to a CD33 protein that does not comprise the CD33 V domain, and / or in that the antibody does not bind to a CD33 protein having the sequence CD33_HUMAN_ECD_Cdomain_His_007. Petition 870250089689, dated 10 / 02 / 2025, p. 161 / 221 4 / 12 9. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody comprises a variable heavy chain (VH) domain with the following CDRs: CDRH1 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; CDRH2 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; CDRH3 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or a set of CDRs containing zero, one, or two amino acid substitutions in each CDR, compared to the set of CDRs above.

10. Isolated antibody, according to claim 9, CHARACTERIZED in that the antibody comprises a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising an amino acid sequence selected from: HCDR1_ATL_0005802; HCDR1_ATL_0005807; HCDR1_ATL_0005808: GYSFTSYW (SEQ ID NO:44) HCDR1_ATL_0005853: GYKFNNNW (SEQ ID NO:47) HCDR1_ATL_0005854: GYKFSNNW (SEQ ID NO: 48) or an amino acid sequence with 1 or 2 mutations compared to the above sequences; a CDRH2 comprising an amino acid sequence selected from: Petition 870250089689, dated 02 / 10 / 2025, page.162 / 221 5 / 12 HCDR2_ATL_0005802; HCDR2_ATL_0005807; HCDR2_ATL_0005808; HCDR2_ATL_0005853; HCDR2_ATL_0005854; IYPGDSDT (SEQ ID NO: 52) or an amino acid sequence with 1 or 2 mutations compared to the sequences above; and a CDRH3 comprising an amino acid sequence selected from: HCDR3_ATL_0005802; ARPRFGEYYFDY (SEQ ID NO: 59) HCDR3_ATL_0005853 ARHSGGLDGYTAAALDY (SEQ ID NO:64) HCDR3_ATL_0005854 ATWGGSNWFVD (SEQ ID NO: 65) or an amino acid sequence with 1 or 2 mutations compared to the sequences above.

11. Isolated antibody, according to claim 9 or 10, CHARACTERIZED in that the antibody comprises a variable heavy chain (VH) domain with the following CDRs: a CDRH1 comprising the sequence HCDR1_ATL_0005802, a CDRH2 comprising the sequence HCDR2_ATL_0005802 and a CDRH3 comprising the sequence HCDR3_ATL_0005802, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences; or a CDRH1 comprising the sequence HCDR1_ATL_0005853, a CDRH2 comprising the sequence HCDR2_ATL_0005853 and a CDRH3 comprising the sequence HCDR3_ATL_0005853, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences; or Petition 870250089689, dated 02 / 10 / 2025, page.163 / 221 6 / 12 a CDRH1 comprising the sequence HCDR1_ATL_0005854, a CDRH2 comprising the sequence HCDR2_ATL_0005854 and a CDRH3 comprising the sequence HCDR3_ATL_0005854, or a set of CDRs comprising 1 or 2 mutations in CDRH1 and CDRH2 compared to these sequences and / or 1, 2 or 3 mutations in CDRH3 compared to these sequences.

12. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody has a variable heavy chain (VH) domain with the following structural sequences: HFWR1 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR2 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, HFWR3 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 and HFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 or structure sequences with one to six substitutions compared to the structure sequences above.

13. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody has a variable heavy chain (VH) domain with an HFWR2 structure sequence of ATL_0005802 and / or in that the antibody has a variable heavy chain (VH) domain with a structure sequence comprising an A at position 40 in the standard IMGT numbering.

14. Isolated antibody, according to claim 12 or 13, CHARACTERIZED in that the substitutions in the sequences of the variable domain structure of the heavy chain are located at any position other than position 40 in the standard IMGT numbering.

15. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody has a variable heavy chain (VH) domain with the following structure sequences: 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. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody has a variable heavy chain (VH) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from the VH sequence of the 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 variable heavy chain (VH) domain comprising a sequence that has at most 2 mutations in each HCR and at most 3 mutations in each structure region compared to a sequence selected from said VH sequences;Optionally, wherein the antibody has a variable heavy chain (VH) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from the VH sequence of antibodies ATL_0005802, ATL_0005853 or ATL_0005854 or a sequence that has at most 2 mutations in each HCDR and at most 3 mutations in each structure region compared to a sequence selected from the aforementioned VH sequences. Petition 870250089689, dated 02 / 10 / 2025, page 165 / 221 8 / 12; 17. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody: (i) has a variable heavy chain (VH) domain comprising CDRH1, CDRH2 and CDRH3 within a germline structure, provided that position 40 in the standard IMGT numbering is A, and / or (ii) is an scFv antibody molecule, a nanobody or a complete antibody, and / or wherein the antibody comprises a constant antibody region, and / or wherein the antibody is a complete antibody and / or wherein the antibody is an IgG1 or variant thereof, optionally wherein the antibody is a variant of IgG1 L234A / L235A (LALA).

18. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that 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 (such as plated rhCD33 binding), and / or wherein the antibody binds selectively to CD33 with respect to one or more siglecs, optionally, wherein the antibody binds selectively to CD33 with respect to one or more (or all): siglec-6, siglec-7, siglec-8 and siglec-9, and / or wherein the antibody binds selectively to human CD33 with respect to one or more homologs, optionally, wherein the antibody binds selectively to human CD33 with respect to mouse CD33 and canine CD33.

19. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody depletes CD33 on the cell surface of human monocytes by less than 50%, or less than 80% after 5 hours of incubation with the antibody, and / or in that the antibody depletes CD33 on the cell surface of human monocytes after 5 hours of incubation with the antibody to a lesser extent than a comparative antibody at the same concentration. Petition 870250089689, dated 10 / 02 / 2025, pp. 166 / 221 9 / 12 20. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody comprises a variable light chain (VL) domain with the following CDRs: CDRL1 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810, CDRL2 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 and CDRL3 comprising the amino acid sequence of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810 or a set of CDRs that contains zero, one, or two amino acid substitutions in each CDR, compared to the set of CDRs above.

21. Isolated antibody, according to claim 20, CHARACTERIZED in that the CDRL1, CDRL2 and CDRL3 of the VL domain are within a germline structure and / or in that the antibody has a variable light chain (VL) domain with the following structure sequences: LFWR1 of any one of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; LFWR2 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; LFWR3 from any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; and LFWR4 of any of the antibodies ATL_0005802, ATL_0005853, ATL_0005854, ATL_0005807, ATL_0005808, ATL_0005809, ATL_0005810; or Petition 870250089689, dated 10 / 02 / 2025, page 167 / 221 10 / 12 a set of FWRs containing one to six amino acid substitutions compared to the FWR set above.

22. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody has a variable light chain (VL) domain comprising a selected sequence that has at least 95% sequence identity with a sequence selected from the VL sequence of the 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 in that the antibody has a variable light chain (VL) domain comprising a sequence that has at most 2 mutations in each LCDR and at most 3 mutations in each structure region compared to a sequence selected from said VL sequences;Optionally, the antibody has a variable light chain (VL) domain comprising a sequence that has at least 95% sequence identity with a sequence selected from the VL sequence of antibodies ATL_0005802, ATL_0005853 or ATL_0005854 or a sequence that has at most 2 mutations in each LCDR and at most 3 mutations in each structure region compared to a sequence selected from said VL sequences.

23. Isolated antibody, according to any of the preceding claims, CHARACTERIZED in that the antibody exhibits lower peripheral clearance when administered to an individual, compared to a comparative anti-CD33 antibody, and / or in that the antibody increases Aβ phagocytosis by microglia cells in vivo compared to a control, and / or increases tau aggregate phagocytosis by microglia with an inflammatory phenotype (e.g., LPS-treated iPSC microglia) compared to a control, and / or increases tau aggregate phagocytosis by microglia with an inflammatory phenotype. Petition 870250089689, dated 10 / 02 / 2025, p.168 / 221 11 / 12 to a greater extent than a comparative anti-CD33 antibody, and / or does not induce the 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 levels of IL-6 and / or MCP-1 released by microglia with an inflammatory phenotype in vitro (e.g., LPS-treated human iPSC-derived microglia) and / or in vivo, and / or reduces the inflammation-induced release of one or more inflammatory markers in a human neural cell culture assay and / or in the central nervous system of an individual, optionally, wherein one or more inflammatory markers are selected from: MCP-1, IP-10, GFAP, and IL-6.

24. Isolated nucleic acid, vector or set of vectors, CHARACTERIZED in that it comprises a nucleotide sequence encoding an antibody, including a VH or VL domain, according to any of the preceding claims, or a fragment thereof.

25. Host cell, CHARACTERIZED in that it comprises the vector(s) according to claim 24, or a host cell transformed in vitro with a nucleic acid according to claim 24.

26. Antibody, according to any one of claims 1 to 23, CHARACTERIZED in that it is for use in the treatment of a disease or disorder selected from: (i) a disease associated with microglia cell dysfunction; (ii) a neurodegenerative disease or disorder, optionally, wherein the neurodegenerative disease or disorder is a tauopathy or a disease or disorder 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 diseases, Lewy body disease, spinal muscular atrophy (SMA), motor neuron disease (MND), such as amyotrophic lateral sclerosis (ALS), supranuclear palsy Petition 870250089689, dated 02 / 10 / 2025, p.169 / 221 12 / 12 progressive (PSP), spinocerebellar ataxias (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral pallidal-Luisian atrophy (DRPLA), X-linked spinal bulbar muscular atrophy type 1 (SMAX1 / SBMA), AndersonFabry (X-linked Fabry disease) and DNAJB6 myopathies, optionally, wherein the neurodegenerative disease is selected from FTD, AD, HD and PD; (iii) a cancer, optionally, wherein the cancer is either AML or a cancer associated with hypersalination of tumor cells and / or overexpression of CD33 by tumor cells; 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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