Single domain antibody targeting LILRB2

Abstract

The present invention relates to single domain antibodies (sdAbs) against leukocyte immunoglobulin-like receptor subfamily B, member 2 (LILRB2), pharmaceutical compositions comprising the same, and uses thereof in diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy and immunodiagnosis. The present invention provides a single domain antibody (sdAb) against leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2). Technical Background

[0002] Leukocyte immunoglobulin (Ig)-like receptors (LILBRs) are inhibitory receptors whose cytoplasmic tails consist of an ITIM (immunoreceptor tyrosine-based inhibitory motif). While LILRB1 is expressed on all immune cell subsets, LILRB2 expression is restricted to antigen-presenting cells (APCs), such as monocytes, dendritic cells, and macrophages.

[0003] LILRB2 interacts with CD1d, several molecules in the complement cascade (C4d, C3d, C4b, C3b and iC3b), angiopoietin-like 2 and 5 (ANGPTL2 / 5) proteins, B-amyloid 1-42 and myelin-derived inhibitor (Nogo66, MAG) and with classical (HLA-A, -B and -C) or non-classical MHC class I molecules (HLA-E, F and G). In particular, it has been demonstrated that the interaction between LILRB2 expressed on immune cells and HLA-G inhibits cell function and is able to induce immunosuppressive cells. In fact, the interaction between HLA-G present in dendritic cells (DCs) and LILRB2 inhibits their maturation and makes them tolerant.

[0004] Interestingly, the LILRB2 receptor was shown to be expressed in several types of cancer and is frequently associated with metastasis. Although LILRB2 is an inhibitory receptor, its expression in tumors was shown to increase the proliferation and motility of tumor cells. In fact, upon binding to HLA-G or ANGPTL2, the LILRB2 receptor inhibits pathways that curb tumor cell proliferation, growth and dissemination. Notably, LILRB2 is expressed by tumor-associated macrophages (TAMs), especially in the case of solid tumors. These macrophages display an M2 phenotype associated with functions that inhibit immune cell infiltration and favor cancer cell proliferation. As LILRB2 receptor expression is restricted to APCs in healthy individuals, its novel expression in tumors and its strong upregulation by tolerogenic DCs and TAMs make the LILRB2 receptor an excellent tumor-associated antigen (TAA) to target with immunotherapy.

[0005] However, to date, there are no effective immunotherapeutic agents that are able to block LILRB2. The generation of blocking anti-LILRB2 monoclonal antibodies (mAbs) would pave the way for new immunotherapies. However, the large size of mAbs (approximately 150 kDa) is a major disadvantage, as it inhibits their tumor penetration and thus limits their use in solid cancers, which remain the most difficult cancers to treat. Therefore, there remains a great need in the art for new and improved agents that target such cancers.

[0006] Members of the Camelidae family naturally produce different classes of antibodies: (i) conventional heavy chain antibodies containing two light chains and two heavy chains (about 150 kDa), (ii) homodimeric heavy chain antibodies (HcAb; about 95 kDa) comprising only the H chain, and (iii) other IgG isotypes based on unique heavy chains. These heavy chain-only antibodies have been shown to have high binding affinity and specificity for their antigens, similar to traditional mAbs.

[0007] The heavy chain variable domain from HcAb (i.e., single domain antibody (sdAb) or (Nb)) are responsible for antigen binding and specificity and can be separated from HcAb without losing their binding properties. Their smaller size, usually around 15-20 kDa, is an important advantage when targeting solid tumors. In fact, they should be able to penetrate the fibrous microenvironment surrounding cancer cells more effectively and reach target cells such as macrophages that reside in this matrix. Then, sdAbs are excellent candidates in terms of targeting the LILRB2 receptor displayed on solid tumors and TAMs.

[0008] The present inventors have now made a significant technical contribution to the art in developing anti-LILRB2 single domain antibodies (sdAbs). Summary of the invention

[0009] The present invention relates to a single domain antibody (sdAb) that specifically binds to or specifically recognizes leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), preferably human LILRB2.

[0010] Preferably, the sdAb anti-LILRB2 does not bind to leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), preferably human LILRB1.

[0011] In one aspect, the sdAb according to the invention comprises at least one complementarity determining region (CDR) comprising or consisting of the sequence shown in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33, or comprising or consisting of an amino acid sequence that differs from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33 due to one, two or three amino acid modifications.

[0012] Preferably, the sdAb according to the invention comprises three CDRs, wherein:

[0013] (a) CDR1 comprises or has SEQ ID NO: 1, or has an amino acid sequence that differs from SEQ ID NO: 1 due to one, two or three amino acid modifications, and

[0014] CDR2 comprises or has SEQ ID NO: 2, or has an amino acid sequence that differs from SEQ ID NO: 2 due to one, two or three amino acid modifications, and

[0015] CDR3 comprises or has SEQ ID NO: 3, or has an amino acid sequence that differs from SEQ ID NO: 3 due to one, two, three or four amino acid modifications; or

[0016] (b) CDR1 comprises or has SEQ ID NO: 4, or has an amino acid sequence that differs from SEQ ID NO: 4 due to one, two or three amino acid modifications, and

[0017] CDR2 comprises or has SEQ ID NO: 5, or has an amino acid sequence that differs from SEQ ID NO: 5 due to one, two or three amino acid modifications, and

[0018] CDR3 comprises or has SEQ ID NO: 6, or has an amino acid sequence that differs from SEQ ID NO: 6 due to one, two, three or four amino acid modifications; or

[0019] (c) CDR1 comprises or has SEQ ID NO: 7, or has an amino acid sequence that differs from SEQ ID NO: 7 due to one, two or three amino acid modifications, and

[0020] CDR2 comprises or has SEQ ID NO: 8, or has an amino acid sequence that differs from SEQ ID NO: 8 due to one, two or three amino acid modifications, and

[0021] CDR3 comprises or has SEQ ID NO: 9, or has an amino acid sequence that differs from SEQ ID NO: 9 due to one, two, three or four amino acid modifications; or

[0022] (d) CDR1 comprises or has SEQ ID NO: 10, or has an amino acid sequence that differs from SEQ ID NO: 10 due to one, two or three amino acid modifications, and

[0023] CDR2 comprises or has SEQ ID NO: 11, or has an amino acid sequence that differs from SEQ ID NO: 11 due to one, two or three amino acid modifications, and

[0024] CDR3 comprises or has SEQ ID NO: 12, or has an amino acid sequence that differs from SEQ ID NO: 12 due to one, two, three or four amino acid modifications; or

[0025] (e) CDR1 comprises or has SEQ ID NO: 13, or has an amino acid sequence that differs from SEQ ID NO: 13 due to one, two or three amino acid modifications, and

[0026] CDR2 comprises or has SEQ ID NO: 14, or has an amino acid sequence that differs from SEQ ID NO: 14 due to one, two or three amino acid modifications, and

[0027] CDR3 comprises or has SEQ ID NO: 15, or has an amino acid sequence that differs from SEQ ID NO: 15 due to one, two, three or four amino acid modifications; or

[0028] (f) CDR1 comprises or has SEQ ID NO: 16, or has an amino acid sequence that differs from SEQ ID NO: 16 due to one, two or three amino acid modifications, and

[0029] CDR2 comprises or has SEQ ID NO: 17, or has an amino acid sequence that differs from SEQ ID NO: 17 due to one, two or three amino acid modifications, and

[0030] CDR3 comprises or has SEQ ID NO: 18, or has an amino acid sequence that differs from SEQ ID NO: 18 due to one, two, three or four amino acid modifications; or

[0031] (g) CDR1 comprises or has SEQ ID NO: 19, or has an amino acid sequence that differs from SEQ ID NO: 19 due to one, two or three amino acid modifications, and

[0032] CDR2 comprises or has SEQ ID NO: 20, or has an amino acid sequence that differs from SEQ ID NO: 20 due to one, two or three amino acid modifications, and

[0033] CDR3 comprises or has SEQ ID NO: 21, or has an amino acid sequence that differs from SEQ ID NO: 21 due to one, two, three or four amino acid modifications; or

[0034] (h) CDR1 comprises or has SEQ ID NO: 22, or has an amino acid sequence that differs from SEQ ID NO: 22 due to one, two or three amino acid modifications, and

[0035] CDR2 comprises or has SEQ ID NO: 23, or has an amino acid sequence that differs from SEQ ID NO: 23 due to one, two or three amino acid modifications, and

[0036] CDR3 comprises or has SEQ ID NO: 24, or has an amino acid sequence that differs from SEQ ID NO: 24 due to one, two, three or four amino acid modifications; or

[0037] (i) CDR1 comprises or has SEQ ID NO: 25, or has an amino acid sequence that differs from SEQ ID NO: 25 due to one, two or three amino acid modifications, and

[0038] CDR2 comprises or has SEQ ID NO: 26, or has an amino acid sequence that differs from SEQ ID NO: 26 due to one, two or three amino acid modifications, and

[0039] CDR3 comprises or has SEQ ID NO: 27, or has an amino acid sequence that differs from SEQ ID NO: 27 due to one, two, three or four amino acid modifications; or

[0040] (j) CDR1 comprises or has SEQ ID NO: 28, or has an amino acid sequence that differs from SEQ ID NO: 28 due to one, two or three amino acid modifications, and

[0041] CDR2 comprises or has SEQ ID NO: 29, or has an amino acid sequence that differs from SEQ ID NO: 29 due to one, two or three amino acid modifications, and

[0042] CDR3 comprises or has SEQ ID NO: 30, or has an amino acid sequence that differs from SEQ ID NO: 30 due to one, two, three or four amino acid modifications; or

[0043] (k) CDR1 comprises or has SEQ ID NO: 31, or has an amino acid sequence that differs from SEQ ID NO: 31 due to one, two or three amino acid modifications, and

[0044] CDR2 comprises or has SEQ ID NO: 32, or has an amino acid sequence that differs from SEQ ID NO: 32 due to one, two or three amino acid modifications, and

[0045] CDR3 comprises or has SEQ ID NO: 33, or has an amino acid sequence that differs from SEQ ID NO: 33 due to one, two, three or four amino acid modifications.

[0046] In particular, the anti-LILRB2 sdAb comprises three CDRs, wherein CDR1 comprises or has SEQ ID NO:1, CDR2 comprises or has SEQ ID NO:2, and CDR3 comprises or has SEQ ID NO:3.

[0047] In a specific aspect, the sdAb anti-LILRB2 comprises or consists of a sequence defined in any one of SEQ ID No:34 to SEQ ID No:44, or a sequence having at least 80% sequence identity thereto, preferably a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity thereto.

[0048] In particular, the anti-LILRB2 sdAb comprises or consists of the sequence defined in SEQ ID No:34.

[0049] Preferably, the sdAb anti-LILRB2 according to the invention inhibits the interaction between LILRB2 and human leukocyte antigen-G (HLA-G) and / or the interaction between LILRB2 and angiopoietin-like 2 (ANGPTL2).

[0050] In another aspect, the present invention relates to an isolated nucleic acid comprising a sequence encoding a sdAb anti-LILRB2 according to the present invention, preferably defined by a sequence selected from the group consisting of SEQ ID: 45-55.

[0051] The present invention further relates to a vector comprising the isolated nucleic acid according to the present invention and also to a chimeric antigen receptor (CAR) comprising the sdAb or the isolated nucleic acid according to the present invention.

[0052] In a specific aspect, the present invention relates to a cell comprising an isolated nucleic acid or vector according to the present invention or expressing a CAR disclosed herein. Preferably, the cell is selected from the group consisting of: T cells, CD4 + T cells, CD8 + T cells, B cells, NK cells, NKT cells, monocytes and dendritic cells. Preferably, the cells are T cells, B cells or NK cells.

[0053] The present invention also relates to a pharmaceutical composition comprising an sdAb, an isolated nucleic acid, a vector, a CAR or a cell expressing a CAR according to the present invention, and optionally a pharmaceutically acceptable carrier.

[0054] In one aspect, the sdAb, isolated nucleic acid, vector, CAR, cell or pharmaceutical composition according to the present invention is used to treat cancer, preferably wherein the cancer overexpresses LILBR2, more preferably the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, pancreatic ductal carcinoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), endometrial cancer, hepatocellular carcinoma, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, kidney cancer, testicular cancer, esophageal cancer, cervical cancer, mouse Lewis lung carcinoma, leukemia, thyroid cancer, liver cancer, urothelial carcinoma and head and neck cancer.

[0055] The present invention finally relates to the use of an sdAb anti-LILRB2 according to the present invention for detecting LILRB2 on tumor cells or tissues in vitro or ex vivo.

[0056] Attached photos

[0057] Figure 1 Alpaca immunization and VHH specificity characterization. A) Alpaca immunization scheme using rhLILRB2-Fc protein. B) Sera from immunized alpacas were tested in ELISA at different dilutions. C) VHHs were selected using phage display vectors and biopanning techniques and evaluated against rhLILRB2-Fc. Positive anti-LILRB2 VHHs are circled with solid circles, while negative ones are circled with dashed circles.

[0058] Figure 2.B8, C7 and C9 Nb recognize the linear epitope of rhLILRB2. Denatured rhLILRB2-Fc (rhILT4-Fc), rhLILRB2 (rhILT4) and rhLILRB1 (rhILT2) proteins were transferred to membranes by Western blotting. A) rhLILRB2-Fc, rhLILRB2 and rhLILRB1 proteins were incubated with control anti-LILRB2 Ab (H-300 and 42D1), control anti-LILRB1 (GHI / 75 and HP-F1). B) rhLILRB2-Fc, rhLILRB2 and rhLILRB1 proteins were incubated with B8, C7 and C9 Nb. Ab binding was detected using HRP-labeled goat anti-rat Ab against H-300 and HRP-labeled goat anti-mouse Ab against 42D1, GHI / 75 and HP-F1, and HRP-labeled mouse anti-c-Myc tag Nb.

[0059] Figure 3 .Binding of Nb(B8, C7, C9) to rhILT4 variant (D1-D4 domains) and absence or binding of Nb(B8, C7, C9) to rhILT2 variant (D1-D4).

[0060] Figure 4 Nb specificity of the LILRB2-transduced D1.1 cell line. LILRB2 D1.1 cell line was co-cultured with 42D1 control antibody or anti-LILRB2 Nb and analyzed by flow cytometry.

[0061] Figure 5 .Nb specificity for LILRB2 receptor on PBMC monocytes. Monocytes were isolated from PBMCs and then stained for LILRB2 receptor expression with 42D1 control antibody and Nb, compared to an irrelevant control Nb, and analyzed by flow cytometry. Monocytes were identified from other leukocytes using anti-CD14 and anti-LILRB1 Abs.

[0062] Figure 6 . Blocking ability of anti-LILRB2 Nbs on LILRB2 / HLA-G6 interaction. As shown in the study design diagram (top right), microtiter plates were coated with rhLILRB2-Fc protein before co-incubation with individual Nbs. HLA-G6 V5 labeled protein was then added, and HLA-G6-V5 protein was detected using HRP-conjugated anti-V5 Ab. Values ​​were normalized to the mean absorbance intensity of the negative control (rhLILRB2-Fc incubated with HLA-G6-V5 protein alone in the absence of Nbs or control Ab) (n=3).

[0063] Figure 7Blocking ability of anti-LILRB2 Nbs on LILRB2 / ANGPTL2 interaction. As shown in the study design diagram (top right), microtiter plates were coated with rhLILRB2-Fc protein prior to co-incubation with individual Nbs. ANGPTL2 protein was then added and detection of ANGPTL2 was performed using anti-ANGPTL2 purified Ab followed by HRP-conjugated anti-rabbit Ab. Values ​​were normalized to the mean absorbance intensity (MFI) of the negative control (rhLILRB2-Fc alone incubated with ANGPLT2 protein in the absence of Nbs or control Ab (n=1)). DETAILED DESCRIPTION OF THE INVENTION

[0065] ■Definition

[0066] As used herein, "leukocyte immunoglobulin-like receptor subfamily B member 2" or "LILRB2" refers to a member of the leukocyte immunoglobulin-like receptor (LIR) family, particularly a member of the LIR receptor subfamily B class that contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB2 is expressed on immune cells where it binds to MHC class I molecules on antigen-presenting cells and transduces negative signals that inhibit immune response stimulation. It is thought to control inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity. LILRB2 has known alternative names, such as LIR2, CD85 antigen-like family member D, CD85D, immunoglobulin-like transcript 4, ILT4, monocyte / macrophage immunoglobulin-like receptor 10, or MIR-10. In the context of the present invention, this term particularly refers to human LILRB2. Human LILRB2 is known in the art, for example under UniProt accession number Q8N423. For example, the human LILRB2 amino acid sequence is approximately 598 amino acids, and the gene is located in a cluster in chromosome region 19q13.4. Human LILRB2 has four known isoforms produced by alternative splicing. Isoform 1 has been selected as the canonical sequence and is described under accession number Uniprot Q8N423-1, isoform 2 differs from isoform 1 by the deletion of the amino acid at position 437 and is described under accession number Uniprot Q8N423-2, isoform 3 differs from isoform 1 by the deletion of the amino acids at positions 495-510 and 511-598 and is described under accession number Uniprot Q8N423-3, isoform 4 differs from isoform 1 by the deletion of the amino acids at positions 1-116 and is described under accession number Uniprot Q8N423-4. In the context of the present invention, the term "LILRB2" includes all isoforms of LILRB2.

[0067] As used herein, "heavy chain antibody" (HCAb) refers to an immunoglobulin that does not contain a light chain and is composed of two heavy chains. Each heavy chain includes a constant region (CH) and a variable region (VH), which enables binding to a specific antigen, epitope or ligand. As used herein, HCAb includes camelid type heavy chain antibodies, wherein each heavy chain includes a variable domain called VHH and two constant domains (CH2 and CH3). It is worth noting that camelid HCAb lacks the first constant domain (CH1). Such heavy chain antibodies for specific antigens can be obtained from immunized camelids. As used herein, "camelids" include dromedaries, camels, llamas (lama) and alpacas. Hamers-Casterman et al., Nature, 1993, 363:446 describes camelid HCAb. Other examples of HCAbs are immunoglobulin-like structures (Ig-NARs) from cartilaginous fishes, such as the nurse shark (Ginglymostoma cirratum) and the wobbegong shark (Orectolobus maculates).

[0068] As used herein, "single domain antibody" (sdAb or Nb) refers to a single variable domain derived from a heavy chain antibody alone, which is capable of binding to an antigen, epitope or ligand alone, that is, without the need for another binding domain. Single domain antibodies can be derived from VHH or V-NAR or consist of VHH or V-NAR. VHH refers to the variable domains found in HCAb of Camelidae. V-NAR refers to the variable domains found in immunoglobulin-like structures (Ig-NAR) found in cartilaginous fish. As an alternative, single domain antibodies can be obtained from an initial synthetic library. For a review of single domain antibodies, reference may be made to Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, Muyldermans et al., Vet Immunol Immunopathol. 2009 Mar 15; 128(1-3): 178-83, and / or Muyldermans 2013, Annu Rev Biochem. 2013; 82: 775-97, the disclosures of which are incorporated by reference.

[0069] As used herein, "bind" or "binding" refers to peptides, polypeptides, proteins, fusion proteins and antibodies (including sdAbs) that recognize and contact an antigen. "Specific binding" or "immunospecific binding" means that the antibody recognizes a specific antigen, but does not substantially recognize or bind to other molecules or antigens in the sample. In some cases, the term "specific binding" or "specific binding" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance to indicate that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope). As used herein, the term "specific binding" means that the antibody and the antigen are bound by at least 10 -6 or 10 -7 In certain aspects, the antibody is contacted with a binding affinity of at least about 10 -8 M, and preferably 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Affinity binding of M.

[0070] As used herein, the term "sdAb that specifically binds to LILRB2" and similar terms refer to an sdAb that specifically recognizes LILRB2 and does not recognize or weakly recognizes other antigens (including other members of the LILR family, such as LILRB1). Preferably, when compared to the affinity for other antigens or fragments thereof (including other LILR family members, such as LILRB1), the sdAb that specifically binds to LILRB2 has a higher affinity for this antigen, preferably at least 10, 100 or 1000 times.

[0071] The affinity of an antibody or sdAb is a measure of its binding to a specific antigen at a single antigen-antibody site, and is essentially the sum of all the attractive and repulsive forces present in the interaction between the antigen binding site of the antibody and a specific epitope. The affinity of an antibody or sdAb for a specific antigen (e.g., LILRB2) can be represented by the dissociation equilibrium constant K, defined by the equation Kd = [Ag][Ab] / [Ag Ab], which represents the affinity of the antibody binding site; wherein [Ag] is the concentration of free antigen (M), [Ab] is the concentration of free antibody (M), and [Ag Ab] is the concentration of antigen-antibody complex (M). When the antigen and antibody or sdAb react strongly together, there will be very little free antigen or free antibody or sdAb, and therefore the equilibrium constant or affinity of the antibody or sdAb will be lower.

[0072] "Percentage identity" or "identity" between two amino acid sequences (A) and (B) is determined by comparing two sequences that are optimally aligned by a comparison window. The sequence alignment can be performed by well-known methods, such as using the Needleman-Wunsch global alignment algorithm. Protein analysis software uses similarity measures assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions to match similar sequences. Once a total alignment is obtained, the identity percentage can be obtained by dividing the total number of the aligned identical amino acid residues by the total number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. In order to compare two amino acid sequences, the "Emboss needle" tool can be used to perform protein paired sequence alignment, which is provided by EMBL-EBI and available at the following website:

[0073] http: / / www.ebi.ac.uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein with the following default settings: (i) Matrix: BLOSUM62, (ii) Gap Opening: 10, (iii) Gap Extension: 0.5, (iv) Output Format: Pairwise, (v) End Gap Penalty: False, (vi) End Gap Opening: 10, (vii) End Gap Extension: 0.5.

[0074] As used herein, "amino acid modification" means a change in the amino acid sequence of a polypeptide. "Amino acid modification" may also be referred to herein as "amino acid change", including amino acid mutations, such as substitutions, insertions and / or deletions in a polypeptide sequence. "Amino acid substitution" or "substitution" herein means replacing an amino acid at a specific position in a parent polypeptide sequence with another amino acid. Preferably, substitution is a silent substitution. "Amino acid insertion" or "insertion" means adding an amino acid at a specific position in a parent polypeptide sequence. "Amino acid deletion" or "deletion" means removing an amino acid at a specific position in a parent polypeptide sequence. Amino acid substitutions may be conservative. Conservative substitutions are substitutions of a given amino acid residue with another residue of a side chain ("R-group") having similar chemical properties (e.g., charge, volume and / or hydrophobicity). In general, conservative amino acid substitutions do not significantly change the functional properties of a protein. Conservative substitutions and corresponding rules are well described in the prior art.

[0075] As used herein, "parent polypeptide" or "polypeptide parent" refers to an unmodified polypeptide that is subsequently modified to generate a variant. In the context of the present invention, the parent polypeptide may be a VHH from a naturally occurring HCAb.

[0076] As used herein, "variant polypeptide", "polypeptide variant" or "variant" refers to a polypeptide sequence that is different from a parent polypeptide sequence due to at least one amino acid modification. For example, in the context of the present invention, a variant can be a variant of a VHH from a naturally occurring HCAb. Typically, a variant includes 1 to 50 amino acid modifications, preferably 1 to 40 amino acid modifications. In particular, a variant can have 1 to 30 amino acid changes compared to its parent, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid changes. A variant can include one or more amino acid substitutions, and / or one or more amino acid insertions, and / or one or more amino acid deletions. In some embodiments, a variant can include one or more conservative substitutions, such as shown above. In some further embodiments, the variant of sdAb may include one or more amino acid modifications in the CDR domain of the parent sdAb. Since CDR3 is generally used to define a sdAb family with the same recognition pattern, such modifications in CDR3 may result in a new sdAb family having different binding properties (e.g., increased binding properties) compared to the parent sdAb, while modifications in CDR1 or CDR2 may result in defining different members of the same family (i.e., having the same CDR3 but different CDR1 and / or CDR2). In some other embodiments, the variant of the parent sdAb may include one or more amino acid modifications in at least one framework domain.

[0077] The term "treatment" refers to any behavior intended to improve the patient's health status, such as the treatment, prevention, control and delay of a disease or disease symptom. It represents both the curative treatment and / or the preventive treatment of a disease. Curative treatment is defined as a treatment that causes a cure or a treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes a disease or disease symptom or the pain caused directly or indirectly by it. Preventive treatment includes treatment that causes disease prevention and treatment that reduces and / or delays disease progression and / or disease incidence or the risk of its occurrence. In certain embodiments, such terms refer to the improvement or eradication of a disease, illness, infection or symptoms associated therewith. In other embodiments, this term refers to minimizing the spread or deterioration of cancer. Treatment according to the present invention does not necessarily mean 100% or complete treatment. More specifically, there are treatments of varying degrees that a person of ordinary skill in the art would consider to have potential benefits or therapeutic effects.

[0078] As used herein, the terms "disorder" or "disease" refer to abnormal functioning of a body organ, part, structure or system due to genetic or developmental errors, infection, poisons, nutritional deficiencies or imbalances, toxicity or adverse environmental factors. Preferably, these terms refer to health conditions or diseases, such as illnesses that disrupt normal physical or mental function. More preferably, the term disorder refers to immune and / or inflammatory diseases affecting animals and / or humans, such as cancer.

[0079] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body, such as metastasis.

[0080] As used herein, the term "subject", "host", "individual" or "patient" refers to human and veterinary subjects, particularly animals, preferably mammals, even more preferably humans, including adults and children. However, the term "subject" also includes non-human animals, particularly mammals, such as dogs, cats, horses, cows, pigs, sheep and non-human primates, etc.

[0081] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active agents, for example, including an antigen binding domain of an anti-LILRB2 antibody or sdAb according to the present invention, and optionally other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical or veterinary composition is to facilitate the administration of the active agent to an organism. The composition of the present invention may be in a form suitable for any conventional route of administration or use. In one embodiment, a "pharmaceutical composition" typically refers to a combination of an active agent (e.g., a compound or composition) and a naturally occurring or non-naturally occurring carrier, which is inert (e.g., a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and includes a pharmaceutically acceptable carrier.

[0082] As referred to herein, an "acceptable vehicle" or "acceptable carrier" is any known compound or combination of compounds known to those skilled in the art to be useful in formulating a pharmaceutical or veterinary composition.

[0083] A "therapeutically effective amount" is the amount of active agent required to treat a target disease or condition or produce a desired effect when administered to a subject. An "effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, and characteristics of the subject to be treated.

[0084] As used herein, the term "pharmaceutical agent" refers to any substance or composition having therapeutic or preventive properties against a condition and / or disease.

[0085] ■Single domain antibody targeting LILRB2

[0086] As described above, sdAb molecules correspond to only the variable regions of an antibody heavy chain naturally devoid of light chains.The antigen-binding surface of an sdAb is generally more convex (or protruding) than the surface of a conventional antibody, which is generally flat or concave.

[0087] Single domain antibodies according to the present invention include a single variable domain derived from an antibody capable of binding to an antigen or epitope (e.g., LILBR2) alone, that is, without the need for another binding domain. In particular, single domain antibodies according to the present invention lack light chains or fragments thereof. The sdAb molecule according to the present invention is a polypeptide comprising, or consisting of, or essentially consisting of, an antigen binding domain of only a heavy chain antibody (HcAb) that can be separated from an engineered form of a heavy variable domain of a camelid, a cartilaginous fish, an initial library, or an antibody. Preferably, the sdAb is derived from a camelid HCAb, preferably from an alpaca HCAb.

[0088] In some preferred embodiments, the single domain antibody is selected from the group consisting of VHH, V-NAR from Ig-NAR, engineered V-NAR, VHH variants, in particular humanized VHH or optimized VHH, and combinations thereof.

[0089] In one embodiment, the sdAb against LILRB2 is an optimized sdAb. An optimized sdAb refers to an sdAb variant derived from an isolated HCAb, and compared with a naturally occurring sdAb, the optimized sdAb includes one or more amino acid modifications, which can, for example, increase the stability of the sdAb or increase the affinity and / or selectivity of the sdAb variant for LILRB2.

[0090] In another or further embodiment, the sdAb against LILRB2 is a humanized sdAb. A humanized sdAb refers to an sdAb variant comprising one or more amino acid modifications compared to a naturally occurring sdAb, which modification is capable of reducing its immunogenicity to human subjects without significantly reducing its affinity for LILRB2. A humanized sdAb according to the present invention can be prepared by replacing one or more amino acids in a camelid or cartilaginous fish sdAb sequence with its human counterpart, preferably as found in a human common sequence, provided that the amino acid modification does not significantly affect the antigen binding ability of the resulting sdAb, nor does it significantly affect its properties, such as the ability to inhibit the interaction between LILRB2 and human leukocyte antigen-G (HLA-G). Such methods are well known to those skilled in the art. The prior art in the art provides several examples of humanized scaffolds of VHHs that can be used in the context of the present invention. Humanized sdAbs include partially humanized sdAbs and fully humanized sdAbs.

[0091] Potentially useful humanized amino acid modifications, in particular substitutions, can be determined by comparing the framework region sequence of a naturally occurring VHH sequence with the corresponding framework region sequence of one or more closely related human VH sequences, after which one or more of the thus determined potentially useful humanized substitutions (or combinations thereof) can be introduced into the VHH sequence (in any manner known per se), and the resulting humanized VHH sequence can be tested for affinity to the target, stability, ease of expression and expression level, and / or other desired properties. In this way, by a limited degree of trial and error, a person skilled in the art can determine suitable humanized substitutions (or suitable combinations thereof). As an alternative, a person skilled in the art can transplant the CDRs of a VHH into a VHH humanized scaffold described in the prior art to obtain the desired humanized sdAb against LILRB2. Methods for humanizing sdAbs and humanized sdAb scaffolds are provided in, for example, patent applications US 2010 / 0215664, WO 2011 / 117423 or publications such as Conrath et al., Journal of Molecular Biology, 2005, 350: 112-125 and Vincke, Journal of Biological Chemistry, 2009, 284, 3273-3284.

[0092] As an alternative, one skilled in the art can transplant the CDRs into the sdAb universal scaffold described in the prior art (Saerens et al., J. Mol. Biol. (2005) 352, 597-607), thereby obtaining the desired sdAb against LILRB2. The sdAb of the present invention can be a VHH comprising a universal framework scaffold, such as shown in Saerens et al., and comprising at least one CDR, preferably three CDRs as defined below.

[0093] The single domain antibody of the present invention comprises at least one, preferably three complementary determining regions (CDRs) that determine its binding specificity. Preferably, the single domain antibody comprises several, preferably three CDRs, which are distributed between the framework regions (FRs). CDRs and FRs are preferably fragments, variants or derivatives from naturally occurring antibody variable domains. CDRs are generally 5 to 30 amino acids in length and exhibit high variability in both sequence content and structural conformation, which participate in antigen binding and provide antigen specificity.

[0094] Preferably, a single domain antibody includes four framework regions or "FRs", which are referred to in the art and herein as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively. These framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. These framework regions and complementary determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxyl terminus).

[0095] The CDRs of a given sdAb can be determined by any method available to those skilled in the art. For example, and in a non-limiting manner, the Chlothia or Kabat methods can be used to determine CDRs (Chothia et al., Nature 342, 877-883; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda). Alternative methods for determining CDRs can also be used, such as an intermediate method between Chlothia and Kabat, called AbM (Oxford Molecular AbM antibody modeling software) or the so-called "Contact" method based on analysis of available complex structures (Saerens et al., Mol Biol. 2005) or the IMGT method, such as disclosed in Lefranc et al., Dev. Comp. Immunol., 2003, 27: 55-77 ("IMGT" numbering scheme).

[0096] Compared with traditional human antibody VH, several amino acids in the FR2 region and CDR of sdAb can be substituted. For example, highly conserved hydrophobic amino acids in the FR2 region (such as Val47, Gly49, Leu50 and / or Trp52) are often replaced by hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52), thereby making the overall structure more hydrophilic and contributing to high stability, solubility and anti-aggregation.

[0097] In some specific embodiments, the single domain antibody of the present invention comprises a CDR3 comprising or consisting of a sequence as shown in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33, or comprising or consisting of an amino acid sequence different from 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33 due to one, two or three amino acid modifications. Preferably, the single domain antibody of the present invention comprises a CDR3 comprising or consisting of a sequence as shown in SEQ ID NO: 3, 6 or 9, or comprising or consisting of an amino acid sequence different from SEQ ID NO: 3, 6 or 9 due to one, two or three amino acid modifications. Preferably, such amino acid modifications do not significantly affect the antigen binding ability of the resulting sdAb, nor do they significantly affect its properties, such as the ability to inhibit the interaction between LILRB2 and human leukocyte antigen-G (HLA-G). Preferably, such amino acid modifications are substitutions, such as silent substitutions.

[0098] In some specific embodiments, the single domain antibody of the present invention comprises a CDR2 comprising or consisting of a sequence as shown in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32, or having an amino acid sequence different from SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32 due to one, two or three amino acid modifications. Preferably, the single domain antibody of the present invention comprises a CDR2 comprising or consisting of a sequence as shown in SEQ ID NO: 2, 5 or 8, or comprising or consisting of an amino acid sequence different from SEQ ID NO: 2, 5 or 8 due to one, two or three amino acid modifications. Preferably, such amino acid modifications do not significantly affect the antigen binding ability of the resulting sdAb, nor do they significantly affect its properties, such as the ability to inhibit the interaction between LILRB2 and human leukocyte antigen-G (HLA-G). Preferably, such amino acid modifications are substitutions, such as silent substitutions.

[0099] In some specific embodiments, the single domain antibody of the present invention comprises a CDR1 comprising or consisting of a sequence as shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31, or having an amino acid sequence different from 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 or 31 due to one, two or three amino acid modifications. Preferably, the single domain antibody of the present invention comprises a CDR1 comprising or consisting of a sequence as shown in SEQ ID NO: 1, 4 or 7, or comprising or consisting of an amino acid sequence different from SEQ ID NO: 1, 4 or 7 due to one, two or three amino acid modifications. Preferably, such amino acid modifications do not significantly affect the antigen binding ability of the resulting sdAb, nor do they significantly affect its properties, such as the ability to inhibit the interaction between LILRB2 and human leukocyte antigen-G (HLA-G). Preferably, such amino acid modifications are substitutions, such as silent substitutions.

[0100] In some embodiments, the single domain antibodies of the invention comprise three CDRs, which include or consist of:

[0101] (a) CDR1 comprises or has SEQ ID NO: 1, or has an amino acid sequence that differs from SEQ ID NO: 1 due to one, two or three amino acid modifications, and

[0102] CDR2 comprises or has SEQ ID NO: 2, or has an amino acid sequence that differs from SEQ ID NO: 2 due to one, two or three amino acid modifications, and

[0103] CDR3 comprises or has SEQ ID NO: 3, or has an amino acid sequence that differs from SEQ ID NO: 3 due to one, two, three or four amino acid modifications; or

[0104] (b) CDR1 comprises or has SEQ ID NO: 4, or has an amino acid sequence that differs from SEQ ID NO: 4 due to one, two or three amino acid modifications, and

[0105] CDR2 comprises or has SEQ ID NO: 5, or has an amino acid sequence that differs from SEQ ID NO: 5 due to one, two or three amino acid modifications, and

[0106] CDR3 comprises or has SEQ ID NO: 6, or has an amino acid sequence that differs from SEQ ID NO: 6 due to one, two, three or four amino acid modifications; or

[0107] (c) CDR1 comprises or has SEQ ID NO: 7, or has an amino acid sequence that differs from SEQ ID NO: 7 due to one, two or three amino acid modifications, and

[0108] CDR2 comprises or has SEQ ID NO: 8, or has an amino acid sequence that differs from SEQ ID NO: 8 due to one, two or three amino acid modifications, and

[0109] CDR3 comprises or has SEQ ID NO: 9, or has an amino acid sequence that differs from SEQ ID NO: 9 due to one, two, three or four amino acid modifications; or

[0110] (d) CDR1 comprises or has SEQ ID NO: 10, or has an amino acid sequence that differs from SEQ ID NO: 10 due to one, two or three amino acid modifications, and

[0111] CDR2 comprises or has SEQ ID NO: 11, or has an amino acid sequence that differs from SEQ ID NO: 11 due to one, two or three amino acid modifications, and

[0112] CDR3 comprises or has SEQ ID NO: 12, or has an amino acid sequence that differs from SEQ ID NO: 12 due to one, two, three or four amino acid modifications; or

[0113] (e) CDR1 comprises or has SEQ ID NO: 13, or has an amino acid sequence that differs from SEQ ID NO: 13 due to one, two or three amino acid modifications, and

[0114] CDR2 comprises or has SEQ ID NO: 14, or has an amino acid sequence that differs from SEQ ID NO: 14 due to one, two or three amino acid modifications, and

[0115] CDR3 comprises or has SEQ ID NO: 15, or has an amino acid sequence that differs from SEQ ID NO: 15 due to one, two, three or four amino acid modifications; or

[0116] (f) CDR1 comprises or has SEQ ID NO: 16, or has an amino acid sequence that differs from SEQ ID NO: 16 due to one, two or three amino acid modifications, and

[0117] CDR2 comprises or has SEQ ID NO: 17, or has an amino acid sequence that differs from SEQ ID NO: 17 due to one, two or three amino acid modifications, and

[0118] CDR3 comprises or has SEQ ID NO: 18, or has an amino acid sequence that differs from SEQ ID NO: 18 due to one, two, three or four amino acid modifications; or

[0119] (g) CDR1 comprises or has SEQ ID NO: 19, or has an amino acid sequence that differs from SEQ ID NO: 19 due to one, two or three amino acid modifications, and

[0120] CDR2 comprises or has SEQ ID NO: 20, or has an amino acid sequence that differs from SEQ ID NO: 20 due to one, two or three amino acid modifications, and

[0121] CDR3 comprises or has SEQ ID NO: 21, or has an amino acid sequence that differs from SEQ ID NO: 21 due to one, two, three or four amino acid modifications; or

[0122] (h) CDR1 comprises or has SEQ ID NO: 22, or has an amino acid sequence that differs from SEQ ID NO: 22 due to one, two or three amino acid modifications, and

[0123] CDR2 comprises or has SEQ ID NO: 23, or has an amino acid sequence that differs from SEQ ID NO: 23 due to one, two or three amino acid modifications, and

[0124] CDR3 comprises or has SEQ ID NO: 24, or has an amino acid sequence that differs from SEQ ID NO: 24 due to one, two, three or four amino acid modifications; or

[0125] (i) CDR1 comprises or has SEQ ID NO: 25, or has an amino acid sequence that differs from SEQ ID NO: 25 due to one, two or three amino acid modifications, and

[0126] CDR2 comprises or has SEQ ID NO: 26, or has an amino acid sequence that differs from SEQ ID NO: 26 due to one, two or three amino acid modifications, and

[0127] CDR3 comprises or has SEQ ID NO: 27, or has an amino acid sequence that differs from SEQ ID NO: 27 due to one, two, three or four amino acid modifications; or

[0128] (j) CDR1 comprises or has SEQ ID NO: 28, or has an amino acid sequence that differs from SEQ ID NO: 28 due to one, two or three amino acid modifications, and

[0129] CDR2 comprises or has SEQ ID NO: 29, or has an amino acid sequence that differs from SEQ ID NO: 29 due to one, two or three amino acid modifications, and

[0130] CDR3 comprises or has SEQ ID NO: 30, or has an amino acid sequence that differs from SEQ ID NO: 30 due to one, two, three or four amino acid modifications; or

[0131] (k) CDR1 comprises or has SEQ ID NO: 31, or has an amino acid sequence that differs from SEQ ID NO: 31 due to one, two or three amino acid modifications, and

[0132] CDR2 comprises or has SEQ ID NO: 32, or has an amino acid sequence that differs from SEQ ID NO: 32 due to one, two or three amino acid modifications, and

[0133] CDR3 comprises or has SEQ ID NO: 33, or has an amino acid sequence that differs from SEQ ID NO: 33 due to one, two, three or four amino acid modifications.

[0134] Preferably, the anti-LILRB2 sdAb comprises three CDRs, wherein:

[0135] (a) CDR1 comprises or has SEQ ID NO: 1, or has an amino acid sequence that differs from SEQ ID NO: 1 due to one, two or three amino acid modifications, and

[0136] CDR2 comprises or has SEQ ID NO: 2, or has an amino acid sequence that differs from SEQ ID NO: 2 due to one, two or three amino acid modifications, and

[0137] CDR3 comprises or has SEQ ID NO: 3, or has an amino acid sequence that differs from SEQ ID NO: 3 due to one, two, three or four amino acid modifications; or

[0138] (b) CDR1 comprises or has SEQ ID NO: 4, or has an amino acid sequence that differs from SEQ ID NO: 4 due to one, two or three amino acid modifications, and

[0139] CDR2 comprises or has SEQ ID NO: 5, or has an amino acid sequence that differs from SEQ ID NO: 5 due to one, two or three amino acid modifications, and

[0140] CDR3 comprises or has SEQ ID NO: 6, or has an amino acid sequence that differs from SEQ ID NO: 6 due to one, two, three or four amino acid modifications; or

[0141] (c) CDR1 comprises or has SEQ ID NO: 7, or has an amino acid sequence that differs from SEQ ID NO: 7 due to one, two or three amino acid modifications, and

[0142] CDR2 comprises or has SEQ ID NO: 8, or has an amino acid sequence that differs from SEQ ID NO: 8 due to one, two or three amino acid modifications, and

[0143] CDR3 includes or has SEQ ID NO: 9, or has an amino acid sequence that differs from SEQ ID NO: 9 due to one, two, three or four amino acid modifications.

[0144] Preferably, such amino acid modifications do not significantly affect the antigen binding ability of the resulting sdAb, nor do they significantly affect its properties, such as the ability to inhibit the interaction between LILRB2 and human leukocyte antigen-G (HLA-G). Preferably, such amino acid modifications are substitutions, such as silent substitutions.

[0145] Even more preferably, the anti-LILRB2 sdAb comprises three CDRs, wherein CDR1 comprises or has SEQ ID NO: 1, or has an amino acid sequence which differs from SEQ ID NO: 1 by one, two or three amino acid modifications, preferably one, two or three silent mutations, even more preferably one, two or three silent substitutions; and CDR2 comprises or has SEQ ID NO: 2, or has an amino acid sequence which differs from SEQ ID NO: 2 by one, two or three amino acid modifications, preferably one, two or three silent mutations, even more preferably one, two or three silent substitutions; and CDR3 comprises or has SEQ ID NO: 3, or has an amino acid sequence which differs from SEQ ID NO: 3 by one, two or three amino acid modifications, preferably one, two or three silent mutations, even more preferably one, two or three silent substitutions.

[0146] In some embodiments, the anti-LILRB2 sdAb comprises or essentially consists of a sequence defined in any one of SEQ ID No:34 to SEQ ID No:44, or a sequence having at least 80% sequence identity thereto, preferably a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity thereto.

[0147] Preferably, the anti-LILRB2 sdAb comprises or consists of a sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, or a sequence having at least 80% sequence identity thereto, preferably at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity thereto.

[0148] In one embodiment, the anti-LILRB2 sdAb comprises or consists essentially of a sequence defined in SEQ ID No: 34 or having at least 80% sequence identity thereto, preferably a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity thereto. Preferably, the anti-LILRB2 sdAb comprising or consisting of a sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity to SEQ ID NO: 34 is still capable of binding to LILRB2, preferably with a similar affinity as the anti-LILRB2 sdAb comprising or consisting of the sequence defined in SEQ ID NO: 34, and retains the same properties, such as the ability to inhibit the interaction of LILRB2 with human leukocyte antigen-G (HLA-G).

[0149] In some specific embodiments, the sdAb of the invention has a molecular weight of about 11 kDa to about 18 kDa, eg, 11 kDa to 17 kDa, such as 14 to 16 kDa or 14.5 to 15.5 kDa, such as about 15 kDa.

[0150] In certain aspects, the sdAb is present in an amount of at least about 10 -6 M or 10 -7 M, and preferably at least 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 The affinity of M for binding to LILRB2. In particular, the apparent K d Between 0.1 nM and 10 μM, in particular between 1 μM and 1 nM. The binding affinity may be measured by any method available to the skilled person, in particular by surface plasmon resonance (SPR).

[0151] In a preferred embodiment, the anti-LILRB2 sdAb does not recognize other members of the LILBR family besides LILRB2. Preferably, the anti-LILRB2 sdAb does not recognize LILRB1. Alternatively, the anti-LILRB2 sdAb weakly recognizes LILRB1. Preferably, the anti-LILRB2 sdAb recognizes LILRB1 less than LILRB2, in particular 10, 100 or 1000 times less.

[0152] In a specific embodiment, the anti-LILRB2 sdAb competitively inhibits the interaction between LILRB2 and human leukocyte antigen-G (HLA-G) or competitively inhibits the binding of human leukocyte antigen-G (HLA-G) to LILRB2.

[0153] The term "competitive inhibition" means that the sdAb according to the present invention can reduce or inhibit or displace the binding of a protein, antibody or ligand to LILRB2, or the interaction between any protein, antibody or ligand and LILRB2, particularly in vitro, ex vivo or in vivo. Competition assays can be performed using standard techniques, such as competitive ELISA or other binding assays. An sdAb is considered competitive when it inhibits or displaces at least 30%, 40%, 50%, 60%, 70% or 80% of the binding of a protein, antibody or ligand to LILRB2. Preferred competitive sdAbs bind to an epitope that has common amino acid residues with the epitope on LILRB2 that is recognized or bound by the protein, antibody or ligand.

[0154] As used herein, the term "HLA-G" refers to human leukocyte antigen G, which includes at least seven isoforms, four of which are membrane-bound (HLA-G1, HLA-G2, HLA-G3, and HLA-G4) and three of which are soluble (HLA-G5, HLA-G6, and HLA-G7). HLA-G human isoforms are described, for example, under the following Uniprot accession numbers: P17693-1 for HLA-G1, P17693-2 for HLA-G2, P17693-3 for HLA-G3, P17693-4 for HLA-G4, P17693-5 for HLA-G5, P17693-6 for HLA-G6, and P17693-7 for HLA-G7.

[0155] In a specific embodiment, the anti-LILRB2 sdAb competitively inhibits the interaction between LILRB2 and HLA-G6 or competitively inhibits the binding of HLA-G6 to LILRB2.

[0156] In another embodiment, the sdAb according to the invention competitively inhibits the binding of angiopoietin-like 2 (ANGPTL2) to LILRB2 or competitively inhibits the interaction between LILRB2 and ANGPTL2. As used herein, "ANGPTL2" is a member of the vascular endothelial growth factor family known in the art for its pro-angiogenic and anti-apoptotic abilities. This term preferably refers to human ANGPTL2. Human ANGPTL2 is described, for example, under Uniprot Accession No. 015123.

[0157] The present invention also relates to chimeric agents (also interchangeably referred to herein as "conjugates") comprising one or more anti-LILRB2 sdAbs as defined above bound to at least one molecule. The molecule bound to the sdAb can be, for example, any active compound that can be used in medicine, such as a drug, an imaging molecule, a diagnostic agent, a tracer, a label or a dye. In addition to or in place of the active compound, the chimeric agent may also contain a stabilizing group (e.g., Fc or IgG) to increase the plasma half-life of the sdAb or the conjugate. Such chimeric agents can be prepared by any method known in the art, preferably by chemical, biochemical or enzymatic pathways, or by genetic engineering, using coupling between the sdAb and the molecule.

[0158] In a specific embodiment, the anti-LILRB2 sdAb of the invention can be fused or conjugated to a labeling substance, such as a molecule or protein selected from the following: an enzyme, such as horseradish peroxidase or alkaline phosphatase; a fluorescent protein, such as GFP; a fluorescent marker, such as fluorescein rhodamine; a label; a chemiluminescent marker or a bioluminescent marker, such as luminal; a chromophore; a radioisotope, such as suitable for in vivo, ex vivo or in vitro imaging or diagnosis.

[0159] In another specific embodiment, the sdAb according to the present invention is included in the CAR construct. As used herein, the term "chimeric antigen receptor" (CAR), "engineered cell receptor" or "chimeric immune receptor" (ICR) refers to an engineered receptor that transplants antigen binding specificity to immune cells, thereby combining the antigen binding properties of the antigen binding domain with the immunogenic activity of immune cells, such as the lysis ability and self-renewal of T cells. In particular, CAR refers to a fusion protein comprising an optional signal peptide, an extracellular domain capable of binding to an antigen, a transmembrane domain, an optional hinge domain, and at least one intracellular domain. In a preferred embodiment, CAR includes an anti-LILRB2 sdAb as disclosed herein as an extracellular domain or antigen binding domain, a transmembrane domain, an optional hinge domain, and at least one intracellular domain.

[0160] ■Nucleic acids, vectors and host cells

[0161] Another aspect of the invention relates to an isolated nucleic acid construct or polypeptide construct encoding an sdAb as defined above. The nucleic acid can be single-stranded or double-stranded or a mixture of the two. The nucleic acid can be DNA (cDNA or gDNA), RNA or a mixture thereof. It can include modified nucleotides, for example, modified bonds, modified purine or pyrimidine bases or modified sugars. It can be prepared by any method known to those skilled in the art, including chemical synthesis, recombination and / or mutagenesis.

[0162] The nucleic acid according to the invention can be derived from the amino acid sequence of the sdAb molecule according to the invention, and the codon usage can be adjusted according to the host cell in which the nucleic acid should be transcribed. These steps can be carried out according to methods well known to those skilled in the art, some of which are described in the reference manual Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edition Cold Spring Harbor). Specific examples of such nucleic acid sequences include sequences containing any one of SEQ ID NO: 61-75, and sequences complementary thereto.

[0163] The present invention also relates to vectors containing such isolated nucleic acids, optionally under the control of regulatory sequences such as promoters, terminators, etc. The vector may be, for example, a plasmid, a virus, a cosmid, a phagemid or an artificial chromosome.

[0164] The invention further relates to the use of a nucleic acid or a vector according to the invention for transforming, transfecting or transducing a host cell.

[0165] Therefore, the present invention also provides host cells comprising one or several nucleic acids of the present invention and / or one or several vectors of the present invention and / or one or several polypeptides encoding sdAbs of the present invention.

[0166] The host cell can be any host cell capable of expressing or producing the sdAb of the present invention, including, for example, prokaryotic host cells, such as Escherichia coli, or (cultured) mammalian, plant, insect, fungal or yeast host cells, including, for example, CHO-cells, BHK-cells, human cell lines (including HeLa, COS and PER C6), Sf9 cells and Sf+ cells. Suitable host cells include eukaryotic microorganisms, such as cells of yeast and filamentous fungi. Preferred yeast host cells include Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha and Kluyveromyces lactis. The term "host cell" also includes any progeny of a parent host cell that is different from the parent host cell due to mutations that occur during replication. Preferably, the cell is not a human embryonic stem cell.

[0167] Another object of the present invention is a method for producing a sdAb according to the present invention, wherein said method comprises the following steps:

[0168] a) cultivating a host cell as defined previously, and

[0169] b) recovering said nucleic acid, vector or polypeptide encoding the sdAb as defined above from the cell culture.

[0170] It goes without saying that step a) is carried out under conditions that allow the host cell to express the desired nucleic acid, vector or polypeptide. Suitable expression conditions may include the use of a suitable culture medium, a suitable food source and / or the presence of a suitable nutrient, a suitable temperature and optionally the presence of a suitable induction factor or compound (e.g. when the nucleotide sequence of the present invention is controlled by an inducible promoter); all of which may be selected by a person skilled in the art.

[0171] Under such conditions, the sdAbs of the invention may be expressed constitutively, in a transient manner, or only upon appropriate induction.

[0172] The sdAb of the invention can then be isolated from the host cells and / or from the culture medium in which the host cells are cultured using protein separation and / or purification techniques known per se, such as chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques, etc. The sdAb may also include a tag, such as a histidine or streptavidin tag, for purification purposes.

[0173] The present invention also provides a method for obtaining an sdAb for LILRB2 as defined herein. The method for obtaining and / or selecting sdAb according to the present invention can be based on protein selection technology, such as but not limited to cell display, phage display, ribosome display, mRNA display, DNA display or plasmid display. These technologies are fully described in the prior art. For example, in order to generate a VHH library displayed on phage, the technician can refer to Muydermans et al., Molecular Biotechnology, 2001, 74, 277-302, in particular the section entitled Recombinant VHH, the disclosure of which is incorporated herein by reference. In order to generate a V-NAR library displayed on phage, the technician can refer to Dooley et al., Mol Immunol, 2003, 40: 25-30. In certain embodiments, the methods of the present invention may include one or more steps enabling the selection of functional sdAbs, in particular sdAbs capable of recognizing LILRB2 or competitively inhibiting the interaction between LILRB2 and HLA-G and / or competitively inhibiting the interaction between LILRB2 and ANGPTL2.

[0174] In a specific embodiment, the CAR comprising the sdAb according to the present invention is expressed by a cell. The cell may be a prokaryotic cell or a eukaryotic cell. Preferably, the cell is a eukaryotic cell, such as a mammalian cell. Preferably, the cell expressing the CAR comprising the sdAb according to the present invention is an immune cell. The cell may be selected from macrophages, T cells, B cells, NK cells, NKT, monocytes and dendritic cells. Preferably, the cell is not a human embryonic stem cell.

[0175] ■Drug composition

[0176] The present invention also relates to a pharmaceutical composition, characterized in that it comprises at least one sdAb, CAR or cell as defined above and optionally one or more pharmaceutically acceptable excipients.

[0177] The pharmaceutical compositions of the present invention can be formulated according to standard methods, such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; Twenty-first Edition, 2005). Useful pharmaceutically acceptable excipients are particularly described in Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; Sixth Revised Edition, 2009).

[0178] In one aspect, the composition of the present invention advantageously comprises a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers can be selected from carriers classically used according to each mode of administration, such as (a) fillers or diluents, such as starch, lactose, sucrose, glucose, mannitol, microcrystalline cellulose and silicic acid; (b) binders, such as carboxymethyl cellulose, gelatin, polyvinyl pyrrolidone, sucrose; (c) humectants, such as glycerol; (d) disintegrators, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, cross-linked carboxymethyl cellulose sodium and sodium carbonate; (e) solution retarders, such as paraffin; (f) absorption promoters, such as quaternary ammonium compounds; (g) wetting agents, such as glycerol monostearate; (h) adsorbents, such as kaolin and bentonite; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate; (j) antioxidants; (k) buffers, such as sodium citrate or sodium phosphate; (l) preservatives; (m) flavors and fragrances, etc.

[0179] The pharmaceutical composition of the present invention can be obtained by mixing the sdAb, CAR, cell or polypeptide of the present invention with at least one conventional excipient (or carrier) as described above with an appropriate purity. Specifically, the sdAb, CAR, cell or polypeptide of the present invention is the active ingredient of the composition.

[0180] It goes without saying that the excipients to be combined with the active ingredient may vary depending on (i) the physicochemical properties of the active ingredient, including stability, (ii) the desired pharmacokinetic profile of the active ingredient, (iii) the galenic form and (iv) the route of administration.

[0181] The pharmaceutical composition typically comprises an effective dose of the sdAb, CAR or cell of the present invention. As described herein, a "therapeutically effective dose" refers to a dose that produces a therapeutic effect on a given condition and administration regimen. A "therapeutically effective dose" of an active substance does not necessarily cure a disease or condition, but provides treatment for such a disease or condition, thereby delaying, hindering or preventing its occurrence, or alleviating its symptoms, or changing its duration or alleviating its severity, or accelerating patient recovery.

[0182] The pharmaceutical compositions of the present invention can be formulated to be suitable for administration by any conventional route, including enteral routes (i.e., oral), for example in the form of tablets, capsules, parenteral, intramuscular, transdermal, intravenous routes, for example in the form of injectable solutions or suspensions, and topical routes, for example in the form of gels, ointments, gels, lotions, patches, suppositories, etc.

[0183] In some embodiments, the pharmaceutical composition may be a lyophilisate or lyophilized powder, which may be dissolved in a suitable vehicle immediately prior to administration to a subject.

[0184] The invention also relates to a diagnostic composition, characterised in that it comprises a sdAb or a sdAb diagnostic or medical imaging agent conjugate compound as defined above.

[0185] ■Use according to the invention

[0186] The sdAb, CAR, cell, composition and construct (ie, isolated nucleic acid, polypeptide and / or vector) according to the present invention can be used in various fields, including biological research, biochemical industry or medicine.

[0187] In particular, the sdAbs, CARs, cells, compositions and constructs of the invention find use in subjects having or suspected of having cancer, particularly for reducing the size of a tumor or preventing the growth or regrowth of a tumor or preventing the induction of an immunosuppressive microenvironment in these subjects.

[0188] In one embodiment, the subject to be treated is a non-human animal, particularly a mammal, such as a dog, cat, horse, cow, pig, sheep, and non-human primate. Alternatively, the subject to be treated can be a human, particularly a human of any age, including a child, teenager, or adult.

[0189] In particular, the subject suffers from a disease involving LILBR2 expression, in particular LILBR2 overexpression. In one embodiment, the subject suffers from cancer, an inflammatory disorder, such as an infectious disease caused by bacteria, virus or fungus, or suffers from an autoimmune disease.

[0190] Preferably, the subject suffers from cancer, even more preferably suffers from LILBR2-positive cancer. For example, a subject suitable for treatment of a disease, such as cancer, can be identified by examining whether such a subject carries LILRB2-positive cells, particularly LILRB2-positive cancer cells, preferably such cells that overexpress LILRB2. Examples of diseases and cancers are described in more detail below.

[0191] Another object of the present invention is an sdAb, CAR, cell, polypeptide construct or pharmaceutical composition according to the present invention for treating a condition or disease involving the LILRB2 receptor, preferably cancer, and / or for use as a medicament or vaccine. Thus, described herein are methods for inhibiting tumor growth or metastatic spread in a subject in need thereof and / or treating cancer in a patient in need thereof. The tumor may be a solid tumor or a liquid tumor, preferably a solid tumor. In some embodiments, the tumor or cancer expresses or overexpresses LILBR2.

[0192] In certain embodiments, these methods include, or alternatively consist essentially of, or further consist of: administering a therapeutically effective amount of the sdAb, CAR, cell, composition, and construct of the invention to a subject or patient. On the other hand, the subject has previously been selected for treatment by diagnosis, preferably assessing whether the tumor expresses or overexpresses LILBR2.

[0193] Since human LILRB2 is a relevant target for treating a disease or disorder, particularly cancer, anti-LILRB2 sdAbs can be used as drugs, medicaments or vaccines. The sdAbs, CARs, cells or polypeptide constructs according to the present invention can be used as medicaments or vaccines, or used to manufacture medicaments or vaccines for treating a disease, disorder or condition in a subject. In some embodiments, such drugs or vaccines can be used to treat cancer.

[0194] In one embodiment, the sdAbs, CARs, cells, compositions and constructs of the invention are used to treat pathologies, diseases and / or conditions that can be prevented or treated by inhibiting the binding of HLAG and / or ANGPTL2 to LILRB2. Thus, the present invention relates to methods of treating pathologies, diseases and / or conditions that can be prevented or treated by inhibiting the binding of HLAG and / or ANGPTL2 to LILRB2.

[0195] The present invention also relates to a method of treating a subject suffering from a disorder or disease involving the LILRB2 receptor, wherein the method comprises administering to the subject a therapeutically effective amount of an sdAb, CAR, cell, construct or pharmaceutical composition according to the present invention.

[0196] In a particular embodiment, the disease or disorder is cancer, preferably a solid tumor, even more preferably selected from the group consisting of lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, pancreatic ductal carcinoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), endometrial cancer, hepatocellular carcinoma, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, kidney cancer, testicular cancer, esophageal cancer, cervical cancer, mouse Lewis lung carcinoma, leukemia, thyroid cancer, liver cancer, urothelial carcinoma and head and neck cancer.

[0197] Therefore, the present invention also relates to methods of inhibiting tumor growth and / or inhibiting metastatic growth and / or spread in a subject in need thereof. The tumor can be a solid tumor or a liquid tumor. In some embodiments, the tumor or cancer expresses or overexpresses LILRB2.

[0198] The sdAb, CAR, cell or pharmaceutical composition described herein can be administered simultaneously or sequentially with other therapeutic agents, including, for example, small molecules, radiotherapy, chemotherapy, surgery, and particularly anticancer agents. "Anticancer" agents can have a negative impact on the cancer of a subject, for example, by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting immune responses to cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the life span of subjects with cancer. More generally, these other compositions can be provided in a combined amount that effectively kills cells or inhibits cell proliferation.

[0199] Conventional methods known to those of ordinary skill in the medical field can be used to administer sdAb, compositions, constructs or CAR disclosed herein to a subject, depending on the type of disease to be treated or the disease site. This composition can be administered by conventional routes, such as parenteral administration (e.g., by intravenous, subcutaneous, intradermal or intramuscular routes), or by oral, nasal or pulmonary routes.

[0200] For diagnosis and prognosis

[0201] Single domain antibodies can be used as ligands to purify LILRB2. They can also be used as crystallization chaperones to facilitate crystallization of the LILRB2 receptor.

[0202] The sdAbs and polypeptides of the invention can also be used for cell immunostaining, in vivo or in vitro imaging and for diagnostic purposes. The invention also relates to sdAbs, conjugates or compositions as described above for use in diagnosing, imaging or treating cells expressing LILRB2, preferably overexpressing LILRB2, such as cancer cells.

[0203] They can also be used as biological reagents in in vitro assays, for example as test compounds or competitive binding agents for identifying, screening or characterizing potential drugs targeting the LILRB2 receptor.

[0204] The anti-LILRB2 sdAbs disclosed herein can be used in a diagnostic manner to monitor LILRB2 expression levels in tissues or cells, as part of in vitro or ex vivo, and in vivo clinical testing procedures, for example, to determine the efficacy of a given treatment regimen.

[0205] The detection methods of the present invention can be used to detect the expression level of LILRB2 in biological samples in vitro or ex vivo and in vivo, for example, after an organ or tissue biopsy to test whether the cells are cancerous. In vitro or ex vivo techniques for detecting LILRB2 by the sdAb of the present invention include enzyme-linked immunosorbent assay (ELISA), RIA, EIA and other "sandwich assays", Western blots, flow cytometry, immunoprecipitation, radioimmunoassay and immunofluorescence (e.g., IHC). In addition, in vivo techniques for detecting LILRB2 polypeptides include introducing a labeled anti-LILRB2 sdAb into a subject. In the in vivo techniques for detecting LILRB2 by the sdAb of the present invention, the sdAb can be labeled with a radioactive label, the presence and location of which in the subject can be detected by standard imaging techniques.

[0206] The present invention also provides diagnostic, prognostic or predictive assays for determining whether a subject is at risk for a medical disease or condition associated with increased LILRB2 expression or activity (e.g., detecting precancerous or cancerous cells that overexpress LILRB2). Such assays can be used for prognostic or predictive purposes to preventively treat an individual prior to the onset of a medical disease or condition characterized by or associated with LILBR2 expression or overexpression.

[0207] The invention also provides diagnostic, prognostic or predictive assays, wherein an sdAb according to the invention is used to select subjects suitable for treatment with an anti-LILRB2 sdAb, e.g., wherein LILRB2 is a biomarker for selecting patients, wherein LILRB2 is overexpressed in cells, such as tumor cells.

[0208] ■Test kit

[0209] Any sdAb, composition, CAR, cell, vector, polypeptide or nucleic acid construct described herein can be included in the kit provided by the present invention.

[0210] In certain embodiments, the kit includes, for example, suitable container components, cells, buffers, cell culture media, vectors, primers, restriction enzymes, salts, etc. The kit may also include components for containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0211] In some embodiments, components for collecting a sample from an individual and / or analyzing a sample may be provided in a kit.

[0212] In some embodiments, the kit further includes an additional agent for treating cancer or an infectious disease, and the additional agent can be combined with the sdAb, composition, CAR, cell, vector, polypeptide or nucleic acid construct or other components of the kit of the present invention or can be provided separately in the kit.

[0213] In some cases of the invention, the kit also includes a second cancer therapy, such as chemotherapy and / or other immunotherapy.The kit can be customized for a specific cancer, such as a cancer that expresses or overexpresses LILRB2.

[0214] The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. In one embodiment, the present invention relates to a kit for a single-dose administration unit as defined above. The kit of the present invention may also contain a first container comprising a dried / lyophilized bifunctional molecule and a second container comprising an aqueous formulation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilizing syringe) is provided. The kit of the present invention uses suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film (Mylar) or plastic bags), etc.

[0215] Instructions related to the use of sdAb, compositions, CARs, cells, vectors, polypeptides or nucleic acid constructs described herein generally include information about dosage, dosing regimen, routes of administration for intended treatment, or methods for reconstitution or dilution of such components. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., a sheet included in the kit in the form of a leaflet or instruction manual). In some embodiments, the kit may include instructions for use according to any method described herein. The included instructions may include a description of the administration of sdAb, compositions, CARs, cells, vectors, polypeptides or nucleic acid constructs described herein, particularly in the context of treating a disease as described herein, such as cancer. The kit may further include a description of selecting an individual suitable for treatment based on identifying whether the individual suffers from a disease associated with LILBR2 (e.g., those described herein).

[0216] Other aspects and advantages of the present invention will become apparent upon consideration of the following examples, which are intended to be illustrative only and not to limit the scope of the present application. Example

[0217] Identification of LILRB2-Fc-specific VHHs.

[0218] Alpacas were first immunized with complete Freund's adjuvant containing LILRB2-Fc protein, and then boosted twice with incomplete Freund's adjuvant containing LILRB2-Fc protein. Conventional antibody subclasses (i.e., IgG1) and VHH were isolated from alpaca serum. The serum was serially diluted and tested for LILRB2-Fc protein by ELISA. Then, B lymphocytes were purified from the alpacas and a sample containing 3,5.10 7 A library of 10 clones was prepared. Biopanning was performed using display against LILRB2-Fc and pre-plasma (PE)-ELISA was performed on selected VHHs. 400 colonies were tested and 130 positive clones against LILRB2 were identified, which are circled with solid circles, while negative clones are circled with dashed circles ( Figure 1 ). All positive clones were sequenced and 12 unique VHH sequences were generated and purified.

[0219] B8, C7 and C9 recognize a linear epitope of rhLILRB2.

[0220] The inventors first investigated whether the generated Nbs were specific for the LILRB2 receptor without any cross-reactivity to the LILRB1 receptor. To this end, Western blotting was performed under reducing conditions. Purified dimeric rhLILRB2-Fc, monomeric rhLILRB2 and monomeric rhLILRB1 proteins were used to compare the antigenic specificity of the different Nbs. Nb specificity was evaluated against control antibodies H-300 (specific for LILRB1, -2, -4, -5, -6), 42D1 (specific for LILRB2), GHI / 75 (specific for LILRB1) and HP-F1 (specific for LILRB1). Membranes labeled with the H-300 polyclonal antibody (specific for LILRB1, -2, -4, -5 and -6 proteins) showed bands of approximately 105 kDa and at 77 kDa, corresponding to the sizes of rhLILRB2-Fc and rhLILRB2, respectively ( Figure 2). The membrane incubated with 42D1 mAb (specific for LILRB2-Fc receptor) showed a unique band at 105 kDa corresponding to the size of rhLILRB2-Fc and no band at 77 kDa, indicating that 42D1 mAb does not recognize rhLILRB2. The membrane incubated with GHI / 75 and HP-F1 mAbs showed a band at 84 kDa, corresponding to the size of rhLILRB1. Among the 15 Nbs, only B8, C7, and C9 showed a band of approximately 105 kDa corresponding to the molecular weight of rhLILRB2-Fc ( Figure 3 ), as well as a band of approximately 77 kDa corresponding to the molecular weight of the D1 and D2 domains of rhLILRB2. In addition, incubation with B8 and C7 Nb did not show any band near 84 kDa. This means that B8 and C7 Nb do not bind to rhLILRB1. However, C9 Nb showed a weak band near 84 kDa, indicating that C9 specificity is not completely limited to the rhLILRB2 receptor. In summary, these data indicate that Nb is able to recognize the denatured dimeric LILRB2-Fc (D1-D2-Fc) protein, which is the immunogen used to induce Nb, as well as the denatured monomeric LILRB2 (D1-D2-D3-D4 domains) protein. For western blot experiments, c-Myc ".9E10" pure (E-Bioscience, Ref 14-6784-82) antibody was used.

[0221] Specificity of Nb for the LILRB2 receptor on the LILRB2-transduced D1.1 cell line.

[0222] The inventors then sought to determine whether the obtained Nbs were able to bind to the conformational LILRB2 receptor. They evaluated the binding specificity of 15 Nbs to the conformational LILRB2 receptor. To this end, the inventors evaluated the specificity of the Nbs to the LILRB2-D1.1 transduced cell line generated by Invectys. To this end, the LILRB2-D1.1 cell line was incubated with the Nbs and compared to the 42D1 control Ab. Figure 4 As shown, 62.6% of LILRB2-D1.1 cells were labeled by the 42D1 control Ab. Interestingly, 93.2%, 76.4%, and 75.2% of the LILRB2-D1.1 cell line were labeled by the B8, C9, and C7 Nbs, respectively ( Figure 7 ), while less than 40% of the LILRB2-D1.1 cell line was labeled by other Nbs, such as A2 (data not shown). The inventors hypothesized that the epitopes recognized by B8, C7 and C9 are more accessible than those of the 42D1 control antibody and other Nbs. For flow cytometry, a mouse monoclonal antibody against Myc tag-phycoerythrin (Abcam, Ref: ab72468) was used [9E10].

[0223] Specificity of Nb for the LILRB2 receptor expressed on monocytes

[0224] Monocytes strongly express monomeric or dimeric LILRB2 receptors on their surface and are a relevant model to study macrophage LILRB2 expression. Therefore, the inventors evaluated the specificity of anti-LILRB2 Nbs for monocytes purified from healthy donor PBMCs. Monocytes were phenotyped by labeling with anti-CD14, anti-LILRB1 Ab and anti-LILRB2 Nbs. 38% of monocytes were positive for the 42D1 control Ab and 5% were positive for irrelevant Nbs (e.g., Nbs raised in alpacas against non-ILT4 antigens). Using anti-LILRB2 Nbs, more than 50% of monocytes were labeled: 68.3% for A2 Nb, 50.8% for B8 Nb, 62% for C7 Nb, 58.1% for C9 Nb, 53.5% for D8 Nb, 57% for G3 Nb and 46.7% for G10 Nb ( Figure 5 ). However, monocytes were negative for D12, F5 and H12 Nbs (data not shown). In summary, the inventors determined that B8, C7 and C9 Nbs all have strong specificity for linear epitopes within monomeric or dimeric LILRB2 receptors. Compared to Nbs, the LILRB2 epitope of control Ab 42D1 may be less accessible to in vitro LILRB2-D1.1 generated cell lines or ex vivo monocytes. This weak binding of the 42D1 monoclonal antibody may be related to steric hindrance that does not affect anti-LILRB2 Nbs, especially B8, C7 and C9 Nbs.

[0225] Nb anti-LILRB2 inhibits LILRB2 / HLA-G interaction.

[0226] The LILRB2 receptor interacts with HLA-G and ANGPTL2 to inhibit immune cell responses and induce tumor development, respectively. The inventors then investigated whether anti-LILRB2 could block these interactions to restore immune cell function and prevent tumor growth. In order to investigate the inhibition of the LILRB2 / HLA-G interaction, the inventors first designed an ELISA assay to evaluate the blocking ability of Nb. To this end, rhLILRB2-Fc protein was coated on microtiter plates and then co-incubated with HLA-G6 protein in the presence or absence of Nb. The rh-LILRB2-Fc receptor has been shown to have a strong affinity for soluble HLA-G6 isoforms. As Figure 6As shown, the isotype control monoclonal antibody interfered with the HLA-G6 / LILRB2 interaction (24% blocking) as well as the H-300 polyclonal antibody which was not reported as blocking (26% blocking). However, the anti-LILRB2 monoclonal blocking antibody 27D6 strongly abolished the interaction between HLA-G6 and the LILRB2 receptor (100% blocking). Regarding the anti-LILRB2 Nbs, the inventors determined that 7 Nbs (A2, C7, C9, D8, E7, F5 and G10) weakly inhibited the interaction (<30%), 3 Nbs showed partial inhibition: D12 (44.8%), G3 (39.4%) and H12 (50%), while the B8 Nb completely inhibited the HLA-G6 / LILRB2 interaction (100% blocking).

[0227] B8 Nb partially inhibits LILRB2 / ANGPTL2 interaction

[0228] It has been demonstrated that the LILRB2 / ANGPTL2 interaction promotes tumor development. Indeed, the interaction between the LILRB2 receptor expressed by cancer cells and the autocrine expressed ANGPTL2 protein leads to tumor proliferation, inhibition of tumor apoptosis, and tumor cell differentiation. To determine whether anti-LILRB2 Nb could block this interaction, the inventors established an ELISA to evaluate the interaction between rhLILRB2-Fc and ANGPTL2 protein. As described previously, rhLILRB2-Fc protein was coated on microtiter plates and then co-incubated with rhANGPTL2 in the presence or absence of anti-LILRB2 antibodies or Nb. Some Nbs partially blocked the LILRB2 / ANGPTL2 interaction (<24% binding inhibition) (data not shown). Compared with the control without Nb, B8Nb strongly blocked this interaction (51.4% binding inhibition), while A2, H12 and G10 showed weak blocking (24%, 20% and 8%, respectively) ( Figure 7 ). Sequence Listing <110> Invectys <120> Single domain antibody targeting LILRB2 <130> B3095PC00 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial <220> <223> ILT4-B8 CDR1 <400> 1 Gly Phe Thr Leu Asp Tyr Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-B8 CDR2 <400> 2 Ile Gly Asn Ser Gly Asp Ser Thr 1 5 <210> 3 <211> 27 <212> PRT <213> artificial <220> <223> ILT4-B8 CDR3 <400> 3 Ala Ala Arg Lys Gly Phe Ala Ser Ser Cys His Gly Leu Gly Ala Ala 1 5 10 15 Tyr Asp Ser Asp Tyr Glu Ser Leu Tyr Asp Tyr 20 25 <210> 4 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-C7 CDR1 <400> 4 Gly Phe Ala Leu Glu His Tyr Ser 1 5 <210> 5 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-C7 CDR2 <400> 5 Ile Ser Asn Ser Gly His Thr Thr 1 5 <210> 6 <211> 16 <212> PRT <213> artificial <220> <223> ILT4-C7 CDR3 <400> 6 Ala Ala Thr Pro Arg Gly Trp Gly Leu Thr Ser Asn Gln Tyr Glu Tyr 1 5 10 15 <210> 7 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-C9 CDR1 <400> 7 Gly Arg Thr Leu Asn Gly Tyr Thr 1 5 <210> 8 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-C9 CDR2 <400> 8 Ile Arg Ala Ile Asp Gly Ser Thr 1 5 <210> 9 <211> 19 <212> PRT <213> artificial <220> <223> ILT4-C9 CDR3 <400> 9 Ala Ala Arg Leu Arg Val Ser Asn Gly Asn Ala Trp Ser Ser Ser Ser 1 5 10 15 Tyr Leu His <210> 10 <211> 8 <212> PRT <213> artificial <220> <223> ILT4 - A2 CDR1 <400> 10 Gly Phe Asn Val Asn Ala Tyr His 1 5 <210> 11 <211> 7 <212> PRT <213> artificial <220> <223> ILT4 - A2 CDR2 <400> 11 Ile Ser Ser Asp His Ser Thr 1 5 <210> 12 <211> 16 <212> PRT <213> artificial <220> <223> ILT4 - A2 CDR3 <400> 12 Ala Ala Thr Ala Asn Gln Leu Trp Gln Val Trp Met Gly Ile Thr Ser 1 5 10 15 <210> 13 <211> 8 <212> PRT <213> artificial <220> <223> ILT4 - D8 CDR1 <400> 13 Gly Leu Thr Ser Arg Pro Tyr Tyr 1 5 <210> 14 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-D8 CDR2 <400> 14 Ile Ser Ser Arg Glu Gly Ser Thr 1 5 <210> 15 <211> 16 <212> PRT <213> artificial <220> <223> ILT4-D8 CDR3 <400> 15 Ala Gly Arg Tyr Ser Thr Cys Ser Gly Met Ala Arg Asp Tyr Asn Tyr 1 5 10 15 <210> 16 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-D12 CDR1 <400> 16 Gly Phe Thr Leu Asp His Tyr Ala 1 5 <210> 17 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-D12 CDR2 <400> 17 Ile Asn Val Asn Arg Gly Thr Ser 1 5 <210> 18 <211> 23 <212> PRT <213> artificial <220> <223> ILT4-D12 CDR3 <400> 18 Ala Ala Leu Leu Glu Gly Ala Ser Gly Asp Val Cys Asp Asp Val Asn 1 5 10 15 Gln Asn Tyr Gly Met Asp Tyr 20 <210> 19 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-E7 CDR1 <400> 19 Gly Ser Thr Ser Arg His Tyr Tyr 1 5 <210> 20 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-E7 CDR2 <400> 20 Ile Thr Ser Arg Glu Gly Ser Thr 1 5 <210> 21 <211> 16 <212> PRT <213> artificial <220> <223> ILT4-E7 CDR3 <400> 21 Ala Ala Arg Tyr Ser Ser Cys Ser Gly Met Pro Ser Asp Tyr Asn Tyr 1 5 10 15 <210> 22 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-F5 CDR1 <400> 22 Gly Phe Asn Val Asn Ala Tyr His 1 5 <210> 23 <211> 7 <212> PRT <213> artificial <220> <223> ILT4-F5 CDR2 <400> 23 Ile Ser Ser Asp Arg Glu Thr 1 5 <210> 24 <211> 16 <212> PRT <213> artificial <220> <223> ILT4-F5 CDR3 <400> 24 Ala Ala Thr Ala Asn Gln Leu Trp Arg Leu Trp Met Gly Ile Ser Ser 1 5 10 15 <210> 25 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-G3 CDR1 <400> 25 Gly Phe Thr Ser Arg His Tyr Tyr 1 5 <210> 26 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-G3 CDR2 <400> 26 Ile Ser Ser Arg Glu Gly Ser Thr 1 5 <210> 27 <211> 16 <212> PRT <213> artificial <220> <223> ILT4-G3 CDR3 <400> 27 Ala Gly Arg Tyr Ser Ser Cys Ser Gly Met Pro Arg Asp Tyr Asn Tyr 1 5 10 15 <210> 28 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-G10 CDR1 <400> 28 Gly Phe Thr Phe Phe Ala Tyr Thr 1 5 <210> 29 <211> 8 <212> PRT <213> artificial <220> <223> ILT4-G10 CDR2 <400> 29 Ile Asp Trp Thr Gly Ser Ser Ser 1 5 <210> 30 <211> 20 <212> PRT <213> Artificial <220> <223> ILT4-G10 CDR3 <400> 30 Ala Thr Leu Cys Ser Leu Leu Leu Arg Val Ala Arg Pro Ile Ile Ala 1 5 10 15 Asp Gly Gly Val 20 <210> 31 <211> 8 <212> PRT <213> Artificial <220> <223> ILT4-H12 CDR1 <400> 31 Gly Phe Thr Leu Asp Ser Tyr Ala 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial <220> <223> ILT4-H12 CDR2 <400> 32 Ile Ser Ser Ser Gly Gly Ser Thr 1 5 <210> 33 <211> twenty one <212> PRT <213> Artificial <220> <223> ILT4-H12 CDR3 <400> 33 Ala Ala Leu Arg Glu Gly Ser Tyr Tyr Pro Asp Asp Asp Ala Cys Arg 1 5 10 15 Asp Gly Met Asp Tyr 20 <210> 34 <211> 134 <212> PRT <213> artificial <220> <223> >ILT4 - B8 <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Ser Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Asn Ser Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Lys Gly Phe Ala Ser Ser Cys His Gly Leu Gly Ala Ala 100 105 110 Tyr Asp Ser Asp Tyr Glu Ser Leu Tyr Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Gln Val Thr Val Ser Ser 130 <210> 35 <211> 125 <212> PRT <213> artificial <220> <223> >ILT4-C7 <400> 35 Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Leu Glu 20 25 30 His Tyr Ser Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Ser Asn Ser Gly His Thr Thr Lys Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala Thr Pro Arg Gly Trp Gly Leu Thr Ser Asn Gln Tyr 100 105 110 Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 126 <212> PRT <213> artificial <220> <223> >ILT4-C9 <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Asn Gly Tyr 20 25 30 Thr Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Arg Ala Ile Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Ser Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Arg Leu Arg Val Ser Asn Gly Asn Ala Trp Ser Ser Ser Ser 100 105 110 Tyr Leu His Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 120 <212> PRT <213> artificial <220> <223> ILT4 - A2 <400> 37 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Val Asn Ala Tyr 20 25 30 His Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Phe Ile Ser Ser Asp His Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Gly Thr Val Tyr Leu Gln Met 65 70 75 80 Asn Arg Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Ala Thr 85 90 95 Ala Asn Gln Leu Trp Gln Val Trp Met Gly Ile Thr Ser Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 38 <211> 123 <212> PRT <213> artificial <220> <223> >ILT4-D8 <400> 38 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Ser Arg Pro Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Arg Glu Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Ser Met Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Arg Tyr Ser Thr Cys Ser Gly Met Ala Arg Asp Tyr Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 39 <211> 130 <212> PRT <213> artificial <220> <223> > ILT4-D12 <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp His Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Glu Lys Arg Glu Gly Val 35 40 45 Ser Cys Ile Asn Val Asn Arg Gly Thr Ser Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Asn Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Leu Leu Glu Gly Ala Ser Gly Asp Val Cys Asp Asp Val Asn 100 105 110 Gln Asn Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val 115 120 125 Ser Ser 130 <210> 40 <211> 123 <212> PRT <213> artificial <220> <223> > ILT4-E7 <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Arg His Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Thr Ser Arg Glu Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Met Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Tyr Ser Ser Cys Ser Gly Met Pro Ser Asp Tyr Asn Tyr 100 105 110 Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 41 <211> 122 <212> PRT <213> artificial <220> <223> > ILT4-F5 <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Val Asn Ala Tyr 20 25 30 His Ile Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Gly Val 35 40 45 Ser Phe Ile Ser Ser Asp Arg Glu Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Thr Ala Asn Gln Leu Trp Arg Leu Trp Met Gly Ile Ser Ser Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 42 <211> 123 <212> PRT <213> artificial <220> <223> > ILT4-G3 <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Arg His Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gly Leu 35 40 45 Ser Cys Ile Ser Ser Arg Glu Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Gln Asp Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Met Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Arg Tyr Ser Ser Cys Ser Gly Met Pro Arg Asp Tyr Asn Tyr 100 105 110 Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 43 <211> 127 <212> PRT <213> artificial <220> <223> >ILT4-G10 <400> 43 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Ala Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Leu 35 40 45 Ser Gly Ile Asp Trp Thr Gly Ser Ser Ser Asn Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg His Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Leu Cys Ser Leu Leu Leu Arg Val Ala Arg Pro Ile Ile Ala 100 105 110 Asp Gly Gly Val Trp Ser Gln Val Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 44 <211> 128 <212> PRT <213> artificial <220> <223> >ILT4-H12 <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ser Tyr 20 25 30 Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Ser Tyr Tyr Cys 85 90 95 Ala Ala Leu Arg Glu Gly Ser Tyr Tyr Pro Asp Asp Asp Ala Cys Arg 100 105 110 Asp Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 402 <212> DNA <213> artificial <220> <223> >ILT4-B8 <400> 45 gaggtgcagc tggttgagtc ggggggaggt ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgtag cctctggatt cactttggat tattatgcca taagctggtt ccgccgggcc 120 ccagggaagg agcgtgaggg tgtctcatgt attggtaata gtggtgatag cacaaactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtttat 240 ctgcaaatga acagcctgaa acctgaggaa acagccgttt attactgcgc agcgcgaaaa 300 gggttcgcta gttcctgtca cggcctcggg gctgcatacg atagtgacta tgaatcgttg 360 tatgactact ggggccaggg gacccaggtc accgtctcct ca 402 <210> 46 <211> 370 <212> DNA <213> artificial <220> <223> >ILT4-C7 <400> 46 tcaggtgcag ctcgtggagt cggggggagg cttggtgcag cctggggggt ctctgagact 60 ctcctgtgca gcctctggat tcgctttgga acattattcc ataggctggt tccgccaggc 120 cccagggaag gagcgtgagg gggtctcatg tattagtaat agtgggcata ccacaaagta 180 tgcagactcc gtgaagggcc gattcaccgt ctccagagac aacgccaaga acacggtgta 240 tctgcaaatg aacagcctga agcctgagga cacagccgtt tattactgtg cagcgacacc 300 aaggggttgg ggcctaacgt cgaatcagta tgaatactgg ggccagggga cccaggtcac 360 cgtctcatca 370 <210> 47 <211> 378 <212> DNA <213> artificial <220> <223> >ILT4-C9 <400> 47 gaggtgcagc tggtagagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctggacg caccctcaat ggttatacca ctggctggtt ccgccaggct 120 ccagggaagg agcgcgagtt tgtagccact attcgcgcga ttgatggtag cacatcctat 180 gcagactccg tgatgggccg attcaccatc tccagagaca acgccaaaaa tacgctctat 240 cttgaaatga acagactgaa acctgaggac tcggccctgt attattgtgc ggcgcggctc 300 agagtgagca atggtaacgc atggtcatca tctagctatc tccactgggg ccgggggacc 360 caggtcaccg tctcctca 378 <210> 48 <211> 361 <212> DNA <213> artificial <220> <223> ILT4-A2 <400> 48 tcagttgcag ctcgtggagt ctgggggagg cttggtgcag cctggggggt ctctgagact 60 ctcctgtgca gcctctggat tcaacgtgaa tgcttatcac ataggctggt tccgccaggc 120 cccaggaaag gagcgtgagg gggtctcatt cattagtagt gatcatagca caaactatgc 180 agactccgtg aagggacgat tcaccatctc cagagacagc ggtacggtgt atctgcaaat 240 gaacagactg aaacctgagg acacagccac ttactattgt gcagcaaccg ccaatcaact 300 gtggcaagta tggatgggca ttacgtcctg gggccagggg acccaggtca ccgtctcctc 360 g 361 <210> 49 <211> 370 <212> DNA <213> artificial <220> <223> >ILT4-D8 <400> 49 tcagttgcag ctcgtggagt cggggggagg cttggtgcag cctggggggt ctctgagact 60 ctcctgtgca gcctctggat tgacttcgcg cccttattac ataggttggt ttcgccagtc 120 cccagggaag gagcgtgagg gagtctcatg tattagtagt agggaaggta gcacaaacta 180 cgcagactcc gtgaaggatc gattcaccat ctccagagat aacgctcaga gtatgtttta 240 tctgcaaatg aacagcctga aatctgagga cacagccgtt tattactgtg caggacgtta 300 ttcgacatgt tcaggaatgg cccgcgatta taactactgg ggtcagggga cccaggtcac 360 cgtctcctca 370 <210> 50 <211> 390 <212> DNA <213> artificial <220> <223> > ILT4-D12 <400> 50 gaggtgcagc tggtagagtc tgggggaggc ttggtgcagc ctggggggtc tctgaaactc 60 tcctgtgcag cctctggatt cactttggat cattatgcca taggctggtt ccgccaggcc 120 ccaggcgaaa agcgtgaagg cgtctcatgt attaatgtta atcgtggtac ctccgcctat 180 gcagactccg tgaagggccg tttcaccatc tcgcgagaca acgccaagaa cacggtgtat 240 ctgcaaatgg acaacctgag acctgaggac acagccgttt attactgtgc agccctactc 300 gagggggcct caggcgatgt gtgtgatgac gtcaatcaaa actacggcat ggactactgg 360 ggcaaaggga ccctggtcac cgtctcctca 390 <210> 51 <211> 369 <212> DNA <213> artificial <220> <223> > ILT4-E7 <400> 51 gaggtgcagc tggttgagtc ggggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggatc gacttcgcgc cattattaca taggctggtt ccgccaggcc 120 ccaggaaagg agcgtgaagg tgtctcatgt attactagta gggaaggtag cacaaactac 180 gcagactccg tgaaggaccg attcaccatc tccagagaca acgctcagaa tatgttttat 240 ttgcaaatga acagcctgaa atctgaggac acagccgttt attactgtgc agcacgttat 300 tcgagttgtt caggaatgcc ctcagattat aactactggg gtcgggggac ccaggtcacc 360 gtctcctca 369 <210> 52 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> > ILT4-F5 <400> 52 gaggtgcagc tggtagagtc ggggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggatt caacgtgaat gcttatcaca taggctggtt ccgccaggcc 120 ccaggagagg agcgtgaagg ggtctcattc attagtagtg atcgtgagac aaactatgca 180 gactccgtga agggccgatt caccatctcc agagacagcg ccaagaacac ggtgtatctg 240 caaatgaaca gactgaaacc tgaggacaca gccgtttatt actgtgcagc aaccgccaat 300 caactgtggc gactatggat gggcattagt tcctggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 53 <211> 369 <212> DNA <213> artificial <220> <223> > ILT4-G3 <400> 53 gaggtgcagc tggtagagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggatt cacttcgcgc cattattaca taggttggtt tcgccagtcc 120 ccagggaagg agcgtgaggg gctctcatgt attagtagta gggaaggtag cacaaactac 180 gcagactccg tacaggaccg attcaccatc tccagagaca acactcagaa tatgttttat 240 ttgcaaatga acagcctgaa atctgaggac acagccgttt attactgtgc aggacgttat 300 tcgagttgtt caggaatgcc ccgggattat aactactggg gtcgggggac ccaggtcacc 360 gtctcctca 369 <210> 54 <211> 381 <212> DNA <213> artificial <220> <223> >ILT4-G10 <400> 54 caggtgcagc tcgtggagtc cgggggaggc ttggtgcacc ctggggggtc tctgagactc 60 tcctgtgcag cctctggatt cacttttttt gcgtatacca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgaggt tctctcaggt attgactgga ccgggagtag ctcaaactat 180 gcagactccg tgaggggccg attcaccatc tccagacaca acgccaagaa tacgctgtat 240 ctacaaatga acagcctgaa acctgacgac acggccgtgt attactgtgc gacactttgt 300 tctctactgt tacgggtcgc gcggccaatt attgctgatg ggggagtttg gagccaggtg 360 acccaggtca ccgtctcctc a 381 <210> 55 <211> 384 <212> DNA <213> artificial <220> <223> >ILT4-H12 <400> 55 gaggtgcagc tggtagagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggatt cactttggat tcatatgccg taggctggtt ccgccaggcc 120 ccagggaagg agcgtgaggg agtctcatgt attagtagta gtggtggtag cacacactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctgcaaatga gcagcctgaa acctgaggac acagccagtt attactgtgc agcacttcgc 300 gaaggttctt actatcccga cgatgacgcg tgtcgtgacg gcatggacta ctggggcaaa 360 gggacccagg tcaccgtctc ctca 384

Claims

1. A single domain antibody (sdAb) that specifically binds to leukocyte immunoglobulin-like receptor subfamily B, member 2 (LILRB2), wherein the sdAb comprises three complementarity determining regions (CDRs), wherein CDR1 consists of the amino acid sequence of SEQ ID NO: 1, CDR2 consists of the amino acid sequence of SEQ ID NO: 2, and CDR3 consists of the amino acid sequence of SEQ ID NO:

3.

2. The sdAb of claim 1, wherein the LILRB2 is human LILRB2. 3 . The sdAb of claim 1 , wherein the sdAb does not bind to leukocyte immunoglobulin-like receptor subfamily B, member 1 (LILRB1). The sdAb of claim 3 , wherein the LILRB1 is human LILRB1.

5. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 90% amino acid sequence identity thereto.

6. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 92% amino acid sequence identity thereto.

7. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 94% amino acid sequence identity thereto.

8. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 95% amino acid sequence identity thereto.

9. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 96% amino acid sequence identity thereto.

10. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 97% amino acid sequence identity thereto.

11. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 98% amino acid sequence identity thereto.

12. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 99% amino acid sequence identity thereto.

13. The sdAb of claim 1, comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having greater than 99% amino acid sequence identity thereto. The sdAb of claim 1 , comprising the amino acid sequence of SEQ ID NO:

34.

15. The sdAb of claim 1, wherein the sdAb inhibits the interaction between LILRB2 and human leukocyte antigen-G (HLA-G).

16. The sdAb of claim 1, wherein the sdAb inhibits the interaction between LILRB2 and angiopoietin-like 2 (ANGPTL2).

17. The sdAb of any one of claims 1-16, wherein the sdAb is conjugated to at least one molecule, wherein the at least one molecule is a drug, an imaging molecule, a diagnostic agent, a tracer, a label, or a dye.

18. The sdAb of any one of claims 1-16, wherein the sdAb is conjugated to a stabilizing group.

19. The sdAb of claim 18, wherein the stabilizing group is Fc or IgG.

20. An isolated nucleic acid comprising a nucleic acid sequence encoding the sdAb of any one of claims 1 to 19.

21. The isolated nucleic acid of claim 20, wherein the isolated nucleic acid comprises the sequence of SEQ ID NO:

45.

22. A vector comprising the isolated nucleic acid according to claim 20 or 21.

23. A chimeric antigen receptor (CAR) comprising the sdAb according to any one of claims 1 to 19.

24. A cell comprising the isolated nucleic acid according to claim 20 or 21.

25. The cell of claim 24, wherein the cell is selected from the group consisting of: a T cell, a B cell, a NK cell, a monocyte, and a dendritic cell.

26. The cell according to claim 25, wherein the T cell is a CD4 + T cells, CD8 + T cells or NKT cells.

27. A pharmaceutical composition comprising the sdAb according to any one of claims 1 to 19, and a pharmaceutically acceptable carrier.

28. Use of the pharmaceutical composition according to claim 27 in the preparation of a medicament for treating cancer, wherein the cancer overexpresses LILRB2.

29. Use of the pharmaceutical composition according to claim 27 in the preparation of a reagent for detecting LILRB2 on tumor cells or tissues, wherein the tumor overexpresses LILRB2.

30. The use according to claim 28 or 29, wherein the cancer or tumor is selected from: lung cancer, pancreatic cancer, endometrial cancer, hepatocellular carcinoma, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, kidney cancer, testicular cancer, esophageal cancer, cervical cancer, leukemia, thyroid cancer, urothelial carcinoma and head and neck cancer.

31. The use according to claim 30, wherein the lung cancer is mouse Lewis lung cancer.

32. The use according to claim 28 or 29, wherein the cancer or tumor is selected from: non-small cell lung cancer (NSCLC), pancreatic ductal carcinoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and liver cancer.

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