WO2024261729A1

WO2024261729A1 - Lilrb2 binding proteins and uses thereof

Info

Publication number: WO2024261729A1
Application number: PCT/IB2024/056100
Authority: WO
Inventors: Des C. JONES; Lorraine IRVING; Alan SANDERCOCK; Robert W. Wilkinson; Marcin D. WOLNY; Simon DOVEDI; Nadia Mohamed LUHESHI; Seraina BLUMLI
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2023-06-23
Filing date: 2024-06-21
Publication date: 2024-12-26
Anticipated expiration: 2025-12-23
Also published as: GB202309512D0

Abstract

The present disclosure relates to binding proteins (e.g. antibodies) that specifically bind LILRB2. The present disclosure also provides methods of treatment, uses, pharmaceutical compositions and kits comprising the binding proteins (e.g. antibodies).

Description

LILRB2 BINDING PROTEINS AND USES THEREOF

1 BACKGROUND

[0001] The treatment of cancer remains a significant problem and whilst chemotherapeutic drugs can reduce cancer cell proliferation and therefore inhibit tumour growth and metastasis, the non-specific nature of chemotherapeutics means that they come with many major side effects due to off-target effects. As such, there has been an emphasis on the identification of alternatives to chemotherapeutics, with equally high efficacy but with an increased specificity for targeting cancer cells to reduce the incidence and severity of adverse events. One such novel anti-cancer therapy is monoclonal antibodybased immunotherapy which presents the opportunity for targeted cancer therapies, in particular those that stimulate the host immune system to encourage immune-mediated attack of cancerous cells.

[0002] The immune system can identify and kill cancerous cells, for example T cells can be primed by antigen presenting cells against mutated, cancer-specific antigens (Houghton, A.N., J Clin. Invest., (2004), 114(4), 468-471), which can aid in the regression or stasis of cancers (Vinay, D.S. et al., Seminars in Cancer Bio., 35, 185-198). However, many cancers develop immune evasion mechanisms to modulate the immune response. One such immuno-evasive method concerns the creation of an immunosuppressive microenvironment, with tumour-associated myeloid cells (TMCs) playing a key role in the maintenance of the immunosuppressive niche. Circulating myeloid cells can be recruited to the tumour site by the secretion of chemokines such as CCL2 and CCL5 (Mantovani, A. et al., Nat Rev Clin Oncol., (2017), 14(7), 399-416). Indeed, the degree of TMC recruitment has been linked to patient outcome, with increased amounts of TMCs being linked to poor prognosis (Nakamura, K. et al., Cell Mol Immunol., (2020), 17(1), 1-12). Moreover, TMCs can contribute to cancer metastasis, proliferation, and suppression of the immune system (Mantovani, A., et al., Cellular and Molecular Immunology, (2021), 18, 566-578). It is thought that most of these effects are governed by the anti-inflammatory phenotype of most TMCs. Therefore, shifting the phenotype of TMCs from anti-inflammatory to proinflammatory has gained traction as a potential method of immunotherapy in cancer. It has been observed in some cancers that maintenance of the anti-inflammatory phenotype of TMCs may be governed, in part, by agonism of the myeloid LILRB2 receptor by cancer cells (Liu, X. et al., Oncotarget, (2015), 6(25), 21004-21015).

[0003] Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is a member of the leukocyte immunoglobulin-like receptor (LILR) family. The LILR family contains activating and inhibitory members that can up- or down-regulate immune cell activity (see e.g. Marffy and McCarthy (2020) Front. Immunol., 11 , 857). LILRB2 is expressed on myeloid cells, including monocytes, macrophages, dendritic cells and neutrophils (Zhao, P. et al., Molecular Neurodegeneration, (2022), 17(44)), but has also been found to be expressed on non-haematopoietic cells, such as neurons and osteoclasts. While LILRB2 is constitutively expressed on myeloid cells, its expression is thought to increase in response to inflammation, with cell surface expression of LILRB2 increasing in response to inflammatory signals such as proinflammatory cytokine release (Deng, M. Antibody Therapeutics., (2020), 4(1), 16-33). [0004] The extracellular domain of LILRB2 consists of four immunoglobulin-like domains, connected to an intracellular cytoplasmic domain, containing an immunoreceptor tyrosine-based inhibitory motif which acts to inhibit activating signals (Wang, Q., Cell Mol Immunol., (2020), 17(9), 966-975). The extracellular domain of LILRB2 can bind to multiple ligands, including HLA-G, HLA-A, HLA-B, HLA-C, HLA-E, CD1 c, CD1d, Beta-Amyloid and angiopoietin-like proteins, including ANGPTL2 and 5 (Zhang, J. J Leukoc Biol., (2017), 102(2), 351-360). Due to its ability to bind multiple ligands, LILRB2 has many functions, including acting as an immune checkpoint inhibitor to prevent the onset of autoimmunity through binding to MHC class I molecules on antigen-presenting cells, transducing a negative signal that inhibits stimulation of an immune response. It is therefore thought to control inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity.

[0005] However, this function can be exploited by cancer cells, to help to maintain an anti-inflammatory tumour microenvironment. Activation of LILRB2 leads to phosphorylation of SHP1/2, governing downstream effects including the increased secretion of anti-inflammatory cytokines and suppressing the production of pro-inflammatory cytokines (Chen, H-M., et al., J. Clin. Investigation, (2018), 128(12), 5647-5662). LILRB2 may also compete with CD8+ T cells for the binding of MHCI on cancer cells, inhibiting T cell-mediated cytotoxicity (Anderson, K.J. et al., Immunology, (2009), 127(1), 8-17). As such, tumour cells may exploit the functions of myeloid cells, via activation of LILRB2.

[0006] Therefore, targeting LILRB2 may present a method of immunotherapy in cancers. Antagonism of LILRB2 has been shown to shift TMC phenotypes, from anti-inflammatory to pro-inflammatory. However, there is a need for highly potent LILRB2-specific binding protein (e.g. antibody) compositions which are suitable for use in the treatment of cancer.

2 SUMMARY

[0007] We have discovered binding proteins (e.g. antibodies) that bind LILRB2 and have properties suitable for development as medicaments. These binding proteins (e.g. antibodies) are suitable for use in treating diseases and conditions associated with increased activity of LILRB2, in particular for use in treating or preventing cancer. Binding proteins (e.g. antibodies) of the disclosure demonstrate a combination of advantageous properties, including strong binding affinity to LILRB2 and/or potency of inhibition of enzymatic activity of LILRB2, that make them suitable for use as therapeutics, in particular for treating cancer.

[0008] Binding proteins (e.g. antibodies) described herein have particularly efficacious properties. For example, binding proteins (e.g. antibodies) described herein, including LILRB0368, have very high potency. In addition, we have shown that blockade of LILRB2 enhances immunostimulatory responses of macrophages in vitro and is efficacious in vivo.

[0009] An aim of the present disclosure is to reduce/ameliorate symptoms of diseases such as cancer through the administration of such binding proteins (e.g. antibodies). Administration of binding proteins (e.g. antibodies) of the disclosure may be used either as a prophylactic or as a therapeutic.

2.1 STATEMENTS OF DISCLOSURE [0010] In a first aspect, the disclosure provides a LILRB2 binding protein which binds to human LILRB2 with an affinity (KD) of <100pM. In some embodiments, the affinity is measured by Biacore. In some embodiments, the LILRB2 binding protein inhibits LILRB2 (e.g. as measured in a co-culture reporter assay). In some embodiments, the LILRB2 binding inhibits HLA-G-mediated downstream signalling (e.g. as measured by co-culture reporter assay). In some embodiments, the LILRB2 binding protein inhibits LILRB2 in myeloid cells (e.g. as measured by a macrophage stimulation assay). In some embodiments, the myeloid cells are macrophages. In some embodiments, the LILRB2 binding protein increases TNF-alpha and/or GM-CSF release in vitro (e.g. as measured by macrophage stimulation assay). In some embodiments, the LILRB2 binding protein decreases VEGF-A in vitro (e.g. as measured by macrophage stimulation assay). In some embodiments, the LILRB2 binding protein induces macrophage repolarisation from tumour-supportive M2 state to tumour-suppressive M1 state, as characterised by the reduction in the cell surface expression of CD163 and increase in the cell surface expression of CD86 and production of TNFalpha (e.g. as measured in a macrophage, T cell, tumour cell co-culture assay, employing antigen-specific T cells). In some embodiments, the LILRB2 binding protein promotes a proinflammatory state in the tumour microenvironment leading to increased T cell lysis of a tumour cell line (e.g. as measured in a macrophage, T cell, tumour cell co-culture assay, employing antigen-specific T cells). In some embodiments, the LILRB2 binding protein decreases tumour volume (e.g. as measured in vivo).

[0011] In some embodiments, the LILRB2 binding protein is a competitive inhibitor of LILRB2 (e.g. as measured by ligand competition assay). In some embodiments, the ligand is HLA-G. In some embodiments, the LILRB2 binding protein does not bind LILRB1 , LILRB3, LILRB4, LILRB5, LILRA1 , LILRA2, LILRA3, LILRA4, LILRA5 or LILRA6. In some embodiments, the LILRB2 is human LILRB2. In some embodiments, the LILRB2 binding protein binds to all human LILRB2 haplotypes with a frequency of greater than 5%.

[0012] In some embodiments, the LILRB2 binding protein has an IC50 of 20pM or lower for human LILRB2 (e.g. as measured by macrophage stimulation assay). In other embodiments, the LILRB2 binding protein has an IC50 of 15pM or lower for human LILRB2 (e.g. as measured by macrophage stimulation assay). In other embodiments, the LILRB2 binding protein has an IC50 of 10pM or lower for human LILRB2 (e.g. as measured by macrophage stimulation assay). In another embodiment, the LILRB2 binding protein has an IC50 of 5pM or lower for human LILRB2 (e.g. as measured by macrophage stimulation assay).

[0013] In some embodiments, the LILRB2 binding protein is an antibody or antigen binding fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an IgG. In some embodiments, the antibody is an lgG1. In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence comprising complementarity determining regions (CDRs) HCDR1 , HCDR2 and HCDR3, and a variable light (VL) domain sequence comprising CDRs LCDR1 , LCDR2 and LCDR3, and wherein HCDR3 is the HCDR3 of SEQ ID NO: 7 (e.g. as determined by Kabat). [0014] In some embodiments, the LILRB2 binding protein comprises a glutamic acid (E), asparagine (N) or aspartic acid (D) residue at heavy chain position 52a (e.g. as determined by Kabat). In some embodiments, the LILRB2 binding protein comprises a HCDR2 sequence of SEQ ID NO: 6, optionally wherein the glutamic acid (E) residue at position 52a is substituted for an asparagine (N) or aspartic acid (D) residue (e.g. as determined by Kabat). In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence comprising HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6 and HCDR3 of SEQ ID NO: 7, and a variable light (VL) domain sequence comprising LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.

[0015] In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3, optionally with 1 , 2, 3, 4 or 5 amino acid alterations, and a variable light (VL) domain sequence of SEQ ID NO: 4, optionally with 1 , 2, 3, 4 or 5 amino acid alterations. In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3, optionally with 1 , 2, 3, 4 or 5 amino acid alterations outside the complementarity determining regions (CDRs), and a variable light (VL) domain sequence of SEQ ID NO: 4, optionally with 1 , 2, 3, 4 or 5 amino acid alterations outside the CDRs. In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and wherein the variable heavy (VH) domain sequence and variable light (VL) domain sequence respectively comprise a sequence having at least 90% identity to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4. In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and wherein the variable heavy (VH) domain sequence and variable light (VL) domain sequence respectively comprise a sequence having at least 90% identity to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, provided that the antibody has the HCDRs of SEQ ID NO: 3 and the LCDRs of SEQ ID NO: 4. In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 and a variable light (VL) domain sequence of SEQ ID NO: 4.

[0016] In some embodiments, the LILRB2 binding protein comprises a Fc domain. In some embodiments, the Fc domain has reduced effector function. In some embodiments, the Fc domain comprises the mutations L234F, L235E and P331 S. In some embodiments, the Fc domain comprises the mutations L234F, L235Q and K322Q. In some embodiments, the Fc domain comprises the mutations L238F, L239E and P335S.

[0017] In some embodiments, the Fc domain comprises at least one half life extension conferring mutation. In some embodiments, the Fc domain comprises the mutations M252Y, S254T and T256E. In some embodiments, the Fc domain has reduced effector function and comprises at least one half life extension conferring mutation. In some embodiments, the Fc domain comprises the mutations L234F, L235E, P331 S, M252Y, S254T and T256E. In some embodiments, the Fc domain comprises the mutations L234F, L235Q, K322Q, M252Y, S254T and T256E.

[0018] In some embodiments, the LILRB2 binding protein comprises kappa light chains. [0019] In some embodiments, the LILRB2 binding protein comprises light chains comprising the sequence SEQ ID NO: 2. In some embodiments, the LILRB2 binding protein comprises heavy chains comprising the sequence SEQ ID NO: 1. In some embodiments, the LILRB2 binding protein comprises light chains comprising the sequence SEQ ID NO: 2 and heavy chains comprising the sequence SEQ ID NO: 1.

[0020] In a further aspect, the disclosure provides a polypeptide comprising one or more domains of a LILRB2 binding protein as described anywhere herein. In some embodiments, the domain is one or more CDRs from the heavy and/or light chain, a VH domain or a VL domain.

[0021] In a further aspect, the disclosure provides a polypeptide comprising one or more chains of a LILRB2 binding protein as described anywhere herein.

[0022] In a further aspect, the disclosure provides a nucleic acid encoding one or more chains of a LILRB2 binding protein as described anywhere herein.

[0023] In a further aspect, the disclosure provides a vector comprising the nucleic acid as described anywhere herein.

[0024] In a further aspect, the disclosure provides a host cell comprising the vector as described anywhere herein.

[0025] In a further aspect, the disclosure provides a pharmaceutical composition comprising a LILRB2 binding protein as described anywhere herein and a pharmaceutically acceptable carrier.

[0026] In a further aspect, the disclosure provides a kit comprising a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein.

[0027] In a further aspect, the disclosure provides a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein for use in therapy. In a further aspect, the disclosure provides a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein for use in treating cancer. In some embodiments, the cancer is colorectal cancer, head and neck cancer, renal cancer, lung cancer, pancreatic cancer, gastric cancer, melanoma, breast cancer or ovarian cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is gastric cancer.

[0028] In a further aspect, the disclosure provides a method of treating cancer, wherein the method comprises administering a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein to a subject in need thereof.

[0029] In a further aspect, the disclosure provides the use of a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein for the manufacture of a medicament for the treatment of cancer.

3 BRIEF DESRIPTION OF THE DRAWINGS Figure 1 : Alignments of LILRB0368 VH and VL sequences, respectively, compared to germline showing the amino acid variation

[0030] Figure 1A-1 B show alignments of LILRB0368 VH and VL sequences, respectively, compared to germline showing the amino acid variations. In the VH sequence of LILRB0368 (as shown in Figure 1A), the VH sequence matches IGHV3-23*04 human germline in framework regions 1-3, and IGHJ2*01 in FW4. As well as the H-CDR3 sequence, non-germline sites elsewhere are N31 in H-CDR1 and E52a in H-CDR2. The E52a site in H-CDR2 has been shown to be critical for antigen binding. Reversion to germline glycine abolishes binding, but we also explored sequences with D or N at this position. In the VL sequence of LILRB0368 (as shown in Figure 1 B), the VL sequence matches human germline IGKV1 -33*01 /IGKV1 D-33*01 in FW1-3 and IGKJ2*02 in FW4. Non-germline sites are D31 in L-CDR1 and Q55 in L-CDR2. Figure 1C shows the VL and VH sequences used to construct anti-LILRB2 antibodies disclosed herein.

Figure 2: HTRF binding assay with LILRA1

[0031] Figure 2A-2G show the results of the HTRF binding assay with LILRA1. The results indicate that none of the LILRB2 mAbs tested show off-target binding to LILRA1. Comparator-N is the only comparator mAb tested that is not specific for LILRB2 and shows binding to LILRA1 .

Figure 3: HTRF binding assay with LILRA2

[0032] Figure 3A-3G show the results of the HTRF binding assay with LILRA2. The results indicate that none of the LILRBXXXX mAbs or the comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I and Comparator-N) show off-target binding to LILRA2.

Figure 4: HTRF binding assay with LILRA3

[0033] Figure 4A-4G show the results of the HTRF binding assay with LILRA3. The results indicate that none of the LILRBXXXX mAbs show off-target binding to LILRA3. Comparator-N is the only comparator mAb tested that is not specific for LILRB2 and shows binding to LILRA3.

Figure 5: HTRF binding assay with LILRB1

[0034] Figure 5A-5G show the results of the HTRF binding assay with LILRB1. The results indicate that none of the LILRBXXXX mAbs show off-target binding to LILRB1. Comparator-N is the only comparator mAb tested that shows binding to LILRB1 .

Figure 6: HTRF binding assay with LILRB2 haplotype 1

[0035] Figure 6A-6G show the results of the HTRF binding assay with LILRB2 haplotype 1 . All of the LILRBXXXX mAbs and the comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I and Comparator-N) show binding to LILRB2 halotype 1 , one of the top four most prevalent haplotypes of LILRB2.

Figure 7: HTRF binding assay with LILRB2 haplotype 2

[0036] Figure 7A-7G show the results of the HTRF binding assay with LILRB2 haplotype 2. All of the LILRBXXXX mAbs and the comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-1 and Comparator-N) show binding to LILRB2 halotype 2, one of the top four most prevalent haplotypes of LILRB2.

Figure 8: HTRF binding assay with LILRB2 haplotype 3

[0037] Figure 8A-8G show the results of the HTRF binding assay with LILRB2 haplotype 3. All of the LILRBXXXX mAbs and the comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-1 and Comparator-N) show binding to LILRB2 halotype 3, one of the top four most prevalent haplotypes of LILRB2.

Figure 9: HTRF binding assay with LILRB2 haplotype 4

[0038] Figure 9A-9G show the results of the HTRF binding assay with LILRB2 haplotype 4. All of the LILRBXXXX mAbs and the comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-1 and Comparator-N) show binding to LILRB2 halotype 4, one of the top four most prevalent haplotypes of LILRB2.

Figure 10: HTRF binding assay with LILRB2 domain 1/domain 2

[0039] Figure 10A-10H show the results of the HTRF binding assay with LILRB2 domain 1/domain 2. The results indicate that all of the LILRBXXXX mAbs and the comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I and Comparator-N) tested show binding to the LILRB2 D1- D2 construct.

Figure 11 : Representative Biacore association and dissociation sensorgrams of IgG binding to human LILRB2 measured by Surface Plasmon Resonance

[0040] Figure 11 shows representative Biacore association and dissociation sensorgrams of IgG binding to human LILRB2 measured by Surface Plasmon Resonance.

Figure 12: All of the LILRB2XXXX mAbs and comparator mAbs tested bind to human LILRB2 as determined by HTRF

[0041] Figure 12A shows that all of the LILRB2XXXX mAbs and comparator mAbs tested bind to human LILRB2 as determined by HTRF. Figure 12B shows that none of the LILRB2XXXX mAbs tested bind to cyno LILRB2, whereas Comparator-N and Comparator-B bind to cyno LILRB2 as determined by HTRF.

Figure 13: RB0368 and Comparator-B LILRB2 Fab affinity on human and cyno LILRB2

[0042] Figure 13 shows LILRB0368 and Comparator-B LILRB2 Fab affinity on human and cyno LILRB2. Comparator B binds to both human and cyno LILRB2, whereas LILRB0368 is not cyno cross reactive.

Figure 14: Flow binding assay to Jurkat-LILRB2 cells

[0043] Figure 14A-14E show the results of the flow binding assay to Jurkat-LILRB2 cells. All tested samples show robust specific binding to Jurkat-NFAT-Luc-LILRB2, with low nanomolar/high picomolar IC50s. None of the tested LILRB2 antibodies bind non-specifically to Jurkat-NFAT-Luc parental cells (data not shown). Figure 15: LILRB0362 epitope competition assay

[0044] Figure 15A-15D show the results of the LILRB0362 epitope competition assay. The results indicate that parent mAbs LILRB0359, LILRB0361 and LILRB0362 share the same epitope, and combinations of features of these mAbs results in variants that retain this epitope. LILRB0359 is 3-fold less potent that LILRB0361 and LILRB0362. All variants containing glycine at position CDR2 52a lost activity (LILRB0369, LILRB0387, LILRB0394, LILRB0395, LILRB0402).

Figure 16: Co-culture reporter assay

[0045] Figure 16A-16D show the results of the co-culture reporter assay. The results indicate that all tested LILRBXXXX mAbs that show activity in the LILRB0362 epitope competition assay, also show functional inhibition of LILRB2-HLA-G signalling in a co-culture reporter assay.

Figure 17: Octet selectivity assays

[0046] Figure 17A-17B show the results of the octet selectivity assays. The results show that none of the tested LILRBXXXX mAbs show off-target binding to related LILR family members. Comparator-J, Comparator-I, Comparator-N and Comparator-M are also specific for LILRB2 and do not show off-target binding to LILR family members. Comparator-B shows strong binding to LILRA5. Comparator-R shows strong binding to LILRB3 and LILRA6

Figure 18: HEK293 binding and an aggregation assay

[0047] Figure 18A-J show the results of the HEK293 binding and an aggregation assay. The results show that none of the tested LILRBXXXX or comparator mAbs show non-specific binding to HEK293 cells, nor do they show propensity for aggregation.

Figure 19: AC-SINS self-association assay

[0048] Figure 19 shows the results of the AC-SINS self-association assay. The results show that none of the tested LILRBXXXX mAbs have a risk of self-association. Comparator-J has a risk of selfassociation in charged buffers (HA), and Comparator-B has a risk of self-association in both neutral (PBS) and charged buffers.

Figure 20: Baculovirus particle (BVP) ELISA

[0049] Figure 20shows the results of the baculovirus particle (BVP) ELISA. The results show that none of the tested LILRBXXXX or comparator mAbs shows a risk of fast clearance due to non-specific binding to baculovirus particles.

Figure 21 : Macrophage stimulation assay

[0050] Figure 21A-21C show the results of the macrophage stimulation assay. TNFa release from CD40L-stimulated macrophages following treatment with our LILRBXXXX mAb (Figure 21A) and Figure 21 B) provide results from two panels). In Figure 21 C, LILRB0368, was compared to several benchmark anti-LILRB2 antibodies.

Figure 22: Macrophage stimulation assay [0051] Figure 22A-C show the results of the macrophage stimulation assay. Levels of Figure 22A) TNFa, Figure 22B) GM-CSF and Figure 22C) VEGF-A released from CD40L-stimulated macrophages following treatment with 50nM of exemplar anti-LILRB2 mAb clone, LILRB0368, are presented. Results are compiled from the fold change values (treatment versus no mAb treatment control) generated from 12 separate donors. Bars represent arithmetic mean and standard deviation. Statistical significance was calculated using a paired T test and two-tailed p-values are presented in the figures as ** p < 0.01 ; *** p < 0.001 ; **** p < 0.0001 .

Figure 23: CD163 and CD206 expression assay

[0052] Figure 23A-B show the results of the CD163 and CD206 expression assay. Expression of CD163 and CD206 is down-regulated on monocyte-derived macrophages differentiated in the presence of 50nM of LILRB0368.

Figure 24: Tumour growth assay

[0053] Figure 24 shows the results of the tumour growth assay. LILRB0361 reduces tumour growth rate in vivo. NSG/SGM3 mice reconstituted with human CD34+ cord blood stem cells were implanted subcutaneously with an MDA-MB-231 tumour xenograft. Mice were treated with 10 mg/kg LILRB0361 (and isotype control) twice weekly for 3 weeks.

Figure 25: T-cell/macrophage/tumour co-culture assay with a EGFR-CD3 (OKT3) T cell engager (TCE)

[0054] Figure 25 shows the results from a T-cell/macrophage/tumour co-culture assay. MDA-MB-231 breast cancer cell line was co-cultured in the presence of polyclonal T cells, with or without autologous macrophages and drugs for three days. Tumour killing and level of expression of CD86 on CD14+ macrophages was assessed by flow cytometry. Results were generated from 8 donors in 3 independent experiments. Arithmetic mean (of triplicates) and SD values are plotted. Statistical analysis was performed by Friedman tests with Dunn’s multiple comparison test comparing all TCE-treated samples to each other (left) or all conditions (right), * p<0.05, and **p<0.01 .

Figure 26: T-cell/macrophage/tumour co-culture assay with HER2 CAR-T cells

[0055] Figure 26 shows the results from a T-cell/macrophage/tumour co-culture assay. The MDA-MB- 231 cell line was co-cultured in the presence of CAR-T cells, with or without non-autologous macrophages and drugs for two days. Tumour killing, was assessed by flow cytometry. Results were generated from two independent runs with one CAR-T cell donor and 6 macrophage donors (averages of triplicates shown). CAR-T cells were used at E:T ratios of 0.5:1 (experiment 1) or 1 :1 (experiment 2). AZD2796 was used at 100nM. Cytotoxicity measured by % tumour cell death using flow cytometry. Statistical analysis was performed by paired t-test showing an increase in tumour cell death in the 3way assay comparing CAR-T cells vs CAR-T cells + AZD2796. Arithmetic mean and SD values are plotted for all conditions. Normality was assessed by Shapiro-Wilk tests. **** P<0.001 .

4 DETAILED DESCRIPTION

4.1 TERMINOLOGY [0056] The term “binding protein” means a protein molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the molecule. As used herein, the term “binding protein” encompasses antibodies. Fragments are also encompassed within the scope of the term “binding protein” as used herein.

[0057] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. Antibody fragments are also encompassed within the scope of the term “antibody” as used herein. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGI, lgG2, lgG3, lgG4, IgAI and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0058] The term “binding protein fragment” refers to a portion of an intact binding protein. The term “antibody fragment” refers to a portion of an intact antibody. An “antigen binding fragment,” “antigenbinding domain,” or “antigen-binding region,” refers to a portion of an intact binding protein (e.g. antibody) that binds to an antigen. An antigen-binding fragment can contain the antigenic determining regions of an intact binding protein (e.g. antibody) (e.g., the complementarity determining regions (CDR)). Examples of antigen-binding fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear binding proteins (e.g. antibodies), and single chain binding proteins (e.g. antibodies). An antigen-binding fragment can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0059] The terms "anti-LILRB2 binding protein" (e.g. "anti-LILRB2 antibody"), "LILRB2 binding protein" (e.g. "LILRB2 antibody") and "binding protein that binds to LILRB2" (e.g. "antibody that binds to LILRB2") are used interchangeably herein to refer to a binding protein (e.g. antibody) that is capable of binding to LILRB2. The extent of binding of a LILRB2 binding protein (e.g. LILRB2 antibody) to a non- LILRB2 protein can be less than about 10% of the binding of the binding protein (e.g. antibody) to LILRB2 as measured, e.g., using ForteBio or Biacore.

[0060] A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0061] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of a binding protein (e.g. antibody), generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among binding proteins (e.g. antibodies) and are used in the binding and specificity of a particular binding protein (e.g. antibody) for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the binding protein (e.g. antibody) with antigen. In some aspects, the variable region is a human variable region. In some aspects, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some aspects, the variable region is a primate (e.g. non-human primate) variable region. In some aspects, the variable region comprises rodent or murine CDRs and primate (e.g. non-human primate) framework regions (FRs).

[0062] The term “complementarity determining region” or “CDR” as used herein refers to each of the regions of a variable domain which are hypervariable in sequence and/or form structurally defined loops (hypervariable loops) and/or contain the antigen-contacting residues. Binding proteins (e.g. antibodies) can comprise six CDRs, e.g., three in the VH and three in the VL.

[0063] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of a binding protein (e.g. antibody).

[0064] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of a binding protein (e.g. antibody).

[0065] The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of a binding protein (e.g. antibody) or an antigen-binding fragment thereof. In some aspects, CDRs can be determined according to the Kabat numbering system (see, e.g. Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within a heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within a light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

[0066] Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end ofthe Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modelling software.

Loop Kabat AbM Chothia

[0067] As used herein, the term “constant region” or “constant domain” are interchangeable and have its meaning common in the art. The constant region is a binding protein (e.g. antibody) portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In some aspects, an antibody or antigenbinding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0068] As used herein, the term “heavy chain” when used in reference to a binding protein (e.g. antibody) can refer to any distinct type, e.g. alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g. lgG1 , lgG2, lgG3, and lgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0069] As used herein, the term “light chain” when used in reference to a binding protein (e.g. antibody) can refer to any distinct type, e.g. kappa (K) or lambda (I) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0070] The term "chimeric" binding proteins (e.g. antibodies) or antigen-binding fragments thereof refers to binding proteins (e.g. antibodies) or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of binding proteins (e.g. antibodies) or antigen-binding fragments thereof derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in binding proteins (e.g. antibodies) or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0071] The term "humanized" binding protein (e.g. antibody) or antigen-binding fragment thereof refers to forms of non-human (e.g. murine) binding proteins (e.g. antibodies) or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized binding proteins (e.g. antibodies) or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (“CDR grafted”) (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues from a non-human species that has the desired specificity, affinity, and capability. The humanized binding protein (e.g. antibody) or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize binding protein (e.g. antibody) or antigen-binding fragment thereof specificity, affinity, and/or capability. In general, the humanized binding protein (e.g. antibody) or antigen-binding fragment thereof will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized binding protein (e.g. antibody) or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91 (3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some aspects, a "humanized binding protein (e.g. antibody)" is a resurfaced binding protein (e.g. antibody).

[0072] The term "human" binding protein (e.g. antibody) or antigen-binding fragment thereof means a binding protein (e.g. antibody) or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, where such binding protein (e.g. antibody) or antigen-binding fragment is made using any technique known in the art. This definition of a human binding protein (e.g. antibody) or antigen-binding fragment thereof includes intact or full-length binding proteins (e.g. antibodies) and fragments thereof.

[0073] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. a binding protein (e.g. antibody) or antigen-binding fragment thereof) and its binding partner (e.g. an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g. binding protein (e.g. antibody) or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of kon/kon, whereas KA is calculated from the quotient of kon/kotr. k_on refers to the association rate constant of, e.g, a binding protein (e.g. antibody) or antigen-binding fragment thereof to an antigen, and kotr refers to the dissociation of, e.g, a binding protein (e.g. antibody) or antigen-binding fragment thereof from an antigen. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as BIACORE® or KinExA.

[0074] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding protein (e.g. antibody) or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In some aspects, the epitope to which a binding protein (e.g. antibody) or antigen-binding fragment thereof binds can be determined by, e.g. NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g. liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g. site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g, Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251 : 6300-6303). Binding protein (e.g. antibody)/ antigen-binding fragment thereof: antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., U.S. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et ah, (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. See, e.g., Champe M et ah, (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0075] A binding protein (e.g. antibody) that “binds to the same epitope” as a reference binding protein (e.g. antibody) refers to a binding protein (e.g. antibody) that binds to the same amino acid residues as the reference binding protein (e.g. antibody). The ability of a binding protein (e.g. antibody) to bind to the same epitope as a reference binding protein (e.g. antibody) can be determined by a hydrogen/deuterium exchange assay (see e.g., Coales et al. Rapid Commun. Mass Spectrom. 2009; 23: 639-647).

[0076] As used herein, the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of binding proteins (e.g. antibodies) or antigen-binding fragments thereof. These terms indicate that the binding protein (e.g. antibody) or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that the binding entails some complementarity between the antigen binding domain and the epitope. Accordingly, in some aspects, a binding protein (e.g. antibody) that “specifically binds” to LILRB2 can also bind to other LILRs, but the extent of binding to an unrelated protein is less than about 10% of the binding of the binding protein (e.g. antibody) to LILRB2 as measured, e.g., using ForteBio or Biacore.

[0077] A binding protein (e.g. antibody) is said to "competitively inhibit" binding of a reference binding protein (e.g. antibody) to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some degree, binding of the reference binding protein (e.g. antibody) to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. A binding protein (e.g. antibody) can be said to competitively inhibit binding of the reference binding protein (e.g. antibody) to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0078] A polypeptide, binding protein (e.g. antibody), polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, binding protein (e.g. antibody), polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, binding proteins (e.g. antibodies), polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, a binding protein (e.g. antibody), polynucleotide, vector, cell, or composition which is isolated is substantially pure. As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0079] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulphide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component. Also included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure are based upon binding proteins (e.g. antibodies), in some aspects, the polypeptides can occur as single chains or associated chains. [0080] “Percent identity” refers to the extent of identity between two sequences (e.g. amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning two sequences, introducing gaps to maximize identity between the sequences. Alignments can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed with the blastn program set at default parameters, and alignment of amino acid sequences can be performed with the blastp program set at default parameters (see National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov).

[0081] As used herein, amino acids with hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tryptophan (W), and tyrosine (Y). Amino acids with aliphatic hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), and valine (V). Amino acids with aromatic hydrophobic side chains include phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0082] As used herein, amino acids with polar neutral side chains include asparagine (N), cysteine (C), glutamine (Q), serine (S), and threonine (T).

[0083] As used herein, amino acids with electrically charged side chains include aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Amino acids with acidic electrically charged side chains include aspartic acid (D) and glutamic acid (E). Amino acids with basic electrically charged side chains include arginine (R), histidine (H), and lysine (K).

[0084] As used herein, the term “host cell” can be any type of cell, e.g. a primary cell, a cell in culture, or a cell from a cell line. In some aspects, the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0085] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0086] As used herein, the terms “subject” and “patient” are used interchangeably. The subject is a human.

[0087] As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0088] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can mean "includes," "including," and the like; "consisting essentially of or "consists essentially" are open- ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0089] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0090] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range (without “about”) are also provided.

[0091] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

4.2 ANTI-LILRB2 BINDING PROTEINS

[0092] The binding proteins (e.g. antibodies) described herein are described with respect to the following concepts, aspects, sentences, arrangements and embodiments. Unless otherwise stated, all concepts, embodiments, sentences, arrangements and aspects are to be read as being able to be combined with any other concept, aspect, sentence, arrangement or embodiment, unless such combination would not make technical sense or is explicitly stated otherwise.

[0093] BINDING - TARGET:

[0094] The disclosure relates particularly to binding proteins (e.g. antibodies) that specifically bind LILRB2. In particular, the disclosure provides binding proteins (e.g. antibodies) comprising a LILRB2 binding domain which specifically binds to LILRB2. In some embodiments, the binding proteins (e.g. antibodies) are monospecific binding proteins (e.g. antibodies). In some embodiments, the binding proteins (e.g. antibodies) are bivalent, i.e. comprising two binding domains each of which specifically binds LILRB2.

[0095] Binding proteins (e.g. antibodies) described herein that specifically bind to LILRB2 are shown herein to be particularly efficacious and thus have particular utility in treating diseases associated with LILRB2 activity, in particular increased levels of LILRB2 activity, such as cancer.

[0096] The disclosure also provides polypeptides that specifically bind to LILRB2. As such, these polypeptides comprise at least one LILRB2 binding domain that specifically binds LILRB2. Such polypeptides may have particular utility when they are comprised in a binding protein (e.g. antibody) (i.e. a binding protein (e.g. antibody) that specifically binds LILRB2). [0097] In some embodiments the LILRB2 is human LILRB2. In some embodiments the LILRB2 is human LILRB2 haplotype 1 . In some embodiments the LILRB2 is human LILRB2 haplotype 2. In some embodiments the LILRB2 is human LILRB2 haplotype 3. In some embodiments the LILRB2 is human LILRB2 haplotype 4. An exemplary amino acid sequence of human LILRB2 protein is described in the Uniprot database as UniProtKB - A0A0G2JMW1 . LILRB2 (Leukocyte immunoglobulin-like receptor subfamily B member 2) may also be referred to as LILRB2, ILT4, MIR10, LIR2, MIR-10, LIR-2, leukocyte immunoglobulin like receptor B2, ILT-4, CD85D.

4.3 BINDING - MEASUREMENT

[0098] Any suitable method may be used to determine whether a binding protein (e.g. antibody) (or a polypeptide) binds to the LILRB2 or LILRB2 protein. Such a method may comprise surface plasmon resonance (SPR), homogenous time resolved fluorescence (HTRF), bio-layer interferometry, or an ELISA to determine specificity of binding proteins (e.g. antibodies). A binding protein (e.g. antibody) may be said to bind its antigen if the level of binding to antigen is at least 2.5 fold greater, e.g. at least 10 fold greater, than binding to a control antigen. Binding between a binding protein (e.g. antibody) and its cognate antigen is often referred to as specific binding. Precise identification of the residues bound by a binding protein (e.g. antibody) can usually be obtained using x-ray crystallography. This technique may be used to determine that a binding protein (e.g. antibody) described herein binds one or more residues of the LILRB2 protein.

[0099] Ability of a binding protein (e.g. antibody) to bind its target antigen, and the specificity and affinity of that binding (KD, Kd and/or Ka) can be determined by any routine method in the art, e.g. using surface plasmon resonance (SPR), such as by BiacoreTM (Cytiva Life Sciences) or using the ProteOn XPR36TM (Bio-Rad®), using KinExA® (Sapidyne Instruments, Inc), or using ForteBio Octet (Pall ForteBio Corp.).

[0100] The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant of a particular binding protein (e.g. antibody)-antigen interaction. Affinity of binding protein (e.g. antibody)- antigen binding may be determined, e.g., by SPR. Affinity may also be determined by bio-layer interferometry.

[0101] In some examples, a binding protein (e.g. antibody) may bind to human LILRB2 with an affinity (KD) of < 100pM. In one embodiment a binding protein (e.g. antibody) may bind to human LILRB2 with an affinity

10 pM, as determined by SPR.

[0102] In one example, the SPR is carried out at 25°C. In brief, the affinity of the binding protein (e.g. antibody) can be determined using SPR by:

1 . Coupling mouse anti-human (or other relevant human, rat or non-human vertebrate binding protein (e.g. antibody) constant region species-matched) IgG to a biosensor chip (e.g. dextran- coated gold chip) such as by primary amine coupling. Thus, an anti-Fc binding protein (e.g. antibody) may be covalently immobilised on the chip surface using amine coupling. 2. Exposing the mouse anti-human IgG (or other matched species binding protein (e.g. antibody)) to the test binding protein (e.g. antibody) (e.g., in human IgG format) to capture the test binding protein (e.g. antibody) on the chip;

3. Passing the test antigen over the chip’s capture surface at a series of concentrations up to a maximum of 100 nM, e.g., at 0.39, 1 .56, 6.25, 25 and 100 nM, and a 0 nM (i.e. buffer alone) control run. The buffer may optionally be 0.01 M HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid), 0.15 M NaCI and 0.05% v/v surfactant P20 in aqueous solution, buffered to pH 7.4; and

4. Determining the affinity of binding of test binding protein (e.g. antibody) to test antigen using surface plasmon resonance. KD, Ka and Kd may then be calculated.

[0103] SPR can be carried out using any standard SPR apparatus, such as by BIACORE or using the ProteOn XPR36TM (BIO-RAD). Regeneration of the capture surface can be carried out with 3 M magnesium chloride solution. This removes the captured test antibody and allows the surface to be used for another interaction. The binding data can be fitted to 1 :1 model inherent using standard techniques, e.g. using analysis software such as Biacore Insight Evaluation Software.

[0104] A suitable protocol for determining binding IC50 of a binding protein (e.g. antibody) to LILRB2 is set out in detail in Example 3 and Example 4.

4.4 CROSS-REACTIVITY

[0105] The LILR family comprises subfamilies of receptors, LILRA family and LILRB family. LILRA subfamily comprises members LILRA1 , LILRA2, LILRA3, LILRA4, LILRA5 and LILRA6. LILRB family comprises members LILRB1 , LILRB2, LILRB3, LILRB4 and LILRB5.

[0106] In some examples, binding proteins (e.g. antibodies) that specifically bind to LILRB2 do not cross-react with other members of the LILR family. Alternatively, in some examples, binding proteins (e.g. antibodies) that specifically bind to LILRB2 may be cross-reactive with other members of the LILR family. In some examples, the other members of the LILR family are human LILR family members.

[0107] In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 does not cross-react with other members of the LILRB family. In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 cross-reacts with other members of the LILRB family. In some examples, the other members of the LILRB family are human LILRB family members.

[0108] In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 does not cross-react with LILRB1. In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 cross-reacts with LILRB1 . In some examples, the LILRB1 is human LILRB1 .

[0109] In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 does not cross-react with other members of the LILRA family. In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 cross-reacts with other members of the LILRA family. In some examples, the other members of the LILRA family are human LILRA family members. [0110] In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 does not cross-react with LILRA1. In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 cross-reacts with LILRA1 . In some examples, the LILRA1 is human LILRA1 .

[0111] In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 does not cross-react with LILRA2. In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 cross-reacts with LILRA2. In some examples, the LILRA2 is human LILRA2.

[0112] In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 does not cross-react with LILRA3. In some examples, a binding protein (e.g. antibody) that specifically binds to LILRB2 cross-reacts with LILRA3. In some examples, the LILRA3 is human LILRA3.

[0113] For binding proteins (e.g. antibodies) that specifically bind to a LILRB2 antigen and cross-react with one or more of the above LILRA or LILRB members, in some examples, the binding protein (e.g. antibody) may bind LILRB2 with at least a 10 fold greater binding affinity than to the other family member (e.g. as measured by SPR). In some examples, the binding protein (e.g. antibody) may bind LILRB2 with at least a 20 fold greater binding affinity than to the other family member (e.g. as measured by SPR). In some examples, the binding protein (e.g. antibody) may bind LILRB2 with at least a 50 fold greater binding affinity than to the other family member (e.g. as measured by SPR).

[0114] Binding proteins (e.g. antibodies) that specifically bind to LILRB2 may be cross-reactive with LILRB2 from other species. Alternatively, in some examples, binding proteins (e.g. antibodies) that specifically bind to LILRB2 do not cross-react with LILRB2 from other species. Thus, in one embodiment, the binding protein (e.g. antibody) does not bind rodent LILRB2. In one embodiment, the binding protein (e.g. antibody) does not bind mouse LILRB2. In one embodiment, the binding protein (e.g. antibody) also binds rodent LILRB2. In one embodiment, the binding protein (e.g. antibody) also binds mouse LILRB2. In one embodiment, the binding protein (e.g. antibody) also binds cynomolgus LILRB2. In one embodiment, the binding protein (e.g. antibody) does not bind cynomolgus LILRB2.

4.5 FUNCTION - INHIBITION

[0115] Binding proteins (e.g. antibodies) described herein are inhibitory binding proteins (e.g. antibodies) that inhibit LILRB2, thus being useful in therapy, “inhibits LILRB2” means “inhibits activity of LILRB2”. Inhibition of activity may occur via multiple mechanisms. In some examples, the binding proteins (e.g. antibodies) inhibit LILRB2 binding to its ligand (such as the ligands HLA-G, HLA-A, HLA- B, HLA-C, HLA-E, CD1 c, CD1d, ANGPT2 and ANGPT5). Inhibition of LILRB2 binding to its ligand may be achieved by a binding protein (e.g. antibody) directly blocking the ligand binding site on LILRB2. Such binding proteins (e.g. antibodies) may compete for binding to LILRB2 protein with its ligand, as described further below. Alternatively, inhibition of LILRB2 binding to its ligand may be achieved by an indirect mechanism, e.g. where a binding protein (e.g. antibody) binds to an epitope on LILRB2 which is outside of the ligand binding site, but which modifies the structure or function of the LILRB2 protein such that binding to the ligand is reduced or prevented. [0116] Inhibition of LILRB2 activity may be measured using a co-culture reporter assay, as detailed in Example 5. Thus in one embodiment, the binding protein (e.g. antibody) inhibits activity of LILRB2 (e.g. as measured in a co-culture reporter assay).

[0117] LILRB2 is highly expressed on myeloid cells, such as macrophages, neutrophils, dendritic cells. Therefore in some embodiments, the binding protein (e.g. antibody) inhibits LILRB2 in myeloid cells (e.g. as measured in a co-culture reporter assay). In some embodiments, the myeloid cells are macrophages.

[0118] Alternative measures may also be used to determine the functional effects of binding proteins (e.g. antibodies) of the disclosure, including the potency of the binding protein (e.g. antibody). Inhibition of LILRB2 leads to TNF-alpha and/or GM-CSF release in vitro and so measurement of TNF-alpha and/or GM-CSF levels can be used as a readout of the efficacy of a LILRB2 binding protein (e.g. antibody). Therefore in some embodiments, the binding protein (e.g. antibody) increases TNF-alpha and/or GM-CSF release in vitro. In some embodiments, the binding protein (e.g. antibody) increases TNF-alpha and/or GM-CSF release from macrophages in vitro (e.g. as measured by macrophage stimulation assay). Further, inhibition of LILRB2 leads to TNF-alpha and/or GM-CSF release while reducing the production of the pro-angiogenic cytokine VEGF-A in vitro and so measurement of TNF- alpha and/or GM-CSF and/or VEGF-A levels can be used as a readout of the efficacy of a LILRB2 binding protein (e.g. antibody). An exemplary macrophage stimulation assay is set out in Example 12.

[0119] LILRB2 activation leads to HLA-G-mediated downstream signalling which includes phosphorylation of SHP1/2. Assays directed to the downstream signalling mechanisms that are induced by LILRB2 activity may be measured as a read out of the efficacy of the LILRB2 binding protein (e.g. antibody). Therefore, in some embodiments, the binding protein (e.g. antibody) inhibits HLA-G- mediated downstream signalling (e.g. as measured by a co-culture reporter assay). In some embodiments, the binding protein (e.g. antibody) inhibits phosphorylation of SHP1/2.

[0120] Inhibition of LILRB2 has also been found to promote macrophage re polarization from a tumour- supportive M2 state to a tumour-suppressive M1 state. In some embodiments, the binding protein (e.g. antibody) induces macrophage repolarisation from tumour-supportive M2 state to tumour-suppressive M1 state, as characterised by the reduction in the cell surface expression of CD163 and increase in the cell surface expression of CD86 and production of TNFalpha (e.g. as measured in a macrophage, T cell, tumour cell co-culture assay, employing antigen-specific T cells). In some embodiments, the binding protein (e.g. antibody) promotes a proinflammatory state in the tumour microenvironment leading to increased T cell lysis of a tumour cell line (e.g. as measured in a macrophage, T cell, tumour cell co-culture assay, employing antigen-specific T cells).

[0121] We have also shown that inhibition of LILRB2 can reduce tumour volume in mouse xenograft models. Therefore, in some embodiments, the binding protein (e.g. antibody) decreases tumour volume (e.g. as measured in vivo). An example tumour growth assay is set out in Example 12.

[0122] The concentration of the binding protein (e.g. antibody), expressed as either or both of the binding protein (e.g. antibody) weight (milligrams, or micrograms, or nanograms or picograms) in a given volume (litre or millilitre or microlitre), or as a molarity of the binding protein (e.g. antibody) (millimolar, or micromolar or nanomolar or picomolar), that is required to inhibit 50% of the detectable activity in the assay (the inhibitory concentration for 50%, or IC50) or inhibit 90% of the detectable activity in the assay (the inhibitory concentration for 90%, or IC90) or inhibit 95% of the detectable activity in the assay (the inhibitory concentration for 95%, or IC95) may reported from the assay used. This can be calculated using any of a variety of methods known to the art, including the fitting of inhibition curves mathematically to the experimentally derived data and reporting these as an IC50 or IC90 or IC95. Finally, the binding protein (e.g. antibody) concentration that completely inhibits LILRB2 activity can be determined.

4.6 LILRB2 inhibition - levels (IC50)

[0123] In some examples, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 10pM or lower (e.g. as determined in a macrophage stimulation assay). In some examples, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 5pM or lower (e.g. as determined in a macrophage stimulation assay).

[0124] In one embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 3.0 E- 9 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 2.0 E-9 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 1 .0 E-9 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 10.0 E-10 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 9.0 E-10 M or lower (e.g. as determined by a flow binding assay to Jurkat- NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 8.0 E-10 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc- LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 7.0 E-10 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 6.0 E-10 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of 5.0 E-10 M or lower (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding proteins (e.g. antibodies) inhibit LILRB2 with an IC50 of from 2.0 E-10 M to 3.0 E-9 M (e.g. as determined by a flow binding assay to Jurkat-NFAT-Luc-LILRB2).

[0125] LILRB2 inhibition is calculated relative to a reference binding protein (e.g. antibody):

[0126] In some examples, the binding proteins (e.g. antibodies) may inhibit LILRB2 with an activity level which is greater than a reference binding protein (e.g. antibody). In some examples, the reference binding protein (e.g. antibody) may be any of the comparator antibodies set out herein in the Examples. For example, the binding proteins (e.g. antibodies) may inhibit LILRB2 with an activity level which is greater than the reference binding protein (e.g. antibody) expressed as fold change relative to the reference binding protein (e.g. antibody). In one example, the binding protein (e.g. antibody) may inhibit LILRB2 with an activity level greater than a 2-fold change relative to the reference binding protein (e.g. antibody). In one example, the binding protein (e.g. antibody) may inhibit LILRB2 with an activity level greater than a 25-fold change relative to the reference binding protein (e.g. antibody). In one example, the binding protein (e.g. antibody) may inhibit LILRB2 with an activity level greaterthan a 50-fold change relative to the reference binding protein (e.g. antibody). In one example, the binding protein (e.g. antibody) may inhibit LILRB2 with an activity level greater than a 100-fold change relative to the reference binding protein (e.g. antibody). In one example, the binding protein (e.g. antibody) may inhibit LILRB2 with an activity level greater than a 500-fold change relative to the reference binding protein (e.g. antibody). In one example, the binding protein (e.g. antibody) may inhibit LILRB2 with an activity level greater than a 1000-fold change relative to the reference binding protein (e.g. antibody).

4.7 COMPETITION WITH LIGANDS

[0127] The binding proteins (e.g. antibodies) provided may compete with LILRB2 for binding to one or more of its ligands (i.e. ligands such as HLA-G, HLA-A, HLA-B, HLA-C, HLA-E, CD1 c, CD1d, ANGPT2 and ANGPT5). The binding proteins (e.g. antibodies) provided may specifically bind to the ligand binding domain of LILRB2, wherein the binding protein (e.g. antibody) is an inhibitory binding protein (e.g. antibody) which competes with LILRB2 for binding to its ligand, thereby preventing downstream signalling. In some embodiments, the LILRB2 binding protein is a competitive inhibitor of LILRB2 (e.g. as measured by ligand competition assay). In some embodiments, the LILRB2 binding protein competes with one or more ligands of LILRB2 for binding to LILRB2. In some embodiments the ligand is selected from the group consisting of HLA-G, HLA-A, HLA-B, HLA-C, HLA-E, CD1 c, CD1d, ANGPT2, and ANGPT5. In some embodiments the ligand is HLA-G.

[0128] Other binding proteins (e.g. antibodies), that do not compete with LILRB2 for binding to one or more of its ligands, may alter the ability of the protein to function correctly and thereby also be an inhibitory binding protein (e.g. antibody) even though the binding protein (e.g. antibody) does not block the ligand binding site and therefore ligand interaction directly.

[0129] Whether a binding protein (e.g. antibody) competes with LILRB2 for binding to one or more of its ligands may be measured using a competition assay. Competition may be determined by surface plasmon resonance (SPR), such techniques being readily apparent to the skilled person. SPR may be carried out using Biacore™, Proteon™ or another standard SPR technique. Such competition may be due, for example, to the binding proteins (e.g. antibodies) or fragments binding to identical or overlapping epitopes of LILRB2 to that which the ligand binds. In one example, competition is determined by ELISA, such techniques being readily apparent to the skilled person. In one example, competition is determined by homogenous time resolved fluorescence (HTRF), such techniques being readily apparent to the skilled person. In one example, competition is determined by fluorescence activated cell sorting (FACS), such techniques being readily apparent to the skilled person. In one example, competition is determined by ForteBio Octet® Bio-Layer Interferometry (BLI) such techniques being readily apparent to the skilled person. In one example, competition is determined as set out in Example 6.

[0130] If the epitope to which the inhibitory binding protein (e.g. antibody) binds completely blocks the ligand binding site of LILRB2, then binding between receptor and ligand is completely prevented (which may be a physical blocking - in the case of overlapping epitopes - or steric blocking - where the antagonist is large such that it prevents the ligand binding to its distinct epitope). If the epitope to which the binding protein (e.g. antibody) binds partially blocks the ligand binding site of the receptor, the ligand may be able to bind, but only weakly (in the case of partial inhibition), or in a different orientation to the natural binding interaction.

4.8 BINDING PROTEINS BINDING THE SAME EPITOPE AND BINDING PROTEINS THAT COMPETE FOR BINDING

[0131] Binding proteins (e.g. antibodies) may be provided which bind to the same epitope as a binding protein (e.g. antibody) defined anywhere herein.

[0132] A binding protein (e.g. antibody) of the present disclosure may be one that specifically binds to the same epitope on LILRB2 as the epitope on LILRB2 that is bound by a reference binding protein (e.g. antibody), where the reference binding protein (e.g. antibody) is a binding protein (e.g. antibody) as defined anywhere herein. A binding protein (e.g. antibody) of the present disclosure may be one that specifically binds to the same epitope on LILRB2 as the epitope on LILRB2 that is bound by a reference binding protein (e.g. antibody), where the reference binding protein (e.g. antibody) is a binding protein (e.g. antibody) as defined anywhere herein. In one embodiment, the reference binding protein (e.g. antibody) is LILRB0368. Binding protein (e.g. antibody) binding to the same epitope as a binding protein (e.g. antibody) defined anywhere herein may optionally be determined using X-ray crystallography or other fine mapping techniques such as electron microscopy to identify the contact points between binding protein (e.g. antibody) and antigen. Alternatively, binding protein (e.g. antibody) binding to the same epitope as a binding protein (e.g. antibody) defined anywhere herein may be determined using an HTRF epitope competition assay, e.g. as set out in Example 4. As described elsewhere herein, competition between binding proteins (e.g. antibodies) may also be determined, for example using SPR, and binding proteins (e.g. antibodies) of the present invention may compete for binding to LILRB2 (compete for binding to their epitope) with an IgG binding protein (e.g. antibody) that is any binding protein (e.g. antibody) as described herein. A binding protein (e.g. antibody) may contact the LILRB2 protein with a footprint that fully or partly overlaps with that of binding protein (e.g. antibody) as defined anywhere herein.

[0133] The present disclosure describes binding proteins (e.g. antibodies) of the invention that specifically bind to LILRB2 and which are defined by their function and/or by their structure. The invention also provides binding proteins (e.g. antibodies) that compete with those binding proteins (e.g. antibodies) described herein for binding to LILRB2. In one embodiment, a binding protein (e.g. antibody) of the present disclosure may be one which competes for binding to LILRB2 with any binding protein (e.g. antibody) described herein. Such competition may be due, for example, to the binding protein (e.g. antibody) binding to an identical or overlapping epitope of LILRB2 as the reference binding protein (e.g. antibody). Such a ‘competing’ binding protein (e.g. antibody) may retain the same function as the reference binding protein (e.g. antibody) with which it competes. A binding protein (e.g. antibody) that binds to an identical (same) epitope can be expected to have the same function as the reference binding protein (e.g. antibody) with which it competes.

[0134] In one embodiment, a binding protein (e.g. antibody) that specifically binds to LILRB2 is provided, wherein said binding protein (e.g. antibody) competes for binding to LILRB2 with a reference binding protein (e.g. antibody), where the reference binding protein (e.g. antibody) is a binding protein (e.g. antibody) as described anywhere herein. In one embodiment, a binding protein (e.g. antibody) that specifically binds to LILRB2 is provided, wherein said binding protein (e.g. antibody) competes for binding to LILRB2 with a reference binding protein (e.g. antibody), where the reference binding protein (e.g. antibody) is a binding protein (e.g. antibody) as described anywhere herein. In one embodiment, the reference binding protein (e.g. antibody) is LILRB0368.

4.9 BINDING PROTEIN (E.G. ANTIBODY) TYPES

[0135] In some embodiments, the binding protein may be an antibody.

[0136] In some embodiments, the binding protein (e.g. antibody) may be an isolated binding protein (e.g. antibody). In some embodiments, the binding protein (e.g. antibody) may be a monoclonal binding protein (e.g. antibody).

[0137] The binding proteins (e.g. antibodies) described herein may be, or may be obtained from, a human binding protein (e.g. antibody), a humanized binding protein (e.g. antibody), a non-human binding protein (e.g. antibody), or a chimeric binding protein (e.g. antibody). A “chimeric” binding protein (e.g. antibody) refers to an binding protein (e.g. antibody) or fragment thereof comprising both human and non-human regions. A “humanized” binding protein (e.g. antibody) is a monoclonal binding protein (e.g. antibody) comprising a human binding protein (e.g. antibody) scaffold and at least one CDR obtained or derived from a non-human binding protein (e.g. antibody). Non-human binding proteins (e.g. antibodies) include binding proteins (e.g. antibodies) isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized binding protein (e.g. antibody) can comprise, one, two, or three CDRs obtained or derived from a non-human binding protein (e.g. antibody). A fully human monoclonal binding protein (e.g. antibody) does not contain any amino acid residues obtained or derived from a non-human animal. It will be appreciated that fully human and humanized binding proteins (e.g. antibodies) carry a lower risk for inducing immune responses in humans than mouse or chimeric binding proteins (e.g. antibodies) (see, e.g., Harding et al., mAbs, 2(3): 256-26 (2010)). In one embodiment, binding protein (e.g. antibody) is a fully human binding protein (e.g. antibody).

[0138] A human binding protein (e.g. antibody), a non-human binding protein (e.g. antibody), a chimeric binding protein (e.g. antibody), or a humanized binding protein (e.g. antibody) can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an binding protein (e.g. antibody) recombinantly) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described in, for example, Kohler and Milstein, Eur. J. Immunol., 5: 511 -519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). In certain embodiments, a human binding protein (e.g. antibody) or a chimeric binding protein (e.g. antibody) can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, the Kirin TO MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181 69-97 (2008)). A humanized binding protein (e.g. antibody) can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, N.J. (2009)), including, e.g., grafting of non-human CDRs onto a human binding protein (e.g. antibody) scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144( ): 115-120 (2008)). In one embodiment, a humanized antibody can be produced using the methods described in, e.g., U.S. Patent Application Publication 2011/0287485 A1 .

[0139] Methods of making the binding proteins (e.g. antibodies) described herein are described in Example 1 .

[0140] In some embodiments, the binding protein (e.g. antibody) is monospecific. In some embodiments, the binding protein (e.g. antibody) has two LILRB2 binding domains such that the binding protein (e.g. antibody) is bivalent for LILRB2.

[0141] In some embodiments, the LILRB2 binding domains are comprised in a Fab domain. In some embodiments, the Fab domains are comprised in an IgG (which comprises 2 heavy chains and 2 light chains, wherein the 2 heavy chains and 2 light chains form 2 Fab domains and an Fc domain).

4.10 EXEMPLARY LILRB2 BINDING PROTEINS

[0142] The present disclosure describes a number of exemplary binding proteins (e.g. antibodies) that specifically bind to LILRB2 (collectively referred to as “LILRBXXXX”), the amino acid sequences of which are set out in Table 8 herein.

[0143] In one embodiment, the binding protein (e.g. antibody) is LILRB0368. LILRB0368 is a monoclonal monospecific IgG 1 antibody that specifically binds to LILRB2. The amino acid sequences of LILRB0368 are set out in Table 8 herein as SEQ ID NOs: 1-10.

[0144] LILRB0368 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 3, comprising the CDRH1 amino acid sequence of SEQ ID NO: 5 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 6 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 7 (Kabat). LILRB0368 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 4, comprising the CDRL1 amino acid sequence of SEQ ID NO: 8 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 9 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 10 (Kabat). LILRB0368 is an IgG 1 comprising 2 heavy and 2 light chains, wherein the two heavy chains each comprise SEQ ID NO: 1 and the two light chains each comprise SEQ ID NO: 2. The light chains of LILRB0368 are kappa light chains.

[0145] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence comprising complementarity determining regions (CDRs) HCDR1 , HCDR2 and HCDR3, and a variable light (VL) domain sequence comprising CDRs LCDR1 , LCDR2 and LCDR3, and wherein HCDR3 is the HCDR3 of SEQ ID NO: 7 (e.g. as determined by Kabat).

[0146] In some embodiments, the binding protein (e.g. antibody) comprises a glutamic acid (E) asparagine (N) or aspartic acid (D) residue at position 52a (e.g. as determined by Kabat) in HCDR2. In some embodiments, the binding protein (e.g. antibody) comprises a HCDR2 sequence of SEQ ID NO: 6, optionally wherein the glutamic acid (E) residue at position 52a is substituted for an asparagine (N) or aspartic acid (D) residue (e.g. as determined by Kabat).

[0147] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence comprising HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6 and HCDR3 of SEQ ID NO: 7, and a variable light (VL) domain sequence comprising LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.

[0148] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3, optionally with 1 , 2, 3, 4 or 5 amino acid alterations, and a variable light (VL) domain sequence of SEQ ID NO: 4, optionally with 1 , 2, 3, 4 or 5 amino acid alterations.

[0149] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3, optionally with 1 , 2, 3, 4 or 5 amino acid alterations outside the complementarity determining regions (CDRs), and a variable light (VL) domain sequence of SEQ ID NO: 4, optionally with 1 , 2, 3, 4 or 5 amino acid alterations outside the CDRs.

[0150] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and wherein the variable heavy (VH) domain sequence and variable light (VL) domain sequence respectively comprise a sequence having at least 90% identity to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4.

[0151] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and wherein the variable heavy (VH) domain sequence and variable light (VL) domain sequence respectively comprise a sequence having at least 90% identity to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, provided that the binding protein (e.g. antibody) has the HCDRs of SEQ ID NO: 3 and the LCDRs of SEQ ID NO: 4.

[0152] In some embodiments, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 and a variable light (VL) domain sequence of SEQ ID NO: 4. [0153] The amino acid sequences of other exemplary LILRB2 binding proteins (e.g. antibodies) described in the Examples (denoted LILRBXXXX) are also set out in Table 8 herein as SEQ ID NOs: 11-115. Such binding proteins (e.g. antibodies), including each of the CDR, VH, VL, HC and LC amino acid sequences, are also embodiments of the disclosure.

[0154] LILRB0354 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 13, comprising the CDRH1 amino acid sequence of SEQ ID NO: 15 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 16 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 17 (Kabat). LILRB0354 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 14, comprising the CDRL1 amino acid sequence of SEQ ID NO: 18 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 19 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 20 (Kabat). LILRB0354 comprises a heavy chain amino acid sequence of SEQ ID NO: 11 and a light chain amino acid sequence of SEQ ID NO: 12.

[0155] LILRB0355 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 23, comprising the CDRH1 amino acid sequence of SEQ ID NO: 25 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 26 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 27 (Kabat). LILRB0355 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 24, comprising the CDRL1 amino acid sequence of SEQ ID NO: 28 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 29 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 30 (Kabat). LILRB0355 comprises a heavy chain amino acid sequence of SEQ ID NO: 21 and a light chain amino acid sequence of SEQ ID NO: 22.

[0156] LILRB0356 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 33, comprising the CDRH1 amino acid sequence of SEQ ID NO: 35 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 36 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 37 (Kabat). LILRB0356 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 34, comprising the CDRL1 amino acid sequence of SEQ ID NO: 38 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 39 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 40 (Kabat). LILRB0356 comprises a heavy chain amino acid sequence of SEQ ID NO: 31 and a light chain amino acid sequence of SEQ ID NO: 32.

[0157] LILRB0357 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 43, comprising the CDRH1 amino acid sequence of SEQ ID NO: 45 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 46 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 47 (Kabat). LILRB0357 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 44, comprising the CDRL1 amino acid sequence of SEQ ID NO: 48 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 49 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 50 (Kabat). LILRB0357 comprises a heavy chain amino acid sequence of SEQ ID NO: 41 and a light chain amino acid sequence of SEQ ID NO: 42.

[0158] LILRB0358 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 53, comprising the CDRH1 amino acid sequence of SEQ ID NO: 55 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 56 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 57 (Kabat). LILRB0358 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 54, comprising the CDRL1 amino acid sequence of SEQ ID NO: 58 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 59 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 60 (Kabat). LILRB0358 comprises a heavy chain amino acid sequence of SEQ ID NO: 51 and a light chain amino acid sequence of SEQ ID NO: 52.

[0159] LILRB0359 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 63, comprising the CDRH1 amino acid sequence of SEQ ID NO: 65 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 66 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 67 (Kabat). LILRB0359 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 64, comprising the CDRL1 amino acid sequence of SEQ ID NO: 68 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 69 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 70 (Kabat). LILRB0359 comprises a heavy chain amino acid sequence of SEQ ID NO: 61 and a light chain amino acid sequence of SEQ ID NO: 62.

[0160] LILRB0360 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 73, comprising the CDRH1 amino acid sequence of SEQ ID NO: 75 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 76 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 77 (Kabat). LILRB0360 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 74, comprising the CDRL1 amino acid sequence of SEQ ID NO: 78 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 79 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 80 (Kabat). LILRB0360 comprises a heavy chain amino acid sequence of SEQ ID NO: 71 and a light chain amino acid sequence of SEQ ID NO: 72.

[0161] LILRB0361 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 83, comprising the CDRH1 amino acid sequence of SEQ ID NO: 85 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 86 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 87 (Kabat). LILRB0361 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 84, comprising the CDRL1 amino acid sequence of SEQ ID NO: 88 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 89 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 90 (Kabat). LILRB0361 comprises a heavy chain amino acid sequence of SEQ ID NO: 81 and a light chain amino acid sequence of SEQ ID NO: 82.

[0162] LILRB0362 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 93, comprising the CDRH1 amino acid sequence of SEQ ID NO: 95 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 96 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 97 (Kabat). LILRB0362 comprises a light chain variable region (VL) amino acid sequence of SEQ ID NO: 94, comprising the CDRL1 amino acid sequence of SEQ ID NO: 98 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 99 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 100 (Kabat). LILRB0362 comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 92. [0163] LILRB0363 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 83 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0164] LILRB0364 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 93 and a variable light (VL) domain sequence of SEQ ID NO: 84.

[0165] LILRB0365 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 63 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0166] LILRB0366 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 103 and a variable light (VL) domain sequence of SEQ ID NO: 84.

[0167] LILRB0367 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 107 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0168] LILRB0369 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 105 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0169] LILRB0385 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 108 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0170] LILRB0386 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 110 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0171] LILRB0387 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 102 and a variable light (VL) domain sequence of SEQ ID NO: 84.

[0172] LILRB0388 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 106 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0173] LILRB0389 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 93 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0174] LILRB0390 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 83 and a variable light (VL) domain sequence of SEQ ID NO: 113.

[0175] LILRB0391 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 101 and a variable light (VL) domain sequence of SEQ ID NO: 114.

[0176] LILRB0392 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 109 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0177] LILRB0393 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 109 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0178] LILRB0394 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 111 and a variable light (VL) domain sequence of SEQ ID NO: 94. [0179] LILRB0395 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 111 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0180] LILRB0396 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 83 and a variable light (VL) domain sequence of SEQ ID NO: 115.

[0181] LILRB0397 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 109 and a variable light (VL) domain sequence of SEQ ID NO: 84.

[0182] LILRB0398 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 106 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0183] LILRB0399 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 107 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0184] LILRB0400 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 108 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0185] LILRB0401 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 110 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0186] LILRB0402 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 102 and a variable light (VL) domain sequence of SEQ ID NO: 113.

[0187] LILRB0403 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 103 and a variable light (VL) domain sequence of SEQ ID NO: 113.

[0188] LILRB0404 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 104 and a variable light (VL) domain sequence of SEQ ID NO: 113.

[0189] LILRB0405 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 108 and a variable light (VL) domain sequence of SEQ ID NO: 84.

[0190] LILRB0406 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 110 and a variable light (VL) domain sequence of SEQ ID NO: 84.

4.11 OTHER BINDING PROTEIN FORMA TS

[0191] In other embodiments, the amino acid sequences of LILRB0368 or any of the other exemplified binding proteins (e.g. antibodies) described herein (including but not limited to HCDRs, LCDRs, VH, VL, HC and LC sequences of the exemplified binding proteins (e.g. antibodies) herein) may be incorporated into other binding protein (e.g. antibody) formats. In particular, the HCDRs and LCDRs, and/or VH and VL domains of LILRB0368 may be incorporated into other binding protein (e.g. antibody) formats, such as a bispecific binding protein (e.g. antibody). The data described herein shows that binding proteins (e.g. antibodies) having the CDR sequences and/or VH and VL sequences of LILRB0368 are particularly efficacious at inhibiting LILRB2 activity and thus have particular suitability for use in treating disorders associated with LILRB2 activity, such as cancer. 4.12 SEQUENCE IDENTITY

[0192] In some examples, the binding protein (e.g. antibody) comprises an amino acid sequence which has a high level of sequence identity to the amino acid sequence of one of the exemplary binding proteins (e.g. antibodies) described herein and set out in Table 8.

[0193] In one example, the amino acid sequence is at least 70% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 75% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 95% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 96% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 97% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 98% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 99% identical to the specified SEQ ID No. In one example, the amino acid sequence is at least 99.5% identical to the specified SEQ ID No. In some examples, the variation is not in the CDRs of said sequences.

[0194] In some examples, the binding protein (e.g. antibody) comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence and wherein the variable heavy (VH) domain and variable light (VL) domain sequences respectively comprise a sequence having at least 90% (preferably 95%, more preferably 98%) identity to the variable heavy (VH) and variable light (VL) domain sequences of any one of the binding proteins (e.g. antibodies) described herein and set out in Table 1 , provided that the binding protein (e.g. antibody) has the CDRs of said binding protein (e.g. antibody) described herein and set out in Table 1 .

[0195] As set out in the Terminology section, percentage sequence identity between two amino acid sequences may be determined by any alignment program known in the art. For example, alignment of amino acid sequences to determine percentage sequence identity can be performed with the blastp program set at default parameters (see National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov).

4.13 SUBSTITUTIONS

[0196] In some examples, a variable heavy (VH) domain sequence as defined anywhere herein may optionally have 1 , 2, 3, 4 or 5 amino acid alterations outside the complementarity determining regions (CDRs). Similarly, a variable light (VL) domain sequence as defined anywhere herein may optionally have 1 , 2, 3, 4 or 5 amino acid alterations outside the CDRs. When one or more mutations (whether additions, insertions, substitutions or deletions of one or more amino acids) are made in the variable domain sequence of an binding protein (e.g. antibody) described herein, the resulting binding protein (e.g. antibody) may be tested (e.g., in one or more assays described herein) to confirm that affinity and/or potency are retained.

[0197] Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present disclosure may also be "non-conservative", in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g. substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

[0198] In one embodiment, the conservative amino acid substitutions are as described herein. For example, the substitution may be of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P. In another embodiment, the conservative amino acid substitutions may be wherein Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V.

[0199] In one example, the amino acid substitutions are located outside the CDR sequences.

4.14 LIGHT CHAINS

[0200] In some examples, the binding protein (e.g. antibody) comprises a kappa light chain. Exemplary kappa light chain constant region amino acid sequences are set out in Table 8.

[0201] In some examples, the binding protein (e.g. antibody) comprises a lambda light chain.

4.15 CONSTANT REGIONS:

[0202] In another embodiment, the binding protein (e.g. antibody) comprises a constant region (Fc), such as a human constant region. The Fc may be of any suitable class. In some embodiments, the binding protein (e.g. antibody) comprises a constant region that is based upon wild-type lgG1 , lgG2, or lgG4 binding proteins (e.g. antibodies), or variants thereof. In some embodiments, the binding protein (e.g. antibody) is an lgG1.

[0203] In certain embodiments, the binding protein (e.g. antibody) comprises an altered Fc region, in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the binding molecule. Such alterations may result in altered effector function, reduced immunogenicity, and/or an increased serum half-life.

[0204] For example, an binding protein (e.g. antibody) may have a heavy chain constant region that binds one or more types of Fc receptor but does not induce cellular effector functions, i.e. which does not mediate ADCC, CDC or ADCP activity. Such a constant region may be unable to bind the particular Fc receptor(s) responsible for triggering ADCC, CDC or ADCP activity. An binding protein (e.g. antibody) may have a heavy chain constant region that does not bind Fey receptors.

[0205] In some embodiments, the binding protein (e.g. antibody) comprises an Fc domain having reduced effector function. This reduced effector function of the Fc region may be achieved through the incorporation of one or more amino acid mutations in the Fc region which are known in the art. [0206] In one embodiment, the binding protein (e.g. antibody) comprises an lgG1 triple mutant, referred to herein as lgG1-TM. The lgG1-TM is a human lgG1 isotype containing three single amino acid substitutions, L234F/L235E/P331 S, within the lower hinge and CH2 domain (Oganesyan et al., Acta Crystallogr. D Biol. Crystallogr. 64:700-704, 2008). The TM causes a profound decrease in binding to human FcyRI, FcyRII, FcyRIII, and C1 q, resulting in a human isotype with very low effector function. Thus in one embodiment, the binding protein (e.g. antibody) comprises an Fc domain comprising the mutations L234F, L235E and P331 S.

[0207] In one embodiment, the binding protein (e.g. antibody) comprises an lgG1 triple mutant referred to herein as FQQ. The FQQ mutant comprises three single amino acid substitutions in the Fc domain: L234F, L235Q and K322Q (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.). This combination of substitutions result in a profound decrease in binding to human FcyRI, FcyRII, FcyRIII, and C1q, resulting in a human isotype with very low effector function (see e.g. Borrok et al., J Pharm Sci. 2017 Apr;106(4):1008-1017). Therefore in one embodiment, the binding protein (e.g. antibody) comprises an Fc domain comprising the mutations L234F, L235Q and K322Q.

[0208] Other mutations that reduce effector function are known in the art. See, e.g., Armour et al., Eur. J. Immunol. 29:2613-2624, 1999; Shields et al., J. Biol. Chem. 276:6591-6604, 2001.

[0209] In some embodiments, the binding proteins (e.g. antibodies) disclosed herein are modified to increase or decrease serum half-life. In certain embodiments, a heavy chain constant region or fragment thereof can include one or more amino acid substitutions relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wildtype IgG constant domain. Thus in one embodiment, the binding protein (e.g. antibody) comprises an Fc domain comprising at least one half life extension conferring mutation. In one embodiment, the heavy chain constant region comprises a YTE mutation. The terms “YTE” or “YTE mutant” refer to a mutation in lgG1 Fc that results in an increase in the binding to human FcRn and improves the serum half-life of the binding protein (e.g. antibody) having the mutation. A YTE mutant comprises a combination of three mutations, M252Y, S254T and T256E (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.), introduced into the heavy chain of an IgG 1 . See U.S. Patent No. 7,658,921 , which is incorporated by reference herein. The YTE mutant has been shown to increase the serum half-life of binding proteins (e.g. antibodies) approximately four-times as compared to wild-type versions of the same binding protein (e.g. antibody) (Dall’Acqua et al., J. Biol. Chem. 281 :23514-24 (2006); Robbie et al., Antimicrob. Agents Chemother. 57, 6147-6153 (2013)). See also U.S. Patent No. 7,083,784, which is hereby incorporated by reference in its entirety.

[0210] In some embodiments the binding protein (e.g. antibody) comprises mutations that confer reduced effector function and mutations that extend the half life of the binding protein (e.g. antibody). Thus in one embodiment, the binding protein (e.g. antibody) comprises an Fc domain having reduced effector function and comprises at least one half life extension conferring mutation. In one embodiment, the binding protein (e.g. antibody) comprises an Fc domain comprising the mutations TM-YTE, i.e. comprising the mutations L234F, L235E, P331 S, M252Y, S254T and T256E. Binding proteins (e.g. antibodies) comprising the mutations TM-YTE have been shown to have extended half-life and lack of binding protein (e.g. antibody)-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity, i.e. lacking immune receptor binding (see e.g. Borrok et al., J Pharm Sci. 2017 Apr;106(4):1008-1017). In one embodiment, the binding protein (e.g. antibody) comprises an Fc domain comprising the mutations FQQ-YTE, i.e. comprising the mutations L234F, L235Q, K322Q, M252Y, S254T and T256E. Binding proteins (e.g. antibodies) comprising the mutation combination FQQ-YTE have been shown to have significantly improved conformational and colloidal stability, whilst retaining the same biological activities (extended half-life and lack of immune receptor binding) as TM- YTE. The Fc domain of antibody LILRB0368 includes the TM mutation, the amino acid sequence of which is set out in Table 8.

4.16 POLYPEPTIDES

[0211] In a further aspect, the disclosure provides a polypeptide comprising one or more chains of an binding protein (e.g. antibody) as described anywhere herein.

[0212] The disclosure also provides polypeptides comprising one or more binding domains of the binding proteins (e.g. antibodies) defined anywhere herein.

[0213] The polypeptide may comprise part or all of a LILRB2 binding domain. In some embodiments, the domain is one or more CDRs from the heavy and/or light chain, a VH domain or a VL domain. The polypeptide may comprise binding domains such as one or more CDRs as defined herein, or variable light or variable heavy domains as defined herein. In some embodiments, these polypeptides may comprise binding domains that comprise all three CDRs (CDR1 , CDR2 and CDR3) of a variable heavy domain sequence as defined herein. In some embodiments, these polypeptides may comprise binding domains that comprise all three CDRs (CDR1 , CDR2 and CDR3) of a variable light domain sequence as defined herein. In some examples, the polypeptide may comprise a variable heavy domain of an binding protein (e.g. antibody) as defined herein. In some examples, the polypeptide may comprise a variable light domain of an binding protein (e.g. antibody) as defined herein. In some embodiments, the polypeptide may comprise a full heavy chain of an binding protein (e.g. antibody) as defined herein. In some embodiments, the polypeptide may comprise a full light chain of an binding protein (e.g. antibody) as defined herein. In some embodiments, the polypeptide is an isolated polypeptides. In one embodiment, polypeptides comprising any one or more of the amino acid sequences set out in Table 1 are provided. For example, the polypeptide may comprise any one or more amino acid sequences from antibody LILRB0368, the sequences of which are set out in Table 8 herein.

4.17 NUCLEIC A CIDS, VECTORS, HOST CELLS

[0214] Nucleic acids that encode any one or more of the amino acid sequences (e.g. any one or more CDRs from the VL and/or VH domains, a VH domain and/or a VL domain, full heavy chain and/or full light chain) of any one of the binding proteins (e.g. antibodies) described herein are also provided. [0215] In one example the nucleic acid encodes one or more chains of binding protein (e.g. antibody) as defined anywhere herein. In one example, the nucleic acid encodes a heavy chain of any one of the binding proteins (e.g. antibodies) described herein. In another example, the nucleic acid encodes a light chain of any one of the binding proteins (e.g. antibodies) described herein.

[0216] In one embodiment, nucleic acids that encode any one or more of the amino acid sequences set out in Table 8 are provided. In some embodiments, nucleic acids that encode any one or more of the amino acid sequences of antibody LILRB0368 are provided.

[0217] Nucleic acids that encode a polypeptide as defined anywhere herein are also provided.

[0218] In one example, the nucleic acid is an isolated and purified nucleic acid.

[0219] The term “nucleic acid sequence” is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g. phosphorothioates, boranophosphates, and the like).

[0220] Vectors comprising the nucleic acids described above are also provided. In one embodiment, the vector comprises one or more nucleic acid sequences encoding an binding protein (e.g. antibody) as defined anywhere herein. In one embodiment, the vector comprises one or more nucleic acid sequences encoding a polypeptide as defined anywhere herein. The vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage. Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)). In one embodiment, the vector may be a CHO vector. In one embodiment, the vector may be a HEK293 vector.

[0221] In addition to the nucleic acid sequence encoding an binding protein (e.g. antibody) as defined anywhere herein or a polypeptide as defined anywhere herein, the vector further comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide forthe expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). [0222] Host cells comprising one or more of the vectors as described anywhere herein or one or more of the nucleic acids as defined anywhere herein are also provided.

[0223] The vector(s) comprising the nucleic acid(s) encoding the amino acid sequence(s) of the binding proteins (e.g. antibodies) or polypeptides described anywhere herein can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby. Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently. The host cell may be a prokaryotic or a eukaryotic cell.

[0224] In some embodiments, the host cells are prokaryotic cells. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include the various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).

[0225] In some embodiments, the host cells are eukaryotic cells. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. In one embodiment, the vector is expressed in mammalian cells. A number of suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR- cells (Urlaub et al, Proc. Natl. Acad. Sci. USA, 97: 4216- 4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). The mammalian cell desirably is a human cell. For example, the mammalian cell can be a human lymphoid or lymphoid derived cell line, such as a cell line of pre-B lymphocyte origin, a PER.C6® cell line (Crucell Holland B.V., The Netherlands), or human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573).

[0226] A nucleic acid sequence encoding amino acids of an binding protein (e.g. antibody) as defined anywhere herein or a polypeptide as defined anywhere herein may be introduced into a cell by “transfection,” “transformation,” or “transduction.” “Transfection,” “transformation,” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al, Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available. 4.18 PHARMACEUTICAL COMPOSITION

[0227] The disclosure also provides a pharmaceutical composition comprising an binding protein (e.g. antibody) as defined anywhere herein and a pharmaceutically acceptable carrier. Also provided is a composition comprising the nucleic acid sequence encoding an binding protein (e.g. antibody) as defined anywhere herein or a polypeptide as defined anywhere herein, or the vector comprising the nucleic acid sequence as defined anywhere herein.

[0228] The pharmaceutical composition comprises an effective amount of the binding protein (e.g. antibody) as defined herein, nucleic acid as defined anywhere herein or vector as defined anywhere herein. An effective amount of the binding protein (e.g. antibody), nucleic acid or vector to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. In one embodiment, the effective amount of binding protein (e.g. antibody) as defined anywhere herein within the pharmaceutical composition is effective to treat or prevent a disease associated with LILRB2 activity, such as cancer.

[0229] The composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. The pharmaceutically acceptable carrier may include one or more excipients. Pharmaceutically acceptable excipients are known and include carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administerthe composition. The physiologically acceptable excipient may be an aqueous pH buffered solution. Examples of physiologically acceptable excipient include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as Ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0230] The composition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0231] The compositions can be administered intravenously. The composition can also be administered parenterally or subcutaneously. When administered systemically, the composition should be sterile, pyrogen-free and in a physiologically acceptable solution having due regard for pH, isotonicity and stability. These conditions are known to those skilled in the art. [0232] Methods of administering a pharmaceutical composition as defined herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific example, a pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Each dose may or may not be administered by an identical route of administration.

[0233] Various delivery systems are known and can be used to administer a prophylactic or therapeutic agent (e.g., an binding protein (e.g. antibody) as disclosed herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding protein (e.g. antibody), receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0234] In a specific example, it may be desirable to administer a prophylactic or therapeutic agent, or a pharmaceutical composition as described herein locally to the area in need of treatment. This may be achieved by, for example, local infusion, by topical administration (e.g., by intranasal spray), by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibres.

[0235] In the case of medicaments that are intended for local and/or topical administration, such as by absorption to epithelial or mucocutaneous linings, an binding protein (e.g. antibody) may be provided as an IgA isotype binding protein (e.g. antibody). For human patients, human lgA1 or human lgA2 binding proteins (e.g. antibodies) are preferred. Medicaments formulated for inhalation and/or for delivery of binding protein (e.g. antibody) (or its encoding nucleic acid, e.g., in a DNA vector) to the upper and/or lower respiratory tract, including formulations for delivery of a nebulised medicament, may comprise an IgA (e.g., human lgA1 or human lgA2) binding protein (e.g. antibody). Inhalers, nebulisers and similar devices may thus be provided containing a medicament comprising an IgA binding protein (e.g. antibody) or its encoding nucleic acid, together with any buffers or other excipients suitable for stabilisation of the medicament and/or for promoting its delivery to the target tissue.

4.19 THERAPEUTIC USE

[0236] We have discovered monoclonal binding proteins (e.g. antibodies) that specifically bind LILRB2 and have advantageous properties suitable for development as medicaments for treating or preventing disorders associated with LILRB2 activity, such as cancer. Binding proteins (e.g. antibodies) of the disclosure demonstrate a combination of advantageous properties, including high specificity for LILRB2, strong binding affinity to LILRB2 and/ or potency of inhibition of LILRB2 activity. [0237] Binding proteins (e.g. antibodies) described herein or pharmaceutical compositions thereof may be used in therapy. In particular, binding proteins (e.g. antibodies) or pharmaceutical compositions thereof may be used in treating diseases associated with LILRB2 activity, such as cancer. Binding proteins (e.g. antibodies) or pharmaceutical compositions thereof may also be used in preventing diseases associated with LILRB2 activity, such as cancer.

[0238] Therefore the disclosure provides an binding protein (e.g. antibody) as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for use in therapy.

[0239] The disclosure also provides a binding protein (e.g. antibody) as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for use in treating a disease or disorder or condition associated with LILRB2 activity, in particular associated with increased LILRB2 activity.

[0240] The disclosure also provides a binding protein (e.g. antibody) as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for use in treating cancer.

[0241] The disclosure also provides a method of treating cancer, wherein the method comprises administering a binding protein (e.g. antibody) as defined anywhere herein or a pharmaceutical composition as defined anywhere herein to a subject in need thereof.

[0242] The disclosure also provides the use of a binding protein (e.g. antibody) as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for the manufacture of a medicament for the treatment of cancer.

[0243] In one embodiment, the subject is a human.

[0244] In some embodiments, the disease or condition to be treated is cancer. In one embodiment, one or more symptoms of cancer are reduced. In one example, the progression of cancer is reduced. In one example, the risk of developing cancer is reduced.

[0245] Some types of cancer may have particularly high levels of LILRB2 activity, which may be due to the presence of a particularly high level of macrophages expressing LILRB2 within the tumour(s). In such cancers, therapy using a binding protein (e.g. antibody) as described anywhere herein may be particularly efficacious. Cancers displaying particularly high levels of LILRB2 activity due to the presence of a particularly high level of macrophages include renal cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, melanoma, breast cancer or ovarian cancer. Renal cancer in particular displays a high level of macrophages within the tumour(s) and thus high level of LILRB2 expression.

[0246] Therefore in some embodiments, the binding protein (e.g. antibody) is for use in treating cancer wherein the cancer is renal cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, melanoma, breast cancer or ovarian cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer.

[0247] The disclosure also relates to the use of the binding protein (e.g. antibody) for use in the treatment of cancer in combination with a T-cell engager. The disclosure also relates to the use of the binding protein (e.g. antibody) for use in the treatment of cancer in combination with a CAR-T cell.

4.20 KIT

[0248] The disclosure also provides a kit comprising a binding protein (e.g. antibody) as described anywhere herein or a pharmaceutical composition as described anywhere herein. In some embodiments, the kit includes a binding protein (e.g. antibody) as defined anywhere herein and instructions to administer the binding protein (e.g. antibody) to a subject in need of treatment.

[0249] There is also provided a pharmaceutical or diagnostic pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions as disclosed herein, such as one or more binding proteins (e.g. antibodies) provided herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration, e.g., an authorisation number.

[0250] In another example, an article of manufacture that includes a container in which a composition containing a binding protein (e.g. antibody) as described herein and a packaging insert or label indicating that the composition can be used to treat a disease associated with LILRB2 activity, such as cancer, is provided. In one example, there is provided a kit for treating and/or preventing a disease associated with LILRB2 activity, such as cancer, the kit comprising a binding protein (e.g. antibody) as disclosed herein in any example or combination of examples optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number); optionally wherein the kit comprises an IV or injection device that comprises the binding protein (e.g. antibody). In another example, the kit comprises a binding protein (e.g. antibody) contained within a container or an IV bag. In another example, the container or IV bag is a sterile container or a sterile IV bag. In another example, the binding protein (e.g. antibody) is formulated into a pharmaceutical composition contained within a (sterile) container or contained within a (sterile) IV bag. In a further example, the kit further comprises instructions for use.

5 EXAMPLES

[0251] Here we describe binding proteins (e.g. antibodies) binding to LILRB2 which are highly efficacious at inhibiting LILRB2 activity and thus are suitable for use in therapy, in particular for use in treating diseases or conditions associated with increased LILRB2 activity, such as cancer. The following examples further illustrate the disclosure but should not be construed as in any way limiting its scope.

6 Example 1. Generation of anti-LILRB2 antibodies [0252] Antibodies specifically binding to LILRB2 (denoted LILRBXXXX) were generated according to the following protocol. Amino acid sequences, including CDR sequences, VL and VH sequences and full light and heavy chain sequences, are set out in Table 8.

[0253] Antibodies specifically binding to LILRB2 (denoted LILRBXXXX) were generated using Immune Replica technology (Rajan et al. Commun. Biol. 2018) which creates natively-paired immune libraries for phage display selections. Humanised Ablexis transgenic mice were immunized with recombinant LILRB2 protein, B-cells were enriched, natively-paired scFv library generated and screened for LILRB2 selectivity. A lead panel of 3 LILRB2-specific IgG were selected based on potency in primary macrophage stimulation assays, biacore affinity and developability: LILRB0361 , LILRB0362 & LILRB0359.

[0254] After identifying LILRB0359, 0361 and 0362 as promising leads in functional assays, and realising that these were closely related sequences, we looked more closely at the differences between them, and generated a set of hybrid molecules that swapped sequences features between the group. Features that were investigated include:

• Combinations of different permutations of the heavy and light chain sequences.

• All three differ in sequence at site 52a in H-CDR2 (D, E or N observed), and are different from germline (G). This site proved to be critical for binding.

• LILRB0362 has an unusual non-germline R in FW4 in a Vernier site.

• Combining light chain CDR features of LILRB0361 and LILRB0362.

• Germlining of frameworks of LILRB0359 and LILRB0362. (LILRB0361 is already germline in its framework regions. LILRB0362 VH germlining retained the unusual Vernier residue in FW4.)

[0255] Note that the light and heavy chain vectors are separate, so various LC or HC variant sequences were generated, then co-expressed in different combinations, as shown in Figure 1C.

[0256] These three leads had identical H-CDR3 sequences and did not require further affinity optimization. Amino acid substitutions were made at select positions of VH and VL CDRs for germlining and to de-risk potential sequence liabilities. A new panel of 29 additional antibodies variants (denoted LILRB0363-LILRB0406) were generated and screened for specificity, potency, affinity and developability.

[0257] Parent clone LILRB0361 was fully germline in both the VH and VL framework regions but contained a deamidation liability risk in H-CDR2 at Kabat position N52a. Germlining of this site to glycine (N52aG) resulted in complete loss of potency (observed in LILRB0369, LILRB387, LILRB394, LILRB395 and LILRB402). Mutation to either aspartate i.e. N52aD (observed in LILRB0362 sequence) or glutamate i.e. N52aE (observed in LILRB0359) retained potency, indicating that the residue at position 52a in VH-CDR2 is critical for binding. Asparagine (Asn) to Glutamic acid (Glu) substitution (N52aE) in LILRB0368 mitigates potential sequence liabilities of parent LILRB0361. LILRB0368 retains specificity, potency and has stronger affinity. [0258] HCDRs 1-3 and LCDRs1-3, VH, VL, heavy chain and light chain amino acids sequences for the exemplified antibodies described in Example 1 are set out in Table 9.

6.1 LILRB0368

[0259] Heavy chain (HC), light chain (LC), VH, VL, HCDRs 1-3 and LCDRs 1-3 sequences set out in Table 9. The heavy chain constant region is human lgG1 m(f) allotype with Fc-dampening triple mutations in the CH2 domain. Light chain is human kappa isotype.

6.2 Comparison to germline (Figure 1A and Figure 1B):

[0260] VH sequence matches IGHV3-23*04 human germline in framework regions 1-3, and IGHJ2*01 in FW4. As well as the H-CDR3 sequence, non-germline sites elsewhere include N31 in H-CDR1 and E52a in H-CDR2. VL sequence matches human germline IGKV1-33*01/IGKV1 D-33*01 in FW1-3 and IGKJ2*02 in FW4. Non-germline sites include D31 in L-CDR1 and Q55 in L-CDR2.

7 Example 2. Biochemical HTRF assay to determine binding to recombinant biotinylated LILR proteins, LILRB2 haplotypes and LILRB2 domain 1 -2 construct:

[0261] Recombinant proteins bearing C-terminal Avi-FLAG tags were generated and biotinylated in house. The following constructs were made: LILRA1 , LILRA2, LILRA3, LILRB1 , LILRB2 haplotype 1 , LILRB2 haplotype 2, LILRB2 haplotype 3, LILRB2 haplotype 4, LILRB2 domains 1 -2. The assay buffer consists of 0.4M potassium fluoride (VWR, 26820.236) and 0.1 % bovine serum albumin (BSA, Sigma, A9576) in phosphate buffered saline (PBS, Invitrogen, 14190). An 11 -point, 1 in 3 serial dilution of test and control antibodies was performed in assay buffer, and 2.5 pL was added to a white, shallow well 384 well assay plate (Corning, 4513). Positive control for LILRB2 haplotype and domain constructs was Comparator-M (MAB6364), for LILRA1 , LILRA3 and LILRB1 was Comparator-H (HPF1 HuIgGITM), and for LILRA2 was Comparator-R (R&D systems LILRB2 IgG clone 287219). Non-specific binding (NSB) controls contained 2.5 pL of assay buffer instead of antibody sample. 5 pL of a solution containing 40 nM AF647-labelled anti-human Fey (Jackson ImmunoResearch, 109-605-008) or 40 nM AF647- labelled anti-mouse Fc (Jackson ImmunoResearch, 115-605-164), and 2 nM streptavidin cryptate (Cisbio, 610SAKLB) in assay buffer was added to the assay plate. 2.5 pL of recombinant biotinylated LILR construct at 4 nM in assay buffer was added to the assay plate. The plate was sealed with an optically clear seal (BioAnalitik, 900510) and incubated in the dark for 4 h at room temperature. Time resolved fluorescence at 620 nm and 665 nm emission wavelengths were determined using an EnVision plate reader (Perkin Elmer). Data was analysed by calculating the 665/620 nm sample ratio followed by the % deltaF values for each sample. 0 100

[0262] As shown in Figure 2-Figure 5, the results indicate that none of the LILRBXXXX mAbs show off-target binding to LILRB1 , LILRA1 , LILRA2 or LILRA3. As shown in Figure 6-Figure 10, all of the LILRBXXXX mAbs show binding to the top four most prevalent haplotypes of LILRB2, and also bind to the LILRB2 D1-D2 construct. All of the comparator mAbs tested (Comparator-M, Comparator-J, Comparator-B, Comparator-I and Comparator-N) show binding to the D1-D2 LILRB2 construct as well as all four LILRB2 haplotypes. Comparator-N is the only mAb that is not specific for LILRB2 over LILRB1 , LILRA1 and LILRA3.

Table 1 : List of samples tested, antigen and relevant plate barcode for all HTRF binding assays:

8 Example 3. Surface Plasmon Resonance to determine LILRBXXXX and comparator LILRB2 IgG panel affinity on human LILRB2

[0263] The kinetic rate constants (ka and kd) and binding affinity (equilibrium dissociation constant, KD) for biotinylated LILRB2 IgG binding recombinant human LILRB2 was determined at 25°C by Surface Plasmon Resonance (SPR) on a Biacore 8K instrument (Cytiva). Minimally amine biotinylated human lgG1s was titrated onto C1 chip streptavidin surfaces on 8K Biacore. Dilutions of Human LILRB2 H1 haplotype was flowed over this surface at 25°C. Multi-Cycle Kinetics (MCK) was performed with 4 M MgCh regeneration.

Table 2: LILRB2 IgG affinity on human LILRB2:

[0264] Comparator-B comprises the variable domains of BMS-986406 (with the exception of a single amino acid difference in FW1 of the VH, which likely comes from a BET standard PCR primer used in the conversions); Comparator-1 comprises the variable domains of 10-108; Comparator-J comprises the variable domains of JTX-8064; and Comparator-M comprises the variable domains of MK-4860. Heavy chain constant domains were the same sequence as LILRB0368, and for the light chains, standard kappa or lambda sequences were used as appropriate; to ensure we had consistent Fc region formats for comparison.

Table 3: LILRB2 IgG affinity data is consistent with published patent affinity:

[0265] Figure 11 shows Representative Biacore Sensorgram of IgG Binding to Human LILRB2 measured by Surface Plasmon Resonance.

9 Example 4. LILRB2 Fab affinity on human and cyno LILRB2

[0266] Figure 12 shows that LILRB0368 and 3 benchmarks LILRB2 IgG (Comparator-J, Comparator- M & Comparator-I) do not bind cyno LILRB2. Comparator-B & Comparator-N bind to cyno LILRB2 proteins. The kinetic rate constants (ka and kd) and binding affinity (equilibrium dissociation constant, KD = kd/ka) for LILRB2 Fab binding recombinant biotinylated human or cynomolgus monkey LILRB variants was determined at 25°C by Surface Plasmon Resonance (SPR) on a Biacore 8K instrument (Cytiva). The biotinylated-LILRB chip surfaces were challenged with LILRB2 Fab titration (1 , 5, 25 and 125 nM) in order to observe binding kinetics and affinity using Single-Cycle Kinetic assays (SCK, Karlsson et al., 2006). Figure 13 shows LILRB0368 and Comparator-B LILRB2 Fab affinity on human and cyno LILRB2.

Table 4: LILRB0368 and Comparator-B LILRB2 Fab affinity on human and cyno LILRB2:

10 Example 5. FLow ASSAY to determine binding to Jurkat cells overexpression full-length LILRB2:

[0267] Jurkat-NFAT-Luc-LILRB2 and Jurkat-NFAT-Luc parental cells were harvested, resuspended to 1 e6 cells/mL in flow buffer (eBioscience, 00-4222-26), and stained for 30 min with live/dead stain (Thermo Fisher Scientific, L34962) according to manufacturer’s instructions. Cells were washed twice in phosphate buffered saline (PBS, Invitrogen, 14190), then resuspended to 2e6 cells/mL in flow buffer. A 6-point, 1 in 3 (or 1 in 5) serial dilution of test antibodies was performed in flow buffer, and 50 pL was added to a 96 well polypropylene assay plate (Greiner, 650201). 50 pL of cell suspension was added to the assay plate. The plates were sealed (Perkin Elmer, 6005250) and incubated on ice for 1 h. The cells were collected by centrifugation (300g, 3 min, 4°C), the supernatant was discarded and the cells were resuspended in residual buffer by vortexing. The cells were washed once in flow buffer, then 50 pL of 15 nM AF647-labelled anti-human Fey (Jackson ImmunoResearch, 109-605-008) was added to each well. The plates were incubated on ice for 1 h in the dark, followed by three washes with flow buffer. 50 pL of CellFix (Becton Dickinson, 340181) was added to each well, and the plates were incubated on ice for 10 min in the dark. A further 150 pL of flow buffer was added to each well, and the plates were stored at 4°C. Data was acquired on a MACSQuant 10 (Miltenyi Biotech) flow cytometer using the V1 (VioBlue) and R1 (APC) channels.

[0268] As shown in Figure 14A - Figure 14E, the results indicate that none of the tested LILRBXXXX bind non-specifically to Jurkat-NFAT-Luc parental cells. All tested samples show robust binding to Jurkat-NFAT-Luc-LILRB2, with low nanomolar/high picomolar IC50s.

Table 5: IC50 values for flow binding of mAbs to Jurkat-NFAT-Luc-LILRB2:

11 Example 6. HTRF EPITOPE COMPETITION ASSAY AGAINST LILRB0362

[0269] An 11 -point, 1 in 3 serial dilution of test antibodies was performed in assay buffer consisting of 0.4 M potassium fluoride (VWR, 26820.236) and 0.1 % bovine serum albumin (BSA, Sigma, A9576) in phosphate buffered saline (PBS, Invitrogen, 14190), and 2.5 pL of sample was added to a white, shallow well 384 well assay plate (Corning, 4513). Non-specific binding (NSB) controls contained 2.5 pL of 200 nM LILRB0362 IgG instead of antibody sample. Total binding controls contained 2.5 pL of assay buffer instead of antibody sample. 5 pL of a solution containing 1.3 nM recombinant biotinylated LILRB2 haplotype 1 (generated in-house) and 2 nM streptavidin cryptate (Cisbio, 610SAKLB) in assay buffer was added to the assay plate. 2.5 pL of Dylight650-labelled LILRB0362 at 10 nM in assay buffer was added to the assay plate. The plate was sealed with an optically clear seal (BioAnalitik, 900510) and incubated in the dark for 4 h at room temperature. Time resolved fluorescence at 620 nm and 665 nm emission wavelengths were determined using an EnVision plate reader (Perkin Elmer). Data was analysed by calculating the 665/620 nm sample ratio followed by the normalised % deltaF (% specific binding) values for each sample. 100

[0270] As shown in Figure 15A - Figure 15D, the results indicate that parent mAbs LILRB0359, LILRB0361 and LILRB0362 share the same epitope, and combinations of features of these mAbs results in variants that retain this epitope. LILRB0359 is 3-fold less potent that LILRB0361 and LILRB0362. All variants containing glycine at position CDR2 52a lost activity (LILRB0369, LILRB0387, LILRB0394, LILRB0395, LILRB0402).

Table 6: IC50 values for mAbs in the LILRB0362 epitope competition assay, and correlation to the residue at position CDR2 52a:

12 Example 7. Activated Jurkat-NFAT-Luc-: LILRB2 and Raji-HLA-G co-culture reporter assay to determine inhibition of LILRB2 signalling:

[0271] Jurkat-NFAT-Luc cells overexpressing LILRB2(ECD)-CD3z-CD137 and Raji cells overexpressing HLA-G were generated in house. Cells were harvested and washed twice in assay medium consisting of 10% foetal bovine serum (FBS, Gibco, 01190-015) in RPMI medium (Gibco, 61870-010) to remove traces of selection antibiotics. Cells were resuspended to 3.33e5 cells/mL in assay medium. 30 pL of Jurkat-NFAT-Luc-LILRB2(ECD)-CD3z-CD137 cells were added to each well of a 96 well TC-treated U-bottom plate (Corning, 8797BC). 30 pL of Raji-HLA-G cells were added to all test wells and stimulated control wells. 30 pL of assay medium was added to unstimulated control wells. An 8-point 1 in 4 serial dilution of test antibodies was performed in assay medium, and 30 pL were added to the test wells. 30 pL of assay medium was added to stimulated and unstimulated control wells. The plate was briefly centrifuged (300g, 30 sec, room temperature), then incubated for 16 h in a humidified incubator set to 37°C, 5% CO2. 85 pL of SteadyGlo reagent (Promega, E2520) was added to each well of the cell plate, and incubated for 5 min at room temperature in the dark. The samples were mixed by pipette and transferred to a white 96 well assay plate (Corning, 3917), and incubated for a further 30 min in the dark at room temperature. Luminescence was measured on an EnVision plate reader (Perkin Elmer) with a 0.1 sec read. [0272] As shown in Figure 16A-Figure 16D, the results indicate that all tested LILRBXXXX mAbs that show activity in the LILRB0362 epitope competition assay also show functional inhibition of LILRB2- HLA-G signalling in a co-culture reporter assay.

Table 7: IC50 values for mAbs tested in a co-culture reporter assay, looking at signalling between Jurkat-NFAT-Luc-LILRB2(ECD)-CD3z-CD137 reporter cells and Raji-HLA-G target cells'.

13 Example 8. Testing for binding to selected LILR family receptors by biolayer interferometry (OCTET ASSAY):

[0273] Streptavidin (Sartorius, 18-5118) and Ni-NTA (Sartorius, 18-5102) biosensors were rehydrated in assay buffer consisting of 0.02% Tween20 (Thermo Fisher Scientific, 85113) and 0.1 % bovine serum albumin (BSA, Sigma, A9576) in phosphate buffered saline (PBS, Invitrogen, 14190) for at least 10min prior to loading. Biotinylated LILRB2 (in-house), LILRA4-6xHis (R&D, 8914-T4-050), LILRA5-6xHis (R&D, 8956-T4-050), LILRA6-6xHis (R&D, 9088-T4-050), LILRB3-6xHis (R&D, 9159-T5-050), LILRB4- 6xHis (R&D, 8488-T4-025), LILRB5-6xHis (R&D, 8487-T4-025), and LAIR1-6xHis (R&D, 2664-LR-050) were diluted in assay buffer to 5-10 pg/mL and loaded onto the appropriate biosensors. A 7-point 1 in 3 serial dilution of test and control antibodies was performed in assay buffer. All control antibodies were obtained from R&D Systems: LILRA4 (MAB6287), LILRA5 (MAB6754), LILRA6 (MAB8656), LILRB3 (MAB1806), LILRB4 (MAB24251), LILRB5 (AF3065) and LAIR1 (MAB2664). The assay protocol was as follows: baseline (assay buffer, 60 s), antigen load (antigen, 300 s), baseline (assay buffer, 60 s), sample association (antibody, 60 s), sample dissociation (assay buffer, 180 s); all steps were performed at 25°C.

[0274] As shown in Figure 17A and Figure 17B, the results show that none of the tested LILRBXXXX mAbs show off-target binding to related LILR family members. Comparator-J, Comparator-I, Comparator-N and Comparator-M are also specific for LILRB2 and do not show off-target binding to LILR family members. Comparator-B shows strong binding to LILRA5, and clone 287219 (R&D Systems) shows strong binding to LILRB3.

14 Example 9. Mirrorball assay to assess non-specific binding to HEK293 cells:

[0275] Frozen HEK293 cells (ECACC) were rapidly thawed, added to 10 mL of assay medium containing 0.5% bovine serum albumin (BSA, Sigma, A9576) in Hanks Balanced Salt Solution (HBSS, Sigma, H8264), and collected by centrifugation (300g, 5 min, room temperature). The cells were resuspended in assay buffer to 2.5e5 cells/mL. An 8-point, 1 in 5 serial dilution of test and control antibodies was performed in assay buffer, and 10 pL was added in duplicate to black 384well assay plates (Corning, 3766). 10 pL of a solution containing 16 nM AF647-labelled anti-human H+L (Invitrogen, A21445) was added to all assay plates. 20 pL of cells was added to one set of assay plates to assess non-specific cell binding. 20 pL of assay buffer was added to the second set of assay plates to assess IgG aggregation. The plates were sealed (Perkin Elmer, 6005250) and incubated in the dark for 2 h at room temperature. The plates were read using a Mirrorball plate reader (TTP Labtech) in the FL3 channel (640 nm) at 4 pm resolution and 500 V. The signal was gated to remove low intensity background fluorescence based on the buffer only controls and high intensity fluorescence due to aggregation.

[0276] As shown in Figure 18A - Figure 18J, the results show that none of the tested LILRBXXXX or comparator mAbs show non-specific binding to HEK293 cells, nor do they show propensity for aggregation.

15 Example 10. AC-SINS (affinity capture self-interaction nanoparticle spectrometry) assay to assess propensity for self-association:

[0277] This method was adapted from the following sources: Sule et al, Mol. Pharmaceutics. 2013, 10, 1322-1331 , and Liu et al, mAbs. 2014, 6(2), 483-492. [0278] 9mL gold nanoparticles (Abeam, ab269935) are coated with 400pg of a 1 :4 mixture of whole goat IgG (Jackson ImmunoResearch, 005-000-003) and goat anti-human Fey (Jackson ImmunoResearch, 109-005-098) in binding buffer (20 mM potassium acetate, pH 4.3) for 1-16 h at room temperature. The nanoparticles are blocked with 100 nM PEG (Sigma, 729140) for 1-2 h at room temperature, collected by centrifugation (13,000 rpm, 6 min, RT) and resuspended in 800 pL binding buffer. The OD of a 1 :10 dilution of prepared nanoparticles should be 0.4-0.5 AU at 535 nm. Test antibodies are diluted to 45 pg/mL in 108 pL of phosphate buffered saline (PBS, Invitrogen, 14190) or histidine-arginine buffer (20 mM histidine, 200 mM arginine, pH 6) in a 96 well polypropylene plate (Greiner, 650201). 12 pL of prepared gold nanoparticles are added to each well, mixed by pipetting, and the samples are incubated for 20 min at room temperature. The samples are transferred in duplicate to a Nunc 384 well assay plate (Thermo Fisher Scientific, 242757) and absorbance values between 450 nm and 700 nm are detected using a PHERAstar plate reader (BMG Labtech). The wavelength at maximum absorbance is determined using MARS software (BMG Labtech) and the redshift over the buffer control is calculated; a redshift >10 nm is indicative of self-association.

[0279] As shown in Figure 19, the results show that none of the tested LILRBXXXX mAbs has a risk of self-association. Comparator-J has a risk of self-association in charged buffers (HA), and Comparator-B has a risk of self-association in both neutral (PBS) and charged buffers.

16 Example 11. Baculovirus particle ELISA to assess risk of fast in-vivo clearance:

[0280] This method was adapted from the following source: Hotzel et al, mAbs, 2012, 4(6), 753-60.

[0281] 96 well Nunc maxisorp F plates (Thermo Fisher Scientific, 44-2404-21) are coated overnight at 4°C with either 50pL baculovirus particles at 1 e8 particles/mL in sodium carbonate buffer (50 mM, pH 9.6), or 50pL sodium carbonate buffer (50 mM, pH 9.6) only. The assay plates are washed once with phosphate buffered saline (PBS), then blocked for 1 h at room temperature with 300 pL assay buffer consisting of 0.5% bovine serum albumin (BSA, Sigma, A9576) in PBS (Invitrogen, 14190). The plates are washed three times with PBS. Test antibodies are diluted to 10 nM and 100 nM in assay buffer, 50 pL of sample was added to the assay plates and incubated for 1 h at room temperature. The plates are washed three times with PBS, then incubated with 50 pL of a solution of HRP-conjugated anti-human Fey (1 :5000 dilution, Sigma, A0170) for 30 min at room temperature. The plates are washed three times with PBS, incubated with 50 pL TMB SureBlue Reserve (KPL, 53-00-03) for 2-5 min and quenched with 50 pL 0.5 M sulfuric acid (H2SO4). The absorbance at 450 nm was measured on an EnVision plate reader (Perkin Elmer). The BV score was determined by averaging the absorbance values at both 10 nM and 100 nM sample, and dividing this by the buffer only control; a BV score above 5 indicates a risk of fast in-vivo clearance.

[0282] As shown in Figure 20, the results show that none of the tested LILRBXXXX or comparator mAbs shows a risk of fast clearance due to non-specific binding to baculovirus particles.

17 Example 12. Macrophage Stimulation assay: [0283] This in vitro assay was performed to assess the ability of LILRB2 antibodies to enhance pro- inflammatory response from immune-stimulated macrophages, as measured through the release of the pro-inflammatory cytokine TNFa into culture supernatants.

[0284] Monocytes were isolated from human PBMCs using a CD14+ negative selection kit (Stemcell #19058) and cultured in RPMI 1640 + Glutamax (Invitrogen, 61870), 10% HI FBS (ThermoFisher, 10082147), penicillin/streptomycin (Invitrogen, #15140122) and supplemented with 100ng/ml recombinant human M-CSF (Peprotech, #300-25) in a Corning® T175 Flask (Corning, #431080) for 6- days at 37°C, 5% CO₂.

[0285] Macrophages were detached from culture flasks using STEMPRO ACCUTASE (ThermoFisher, #A1 110501), washed in PBS and resuspended at 2e5/ml in RPM1 1640 + Glutamax (Invitrogen, 61870), 10% HI FBS (ThermoFisher, 10082147), penicillin/streptomycin (Invitrogen, #15140122), supplemented with 100ng/ml recombinant human M-CSF (Peprotech, #300-25). Cells were seeded at 20,000 cells I well into a CORNING sterile, flat-bottom 96 well-plate (Corning, #10695951) and incubated overnight at 37°C, 5% CO₂.

[0286] Antibody solutions were prepared to a range of concentrations, added to the plate and incubated at RT for 30 minutes. The cells were stimulated using 500ng/ml MEGACD40L® (Enzo, #ALX- 522-110-C010) and incubated overnight at 37°C, 5% CO2. Supernatants were collected and assessed for TNFa protein levels by ELISA (R&D Systems, #DY210). The results were analysed as following: Each value from a given donor were normalised in Graphpad whereby the top mean average value achieved by that donor following treatment with the anti-LILRB2 mAb LILRB0361 was defined as 100%, while zero was defined as 0%. These normalised values were then combined across all donors. The number of donors assessed are provided in the figures. Dose-response curves were generated in Graphpad using log(agonist) vs. response -- Variable slope (four parameters) non-linear regression model.

[0287] Blockade of LILRB2 on CD40L-stimulated macrophages enhances robustly the release of the pro-inflammatory cytokine TNFa. All our LILRBXXXX mAb clones screened against CD40L-stimulated macrophages increased TNFa release (as shown in Figure 21 A - Figure 21 C), although some variation in potency was observed (as shown in Figure 21A). LILRB0368 was found to be more potent in this assay, compared to the Comparators tested (Figure 21 C)

Table 8 IC50 values for LILRB0368 and Comparator mAbs in the macrophage stimulation assay:

[0288] In a separate experiment, blockade of LILRB2 using LILRB0368 on CD40L-stimulated macrophages enhanced the release of the pro-inflammatory cytokines GM-CSF and TNFa, while reducing the production of the pro-angiogenic cytokine VEGF-A (as shown in Figure 22A to Figure 22C), compared to isotype control.

18 Example 13. CD163 and CD206 Expression assay:

[0289] This in vitro assay was performed to assess whether monocytes treated with LILRB0368 differentiate into macrophages with a heightened proinflammatory phenotype, as determined by a reduction in the cell surface expression of the suppressive M2 markers CD163 and CD206.

[0290] Monocytes were isolated from human PBMCs using a CD14+ negative selection kit (Stemcell #19058) and cultured in the presence of 50nM of either our exemplar anti-LILRB2 mAb clone, LILRB0368 or an isotype control antibody, in culture media [RPM1 1640 + Glutamax (Invitrogen, 61870), 10% HI FBS (ThermoFisher, 10082147), penicillin/streptomycin (Invitrogen, #15140122) and supplemented with 100ng/ml recombinant human M-CSF (Peprotech, #300-25)] in a flat-bottomed 96 well tissue culture plate (Corning, #3527) for 7-days at 37°C, 5% CO2. The resulting macrophages were detached from culture plates using STEMPRO ACCUTASE (ThermoFisher, #A1110501), transferred to a 96 well v-bottom plate (Greiner, #651201), washed twice in PBS in preparation for staining for flow cytometry. Cells were resuspended in ROBOSEP Buffer (StemCell, #20104) containing Fc block (Rockland, #009-0103), BV785-conjugated anti-CD206 antibody (Biolegend, #321142) and BV605- conjugated anti-CD163 antibody (Biolegend #333616). Cells were then incubated on ice for 30 minutes. The staining solution was then removed and cells were washed once in ROBOSEP Buffer. Cells were fixed following 10-minute incubation in Cytofix solution (BD Biosciences #554655), pelleted, and resuspended in PBS. Cells were then acquired on a Fortessa flow cytometer (BD Biosciences), the expression levels of CD163 and CD206 on macrophages were analysed using Flowjo v10 software package (BD Biosciences).

[0291] Figure 23A - Figure 23B show the results of the CD163 and CD206 expression assay. Expression of CD163 and CD206 is down-regulated on monocyte-derived macrophages differentiated in the presence of 50nM of LILRB0368.

19 Example 14. Tumour growth assay

[0292] NSG/SGM3 mice reconstituted with human CD34+ cord blood stem cells were implanted subcutaneously with 1x10e6 MDA-MB-231 tumour cells in their right hind flank. Mice were randomised based on tumour size 14 days post tumour implantation into two groups, each consisting of four mice. One group was treated with 10 mg/kg LILRB0361 , the other treated with 10 mg/kg of an isotype control antibody. Both test articles were co-administered with 20mg/kg anti-Mouse CD16/CD32 (BioXcell, FcBlock) via intraperitoneal injection twice weekly for 3 weeks. Tumour volumes were measured twice weekly for the duration of the study, the mean tumour volume (mm³) and standard deviation over time are plotted in Figure 23. [0293] Figure 24 shows the results of the tumour growth assay. The anti-LILRB2 mAb clone, LILRB0361 reduces tumour growth rate in vivo. NSG/SGM3 mice reconstituted with human CD34+ cord blood stem cells were implanted s.c. with an MDA-MB-231 tumour xenograft. Mice were treated with 10 mg/kg LILRB0361 (and isotype control) twice weekly for 3 weeks.

20 Example 14. Combination therapy with T cell engagers

[0294] Results for an anti-OKT3-EGFR T cell engager (TCE) are shown in Figure 25. T cells from eight donors were co-cultured with tumour cells and autologous macrophages. The addition of macrophages to the co-culture significantly reduced the level of tumour killing by T cells stimulated with OKT3-EGFR T cell engager from a mean of 31.8% to 8.1 % (Figure 25A). This reduction demonstrates the suppressive effect of macrophages on T cell effector function. In the presence of these suppressive macrophages, anit-LILRB2 antibody increased TCE-driven tumour killing from 8.1 % to 21.8%, indicating partial restoration of killing activity. In parallel, the addition of AZD2796 into the co-culture significantly enhanced expression of activation marker CD86 on macrophages in the presence ofT cells activated by the T cell engager (Figure 25B).

[0295] These data show that T cell engager-directed tumour killing can be effectively suppressed by macrophages in an in vitro co-culture assay. The anti-LILRB2 antibody significantly enhanced tumour killing induced by a T cell engagers in the presence of suppressive macrophages. This increase in killing was accompanied by a repolarization of macrophages indicated by an increase in expression of pro- inflammatory macrophage marker CD86.

21 Example 14. Combination therapy with CAR-T cells

[0296] Results for HER2 CAR-T cells are shown in Figure 26, where CAR-T cells (derived from one donor) were co-cultured with tumour cells (2way) or tumour cells and macrophages (3way) from 6 different non-autologous donors. The addition of macrophages to the co-culture significantly reduced the level of tumour killing by HER2-CAR T cells from a mean of 59.8% to 14.3% (Figure 26). This reduction demonstrates the suppressive effect of macrophages on CAR-T cell effector function. In the presence of these suppressive macrophages, the anti-LILRB2 antibody increased CAR-T-driven tumour killing from 14.3% to 34.7%, indicating partial restoration of killing activity.

[0297] These data show that CAR-T-directed tumour killing can be effectively suppressed by macrophages in an in vitro co-culture assay. The addition of an anti-LILRB2 antibody significantly enhanced tumour killing induced CAR-T cells in the presence of suppressive macrophages

22 SEQUENCES

Table 9: Amino acid sequences of antibodies described in this specification.

[0298] All heavy chains, light chains, VH domains, VL domains, CDRs, antibodies comprising them, as well as their encoding nucleic acids, represent examples of the present disclosure. CDRs are determined according to the Kabat method.

Table 10: Amino acid sequences of comparator antibodies described in this specification.

Claims

1 . A LILRB2 binding protein which binds to human LILRB2 with an affinity (KD) of < 10OpM.

2. The LILRB2 binding protein according to claim 1 , wherein the affinity is measured by Biacore.

3. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein inhibits LILRB2 (e.g. as measured in a co-culture reporter assay).

4. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein inhibits HLA-G-mediated downstream signalling (e.g. as measured by co-culture reporter assay).

5. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein inhibits LILRB2 in myeloid cells (e.g. as measured in a macrophage stimulation assay).

6. The LILRB2 binding protein according to claim 5, wherein the myeloid cells are macrophages.

7. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein increases TNF-alpha and/or GM-CSF release in vitro (e.g. as measured by macrophage stimulation assay).

8. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein decreases VEGF in vitro (e.g. as measured by macrophage stimulation assay).

9. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein induces macrophage repolarisation from tumour-supportive M2 state to tumoursuppressive M1 state, as characterised by the reduction in the cell surface expression of CD163 and increase in the cell surface expression of CD86 and production of TNFalpha (e.g. as measured in a macrophage, T cell, tumour cell co-culture assay, employing antigen-specific T cells).

10. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein promotes a proinflammatory state in the tumour microenvironment leading to increased T cell lysis of a tumour cell line (e.g. as measured in a macrophage, T cell, tumour cell coculture assay, employing antigen-specific T cells).

11. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein decreases tumour volume (e.g. as measured in vivo).

12. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein is a competitive inhibitor of LILRB2 (e.g. as measured by ligand competition assay).

13. The LILRB2 binding protein according to claim 12, wherein the ligand is HLA-G.

14. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein does not bind LILRB1 , LILRB3, LILRB4, LILRB5, LILRA1 , LILRA2, LILRA3, LILRA4, LILRA5 or LILRA6.

15. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 is human LILRB2.

16. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein binds to all human LILRB2 haplotypes with a frequency of greater than 5%.

17. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein has an IC50 of 5pM or lower for human LILRB2 (e.g. as measured by macrophage stimulation assay).

18. The LILRB2 binding protein according to any preceding claim, wherein the binding protein is an antibody or antigen binding fragment thereof.

19. The LILRB2 binding protein according to claim 18, wherein the antibody is a monoclonal antibody.

20. The LILRB2 binding protein according to claim 18 or 19, wherein the antibody is an IgG.

21. The LILRB2 binding protein according to any one of claims 18-20, wherein the antibody is an igGi .

22. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence comprising complementarity determining regions (CDRs) HCDR1 , HCDR2 and HCDR3, and a variable light (VL) domain sequence comprising CDRs LCDR1 , LCDR2 and LCDR3, and wherein HCDR3 is the HCDR3 of SEQ ID NO:7 (e.g. as determined by Kabat).

23. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a glutamic acid (E), asparagine (N) or aspartic acid (D) residue at heavy chain position 52a (e.g. as determined by Kabat).

24. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a HCDR2 sequence of SEQ ID NO: 6, optionally wherein the glutamic acid (E) residue at heavy chain position 52a is substituted for an asparagine (N) or aspartic acid (D) residue (e.g. as determined by Kabat).

25. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence comprising HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6 and HCDR3 of SEQ ID NO: 7, and a variable light (VL) domain sequence comprising LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.

26. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3, optionally with 1 , 2, 3, 4 or 5 amino acid alterations, and a variable light (VL) domain sequence of SEQ ID NO: 4, optionally with 1 , 2, 3, 4 or 5 amino acid alterations.

27. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3, optionally with 1 , 2, 3, 4 or 5 amino acid alterations outside the complementarity determining regions (CDRs), and a variable light (VL) domain sequence of SEQ ID NO: 4, optionally with 1 , 2, 3, 4 or 5 amino acid alterations outside the CDRs.

28. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and wherein the variable heavy (VH) domain sequence and variable light (VL) domain sequence respectively comprise a sequence having at least 90% identity to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4.

29. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and wherein the variable heavy (VH) domain sequence and variable light (VL) domain sequence respectively comprise a sequence having at least 90% identity to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, provided that the antibody has the HCDRs of SEQ ID NO: 3 and the LCDRs of SEQ ID NO: 4.

30. The LILRB2 binding protein according to any preceding claim, wherein the antibody comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 and a variable light (VL) domain sequence of SEQ ID NO: 4.

31. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises a Fc domain.

32. The LILRB2 binding protein according to claim 31 , wherein the Fc domain has reduced effector function.

33. The antibody according to claim 32, wherein the Fc domain comprises the mutations L234F, L235E and P331 S.

34. The antibody according to claim 32, wherein the Fc domain comprises the mutations L234F, L235Q and K322Q.

35. The antibody according to claim 32, wherein the Fc domain comprises the mutations L238F, L239E and P335S.

36. The antibody according to claim 31 , wherein the Fc domain comprises at least one half-life extension conferring mutation.

37. The antibody according to claim 36, wherein the Fc domain comprises the mutations M252Y, S254T and T256E.

38. The antibody according to claim 31 , wherein the Fc domain has reduced effector function and comprises at least one half-life extension conferring mutation.

39. The antibody according to claim 38, wherein the Fc domain comprises the mutations L234F, L235E, P331 S, M252Y, S254T and T256E.

40. The antibody according to claim 38, wherein the Fc domain comprises the mutations L234F, L235Q, K322Q, M252Y, S254T and T256E.

41. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises kappa light chains.

42. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises light chains comprising the sequence SEQ ID NO: 2.

43. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises heavy chains comprising the sequence SEQ ID NO: 1 .

44. The LILRB2 binding protein according to any preceding claim, wherein the LILRB2 binding protein comprises light chains comprising the sequence SEQ ID NO: 2 and heavy chains comprising the sequence SEQ ID NO: 1 .

45. A polypeptide comprising one or more domains of a LILRB2 binding protein as defined in any one of claims 1-44.

46. A polypeptide according to claim 45, wherein the domain is one or more CDRs from a heavy and/or light chain, a VH domain or a VL domain.

47. A polypeptide comprising one or more chains of a LILRB2 binding protein as defined in any one of claims 1-44.

48. A nucleic acid encoding one or more chains of a LILRB2 binding protein as defined in any one of claims 1-44.

49. A vector comprising the nucleic acid of claim 48.

50. A host cell comprising the vector of claim 49.

51. A pharmaceutical composition comprising a LILRB2 binding protein according to any one of claims 1-44 and a pharmaceutically acceptable carrier.

52. A kit comprising a LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 51 .

53. A LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 51 for use in therapy.

54. A LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 50 for use in treating cancer.

55. The LILRB2 binding protein for use according to claim 54, wherein the cancer is colorectal cancer, head and neck cancer, renal cancer, lung cancer, pancreatic cancer, gastric cancer, melanoma, breast cancer or ovarian cancer.

56. The LILRB2 binding protein for use according to claim 54, wherein the cancer is renal cancer.

57. The LILRB2 binding protein for use according to claim 54, wherein the cancer is lung cancer.

58. The LILRB2 binding protein for use according to claim 54, wherein the cancer is gastric cancer.

59. A method of treating cancer, wherein the method comprises administering a LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 50 to a subject in need thereof.

60. A method of treating cancer, wherein the method comprises administering a LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 50 to a subject in need thereof, in combination with a chimeric antigen receptor (CAR)-T cell.

61 . A method of treating cancer, wherein the method comprises administering a LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 50 to a subject in need thereof, in combination with a T cell engager.

62. The method according to any of claims 59-61 , wherein the cancer is colorectal cancer, head and neck cancer, renal cancer, lung cancer, pancreatic cancer, gastric cancer, melanoma, breast cancer or ovarian cancer.

63. The method according to any of claims 59-61 , wherein the cancer is renal cancer.

64. The method according to any of claims 59-61 , wherein the cancer is lung cancer.

65. The method according to any of claims 59-61 , wherein the cancer is gastric cancer.

66. The use of a LILRB2 binding protein according to any one of claims 1-44 or a pharmaceutical composition according to claim 51 for the manufacture of a medicament for the treatment of cancer.

67. The use according to claim 66, wherein the cancer is colorectal cancer, head and neck cancer, renal cancer, lung cancer, pancreatic cancer, gastric cancer, melanoma, breast cancer or ovarian cancer.

68. The use according to claim 66, wherein the cancer is renal cancer.

69. The use according to claim 66, wherein the cancer is lung cancer.

70. The use according to claim 66, wherein the cancer is gastric cancer.