Lilrb2 binding proteins and uses thereof

Abstract

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

Description

1. Background Technology

[0001] Cancer treatment remains a significant challenge, and while chemotherapy drugs can reduce cancer cell proliferation and thus inhibit tumor growth and metastasis, their non-specificity means they are accompanied by numerous serious side effects due to off-target effects. Therefore, there has been a strong emphasis on identifying alternatives to chemotherapy drugs that possess the same high efficacy but with increased specificity targeting cancer cells to reduce the incidence and severity of adverse events. One such novel anticancer therapy is monoclonal antibody-based immunotherapy, which offers opportunities for targeted cancer therapies, particularly those that stimulate the host's immune system to promote immune-mediated attack on cancer cells.

[0002] The immune system can recognize and kill cancer cells; for example, T cells can be sensitized by antigen-presenting cells against mutated cancer-specific antigens (Houghton, AN, J Clin.Invest., (2004), 114(4), 468-471), which can contribute to cancer regression or arrest (Vinay, DS et al., Seminars in Cancer Bio., 35, 185-198). However, many cancers develop immune evasion mechanisms to modulate the immune response. One such immune evasion approach involves the creation of an immunosuppressive microenvironment in which tumor-associated myeloid cells (TMCs) play a key role in maintaining the immunosuppressive niche. Circulating myeloid cells can be recruited to the tumor site by secreting chemokines such as CCL2 and CCL5 (Mantovani, A. et al., Nat Rev Clin Oncol., (2017), 14(7), 399-416). In fact, the extent of TMC recruitment is associated with patient outcomes, with increased TMC levels associated with poor prognosis (Nakamura, K. et al., Cell Mol Immunol., (2020), 17(1), 1-12). Furthermore, TMCs can promote cancer metastasis, proliferation, and immunosuppression (Mantovani, A. et al., Cellular and Molecular Immunology, (2021), 18, 566-578). It is believed that most of these effects are controlled by the anti-inflammatory phenotype of most TMCs. Therefore, shifting the phenotype of TMCs from anti-inflammatory to pro-inflammatory has gained attention as a potential approach for cancer immunotherapy. In some cancers, the maintenance of the anti-inflammatory phenotype of TMCs has been observed to be partially controlled by the agonistic effect of cancer cells on myeloid LILRB2 receptors (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 both activating and inhibitory members that can upregulate or downregulate 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-hematopoietic 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 pro-inflammatory cytokine release (Deng, M. Antibody Therapeutics., (2020), 4(1), 16-33).

[0004] The extracellular domain of LILRB2 is composed of four immunoglobulin-like domains that are linked to a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif that acts to inhibit activating signals (Wang, Q., Cell Mol Immunol., (2020), 17(9), 966-975). The extracellular domain of LILRB2 can bind to a variety of ligands, including HLA-G, HLA-A, HLA-B, HLA-C, HLA-E, CD1c, CD1d, beta-amyloid protein, and angiopoietin-like proteins, including ANGPTL2 and 5 (Zhang, J. J Leukoc Biol., (2017), 102(2), 351-360). Due to its ability to bind to a variety of ligands, LILRB2 has many functions, including acting as an immune checkpoint inhibitor to prevent the onset of autoimmunity by binding to MHC class I molecules on antigen-presenting cells, transducing a negative signal that inhibits stimulation of the immune response. As such, it is thought to control inflammatory responses and cytotoxicity to help focus the immune response and limit self-reactivity.

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

[0006] Accordingly, targeting LILRB2 can provide a method for cancer immunotherapy. Antagonism of LILRB2 has been shown to shift the TMC phenotype from anti-inflammatory to pro-inflammatory. However, there is a need for high potency LILRB2 specific binding protein (e.g. antibody) compositions suitable for the treatment of cancer. 2. SUMMARY

[0007] We have discovered binding proteins (e.g. antibodies) that bind to LILRB2 and have properties suitable for development as pharmaceuticals. These binding proteins (e.g. antibodies) are suitable for use in the treatment of diseases and conditions associated with increased activity of LILRB2, in particular for use in the treatment or prevention of cancer. The binding proteins (e.g. antibodies) of the present disclosure exhibit a combination of advantageous properties, including strong binding affinity for LILRB2 and / or potency in inhibiting the enzymatic activity of LILRB2, which make them suitable for use as therapeutic agents, in particular for use in the treatment of cancer.

[0008] The binding proteins (e.g. antibodies) described herein have particularly potent properties. For example, the binding proteins (e.g. antibodies) described herein, including LILRB0368, have very high potency. Furthermore, we have shown that blocking LILRB2 enhances the immune stimulatory response of macrophages in vitro and is effective in vivo.

[0009] It is an object of the present disclosure to reduce / improve the symptoms of a disease, such as cancer, by administration of such binding proteins (e.g. antibodies). Administration of the binding proteins (e.g. antibodies) of the present disclosure can be used as a prophylactic or therapeutic agent.

[0010] 2.1 Statement of the disclosure

[0011] In a first aspect, the present disclosure provides a LILRB2 binding protein that binds to human LILRB2 with an affinity (KD) of < 100 pM. 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 gene assay). In some embodiments, the LILRB2 binding inhibits HLA-G mediated downstream signaling (e.g., as measured by a co-culture reporter gene 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-a and / or GM-CSF release in vitro (e.g., as measured by a macrophage stimulation assay). In some embodiments, the LILRB2 binding protein decreases VEGF-A in vitro (e.g., as measured by a macrophage stimulation assay). In some embodiments, the LILRB2 binding protein induces macrophages to polarize from a tumor-supportive M2 state to a tumor-inhibitory Ml state characterized by decreased cell surface expression of CD163 and increased cell surface expression of CD86 and production of TNFa (e.g., as measured in a macrophage, T cell, tumor cell co-culture assay using antigen-specific T cells). In some embodiments, the LILRB2 binding protein promotes a pro-inflammatory state in the tumor microenvironment resulting in increased T cell lysis of tumor cell lines (e.g., as measured in a macrophage, T cell, tumor cell co-culture assay using antigen-specific T cells). In some embodiments, the LILRB2 binding protein reduces tumor volume (e.g., as measured in vivo).

[0012] In some embodiments, the LILRB2 binding protein is a competitive inhibitor of LILRB2 (e.g., as measured by a 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 greater than 5%.

[0013] In some embodiments, the LILRB2 binding protein has an IC50 of 20 pM or less for human LILRB2 (e.g., as measured by the macrophage stimulation assay). In other embodiments, the LILRB2 binding protein has an IC50 of 15 pM or less for human LILRB2 (e.g., as measured by the macrophage stimulation assay). In other embodiments, the LILRB2 binding protein has an IC50 of 10 pM or less for human LILRB2 (e.g., as measured by the macrophage stimulation assay). In another embodiment, the LILRB2 binding protein has an IC50 of 5 pM or less for human LILRB2 (e.g., as measured by the macrophage stimulation assay).

[0014] 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 IgGl. 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 HCDR3 of SEQ ID NO: 7 (e.g., as determined by Kabat).

[0015] In some embodiments, the LILRB2 binding protein comprises an 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.

[0016] In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 optionally having 1, 2, 3, 4, or 5 amino acid changes, and a variable light (VL) domain sequence of SEQ ID NO: 4 optionally having 1, 2, 3, 4, or 5 amino acid changes. In some embodiments, the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 optionally having 1, 2, 3, 4, or 5 amino acid changes outside the complementarity determining regions (CDRs), and a variable light (VL) domain sequence of SEQ ID NO: 4 optionally having 1, 2, 3, 4, or 5 amino acid changes 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 the variable light (VL) domain sequence comprise a sequence that is at least 90% identical to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, respectively. 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 the variable light (VL) domain sequence comprise a sequence that is at least 90% identical to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, respectively, 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.

[0017] In some embodiments, the LILRB2 binding protein comprises an Fc domain. In some embodiments, the Fc domain has reduced effector function. In some embodiments, the Fc domain comprises mutations L234F, L235E, and P331S. In some embodiments, the Fc domain comprises mutations L234F, L235Q, and K322Q. In some embodiments, the Fc domain comprises mutations L238F, L239E, and P335S.

[0018] In some embodiments, the Fc domain comprises at least one mutation that confers extended half-life. In some embodiments, the Fc domain comprises mutations M252Y, S254T, and T256E. In some embodiments, the Fc domain has reduced effector function and comprises at least one mutation that confers extended half-life. In some embodiments, the Fc domain comprises mutations L234F, L235E, P331S, M252Y, S254T, and T256E. In some embodiments, the Fc domain comprises mutations L234F, L235Q, K322Q, M252Y, S254T, and T256E.

[0019] In some embodiments, the LILRB2 binding protein comprises a kappa light chain.

[0020] In some embodiments, the LILRB2 binding protein comprises a light chain comprising the sequence of SEQ ID NO: 2. In some embodiments, the LILRB2 binding protein comprises a heavy chain comprising the sequence of SEQ ID NO: 1. In some embodiments, the LILRB2 binding protein comprises a light chain comprising the sequence of SEQ ID NO: 2 and a heavy chain comprising the sequence of SEQ ID NO: 1.

[0021] In another aspect, the present 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 a heavy chain and / or light chain, a VH domain, or a VL domain.

[0022] In another aspect, the present disclosure provides a polypeptide comprising one or more chains of a LILRB2 binding protein as described anywhere herein.

[0023] In another aspect, the present disclosure provides a nucleic acid encoding one or more chains of a LILRB2 binding protein as described anywhere herein.

[0024] In another aspect, the present disclosure provides a vector comprising a nucleic acid as described anywhere herein.

[0025] In another aspect, the present disclosure provides a host cell comprising a vector as described anywhere herein.

[0026] In another aspect, the present disclosure provides a pharmaceutical composition comprising a LILRB2 binding protein as described anywhere herein and a pharmaceutically acceptable carrier.

[0027] In another aspect, the present disclosure provides a kit comprising a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein.

[0028] In another aspect, the present disclosure provides a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein for use in therapy. In another aspect, the present disclosure provides a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein for use in the treatment of 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.

[0029] In another aspect, the present disclosure provides a method of treating cancer, wherein the method comprises administering to a subject in need thereof a LILRB2 binding protein as described anywhere herein or a pharmaceutical composition as described anywhere herein.

[0030] In another aspect, the present 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 DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Alignment of LILRB0368 VH and VL sequences compared to germline showing amino acid variations, respectively

[0032] Figures 1A-1B An alignment of LILRB0368 VH and VL sequences compared to germline showing amino acid variations, respectively, is shown. In the VH sequence of LILRB0368 (as shown in Figure 1A the VH sequence matches the IGHV3-23*04 human germline in framework regions 1-3 and IGHJ2*01 in FW4. As with the H-CDR3 sequence, the non-germline sites elsewhere are also N31 in H-CDR1 and E52a in H-CDR2. The E52a site in H-CDR2 has been shown to be critical for antigen binding. Backing to the germline glycine eliminated binding, but we also explored sequences with D or N at this position. In the VL sequence of LILRB0368 (as shown in Figure 1B the VL sequence matches the human germline IGKV1-33*01 / IGKV1D-33*01 in FW1-3 and IGKJ2*02 in FW4. The non-germline sites are D31 in L-CDR1 and Q55 in L-CDR2. Figure 1CVL and VH sequences used to construct the anti-LILRB2 antibodies disclosed herein are shown.

[0033] Figure 2: HTRF binding assay using LILRA1

[0034] Figures 2A-2G Results of the HTRF binding assay using LILRA1 are shown. The results indicate that none of the LILRB2 mAbs tested showed off-target binding to LILRA1. Comparator-N is the only comparator mAb tested that does not have specificity for LILRB2 and shows binding to LILRA1.

[0035] Figure 3: HTRF binding assay using LILRA2

[0036] Figures 3A-3G Results of the HTRF binding assay using LILRA2 are shown. The results indicate that none of the LILRBXXXX mAbs or comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed off-target binding to LILRA2.

[0037] Figure 4: HTRF binding assay using LILRA3

[0038] Figures 4A-4G Results of the HTRF binding assay using LILRA3 are shown. The results indicate that none of the LILRBXXXX mAbs showed off-target binding to LILRA3. Comparator-N is the only comparator mAb tested that does not have specificity for LILRB2 and shows binding to LILRA3.

[0039] Figure 5: HTRF binding assay using LILRB1

[0040] Figures 5A-5G Results of the HTRF binding assay using LILRB1 are shown. The results indicate that none of the LILRBXXXX mAbs showed off-target binding to LILRB1. Comparator-N is the only comparator mAb tested that shows binding to LILRB1.

[0041] Figure 6: HTRF binding assay using LILRB2 haplotype 1

[0042] Figures 6A-6G Results of the HTRF binding assay using LILRB2 haplotype 1 are shown. All of the LILRBXXXX mAbs and comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed binding to LILRB2 haplotype 1, one of the four most common haplotypes of LILRB2.

[0043] Figure 7: HTRF binding assay using LILRB2 haplotype 2

[0044] Figures 7A-7G Results from the HTRF binding assay using LILRB2 haplotype 2 are shown. All LILRBXXXX mAbs and comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed binding to LILRB2 haplotype 2, one of the four most common haplotypes of LILRB2.

[0045] Figure 8: HTRF binding assay using LILRB2 haplotype 3

[0046] Figures 8A-8G Results from the HTRF binding assay using LILRB2 haplotype 3 are shown. All LILRBXXXX mAbs and comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed binding to LILRB2 haplotype 3, one of the four most common haplotypes of LILRB2.

[0047] Figure 9: HTRF binding assay using LILRB2 haplotype 4

[0048] Figures 9A-9G Results from the HTRF binding assay using LILRB2 haplotype 4 are shown. All LILRBXXXX mAbs and comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed binding to LILRB2 haplotype 4, one of the four most common haplotypes of LILRB2.

[0049] Figure 10: HTRF binding assay using LILRB2 domain 1 / domain 2

[0050] Figures 10A-10H Results from the HTRF binding assay using LILRB2 domain 1 / domain 2 are shown. Results indicate that all tested LILRBXXXX mAbs and comparator mAbs (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed binding to the LILRB2 D1-D2 construct.

[0051] Figure 11: Representative Biacore association and dissociation sensorgrams of IgG binding to human LILRB2 measured by surface plasmon resonance

[0052] Figure 11 shows representative Biacore association and dissociation sensorgrams of IgG binding to human LILRB2 measured by surface plasmon resonance.

[0053] Figure 12: All tested LILRB2 XXXX mAbs and comparator mAbs bind to human LILRB2 as determined by HTRF

[0054] Figure 12A All tested LILRB2 XXXX mAbs and comparator mAbs bind to human LILRB2 as determined by HTRF are shown. Figure 12B The tested LILRB2 XXXX mAbs do not bind to cynomolgus LILRB2, while comparator-N and comparator-B bind to cynomolgus LILRB2 as determined by HTRF are shown.

[0055] Figure 13: Affinity of RB0368 and comparator-B LILRB2 Fabs to human and cynomolgus LILRB2

[0056] Figure 13 shows the affinity of LILRB0368 and comparator-B LILRB2 Fabs to human and cynomolgus LILRB2. Comparator B binds to both human and cynomolgus LILRB2, while LILRB0368 does not have cynomolgus cross-reactivity.

[0057] Figure 14: Flow cytometry binding assay on Jurkat-LILRB2 cells

[0058] Figures 14A-14E Results of the flow cytometry binding assay on Jurkat-LILRB2 cells are shown. All tested samples show robust specific binding to Jurkat-NFAT-Luc-LILRB2 with low nanomolar to high picomolar IC50. None of the tested LILRB2 antibodies bind non-specifically to Jurkat-NFAT-Luc parental cells (data not shown).

[0059] Figure 15: LILRB0362 epitope competition assay

[0060] Figures 15A-15D Results of the LILRB0362 epitope competition assay are shown. The results indicate that parental mAbs LILRB0359, LILRB0361, and LILRB0362 share the same epitope, and that a combination of features of these mAbs produces variants that retain that epitope. LILRB0359 is 3-fold less potent than LILRB0361 and LILRB0362. All variants containing a glycine at position CDR2 52a lose activity (LILRB0369, LILRB0387, LILRB0394, LILRB0395, LILRB0402).

[0061] Figure 16: Co-culture reporter assay

[0062] Figures 16A-16D Results of the co-culture reporter assay are shown. Results indicate that all tested LILRBXXXX mAbs that showed activity in the LILRB0362 epitope competition assay also showed functional inhibition of LILRB2-HLA-G signaling in the co-culture reporter assay.

[0063] Figure 17: Octet selectivity assay

[0064] Figures 17A-17B Results of the octet selectivity assay are shown. Results indicate that none of the tested LILRBXXXX mAbs showed 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.

[0065] Figure 18: HEK293 binding and aggregation assay

[0066] Figure 18A Results of the HEK293 binding and aggregation assay are shown. Results show that none of the tested LILRBXXXX or comparator mAbs showed non-specific binding to HEK293 cells, and they also did not show a tendency to aggregate.

[0067] Figure 19 AC-SINS self-association assay

[0068] Figure 19 Results of the AC-SINS self-association assay are shown. Results show that none of the tested LILRBXXXX mAbs have a risk of self-association. Comparator-J has a risk of self-association in the charged buffer (HA), and Comparator-B has a risk of self-association in both the neutral buffer (PBS) and the charged buffer.

[0069] Figure 20 Baculovirus particle (BVP) ELISA

[0070] Figure 20 Results of the baculovirus particle (BVP) ELISA are shown. Results show that none of the tested LILRBXXXX or comparator mAbs showed a risk of rapid clearance due to non-specific binding to baculovirus particles.

[0071] Figure 21: Macrophage stimulation assay

[0072] Figures 21A-21CResults of macrophage stimulation assay. TNFa release from CD40L stimulated macrophages after treatment with our LILRBXXXX mAbs Figure 21A ) and Figure 21B ) provide results of two graphs). In Figure 21C LILRB0368 was compared to several benchmark anti-LILRB2 antibodies.

[0073] Figure 22: Macrophage stimulation assay

[0074] Figure 22A -C shows results of macrophage stimulation assay. Levels of Figure 22A ) TNFa, Figure 22B ) GM-CSF and Figure 22C ) VEGF-A released from CD40L stimulated macrophages after treatment with 50 nM of the exemplary anti-LILRB2 mAb clone LILRB0368 are presented. Results compiled from fold change values (treatment vs. no mAb treatment control) generated from 12 individual donors. Bar represents arithmetic mean and standard deviation. Statistical significance was calculated using paired T-test and two-tailed p values are presented in the graph as ** p<0.01; *** p<0.001; **** p<0.0001.

[0075] Figure 23: CD163 and CD206 expression assay

[0076] Figure 23A -B shows results of CD163 and CD206 expression assay. Expression of CD163 and CD206 on differentiated monocyte-derived macrophages was downregulated in the presence of 50 nM LILRB0368.

[0077] Figure 24 : Tumor growth assay

[0078] Figure 24 Results of tumor growth assay are shown. LILRB0361 reduced tumor growth rate in vivo. NSG / SGM3 mice reconstituted with human CD34+ cord blood stem cells were subcutaneously implanted with MDA-MB-231 tumor xenografts. Mice were treated with 10 mg / kg LILRB0361 (and isotype control) twice weekly for 3 weeks.

[0079] Figure 25: T cell / macrophage / tumor co-culture assay using EGFR-CD3 (OKT3) T cell engager (TCE)

[0080] Figure 25 shows results from a T cell / macrophage / tumor co-culture assay. MDA-MB-231 breast cancer cell line was co-cultured in the presence of polyclonal T cells for three days with or without autologous macrophages and drugs. Tumor killing and CD86 expression levels on CD14+ macrophages were assessed by flow cytometry. Results were generated from 8 donors in 3 independent experiments. Arithmetic mean (triplicates) and SD values were plotted. Statistical analysis was performed by Friedman test with Dunn's multiple comparison test comparing all TCE treated samples to each other (left) or comparing all conditions (right), * p < 0.05 and ** p < 0.01.

[0081] Figure 26 T cell / macrophage / tumor co-culture assay with HER2 CAR-T cells

[0082] Figure 26 Results from a T cell / macrophage / tumor co-culture assay are shown. MDA-MB-231 cell line was co-cultured in the presence of CAR-T cells for two days with or without non-autologous macrophages and drugs. Tumor killing was assessed by flow cytometry. Results were generated from two independent runs using one CAR-T cell donor and 6 macrophage donors (average of three replicates is shown). CAR-T cells were used at E:T ratio of 0.5:1 (experiment 1) or 1 :1 (experiment 2). AZD2796 was used at 100 nM. Cytotoxicity was measured by tumor cell death % using flow cytometry. Statistical analysis was performed by paired t-test showing an increase in tumor cell death in the 3 way assay comparing CAR-T cells to CAR-T cells + AZD2796. Arithmetic mean and SD values for all conditions were plotted. Normality was assessed by Shapiro-Wilk test. **** P < 0.001. 4. DETAILED DESCRIPTION

[0083] 4.1. Terminology

[0084] 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 a variable region of the molecule. The term "binding protein" as used herein encompasses antibodies. Fragments are also encompassed within the scope of the term "binding protein" as used herein.

[0085] 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 a combination 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 incorporating an antibody, and any other modified configuration of the immunoglobulin molecule that exhibits the desired biological activity. Antibody fragments are also included within the scope of the term "antibody" as used herein. An antibody can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy chain constant domains (a, d, e, g, and m, respectively). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. An antibody can be naked or conjugated to other molecules, such as toxins, radioisotopes, and the like.

[0086] 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," "antigen-binding 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 an antigenic determinant (e.g., a complementarity-determining region (CDR)) of an intact binding protein (e.g., antibody). Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, straight chain 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., mice, rats, or hamsters) and humans, or can be artificially produced.

[0087] 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 non-LILRB2 proteins 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.

[0088] 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 and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins including an antibody portion, and any other modified immunoglobulin molecule that includes 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.

[0089] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. Variable region generally refers to the portion of a binding protein (e.g., an antibody), typically a portion of a light or heavy chain, typically about the first 110 to 120 amino acids or 110 to 125 amino acids of the amino terminal end in a mature heavy chain, and about 90 to 115 amino acids in a mature light chain, that vary widely in sequence between binding proteins (e.g., antibodies) and are used for the binding and specificity of a particular binding protein (e.g., antibody) to its particular antigen. The variability of sequence is concentrated in those regions known as complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). 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 a binding protein (e.g., an antibody) with an 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).

[0090] As used herein, the term “complementarity determining region” or “CDR” refers to each of the regions of a variable domain that are hypervariable in sequence and / or form structurally defined loops (hypervariable loops) and / or contain antigen contact residues. A binding protein (e.g., an antibody) can comprise six CDRs, e.g., three in the VH and three in the VL.

[0091] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of a binding protein (e.g., an antibody).

[0092] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of a binding protein (e.g., an antibody).

[0093] The terms "Kabat numbering" and like terms are art-recognized and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of binding proteins (e.g., antibodies) or antigen-binding fragments 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 generally found at amino acid positions 31-35, which can optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, CDRs within a light chain molecule are generally found at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3).

[0094] Chothia, on the other hand, refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop varies between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if both 35A and 35B are not present, the loop ends at 32; if only 35A 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 modeling software.

[0095]

[0096] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of a binding protein (e.g., an antibody), e.g., the carboxy-terminal portion of the light and / or heavy chains, that is not directly involved in binding of the antibody to an 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 the immunoglobulin variable domain. In some aspects, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0097] As used herein, the term "heavy chain" when used in reference to a binding protein (e.g., an antibody) can refer to any of the different types based on the amino acid sequence of the constant domain, e.g., alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), which give rise to IgA, IgD, IgE, IgG, and IgM class antibodies, respectively, including the subclasses of IgG, e.g., IgGl, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0098] As used herein, the term "light chain" when used in reference to a binding protein (e.g., an antibody) can refer to any of the different types, e.g., kappa (K) or lambda (l), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0099] The term "chimeric" binding protein (e.g., an antibody) or antigen-binding fragment thereof refers to a binding protein (e.g., an antibody) or antigen-binding fragment thereof in which the amino acid sequence is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to those of a binding protein (e.g., an antibody) or antigen-binding fragment thereof having the desired specificity, affinity, and capability derived from one mammal (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to those in a binding protein (e.g., an antibody) or antigen-binding fragment thereof derived from another mammal (usually human) to avoid eliciting an immune response in that species.

[0100] 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 contain specific immunoglobulin chains, chimeric immunoglobulins or fragments thereof that contain minimal sequence derived from non-human (e.g., murine) sequences. Typically, a humanized binding protein (e.g., antibody) or antigen-binding fragment thereof is a human immunoglobulin in which residues from the complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capacity ( "CDR grafting" ) (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 examples, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues from a non-human species. 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 improve and optimize the specificity, affinity, and / or capacity of the binding protein (e.g., antibody) or antigen-binding fragment thereof. In general, a 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, while 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 for generating humanized antibodies are described in U.S. Patent 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).

[0101] 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 locus, wherein 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.

[0102] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule, e.g., a binding protein (e.g., an antibody) or antigen binding fragment thereof, and its binding partner, e.g., an antigen. Unless otherwise indicated, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects 1 : 1 interaction between members of a binding pair, e.g., a binding protein (e.g., an antibody) or antigen binding fragment thereof and an antigen. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and the equilibrium association constant (KA). K D is calculated from the quotient of k 0ff / k 0n , while KA is calculated from the quotient of k on / k off . k on refers to the association rate constant of, e.g., a binding protein (e.g., an antibody) or antigen binding fragment thereof and an antigen, and k 0ff refers to the dissociation of, e.g., a binding protein (e.g., an antibody) or antigen binding fragment thereof and an antigen. k on and k off can be determined by techniques known to one of ordinary skill in the art, such as BIACORE ® or KinExA.

[0103] As used herein, an "epitope" is a term of art and refers to a localized region of an antigen to which a binding protein (e.g., an antibody) or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, a contiguous amino acid of a polypeptide (linear or contiguous epitope), or an epitope can be, for example, clustered together from two or more noncontiguous regions of one or more polypeptides (conformational, nonlinear, discontinuous, or noncontiguous epitope). In some aspects, an epitope to which a binding protein (e.g., an antibody) or antigen-binding fragment thereof binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange coupled mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any known method 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) Vols. 114 and 115, Wyckoff HW, et al., eds., 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, Carter CW, ed.; Roversi P, et al., (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.Mutagenesis techniques, including alanine scanning mutagenesis, are described, for example, in Champe M et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0104] 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 hydrogen / deuterium exchange assay (see, e.g., Coales et al. Rapid Commun. Mass Spectrom. 2009; 23: 639-647).

[0105] As used herein, the terms "immunospecifically binds," "immunospecifically recognizes," "specifically binds," and "specifically recognizes" are analogous terms in the context of a binding protein (e.g., antibody) or antigen-binding fragment thereof. These terms indicate that the binding protein (e.g., antibody) or antigen-binding fragment thereof binds via its antigen-binding domain to an epitope, and that the binding requires some complementarity between the antigen-binding domain and the epitope. Thus, in some aspects, a binding protein (e.g., antibody) that "specifically binds" to LILRB2 can also bind to other LILRs, but to a lesser extent than the binding of the binding protein (e.g., antibody) to LILRB2, e.g., as measured using ForteBio or Biacore, to an unrelated protein.

[0106] A binding protein (e.g., antibody) is considered to "compete with" a reference binding protein (e.g., antibody) for binding to a given epitope or overlapping epitopes to the extent that it preferentially binds to that epitope to the extent that it blocks binding of the reference binding protein (e.g., antibody) to that epitope to some extent. Competition for inhibition can be determined by any method known in the art, e.g., a competition ELISA assay. A binding protein (e.g., antibody) can be considered to competitively inhibit binding of a 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%.

[0107] An "isolated" polypeptide, binding protein (e.g., antibody), polynucleotide, vector, cell, or composition is a polypeptide, binding protein (e.g., antibody), polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, binding proteins (e.g., antibodies), polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form in which they are found in nature. In some aspects, an isolated binding protein (e.g., antibody), polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0108] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer 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 by

[0109] "Percent identity" means the extent to which two sequences (e.g., amino acid or nucleic acid sequences) are identical. Percent identity can be determined by aligning the two sequences, introducing gaps to maximize sequence identity. Alignment can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed with the blastn program set to default parameters, and alignment of amino acid sequences can be performed with the blastp program set to default parameters (see National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov).

[0110] 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 aliphatically 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).

[0111] As used herein, amino acids with polar neutral side chains include asparagine (N), cysteine (C), glutamine (Q), serine (S), and threonine (T).

[0112] As used herein, amino acids with charged side chains include aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Amino acids with acidic charged side chains include aspartic acid (D) and glutamic acid (E). Amino acids with basic charged side chains include arginine (R), histidine (H), and lysine (K).

[0113] As used herein, the term "host cell" can be any type of cell, such as 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 can not be identical to the parent cell from which they were replicated, such as due to mutations that can occur during replication or environmental influences, or integration of the nucleic acid molecule into the host cell genome.

[0114] 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 the subject to which the formulation will be administered. The formulation can be sterile.

[0115] As used herein, the terms "subject" and "patient" are used interchangeably. The subject is a human.

[0116] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0117] It should be understood that wherever aspects are described herein with the language "comprising" or "including" or "consisting of it is intended that the aspects can also consist of "consisting essentially of or "consist of the features next listed and not other features. In the disclosure, "comprises," "comprising," "containing" and "containing" and "having" and the like can mean "includes," "including," or the like; "consisting essentially of or "consisting of are open-ended, allowing for the presence of more than the recited components, but precluding presence of other, non-recited components.

[0118] The term "or" as used herein, unless otherwise indicated, is to be understood to mean both "and" and "or." The term "and / or" as used in a phrase such as "A and / or B" is intended to cover the aspects "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).

[0119] As used herein, the terms "about" and "approximately" when used in relation to a numerical value or a range of numbers, indicate that a deviation of up to 10% above the stated numerical value or range and up to 10% below the stated numerical value or range is still within the intended meaning of the recited numerical value or range. It is understood that wherever aspects are described herein with language such as "about" or "approximately" a numerical value or range of values, other aspects are provided in which the terms are replaced with the specific numerical value or range (without "about").

[0120] Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0121] 4.2 Anti-LILRB2 binding proteins

[0122] The binding proteins (e.g., antibodies) described herein are described in relation to the following concepts, aspects, sentences, arrangements, and embodiments. Unless otherwise indicated, all concepts, embodiments, sentences, arrangements, and aspects are to be understood as being combinable with any other concept, aspect, sentence, arrangement, or embodiment, unless such combination would not be technically meaningful or is otherwise explicitly stated.

[0123] Binding-target:

[0124] The present disclosure relates, inter alia, to binding proteins (e.g., antibodies) that specifically bind LILRB2. In particular, the present disclosure provides binding proteins (e.g., antibodies) comprising a LILRB2 binding domain that specifically binds 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., comprise two binding domains, each of which specifically binds LILRB2.

[0125] The binding proteins (e.g., antibodies) described herein that specifically bind LILRB2 are shown herein to be particularly effective and thus have particular utility in the treatment of diseases associated with LILRB2 activity, in particular increased levels of LILRB2 activity, such as cancer.

[0126] The present disclosure also provides polypeptides that specifically bind to LILRB2. Accordingly, these polypeptides comprise at least one LILRB2 binding domain that specifically binds to LILRB2. Such polypeptides can have particular utility when comprised in a binding protein (e.g., an antibody), i.e., a binding protein (e.g., an antibody) that specifically binds to LILRB2.

[0127] 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 a human LILRB2 protein is described in the Uniprot database as UniProtKB - A0A0G2JMW1. LILRB2 (leukocyte immunoglobulin-like receptor subfamily B member 2) can also be referred to as LILRB2, ILT4, MIR10, LIR2, MIR-10, LIR-2, leukocyte immunoglobulin-like receptor B2, ILT-4, CD85D.

[0128] 4.3 Binding - Measurement

[0129] Any suitable method can be used to determine whether a binding protein (e.g., an antibody) (or polypeptide) binds to LILRB2 or LILRB2 protein. Such methods can include surface plasmon resonance (SPR), homogenous time resolved fluorescence (HTRF), bio-layer interferometry, or ELISA to determine the specificity of the binding protein (e.g., an antibody). A binding protein (e.g., an antibody) can be said to bind its antigen if the level of binding to the antigen is at least 2.5-fold greater, e.g., at least 10-fold greater, than the binding to a control antigen. Binding between a binding protein (e.g., an antibody) and its cognate antigen is often referred to as specific binding. The precise identification of the residues bound by a binding protein (e.g., an antibody) can often be obtained using x-ray crystallography. This technique can be used to determine which residue(s) of the LILRB2 protein is bound by a binding protein (e.g., an antibody) as described herein.

[0130] The ability of a binding protein (e.g., an antibody) to bind to its target antigen, as well as the specificity and affinity (KD, Kd, and / or Ka) of that binding, can be determined by any routine method in the art, for example, using surface plasmon resonance (SPR), for example, by Biacore™ (Cytiva Life Sciences) or using ProteOn XPR36™ (Bio-Rad ® ), using KinExA ®(Sapidyne Instruments, Inc) or using ForteBio Octet (Pall ForteBio Corp.).

[0131] As used herein, the term "KD" is intended to refer to the equilibrium dissociation constant of a particular binding protein (e.g., antibody)-antigen interaction. The affinity of a binding protein (e.g., antibody)-antigen binding can be determined, for example, by SPR. Affinity can also be determined by biolayer interferometry.

[0132] In some examples, a binding protein (e.g., antibody) can bind to human LILRB2 with an affinity (KD) of < 100 pM. In one embodiment, a binding protein (e.g., antibody) can bind to human LILRB2 with an affinity (KD) of < 90 pM, < 80 pM, < 70 pM, < 60 pM, < 50 pM, < 40 pM, < 30 pM, < 20 pM, or < 10 pM, as determined by SPR.

[0133] In one example, SPR is performed at 25 °C. Briefly, the affinity of a binding protein (e.g., antibody) can be determined using SPR by:

[0134] 1. Coupling of 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., a dextran-coated gold chip), for example, by primary amine coupling. Thus, anti-Fc binding protein (e.g., antibody) can be covalently immobilized on the chip surface using amine coupling.

[0135] 2. Exposing 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;

[0136] 3. Passing the test antigen through the capture surface of the chip at a range of concentrations up to a maximum of 100 nM, for example, at 0.39 nM, 1.56 nM, 6.25 nM, 25 nM, and 100 nM, with a 0 nM (i.e., buffer only) control run. The buffer can optionally be an aqueous solution of 0.01 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.15 M NaCl, and 0.05% v / v surfactant P20, buffered to pH 7.4; and

[0137] 4. Determining the binding affinity of the test binding protein (e.g., antibody) to the test antigen using surface plasmon resonance. KD, Ka, and Kd can then be calculated.

[0138] SPR can be performed using any standard SPR device, such as by BIACORE or using ProteOn XPR36™ (BIO-RAD). Regeneration of the capture surface can be performed with 3M magnesium chloride solution. This removes the captured test antibody and allows the surface to be used for another interaction. Binding data can be fitted to a 1 : 1 intrinsic model using standard techniques, for example using analysis software such as Biacore Insight Evaluation Software.

[0139] Suitable protocols for determining the binding IC50of a binding protein (e.g. an antibody) to LILRB2 are set out in detail in Example 3 and Example 4.

[0140] 4.4 Cross-reactivity

[0141] The LILR family includes a subfamily of receptors, the LILRA family and the LILRB family. The LILRA subfamily includes members LILRA1, LILRA2, LILRA3, LILRA4, LILRA5 and LILRA6. The LILRB family includes members LILRB1, LILRB2, LILRB3, LILRB4 and LILRB5.

[0142] In some examples, a binding protein (e.g. an antibody) that specifically binds to LILRB2 does not cross-react with other members of the LILR family. Alternatively, in some examples, a binding protein (e.g. an antibody) that specifically binds to LILRB2 can cross-react with other members of the LILR family. In some examples, the other members of the LILR family are human LILR family members.

[0143] In some examples, a binding protein (e.g. an 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. an 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.

[0144] In some examples, a binding protein (e.g. an antibody) that specifically binds to LILRB2 does not cross-react with LILRB1. In some examples, a binding protein (e.g. an antibody) that specifically binds to LILRB2 cross-reacts with LILRB1. In some examples, LILRB1 is human LILRB1.

[0145] 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.

[0146] 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, LILRA1 is human LILRA1.

[0147] 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, LILRA2 is human LILRA2.

[0148] 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, LILRA3 is human LILRA3.

[0149] For a binding protein (e.g., antibody) that specifically binds to a LILRB2 antigen and cross-reacts with one or more of the LILRA or LILRB members described above, in some examples, the binding protein (e.g., antibody) can bind to LILRB2 with a binding affinity that is at least 10-fold higher than the binding affinity to the other family member (e.g., as measured by SPR). In some examples, the binding protein (e.g., antibody) can bind to LILRB2 with a binding affinity that is at least 20-fold higher than the binding affinity to the other family member (e.g., as measured by SPR). In some examples, the binding protein (e.g., antibody) can bind to LILRB2 with a binding affinity that is at least 50-fold higher than the binding affinity to the other family member (e.g., as measured by SPR).

[0150] Binding proteins (e.g., antibodies) that specifically bind to LILRB2 can cross-react 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 to a rodent LILRB2. In one embodiment, the binding protein (e.g., antibody) does not bind to a mouse LILRB2. In one embodiment, the binding protein (e.g., antibody) also binds to a rodent LILRB2. In one embodiment, the binding protein (e.g., antibody) also binds to a mouse LILRB2. In one embodiment, the binding protein (e.g., antibody) also binds to a cynomolgus LILRB2. In one embodiment, the binding protein (e.g., antibody) does not bind to a cynomolgus LILRB2.

[0151] 4.5 Function - Inhibition

[0152] The binding proteins (e.g., antibodies) described herein are inhibitory binding proteins (e.g., antibodies) that inhibit LILRB2, and thus are useful in therapy. By “inhibit LILRB2” is meant “inhibit an activity of LILRB2”. Inhibition of activity can occur via a variety of mechanisms. In some examples, the binding protein (e.g., antibody) inhibits binding of LILRB2 to its ligands, such as ligands HLA-G, HLA-A, HLA-B, HLA-C, HLA-E, CD1c, CD1d, ANGPT2, and ANGPT5. Inhibition of LILRB2 binding to its ligands can be achieved by the binding protein (e.g., antibody) directly blocking the ligand binding site on LILRB2. As described further below, such binding proteins (e.g., antibodies) can compete with their ligands for binding to the LILRB2 protein. Alternatively, inhibition of LILRB2 binding to its ligands can be achieved by indirect mechanisms, for example where the binding protein (e.g., antibody) binds to an epitope on LILRB2 that 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.

[0153] Inhibition of LILRB2 activity can be measured using a co-culture reporter assay, as detailed in Example 5. Thus, in one embodiment, the binding protein (e.g., antibody) inhibits an activity of LILRB2 (e.g., as measured in a co-culture reporter assay).

[0154] LILRB2 is highly expressed on myeloid cells, such as macrophages, neutrophils, dendritic cells. Thus, 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 cell is a macrophage.

[0155] Alternative measurements can also be used to determine the functional effect of a binding protein (e.g., an antibody) of the present disclosure, including potency of the binding protein (e.g., an antibody). Inhibition of LILRB2 leads to TNF-a and / or GM-CSF release in vitro, and thus measurement of TNF-a and / or GM-CSF levels can be used as a readout of efficacy of a LILRB2 binding protein (e.g., an antibody). Thus, in some embodiments, a binding protein (e.g., an antibody) increases TNF-a and / or GM-CSF release in vitro. In some embodiments, a binding protein (e.g., an antibody) increases TNF-a and / or GM-CSF release in vitro from macrophages (e.g., as measured by a macrophage stimulation assay). Furthermore, inhibition of LILRB2 leads to TNF-a and / or GM-CSF release while decreasing production of the pro-angiogenic cytokine VEGF-A in vitro, and thus measurement of TNF-a and / or GM-CSF and / or VEGF-A levels can be used as a readout of efficacy of a LILRB2 binding protein (e.g., an antibody). Exemplary macrophage stimulation assays are set forth in Example 12.

[0156] LILRB2 activation leads to HLA-G-mediated downstream signaling, which includes phosphorylation of SHP1 / 2. Assays directed to the downstream signaling mechanisms induced by LILRB2 activity can be measured as a readout of efficacy of a LILRB2 binding protein (e.g., an antibody). Thus, in some embodiments, a binding protein (e.g., an antibody) inhibits HLA-G-mediated downstream signaling (e.g., as measured by a co-culture reporter assay). In some embodiments, a binding protein (e.g., an antibody) inhibits phosphorylation of SHP1 / 2.

[0157] Inhibition of LILRB2 was also found to promote repolarization of macrophages from tumor-supportive M2 state to tumor-inhibitory M1 state. In some embodiments, a binding protein (e.g., an antibody) induces repolarization of macrophages from tumor-supportive M2 state to tumor-inhibitory M1 state, characterized by decreased cell surface expression of CD163 and increased cell surface expression of CD86 and production of TNFa (e.g., as measured in a macrophage, T cell, tumor cell co-culture assay using antigen-specific T cells). In some embodiments, a binding protein (e.g., an antibody) promotes a pro-inflammatory state in the tumor microenvironment, resulting in increased T cell lysis of tumor cell lines (e.g., as measured in a macrophage, T cell, tumor cell co-culture assay using antigen-specific T cells).

[0158] We have also shown that inhibition of LILRB2 can reduce tumor volume in a mouse xenograft model. Thus, in some embodiments, the binding protein (e.g., antibody) reduces tumor volume (e.g., as measured in vivo). Exemplary tumor growth assays are set forth in Example 12.

[0159] The concentration of the binding protein (e.g., antibody) can be reported in terms of either or both of binding protein (e.g., antibody) weight (milligrams or micrograms or nanograms or picograms) in a given volume (liters or milliliters or microliters), or in terms of molarity (millimolar or micromolar or nanomolar or picomolar) of the binding protein (e.g., antibody), which is the concentration required for 50% of detectable activity in an inhibition assay (50% inhibition concentration, or IC50), or 90% of detectable activity in an inhibition assay (90% inhibition concentration, or IC90), or 95% of detectable activity in an inhibition assay (95% inhibition concentration, or IC95). This can be calculated using any of a variety of methods known in the art, including mathematical fitting of inhibition curves to experimentally derived data and reporting these as IC50 or IC90 or IC95. Finally, the concentration of binding protein (e.g., antibody) that results in complete inhibition of LILRB2 activity can be determined.

[0160] 4.6 LILRB2 Inhibition - Level (IC50)

[0161] In some examples, the binding protein (e.g., antibody) inhibits LILRB2 (e.g., as determined in a macrophage stimulation assay) with an IC50 of 10 pM or less. In some examples, the binding protein (e.g., antibody) inhibits LILRB2 (e.g., as determined in a macrophage stimulation assay) with an IC50 of 5 pM or less.

[0162] In one embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 3.0 E-9 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 2.0 E-9 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 1.0 E-9 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 10.0 E-10 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 9.0 E-10 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 8.0 E-10 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 7.0 E-10 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 6.0 E-10 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 5.0 E-10 M or less (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2). In another embodiment, the binding protein (e.g., antibody) inhibits LILRB2 with an IC50 of 2.0 E-10 M to 3.0 E-9 M (e.g., as determined by a flow binding assay on Jurkat-NFAT-Luc-LILRB2).

[0163] LILRB2 inhibition is calculated relative to a reference binding protein (e.g., antibody):

[0164] In some examples, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than a reference binding protein (e.g., an antibody). In some examples, the reference binding protein (e.g., an antibody) can be any of the comparator antibodies listed in the examples herein. For example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than a reference binding protein (e.g., an antibody) expressed as a fold change relative to the reference binding protein (e.g., an antibody). In one example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than 2-fold change relative to a reference binding protein (e.g., an antibody). In one example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than 25-fold change relative to a reference binding protein (e.g., an antibody). In one example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than 50-fold change relative to a reference binding protein (e.g., an antibody). In one example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than 100-fold change relative to a reference binding protein (e.g., an antibody). In one example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than 500-fold change relative to a reference binding protein (e.g., an antibody). In one example, a binding protein (e.g., an antibody) can inhibit LILRB2 at an activity level greater than 1000-fold change relative to a reference binding protein (e.g., an antibody).

[0165] 4.7 Competition with ligands

[0166] A provided binding protein (e.g., an antibody) can compete with LILRB2 for binding to one or more of its ligands (i.e., such as HLA-G, HLA-A, HLA-B, HLA-C, HLA-E, CD1c, CD1d, ANGPT2, and ANGPT5). A provided binding protein (e.g., an antibody) can specifically bind to the ligand binding domain of LILRB2, wherein the binding protein (e.g., an antibody) is an inhibitory binding protein (e.g., an antibody) that competes with LILRB2 for binding to its ligands, thereby preventing downstream signaling. In some embodiments, the LILRB2 binding protein is a competitive inhibitor of LILRB2 (e.g., as measured by a 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, CD1c, CD1d, ANGPT2, and ANGPT5. In some embodiments, the ligand is HLA-G.

[0167] Other binding proteins (e.g. antibodies) that do not compete with LILRB2 for binding to one or more of its ligands can alter the ability of the protein to function correctly, and thus are inhibitory binding proteins (e.g. antibodies) even if the binding protein (e.g. antibody) does not block the ligand binding site, and thus does not directly block ligand interaction.

[0168] Whether a binding protein (e.g. antibody) competes with LILRB2 for binding to one or more of its ligands can be measured using a competition assay. Competition can be determined by surface plasmon resonance (SPR), such techniques being apparent to the skilled person. SPR can be performed using Biacore ™ , Proteon ™ or another standard SPR technique. Such competition can be due to, for example, the binding protein (e.g. antibody) or fragment binding to the same or overlapping epitope of LILRB2 as the ligand. In one example, competition is determined by ELISA, such techniques being apparent to the skilled person. In one example, competition is determined by homogenous time resolved fluorescence (HTRF), such techniques being apparent to the skilled person. In one example, competition is determined by fluorescence activated cell sorting (FACS), such techniques being apparent to the skilled person. In one example, competition is determined by ForteBio Octet ® biolayer interferometry (BLI), such techniques being apparent to the skilled person. In one example, competition is determined as described in Example 6.

[0169] If the epitope bound by the inhibitory binding protein (e.g. antibody) completely blocks the ligand binding site of LILRB2, then binding between the receptor and the ligand is completely prevented (this can be physical blocking - in the case of overlapping epitopes - or steric blocking - where the antagonist is large such that it prevents the ligand from binding to its different epitope). If the epitope bound by the binding protein (e.g. antibody) partially blocks the ligand binding site of the receptor, then the ligand can be able to bind, but only weakly (in the case of partial inhibition), or bind in a different orientation to the native binding interaction.

[0170] 4.8 Binding proteins that bind the same epitope and compete for binding

[0171] Binding proteins (e.g. antibodies) can be provided which bind to the same epitope as a binding protein (e.g. antibody) defined anywhere herein.

[0172] A binding protein (e.g., antibody) of the present disclosure can be one that specifically binds to the same epitope on LILRB2 as an epitope on LILRB2 bound by a reference binding protein (e.g., antibody), wherein 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 can be one that specifically binds to the same epitope on LILRB2 as an epitope on LILRB2 bound by a reference binding protein (e.g., antibody), wherein 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 proteins (e.g., antibodies) that bind to the same epitope as a binding protein (e.g., antibody) as defined anywhere herein can optionally be determined using X-ray crystallography or other fine mapping techniques such as electron microscopy to identify points of contact between the binding protein (e.g., antibody) and antigen. Alternatively, binding proteins (e.g., antibodies) that bind to the same epitope as a binding protein (e.g., antibody) as defined anywhere herein can be determined using, for example, the HTRF epitope competition assay described in Example 4. Competition between binding proteins (e.g., antibodies) can also be determined, for example, using SPR as described elsewhere herein, and a binding protein (e.g., antibody) of the present invention can compete with an IgG binding protein (e.g., antibody) for binding to LILRB2 (for binding to its epitope), the IgG binding protein being any binding protein (e.g., antibody) as described herein. A binding protein (e.g., antibody) can contact the LILRB2 protein with a footprint that fully or partially overlaps with the footprint of a binding protein (e.g., antibody) as defined anywhere herein.

[0173] The present disclosure describes binding proteins (e.g., antibodies) of the present invention that specifically bind to LILRB2 and are defined by their function and / or their structure. The present invention also provides binding proteins (e.g., antibodies) that compete for binding to LILRB2 with those binding proteins (e.g., antibodies) described herein. In one embodiment, a binding protein (e.g., antibody) of the present disclosure can be one that competes for binding to LILRB2 with any binding protein (e.g., antibody) described herein. Such competition can be due to, for example, the binding protein (e.g., antibody) binding to the same or overlapping epitope on LILRB2 as a reference binding protein (e.g., antibody). Such a 'competing' binding protein (e.g., antibody) can retain the same function as the reference binding protein (e.g., antibody) with which it competes. It can be expected that a binding protein (e.g., antibody) that binds to the same (identical / same) epitope as a reference binding protein (e.g., antibody) has the same function as the reference binding protein (e.g., antibody) with which it competes.

[0174] In one embodiment, provided is a binding protein (e.g., antibody) that specifically binds to LILRB2, wherein the binding protein (e.g., antibody) competes for binding to LILRB2 with a reference binding protein (e.g., antibody), wherein the reference binding protein (e.g., antibody) is a binding protein (e.g., antibody) as described anywhere herein. In one embodiment, provided is a binding protein (e.g., antibody) that specifically binds to LILRB2, wherein the binding protein (e.g., antibody) competes for binding to LILRB2 with a reference binding protein (e.g., antibody), wherein 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.

[0175] 4.9 Binding protein (e.g., antibody) types

[0176] In some embodiments, the binding protein can be an antibody.

[0177] In some embodiments, the binding protein (e.g., antibody) can be an isolated binding protein (e.g., antibody). In some embodiments, the binding protein (e.g., antibody) can be a monoclonal binding protein (e.g., antibody).

[0178] The binding proteins (e.g., antibodies) described herein can be or can 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 a binding protein (e.g., antibody) or fragment thereof that comprises a human and a non-human region. A “humanized” binding protein (e.g., antibody) is a monoclonal binding protein (e.g., antibody) that comprises 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 a 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) contains no 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) have a lower risk of inducing an immune response 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, the binding protein (e.g., antibody) is a fully human binding protein (e.g., antibody).

[0179] A human binding protein (e.g., antibody), non-human binding protein (e.g., antibody), chimeric binding protein (e.g., antibody), or humanized binding protein (e.g., antibody) can be obtained by any means, including via in vitro sources (e.g., hybridomas or recombinantly produced binding protein (e.g., antibody) cell lines) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and 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, 5thEd., Garland Publishing, New York, N.Y. (2001). In certain embodiments, a human binding protein (e.g., antibody) or chimeric binding protein (e.g., antibody) can be generated using a transgenic animal (e.g., mouse) in which one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, Medarex HUMAB-MOUSE ™ , Kirin TC MOUSE ™ , and 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, for example, grafting 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(1): 115-120 (2008)). In one embodiment, a humanized antibody can be produced using, for example, the methods described in U.S. Patent Application Publication 2011 / 0287485 Al.

[0180] Methods of making the binding proteins (e.g., antibodies) described herein are described in Example 1.

[0181] 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.

[0182] In some embodiments, the LILRB2 binding domain is comprised in a Fab domain. In some embodiments, the Fab domain is 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).

[0183] 4.10 Exemplary LILRB2 binding proteins

[0184] 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 forth in Table 8 herein.

[0185] In one embodiment, the binding protein (e.g., antibody) is LILRB0368. LILRB0368 is a monoclonal monospecific IgG1 antibody that specifically binds to LILRB2. The amino acid sequences of LILRB0368 are set forth in Table 8 herein as SEQ ID NOs: 1-10.

[0186] LILRB0368 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 3, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 5 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 6 (Kabat), and a 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, which comprises a CDRL1 amino acid sequence of SEQ ID NO: 8 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 9 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 10 (Kabat). LILRB0368 is an IgG1 comprising 2 heavy chains 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 chain of LILRB0368 is a kappa light chain.

[0187] 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 HCDR3 of SEQ ID NO: 7 (e.g., as determined by Kabat).

[0188] 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 HCDR3 of SEQ ID NO: 7 (e.g., as determined by Kabat).

[0189] 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.

[0190] In some embodiments, the binding protein (e.g., antibody) comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 optionally having 1, 2, 3, 4, or 5 amino acid changes, and a variable light (VL) domain sequence of SEQ ID NO: 4 optionally having 1, 2, 3, 4, or 5 amino acid changes.

[0191] In some embodiments, the binding protein (e.g., antibody) comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 optionally having 1, 2, 3, 4, or 5 amino acid changes outside of the complementarity determining regions (CDRs), and a variable light (VL) domain sequence of SEQ ID NO: 4 optionally having 1, 2, 3, 4, or 5 amino acid changes outside of the CDRs.

[0192] 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 the variable light (VL) domain sequence comprise sequences that are at least 90% identical to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, respectively.

[0193] 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 the variable light (VL) domain sequence comprise sequences that are at least 90% identical to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, respectively, provided that the binding protein (e.g., antibody) has the HCDRs of SEQ ID NO: 3 and the LCDRs of SEQ ID NO: 4.

[0194] 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.

[0195] The amino acid sequences of other exemplary LILRB2 binding proteins (e.g., antibodies) described in the Examples, denoted as LILRBXXXX, are also set forth in Table 8 herein as SEQ ID NOS: 11-115. Such binding proteins (e.g., antibodies) that include each of the CDR, VH, VL, HC, and LC amino acid sequences are also embodiments of the present disclosure.

[0196] LILRB0354 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 13 comprising a CDRH1 amino acid sequence of SEQ ID NO: 15 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 16 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 18 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 19 (Kabat), and a 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.

[0197] LILRB0355 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 23 comprising a CDRH1 amino acid sequence of SEQ ID NO: 25 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 26 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 28 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 29 (Kabat), and a 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.

[0198] LILRB0356 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 33 comprising a CDRH1 amino acid sequence of SEQ ID NO: 35 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 36 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 38 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 39 (Kabat), and a 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.

[0199] LILRB0357 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 43 comprising a CDRH1 amino acid sequence of SEQ ID NO: 45 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 46 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 48 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 49 (Kabat), and a 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.

[0200] LILRB0358 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 53 comprising a CDRH1 amino acid sequence of SEQ ID NO: 55 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 56 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 58 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 59 (Kabat), and a 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.

[0201] LILRB0359 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 63 comprising a CDRH1 amino acid sequence of SEQ ID NO: 65 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 66 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 68 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 69 (Kabat), and a 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.

[0202] LILRB0360 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 73 comprising a CDRH1 amino acid sequence of SEQ ID NO: 75 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 76 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 78 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 79 (Kabat), and a 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.

[0203] LILRB0361 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 83 comprising a CDRH1 amino acid sequence of SEQ ID NO: 85 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 86 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 88 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 89 (Kabat), and a 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.

[0204] LILRB0362 comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 93 comprising a CDRH1 amino acid sequence of SEQ ID NO: 95 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 96 (Kabat), and a 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 a CDRL1 amino acid sequence of SEQ ID NO: 98 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 99 (Kabat), and a 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] LILRB0 392 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 109 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0219] LILRB0 393 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 109 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0220] LILRB0 394 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 111 and a variable light (VL) domain sequence of SEQ ID NO: 94.

[0221] LILRB0 395 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 111 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0222] LILRB0 396 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 83 and a variable light (VL) domain sequence of SEQ ID NO: 115.

[0223] LILRB0 397 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 109 and a variable light (VL) domain sequence of SEQ ID NO: 84.

[0224] LILRB0 398 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 106 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0225] LILRB0 399 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 107 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0226] LILRB0 400 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 108 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0227] LILRB0 401 comprises a variable heavy (VH) domain sequence of SEQ ID NO: 110 and a variable light (VL) domain sequence of SEQ ID NO: 112.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 4.11 Other binding protein formats

[0234] 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, the HCDRs, LCDRs, VH, VL, HC, and LC sequences of the exemplified binding proteins (e.g., antibodies) described herein) can be incorporated into other binding protein (e.g., antibody) formats. In particular, the HCDRs and LCDRs and / or VH and VL domains of LILRB0368 can be incorporated into other binding protein (e.g., antibody) formats, such as bispecific binding proteins (e.g., antibodies). The data described herein show that binding proteins (e.g., antibodies) having the CDR sequences and / or VH and VL sequences of LILRB0368 are particularly effective at inhibiting LILRB2 activity, and are thus particularly useful for treating disorders associated with LILRB2 activity, such as cancer.

[0235] 4.12 Sequence identity

[0236] In some examples, the binding protein (e.g., antibody) comprises an amino acid sequence having a high level of sequence identity to the amino acid sequence of one of the exemplary binding proteins (e.g., antibodies) described herein and listed in Table 8.

[0237] 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 a CDR of the sequence.

[0238] 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 comprise sequences that are at least 90% (preferably 95%, more preferably 98%) identical to the variable heavy (VH) domain sequence and variable light (VL) domain sequence, respectively, of any one of the binding proteins (e.g., antibodies) described herein and listed in Table 1, provided that the binding protein (e.g., antibody) has the CDRs of the binding protein (e.g., antibody) described herein and listed in Table 1.

[0239] As described in the Glossary, the percent sequence identity between two amino acid sequences can be determined by any alignment program known in the art. For example, amino acid sequence alignments to determine percent sequence identity can be performed using the blastp program with default parameters (see National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov).

[0240] 4.13 Substitutions

[0241] In some examples, a variable heavy (VH) domain sequence as defined anywhere herein can optionally have 1, 2, 3, 4, or 5 amino acid changes outside of the complementarity determining regions (CDRs). Similarly, a variable light (VL) domain sequence as defined anywhere herein can optionally have 1, 2, 3, 4, or 5 amino acid changes outside of the CDRs. When one or more mutations (whether additions, insertions, substitutions, or deletions of one or more amino acids) are made in a variable domain sequence of a binding protein (e.g., antibody) described herein, the resulting binding protein (e.g., antibody) can be tested (e.g., in one or more assays described herein) to confirm that affinity and / or potency are retained.

[0242] Amino acid substitutions include alterations wherein an amino acid is replaced by a different naturally occurring amino acid residue. Such substitutions can be classified as "conservative", in which case the amino acid residue contained in the polypeptide is replaced with another naturally occurring amino acid that has similar properties in terms of polarity, charge and size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present disclosure can also be "non-conservative", in which case an amino acid residue present in the peptide is replaced with an amino acid having different properties, such as from a different group of naturally occurring amino acids (e.g. replacing a charged or hydrophobic amino; with an acid such as alanine), or alternatively, in which a naturally occurring amino acid is replaced with a non- conventional amino acid.

[0243] In one embodiment, conservative amino acid substitutions are as described herein. For example, the substitution can be Y replaced with F, T replaced with S or K, P replaced with A, E replaced with D or Q, N replaced with D or G, R replaced with K, G replaced with N or A, T replaced with S or K, D replaced with N or E, I replaced with L or V, F replaced with Y, S replaced with T or A, R replaced with K, G replaced with N or A, K replaced with R, A replaced with S, K or P. In another embodiment, conservative amino acid substitutions can be where Y is replaced with F, T is replaced with A or S, I is replaced with L or V, W is replaced with Y, M is replaced with L, N is replaced with D, G is replaced with A, T is replaced with A or S, D is replaced with N, I is replaced with L or V, F is replaced with Y or L, S is replaced with A or T and A is replaced with S, G, T or V.

[0244] In one example, the amino acid substitution is outside of the CDR sequences.

[0245] 4.14 Light chains

[0246] In some examples, the binding protein (e.g., antibody) comprises a kappa light chain. Exemplary kappa light chain constant region amino acid sequences are listed in Table 8.

[0247] In some examples, the binding protein (e.g., antibody) comprises a lambda light chain.

[0248] 4.15 Constant regions :

[0249] In another embodiment, the binding protein (e.g., antibody) comprises a constant region (Fc), such as a human constant region. The Fc can be of any suitable class. In some embodiments, the binding protein (e.g., antibody) comprises a constant region based on a wild-type IgGl, IgG2 or IgG4 binding protein (e.g., antibody) or a variant thereof. In some embodiments, the binding protein (e.g., antibody) is an IgGl.

[0250] In certain embodiments, the binding protein (e.g., antibody) comprises an altered Fc region, wherein one or more alterations are made in the Fc region to alter the functional and / or pharmacokinetic properties of the binding molecule. Such alterations can result in altered effector function, reduced immunogenicity, and / or increased serum half-life.

[0251] For example, the binding protein (e.g., antibody) can have a heavy chain constant region that binds one or more types of Fc receptors but does not induce cellular effector functions (i.e., it does not mediate ADCC, CDC, or ADCP activity). Such a constant region can not be able to bind the specific Fc receptors responsible for triggering ADCC, CDC, or ADCP activity. The binding protein (e.g., antibody) can have a heavy chain constant region that does not bind Fc receptors.

[0252] In some embodiments, the binding protein (e.g., antibody) comprises an Fc domain with reduced effector function. Such reduced effector function of the Fc region can be achieved by incorporating one or more amino acid mutations in the Fc region known in the art.

[0253] In one embodiment, the binding protein (e.g., antibody) comprises an IgGl triple mutant, referred to herein as IgGl-TM. IgGl-TM is a human IgGl isotype that contains three single amino acid substitutions L234F / L235E / P331S within the lower hinge and CH2 domain (Oganesyan et al., Acta Crystallogr. D Biol. Crystallogr. 64:700-704, 2008). TM results in significantly reduced binding to human FcyRI, FcyRII, FcyRIII, and Clq, yielding 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 P331S.

[0254] ​In one embodiment, the binding protein (e.g., antibody) comprises an IgGl 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.). The combination of these substitutions results in significantly reduced binding to human FcyRI, FcyRII, FcyRIII, and Clq, 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). Thus, in one embodiment, the binding protein (e.g., antibody) comprises an Fc domain comprising the mutations L234F, L235Q, and K322Q.

[0255] 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.

[0256] In some embodiments, the binding proteins (e.g., antibodies) disclosed herein are modified to increase or decrease serum half-life. In certain embodiments, the 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 half-life of the modified IgG is increased compared to the half-life of an IgG having a wild-type IgG constant domain. Thus, in one embodiment, the binding protein (e.g., antibody) comprises an Fc domain comprising at least one mutation that confers an extended half-life. In one embodiment, the heavy chain constant region comprises a YTE mutation. The term "YTE" or "YTE mutant" refers to a mutation in an IgGl Fc that results in increased binding to human FcRn and improves the serum half-life of the binding protein (e.g., antibody) having the mutation. The YTE mutant comprises a combination of three mutations M252Y, S254T, and T256E introduced into the heavy chain of IgGl (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.). See U.S. Patent 7,658,921, which is incorporated herein by reference. The YTE mutant has been shown to increase the serum half-life of the binding protein (e.g., antibody) by about four-fold compared to a wild-type version 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, e.g., U.S. Patent 7,083,784, which is hereby incorporated by reference in its entirety.

[0257] In some embodiments, the binding protein (e.g., antibody) comprises a mutation that imparts reduced effector function and a mutation that prolongs the half-life of the binding protein (e.g., antibody). Thus, in one embodiment, the binding protein (e.g., antibody) comprises an Fc domain with reduced effector function and comprises at least one mutation that imparts half-life prolongation. In one embodiment, the binding protein (e.g., antibody) comprises an Fc domain comprising the mutation TM-YTE, i.e., comprising the mutations L234F, L235E, P331S, M252Y, S254T, and T256E. Binding proteins (e.g., antibodies) comprising the mutation TM-YTE have been shown to have prolonged half-life and lack binding protein (e.g., antibody)-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity, i.e., lack 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 mutation 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 while retaining the same biological activity as TM-YTE (half-life prolongation and lack of immune receptor binding). The Fc domain of antibody LILRB0368 includes the TM mutations, the amino acid sequence of which is set forth in Table 8.

[0258] 4.16 Polypeptides

[0259] In another aspect, the present disclosure provides a polypeptide comprising one or more chains of a binding protein (e.g., antibody) as described anywhere herein.

[0260] The present disclosure also provides a polypeptide comprising one or more binding domains of a binding protein (e.g., antibody) as defined anywhere herein.

[0261] A polypeptide can comprise part or all of a LILRB2 binding domain. In some embodiments, the domain is one or more CDRs from a heavy chain and / or light chain, a VH domain or a VL domain. A polypeptide can comprise a binding domain, such as one or more CDRs as defined herein, or a variable light domain or a variable heavy domain as defined herein. In some embodiments, these polypeptides can comprise binding domains comprising all three CDRs (CDR1, CDR2 and CDR3) of a variable heavy domain sequence as defined herein. In some embodiments, these polypeptides can comprise binding domains comprising all three CDRs (CDR1, CDR2 and CDR3) of a variable light domain sequence as defined herein. In some examples, a polypeptide can comprise a variable heavy domain of a binding protein (e.g. an antibody) as defined herein. In some examples, a polypeptide can comprise a variable light domain of a binding protein (e.g. an antibody) as defined herein. In some embodiments, a polypeptide can comprise a complete heavy chain of a binding protein (e.g. an antibody) as defined herein. In some embodiments, a polypeptide can comprise a complete light chain of a binding protein (e.g. an antibody) as defined herein. In some embodiments, a polypeptide is an isolated polypeptide. In one embodiment, there is provided a polypeptide comprising any one or more of the amino acid sequences listed in Table 1. For example, a polypeptide can comprise any one or more of the amino acid sequences from antibody LILRB0368, the sequences of which are listed in Table 8 herein.

[0262] 4.17 Nucleic acids, vectors, host cells

[0263] Also provided are nucleic acids encoding any one or more of the amino acid sequences of any of the binding proteins (e.g. antibodies) described herein (e.g. any one or more CDRs from a VL and / or VH domain, a VH domain and / or a VL domain, a complete heavy chain and / or a complete light chain).

[0264] In one example, a nucleic acid encodes one or more chains of a binding protein (e.g. an antibody) as defined anywhere herein. In one example, a nucleic acid encodes a heavy chain of any of the binding proteins (e.g. antibodies) described herein. In another example, a nucleic acid encodes a light chain of any of the binding proteins (e.g. antibodies) described herein.

[0265] In one embodiment, there is provided a nucleic acid encoding any one or more of the amino acid sequences listed in Table 8. In some embodiments, there is provided a nucleic acid encoding any one or more of the amino acid sequences of antibody LILRB0368.

[0266] Also provided are nucleic acids encoding a polypeptide as defined anywhere herein.

[0267] In one example, the nucleic acid is an isolated and purified nucleic acid.

[0268] The term "nucleic acid sequence" is intended to encompass polymers 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 polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and therefore include double- and single-stranded DNA, as well as double- and single-stranded RNA. These terms also include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, but not limited to, methylated

[0269] Also provided are vectors comprising the above-described nucleic acids. In one embodiment, the vector comprises one or more nucleic acid sequences encoding a binding protein (e.g., an 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, an episome, a cosmid, a viral vector (e.g., a retrovirus or adenovirus), or a phage. Suitable vectors and methods of making vectors are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rded., 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 can be a CHO vector. In one embodiment, the vector can be a HEK293 vector.

[0270] In addition to a nucleic acid sequence encoding a binding protein (e.g., an antibody) as defined anywhere herein or a polypeptide as defined anywhere herein, a vector comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, which provide for expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and described in, e.g., Goeddel, Gene Expression Technology: Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990).

[0271] Also provided are 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.

[0272] A vector comprising a nucleic acid encoding an amino acid sequence of a binding protein (e.g., an antibody) or polypeptide as described anywhere herein can be introduced into a host cell capable of expressing the polypeptide encoded thereby. Preferred host cells are those that can be easily and reliably grown, have a reasonably fast growth rate, have a well-characterized expression system, and can be readily and efficiently transformed or transfected. The host cell can be a prokaryotic cell or a eukaryotic cell.

[0273] In some embodiments, the host cell is a prokaryotic cell. 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 various strains of E. coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).

[0274] In some embodiments, the host cell is a eukaryotic cell. 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 a mammalian cell. Many 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 monkey COS-1 (ATCC No. CRL1650) and COS-7 (ATCC No. CRL1651), and the CV-1 (ATCC No. CCL70). Mammalian cells are ideally human cells. For example, mammalian cells can be human lymphocytes or lymphocyte-derived cell lines, such as pre-B lymphocyte-derived cell lines, PER.C6, etc. ® The cell line (Crucell Holland BV, The Netherlands) or human embryonic kidney (HEK) 293 or 293T cells (ATCC number CRL1573).

[0275] A nucleic acid sequence encoding an amino acid of a binding protein (e.g., an antibody) as defined anywhere herein or a polypeptide as defined anywhere herein can be introduced into a cell by "transfection," "transformation," or "transduction." As used herein, "transfection," "transformation," or "transduction" refer to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Numerous 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)). Bacteriophage or viral vectors can be introduced into host cells after the infectious particles are grown in suitable packaging cells, many of which are commercially available.

[0276] 4.18 Pharmaceutical compositions

[0277] The present disclosure also provides a pharmaceutical composition comprising a binding protein (e.g., an antibody) as defined anywhere herein and a pharmaceutically acceptable carrier. Also provided is a composition comprising a nucleic acid sequence encoding a binding protein (e.g., an antibody) as defined anywhere herein or a polypeptide as defined anywhere herein, or a vector comprising a nucleic acid sequence as defined anywhere herein.

[0278] A pharmaceutical composition comprises an effective amount of a binding protein (e.g., an antibody) as defined herein, a nucleic acid as defined anywhere herein, or a vector as defined anywhere herein. An effective amount of a binding protein (e.g., an antibody), a nucleic acid, or a vector to be used therapeutically will depend, for example, on the therapeutic goal, the route of administration, and the condition of the patient. In one embodiment, an effective amount of a binding protein (e.g., an antibody) as defined anywhere herein within a pharmaceutical composition is capable of effectively treating or preventing a disease associated with LILRB2 activity, such as a cancer.

[0279] The composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which includes a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. Pharmaceutically acceptable carriers can include one or more excipients. Pharmaceutically acceptable excipients are known and include carriers, excipients, or stabilizers that are nontoxic to the cells or mammals being exposed thereto at the dosages and concentrations employed. In the context of the present disclosure, any suitable carrier can be used, and such carriers are well known in the art. The selection of carrier will be determined in part by the particular site where the composition is able to be administered, as well as the particular method used to administer the composition. Physiologically acceptable excipients can be aqueous pH buffered solutions. Examples of physiologically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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 nonionic surfactants such as TWEEN ™ , polyethylene glycol (PEG), and PLURONICS ™ .

[0280] The composition can optionally be sterile. The composition can be stored refrigerated or lyophilized, and reconstituted in a suitable sterile vehicle prior to use. The composition can be generated according to conventional techniques described, for example, in Remington: The Science and Practice of Pharmacy, 21stEdition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0281] The composition 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, which should take proper account of pH, isotonicity, and stability. These conditions are known to those skilled in the art.

[0282] 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 particular example, the pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The composition can be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous membranes (e.g., the mouth, nose, throat, rectum, and the like) and can be administered, for example, with other biologically active agents. Administration can be systemic or local. Each dose can or can not be administered by the same route of administration.

[0283] Various delivery systems are known and can be used to administer the prophylactic or therapeutic agent (e.g., a binding protein (e.g., antibody) as disclosed herein) including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing binding protein (e.g., antibody), receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of the nucleic acid to be expressed to be part of a retroviral or other vector, and the like. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosol or other convenient delivery means.

[0284] In a particular example, it can be desirable to administer a prophylactic or therapeutic agent or a pharmaceutical composition as described herein locally to an area in need of treatment. This can be achieved by, for example, local infusion during surgery, by injection, by means of a minipump, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

[0285] In the case of drugs intended for local and / or topical administration, such as by absorption into the inner layer of the epithelial or skin mucosa, the binding protein (e.g., antibody) can be provided as an IgA isotype binding protein (e.g., antibody). For human patients, it is preferred that the binding protein (e.g., antibody) be a human IgAl or human IgA2 binding protein (e.g., antibody). Drugs formulated for inhalation and / or for delivery of binding proteins (e.g., antibodies) (or encoding nucleic acids thereof, e.g., in a DNA vector) to the upper and / or lower respiratory tract, including formulations for delivery of aerosolized drugs, can comprise IgA (e.g., human IgAl or human IgA2) binding proteins (e.g., antibodies). Thus, inhalers, nebulizers, and similar devices containing drugs comprising IgA binding proteins (e.g., antibodies) or encoding nucleic acids thereof, as well as any buffers or other excipients suitable for stabilizing the drugs and / or facilitating their delivery to target tissues, can be provided.

[0286] 4.19 Therapeutic uses

[0287] We have discovered monoclonal binding proteins (e.g., antibodies) that specifically bind LILRB2 and have advantageous properties suitable for development as pharmaceuticals for treating or preventing a disorder associated with LILRB2 activity, such as cancer. The binding proteins (e.g., antibodies) of the present disclosure exhibit a combination of advantageous properties, including high specificity for LILRB2, strong binding affinity for LILRB2, and / or potency in inhibiting LILRB2 activity.

[0288] The binding proteins (e.g., antibodies) described herein, or pharmaceutical compositions thereof, can be used in therapy. In particular, the binding proteins (e.g., antibodies), or pharmaceutical compositions thereof, can be used to treat a disease associated with LILRB2 activity, such as cancer. The binding proteins (e.g., antibodies), or pharmaceutical compositions thereof, can also be used to prevent a disease associated with LILRB2 activity, such as cancer.

[0289] Accordingly, the present disclosure provides a binding protein (e.g., antibody) as defined anywhere herein, or a pharmaceutical composition as defined anywhere herein, for use in therapy.

[0290] The present disclosure also provides a binding protein (e.g., antibody) as defined anywhere herein, or a pharmaceutical composition as defined anywhere herein, for use in the treatment of a disease or disorder or condition associated with LILRB2 activity, in particular with increased LILRB2 activity.

[0291] The present disclosure also provides a binding protein (e.g., antibody) as defined anywhere herein, or a pharmaceutical composition as defined anywhere herein, for use in the treatment of cancer.

[0292] The present disclosure also provides a method of treating cancer, wherein the method comprises administering to a subject in need thereof a binding protein (e.g., antibody) as defined anywhere herein, or a pharmaceutical composition as defined anywhere herein.

[0293] The present 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.

[0294] In one embodiment, the subject is a human.

[0295] 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, progression of cancer is reduced. In one example, the risk of developing cancer is reduced.

[0296] Certain types of cancer can have particularly high levels of LILRB2 activity, which can be due to the presence of particularly high levels of LILRB2-expressing macrophages within the tumor. In such cancers, therapy using a binding protein (e.g., an antibody) as described anywhere herein can be particularly effective. Cancers that show particularly high levels of LILRB2 activity due to the presence of particularly high levels of macrophages include renal carcinoma, lung adenocarcinoma, pancreatic carcinoma, gastric carcinoma, melanoma, breast carcinoma, or ovarian carcinoma. Renal carcinoma in particular shows high levels of macrophages within the tumor, and thus high levels of LILRB2 expression.

[0297] Thus, in some embodiments, a binding protein (e.g., an antibody) is used to treat a cancer, wherein the cancer is renal carcinoma, lung adenocarcinoma, pancreatic carcinoma, gastric carcinoma, melanoma, breast carcinoma, or ovarian carcinoma. In some embodiments, the cancer is renal carcinoma. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is pancreatic carcinoma. In some embodiments, the cancer is gastric carcinoma. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is breast carcinoma. In some embodiments, the cancer is ovarian carcinoma.

[0298] The present disclosure also relates to the use of a binding protein (e.g., an antibody) in combination with a T cell engager for the treatment of a cancer. The present disclosure also relates to the use of a binding protein (e.g., an antibody) in combination with a CAR-T cell for the treatment of a cancer.

[0299] 4.20 Kits

[0300] The present disclosure also provides a kit comprising a binding protein (e.g., an antibody) as described anywhere herein or a pharmaceutical composition as described anywhere herein. In some embodiments, the kit comprises a binding protein (e.g., an antibody) as defined anywhere herein and instructions for administering the binding protein (e.g., an antibody) to a subject in need of treatment.

[0301] Also provided is 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., an antibody) provided herein. Optionally associated with such container(s) can be 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 authorization number.

[0302] In another example, there is provided an article of manufacture comprising a container having a composition comprising a binding protein (e.g., an antibody) as described herein, and a package insert or label indicating that the composition can be used to treat a disease associated with LILRB2 activity, such as a cancer. In one example, there is provided a kit for use in treating and / or preventing a disease associated with LILRB2 activity, such as a cancer, the kit comprising a binding protein (e.g., an antibody) as disclosed herein in any example or combination of examples, optionally in combination with a label or instructions for use in treating and / or preventing the disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorization number (e.g., an FDA or EMA authorization number); optionally wherein the kit comprises an IV or injection device comprising the binding protein (e.g., an antibody). In another example, the kit comprises the binding protein (e.g., an antibody) contained in a container or 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., an antibody) is formulated as a pharmaceutical composition contained within a (sterile) container or contained within a (sterile) IV bag. In another example, the kit further comprises instructions for use.

[0303] 5 Examples

[0304] Herein, we describe binding proteins (e.g., antibodies) that bind to LILRB2, which are highly effective in inhibiting LILRB2 activity, and are thus suitable for use in therapy, in particular for use in treating a disease or condition associated with increased LILRB2 activity, such as a cancer. The following examples further illustrate the disclosure, but should not be construed as limiting the scope of the disclosure in any way.

[0305] 6 Example 1. Generation of Anti-LILRB2 Antibodies

[0306] Antibodies that specifically bind to LILRB2 (denoted LILRBXXXX) were generated according to the following protocol. Amino acid sequences, including CDR sequences, VL and VH sequences, and complete light and heavy chain sequences, are listed in Table 8.

[0307] Antibodies that specifically bind to LILRB2 (denoted LILRBXXXX) were generated using an immunorepertoire technology (Rajan et al. Commun. Biol. 2018), which produces naturally paired immune libraries for phage display selection. Humanized Ablexis transgenic mice were immunized with recombinant LILRB2 protein, B cells were enriched, a naturally paired scFv library was generated and screened for LILRB2 selectivity. A lead set of 3 LILRB2 specific IgGs, LILRB0361, LILRB0362 and LILRB0359, was selected based on potency in a primary macrophage stimulation assay, biacore affinity and developability.

[0308] After identifying LILRB0359, 0361 and 0362 as promising lead sequences in functional assays and realizing that these are closely related sequences, we looked more closely at the differences between them and generated a set of hybrid molecules that swap sequence features between the groups. Features studied include:

[0309] • Combinations of different arrangements of heavy and light chain sequences.

[0310] • The sequence at position 52a in H-CDR2 is different in all three antibodies (D, E or N observed) and is different from germline (G). This site proved to be critical for binding.

[0311] • LILRB0362 has an unusual non-germline R in FW4 in the Vernier site.

[0312] • Combining the light chain CDR features of LILRB0361 and LILRB0362.

[0313] • Germlining of the framework of LILRB0359 and LILRB0362. (LILRB0361 is already germlined in its framework regions. Germlining of LILRB0362 VH preserves the unusual Vernier residue in FW4.)

[0314] Note that the light chain and heavy chain vectors are separate, so various LC or HC variant sequences are generated and then co-expressed in different combinations, as shown in Figure 1C .

[0315] These three lead sequences have the same H-CDR3 sequence and do not require further affinity optimization. Amino acid substitutions are made at selected positions in the VH and VL CDRs for germlining and to reduce the risk of potential sequence liabilities. A new set of 29 additional antibody variants (designated LILRB0363-LILRB0406) is generated and screened for specificity, potency, affinity and developability.

[0316] Parental clone LILRB0361 is completely germlined in both VH and VL framework regions, but contains a deamidation-prone risk in H-CDR2 at Kabat position N52a. Germlining of this site with a glycine (N52aG) results in complete loss of potency (observed in LILRB0369, LILRB387, LILRB394, LILRB395, and LILRB402). Mutation to either aspartic acid (i.e., N52aD) (observed in LILRB0362 sequence) or glutamic acid (i.e., N52aE) (observed in LILRB0359) preserves potency, indicating that the residue at position 52a in VH-CDR2 is critical for binding. The substitution of asparagine (Asn) to glutamic acid (Glu) in LILRB0368 (N52aE) mitigates the potential sequence liability of parental LILRB0361. LILRB0368 retains specificity, potency, and has stronger affinity.

[0317] The HCDR 1-3 and LCDR 1-3, VH, VL, heavy chain, and light chain amino acid sequences of the exemplified antibodies described in Example 1 are listed in Table 9.

[0318] 6.1 LILRB0368

[0319] The heavy chain (HC), light chain (LC), VH, VL, HCDR 1-3, and LCDR 1-3 sequences are listed in Table 9. The heavy chain constant region is a human IgGl m(f) allotype with Fc-dampening triple mutations in the CH2 domain. The light chain is a human kappa isotype.

[0320] 6.2 Comparison with germline (Figure 6) Figure 1A and Figure 1B ) :

[0321] The VH sequence matches the IGHV3-23*04 human germline in framework regions 1-3 and IGHJ2*01 in FW4. As with the H-CDR3 sequence, non-germlined sites elsewhere include N31 in H-CDR1 and E52a in H-CDR2. The VL sequence matches the human germline IGKV1-33*01 / IGKV1D-33*01 in FW1-3 and IGKJ2*02 in FW4. Non-germlined sites include D31 in L-CDR1 and Q55 in L-CDR2.

[0322] 7Example 2. Biochemical HTRF assay to determine binding to recombinant biotinylated LILR proteins, LILRB2 haplotypes, and LILRB2 domains 1-2 constructs:

[0323] Recombinant proteins with C-terminal Avi-FLAG tag 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. Assay buffer consisted of phosphate buffered saline (PBS, Invitrogen, 14190) containing 0.4M potassium fluoride (VWR, 26820.236) and 0.1% bovine serum albumin (BSA, Sigma, A9576). 11-point, 1:3 serial dilutions of test and control antibodies were performed in assay buffer and 2.5 μΐ, was added to white low volume 384 well assay plates (Corning, 4513). Positive control for LILRB2 haplotypes and domain constructs was Comparator-M (MAB6364), positive control for LILRA1, LILRA3 and LILRB1 was Comparator-H (HPF1 HuIgG1™) and positive control for LILRA2 was Comparator-R (R&D systems LILRB2 IgG clone 287219). Non-specific binding (NSB) control contained 2.5 μΐ, assay buffer instead of antibody sample. 5 μΐ, of a solution containing 40 nM AF647 labeled anti-human Fc gamma (Jackson ImmunoResearch, 109-605-008) or 40 nM AF647 labeled anti-mouse Fc (Jackson ImmunoResearch, 115-605-164) and 2 nM streptavidin cryptate (Cisbio, 610SAKLB) in assay buffer was added to the assay plates. 2.5 μΐ, of assay buffer containing 4 nM of recombinant biotinylated LILR construct was added to the assay plates. Plates were sealed with optical clear sealant (BioAnalitik, 900510) and incubated for 4 hours at room temperature in the dark. Time resolved fluorescence at 620 nm and 665 nm emission wavelengths was measured using an EnVision plate reader (Perkin Elmer). Data was analyzed by calculating the 665 / 620 nm sample ratio followed by calculating the AF% value for each sample.

[0324]

[0325]

[0326] As shown in FIGS. 2-5, the results indicated that none of the LILRBXXXX mAbs showed off-target binding to LILRB1, LILRA1, LILRA2, or LILRA3. As shown in FIGS. 6-10, all LILRBXXXX mAbs showed binding to the four most prevalent haplotypes of LILRB2, and also bound to the LILRB2 D1-D2 construct. All of the comparator mAbs tested (Comparator-M, Comparator-J, Comparator-B, Comparator-I, and Comparator-N) showed binding to the D1-D2 LILRB2 construct, as well as all four LILRB2 haplotypes. Comparator-N was the only mAb that was not specific for LILRB2, but not LILRB1, LILRA1, and LILRA3.

[0327] Table 1: List of samples, antigens, and associated plate barcodes tested for all HTRF binding assays :

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] 8Example 3. Surface Plasmon Resonance to Determine Affinity of LILRBXXXX and Comparator LILRB2 IgG Panels for Human LILRB2

[0335] Kinetic rate constants (kaand kd) and binding affinity (equilibrium dissociation constant, KD) of biotinylated LILRB2 IgG binding to recombinant human LILRB2 were determined by surface plasmon resonance (SPR) at 25 °C on a Biacore 8K instrument (Cytiva). Minimal amine biotinylated human IgG1 was titrated onto a C1 chip streptavidin protein surface on an 8K Biacore. Dilutions of human LILRB2 H1 haplotype were flowed over the surface at 250 °C. Multi-cycle kinetics (MCK) was performed with 4M MgCl2regeneration.

[0336] Table 2: LILRB2 IgG affinity to human LILRB2 :

[0337]

[0338] Comparator-B contains the variable domains of BMS-986406 (except for a single amino acid difference in FW1 of VH, which can have come from the use of BET standard PCR primers in the transformation); Comparator-I contains the variable domains of IO-108; Comparator-J contains the variable domains of JTX-8064; and Comparator-M contains the variable domains of MK-4860. The heavy chain constant domain is the same sequence as LILRB0368, and for the light chain, either the standard kappa or lambda sequence is used as appropriate; to ensure we have a consistent Fc region format for comparison.

[0339] Table 3: LILRB2 IgG affinity data is consistent with published patent affinities :

[0340]

[0341] Figure 11 shows representative Biacore sensorgrams of IgG binding to human LILRB2 measured by surface plasmon resonance.

[0342] 9Example 4. LILRB2 Fab affinity to human and cynomolgus LILRB2

[0343] Figure 12 shows that LILRB0368 and 3 benchmark LILRB2 IgGs (Comparator-J, Comparator-M, and Comparator-I) do not bind cynomolgus LILRB2. Comparator-B and Comparator-N bind to cynomolgus LILRB2 protein. Kinetic rate constants (kaand kd) and binding affinities (equilibrium dissociation constant, KD= kd / ka) of LILRB2 Fabs binding to recombinant biotinylated human or cynomolgus LILRB variants were determined by surface plasmon resonance (SPR) at 25 °C on a Biacore 8K instrument (Cytiva). The biotinylated LILRB chip surface was activated with LILRB2 Fab titrations (1 nM, 5 nM, 25 nM, and 125 nM) in order to observe binding kinetics and affinities using single cycle kinetics assay (SCK, Karlsson et al., 2006). Figure 13 shows the affinity of LILRB0368 and Comparator-B LILRB2 Fabs to human and cynomolgus LILRB2.

[0344] Table 4: LILRB0368 and comparator-B LILRB2 Fab affinity to human and cynomolgus LILRB2 :

[0345]

[0346] 10Example 5. Flow assay to determine binding to Jurkat cells overexpressing full length LILRB2:

[0347] 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 with live / dead stain (Thermo Fisher Scientific, L34962) for 30 minutes according to the manufacturer’s instructions. Cells were washed twice in phosphate buffered saline (PBS, Invitrogen, 14190) and then resuspended to 2 e6 cells / mL in flow buffer. Six-point, 1 :3 (or 1 :5) serial dilutions of test antibodies were performed in flow buffer and 50 pL added to 96-well polypropylene assay plates (Greiner, 650201). 50 pL of cell suspension was added to the assay plates. Plates were sealed (Perkin Elmer, 6005250) and incubated on ice for 1 hour. Cells were collected by centrifugation (300g, 3 minutes, 4°C), supernatant discarded and cells resuspended in residual buffer by vortexing. Cells were washed once in flow buffer and then 50 pL of 15nM A647-labelled anti-human Fc gamma (Jackson ImmunoResearch, 109-605-008) was added to each well. Plates were incubated on ice for 1 hour in the dark, followed by three washes with flow buffer. 50 pL of CellFix (Becton Dickinson, 340181) was added to each well and plates incubated on ice for 10 minutes in the dark. A further 150 pL of flow buffer was added to each well and plates stored at 4°C. Data was acquired on a MACSQuant 10 (Miltenyi Biotech) flow cytometer using V1 (VioBlue) and R1 (APC) channels.

[0348] As shown in Figures 14A-14E the results indicate that none of the LILRBXXXX tested bound non-specifically to Jurkat-NFAT-Luc parental cells. All samples tested showed robust binding to Jurkat-NFAT-Luc-LILRB2 with low nanomolar / high picomolar IC50.

[0349] Table 5: IC50 values of mAbs for flow binding to Jurkat-NFAT-Luc-LILRB2 :

[0350]

[0351] 11 Example 6. HTRF epitope competition assay against LILRB0362

[0352] Eleven point, 1 :3 serial dilutions of test antibodies were 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 μΐ, of sample was added to white low volume 384 well assay plates (Corning, 4513). Non-specific binding (NSB) controls contained 2.5 μΐ, of 200 nM LILRB0362 IgG instead of antibody sample. Total binding controls contained 2.5 μΐ, of assay buffer instead of antibody sample. 5 μΐ, 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 plates. 2.5 μΐ, of assay buffer containing 10 nM Dylight650 labeled LILRB0362 was added to the assay plates. Plates were sealed with optical clear seals (BioAnalitik, 900510) and incubated at room temperature for 4 hours in the dark. Time resolved fluorescence at 620 nm and 665 nm emission wavelengths was measured using an EnVision plate reader (Perkin Elmer). Data was analyzed by calculating the 665 / 620 nm sample ratio followed by calculating the normalized AF% (percent specific binding) value for each sample.

[0353]

[0354]

[0355]

[0356] As shown in Figures 15A-15D the results indicate that the parental mAbs LILRB0359, LILRB0361 and LILRB0362 share the same epitope and that a combination of the characteristics of these mAbs results in variants that retain this epitope. LILRB0359 is 3-fold less potent than LILRB0361 and LILRB0362. All variants containing a glycine at position CDR2 52a lose activity (LILRB0369, LILRB0387, LILRB0394, LILRB0395, LILRB0402).

[0357] Table 6: IC50 values of mAbs in LILRB0362 epitope competition assay, and correlation with residues at position CDR2 52a Figures 16A-16D :

[0358]

[0359] 12Example 7. Activation of Jurkat-NFAT-Luc-: LILRB2 and Raji-HLA-G co-culture reporter assay to determine inhibition of LILRB2 signaling

[0360] 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 media consisting of RPMI media (Gibco, 61870-010) containing 10% fetal bovine serum (FBS, Gibco, 01190-015) to remove trace amounts of selection antibiotics. Cells were resuspended to 3.33e5 cells / mL in assay media. 30 pL 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 Raji-HLA-G cells were added to all test and stimulated control wells. 30 pL assay media was added to unstimulated control wells. An 8-point, 1:4 serial dilution of test antibodies was performed in assay media and 30 pL was added to test wells. 30 pL assay media was added to stimulated and unstimulated control wells. The plate was briefly centrifuged (300g, 30 seconds, room temperature) and then incubated in a humidified incubator set at 37°C, 5% CO2for 16 hours. 85 pL SteadyGlo reagent (Promega, E2520) was added to each well of the cell plate and incubated at room temperature in the dark for 5 minutes. Samples were mixed by pipette and transferred to a white 96-well assay plate (Corning, 3917) and incubated at room temperature in the dark for an additional 30 minutes. Luminescence was measured on an EnVision plate reader (PerkinElmer) at 0.1 second read.

[0361] As shown in Table 7: IC50 values of mAbs tested in co-culture reporter assay, observing signaling between Jurkat-NFAT-Luc- LILRB2(ECD)-CD3z-CD137 reporter cells and Raji-HLA-G target cells , the results indicate that all of the tested LILRBXXXX mAbs that showed activity in the LILRB0362 epitope competition assay also showed functional inhibition of LILRB2-HLA-G signaling in the co-culture reporter assay.

[0362] Figure 17A Figure 17B :

[0363]

[0364]

[0365] 13Example 8. Testing of binding to selected LILR family receptors by BioLayer Interferometry (OCTET assay):

[0366] Prior to loading, streptavidin (Sartorius, 18-5118) and Ni-NTA (Sartorius, 18-5102) biosensors were rehydrated in assay buffer consisting of phosphate buffered saline (PBS, Invitrogen, 14190) with 0.02% Tween20 (Thermo Fisher Scientific, 85113) and 0.1% bovine serum albumin (BSA, Sigma, A9576) for at least 10 minutes. 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 to 5 µg / mL - 10 µg / mL in assay buffer and loaded onto the appropriate biosensor. Seven-point, 1:3 serial dilutions of test and control antibodies were 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 seconds), antigen loading (antigen, 300 seconds), baseline (assay buffer, 60 seconds), sample association (antibody, 60 seconds), sample dissociation (assay buffer, 180 seconds); all steps were performed at 25 °C.

[0367] As shown in Figures 18A-18J and Figure 19 , the results indicate that none of the LILRBXXXX mAbs tested showed off-target binding to the relevant 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.

[0368] 14 Example 9. Mirrorball assay to assess non-specific binding to HEK293 cells:

[0369] Frozen HEK293 cells (ECACC) were quickly thawed, added to 10 mL assay medium of Hank’s Balanced Salt Solution (HBSS, Sigma, H8264) containing 0.5% Bovine Serum Albumin (BSA, Sigma, A9576) and collected by centrifugation (300 g, 5 min, room temperature). Cells were resuspended to 2.5e5 cells / mL in assay buffer. An 8-point, 1 :5 serial dilution of test and control antibodies was performed in assay buffer and 10 pL was added in duplicate to black 384-well 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 a second set of assay plates to assess IgG aggregation. Plates were sealed (Perkin Elmer, 6005250) and incubated for 2 hours at room temperature in the dark. Plates were read using a Mirrorball plate reader (TTP Labtech) in the FL3 channel (640 nm) at 4 pm resolution and 500 V. Signals were gated to remove low intensity background fluorescence based on buffer only controls and high intensity fluorescence due to aggregation.

[0370] As shown in Figure 20 the results show that neither the LILRBXXXX nor the comparator mAb tested showed non-specific binding to HEK293 cells and they also did not show a propensity to aggregate.

[0371] 15 Example 10. AC-SINS (Affinity Capture Self-Interaction Nanoparticle Spectroscopy) assay to assess propensity to self-associate:

[0372] The 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.

[0373] Nine mL of gold nanoparticles (Abeam, ab269935) were coated with a 1 :4 mix of 400 pg of intact goat IgG (Jackson ImmunoResearch, 005-000-003) and goat anti-human Fc gamma (Jackson ImmunoResearch, 109-005-098) in binding buffer (20 mM potassium acetate, pH 4.3) for 1-16 hours at room temperature. Nanoparticles were blocked with 100 nM PEG (Sigma, 729140) for 1-2 hours at room temperature, collected by centrifugation (13,000 rpm, 6 minutes, room temperature), and resuspended in 800 pL of binding buffer. A 1 :10 dilution of the prepared nanoparticles should have an OD at 535 nm of 0.4-0.5 AU. Test antibodies were 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). Twelve pL of prepared gold nanoparticles were added to each well, mixed by pipetting, and the samples were incubated for 20 minutes at room temperature. Samples were transferred in duplicate to Nunc 384-well assay plates (Thermo Fisher Scientific, 242757) and absorbance values from 450 nm to 700 nm were detected using a PHERAstar plate reader (BMG Labtech). The wavelength at the maximum absorbance was determined using MARS software (BMG Labtech), and the red shift relative to the buffer control was calculated; a red shift >10 nm indicates self-association.

[0374] As Figures 21A-21C indicated in the results, none of the LILRBXXXX mAbs tested had a risk of self-association. Comparator-J had a risk of self-association in the charged buffer (HA), and comparator-B had a risk of self-association in both the neutral buffer (PBS) and the charged buffer.

[0375] 16Example 11. Assessment of baculovirus particle ELISA for risk of rapid in vivo clearance:

[0376] This method was adapted from the following source: Hotzel et al., mAbs, 2012, 4(6), 753-60.

[0377] Nunc maxisorp F plates (Thermo Fisher Scientific, 44-2404-21) were coated overnight at 4°C with 50 µL of baculovirus particles at 1e8 particles / mL in sodium carbonate buffer (50 mM, pH 9.6), or with 50 µL of sodium carbonate buffer (50 mM, pH 9.6) only. The assay plates were washed once with phosphate buffered saline (PBS) and then blocked for 1 hour at room temperature with 300 µL of assay buffer consisting of PBS (Invitrogen, 14190) containing 0.5% bovine serum albumin (BSA, Sigma, A9576). The plates were washed three times with PBS. Test antibodies were diluted to 10 nM and 100 nM in assay buffer, and 50 µL of sample was added to the assay plates and incubated for 1 hour at room temperature. The plates were washed three times with PBS and then incubated with 50 µL of HRP-conjugated anti-human Fc gamma solution (1:5000 dilution, Sigma, A0170) for 30 minutes at room temperature. The plates were washed three times with PBS, incubated with 50 µL of TMB SureBlue Reserve (KPL, 53-00-03) for 2-5 minutes, and quenched with 50 µL of 0.5 M sulfuric acid (H2SO4). The absorbance at 450 nm was measured on an EnVision plate reader (Perkin Elmer). BV scores were determined by averaging the absorbance values for the 10 nM and 100 nM samples and dividing by the buffer-only control: BV scores above 5 indicated a risk of rapid in vivo clearance.

[0378] As shown in Figure 21A the results showed that neither the LILRBXXXX nor the comparator mAb tested showed a risk of rapid clearance due to non-specific binding to baculovirus particles.

[0379] 17Example 12. Macrophage stimulation assay:

[0380] This in vitro assay was performed to assess the ability of LILRB2 antibodies to enhance the pro-inflammatory response of macrophages from immune stimulation, as measured by the release of the pro-inflammatory cytokine TNFa into the culture supernatant.

[0381] Monocytes were isolated from human PBMC using CD14+ negative selection kit (Stemcell #19058) and cultured in RPMI 1640 + Glutamax (Invitrogen, 61870), 10% HIFBS (ThermoFisher, 10082147), Penicillin / Streptomycin (Invitrogen, #15140122) at 370C, 5% C02 in Corning ® T175 flasks (Corning, #431080) supplemented with 100 ng / ml recombinant human M-CSF (Peprotech, #300-25) for 6 days.

[0382] Macrophages were isolated from culture flasks using STEMPRO ACCUTASE (ThermoFisher, #A1110501), washed in PBS and resuspended at 2e5 / ml in RPMI1640 + Glutamax (Invitrogen, 61870), 10% HIFBS (ThermoFisher, 10082147), Penicillin / Streptomycin (Invitrogen, #15140122) supplemented with 100 ng / ml recombinant human M-CSF (Peprotech, #300-25). Cells were seeded at 20,000 cells / well in CORNING sterile flat bottom 96-well plates (Corning, #10695951) and incubated overnight at 37C, 5% C02.

[0383] Antibody solutions were prepared at a range of concentrations, added to the plates and incubated for 30 minutes at room temperature. Cells were stimulated using 500 ng / ml MEGACD40L ® (Enzo, #ALX-522-110-C010) and incubated overnight at 37C, 5% C02. Supernatants were collected and TNFa protein levels assessed by ELISA (R&D Systems, #DY210). Results were analyzed as follows: each value from a given donor was normalized in Graphpad, whereby the highest mean value obtained for this donor after treatment with anti-LILRB2 mAb LILRB0361 was defined as 100% and zero as 0%. These normalized values were then combined across all donors. The number of donors evaluated is provided in the figure. Dose response curves were generated using a log(agonsist) vs response - variable slope (four parameters) nonlinear regression model in Graphpad.

[0384] Blockade of LILRB2 on CD40L-stimulated macrophages strongly enhanced the release of the pro-inflammatory cytokine TNFa. All of our LILRBXXXX mAb clones screened against CD40L-stimulated macrophages increased TNFa release (as shown in Figure 21C Table 2), although some variation in potency was observed (as shown in Table 8 IC50 values of LILRB0368 and comparator mAbs in macrophage stimulation assay LILRB0368 was found to be more potent in this assay compared to the comparator tested ( Figures 22A to 22C ).

[0385] Figures 23A-23B :

[0386]

[0387] In a separate experiment, blockade of LILRB2 on CD40L-stimulated macrophages using LILRB0368 enhanced the release of the pro-inflammatory cytokines GM-CSF and TNFa, while reducing the production of the pro-angiogenic cytokine VEGF-A, compared to isotype control (as shown in Figure 24

[0388] 18Example 13. CD163 and CD206 expression assay:

[0389] This in vitro assay was performed to assess whether monocytes treated with LILRB0368 differentiated into macrophages with an enhanced pro-inflammatory phenotype, as determined by a reduction in cell surface expression of the inhibitory M2 markers CD163 and CD206.

[0390] ​Monocytes were isolated from human PBMCs using CD14+ negative selection kit (Stemcell #19058) and cultured in the presence of 50nM of our exemplary anti-LILRB2 mAb clone, LILRB0368, or isotype control antibody in culture medium [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)] at 37°C, 5% CO2 in flat bottom 96 well tissue culture plates (Corning, #3527) for 7 days. Resulting macrophages were detached from culture plates using STEMPRO ACCUTASE (ThermoFisher, #A1110501), transferred to 96 well v-bottom plates (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. Staining solution was then removed and cells were washed once in ROBOSEP buffer. Cells were fixed following incubation in Cytofix solution (BD Biosciences #554655) for 10 minutes, pelleted and resuspended in PBS. Cells were then acquired on a Fortessa flow cytometer (BD Biosciences) and analyzed for expression levels of CD163 and CD206 on macrophages using Flowjo v10 software package (BD Biosciences).

[0391] Figure 25A Results of CD163 and CD206 expression assay are shown. Expression of CD163 and CD206 on differentiated monocyte-derived macrophages was downregulated in the presence of 50nM LILRB0368.

[0392] 19Example 14. Tumor growth assay

[0393] NSG / SGM3 mice reconstituted with human CD34+ cord blood stem cells were implanted s.c. in their right posterior flank with 1 x 10e6 MDA-MB-231 tumor cells. Mice were randomized into two groups of four mice each based on tumor size 14 days after tumor implantation. One group was treated with 10 mg / kg LILRB0361 and the other group was treated with 10 mg / kg isotype control antibody. Both test articles were co-administered with 20 mg / kg anti-mouse CD16 / CD32 (BioXcell, FcBlock) via intraperitoneal injection twice weekly for 3 weeks. Tumor volume was measured twice weekly during the study and the mean tumor volume (mm3) and standard deviation over time are plotted in FIG. 23. 3

[0394] Figure 25B Results from the tumor growth assay are shown. Anti-LILRB2 mAb clone LILRB0361 reduced the rate of tumor growth in vivo. NSG / SGM3 mice reconstituted with human CD34+ cord blood stem cells were implanted s.c. with MDA-MB-231 tumor xenografts. Mice were treated twice weekly for 3 weeks with 10 mg / kg LILRB0361 (and isotype control).

[0395] 20Example 14. Combination therapy with T cell engagers

[0396] Results from anti-OKT3-EGFR T cell engagers (TCEs) are shown in FIG. 25. T cells from eight donors were co-cultured with tumor cells and autologous macrophages. The addition of macrophages to the co-culture significantly reduced the level of tumor killing by T cells stimulated with OKT3-EGFR T cell engagers from an average of 31.8% to 8.1% ( Figure 26 ). This reduction demonstrates the inhibitory effect of macrophages on T cell effector function. In the presence of these suppressive macrophages, anti-LILRB2 antibody increased TCE-driven tumor killing from 8.1% to 21.8%, indicating a partial restoration of killing activity. In parallel, the addition of AZD2796 to the co-culture significantly enhanced the expression of the activation marker CD86 on macrophages in the presence of T cells activated by T cell engagers ( Figure 26 ).

[0397] These data show that macrophages can effectively inhibit T cell engager directed tumor killing in an in vitro co-culture assay. Anti-LILRB2 antibody significantly enhanced tumor killing induced by T cell engagers in the presence of suppressive macrophages. This increase in killing was accompanied by repolarization of macrophages, indicated by an increase in expression of the pro-inflammatory macrophage marker CD86.

[0398] ​21 Example 14. Combination therapy with CAR-T cells

[0399] Results for HER2 CAR-T cells are shown in Table 9: Amino acid sequences of antibodies described in this specification where CAR-T cells (derived from one donor) were co-cultured with tumor cells from 6 different non-autologous donors (2way) or tumor cells and macrophages (3way). The addition of macrophages to the co-culture significantly reduced the level of tumor killing by HER2-CAR T cells from an average of 59.8% to 14.3% ( Table 10: Amino acid sequences of comparator antibodies described in this specification ). This reduction demonstrates the inhibitory effect of macrophages on CAR-T cell effector function. In the presence of these suppressive macrophages, anti-LILRB2 antibody increased CAR-T driven tumor killing from 14.3% to 34.7%, indicating a partial restoration of killing activity.

[0400] These data show that macrophages can effectively inhibit CAR-T directed tumor killing in an in vitro co-culture assay. In the presence of suppressive macrophages, the addition of anti-LILRB2 antibody significantly enhanced tumor killing induced CAR-T cells.

[0401] 22 Sequences

[0402] ​ .

[0403] All heavy chains, light chains, VH domains, VL domains, CDRs, antibodies comprising them, and their encoding nucleic acids represent examples of the present disclosure. CDRs are determined according to the Kabat method.

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423] ​ 。

[0424]

[0425]

[0426]

[0427]

Claims

1. A LILRB2 binding protein that binds to human LILRB2 with an affinity (KD) of < 100 pM.

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

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

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

5. The LILRB2 binding protein of 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 of claim 5, wherein the myeloid cells are macrophages.

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

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

9. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein induces macrophages to polarize from a tumor-supportive M2 state to a tumor- inhibitory Ml state, characterized by CD163 cell surface expression is reduced and CD86 cell surface expression and TNFa production is increased (e.g., as measured in a macrophage, T cell, tumor cell co-culture assay using antigen specific T cells).

10. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein promotes a pro-inflammatory state in the tumor microenvironment, resulting in increased T cell lysis of tumor cell lines (e.g., as measured in a macrophage, T cell, tumor cell co-culture assay using antigen specific T cells).

11. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein reduces tumor volume (e.g., as measured in vivo).

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

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

14. The LILRB2 binding protein of 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 of any preceding claim, wherein the LILRB2 is human LILRB2.

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

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

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

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

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

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

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

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

27. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein comprises a variable heavy (VH) domain sequence of SEQ ID NO: 3 optionally having 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 having 1, 2, 3, 4, or 5 amino acid alterations outside the CDRs.

28. The LILRB2 binding protein of 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 the variable light (VL) domain sequence comprise a sequence that is at least 90% identical to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, respectively.

29. The LILRB2 binding protein of 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 the variable light (VL) domain sequence comprise a sequence that is at least 90% identical to the variable heavy (VH) sequence of SEQ ID NO: 3 and the variable light (VL) sequence of SEQ ID NO: 4, respectively, 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 of 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 of any preceding claim, wherein the LILRB2 binding protein comprises an Fc domain.

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

33. The antibody of claim 32, wherein the Fc domain comprises mutations L234F, L235E, and P331S.

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

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

36. The antibody of claim 31, wherein the Fc domain comprises at least one mutation that confers extended half-life.

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

38. The antibody of claim 31, wherein the Fc domain has reduced effector function and comprises at least one mutation that confers extended half-life.

39. The antibody of claim 38, wherein the Fc domain comprises mutations L234F, L235E, P331S, M252Y, S254T, and T256E.

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

41. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein comprises a kappa light chain.

42. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein comprises a light chain comprising the sequence SEQ ID NO:

2.

43. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein comprises a heavy chain comprising the sequence SEQ ID NO:

1.

44. The LILRB2 binding protein of any preceding claim, wherein the LILRB2 binding protein comprises a light chain comprising the sequence SEQ ID NO: 2 and a heavy chain comprising the sequence SEQ ID NO:

1.

45. A polypeptide comprising one or more domains of the LILRB2 binding protein of any one of claims 1 to 44.

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

47. A polypeptide comprising one or more chains of the LILRB2 binding protein of any one of claims 1 to 44.

48. A nucleic acid encoding one or more chains of the LILRB2 binding protein of any one of claims 1 to 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 the LILRB2 binding protein of any one of claims 1 to 44, and a pharmaceutically acceptable carrier.

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

53. The LILRB2 binding protein of any one of claims 1 to 44 or the pharmaceutical composition of claim 51 for use in therapy.

54. The LILRB2 binding protein of any one of claims 1 to 44 or the pharmaceutical composition of 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, kidney 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 kidney 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 to a subject in need thereof a LILRB2 binding protein of any one of claims 1 to 44 or a pharmaceutical composition of claim 50.

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

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

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

63. The method of any one of claims 59 to 61, wherein the cancer is kidney cancer.

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

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

66. Use of a LILRB2 binding protein of any one of claims 1 to 44 or a pharmaceutical composition of claim 51 for the manufacture of a medicament for treating cancer.

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

68. The use of claim 66, wherein the cancer is kidney cancer.

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

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

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