Therapeutic agents and methods for targeting myeloid cell subtypes

Abstract

Provided herein are agents and methods suitable for the treatment of fibrosis, such as liver fibrosis.

Description

[0001] THERAPEUTIC AGENTS AND METHODS FOR TARGETING MYELOID CELL SUBTYPES

[0002] FIELD OF THE INVENTION

[0003] Provided herein are agents and methods suitable for the treatment of fibrosis, such as liver fibrosis.

[0004] BACKGROUND OF THE INVENTION

[0005] Myeloid lineage cells, including dendritic cells and macrophages, are highly diverse. This diversity arises due to differences in their origin (e.g. bone marrow -derived vs embryonic), and their adaptation to specific tissue environments, leading to macrophage subtypes with unique characteristics (e.g. dendritic cells, Kupffer cells in the liver, or alveolar macrophages in the lung). This specialization enables macrophages to perform a wide range of tasks, including, but not limited to, maintaining tissue homeostasis, orchestrating immune responses, and facilitating wound healing. Given this complexity, therapeutically suppressing the function of, or deleting macrophage populations broadly could potentially dismpt many essential physiological processes. Therefore, there is a need in the art to develop therapeutics that selectively target macrophage subsets that play pathogenic roles in certain diseases, without compromising the beneficial roles played by the broader macrophage population in health and disease.

[0006] SUMMARY OF THE INVENTION

[0007] In an aspect, there is provided a multispecific molecule comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for any one of CSF1R, MSR1, or CLEC7A.

[0008] The multispecific molecule may bind to SAMac cells and / or may be selective for SAMac cells. The multispecific may have cytotoxic activity or may promote cytotoxic activity towards SAMac cells.

[0009] When the second antigen-binding domain is specific for CSF1R it may comprise or may be CSF1 or a mutant CSF1, a variant, or portion thereof. The second antigen-binding domain may block CSF1R signalling. The second antigen-binding domain may prevent CSF-1 binding to CSF1R or may inhibit dimerisation of CSF1R.

[0010] The first and / or second antigen binding domain may be an antigen-binding portion of an antibody. For instance, a single-chain fragment variable (scFv).

[0011] The multispecific molecule may be bispecific. The multispecific molecule may be a bispecific antibody.

[0012] The first antigen-binding domain may comprise a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein (a) the VH domain, or portion thereof, comprises a heavy chain complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 2, 5, or 8; a heavy chain complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 3, 6, or 9; and a heavy chain complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 4 or 7 and the VL domain, or portion thereof, comprises a light chain complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 11 or 14; a light chain complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 12 or 15; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 13; or (b) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 17, 20, or 23; an HCDR2 comprising any one of SEQ ID NOs: 18, 21, or 24; and an HCDR3 comprising any one of SEQ ID NOs: 19 or 22 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 26 or 29; an LCDR2 comprising any one of SEQ ID NOs: 27 or 30; and an LCDR3 comprising SEQ ID NO: 28. In some embodiments, the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; or (b) the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25. The second antigen-binding domain may comprise a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein (a) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 32, 35, or 38; an HCDR2 comprising any one of SEQ ID NOs: 33, 36, or 39; and an HCDR3 comprising any one of SEQ ID NOs: 34 or 37 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 41 or 44; an LCDR2 comprising any one of SEQ ID NOs: 42 or 45; and an LCDR3 comprising SEQ ID NO: 43; or (b) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 49 or 52 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 56 or 59; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 58; or (c) the VH domain, or portion thereof, comprises an HCDRl comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 62 or 63 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 65 or 67; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 66; or (d) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 69 or 70 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 72 or 75; an LCDR2 comprising any one of SEQ ID NOs: 60 or 73; and an LCDR3 comprising SEQ ID NO: 74. In some embodiments, the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40; or (b) the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55; or (c) the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64; or (d) the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0013] In an aspect, there is provided an immunoconjugate comprising any multispecific molecule disclosed herein conjugated to an agent.

[0014] In an aspect, there is provided one or more nucleic acid molecules encoding any multispecific molecule disclosed herein.

[0015] In an aspect, there is provided one or more vectors comprising any of the one or more nucleic acid molecules disclosed herein.

[0016] In an aspect, there is provided a cell comprising any of the one or more nucleic acid molecules or any of the one or more vectors disclosed herein.

[0017] In an aspect, there is provided a pharmaceutical composition comprising any of the multispecific molecules, any of the immunoconjugates, any of the one or more nucleic acid molecules, any of the one or more vectors, or any of the cells disclosed herein.

[0018] In an aspect, there is provided any of the multispecific molecules, any of the immunoconjugates, any of the one or more nucleic acid molecules, any of the one or more vectors, any of the cells, or any of the pharmaceutical compositions disclosed herein, for use in a method of treatment.

[0019] In an aspect, there is provided any of the multispecific molecules, any of the immunoconjugates, any of the one or more nucleic acid molecules, any of the one or more vectors, any of the cells, or any of the pharmaceutical compositions disclosed herein, for use in a method of treating fibrosis. In some embodiments, the fibrosis affects the liver, lung, kidney, intestine, skin, or heart. In a particular embodiment, the fibrosis is or includes liver fibrosis.

[0020] In an aspect, there is provided any of the multispecific molecules, any of the immunoconjugates, any of the one or more nucleic acid molecules, any of the one or more vectors, any of the cells, or any of the pharmaceutical compositions disclosed herein, for use in a method of treating idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease, or scleroderma.

[0021] In an aspect, there is provided any of the multispecific molecules, any of the immunoconjugates, any of the one or more nucleic acid molecules, any of the one or more vectors, any of the cells, or any of the pharmaceutical compositions disclosed herein, for use in a method of treating fibrotic liver disease, cirrhosis of the liver, alcohol- associated liver disease, metabolic dysfunction-associated steatohepatitis, or primary sclerosing cholangitis.

[0022] In an aspect, there is provided a method of treating fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of any of the multispecific molecules, any of the immunoconjugates, any of the one or more nucleic acid molecules, any of the one or more vectors, any of the cells, or any of the pharmaceutical compositions disclosed herein to the subject.

[0023] In an aspect, there is provided a method of inhibiting the fibrotic activity, reducing the proliferation, or reducing the survival of SAMac cells, the method comprising contacting the cells with any of the multispecific molecules disclosed herein. In some embodiments, the method is in vitro, in vivo, or ex vivo.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1: Schematic representation of a cis-acting bispecific antibody (BsAb) targeting SAMac cells. The BsAb is engineered with two distinct binding sites: Binding Site A (dark grey) and Binding Site B (light grey), which correspond to Receptor A and Receptor B, respectively. The top panels demonstrate no or reduced binding of the BsAb's with individual Receptor A and Receptor B when they are not co-expressed on the same cell surface, leading to low-affinity or no binding. The bottom right panel shows the BsAb binding simultaneously to both Receptor A and Receptor B when co-expressed on the same cell, thereby exploiting the principle of avidity to achieve high-affinity binding. In embodiments of the invention, Receptor A may be TREM2 and Receptor B may be any one of MSR1, CLEC7A, or CSF1R.

[0026] Figure 2. A) Dotplot showing fraction of cells and mean expression of known and selected surface receptor targets for SAMac annotated as MPs (5) and MPs (4). B) Uniform Manifold Approximation and Projection (UMAP) visualisation of cells with any transcripts present that map to single receptors (grey) & cells with transcripts captured from both receptor genes (black).

[0027] Figure 3. A) Dotplot showing fraction of cells and mean expression of known and selected surface receptor targets for SAMac. B) UMAP visualisation of cells with any transcripts present that map to single receptors (grey) & cells with transcripts captured from both receptor genes (black).

[0028] Figure 4: Cell-cell interaction analysis using scRNA-seq dataset from Ramachandran et al., 2019. A) Analysis was performed to identify cell-cell interactions that induce expression observed in disease SAMac cells. Heatmap shows top ligand-receptor pairs, where receptor is expressed on disease SAMacs and where ligand is expressed by other cells in the tissue. Colour indicates the significance of the interaction (low = more significant) as determined by p value calculated using CellphoneDB (v5) method (Efremova et al., Nat Protoc., 2020) B) Barplot shows NicheNet predicted regulatory potential of key ligands. To calculate this, NicheNet uses integrated networks of signalling, protein-protein and gene-regulatory interactions from publicly available datasets to predict regulatory potential of a ligand (Browaeys et al., Nature Methods, 2019). Regulatory potential signifies the ability of a ligand to induce gene expression observed in SAMac cells. These data indicate that SAMac cells are capable of receiving a CSF1 signal. C) Heatmaps showing the expression of CSF1 across liver cell types from uninjured and cirrhotic liver scRNA-seq dataset from Ramachandran et al., 2019. CSF1 gene is expressed in cholangiocyte, endothelial, mast, and mesenchymal cells in uninjured and cirrhotic liver. CSF1 is expressed more highly in endothelial cells and there is greater signal in cirrhotic liver.

[0029] Figure 5: Systemic expression of the individual five receptors in single cell data from multiple human organs in CZI CellxGene Discover database. In total we considered 3,226,515 cells (from 40 organs and tissues) and assess the expression across myeloid, lymphoid, endothelial, lymphatic endothelial, epithelial, fibroblast cell lineages. The dotplot shows mean expression (colour intensity) and fraction of cells (dot size) per lineage and organ expressing individual receptors. Bar plot shows the number of cells considered per organ and indicates the % of total cells per organ considered in this analysis.

[0030] Figure 6: SAMacs spatially localize to scar regions in cirrhotic liver. Spatial transcriptomics data showing scar localization of cells labelled as SAMacs (as defined by Ramachandran et al.) in control (A) and cirrhotic (B) human liver samples. Scar regions and non-scar regions are annotated based on scar-associated gene expression, and distribution of SAMacs within each region is shown with dots.

[0031] Figure 7: SAMacs spatially localize to scar regions in fibrotic liver and increase with fibrosis stage.

[0032] Quantification of spatial transcriptomics data showing scar localization of cells labelled as SAMacs (as defined by Ramachandran et al.) in human liver samples at differing fibrosis stages. (A) shows the distribution of SAMacs in scar vs non-scar regions. (B) shows the distribution of SAMacs in scar vs non-scar regions adjusted for total region cell count.

[0033] Figure 8: SAMacs proliferate within scar regions. Proliferating SAMacs were defined as expressing the proliferation gene marker, MKI67. Percentage proportion of MKI67+ SAMacs in scar regions and non-scar regions was then quantified in samples spanning different stages of fibrosis (F0-F4), with absolute number of SAMacs indicated in the bar chart. The majority of SAMacs in scar regions are proliferating. Across 5 liver fibrosis Xenium samples, 3.8% SAMacs have expression ofMK167, and ~50% of MK167+ SAMacs are located in scar regions.

[0034] Figure 9: Expression of CSF1 is enriched in fibrotic scar regions. Quantification of spatial transcriptomics data showing proportion of cells expressing CSF1, a growth factor essential for the differentiation of circulating monocytes into macrophages, is localized to scar regions and increases with disease severity. As SAMacs have been shown to differentiate from infiltrating monocytes, this data illustrates that the fibrotic scar becomes an environment suited for macrophage differentiation. (A) Distribution of CSF1+ population in scar vs non-scar regions adjusted for total region cell count. (B) Distribution of CSF 1+ population in scar vs non-scar regions.

[0035] Figure 10: In vitro SAMac models shows TREM2 / CSF1R expression and similar transcriptional profile to in vivo SAMac. (A) is a barplot showing the surface expression of human TREM2 measured by flow cytometry in human monocyte-derived macrophages differentiated with different cytokine cocktails. Macrophages differentiated in the presence of M-CSF show the highest surface expression of TREM2. (B) is a barplot showing the surface expression of human CSF1R measured by flow cytometry in human monocyte-derived macrophages differentiated with different cytokine cocktails. (C) is a heatmap showing the proportions of human monocyte- derived macrophages assigned to each in vitro differentiated cell type. Monocyte-derived macrophages were differentiated in vitro with different cytokine cocktails and subjected to scRNA-seq. A regularized linear classifier was trained on human primary liver cells to predict cell conditions, and subsequently applied to the human primary cells shown here (Ramachandran et al., Nature, 2019). Figure 11: In vitro differentiated SAMac model induces fibrosis in hepatic stellate cell-based fibrosis assay.

[0036] Boxplot showing the expression of COL1A1 normalized to GAPDFFby qPCR in human primary hepatic stellate cells that were treated for 24h with the supernatants from human monocyte-macrophages differentiated with different cytokine cocktails, Ing / mL TGFp alone, DMEM media or RPMI + 10% FBS media as controls. To obtain supernatants from monocyte-derived macrophages, human monocytes were differentiated in the presence of different cytokines. The cytokine-containing media was removed at the end of the differentiation and exchanged to RPMI + 10% FBS media. After 24h of incubation, macrophage supernatants were collected and used for the assay.

[0037] Figure 12: CSF1R antibodies (clones 1B7, 1B6, 1H8, and 1H9) inhibit CSF1R activity in vitro. CSF1R dimerizes upon binding to ligands like CSF-1, triggering autophosphorylation of tyrosine residues and downstream signalling. We used a commercial assay (PathHunter) which CSF1R subunits tagged with complementary fragments of P-galactosidase. Ligand binding induces receptor dimerization, reconstituting the active enzyme and generating a measurable chemiluminescent signal. CSF1R antibodies (clones 1B7, 1B6, 1H8, and 1H9) showed a dose dependent inhibition of CSF1R activity, providing evidence for CSF1R dimerization blockade.

[0038] Figure 13: CSF1R antibodies (clones 1B7, 1B6, 1H8, and 1H9) inhibit monocyte proliferation in vitro.

[0039] Inhibition of CSF1R downstream signalling leads to a decrease of monocytes proliferation and an increase in apoptosis. PBMCs-derived monocytes were treated for 5 days with increasing doses on CSF1R antibodies (clones 1B7, 1B6, 1H8, and 1H9) in the presence of lOOng / ml CSF-1. The viability of the cells was assessed using CellTiterGlo which determines the viable cells by quantifying the amount of ATP present in the cells. CSF1R antibodies (clones 1B7, 1B6, 1H8, and 1H9) showed a reduction of viable cells of 50% at the highest dose, suggesting an inhibition of monocytes proliferation.

[0040] Figure 14: TREM2+CSF1R+cells are localized to fibrotic scar regions. Within each sample spanning different stages of fibrosis (F0-F4), the proportion of cells that express only TREM2 or both TREM2 and CSF1R, as well as their distribution in scar vs non-scar regions, was quantified. A general increase in the localization of TREM2+and TREM2+CSF1R+cells to scar regions was observed with increasing disease severity.

[0041] Figure 15: SAMacs express profibrotic factors. The publicly available scRNA-seq dataset from Ramachandran et al. was analysed to interrogate expression of well-defined profibrotic factors within their SAMac cell cluster. These genes were then ranked by the proportion of SAMacs that express them (Expressing Cell Fraction). Of note, over 50% of SAMacs expressed important profibrotic genes (LGALS3, TGFB1, ITGB1).

[0042] Figure 16: Sensorgrams illustrating the binding of anti-TREM2 mAb to huTREM2 (ECD). (A) shows the binding of 1F7 to huTREM2 (ECD). (B) shows the binding of 2G4 to huTREM2 (ECD). Numerical results are presented in Table 10.

[0043] Figure 17: Sensorgrams illustrating the binding of anti-TREM2 mAb to cynoTREM2 (ECD). (A) shows the binding of 1F7 to cynoTREM2 (ECD). (B) shows the binding of 2G4 to cynoTREM2 (ECD). Numerical results are presented in Table 11.

[0044] Figure 18: Sensorgram illustrating the binding of anti-TREM2 mAb to muTREM2 (ECD). The binding of 2G4 to mouse TREM2 (ECD) is illustrated. Numerical results are presented in Table 12.

[0045] Figure 19: Results from an ELISA to show binding of anti-TREM2 mAb to human TREM2. Numerical results are presented in Table 13. Figure 20: Results from an ELISA to show binding of anti-TREM2 mAh to cyno TREM2. Numerical results are presented in Table 14.

[0046] Figure 21: Results from an ELISA to show binding of anti-TREM2 mAh to mouse TREM2. Numerical results are presented in Table 15.

[0047] Figure 22: Sensorgrams illustrating the binding of anti-CSFIR mAh to human CSF1R (ECD). (A) shows the binding of 1B7 to human CSF1R (ECD). (B) shows the binding of 1B6 to human CSF1R (ECD). (C) shows the binding of 1H8 to human CSF1R (ECD). (D) shows the binding of 1H9 to human CSF1R (ECD). Numerical results are presented in Table 17.

[0048] Figure 23: Sensorgrams illustrating the binding of anti-CSFIR mAh to cyno CSF1R (ECD). (A) shows the binding of 1B7 to cyno CSF1R (ECD). (B) shows the binding of 1B6 to cyno CSF1R (ECD). (C) shows the binding of 1H8 to cyno CSF1R (ECD). (D) shows the binding of 1H9 to cyno CSF1R (ECD). Numerical results are presented in Table 18.

[0049] Figure 24: Results from an ELISA to show binding of anti-CSFIR mAh to human CSF1R. Numerical results are presented in Table 20.

[0050] Figure 25: Results from an ELISA to show binding of anti-CSFIR mAh to cyno CSF1R. Numerical results are presented in Table 21.

[0051] Figure 26: Binding of anti-TREM2 antibodies to human TREM2 expressing HEK293F cells.

[0052] Figure 27: Binding of anti-TREM2 antibodies to the human TREM2 expressing THP1 cell line.

[0053] Figure 28: Binding of anti TREM2 antibodies to human TREM2 expressing HEK293F cells.

[0054] Figure 29: Binding of anti-TREM2 antibodies to cyno TREM2 expressing HEK293F cells.

[0055] Figure 30: Binding of anti-CSFIR antibodies to human CSF1R expressing HEK293F cells.

[0056] Figure 31: BLI binding data for TREM2 2G4 x CSF1R 1H9, TREM2 1F7 x CSF1R 1H9, TREM2 2G4 x CSF1R 1B6, and TREM2 BMK x CSF1R 1H9 bispecific antibodies.

[0057] Figure 32: BLI binding data for TREM2 2G4 x CSF1R 1H9 bispecific antibody.

[0058] Figure 33: BLI binding data for TREM2 1F7 x CSF1R 1H9 bispecific antibody.

[0059] Figure 34: BLI binding data for TREM2 2G4 x CSF1R 1B6 bispecific antibody.

[0060] Figure 35: BLI binding data for TREM2 BMK x CSF1R 1H9 bispecific antibody.

[0061] Figure 36: Assay to demonstrate binding bispecific antibodies to CSF1R and TREM2.

[0062] Figure 37: Detection of bispecific binding to cell surface CSF1R and soluble TREM2. (A) Light grey is CSF1R 1H9 x TREM2 2G4, dark grey is CSF1R 1H9. (B) Light grey is CSF1R 1H9 x TREM2 1F7, dark grey is CSF1R 1H9. (C) Light grey is CSF1R 1H9 x TREM2 BMK, dark grey is CSF1R 1H9. (D) Light grey is CSF1R 1H8 x TREM2 2G4, dark grey is CSF1R 1H8. Figure 38: Quantification of fluorescence intensity from TREM2 biotin / streptavidinPE.

[0063] Figure 39: Detection of bispecific binding to cell surface TREM2 and soluble CSF1R. A) Light grey is CSF1R 1H9 x TREM2 2G4, dark grey is TREM2 2G4. (B) Light grey is CSF1R 1H8 x TREM2 2G4, dark grey is TREM2 2G4. (C) Light grey is CSF1R 1H9 x TREM2 1F7, dark grey is TREM2 1F7. (D) Light grey is CSF1R 1H9 x TREM2 BMK, dark grey is TREM2 BMK.

[0064] Figure 40: Quantification of fluorescence intensity from CSF1R biotin / streptavidinPE.

[0065] DETAILED DESCRIPTION

[0066] A subset of macrophages known as Scar-Associated Macrophages (SAMac) are dramatically increased in abundance in cirrhotic livers compared to normal livers, are selectively located within regions of fibrosis within the liver, and have pro-fibrotic activity in an in vitro culture system (Ramachandran, P., R. Dobie, J. R. Wilson- Kanamori, E. F. Dora, B. E. P. Henderson, N. T. Luu, J. R. Portman, el al. 2019. “Resolving the Fibrotic Niche of Human Liver Cirrhosis at Single-Cell Level.” Nature 575 (7783): 512-18). Analysis of the transcriptome of SAMac cells by single-cell sequencing (scRNAseq) has identified two cell surface receptors, TREM2 and CD9, that together distinguish SAMac cells from the eight other macrophage subpopulations within the liver as well as from other cell types in the liver.

[0067] The present inventors have noted that it would be desirable to develop therapeutic agents for the targeting of SAMac cells due to their pathogenic and pro-fibrotic activity in the context of fibrotic disease, and their increased abundance in fibrotic disease. SAMac cells are known to be associated with TREM2 and CD9 but, as discussed in the Examples section, it would be preferable to identify receptor pairs for improved targeting of these cells.

[0068] As detailed in the Examples section, the inventors have extensively reviewed 2886 potential cell surface receptor targets in order to identify those that are expressed at a suitable level on the target cells (single cell data analysed for 14,014 cells) but which are not expressed at an unsuitable level on off-target cells (single cell data analysed for 56,944 cells). In particular, the inventors reviewed the mean expression levels in target and off-target cells, the target cell fraction expressing the receptors, the off-target cell fraction expressing the receptors, the receptor class and topology, and the homology in non-human primate and mouse models. Once suitable candidates were identified, the inventors analysed 3,226,515 cells from 40 organs and tissues to determine the systemic expression.

[0069] After synthesising all these data and considerations, the inventors have identified the following receptor pairs as suitable for targeting SAMac cells: triggering receptor expressed on myeloid cells 2 (TREM2) and macrophage scavenger receptor 1 (MSR1), TREM2 and colony stimulating factor 1 receptor (CSF1R), and TREM2 and C-type lectin domain family 7 member A (CLEC7A).

[0070] Thus, in a first aspect, there is provided herein a multispecific molecule comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for any one of CSF1R, MSR1, or CLEC7A.

[0071] In an embodiment, there is provided a multispecific molecule comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CSF1R.

[0072] In an embodiment, there is provided a multispecific molecule comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for MSR1.

[0073] In an embodiment, there is provided a multispecific molecule comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CLEC7A. TREM2 is a protein expressed on various cell types, including myeloid cells such as macrophages. An exemplary human sequence of TREM2 is inUniprot as Q9NZC2-1 (vl - 2000-10-01).

[0074] CSF1R is a transmembrane tyrosine kinase receptor. This receptor is essential for the survival, proliferation, and differentiation of monocytes and macrophages. It binds to colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), leading to the activation of downstream signalling pathways. An exemplary human sequence of CSF1R is inUniprot as P07333-1 (v2 - 1994-06-01).

[0075] MSR1 is a class A macrophage scavenger receptor, also known as CD204. MSR1 protein is homo-trimeric transmembrane glycoprotein consisting of six distinct domains, with the collagen-like domain and the scavenger receptor cysteine-rich (SRCR) domain being most relevant to its function. MSR1 has ability to bind a wide range of molecules, including modified low-density lipoproteins (LDL) and bacterial antigens. This receptor plays a role in host defence mechanisms, including the clearance of pathogens and the metabolism of lipids by macrophages. An exemplary human sequence of MSR1 is in Uniprot as P21757-1 (vl - 1991-05-01).

[0076] CLEC7A is also known as Dectin- 1. It is a type II transmembrane receptor that plays a role in the innate immune system's response to fungal infections. Dectin- 1 recognizes P-glucans present on the surface of fungi, triggering phagocytosis and the production of pro-inflammatory cytokines through its immunoreceptor tyrosine-based activation motif (IT AM). This receptor is primarily expressed on the surface of myeloid cells, including macrophages, neutrophils, and dendritic cells. An exemplary human sequence of CLEC7A is in Uniprot as Q9BXN2-1 (vl - 2001-06-01).

[0077] A multispecific molecule is a molecule that can specifically bind to at least two targets (referred to herein as “antigens”). In some embodiments, the multispecific molecules disclosed herein are bispecific and so specifically bind to two targets. In other embodiments, the multispecific molecules specifically bind to three or more targets.

[0078] The first and / or second antigen-binding domains may be low affinity. The combination of both antigen-binding domains may lead to a higher avidity, and hence the multispecific antibodies may have a reduced binding to cells expressing only one target but a stronger binding to cells expressing both targets. In some embodiments, the multispecific molecules are cis-acting multispecific molecules or are cis-acting bispecific antibodies.

[0079] Exemplary assays to determine whether an agent is cis-acting are discussed in Example 5. In some embodiments, the multispecific molecules bind to cells expressing TREM2 and any one of MSR1, CSF1R, or CLEC7A, but bind weakly - or do not bind - to cells expressing only one of these targets.

[0080] The multispecific molecule may comprise a polypeptide chain. The multispecific molecule may comprise two or more polypeptide chains. The multispecific molecule may be functional when consisting only of one or more polypeptide chains but may optionally be conjugated to other moieties.

[0081] The first and / or second antigen-binding domains may be based on an antibody scaffold. The first antigen-binding domain may be or may comprise an antigen-binding portion of an antibody and / or the second antigen-binding domain may be or may comprise an antigen-binding portion of an antibody. In some examples, the first and / or antigen-binding domains are murine, human, humanized, or a chimeric antibodies or antigen-binding fragments thereof. The antigen-binding domains may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. The antigen-binding domains may be mutated to alter such post-translational modifications, for example by adding, removing or replacing one or more amino acid residues to form or remove a glycosylation site. The antigen-binding domains may be modified for example by amino acid substitution to remove potential proteolytic sites. The antigen-binding domains may comprise one or more substitutions, deletions and / or insertions which remove a post-translational modification (PTM) site, for example a glycosylation site (N-linked or O-linked), a deamination site, a phosphorylation site or an isomerisation / fragmentation site.

[0082] In some examples of full-length antibodies, each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH domain) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable domain (abbreviated herein as VL domain) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH domain and VL domain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0083] The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Such fragments may contain all six CDRs of the antibody. It has been shown that the antigen-binding function of an antibody can be performed by portions or fragments of a full-length antibody. Examples of binding portions encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb (domain antibody) fragment (Ward et al., (1989) Nature 341:544-546; WO 90 / 05144 Al, each herein incorporated by reference in its entirety), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). The disclosure also encompasses a Fab' fragment. Fab' fragments can be formed by the reduction of F(ab')2 fragments. Fab' is derived from F(ab')2; therefore, it may contain a small portion of Fc. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. In some embodiments, scFv molecules may be incorporated into a fusion protein. In some embodiments, provided herein is a single chain camelid antibody. In some embodiments, provided herein is a shark heavy chain antibody (V-NAR). See, English et al. (2020) Antibody Therapeutics, 3(1): 1-9. Examples of antigen-binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.). In some embodiments, provided herein is a single domain antibody.

[0084] The term “humanized” is intended to refer to an antibody, or an antigen-binding portion thereof, that has been engineered to comprise one or more human framework regions in the variable domain together with non-human (e.g., mouse, rat, or hamster) CDRs of the heavy and / or light chain. In some embodiments, a humanized antibody comprises sequences that are entirely human except for the CDRs. An antibody molecule or antigen-binding portion thereof may comprise one or more human variable domain framework scaffolds into which the CDRs have been inserted. In some embodiments, the VH domain, the VL domain, or both the VH domain and the VL domain of an antibody or antigen-binding portion provided herein comprise one or more human framework region amino acid sequences. Examples of humanized antibodies and suitable techniques for their generation are provided in Hwang et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. 5,225,539; U.S. 5,530,101; U.S. 5,585,089; U.S. 5,693,761; U.S. 5,693,762; U.S. 6,180,370; and WO 90 / 07861, each of which is incorporated herein by reference in its entirety. When choosing FR to flank CDRs, for example when humanizing or optimizing an antibody, FRs from antibodies that contain CDR sequences in the same canonical class are preferred.

[0085] The term “chimeric” is intended to refer to an antibody molecule, or an antigen-binding portion thereof, in which the variable domain sequences are derived from one species and at least one constant region sequence is derived from another species. For example, one or all the variable domains of the light chain(s) and / or one or all the variable domains of the heavy chain(s) of a mouse antibody (e.g., a mouse monoclonal antibody) may each be joined to a human constant region, such as, without limitation an IgGl or an IgG4 human constant region. Examples of chimeric antibodies and suitable techniques for their generation are provided in U.S. 4,816,567; U.S. 4,975,369; and U.S. 4,816,397, each of which is incorporated herein by reference in its entirety.

[0086] Examples of suitable formats for antigen-binding domains that are antibody fragments include a Fab, a Fab', an F(ab')2, an Fd, an Fv, an scFv, a single domain antibody (sdAb), a maxibody, an scFv-Fc, an scFc-Fc-scFv, an scFv-CH, an scFv-CH3, an sc-Fv-zipper, an scFab, a minibody, an intrabody, a diabody, a triabody, a tetrabody, a v-NAR, or a bis-scFv.

[0087] The antigen-binding domains of a multispecific molecule disclosed herein may cross-compete with an exemplary antigen-binding domain disclosed herein. The terms “cross-compete”, “cross-competition”, “cross-block”, “crossblocked” and “cross-blocking” are used interchangeably herein to mean the ability of an antibody or an antigenbinding portion thereof to interfere with the binding directly or indirectly through allosteric modulation of the antibodies of the disclosure to the target. The extent to which an antibody or portion thereof is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block or cross-compete, can be determined using competition binding assays. One example of a binding competition assay is Homogeneous Time Resolved Fluorescence (HTRF). One particularly suitable quantitative cross-competition assay uses a FACS- or an Alphascreen-based approach to measure competition between the labelled (e.g., His-tagged, biotinylated or radioactive labelled) antibody or portion thereof and the other antibody or portion thereof in terms of their binding to the target. In general, a cross-competing antibody or portion thereof is, for example, one which will bind to the target in the cross-competition assay such that, during the assay and in the presence of a second antibody or portion thereof, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g. in a FACS-based competition assay) of the maximum theoretical displacement (e.g. displacement by cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked) by the potentially cross-blocking antibody or fragment thereof that is present in a given amount. In some embodiments, cross-competing antibodies or portions thereof have a recorded displacement that is between 10% and 100%, or between 50% and 100%.

[0088] In some embodiments, the multispecific molecule may be a multispecific antibody or a bispecific antibody.

[0089] In some examples, the first antigen-binding domain is a TREM2 specific antibody fragment comprising one or more amino acid sequences of antibody 2G4 or 1F7, for instance a set of CDRs from these clones. This is discussed further in the relevant section herein.

[0090] In some examples, the second antigen-binding domain is a CSF1R specific antibody fragment comprising one or more amino acid sequences of antibody 1B7, 1B6, 1H8, or 1H9, for instance a set of CDRs from these clones. This is discussed further in the relevant section herein.

[0091] As used herein, a CDR is said to be “invariant” when it is maintained without changes in a sequence that is otherwise not 100% identical to the recited variable region sequence. Thus, the CDRs may be invariant where lower degrees of sequence identity are disclosed in combination with V region sequences. Any of the CDR sequences disclosed herein may include one, two, or three additional residues at the N-terminal side and / or the C-terminal side. Any of the CDR sequences disclosed herein may include one to ten additional residues at the N-terminal side and / or the C-terminal side. The additional residues may be those contiguous to the recited CDR within the relevant sequence of the relevant Table herein, i.e. the CDRs may include a longer sequence from the parent clone. In an example, the CDRs grafted onto a framework may be according to the CDRs recited in one of Tables 1 to 6, and may include one, two, or three additional residues at the N-terminal side and / or the C-terminal side, wherein the additional residues are the contiguous residues of the relevant sequence in the Table.

[0092] The first antigen-binding domain may have an affinity that is the same as or similar to the affinity of 2G4 or 1F7 for TREM2. The affinity may be no more than 5-fold higher or lower, 4-fold higher or lower, 3-fold higher or lower, 2-fold higher or lower. The affinity may be measured as Kd and may be no more that ±50%, ±40%, ±30%, ±20%, or ±10%.

[0093] The binding affinity (KD) of the first antigen-binding domain may be from 10-7M to IO10M or from 8xl0-8M to 3xl0-9M. The KD may be from 10-7M to IO-8M, from 9xl0-8M to 7xl0-8M, or approximately 8xl0-8. The KD may be from IO-8M to 10-1°, from 5xl0-9to 3xl0-9, or approximately 4xl0-9. The affinity may be for human TREM2 and may be measured by SPR as described in Example 7.

[0094] The second antigen-binding domain may have an affinity that is the same as or similar to the affinity of 1B7, 1B6, 1H8, or 1H9 for CSF1R. The affinity may be no more than 5-fold higher or lower, 4-fold higher or lower, 3-fold higher or lower, 2-fold higher or lower. The affinity may be measured as Kd and may be no more that ±50%, ±40%, ±30%, ±20%, or ±10%.

[0095] The second antigen-binding domain may have a binding affinity (KD) within the range of IO-5M to IO-12M. For example, the second antigen-binding domain is from IO-6M to 10-1° M, or from 10-7M to 10-11M. The binding affinity of second antigen-binding domain may be from 2xl0-6M to IxlO-9M, 1.5xl0-6M to 5xl0-9M, IxlO-6M to IxlO-8M, or 8xl0-7M to 5xl0-8M. In an example, the binding affinity is from 1.5xl0-6M to IxlO-7M, IxlO-6M to 5xl0-7M, 9xl0-7M to 6xl0-7M, or approximately 8xl0-7M. In an example, the binding affinity is from 1.5xl0-7M to IxlO-8M, IxlO-7M to 5xl0-8M, 9xl0-8M to 6xl0-8M, or approximately 7xl0-8M. In an example, the binding affinity is from IxlO-6M to IxlO-9M, 5xl0-7M to 5xl0-8M, 2xl0-7M to 8xl0-8M, or approximately IxlO-7M. The affinity may be for human CSF1R and may be measured by SPR as described in Example 9.

[0096] In a particular embodiment, the first antigen-binding domain is an scFv specific for TREM2 and / or the second antigen-binding domain is an scFv specific for any one of MSR1, CSF1R, or CLEC7A.

[0097] The first and / or second antigen-binding domains may be an antibody mimetic, for instance a designed ankyrin repeat protein (DARPin) (See chapter 5. "Designed Ankyrin Repeat Proteins (DARPins); From Research to Therapy", Methods in Enzymology, vol 503: 101~ 134 (2012); or "Efficient Selection of DARPins with Sub- nanomolar Affinities using SRP Phage Display", J. Mol. Biol. (2008) 382, 1211-1227).

[0098] The domain that is specific for CSF1R may comprise CSF1, or a variant or portion thereof. CSF1 is a natural ligand for CSF1R. The CSF1 may comprise mutations that prevent dimerization of the receptor and, hence, the CSF1 may function as an antagonist of CSF1R signalling. The mutations may be C3 IS and M27R. An example of a mutant CSF1 is provide in Zur et al. (“Engineering a monomeric variant of macrophage colony-stimulating factor (M-CSF) that antagonizes the c-FMS receptor” Biochem J. 2017 Jul 20;474(15):2601-2617. doi: 10.1042 / BCJ20170276). Thus, in an embodiment, the second antigen-binding domain comprises CSF1, or a portion thereof, wherein the CSF1 or portion thereof comprises the mutations C3 IS and M27R. As used herein, “specific for” a target means that the domain is capable of binding to a particular target to a greater extent than the domain would bind to dissimilar targets. Specificity can be tested by comparison with a control domain that has not been designed or selected for specificity to the target in question. In the context of an antibody, portion thereof, or antibody scaffold, the term “specific for” means that the antibody binds to an antigen whereas an isotype control antibody would not bind or would only minimally bind. An “isotype control antibody” is an antibody, portion thereof, or antibody scaffold that matches the antibody in question but has an irrelevant specificity. For instance, the isotype control may be the same as the antibody or portion in question but may have an entirely different set of CDRs or may have been raised against an irrelevant antigen.

[0099] The multispecific molecule may comprise an immunoglobulin constant region. The multispecific molecule may comprise an immunologically inert constant region. The multispecific molecule may comprise a constant region capable of binding to Fc receptors, or a specific pattern of Fc receptors such as activating Fc receptors. The constant region may be modified to have an increase or decrease in one or more effector activities. In some examples, the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2. In additional examples, the immunoglobulin constant region is IgGl, IgG2, IgG3, IgGlnull, IgG4(S228P), IgAl or IgA2.

[0100] The multispecific molecule may comprise other domains capable of binding or being bound. For instance, as discussed above the molecules may comprise an Fc region and may be capable of interacting with Fc receptors. In this situation, it is common to refer to such molecules as comprising an additional functionality rather than an additional specificity. For instance, bispecific antibodies comprising a functional Fc domain are sometimes referred to as trifunctional.

[0101] In some examples, particularly those where an anti-CSFIR domain blocks CSF1R signalling, the Fc region may be effector-function deficient. For instance, an ADCC-deficient IgG isotype, an Fc region comprising an N297A mutation, or an Fc region comprising the LALA mutations.

[0102] Non-exhaustive examples of bispecific molecule formats include those comprising two monovalent scFvs linked in tandem (e.g. bispecific T cell engagers (BiTEs) are in this format), dual-affinity re-targeting (DART) formats comprising a diabody format stabilized by a C-terminal disulfide bridge, Dock-And-Lock formats that comprise antigen-binding domains linked by dimerization domains and anchoring domains, tandem diabodies (TandAbs), and the like. Other methods of creating bispecific molecules include using orthogonal Fab interface, DuoBody, XmAb, CrossMab, and knobs-into-holes (KiH). Other formats include approaches involving the replacement of one parental mAb's CH1 / CL region with the TCR constant domain, including WuXiBody™ bispecific antibodies (e.g. Guo et al. “A potential downstream platform approach for WuXiBody -based IgG-like bispecific antibodies”, Protein Expression and Purification, Volume 173, 2020, 105647, ISSN 1046-5928, https: / / doi.org / 10.1016 / j.pep.2020.105647). Yet another approach involves moving a disulphide bridge in one of the ‘half mAbs’ from constant domains (CH1 / CL) to variable domains (VH / VL) to favours formation of a heterodimer (e.g. the bYLok™ format).

[0103] Examples ofbispecific molecules and formats for creating such molecules are provided inUS2012 / 0095192Al; Deppisch et al., Mol Cancer Ther; 14(8) August 2015 (trifunctional antibodies); Eissler et al. Mol Med.

[0104] 2013; 19(1):54-61 (Surek); US8,277,806B2; WO2012143524; W02014028560A2 (Dock-and-lock example - anti-CD19 and anti-CD3); Cheadle, Curr Opin Mol Ther. 2006 Feb;8(l):62-8 (MT-103 BiTE); Rossi et al. MAbs. 2014;6(2):381-391 (Dock-and-Lock); Ordonez-Reyes et al. Pharmaceutics. 2022;14(6):1243 (multiple examples of formats - e.g. Fig. 1); Wu and Cheung, Pharmacol Ther. 2018;182:161-175 (multiple examples of formats - e.g. Fig. 1); Goldenberg et al. Journal of Nuclear Medicine January 2008, 49 (1) 158-163 (Dock-and-Lock); and Labrijn et al. Nature Reviews | Drug Discovery 18, pages 585-608 (2019) (each of which is incorporated herein by reference). SAMac cells are further explained in Ramachandran el al. (2019 - herein incorporated by reference), where they are referred to as SAM$.

[0105] The multispecific molecules disclosed herein bind to SAMac cells. This means that, given a naturally occurring population of SAMac cells, the multispecific molecules will bind to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells.

[0106] The multispecific molecules may be selective for SAMac cells.

[0107] As used herein, the term “selective” means that the multispecific molecules bind to a higher proportion of target cells in comparison to the binding to a population of non-target cells from the same organism. In some embodiments, the multispecific molecules bind to a higher proportion of target cells in comparison to the binding to a population of non-target cells from key organs in the same organism, such as the liver, brain, lungs, kidneys, skin, intestines, circulatory system, heart, etc. In some embodiments, the multispecific molecules bind to a higher proportion of target cells in comparison to the binding to a population of non-target cells from the same organ. In some embodiments, the multispecific molecules bind to a higher proportion of SAMac cells in comparison to the binding to a population of non-SAMac macrophages. In some embodiments, the multispecific molecules bind to a higher proportion of SAMac cells in comparison to the binding to a population of non-SAMac macrophages from the same organ. In some embodiments, the multispecific molecules bind to a higher proportion of SAMac cells in comparison to the binding to a population of non-SAMac liver macrophages. The non-target cells for the aforementioned embodiments may be otherwise unsorted cells from the organism in question, from the organ in question, or of the cell type in question. In any of the aforementioned embodiments, “selective” may mean that the multispecific antibody binds to at least 90% of target cells and less than 10% of non-target cells.

[0108] Exemplary assays for determining selectivity are disclosed in Example 5 herein.

[0109] In some embodiments, the multispecific molecules have cytotoxic activity or promote cytotoxic activity towards SAMac cells. For instance, the multispecific molecules may be capable of inducing antibody-directed cellular cytotoxicity (ADCC) or complement — dependent cytotoxicity (CDC). The multispecific molecules may be linked to another agent that contributes to the cytotoxicity, such agents are discussed further herein.

[0110] The multispecific molecules may only have a cytotoxic effect when bound to a cell expressing both targets ( / . e. TREM2; and any one of MSR1, CSF1R, or CLEC7A) and not when bound to a cell expressing only one target. For instance, the ADCC and CDC require aggregation, and so the multispecific molecules will not aggregate and cause cytotoxicity when bound to a cell expressing a single target.

[0111] In embodiments where the multispecific molecules comprise an antigen-binding domain that is specific for CSF1R, the multispecific molecule may block CSF1R signalling. For instance, the binding of the multispecific molecule may prevent or reduce ligand binding to CSF1R, and hence reduce or abolish intracellular signalling caused by the binding of CSF-1 to the cells. In other embodiments, the binding of the multispecific molecule may prevent intracellular signalling from CSF1R, for instance by preventing or reducing CSF1R dimerization. The multispecific molecules may block CSF1R signalling in SAMac cells.

[0112] Signalling from CSF1R can be measured by a cell-based reporter assay, for instance where downstream signalling is linked to a detectable reporter, hence enabling the skilled person to determine if a particular agent affects CSF1R signalling. Non-limiting examples of a suitable reporter cell lines include those where a reporter gene (such as a gene encoding firefly luciferase) is controlled by the MAPK / ERK signalling pathway. Commercially available kits are available from BPS Bioscience or DIscoverX. An alternative approach to determine effects on signalling downstream of CSF1R is to measure functional effects of CSFR1 signalling. For instance, in the presence of a CSF1R agonist, such as the natural ligand, SAMac cells proliferate or the level of proliferation is increased. Thus, whether an agent can block CSF1R signalling may be measured by determining the effect on SAMac cell proliferation in the presence of a CSF1R agonist.

[0113] Other techniques include the use of scRNAseq to detect transcriptional changes associated with CSF1R signalling. Yet further techniques include competitive binding assays, such as Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), or BLI, to determine whether the agent affects CSF-1 to binding to CSF1R.

[0114] In another embodiment, the multispecific molecule may block CSF1R signalling in a manner that prevents the differentiation of monocytes into SAMac cells.

[0115] In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for MSR1. In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CSF1R. In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CLEC7A.

[0116] The multispecific molecule may be an isolated molecule.

[0117] The multispecific molecule may be linked to a pay load or agent. The multispecific molecule may be a part of an immunoconjugate. Thus, in an embodiment, there is provided an immunoconjugate comprising a multispecific molecule comprising a first antigen-binding domain that is specific for TREM2; and a second antigen-binding domain that is specific for any one of MSR1, CSF1R, or CLEC7A. The immunoconjugate may further comprise another agent, such as a label, a therapeutic agent, or a cytotoxic agent.

[0118] The multispecific molecule may be linked to an agent that can induce changes in SAMac cells. For instance, the agent may be cytotoxic or radioactive and so may kill SAMac cells upon binding of the multispecific molecule. Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin, tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, flurouracils, etoposide, taxol, taxol analogs, platins such as cis-platin and carbo-platin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterlins, esperamicins, and maytansinoids.

[0119] The linkage may be any suitable for linking the agent or payload to the multispecific molecule. For instance, the linkage may comprise a covalent or non-covalent bond. The linkage may be via a bi-functional linker. The linkage may be via two moieties capable of forming a non-covalent interaction, such as streptavidin and biotin.

[0120] In another aspect, there is provided one or more nucleic acid molecules encoding a multispecific molecule as disclosed herein.

[0121] The multispecific molecule may be a single polypeptide chain, in which case it may be encoded by a single nucleic acid molecule. In other embodiments, the multispecific molecule may comprise two or more polypeptide chains and one nucleic acid molecule may encode the two or more polypeptide chains. The genes encoding these chains may be under the control of separate promoters. In other examples, the genes encoding these chains may under the control of the same promoter, but separated by a region that can induce ribosomal skipping or by a selfcleaving peptide sequence. Examples of such features include P2A, an internal ribosome entry site (IRES), or an intein.

[0122] In some embodiments, the multispecific molecule may comprise two or more polypeptide chains and each polypeptide chain may be encoded on a different nucleic acid molecule. Thus, in an embodiment, there is provided two or more nucleic acid molecules encoding the multispecific molecule.

[0123] In another aspect, there is provided one or more vectors comprising one or more nucleic acid molecules as disclosed herein. The one or more vectors may be expression vectors or may be vectors designed for delivery to a subject, such as a human patient.

[0124] In certain vectors, a nucleic acid molecule is operatively linked to one or more regulatory sequences suitable for expression of the nucleic acid segment in a host cell. In some cases, an expression vector comprises sequences that mediate replication and comprises one or more selectable markers. The vector may be capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0125] In another aspect, there is provided a cell comprising one or more nucleic acid molecules as disclosed herein or one or more vectors as disclosed herein. The cell may be a recombinant cell. The cell may be a cell suitable for commercial production of recombinant polypeptides and proteins. In some examples, the cell is mammal, bacteria, yeast, or plant derived. For instance, the cells may be CHO cells, NS0 cells, HEK cells, E. coli cells, Pichia pastoris cells, or other suitable cells.

[0126] In another aspect, there is provided a method of producing a multispecific molecule as disclosed herein, comprising culturing a cell disclosed herein under conditions that result in expression and / or production of the multispecific molecule, and isolating the multispecific molecule thereof from the cell or culture.

[0127] In another aspect, there is provided a cell comprising a multispecific molecule as disclosed herein, one or more nucleic acid molecules as disclosed herein, or one or more vectors as disclosed herein. The cell may be a mammalian or human cell. The cell may be an immune cell, such as a B cell. The cell may be suitable for administration to subjects in order to deliver the multispecific molecule in vivo.

[0128] The multispecific molecule as disclosed herein may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised proteins may be re-constituted in sterile water and mixed with saline prior to administration to an individual. A nucleic acid as disclosed herein or a vector as disclosed herein may be provided in a lyophilised form. A cell as disclosed herein may be provided in a stable form for storage, for instance, the cells may be frozen in the presence of a cryoprotectant. In other embodiments, all of these products may be provided in a form suitable for administration.

[0129] Thus, in another aspect, there is a provided a pharmaceutical composition comprising a multispecific molecule as disclosed herein, one or more nucleic acid molecules as disclosed herein, one or more vectors as disclosed herein, or a cell as disclosed herein.

[0130] The multispecific molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the multispecific molecule. Thus, pharmaceutical compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the multispecific or bispecific molecule. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route.

[0131] In an aspect of the invention, there is provided a multispecific molecule of the present disclosure for use as a medicament.

[0132] In another aspect of the invention, there is provided an immunoconjugate, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for use as a medicament.

[0133] In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for MSR1 for use as a medicament. In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CSF1R for use as a medicament. In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CLEC7A for use as a medicament.

[0134] In an embodiment, there is provided a multispecific molecule comprising a first antigen-binding domain that is a TREM2 specific antibody fragment comprising a set of six CDRs of antibody 2G4 or 1F7 as disclosed herein and a second antigen-binding domain that is a CSF1R specific antibody fragment comprising a set of six CDRs of antibody 1B7, 1B6, 1H8, or 1H9 as disclosed herein, for use as a medicament. In some examples, all of the CDRs are determined using the same scheme. Sets of CDRs are disclosed herein in the relevant sections. The first antigen-binding domain may a TREM2 specific antibody fragment comprising a light chain variable domain as disclosed herein and a heavy chain variable domain as disclosed herein, and the second antigen-binding domain may be a CSF1R specific antibody fragment comprising a light chain variable domain as disclosed herein and a heavy chain variable domain as disclosed herein. The first antigen-binding domain may comprise: a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 or 16 and a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 25; and the second antigen-binding domain may comprise: a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, 46, 61, or 76 and a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40, 55, 70, 85. These sequences may be paired as depicted in Tables 1 to 6. The CDRs may be invariant. The CDRs may be as depicted in any one of Tables 1 to 6 and may be determined by the same scheme.

[0135] SAMac have pro-fibrotic properties, and they may be present in tissues other than liver tissue (Ouyang et al. “Systems level identification of a matrisome-associated macrophage polarisation state in multi-organ fibrosis”. Elife. 2023 Sep 14;12:e85530. doi: 10.7554 / eLife.85530. PMID: 37706477; PMCID: PMC10547479). Thus, the multispecific molecules of the present disclosure may be for use in treating fibrosis. The immunoconjugates, one or more nucleic acids, one or more vectors, cells, or pharmaceutical compositions of the present disclosure may be for use in treating fibrosis. In some examples, the fibrosis affects the liver, lung, kidney, intestine, skin, and / or heart.

[0136] SAMac cells are selectively located within the fibrotic niche within cirrhotic livers. Thus, the multispecific molecules, immunoconjugates, one or more nucleic acids, one or more vectors, cells, or pharmaceutical compositions of the present disclosure may be for use in treating fibrosis of the liver.

[0137] In some embodiments, the multispecific antibodies, immunoconjugates, one or more nucleic acids, one or more vectors, cells, or pharmaceutical compositions of the present disclosure are for use in treating idiopathic pulmonary fibrosis (IPF), diabetic nephropathy, lupus nephritis, inflammatory bowel disease, or scleroderma. In particular embodiments, the multispecific antibodies, immunoconjugates, one or more nucleic acids, one or more vectors, cells, or pharmaceutical compositions of the present disclosure are for use in treating fibrotic liver disease, cirrhosis of the liver, alcohol-associated liver disease, metabolic dysfunction-associated steatohepatitis, or primary sclerosing cholangitis.

[0138] In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for MSR1 for use in treating fibrosis of the liver. In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CSF1R for use in treating fibrosis of the liver. In a particular embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CLEC7A for use in treating fibrosis of the liver.

[0139] In another aspect of the invention, there is provided a method of treating fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of an multispecific molecule, an immunoconjugate, one or more nucleic acids, one or more vectors, a cell, or a pharmaceutical composition of the present disclosure to the subject. The method may be any use for which the agents disclosed herein are suitable. As discussed above, the fibrosis may be fibrosis of the liver, lung, kidney, intestine, skin, and / or heart. In particular, the fibrosis may be of the liver. The method may be for the treatment of idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease, or scleroderma. The method may be for the treatment of fibrotic liver disease, cirrhosis of the liver, alcohol-associated liver disease, metabolic dysfunction-associated steatohepatitis, or primary sclerosing cholangitis.

[0140] The methods may be used for the treatment of the human or animal body. The methods may be for treatment of a mammalian subject. The methods may be for treatment of a mouse, rat, rabbit, dog, cat, horse, or pig. Preferably, the methods are for the treatment of a human patient.

[0141] Administration is normally in a “therapeutically effective amount”, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of medical professionals and may depend on the severity of the symptoms and / or progression of a disease being treated. A therapeutically effective amount or suitable dose of a medicament may be determined by comparing in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the medicament is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the medicament and the nature of any detectable label or other molecule attached to the antibody.

[0142] The methods of treatment include prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset).

[0143] The terms “prophylactic”, “preventative”, or “preventing” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. Prevention and the like do not mean preventing a subject from ever getting the specific disease or disorder. Prevention may require the administration of multiple doses. Prevention can include the prevention of a recurrence of a disease in a subject for whom all disease symptoms were eliminated, or prevention of recurrence in a relapsing-remitting disease. Administration may be via any suitable method, such as intravenous or subcutaneous administration, e.g. by injection. Administration may be of a single dose or may be repeated periodically. The agents disclosed herein may be administered as a monotherapy or in combination with other agents.

[0144] In another aspect, there is provided a method of inhibiting the fibrotic activity, reducing the proliferation, or reducing the survival of SAMac cells, the method comprising contacting the cells with a multispecific molecule as disclosed herein. In some embodiments, the method is in vitro, in vitro, or ex vivo.

[0145] The fibrotic activity of the SAMac cells may be measured by determining the expression of fibrillar collagen. A purely exemplary assay is provided below, and further details are found in Ramachandran et al., 2019.

[0146] For assessment of fibrillar collagen gene expression, primary human hepatic stellate cells (HSC) are cultured in stellate cell medium (SteCM, ScienCell, 5301) on Poly-L-Lysine (Sigma, P4832) coated T75 tissue culture flasks, according to the supplier’s protocol. The cells may, for instance, be between passage 3 and 5. HSC can be plated at 75,000 cells per well in 24 well-plates (Costar, 3524) in HSC media consisting of DMEM (Gibco, 21969-035) supplemented with 20 pM HEPES (Sigma, H3375,), 2 mM L-Glutamine (Gibco, 25030-024), 1% Penicillin Streptomycin (Gibco, 15140-122, Gibco) and 2% Foetal Bovine Serum (FBS, Gibco, 10270). HSC are then serum starved overnight (in HSC media without FBS), washed with PBS, then 250pl of conditioned media from the cells for which fibrotic activity is to be measured (e.g. the SAMac cells) added for 24 hours. HSC can then be harvested for RNA analysis.

[0147] The reduction of proliferation may be in comparison to control cells. As the skilled person would know, control cells are cells that are otherwise the same as the target cells, but which have not been contacted with the multispecific molecule. The cells may be contacted with a molecule that is structurally similar but lacks the specificities of the multispecific molecules and the control molecule (e.g. isotype control) may be delivered in the same vehicle and at the same dose as the multispecific molecule. The proliferation may be measured in vitro, e.g. by measuring the incorporation of a marker for measuring proliferation or by measuring the dilution of a pre- loaded marker suitable for measuring proliferation. The proliferation may be measured in vivo, e.g. by administering the agent and measuring the in vivo incorporation of a marker suitable for measuring proliferation or by quantifying the presence of cell cycle markers from cells extracted from the organism subsequent to administration of the agent.

[0148] The reduction of survival may be in comparison to control cells. As the skilled person would know, control cells are cells that are otherwise the same as the target cells, but which have not been contacted with the multispecific molecule. The cells may be contacted with a molecule that is structurally similar but lacks the specificities of the multispecific molecules and the control molecule (e.g. isotype control) may be delivered in the same vehicle and at the same dose as the multispecific molecule. The survival may be measured in vitro, e.g. by measuring the killing of cells that have been contacted with the multispecific molecules. The measurement may include quantifying the presence of apoptosis or cell death markers. The survival may be measured in vivo, e.g. by administering the agent and measuring and quantifying the presence of cell death or apoptosis markers.

[0149] Antigen-binding domains specific for TREM2

[0150] The multispecific molecules of the present disclosure comprise a domain that is specific for TREM2. Exemplary embodiments of TREM2 specific domains that may be components of the multispecific molecules disclosed herein are described in more detail below.

[0151] The antigen-binding domains may be antibodies, or antigen-binding portions thereof. The antibody may be an immunoglobulin (Ig) molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential target binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0152] In some embodiments, the TREM2 specific antibody or antigen-binding portion is human, humanized, or chimeric. In some embodiments, the TREM2 specific antibody or antigen-binding portion is humanized.

[0153] In some embodiments, a TREM2 specific antibody or antigen-binding portion is human (e.g. , fully human). A human antibody comprises human CDR sequences, human framework sequences and human constant region sequences.

[0154] The antigen-binding domain specific for TREM2 may comprise an antibody VH domain, or portion thereof, and an antibody VL domain, or portion thereof. The antigen-binding domain specific for TREM2 may comprise an antibody VH domain and an antibody VL domain.

[0155] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises one or more amino acid sequences of antibody 2G4 or 1F7. The combinations of VH domain, VL domain, and CDR sequences forming these antibodies are provided in Tables 1 and 2. In some embodiments, the VH domain sequence and / or the VL domain sequence comprises a signal sequence (also known as a signal peptide) at the amino-terminus.

[0156] Table 1. Amino acid sequences of anti-TREM2 antibody 2G4

[0157] Table 2. Amino acid sequences of anti-TREM2 antibody 1F7

[0158] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that cross-competes for binding to TREM2 with antibody 2G4 or 1F7 or an antibody that comprises one or more amino acid sequences of antibody 2G4 or 1F7.

[0159] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises the CDRs of antibody 2G4.

[0160] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain, or portion thereof, comprises a heavy chain complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 2, 5, or 8; a heavy chain complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 3, 6, or 9; and a heavy chain complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 4 or 7; and / or the VL domain, or portion thereof, comprises a light chain complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 11 or 14; a light chain complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 12 or 15; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 13.

[0161] The antigen-binding domain specific for TREM2 may be a TREM2 specific antibody or an antigen-binding portion thereof that comprises a set of CDRs from Table 1 defined by Kabat, a set of CDRs from Table 1 defined by IMGT, or a set of CDRs from Table 1 defined by Chothia.

[0162] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein:

[0163] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 2; an HCDR2 that comprises or is SEQ ID NO: 3; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13; or (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 5; an HCDR2 that comprises or is SEQ ID NO: 6; and an HCDR3 that comprises or is SEQ ID NO: 7; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 14; an LCDR2 that comprises or is SEQ ID NO: 15; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0164] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 8; an HCDR2 that comprises or is SEQ ID NO: 9; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13.

[0165] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, and a VL domain, wherein:

[0166] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 2; an HCDR2 that comprises or is SEQ ID NO: 3; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0167] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 5; an HCDR2 that comprises or is SEQ ID NO: 6; and an HCDR3 that comprises or is SEQ ID NO: 7; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 14; an LCDR2 that comprises or is SEQ ID NO: 15; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0168] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 8; an HCDR2 that comprises or is SEQ ID NO: 9; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13.

[0169] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1; and / or the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID

[0170] NO: 10.

[0171] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain wherein: the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1; and / or the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

[0172] The CDRs may be invariant. Thus, the antigen-binding domain specific for TREM2 may a TREM2 specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1; and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 2, 5, or 8; an HCDR2 comprising any one of SEQ ID NOs: 3, 6, or 9; and an HCDR3 comprising any one of SEQ ID NOs: 4 or 7; and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 11 or 14; an LCDR2 comprising any one of SEQ ID NOs: 12 or 15; and an LCDR3 comprising SEQ ID NO: 13. The antigen-binding domain specific for TREM2 may a TREM2 specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and

[0173] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 2; an HCDR2 that comprises or is SEQ ID NO: 3; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0174] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 5; an HCDR2 that comprises or is SEQ ID NO: 6; and an HCDR3 that comprises or is SEQ ID NO: 7; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 14; an LCDR2 that comprises or is SEQ ID NO: 15; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0175] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 8; an HCDR2 that comprises or is SEQ ID NO: 9; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13.

[0176] The antigen-binding domain specific for TREM2 may a TREM2 specific antibody or an antigen-binding portion thereof that comprises a VH domain and a VL domain, wherein the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 and the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and

[0177] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 2; an HCDR2 that comprises or is SEQ ID NO: 3; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0178] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 5; an HCDR2 that comprises or is SEQ ID NO: 6; and an HCDR3 that comprises or is SEQ ID NO: 7; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 14; an LCDR2 that comprises or is SEQ ID NO: 15; and an LCDR3 that comprises or is SEQ ID NO: 13; or

[0179] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 8; an HCDR2 that comprises or is SEQ ID NO: 9; and an HCDR3 that comprises or is SEQ ID NO: 4; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 11; an LCDR2 that comprises or is SEQ ID NO: 12; and an LCDR3 that comprises or is SEQ ID NO: 13.

[0180] In some embodiments, the antigen-binding domain specific for TREM2 comprises a VH domain that is or comprises SEQ ID NO: 1 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions and a VL domain that is or comprises SEQ ID NO: 10 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions. The framework regions are regions of the variable domain outside of the CDRs as defined by any definition or any one definition herein. The substitutions may be conservative substitutions. An example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919).

[0181] In a particular embodiment, the antigen-binding domain specific for TREM2 comprises a VH domain that is SEQ ID NO: 1 and a VL domain that is SEQ ID NO: 10.

[0182] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises the CDRs of antibody 1F7. In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 17, 20, or 23; an HCDR2 comprising any one of SEQ ID NOs: 18, 21, or 24; and an HCDR3 comprising any one of SEQ ID NOs: 19 or 22; and / or the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 26 or 29; an LCDR2 comprising any one of SEQ ID NOs: 27 or 30; and an LCDR3 comprising SEQ ID NO: 28.

[0183] The antigen-binding domain specific for TREM2 may be a TREM2 specific antibody or an antigen-binding portion thereof that comprises a set of CDRs from Table 2 defined by Kabat, a set of CDRs from Table 2 defined by IMGT, or a set of CDRs from Table 2 defined by Chothia.

[0184] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein:

[0185] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 17; an HCDR2 that comprises or is SEQ ID NO: 18; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0186] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 20; an HCDR2 that comprises or is SEQ ID NO: 21; and an HCDR3 that comprises or is SEQ ID NO: 22; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 29; an LCDR2 that comprises or is SEQ ID NO: 30; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0187] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 23; an HCDR2 that comprises or is SEQ ID NO: 24; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28.

[0188] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein:

[0189] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 17; an HCDR2 that comprises or is SEQ ID NO: 18; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0190] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 20; an HCDR2 that comprises or is SEQ ID NO: 21; and an HCDR3 that comprises or is SEQ ID NO: 22; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 29; an LCDR2 that comprises or is SEQ ID NO: 30; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0191] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 23; an HCDR2 that comprises or is SEQ ID NO: 24; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28.

[0192] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16; and / or the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID

[0193] NO: 25.

[0194] In some examples, the antigen-binding domain specific for TREM2 is a TREM2 specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein: the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16; and / or the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25.

[0195] The CDRs may be invariant. Thus, the antigen-binding domain specific for TREM2 may a TREM2 specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16; and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; and the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 17, 20, or 23; an HCDR2 comprising any one of SEQ ID NOs: 18, 21, or 24; and an HCDR3 comprising any one of SEQ ID NOs: 19 or 22; and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 26 or 29; an LCDR2 comprising any one of SEQ ID NOs: 27 or 30; and an LCDR3 comprising SEQ ID NO: 28.

[0196] The antigen-binding domain specific for TREM2 may a TREM2 specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; and

[0197] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 17; an HCDR2 that comprises or is SEQ ID NO: 18; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0198] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 20; an HCDR2 that comprises or is SEQ ID NO: 21; and an HCDR3 that comprises or is SEQ ID NO: 22; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 29; an LCDR2 that comprises or is SEQ ID NO: 30; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0199] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 23; an HCDR2 that comprises or is SEQ ID NO: 24; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28.

[0200] The antigen-binding domain specific for TREM2 may a TREM2 specific antibody or an antigen-binding portion thereof that comprises a VH domain and a VL domain, wherein the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16 and the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; and

[0201] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 17; an HCDR2 that comprises or is SEQ ID NO: 18; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28; or

[0202] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 20; an HCDR2 that comprises or is SEQ ID NO: 21; and an HCDR3 that comprises or is SEQ ID NO: 22; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 29; an LCDR2 that comprises or is SEQ ID NO: 30; and an LCDR3 that comprises or is SEQ ID NO: 28; or (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 23; an HCDR2 that comprises or is SEQ ID NO: 24; and an HCDR3 that comprises or is SEQ ID NO: 19; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 26; an LCDR2 that comprises or is SEQ ID NO: 27; and an LCDR3 that comprises or is SEQ ID NO: 28.

[0203] In some embodiments, the antigen-binding domain specific for TREM2 comprises a VH domain that is or comprises SEQ ID NO: 16 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions and a VL domain that is or comprises SEQ ID NO: 25 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions. The framework regions are regions of the variable domain outside of the CDRs as defined by any definition or any one definition herein. The substitutions may be conservative substitutions. An example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919).

[0204] In a particular embodiment, the antigen-binding domain specific for TREM2 comprises a VH domain that is SEQ ID NO: 16 and a VL domain that is SEQ ID NO: 25.

[0205] Antigen-binding domains specific for CSF1R

[0206] The multispecific molecules of the present disclosure comprise a domain that is specific for CSF1R. Exemplary embodiments of CSF1R specific domains that may be components of the multispecific molecules disclosed herein are described in more detail below.

[0207] The antigen-binding domains may be antibodies, or antigen-binding portions thereof. The antibody may be an immunoglobulin molecule comprising four polypeptide chains, two heavy chains and two light chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential target binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0208] In some embodiments, the CSF1R specific antibody or antigen-binding portion is human, humanized, or chimeric. In some embodiments, a CSF1R specific antibody or antigen-binding portion is humanized.

[0209] In some embodiments, a CSF1R specific antibody or antigen-binding portion is human (e.g., fully human). A human antibody comprises human CDR sequences, human framework sequences and human constant region sequences.

[0210] The antigen-binding domain specific for CSF1R may comprise an antibody VH domain, or portion thereof, and an antibody VL domain, or portion thereof. The antigen-binding domain specific for CSF1R may comprise an antibody VH domain and an antibody VL domain.

[0211] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises one or more amino acid sequences of antibody 1B7, 1B6, 1H8, or 1H9. The combinations of VH domain, VL domain, and CDR sequences forming these antibodies are provided in Tables 3, 4, 5, and 6. In some embodiments, the VH domain sequence and / or the VL domain sequence comprises a signal sequence (also known as a signal peptide) at the amino-terminus.

[0212] Table 3. Amino acid sequences of anti-CSFIR antibody 1B7

[0213] Table 4. Amino acid sequences of anti-CSFIR antibody 1B6

[0214] Table 5. Amino acid sequences of anti-CSFIR antibody 1H8

[0215] Table 6. Amino acid sequences of anti-CSFIR antibody 1H9

[0216] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigen- binding portion thereof that cross-competes for binding to CSF1R with antibody 1B7, 1B6, 1H8, or 1H9 or an antibody that comprises one or more amino acid sequences of antibody 1B7, 1B6, 1H8, or 1H9.

[0217] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises the CDRs of antibody 1B7. In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 32, 35, or 38; an HCDR2 comprising any one of SEQ ID NOs: 33, 36, or 39; and an HCDR3 comprising any one of SEQ ID NOs: 34 or 37; and / or the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 41 or 44; an LCDR2 comprising any one of SEQ ID NOs: 42 or 45; and an LCDR3 comprising SEQ ID NO: 43.

[0218] The antigen-binding domain specific for CSF1R may be a CSF1R specific antibody or an antigen-binding portion thereof that comprises a set of CDRs from Table 3 defined by Kabat, a set of CDRs from Table 3 defined by IMGT, or a set of CDRs from Table 3 defined by Chothia.

[0219] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein:

[0220] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 32; an HCDR2 that comprises or is SEQ ID NO: 33; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0221] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 35; an HCDR2 that comprises or is SEQ ID NO: 36; and an HCDR3 that comprises or is SEQ ID NO: 37; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 44; an LCDR2 that comprises or is SEQ ID NO: 45; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0222] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 38; an HCDR2 that comprises or is SEQ ID NO: 39; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43.

[0223] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein:

[0224] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 32; an HCDR2 that comprises or is SEQ ID NO: 33; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0225] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 35; an HCDR2 that comprises or is SEQ ID NO: 36; and an HCDR3 that comprises or is SEQ ID NO: 37; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 44; an LCDR2 that comprises or is SEQ ID NO: 45; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0226] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 38; an HCDR2 that comprises or is SEQ ID NO: 39; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43.

[0227] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31; and / or the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID

[0228] NO: 40.

[0229] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein: the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31; and / or the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40.

[0230] The CDRs may be invariant. Thus, the antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31; and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40; and the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 32, 35, or 38; an HCDR2 comprising any one of SEQ ID NOs: 33, 36, or 39; and an HCDR3 comprising any one of SEQ ID NOs: 34 or 37; and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 41 or 44; an LCDR2 comprising any one of SEQ ID NOs: 42 or 45; and an LCDR3 comprising SEQ ID NO: 43.

[0231] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40; and

[0232] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 32; an HCDR2 that comprises or is SEQ ID NO: 33; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0233] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 35; an HCDR2 that comprises or is SEQ ID NO: 36; and an HCDR3 that comprises or is SEQ ID NO: 37; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 44; an LCDR2 that comprises or is SEQ ID NO: 45; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0234] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 38; an HCDR2 that comprises or is SEQ ID NO: 39; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43.

[0235] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain and a VL domain, wherein the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 and the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40; and

[0236] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 32; an HCDR2 that comprises or is SEQ ID NO: 33; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43; or

[0237] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 35; an HCDR2 that comprises or is SEQ ID NO: 36; and an HCDR3 that comprises or is SEQ ID NO: 37; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 44; an LCDR2 that comprises or is SEQ ID NO: 45; and an LCDR3 that comprises or is SEQ ID NO: 43; or (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 38; an HCDR2 that comprises or is SEQ ID NO: 39; and an HCDR3 that comprises or is SEQ ID NO: 34; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 41; an LCDR2 that comprises or is SEQ ID NO: 42; and an LCDR3 that comprises or is SEQ ID NO: 43.

[0238] In some embodiments, the antigen-binding domain specific for CSF1R comprises a VH domain that is or comprises SEQ ID NO: 31 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions and a VL domain that is or comprises SEQ ID NO: 40 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions. The framework regions are regions of the variable domain outside of the CDRs as defined by any definition or any one definition herein. The substitutions may be conservative substitutions. An example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919).

[0239] In a particular embodiment, the antigen-binding domain specific for CSF1R comprises a VH domain that is SEQ ID NO: 31 and a VL domain that is SEQ ID NO: 40.

[0240] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises the CDRs of antibody 1B6.

[0241] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 49 or 52; and / or the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 56 or 59; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 58.

[0242] The antigen-binding domain specific for CSF1R may be a CSF1R specific antibody or an antigen-binding portion thereof that comprises a set of CDRs from Table 4 defined by Kabat, a set of CDRs from Table 4 defined by IMGT, or a set of CDRs from Table 4 defined by Chothia.

[0243] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein:

[0244] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0245] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 52; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 59; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0246] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58. In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein:

[0247] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0248] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and anHCDR3 that comprises or is SEQ ID NO: 52; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 59; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0249] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58.

[0250] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46; and / or the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55.

[0251] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein: the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46; and / or the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55.

[0252] The CDRs may be invariant. Thus, the antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46; and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55; and the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; anHCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and anHCDR3 comprising any one of SEQ ID NOs: 49 or 52; and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 56 or 59; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 58.

[0253] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55; and

[0254] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0255] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 52; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 59; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0256] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58.

[0257] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain and a VL domain, wherein the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46 and the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55; and

[0258] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0259] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and anHCDR3 that comprises or is SEQ ID NO: 52; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 59; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 58; or

[0260] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 49; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 56; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 58.

[0261] In some embodiments, the antigen-binding domain specific for CSF1R comprises a VH domain that is or comprises SEQ ID NO: 46 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions and a VL domain that is or comprises SEQ ID NO: 55 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions. The framework regions are regions of the variable domain outside of the CDRs as defined by any definition or any one definition herein. The substitutions may be conservative substitutions. An example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919).

[0262] In a particular embodiment, the antigen-binding domain specific for CSF1R comprises a VH domain that is SEQ ID NO: 46 and a VL domain that is SEQ ID NO: 55.

[0263] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises the CDRs of antibody 1H8.

[0264] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; anHCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and anHCDR3 comprising any one of SEQ ID NOs: 62 or 63; and / or the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 65 or 67; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 66. The antigen-binding domain specific for CSF1R may be a CSF1R specific antibody or an antigen-binding portion thereof that comprises a set of CDRs from Table 5 defined by Kabat, a set of CDRs from Table 5 defined by IMGT, or a set of CDRs from Table 5 defined by Chothia.

[0265] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein:

[0266] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0267] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 63; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 67; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0268] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66.

[0269] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein:

[0270] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0271] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and anHCDR3 that comprises or is SEQ ID NO: 63; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 67; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0272] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66.

[0273] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61; and / or the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64.

[0274] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein: the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61; and / or the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64. The CDRs may be invariant. Thus, the antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61; and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64; and the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 62 or 63 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 65 or 67; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 66.

[0275] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64; and

[0276] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0277] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 63; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 67; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0278] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66.

[0279] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain and a VL domain, wherein the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61 and the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64; and

[0280] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0281] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 63; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 67; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 66; or

[0282] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 62; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 65; an LCDR2 that comprises or is SEQ ID NO: 57; and an LCDR3 that comprises or is SEQ ID NO: 66.

[0283] In some embodiments, the antigen-binding domain specific for CSF1R comprises a VH domain that is or comprises SEQ ID NO: 61 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions and a VL domain that is or comprises SEQ ID NO: 64 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions. The framework regions are regions of the variable domain outside of the CDRs as defined by any definition or any one definition herein. The substitutions may be conservative substitutions. An example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919).

[0284] In a particular embodiment, the antigen-binding domain specific for CSF1R comprises a VH domain that is SEQ ID NO: 61 and a VL domain that is SEQ ID NO: 64.

[0285] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises the CDRs of antibody 1H9.

[0286] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; anHCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and anHCDR3 comprising any one of SEQ ID NOs: 69 or 70; and / or the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 72 or 75; an LCDR2 comprising any one of SEQ ID NOs: 60 or 73; and an LCDR3 comprising SEQ ID NO: 74.

[0287] The antigen-binding domain specific for CSF1R may be a CSF1R specific antibody or an antigen-binding portion thereof that comprises a set of CDRs from Table 6 defined by Kabat, a set of CDRs from Table 6 defined by IMGT, or a set of CDRs from Table 6 defined by Chothia.

[0288] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein:

[0289] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0290] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 70; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 75; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0291] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74.

[0292] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein:

[0293] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0294] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and anHCDR3 that comprises or is SEQ ID NO: 70; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 75; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 74; or (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74.

[0295] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68; and / or the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0296] In some examples, the antigen-binding domain specific for CSF1R is a CSF1R specific antibody or an antigenbinding portion thereof that comprises a VH domain and a VL domain, wherein: the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68; and / or the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0297] The CDRs may be invariant. Thus, the antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein: the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68; and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71; and the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 69 or 70; and / or the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 72 or 75; an LCDR2 comprising any one of SEQ ID NOs: 60 or 73; and an LCDR3 comprising SEQ ID NO: 74.

[0298] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71; and

[0299] (a) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0300] (b) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and an HCDR3 that comprises or is SEQ ID NO: 70; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 75; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0301] (c) the VH domain, or portion thereof, comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain, or portion thereof, comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74.

[0302] The antigen-binding domain specific for CSF1R may a CSF1R specific antibody or an antigen-binding portion thereof that comprises a VH domain and a VL domain, wherein the VH domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68 and the VL domain is at least 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71; and

[0303] (a) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 47; an HCDR2 that comprises or is SEQ ID NO: 48; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0304] (b) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 50; an HCDR2 that comprises or is SEQ ID NO: 51; and anHCDR3 that comprises or is SEQ ID NO: 70; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 75; an LCDR2 that comprises or is SEQ ID NO: 60; and an LCDR3 that comprises or is SEQ ID NO: 74; or

[0305] (c) the VH domain comprises an HCDR1 that comprises or is SEQ ID NO: 53; an HCDR2 that comprises or is SEQ ID NO: 54; and an HCDR3 that comprises or is SEQ ID NO: 69; and the VL domain comprises an LCDR1 that comprises or is SEQ ID NO: 72; an LCDR2 that comprises or is SEQ ID NO: 73; and an LCDR3 that comprises or is SEQ ID NO: 74.

[0306] In some embodiments, the antigen-binding domain specific for CSF1R comprises a VH domain that is or comprises SEQ ID NO: 68 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions and a VL domain that is or comprises SEQ ID NO: 71 with no more than 20, 15, 10, 5, 4, 3, 2, 1, or 0 substitutions, deletions, or insertions in framework regions. The framework regions are regions of the variable domain outside of the CDRs as defined by any definition or any one definition herein. The substitutions may be conservative substitutions. An example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919).

[0307] In a particular embodiment, the antigen-binding domain specific for CSF1R comprises a VH domain that is SEQ ID NO: 68 and a VL domain that is SEQ ID NO: 71.

[0308] Illustrative embodiments of the multispecific molecules

[0309] The presently disclosed multispecific molecules may comprise a first antigen-binding domain that is specific for TREM2 and a second antigen-binding domain that is specific for CSF1R.

[0310] In particular embodiments, the first and second antigen-binding domains are both antigen-binding portions of antibodies. In particular the first antigen-binding domain may a TREM2 specific antibody portion disclosed herein and the second antigen-binding domain may be a CSF1R specific antibody portion disclosed herein.

[0311] The multispecific molecule may be abispecific antibody. The multispecific molecule may comprise the first and second antigen binding domains joined by a linker, which may be a polypeptide. The multispecific molecule may be a fusion protein comprising the first and the second antigen binding domains.

[0312] In some examples, the first antigen-binding domain is a TREM2 specific antibody fragment comprising one or more amino acid sequences of antibody 2G4 or 1F7 and the second antigen-binding domain is a CSF1R specific antibody fragment comprising one or more amino acid sequences of antibody 1B7, 1B6, 1H8, or 1H9.

[0313] The first antigen-binding domain may a TREM2 specific antibody fragment comprising a set of six CDRs of antibody 2G4 or 1F7 as disclosed herein and the second antigen-binding domain may be a CSF1R specific antibody fragment comprising a set of six CDRs of antibody 1B7, 1B6, 1H8, or 1H9 as disclosed herein. In some examples, all of the CDRs are determined using the same scheme. Sets of CDRs are disclosed herein in the relevant sections. The first antigen-binding domain may a TREM2 specific antibody fragment comprising a light chain variable domain as disclosed herein and a heavy chain variable domain as disclosed herein, and the second antigen-binding domain may be a CSF1R specific antibody fragment comprising a light chain variable domain as disclosed herein and a heavy chain variable domain as disclosed herein.

[0314] The first antigen-binding domain may comprise: a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 or 16 and a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 25; and the second antigen-binding domain may comprise: a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, 46, 61, or 76 and a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40, 55, 70, 85. These sequences may be paired as depicted in Tables 1 to 6. The CDRs may be invariant. The CDRs may be as depicted in any one of Tables 1 to 6 and may be determined by the same scheme.

[0315] Sequence comparisons can be conducted with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

[0316] The skilled technician will appreciate how to calculate the percentage identity between two nucleic sequences or two amino acid sequences. In order to calculate the percentage identity, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, the Needleman-Wunsch algorithm (e.g. as applied by Needle(EMBOSS) or Stretcher(EMBOSS), the Smith- Waterman algorithm (e.g. as applied by Water(EMBOSS)), or the LALIGN application (e.g. as applied by Matcher(EMBOSS); and (ii) the parameters used by the alignment method, for example, local versus global alignment, the matrix used, and the parameters applied to gaps. In a particular embodiment, the sequence identities disclosed herein may be calculated based on a global alignment of the relevant feature, for instance the comparison of a complete length of a variable domain to the complete reference sequence recited herein.

[0317] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length-dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0318] A calculation of percentage identities between two nucleic acid sequences may then be calculated from such an alignment as (N / T)* 100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.

[0319] The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In an example, the identity between two amino acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two amino acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (14), gap extend (4), alternative matches (1). In an example, the identity between two nucleic acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1).

[0320] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Other aspects and embodiments of the invention provide the aspects and embodiments described herein with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of’.

[0321] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0322] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the Examples, which are not intended to limit the invention in any way.

[0323] EXAMPLES

[0324] Example 1 - Outline of research

[0325] Recent discoveries in the pathogenesis of liver cirrhosis, a leading cause of mortality worldwide, have highlighted the potential for novel therapeutic approaches targeting the cellular and molecular mechanisms of fibrosis.

[0326] A key advance in this area is the discovery of a subset of macrophages known as Scar- Associated Macrophages (SAMac), which are dramatically increased abundance in cirrhotic livers compared to normal livers, are selectively located within regions of fibrosis within the liver, and have pro-fibrotic activity in an in vitro culture system (Ramachandran et al., 2019). Analysis of the transcriptome of SAMac cells by single-cell sequencing (scRNAseq) has identified two cell surface receptors, TREM2 and CD9, that together distinguish SAMac cells from the eight other macrophage subpopulations within the liver as well as from other cell types in the liver. The present inventors have noted that the pathogenic, pro-fibrotic activity of these cells in the context of fibrotic disease, their increased abundance in fibrotic disease, and the existence of transcriptomic differences that distinguish SAMac cells from other cell types all indicates that reduction of the number of SAMac and profibrotic activities of SAMac renders it desirable to target these cells for the treatment of fibrotic diseases, such as fibrotic liver disease.

[0327] Use of the pseudotime computational method for elucidation of the cell lineage, origin, and the differentiation pathway of cells have indicated that SAMac cells originate from circulating blood monocytes, and undergo proliferation within the cirrhotic liver (Ramachandran et al., 2019).

[0328] SAMac cells could be targeted if a unique cell surface marker were identified that could be targeted by a monoclonal antibody. However, a single surface receptor that defines SAMac cells has not yet been identified and may not exist.

[0329] To overcome this issue, the present inventors have discovered combinations of markers that, in a combinatorial fashion, provide sufficient selectivity to distinguish SAMac from other macrophages and from other human cells.

[0330] These markers may be targeted using agents such as bispecific antibodies (bsAbs) that have two different binding sites, Binding Site A and Binding Site B, that are directed to the different receptors (Fig. 1 - the receptors are represented here in an exemplary fashion as Receptor A and Receptor B). In particular, a bsAb that has low affinity for each of the two individual cognate receptors, Receptor A and Receptor B, can exhibit higher affinity for cells expressing both Receptor A and Receptor B through the principle of avidity, would be of particular interest. Such a bsAb has been referred to as a cis-acting bsAb (Labrijn, Aran F., Maarten L. Janmaat, Janice M. Reichert, and Paul W. H. I. Parren. 2019. “Bispecific Antibodies: A Mechanistic Review of the Pipeline.” Nature Reviews. Drug Discovery 18 (8): 585-608 - herein incorporated by reference).

[0331] Identification of pairs of receptors that combinatorically define SAMac cells is not straightforward. In addition to the challenge of systematically searching through many pairs of receptors within the human “surfaceome” the chosen pair of receptors will optimally have other properties. First, the two members of the receptor pair should have optimal expression levels on the cell surface to increase the probability of engagement of both receptors by the bsAb. Second, while each member of the pair could be expressed on other cell types and in other tissues, if the expression of Receptor A, Receptor B, or both is high in “off-target” cells (i.e., not SAMac cells) or if off-target cells are very abundant, such off-target cells could provide a tissue “sink” that would deplete bsAb from the circulation, driven by the individual specificities of the bsAb, potentially reducing its effectiveness. Third, some classes of receptors present high technical challenges to generating antibodies to those receptors. For instance, generating antibodies to multispan and 7TM receptors, many with short extracellular domains and loops, is more challenging than for Type 1 or Type 2 receptors.

[0332] Herein, the inventors leverage single cell data from both healthy and cirrhotic livers (Ramachandran et al., 2019), alongside comprehensive cellular data from the human body (CellxGene Discover, https: / / cellxgene.cziscience.com / ), to refine and prioritize bsBA surface receptor targets for SAMacs. This strategy minimizes off-target effects and enhances the selectivity of bsAb therapeutics against SAMacs in liver fibrosis.

[0333] In light of their findings, the inventors provide herein the following 4 single receptors (TREM2, CSF1R, MSR1, CLEC7A) and 3 targeting receptor pairs (TREM2 CSF1R, TREM2 MSR1, TREM2 CLEC7A) for targeting SAMac cells and hence for the treatment of fibrosis.

[0334] Example 2 - Identification of receptor pairs for engineering of SAMac-specific bsAb

[0335] Single cell sequencing approaches reveal unique gene expression patterns across different cell types by comparing individual cell transcriptomes. Mean expression and expressing cell fraction (or ECF) are two effective metrics that are used to assess the selectivity of genes for cell types in a single cell experiment. However, single cell data has several features that require specific considerations when interpreting these results. Firstly, the data is relatively sparse owning to a high frequency of dropout events (lack of detection of specific transcripts), which can occur during sample preparation and sequencing. Secondly, transcripts in cells are subject to temporal fluctuations, often referred to as transcriptional bursts, further contributing to number of zero observations in a single cell experiment (Haque, Ashraful, Jessica Engel, Sarah A. Teichmann, and Tapio Lonnberg. 2017. “A Practical Guide to Single-Cell RNA-Sequencing for Biomedical Research and Clinical Applications.” Genome Medicine 9 (1): 75). Therefore, mean expression and ECF calculated using single cell data may underestimate the actual expression due to technical and biological factors. As the inventors consider targeting genes for therapeutic purposes, a delicate balance must be stmck between selecting genes with low target cell expression and high selectivity (presence in other cell types) versus high target cell expression and low selectivity.

[0336] To pinpoint the specific surface receptors expressed on SAMacs, the inventors used the single cell dataset of (Ramachandran et al., 2019) and analyzed the expression of 2,886 known receptor genes, as listed on the Surfaceome Database (Bausch-Fluck, Damaris, Ulrich Goldmann, Sebastian Muller, Marc van Oostrum, Maik Muller, Olga T. Schubert, and Bernd Wollscheid. 2018. “The in Silico Human Surfaceome.” Proceedings of the National Academy of Sciences of the United States of America 115 (46): E10988-97). They compared the proportion of these receptors expressed by the SAMac cells labelled as MPs (5) (referred to as ecf on) to those expressed by other liver cell types (ecf off), effectively distinguishing the receptors unique to SAMacs from those shared with off-target cells. In total, they considered 1,4014 SAMac cells and 56,944 other liver cells. The inventors filtered the table by excluding receptors that are encoded by HLA genes and those with lower than 20% ecf on and then ranked the list based on the ratio of ecf_on:ecf_off.

[0337] Example 3 - The TREM2 / CD9 receptor pair

[0338] The top gene within this list generated in Example 2 is TREM2 with ecf on of 47% and an ecf off of 0.1% TREM2 belongs to a family of receptors referred to as the triggering receptors expressed on myeloid cells (TREM). Members of the TREM family are cell surface transmembrane glycoproteins with V-immuno globulin extra-cellular domains and cytoplasmic tails. The TREM2 gene is located on human chromosome 6p21 and encodes a 230-amino acid protein. TREM2 is a promising target for therapeutic intervention in diseases like Alzheimer's due to its role in microglial function.

[0339] Another noted receptor that distinguishes SAMacs from other macrophages is CD9 (Ramachandran et al., 2019). CD9, or Tspan-29, is a 21-24 kDa member of the tetraspanin protein family. Tetraspanins are structurally characterized as containing four transmembrane domains, which delimit a small extracellular loop, a large extracellular loop, and short intracellular N- and C-terminal tails. In our analysis, CD9 was 125th on the list with 57% ecf on and 20% ecf off. Nevertheless, the expression of TREM2 and CD9 distinguished SAMac from other macrophage populations. The inventors then assessed the expression of TREM2 and CD9 across liver cells using single cell RNA-seq data (Figure 2A). While TREM2 was selective to MPs (5) population, CD9 was also expressed in mast, plasma B, cholangiacytes, hepatocytes, mesothelial, endothelial, and mesenchymal cells (Figure 2A).

[0340] The inventors then calculated the ECF on and ECF off considering co-expression of both TREM2 and CD9 to explore the selectivity of TREM2 x CD9 for SAMacs as a potential receptor pair for bispecific antibody. TREM2 x CD9 ecf on was 27% and ecf off was reduced to 0.3%. They visualised cells co-expressing CD9 and TREM2 in liver using 2-dimensional embedding where all cells with >0 log normalised counts of either receptor is coloured in grey and cells with counts from both receptors are coloured in black (Figure 2B).

[0341] Thus, a TREM2 / CD9 bsAb could have potential for selectively recognizing and targeting SAMac. However, generating mAbs to CD9 could be challenging due to it belonging to the tetraspanin family of receptors. The transmembrane nature of tetraspanins means that generating antibodies often requires the production of extracellular loops as immunogens, which must be properly folded and presented to mimic the natural conformation on the cell surface. A second liability for CD9 as a member of a bsAb receptor pair is its, broad expression in multiple cell types, leading to a potential to “tissue sink” which refers to the phenomenon where a bispecific antibody is sequestered by other cells expressing the same antigen, thus reducing its availability to bind to the intended target cells. However, the significance of this potential tissue sink can only be determined empirically in animal models or in humans in pharmacokinetic studies of bsAb half-life and tissue distribution. These factors led the inventors to search for improved bsAb receptor pairs.

[0342] Example 4 - Discovery of additional SAMac-selective bsAb receptor pairs

[0343] To identify alternative receptor pairs for SAMacs, the inventors analysed the receptor ecf on. They considered the following criteria for the top candidate genes:

[0344] • Mean expression level of individual receptors in target and off target cells

[0345] • ECF on - Expressing cell fraction in the target cells (SAMac)

[0346] • ECF off - Expressing cell fraction in off-target cells (non-SAMac cells in liver)

[0347] • Receptor class and topology

[0348] • Homology in NHP and mice models (which could influence the cross-reactivity and thus the ability to test a bsAb in toxicology and efficacy studies). Four receptors were chosen from this list based on high on target ecf: TREM2, MSR1, CSF1R, CLEC7A. Expression was confirmed in monocyte and macrophage populations (MPs 1-7) for MSR1, CSF1R and in addition in dendritic cells (MPs 8-9) for CLEC7A (Figure 3 A).

[0349] For chosen receptors, the inventors have calculated the percentage of cells co-expressing the receptor and TREM2 in on target cell population vs off target cells (Table 7). Co-expression of both members of the pairs in single cells of the SAMac population was confirmed by inspection of UMAP plots (Figure 3B).

[0350] Table 7

[0351] Systemic expression of the individual four receptors was then determined by CZI CellxGene Discover database. In total, the inventors considered 3,226,515 cells (from 40 organs and tissues). The inventors first annotated cells from the database using CellTypist classification of broad lineages (Myeloid, Endothelial, Epithelial, Fibroblasts, Smooth muscle cells (SMC), and lymphoid cells). Each lineage was also annotated based on the tissue the cells were profiled in (e.g. lung-epithelial, heart-lymphoid). They then assessed the fraction of cells (ecf on) and mean expression of each of prioritised genes in the organ-cell lineage pool and filtered out organ-cell type entries by at least 100 cells considered in the category.

[0352] TREM2 expression was restricted to myeloid cells across organs. More than 20% of myeloid cells expressed TREM2 in lung, exocrine, adrenal, prostate glands, uterus, central nervous system, spinal cord, pancreas. As expected, myeloid cells across organs also showed substantial expression of CLEC7A, CSF1R, MSR1 and CD9. This analysis provides organs that can be targeted including the lungs, kidneys, and skin.

[0353] When selecting genes for bispecific antibody (bsAb) targeting, it is important to consider both length of the extracellular domain (ECD) and its cross-species homology (Table 8). For instance, TREM2, a Type I membrane protein, with ECD positions 19-174, shows high homology with cynomolgus monkeys (96.5%) and lower with mice (50%). CD9, a tetraspan protein, exhibits ECDs at positions 56-75 and 196-221, with very high homology across species (98.7% with cynomolgus monkeys and 89.0% with mice), indicating its broad therapeutic potential due to its conserved nature. The conserved nature of CD9 suggests that further experimentation and pharmacokinetic studies of bsAb half-life and tissue distribution are plausible to assess tissue sink effects in animal models.

[0354] Table 8: Evaluation of the ECD and the cross-species homology of the candidate bsAb targets. N-terminal extracellular domain (ECD), homology and topology results for selected receptors. Figure 4 shows that SAMac cells are capable of receiving a CSF1 signal. Figure 4 also shows that CSF1 is expressed more highly in endothelial cells and there is greater signal in the cirrhotic liver. The parameters used are as follows.

[0355] CellphoneDB (version 5) was ran on all cells from Ramachandran et al., 2019 dataset with default parameters and threshold for % of cells expressing ligand / receptor set to 0.1. NicheNet analysis was performed by first running a differential gene expression (DE) comparison between Cirrhotic MPs (5) (e.i. SAMacs) vs cirrhotis MPs(l,2,3) (e.i. monocytes) and the background genes used were all genes expressed in all considered cells (MPs (1,2, 3, 5)). The method was run with the following parameters: nLigands out: 91 (number of ligands as input), gene_exp_pct_threshold = 0.1 (% cells with expression of gene), sig_DE_pval = 0.05 (pval for DE threshold), sig_DE_log2FC = 1 (log2FC for DE threshold).

[0356] Figure 5 illustrates the determination of systemic expression of the individual receptors by CZI CellxGene Discover database. In total we considered 3,226,515 cells (from 40 organs and tissues). We first annotated cells from the database using CellTypist classification of broad lineages (Myeloid, Endothelial, Epithelial, Fibroblasts, and lymphoid cells). Each lineage was also annotated based on the tissue the cells were profiled in (e.g. lung- epithelial, heart-lymphoid). We then assessed the fraction of cells (ecf on) and mean expression of each of prioritised genes in the organ-cell lineage pool and filtered out organ-cell type entries by at least 100 cells considered in the category.

[0357] Example 5 - Development of agents for targeting SAMacs

[0358] The aforementioned experimental data that have led the inventors to identify receptor pairs that allow SAMacs to be targeted. The agents for targeting the SAMacs include the multispecific molecules disclosed herein.

[0359] The multispecific molecules may bind to SAMac cells but not to non-target cells (or only weakly to non-target cells). This can be determined by an assay making use of a test cell type suitable for cell culture, such as a cell line or suitable primary cell. The test cell type may express comparable amounts of the indicated receptors as SAMac cells. In an example, the test cells of the assay are HEK or CHO cells. The test cells may express TREM2; one of MSR1, CSF1R, and CLEC7A; or both TREM2 and one of MSR1, CSF1R, and CLEC7A (i.e. Receptor A, B, or AB in Figure 1). Binding of the multispecific molecules to the test cells may be determined by flow cytometry (e.g. by labelling the multispecific molecules directly or indirectly). A selective multispecific molecule will bind most strongly to cells expressing both targets.

[0360] Another assay makes use of SAMac cells themselves as the target cells (i.e. the cells expressing AB). The SAMac cells may have been isolated from a human liver or may be SAMac cells that have been differentiated from blood monocytes as described by Fabre et al. (2023). In some embodiments, suitable multispecific molecules bind most strongly to SAMac cells compared to binding to other macrophage subtypes, such as other macrophages from the liver and / or kidney.

[0361] For embodiments where the multispecific molecule is cytotoxic towards SAMac cells, the above-mentioned assays can be altered to measure cytotoxicity of candidate agents towards the test cells.

[0362] Example 6 - Evidence for the pathogenic role of SAMacs

[0363] SAMacs are a specialized subset of macrophages that emerge in fibrotic tissues, playing critical roles in extracellular matrix (ECM) remodeling and fibrosis progression. These macrophages primarily originate from monocytes and are recmited to sites of injury. SAMacs are enriched in fibrotic niches across multiple tissues, including the liver, skin, heart, kidneys, and lungs, contributing to the pro-fibrotic response. By quantifying the number and density of SAMacs within and outside the scar region, we found that these macrophages preferentially localize within the scar (Figure 6), providing evidence for a role in driving scar formation. Additionally, SAMac numbers increase with different stages of fibrotic disease (Figure 7). To characterize this population further, we selected two cell surface receptors, TREM2 and CSF1R, as markers. Our findings indicate that TREM2 and CSF1R double-positive cells are predominantly located within the scar region (Figure 14), confirming that SAMacs express these target receptors. Furthermore, we demonstrated that SAMacs actively proliferate within the scar region, as evidenced by Ki67 expression (Figure 8). This proliferation may be driven by an excessive presence of the macrophage growth factor CSF 1 , which is also preferentially enriched in the scar region (Figure 9). Additionally, we identified the pro-fibrotic mechanisms of SAMacs, showing that they express a range of known fibrosis-promoting factors, including Galectin-3, TGF-fy and PDGF-B (Figure 15).

[0364] To investigate the pro-fibrotic properties of SAMacs, we developed an in vitro SAMac differentiation model using M-CSF and TGF-fy Under these conditions, we observed an increased expression of our target genes, as well as cell surface markers TREM2 and CSF1R (Figure 10). When comparing the transcriptome profiles of in vitro- differentiated SAMacs with in vivo SAMacs, we found significant similarity, suggesting that M-CSF plus TGF-P provides a robust model for studying SAMacs (Figure 10). To determine whether in vitro-differentiated SAMacs could induce fibrosis, we collected conditioned media from these cells and treated primary hepatic stellate cells. This treatment led to increased COL 1 Al expression under M-CSF and TGF-P conditions (Figure 11), further demonstrating the fibrogenic potential of in vitro-differentiated SAMacs.

[0365] Given that SAMacs proliferate within the scar region, targeting their proliferation is a therapeutic strategy. Since CSF 1R is a key growth-related receptor for SAMacs, we aimed to identify an anti-CSF 1R blocking antibody capable of inhibiting macrophage differentiation and proliferation. Using a CSF1R activity assay, we identified four antibody clones (1B7, 1B6, 1H8, and 1H9) that effectively blocked CSF1R dimerization (Figure 12) and suppressed monocyte-to-macrophage differentiation and proliferation (Figure 13). Overall, these findings highlight the role of SAMacs in fibrosis progression and provide evidence supporting CSF1R inhibition as a therapeutic approach to mitigate fibrosis.

[0366] Example 7 - TREM2 SPR Data and Methods

[0367] Two monoclonal antibodies targeting TREM2 were characterized for binding affinity to human and cynomolgus monkey (cyno) TREM2 antigens. The antibodies demonstrated binding in the single and double-digit nanomolar (nM) range. Cross-reactivity analysis revealed that both clones recognized cyno TREM2. Furthermore, clone 2G4 exhibited good cross-reactivity to mouse TREM2.

[0368] Test antibodies were captured on flow cells on CM5 chip (Cytiva) with anti-human Fc IgG (Jackson), with contact time of 30s with a flow rate of 10 ul / min. The extracellular domain (ECD) protein fragment of human, cyno, or mouse TREM2 (ACRO) was used as analyte, at a flow rate of 10 pl / min. Association was measured for 180s and dissociation for 600s. Between injections the capture surface was regenerated by a 30 second injection of 10 mM glycine, pH 1.5 at a flow rate of 30 pl / min. Interactions were assessed at 25° C. in HBS-EP+ buffer (Teknova), at a flow rate of 30 ul / min. Reference data from the reference flow cell and from injection of buffer alone was subtracted prior to kinetic analysis. Kinetic information was calculated by fitting data to a steady state or 1 : 1 binding model. Reference subtraction and data fitting were performed using Biacore evaluation software (Cytiva).

[0369] Table 9 - Materials and methods

[0370] Table 10 - SPR results for huTREM2

[0371] Sensorgrams are illustrated in Figure 16. (A) shows results for 1F7. (B) shows results for 2G4. Table 11 - SPR results for cynoTREM2

[0372] Sensorgrams are illustrated in Figure 17. (A) shows results for 1F7. (B) shows results for 2G4.

[0373] Table 12 - SPR results for muTREM2

[0374] A sensorgram is illustrated in Figure 18 showing results for 2G4.

[0375] Example 8 - TREM2 ELISA Data

[0376] ELISA was used to test antigen binding by selected antibodies. In brief, wells were coated with streptavidin overnight, blocked with casein for 1 hour at room temperature, biotinylated antigen was incubated for 20 minutes before addition of the selected antibodies at 500nM concentration with five-fold dilution series for 1 hour. OD450 was measured after 1 hr incubation of anti-human-Fc-HRP and TMB substrate. The antibodies 1F7 and 2G4 were tested in ELIS As for binding to human TREM2 (Figure 19), cyno TREM2 (Figure 20), and mouse TREM2 (Figure 21).

[0377] The results are summarised in the tables below.

[0378] Table 13 - Results from an ELISA showing binding of anti-TREM2 Abs to human TREM2

[0379] EC50 (nM)

[0380] Table 14 - Results from an ELISA showing binding of anti-TREM2 Abs to cyno TREM2

[0381] EC50 (nM)

[0382] Table 15 - Results from an ELISA showing binding of anti-TREM2 Abs to mouse TREM2

[0383] EC50 (nM)

[0384] The two anti-TREM2 clones were characterized, demonstrating high-affinity binding to human and cyno TREM2, with binding affinities in the single-digit nM range. Cross-reactivity analysis revealed that both anti-TREM2 clones recognized cyno TREM2. Furthermore, clone 2C4 exhibited cross-reactivity to mouse TREM2, while clone 1F7 showed weaker binding.

[0385] Example 9 - CSF1R SPR Data and Methods

[0386] Our panel of monoclonal antibodies targeting CSF1R was generated and characterized for binding kinetics using SPR. All antibodies exhibited modest binding affinities, displaying equilibrium dissociation constants (KDs) in the range of 1 x IO-7M to 7 x IO-8M against both human and cynomolgus monkey (cyno) CSF1R antigens.

[0387] Test antibodies were captured on flow cells on CM5 chip (Cytiva) with anti-human Fc IgG (Jackson), with contact time of 30s with a flow rate of 10 ul / min. The extracellular domain (ECD) protein fragment of human or cyno CSF1R (ACRO) was used as analyte, at a flow rate of 10 pl / min. Association was measured for 180s and dissociation for 600s. Between injections the capture surface was regenerated by a 30 second injection of 10 m glycine, pH 1.5 at a flow rate of 30 pl / min. Interactions were assessed at 25° C. in HBS-EP+ buffer (Teknova), at a flow rate of 30 ul / min. Reference data from the reference flow cell and from injection of buffer alone was subtracted prior to kinetic analysis. Kinetic information was calculated by fitting data to a steady state or 1 : 1 binding model. Reference subtraction and data fitting were performed using Biacore evaluation software (Cytiva). Table 16 - Materials and methods

[0388] Table 17 - SPR results for human CSF1R (ECD)

[0389] Sensorgrams are illustrated in Figure 22. (A) shows results for 1B7, (B) shows results for 1B6, (C) shows results for 1H8, and (D) shows results for 1H9.

[0390] Table 18 - SPR results for cyno CSF1R (ECD)

[0391] Sensorgrams are illustrated in Figure 23. (A) shows results for 1B7, (B) shows results for 1B6, (C) shows results for 1H8, and (D) shows results for 1H9.

[0392] Table 19 - Summary of Anti-CSFIR binding kinetics Example 10 - CSF1R ELISA Data

[0393] ELISA was used to test antigen binding by selected antibodies. In brief, wells were coated with streptavidin overnight, blocked with casein for 1 hour at room temperature, biotinylated antigen was incubated for 20 minutes before addition of the selected antibodies at 500nM concentration with five-fold dilution series for 1 hour. OD450 was measured after 1 hr incubation of anti-human-Fc-HRP and TMB substrate.

[0394] The antibodies 1B7, 1B6, 1H8, and 1H9 were tested in ELISAs for binding to human CSF1R (Figure 24) and, cyno CSF1R (Figure 25).

[0395] The results are summarised in the tables below.

[0396] Table 20 - Results from an ELISA showing binding of anti-CSFIR Abs to human CSF1R

[0397] EC50 (nM)

[0398] Table 21 - Results from an ELISA showing binding of anti-CSFIR Abs to cyno CSF1R

[0399] Screening by ELISA demonstrated significant binding of anti-CSFIR antibodies to both human and cynomolgus monkey (cyno) CSF1R antigens. The benchmark used in these experiments have binding affinity in single digit pM, suggesting our panel of antibodies have very tight binding to human CSF1R. Each clone also exhibited good but weaker binding to cyno CSF1R.

[0400] Example 11 - anti-TREM2 cell binding data

[0401] In order to test specificity of selected antibodies, binding was assessed by flow cytometry on antigen expressing HEK293F cells or THP1 cell line. Cells were incubated with selected anti-TREM2 antibodies (indicated concentrations with five-fold dilution series) for 30-60 minutes at 4° C. After washing, bound antibodies were detected with an APC labeled secondary goat anti-human IgG Fc (Jackson ImmunoReasearch) incubated for 20- 30 minutes at 4° C. Fluorescence intensity was measured by flow cytometry using a Cytoflex LX (Beckman Coulter). Geometric mean of the fluorescence intensity was calculated using Flowjo software (BD). EC50S were calculated using Graphpad (Prism).

[0402] The results are summarised in the tables below and illustrated in Figures 26 to 29.

[0403] Table 22 - Results from binding of anti-TREM2 to human TREM2 expressing HEK293F cells.

[0404] EC50(nM) Table 23 - Results from binding of anti-TREM2 to human TREM2 expressing THP1 cell line.

[0405] Table 24 - Results from binding of anti-TREM2 (alternative format) to human TREM2 expressing HEK293F cells.

[0406] EC50(nM)

[0407] Table 25 - Results from binding of anti-TREM2 to cyno TREM2 expressing HEK293F cells.

[0408] EC50(nM)

[0409] Example 12 - anti-CSFIR cell binding data

[0410] In order to test specificity of selected antibodies, binding was assessed by flow cytometry on antigen expressing HEK293F cells. Cells were incubated with selected anti-CSFIR antibodies (final concentration lOOug / ml with five-fold dilution series) for 30 minutes at 4° C. After washing, bound antibodies were detected with an APC labeled secondary goat anti-human IgG Fc (Jackson ImmunoReasearch) incubated for 20 minutes at 4° C. Fluorescence intensity was measured by flow cytometry using a Cytoflex LX (Beckman Coulter). Geometric mean of the fluorescence intensity was calculated using Flowjo software (BD). EC50S were calculated using Graphpad (Prism).

[0411] The results are summarised in the table below and illustrated in Figure 30.

[0412] Table 26 - Results from binding of anti-CSFIR antibodies to human CSF1R expressing HEK293F cells.

[0413] EC50(nM)

[0414] Example 13 - TREM2xCSFlR bispecific antibody binding data - BLI binding

[0415] Octet Red (Sartorius) Experiment:

[0416] • Anti-TREM2 x CSF1R bispecific antibodies in BLI kinetics buffer (Sartorius) were loaded onto AHC (anti-Fc) Biosensors (Sartorius)

[0417] • After a brief washing step, biosensors loaded with bispecific antibodies were dipped into wells with lOOnM TREM2 (Aero) for first association step • Immediately after association to TREM2, the bispecific antibodies were dipped into wells with lOOnM CSF1R (Aero) for the second association step

[0418] • Lastly, tips were dipped into wells with kinetics buffer for dissociation step

[0419] Bispecific Antibodies Tested:

[0420] 1. TREM2 2G4 x CSF 1R 1H9

[0421] 2. TREM2 1F7 x CSF1R 1H9

[0422] 3. TREM2 2G4 x CSF1R 1B6

[0423] 4. TREM2 BMK x CSF1R 1H9

[0424] The results are illustrated in Figures 31 to 35.

[0425] Example 14 - TREM2xCSFlR bispecific antibody binding data - Cell binding

[0426] Goal: demonstrate binding of bispecific antibody to CSF1R (cell surface) and TREM2 (in solution)

[0427] Experimental setup (illustrated in Figure 36): CSF1R expressing cell incubated with either aCSFIR mAb or

[0428] CSF1R x TREM2 bsAb followed by TREM2 biotin and SA PE

[0429] Or

[0430] TREM2 expressing cells incubated with either aTREM2 mAb or CSF1R x TREM2 bsAb followed by CSF1R biotin and SA PA

[0431] The results are illustrated in Figures 37 to 40.

[0432] Conclusion: CSF1R x TREM2 bsAb binds to CSF1R and TREM2.

Claims

CLAIMS1. A multispecific molecule comprising: a first antigen-binding domain that is specific for TREM2; and a second antigen-binding domain that is specific for any one of CSF1R, MSR1, or CLEC7A.

2. The multispecific molecule of claim 1, wherein the multispecific molecule binds to SAMac cells and / or is selective for SAMac cells.

3. The multispecific molecule of claim 1 or claim 2, wherein the multispecific molecule has cytotoxic activity or promotes cytotoxic activity towards SAMac cells.

4. The multispecific molecule of any preceding claim, wherein the second antigen-binding domain is specific for CSF1R and comprises or is CSF1 or a mutant CSF1, a variant, or portion thereof.

5. The multispecific molecule of any preceding claim, wherein the second antigen-binding domain is specific for CSF1R and blocks CSF1R signalling.

6. The multispecific molecule of claim 5, wherein the second antigen-binding domain prevents CSF-1 binding to CSF1R or inhibits dimerisation of CSF1R.

7. The multispecific molecule of any preceding claim, wherein the first and / or second antigen binding domain is an antigen-binding portion of an antibody.

8. The multispecific molecule of any preceding claim, wherein the first and / or second antigen binding domain is a single-chain fragment variable (scFv).

9. The multispecific molecule of any preceding claim, wherein the multispecific molecule is bispecific.

10. The multispecific molecule of any preceding claim, wherein the multispecific molecule is a bispecific antibody.

11. The multispecific molecule of any preceding claim, wherein the first antigen-binding domain comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein(a) the VH domain, or portion thereof, comprises a heavy chain complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 2, 5, or 8; a heavy chain complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 3, 6, or 9; and a heavy chain complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 4 or 7 and the VL domain, or portion thereof, comprises a light chain complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 11 or 14; a light chain complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 12 or 15; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 13; or(b) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 17, 20, or 23; an HCDR2 comprising any one of SEQ ID NOs: 18, 21, or 24; and an HCDR3 comprising any one of SEQ ID NOs: 19 or 22 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 26 or 29; an LCDR2 comprising any one of SEQ ID NOs: 27 or 30; and an LCDR3 comprising SEQ ID NO: 28.

12. The multispecific molecule of claim 11, wherein:(a) the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; or(b) the VH domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16 and the VL domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25.

13. The multispecific molecule of any preceding claim, wherein the second antigen-binding domain is specific for CSF1R and comprises a VH domain, or portion thereof, and a VL domain, or portion thereof, wherein(a) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 32, 35, or 38; an HCDR2 comprising any one of SEQ ID NOs: 33, 36, or 39; and an HCDR3 comprising any one of SEQ ID NOs: 34 or 37 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 41 or 44; an LCDR2 comprising any one of SEQ ID NOs: 42 or 45; and an LCDR3 comprising SEQ ID NO: 43; or(b) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 49 or 52 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 56 or 59; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 58; or(c) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 62 or 63 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 65 or 67; an LCDR2 comprising any one of SEQ ID NOs: 57 or 60; and an LCDR3 comprising SEQ ID NO: 66; or(d) the VH domain, or portion thereof, comprises an HCDR1 comprising any one of SEQ ID NOs: 47, 50, or 53; an HCDR2 comprising any one of SEQ ID NOs: 48, 51, or 54; and an HCDR3 comprising any one of SEQ ID NOs: 69 or 70 and the VL domain, or portion thereof, comprises an LCDR1 comprising any one of SEQ ID NOs: 72 or 75; an LCDR2 comprising any one of SEQ ID NOs: 60 or 73; and an LCDR3 comprising SEQ ID NO: 74.

14. The multispecific molecule of claim 13, wherein:(a) the VH domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 and the VL domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40; or(b) the VH domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46 and the VL domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55; or(c) the VH domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61 and the VL domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64; or(d) the VH domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68 and the VL domain of the second antigen-binding domain is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

15. An immunoconjugate comprising the multispecific molecule of any preceding claim conjugated to an agent.

16. One or more nucleic acid molecules encoding a multispecific molecule of any one of claims 1 to 14.

17. One or more vectors comprising the one or more nucleic acid molecules of claim 16.

18. A cell comprising the one or more nucleic acid molecules of claim 16 or the one or more vectors of claim17.

19. A pharmaceutical composition comprising a multispecific molecule of any one of claims 1 to 14, the immunoconjugate of claim 15, the one or more nucleic acid molecules of claim 16, the one or more vectors of claim 17, or the cell of claim 18.

20. A multispecific molecule of any one of claims 1 to 14, the immunoconjugate of claim 15, the one or more nucleic acid molecules of claim 16, the one or more vectors of claim 17, the cell of claim 18, or the pharmaceutical composition of claim 19 for use in a method of treatment.

21. A multispecific molecule of any one of claims 1 to 14, the immunoconjugate of claim 15, the one or more nucleic acid molecules of claim 16, the one or more vectors of claim 17, the cell of claim 18, or the pharmaceutical composition of claim 19 for use in a method of treating fibrosis.

22. The multispecific molecule, immunoconjugate, one or more nucleic acid molecules, one or more vectors, cell, or pharmaceutical composition for use of claim 21, wherein the fibrosis affects the liver, lung, kidney, intestine, skin, or heart.

23. The multispecific molecule, immunoconjugate, one or more nucleic acid molecules, one or more vectors, cell, or pharmaceutical composition for use of claim 21, wherein the fibrosis is liver fibrosis.

24. The multispecific molecule, immunoconjugate, one or more nucleic acid molecules, one or more vectors, cell, or pharmaceutical composition for use of claim 20, 21 , or 22, wherein the method is for treating a subject with idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease, or scleroderma.

25. A multispecific molecule of any one of claims 1 to 14, the immunoconjugate of claim 15, the one or more nucleic acid molecules of claim 16, the one or more vectors of claim 17, the cell of claim 18, or the pharmaceutical composition of claim 19 for use in a method of treating fibrotic liver disease, cirrhosis of the liver, alcohol-associated liver disease, metabolic dysfunction-associated steatohepatitis, or primary sclerosing cholangitis.

26. A method of treating fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a multispecific molecule of any one of claims 1 to 14, immunoconjugate of claim 15, one or more nucleic acid molecules of claim 16, one or more vectors of claim 17, cell of claim 18, or pharmaceutical composition of claim 19 to the subject.

27. A method of inhibiting the fibrotic activity, reducing the proliferation, or reducing the survival of SAMac cells, the method comprising contacting the cells with a multispecific molecule of any one of claims 1 to 14.

28. The method of claim 27, wherein the method is in vivo, in vitro, or ex vivo.

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