NOVEL ANTI-LILRB2 ANTIBODIES AND DERIVATIVE PRODUCTS

CROSS-REFERENCE

[001] This application claims priority to U.S. provisional patent applications no.

63/274,511, filed November 01, 2021, the disclosure of which is incorporated herein by reference.

SEQUENCE LISTING

[002] The sequence listing that is contained in the file named “066564-

8016W001_ST26”, which is 554,405 bytes (as measured in Microsoft Windows) and was created on November 01, 2022, is filed herewith by electronic submission and is incorporated by reference herein.

BACKGROUND

[003] I. Field

[004] The present disclosure relates generally to the fields of biochemistry, molecular biology, cell biology, immunology and oncology. More particular, the disclosure relates to antibodies that bind to LILRB2 and their use in detecting LILRB2.

[005] II. Description of Related Art

[006] Leukocyte Immunoglobulin-Like Receptors (LILR), also known as Immunoglobulin-like transcripts (ILTs) and CD85, are a family of receptors that can exert immunomodulatory effects on a wide range of immune cells. The LILRs belong to the Ig superfamily and have either two or four Ig-like extracellular domains. There are inhibitory (LILRB1-5) and activating (LILRA1-6) receptors. The expression of LILRs varies widely on immune cells, but they are found primarily on antigen presenting cells (APC) of the myeloid lineage such as monocytes, macrophages and dendritic cells, as well as on B cells and on subsets of NK and T cells.

[007] LIR subfamily B member 2 (LILRB2), also known as Immunoglobulin-like transcript 4 (ILT4 or ILT-4), Leukocyte Immunoglobulin-like Receptor 2 (LIR2 or LIR-2), and CD85d or CD85D has emerged as a key immune checkpoint mediating the tolerogenic activity of myeloid cells associated with cancer and is thought to be expressed primarily by myeloid cells (monocytes, macrophages, dendritic cells and neutrophils). However, the accurate identification of LILRB2 expression by immunohistochemistry and immunofluorescence staining has been a problem due to the lack of antibodies specifically binding to LILRB2 and not cross-reacting with other LILR family members. Therefore, there is a significant need for novel anti-LILRB2 antibodies to provide highly sensitive and specific detection of LILRB2 by immunohistochemistry.

BRIEF SUMMARY OF THE INVENTION

[008] The present disclosure provides anti-LILRB2 antibodies and antigen-binding fragment thereof, amino acid and nucleotide sequences thereof, anti-LILRB2 chimeric antigen receptors, and uses thereof.

[009] In one aspect, the present disclosure provides an isolated anti-LILRB2 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-LILRB2 antibody or an antigen-binding fragment comprises a heavy chain (HC) variable region (VH) and a light chain (LC) variable region (VL), wherein the VH and VL comprise clone-paired complementarity determining region (CDR) sequences as set forth in Table 1, and variants thereof wherein one or more of the HC-CDRs and/or LC-CDRs has one, two, or three amino acid substitutions, additions, deletions or combination thereof.

[0010] In some embodiments, the VH and VL have amino acid sequences at least 90% or 95% identical to clone-paired sequences of Table 2. In some embodiments, the VH and VL have amino acid sequences identical to clone-paired sequences of Table 2.

[0011] In some embodiments, the isolated antibody is a mouse, a rat, a hamster, a guinea pig, sheep, goat, donkey, horse, llama, or a rabbit antibody.

[0012] In some embodiments, the antigen-binding fragment is a recombinant ScFv (single chain fragment variable) antibody, a Fab fragment, a F(ab’)2 fragment, or a Fv fragment.

[0013] In some embodiments, the isolated antibody or an antigen-binding fragment thereof disclosed herein further comprises an immunoglobulin constant region, optionally a constant region of Ig, or optionally a constant region of IgG.

[0014] In some embodiments, the isolated antibody or an antigen-binding fragment thereof disclosed herein is linked to a detectable label. In some embodiments, the detectable label is a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemo-luminescent molecule, or a dye. [0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated antibody or an antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof disclosed herein.

[0017] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof disclosed herein.

[0018] In another aspect, the present disclosure provides a host cell comprising the vector disclosed herein.

[0019] In yet another aspect, the present disclosure provides a method of producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell disclosed herein under the condition at which the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.

[0020] In another aspect, the present disclosure provides a method for detecting LILRB2 in a sample. In some embodiments, the method comprises: (a) contacting a sample containing LILRB2 with the antibody or an antigen-binding fragment thereof disclosed herein, and (b) detecting binding of the antibody or an antigen-binding fragment thereof to the LILRB2 in the sample.

[0021] In some embodiments, the sample is a cell, tissue or organ. In some embodiments, the sample is obtained from a human subject.

[0022] In some embodiments, the binding of the antibody or an antigen-binding fragment thereof to the LILRB2 is detected by immunohistochemistry, immunofluorescence, flow cytometry, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or other immunoassays.

[0023] In another aspect, the present disclosure provides a method of generating an antibody specifically binding to LILRB2. In some embodiments, the method comprises immunizing a non-human animal with a peptide having an amino acid sequence as set forth in any one of SEQ ID NO: 25-27. In some embodiments, the method further comprises generating from the animal a hybridoma expressing an antibody specifically binding to LILRB2. In some embodiments, the method further comprises obtaining a polynucleotide sequence encoding the antibody specifically binding to LILRB2. [0024] In some embodiments, the non-human animal is a mouse, a rat, a hamster, a guinea pig, llama, goat, sheep, donkey, horse or a rabbit.

[0025] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof disclosed herein, useful in detecting LILRB2.

BRIEF DESCFRIPTION OF FIGURES

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] Figure 1 shows the screening of anti-LILRB2 hybridoma clones in the immunohistochemistry (IHC) assay. Formalin-fixed paraffin-embedded (FFPE) cell pellets of HEK293 stably expressing full length LILRB2 (hereafter designated as HEK293 LILRB2) were processed and stained with supernatant of hybridoma clones generated from mouse immunization with antigen GQIHGTPFI (Figure 1A, 1 : 10 dilution) or MGSSPPPTGPISTP (Figure IB, 1 :5 dilution). Clone 16B9 was used as positive control for results shown in Figure 1A.

[0028] Figure 2 shows the results for binding specificity testing of anti-LILRB2 hybridoma clones in the IHC assay. HEK293 cells cultured on microscope slide coverslips were transiently transfected with LILRB2, LILRB1, LILRB3 or LILRA6. Non-transfected cells were used as negative control. The specificity of 16B9, 29F12, 49G11 (Figure 2A) or 32G8, 37B2 (Figure 2B) were tested using IHC staining with supernatant diluted 1 :5 from each hybridoma clone.

[0029] Figure 3 shows the titration of anti-LILRB2 recombinant antibodies in the IHC assay. FFPE sections of HEK293 or HEK293 LILRB2 cell pellets were processed and stained with a 2-fold dilution series (10-0.15625 pg/mL) of recombinant 16B9 or 37B2. Nuclei were counterstained with hematoxylin.

[0030] Figure 4 shows results of specificity testing for recombinant 16B9 and 37B2 antibodies. (Figure 4A) Sequence alignment of peptides among different members of LILR family compared to peptide sequence of LILRB2 antigen (MGSSPPPTGPISTP). (Figure 4B- 4D) Evaluation of cross-reactivity of recombinant 16B9 (Figure 4B and Figure 4C) and 37B2 (Figure 4D) antibodies to the peptides from different LILR, as assessed by ELISA (Figure 4B) and bio-layer interferometry (BLI) (Figure 4C and Figure 4D). (Figure 4E) HEK293 cells cultured on microscope slide coverslips were transiently transfected with LILRB2, LILRB1 or LILRB3, then the specificity of 16B9 was tested at different concentrations, using IHC staining.

[0031] Figure 5 shows the detection of endogenous LILRB2 in human normal tissues by IHC using recombinant 16B9 and 37B2. FFPE sections of lung, spleen or bone marrow from healthy human donors were stained by IHC using 16B9 (Figure 5 A and Figure 5B) or 37B2 (Figure 5B) at different concentrations. The nuclei were counterstained with hematoxylin.

[0032] Figure 6 shows the detection of endogenous LILRB2 in human normal tissues using recombinant 16B9 by IHC and immunofluorescent staining. (Figure 6A) FFPE tissue sections of human normal lung were stained by IHC with recombinant 16B9 antibody or anti- CDl lb. The nuclei were counterstained with hematoxylin. (Figure 6B) FFPE tissue sections of human normal lung and spleen were stained with recombinant 16B9 and anti-CDl lb antibodies by immunofluorescence staining; nuclei were counterstained with 4’,6-diamidino- 2-phenylindoJe (DAPI).

[0033] Figure 7 shows the detection of endogenous LILRB2 in human tumor tissues by IHC or immunofluorescent staining using recombinant 16B9 and 37B2 antibodies. (Figure 7 A) FFPE sections of human tumors of breast, colon, ovary, liver or lung were stained by IHC with 2.5 pg/mL recombinant 16B9 or anti-CD163. The nuclei were counterstained with hematoxylin. (Figure 7B) FFPE sections of human tumors of colon and ovary were stained with recombinant 16B9 and anti-CD163 antibodies by immunofluorescence; nuclei were counterstained with DAPI. (Figure 7C) FFPE sections of human ovarian tumor were stained by IHC with different concentrations of recombinant 16B9 or 37B2 antibodies. The nuclei were counterstained with hematoxylin.

[0034] Figure 8 shows the identification of the critical LILRB2 amino acids for binding of 16B9 or 37B2 antibodies. (Figure 8A and Figure 8B) Binding sensorgrams obtained by BLI analysis of captured biotinylated peptides interacting with recombinant 16B9 (Figure 8A) or 37B2 (Figure 8B). (Figure 8C) Kinetic rates (k _on and k _O ff) and dissociation constants (K _D ) were determined based on data shown in Figure 8A and Figure 8B, OR = out of range. (Figure 8D) Peptides synthesized to determine which amino acid residues are necessary for antibody specificity to LILRB2 versus LILRA6 or LILRB3. (Figure 8E and Figure 8F) Binding sensorgrams obtained by BLI analysis of captured biotinylated peptide interacting with recombinant 16B9 (Figure 8E) or 37B2 (Figure 8F). [0035] Figure 9 shows that 16B9 recombinant antibody can detect LILRB2 by IHC staining in FFPE samples of HEK293 LILRB2 cells that have been pre-treated with a therapeutic anti-LILRB2, in this case an antagonist antibody (e.g., a B2-19 variant^ Cultured HEK293 LILRB2 cells were detached from tissue culture dishes, resuspended in culture medium, and then treated, or left untreated, with 20 pg/mL anti-LILRB2 antagonist antibody in suspension for 1 hour at 37 °C. The cells were then washed in PBS, processed into FFPE sections and stained with 16B9 recombinant antibody at different concentrations. The nuclei were counterstained with hematoxylin.

[0036] Figure 10 shows the logo plot of enriched NGS sequences after ELISA binding selection of a 16B9 ribosome display scFv NNK walk library on peptide #1, #5 or #8. Sequences with >100 reads were included in the analysis and the total number of reads derived from sequencing of each amplicon sample is shown. Positions indicated by an astrix were changed in IgG variants.

[0037] Figure 11 shows the bilayer interferometry (BLI) sensorgrams of five different versions of the 16B9 antibody. Peptides #1, A6, B3, Bl, #5, #6 and #8 are shown in order from top to bottom. After peptides were captured at a concentration of 7ug/ml on streptavidin sensors, kinetic binding of lOOnM of mouse IgGi kappa antibody was evaluated (association and dissociation).

[0038] Figure 12 shows the IHC staining with 16B9 and its variants in human heart tissue and lung cancer tissues. Human lung cancer and left ventricle FFPE slides were IHC stained using 5 ug/ml 16B9 or 16B9 V2, V3, V4 and V5 for 30 minutes at room temperature. Nuclear counterstain was carried out with haematoxylin. Images were acquired using 40x objective.

[0039] Figure 13 shows the IHC staining with 16B9 and its variants in human lung cancer tissues with different antigen retrieval methods. Human lung cancer FFPE slides were subjected to different antigen retrieval and then IHC stained using 15 ug/ml 16B9 and its variants overnight at 4°C. Nuclear counterstain was carried out with haematoxylin. Images were acquired using 40x objective.

[0040] Figures 14A-14C show the staining and scoring of human cancer samples based on LILRB2, CD3 and CD163 IHC. CD163, CD3 and LILRB2 were stained with respective IHC antibodies in 195 evaluable samples from 19 different human cancer indications. Figure 14A shows the number of samples in each LILRB2 IHC reactivity score (0-5) in macrophages across solid tumor types. Figure 14B shows the average score and correlation between LILRB2, CD 163 and CD3 IHC staining across tumor types. Figure 14C shows the ranking of tumor types stained by IHC based on reactivity scores. Each tissue was evaluated by CRO’s board-certified pathologist separately for LILRB2, CD163, and CD3 reactivity. Positively staining immune cells were assessed and scored within tumor and tumor-induced stroma (TIS). The number of reactive cells for each biomarker in each sample was counted at 20X magnification over multiple fields and the cell counts were converted to IHC reactivity score of 0-5 as defined here: 5: >100 reactive cells; 4: 51-100 reactive cells; 3: 26-50 reactive cells; 2: 11-25 reactive cells; 1 : 1-10 reactive cells; 0: <1 reactive cells.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

[0042] I. Definitions

[0043] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this disclosure, the term “or” is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. As used herein “another” may mean at least a second or more. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both element or component comprising one unit and elements or components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.

[0044] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. [0045] The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multi-specific antibody, or bispecific antibody that binds to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable domain (VH) and a constant region including a first, second, and third constant domain (CHI, CH2, CH3, respectively); mammalian light chains are classified as X or K, while each light chain consists of a variable domain (V _L ) and a constant domain (CL). A typical IgG antibody has a “Y” shape, with the stem of the Y typically consisting of the second and third constant domains of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable domain and first constant domain of a single heavy chain bound to the variable and constant domains of a single light chain. The variable domains of the light and heavy chains are responsible for antigen binding. The variable domains in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for the antibodies and antigenbinding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. (1985) 186(3):651 -63 ; Chothia, C. and Lesk, A.M., J.Mol.Biol. (1987) 196:901; Chothia, C. et al., Nature (1989) 342(6252):877-83; Marie-Paule Lefranc et al., Developmental and Comparative Immunology (2003) 27: 55-77; Marie-Paule Lefranc et al., Immunome Research (2005) 1(3); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). The three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant domains of the heavy and light chains are not involved in antigen-binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (gammal heavy chain), IgG2 (gamma2 heavy chain), IgG3 (gamma3 heavy chain), IgG4 (gamma4 heavy chain), IgAl (alpha 1 heavy chain), or IgA2 (alpha2 heavy chain). [0046] The term “antigen” refers to a substance capable of inducing adaptive immune responses. Specifically, an antigen is a substance specifically bound by antibodies or T lymphocyte antigen receptors. Antigens are usually proteins and polysaccharides, less frequently also lipids. Suitable antigens include without limitation parts of bacteria (coats, capsules, cell walls, flagella, fimbrai, and toxins), viruses, and other microorganisms. Antigens also include tumor antigens, e.g., antigens generated by mutations in tumors. As used herein, antigens also include immunogens and haptens.

[0047] The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab’, a F(ab’) ₂ , an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) ₂ , a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a bispecific antibody, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.

[0048] A “Fab fragment” comprises one light chain and the CHI and variable domains of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0049] A “Fab' fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and C _H 2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab') ₂ molecule.

[0050] A “F(ab') ₂ fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab') ₂ fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[0051] “Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site. An Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain. [0052] “Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable domain and a heavy chain variable domain connected to one another directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA (1988) 85:5879).

[0053] An “Fc” region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C _H 3 domains. The Fc region of the antibody is responsible for various effector functions such as antibody-dependent cell- mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC), but does not function in antigen binding.

[0054] “Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.

[0055] A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable domain of a single light chain and the variable domain of a single heavy chain is a disulfide bond. In some embodiments, a “(dsFv) ₂ ” or “(dsFv-dsFv’)” comprises three peptide chains: two V _H domains linked by a peptide linker (e.g., a long flexible linker) and bound to two VL domains, respectively, via disulfide bridges. In some embodiments, dsFv- dsFv’ is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.

[0056] Camelized single domain antibody,” “heavy chain antibody,” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231 (1 -2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94/04678; WO94/25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature (1993) 363:446-8; Nguyen VK. Et al, Immunogenetics (2002) 54:39-47; Nguyen VK. Et al., Immunology (2003) 109:93-101). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. (2007) 21 :3490-8).

[0057] A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3. [0058] “Diabodies” or “dAbs” include small antibody fragments with two antigenbinding sites, wherein the fragments comprise a VH domain connected to a VL domain in the same polypeptide chain (V _H -V _L or V _L -V _H ) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15;90(14):6444-8 (1993); EP404097; WO93/11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a “bispecific ds diabody” is a diabody target two different antigens (or epitopes).

[0059] In certain embodiments, an “scFv dimer” is divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) that can be engineered by linking two scFvs. A bivalent diabody or bivalent scFv (BsFv, di-scFvs, bi-scFvs) comprising V _H -V _L (linked by a peptide linker) dimerized with another VH-VL moiety such that VH’S of one moiety coordinate with the VL’S of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes). In other embodiments, an “scFv dimer” is a bispecific diabody comprising V _H i-V _L 2 (linked by a peptide linker) associated with VLI-V _H2 (also linked by a peptide linker) such that VHI and VLI coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen specificity.

[0060] A “domain antibody” refers to an antibody fragment containing only the variable domain of a heavy chain or the variable domain of a light chain. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

[0061] A “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens.

[0062] Cancer” as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia. As used herein “solid tumor” refers to a solid mass of neoplastic and/or malignant cells. Examples of cancer or tumors include hematological malignancies, oral carcinomas (for example of the lip, tongue or pharynx), digestive organs (for example esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary passages, pancreas, respiratory system such as larynx or lung (small cell and non-small cell), bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tube, uterus, cervix, testicles, ovary, or prostate), urinary tract (e.g., bladder or kidney), brain and endocrine glands such as the thyroid. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular cancer, and colorectal cancer. In certain embodiments, the cancer is selected from a lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma and B-cell lymphoma.

[0063] The term “chimeric” as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse or rabbit. In some embodiments, the nonhuman animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, a llama, a horse, a donkey or a hamster.

[0064] The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human LILRB2 with a binding affinity (K _D ) of <10' ⁶ M (e.g., <5xl0' ⁷ M, <2xl0' ⁷ M, <10' ⁷ M, <5xl0' ⁸ M, <2xl0' ⁸ M, <10' ⁸ M, <5xl0' ⁹ M, <4xlO' ⁹ M, <3X10' ⁹ M, <2X10' ⁹ M, or <10' ⁹ M). KD used herein refers to the ratio of the dissociation rate to the association rate (k _o ff/k _on ), which may be determined by using any conventional method known in the art, including, but not limited to, BLI, surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. In certain embodiments, the K _D value can be appropriately determined by using flow cytometry.

[0065] Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is a protein that in humans is encoded by the LILRB2 gene. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is found in a gene cluster at chromosomal region 19ql3.4. The encoded protein belongs to the subfamily B class of LIR receptors which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). The receptor is expressed on myeloid cells, binds to MHC class I (MHC-I) and other ligands and transduces a negative signal that inhibits stimulation of an immune response. The receptor can also compete with CD8 to the binding of MHC-I. It is thought to negatively regulate immune inflammatory responses and cytotoxicity to help focus the immune response and limit autoimmunity. Multiple LILRB2 isoform and polymorphic variants have been identified. LILRB2 has been shown to interact with classical and non-classical MHC-I (most notably, human leukocyte antigen G (HLA-G)), angiopoietin-like protein (ANGPTL) 2 and 5, Semaphorin-4A (SEMA4A), complement split products (CSPs), CDlc/d, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (Omgp), P-amyloid protein and RIFIN.

[0066] The term “anti-LILRB2 antibody” refers to an antibody that is capable of specifically binding to LILRB2.

[0067] A “LILRB2-related” disease or condition as used herein refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of LILRB2. In some embodiments, the LILRB2 related condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease.

[0068] A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Vai, Leu, and He), among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gin), among residues with acidic side chains (e.g., Asp, Glu), among amino acids with basic side chains (e.g., His, Lys, and Arg), or among residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

[0069] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment may be considered to bind the same/closely related epitope as the reference antibody. [0070] The term “homologue” and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequence when optimally aligned.

[0071] The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.

[0072] An “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an “isolated antibody or antigen-binding fragment thereof’ refers to the antibody or antigenbinding fragments having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).

[0073] A “leader peptide” or “signal peptide” refers to a peptide having a length of about 5-30 amino acids that is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to proteins that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), are secreted from the cell, or are inserted into a cellular membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of a protein.

[0074] The term “link” as used herein refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and/or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.

[0075] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0076] “Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI), see also, Altschul S.F. et al., J. Mol. Biol. (1990) 215:403-410; Stephen F. et al., Nucleic Acids Res. (1997) 25:3389-3402), ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al., Methods in Enzymology (1996) 266:383-402; Larkin M.A. et al., Bioinformatics (2007) 23:2947-8), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.

[0077] The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3 '-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

[0078] The term “polypeptide” or “protein” means a string of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may include moieties in addition to amino acids (e.g., may be glycosylated) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “polypeptide” or “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a functional portion thereof. Those of ordinary skill will further appreciate that a polypeptide or protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.

[0079] As used herein, the term “sample” or “biological sample” refers to any sample that is taken from a subject (e.g., a human, such as a person with a disease of interest, or a person suspected of having such disease) and contains one or more molecule(s) of interest. The biological sample includes but not limited to cells (e.g., bacteria, yeast, virus, plant cells, animal cells and the like), tissues (e.g., biopsy tissue, paraffin embedded tissue and the like), and body fluids (e.g., blood, plasma, serum, saliva, pleural effusion, ascites, amniocentesis fluid, seroperitoneum and the like).

[0080] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0081] The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-la) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-Gseu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, pl5TV- L, pProl8, pTD, pRSlO, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNAl.l/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXTl, pCDEF3, pSVSPORT, pEF-Bos etc.

[0082] II. Anti-LILRB2 Antibody and Antigen-binding Fragment

[0083] The present disclosure in one aspect provides an anti-LILRB2 antibody and antigen-binding fragment thereof that has a high sensitivity and specificity to LILRB2.

[0084] Binding affinity of the antibody and antigen-binding fragment provided herein can be represented by K _D value, which represents the ratio of dissociation rate to association rate (k _of f/k _on ) when the binding between the antigen and antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g., K _D ) can be appropriately determined using suitable methods known in the art, including, for example, bio-layer interferometry (BLI).

[0085] Binding of the antibodies to LILRB2 can also be represented by “half maximal effective concentration” (EC ₅₀ ) value, which refers to the concentration of an antibody where 50% of its maximal effect (e.g., binding or inhibition etc.) is observed. The EC50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, flow cytometry assay, and other quantitative binding assays.

[0086] Specific anti-LILRB2 antibodies

[0087] The present disclosure in one aspect provides an anti-LILRB2 antibody and antigen-binding fragment thereof comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-LILRB2 antibody disclosed herein. CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in anti-LILRB2 antibody disclosed herein, yet substantially retain the specific binding affinity to LILRB2.

[0088] In certain embodiments, the anti-LILRB2 antibody has CDR sequences as listed in Table 1 below.

[0089] Table 1. CDR sequences of the anti-LILRB2 antibodies

[0090] In some embodiments, the anti-LILRB2 antibodies and antigen-binding fragments thereof provided herein comprise H-CDR sequences derived from clone 16B9, which have the formula as shown below:

[0091] HC-CDR1 : GX ₁ SIX ₂ SX ₃ YAX ₄ X ₅ (SEQ ID NO: 75), wherein Xi is Y, I or H, X ₂ is T or A, X ₃ is D or G, X ₄ is W or R, X ₅ is N, S or T;

[0092] HC-CDR2: X|IX ₂ YNPSLKS (SEQ ID NO: 76), wherein Xi is Y or H, X ₂ is T or A;

[0093] HC-CDR3: X!WX ₂ GX ₃ (SEQ ID NO: 77), wherein X ₄ is S or P, X ₂ is F or L, X ₃ is R, T, S, H, K, N, Q, or P.

[0094] In certain embodiments, the anti-LILRB2 antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can specifically bind to LILRB2. The CDR sequences provided in Table 1 can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, sheep, llama, horse, donkey, guinea pig, hamster, among others, using suitable methods known in the art such as recombinant techniques. [0095] In certain embodiments, the anti-LILRB2 antibodies and antigen-binding fragments thereof provided herein comprise heavy chain and light chain variable region amino acid sequences as provided in Table 2 below.

[0096] Table 2. Clone-Paired VH and VL Sequences

[0097] In some embodiments, the anti-LILRB2 antibodies and the antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain. In one embodiment, the anti- LILRB2 antibodies and the antigen-binding fragments provided herein is a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516).

[0098] In certain embodiments, the anti-LILRB2 antibodies and the fragments thereof provided herein further comprise an immunoglobulin constant region. In some embodiments, an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CHI, hinge, and/or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises CK or Ck.

[0099] The antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti- idiotypic antibody. A recombinant antibody is an antibody prepared in vitro using recombinant methods rather than in animals. [00100] Antigen-binding fragments

[00101] Provided herein are also anti-LILRB2 antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-LILRB2 antibodies provided herein, including for example, the exemplary antibodies whose CDR and variable sequences are provided herein, and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on).

[00102] In certain embodiments, an anti-LILRB2 antigen-binding fragment provided herein is a camelized single domain antibody, a diabody, a single chain Fv fragment (scFv), an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv’, an Fv fragment, a Fab, a Fab’, a F(ab’)2, a bispecific antibody, a ds diabody, a nanobody, a domain antibody, a single domain antibody, or a bivalent domain antibody.

[00103] A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

[00104] Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (21 itteri. 5 x io ⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

[00105] The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

[00106] In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

[00107] Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step- wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

[00108] An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., Nhydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

[00109] It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents. [00110] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

[00111] The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of crosslinker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- 1,3’- dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

[00112] In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[00113] U.S. Patent 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.

[00114] U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. [00115] Various techniques can be used for the production of such antigen-binding fragments. Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods (1992) 24: 107-117; and Brennan et al., Science (1985) 229:81), recombinant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv antibody fragments), screening from a phase display library as discussed above (e.g. for ScFv), and chemical coupling of two Fab’-SH fragments to form F(ab’) ₂ fragments (Carter et al., Bio/Technology (1992) 10: 163-167). Other techniques for the production of antibody fragments will be apparent to a skilled practitioner.

[00116] In certain embodiments, the antigen-binding fragment is a scFv. Generation of scFv is described in, for example, WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. scFv may be fused to an effector protein at either the amino or the carboxyl terminus to provide for a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).

[00117] Conjugates

[00118] In some embodiments, the anti-LILRB2 antibodies and antigen-binding fragments thereof further comprise a conjugate moiety. The conjugate moiety can be linked to the antibodies and antigen-binding fragments thereof. A conjugate moiety is a proteinaceous or non-proteinaceous moiety that can be attached to the antibody or antigenbinding fragment thereof. It is contemplated that a variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.

[00119] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.

[00120] In certain embodiments, the antibodies may be linked to a conjugate moiety indirectly, or through another conjugate moiety. For example, the antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate can be a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), a clearance-modifying agent, or purification moiety.

[00121] Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luciferases, glucoamylase, lysozyme, saccharide oxidases or p-D-galactosidase), radioisotopes (e.g. ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ^m In, ¹¹² In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ LU, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, and ³² P, other lanthanides), luminescent labels, chromophore moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection.

[00122] In certain embodiments, the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody. Illustrative examples include water- soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules.

[00123] In certain embodiments, the conjugate moiety can be a purification moiety such as a magnetic bead.

[00124] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein is used for a base for a conjugate.

[00125] Polynucleotides and Recombinant Methods

[00126] The present disclosure provides isolated polynucleotides that encode the anti- LILRB2 antibodies and antigen-binding fragments thereof disclosed herein. In certain embodiments, the isolated polynucleotides comprise one or more nucleotide sequences listed in Table 1 that encodes the variable region of the exemplary antibodies provided herein. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[00127] The isolated polynucleotide that encodes the anti-LILRB2 antibodies and antigen-binding fragments can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-la), and a transcription termination sequence.

[00128] The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibodies or antigen-binding fragments, at least one promoter (e.g., SV40, CMV, EF-la) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-Gseu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, pl5TV-L, pProl8, pTD, pRSlO, pLexA, pACT2.2, pCMV- SCRIPT.RTM., pCDM8, pCDNAl. l/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXTl, pCDEF3, pSVSPORT, pEF-Bos etc.

[00129] Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[00130] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-LILRB2 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe,' Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus,' yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida,' Trichoderma reesia (EP 244,234); Neurospora crassa, Schwanniomyces such as Schwanniomyces occidentalism and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[00131] Suitable host cells for the expression of glycosylated antibodies or antigenfragment provided here are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, com, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[00132] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. (1977) 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese Hamster Ovary cells (CHO), CHO cells deficient in dihydrofolate reductase (DHFR) activity, CHO-DHFR (Urlaub et al., Proc. Natl. Acad. Sci. USA (1980) 77:4216); mouse 27itteri cells (TM4, Mather, Biol. Reprod. (1980) 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some preferable embodiments, the host cell is 293F cell.

[00133] Host cells are transfected with the above-described expression or cloning vectors for anti-LILRB2 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the antibody may be produced by homologous recombination known in the art.

[00134] The host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle’s Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. (1980) 102:255, U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[00135] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology (1992) 10: 163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. [00136] The anti-LILRB2 antibodies and antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[00137] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma 1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. (1983) 62: 1-13). Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. (1986)5: 1567-75). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[00138] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[00139] Purification

[00140] In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

[00141] Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

[00142] In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[00143] Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies are bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).

[00144] Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

[00145] It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

[00146] III. Methods of Use of Anti-LILRB2 Antibodies

[00147] The present disclosure further provides methods of using the anti-LILRB2 antibodies or antigen-binding fragments thereof to detect presence or amount of LILRB2 in a sample. In some embodiments, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof disclosed herein and determining the presence or the amount of LILRB2 in the sample. The methods of detecting LILRB2 using an anti- LILRB2 antibody are generally immunoassays. An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a sample through the use of an antibody or an antigen. Immunoassays come in many different format and variants, including, without limitation, enzyme linked immunosorbent assay (ELISA), Western-blot, immunohistochemistry, immunofluorescence, flow cytometry and FACS. Additional immunoassays include radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay.

[00148] ELISA

[00149] Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

[00150] In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test sample suspected of containing LILRB2 is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-LILRB2 antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-LILRB2 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[00151] In another exemplary ELISA, the samples suspected of containing the LILRB2 are immobilized onto the well surface and then contacted with the anti- LILRB2 antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-LILRB2 antibodies are detected. Where the initial anti- LILRB2 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-LILRB2 antibody, with the second antibody being linked to a detectable label.

[00152] Irrespective of the format employed, ELIS As have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

[00153] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[00154] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[00155] “Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[00156] The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C or may be overnight at about 4°C or so.

[00157] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[00158] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[00159] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2 ’-azino-di-(3 -ethyl - benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

[00160] Western Blot

[00161] The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ nondenaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[00162] Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

[00163] The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pl), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[00164] In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF but are far more fragile and do not stand up well to repeated probing. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.

[00165] Immunohistochemistry and Immunofluorescence

[00166] The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC) or immunofluorescence staining. The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC and immunofluorescence studies of various prognostic factors and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[00167] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in 70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

[00168] Permanent sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.

[00169] Flow Cytometry and FACS

[00170] The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser- or impedance-based technology employed in many detection assays, including cell counting, cell sorting, biomarker detection and protein engineering. The technology suspends cells in a stream of fluid and passing them through an electronic detection apparatus, which allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials.

[00171] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogenous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell. In general, the technology involves a cell suspension entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescence of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.

[00172] In certain embodiments, to be used in flow cytometry or FACS, the antibodies of the present disclosure are labeled with fluorophores and then allowed to bind to the cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine.

[00173] Immunodetection Kits

[00174] In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to LILRB2, and optionally an immunodetection reagent.

[00175] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtiter plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[00176] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, the second antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

[00177] The kits may further comprise a suitably aliquoted composition of LILRBs, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[00178] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[00179] Diagnostic Methods

[00180] In some embodiments, the present disclosure provides methods of diagnosing a LILRB2 related disease or condition in a subject. In some embodiments, the method comprises: a) contacting a sample obtained from the subject with the antibody or antigenbinding fragment thereof provided herein; b) determining presence or amount of LILRB2 in the sample; and c) correlating the existence or the amount of the LILRB2 to the LILRB2- related disease or condition in the subject.

[00181] In some embodiments, the present disclosure provides kits comprising the antibody or antigen-binding fragment thereof provided herein, optionally conjugated with a detectable moiety. The kits may be useful in detection of LILRB2 or diagnosis of LILRB2 related disease.

[00182] In some embodiments, the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a LILRB2 related disease or condition in a subject, in the manufacture of a diagnostic reagent for diagnosing a LILRB2 related disease or condition.

[00183] IV. Methods of Producing Anti-LILRB2 Antibodies

[00184] In another aspect, the present disclosure provides a method of generating an antibody specifically binding to LILRB2. In some embodiments, the method comprises immunizing a non-human animal with a peptide fragment of LILRB2 that has low homology to other LILR family members. In some embodiments, the peptide has an amino acid sequence as set forth in any one of SEQ ID NO: 25-27. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m- maleimidobencoyl-N-hydroxysuccinimide ester, 38ittering38de and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund’s adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants and aluminum hydroxide adjuvant.

[00185] The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and 38ittering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and polyclonal antibodies specific to LILRB2 can be purified.

[00186] In some embodiments, the method further comprises the steps of generating monoclonal antibodies (mAbs) against LILRB2. The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step is immunization of an appropriate host. When a desired level of immunogenicity is obtained, the immunized animal can be used to generate mAbs.

[00187] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibodyproducing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).

[00188] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10' ⁶ to 1 x 10' ⁸ . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.

[00189] The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain is also used for drug selection of hybrids as EBV- transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

[00190] Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for Mab production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide Mabs in high concentration. The individual cell lines could also be cultured in vitro, where the Mabs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.

[00191] Mabs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[00192] It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10 ⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

[00193] Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates.

[00194] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLE 1

[00195] This example illustrates the generation and sequencing of anti-LILRB2 antibodies.

[00196] Potential peptides for mouse immunization were chosen by analyzing sequences of all human Leukocyte immunoglobulin-like receptors (LILRs) available in UniProt: LILRB1 (Q8NHL6), LILRB2 (Q8N423), LILRB3 (075022), LILRB4 (Q8NHJ6), LILRB5 (075023), LILRA1 (075019), LILRA2 (Q8N149), LILRA3 (Q8N6C8), LILRA4 (P59901), LILRB5 (LILRA5), and LILRA6 (Q6PI73). Specifically, the full-length protein sequences for isoform 1 from each LILR were aligned using XlibraryDisplay (Stafford et al, J Chem Inf Model 2014, 54, 10, 3020-3032) and analyzed by visual inspection to identify peptides from LILRB2 with low sequence homology to all other LILR members. Candidate peptides were then analyzed using NCBI BLAST (Johnson et al 2008 Nucleic Acids Res; 36 web server issue: W5-9) to confirm low match for other proteins in the human and mouse proteomes. DNAStar software was used to calculate their immunogenicity index (Jameson BA and Wolf H 1988 CABIOS, 4, 181-186) to estimate likelihood of eliciting an immune response in mice. Three peptides as listed in Table 3 were chosen for immunization.

[00197] Table 3: Peptides chosen for immunization

[00198] The chosen peptides were synthesized with a C-terminal Cys at 2 mg scale using standard solid-phase methods to >85% purity (GenScript Biotech, Piscataway, NJ). The synthesized peptides were characterized by mass spectrometry (MS) and high-pressure liquid chromatography (HPLC) and then conjugated to keyhole limpet hemocyanin (KLH). Each KLH-peptide conjugate was used for immunizing 3 separate mice (Balb/c). Anti-peptide titers were monitored by ELISA dilution series (1 : 1000 to 1 :512,000). Briefly, 1 pg/mL or peptide was directly coated in PBS, treated with diluted pre-immune serum or antiserum, and detection performed using Peroxidase-AffiniPure Goat Anti-Mouse IgG. Mice immunized with KLH-peptide conjugates Peptide No. 2 and Peptide No. 3 were selected for hybridoma screening and isolation.

[00199] Hybridoma sequencing

[00200] After subcloning, hybridoma cells (5xl0 ⁶ ) for the mouse IgGik isotype monocl onals cryopreserved in FBS with 10% DMSO were used to isolate total RNA. Cells were rapidly thawed in a 37°C water bath, immediately diluted into 5ml of DMEM with 10% FBS, and centrifuged at 300xg for 5 min. Cells were resuspended in ice-cold PBS and centrifuged at 300xg for 5min. The washed pellets were resuspended in TRI reagent with a 1ml syringe and 20-gauge needle, and snap frozen on dry ice. After thawing, lysates were diluted 1 : 1 with absolute ethanol and total RNA purified according to the kit instructions (Direct-zol RNA miniprep kit, Zymo Research, Irvine, CA, USA). Total RNA was quantified on the Nanodrop and 5ug of each used to prepare 5’ RACE-ready cDNA via the SMART (switching mechanism at 5’ end of RNA template) technology (SMART er RACE 573’ kit, Takara Bio, Mountain View, CA, USA). RACE was performed using the Universal primer A (provided in kit) and custom ordered gene-specific primers (IDT DNA, Coralville, Iowa) directed to the constant regions of the mouse IgGi heavy chain (1 : 1 mix of GATTACGCCAAGCTTTCTTGTCCACCTTGGTGCTGCTGGCCGG (SEQ ID NO: 28), GATTACGCCAAGCTTTTTGTCCACCGTGGTGCTGCTGGCTGGT (SEQ ID NO: 29)) or kappa light chain (GATTACGCCAAGCTTGATCAGTCCAACTGTTCAGGACGCC (SEQ ID NO: 30)). Gene-specific primers included a 15bp pRACE vector cloning sequence (GATTACGCCAAGCTT (SEQ ID NO: 31)) at the 5’ ends for downstream fusion cloning. PCR was carried out for 25 cycles of 94°C 30sec, 68°C 30sec and 72°C 3 min. PCR products were run on and extracted from 1.2% agarose gels, followed by elution in 15ul from a NucleoSpin Gel clean-up column (provided in kit). Purified PCR RACE products were cloned into the linearized pRACE vector by In-Fusion HD cloning and 5ul transformed into 50ul of Stellar competent cells (provided in kit). Twelve colonies from each VH and Vk product were sequenced using the standard pucl9 vector M13F primer (Sequetech, Mountain View, CA, USA).

EXAMPLE 2

[00201] This example illustrates the screening of anti-LILRB2 antibodies suitable for immunohistochemistry assay.

[00202] Screening by immunohistochemistry staining

[00203] The inventors screened the anti-LILRB2 hybridoma clones in immunohistochemistry (IHC) assay using HEK293 cells stably expressing LILRB2. Formalin-fixed paraffin-embedded (FFPE) cell pellets of HEK293 expressing full length LILRB2 (HEK293 LILRB2) were stained with supernatant of hybridoma clones generated from mouse immunization with Peptide No. 2 and Peptide No. 3 of Example 1 using the method summarized below.

[00204] HEK293 cells stably expressing LILRB2 were made into FFPE cells pellets samples. FFPE sections mounted on microscope slides were deparaffinized twice in xylene for 10 minutes and then sequentially rehydrated in 100%, 95%, 70% and 50% ethanol baths for 5 minutes each. After rinsing in tap water for 5 minutes, the sections were subjected to antigen-unmasking in a 2100-Retriever (EMS, Cat. No. 62706) using Target Retrieval Solution, Citrate pH 6 (Agilent, Cat. No. S236984-2). Sections were rinsed in tap water for 5 minutes, then blocked and stained using ImmPRESS® HRP Horse Anti-Mouse IgG PLUS Polymer Kit (for mouse antibody; Vector Laboratories, Cat. No. MP-7802-15) or ImmPRESS® HRP Horse Anti-Rabbit IgG PLUS Polymer Kit (for rabbit antibody; Vector Laboratories, Cat. No. MP-7801) according to the manufacturer’s manual. Briefly, the sections were incubated in BLOXALL Blocking Solution for 10 minutes, washed in PBS, blocked with 2.5% horse serum for 20 minutes, then incubated with diluted primary antibody for 30 minutes at room temperature. The sections were then washed in PBS, incubated with ImmPRESS Horse Reagent, washed in PBS. The signal was developed using ImmPACT DAB Eqv working solution. The cell nuclei were counterstained with hematoxylin for 30 seconds. The sections were then rinsed in tap water for 5 minutes and dehydrated in 50%, 70%, 95%, 100% ethanol baths and twice in xylene baths for 5 minutes each. The sections were air-dried, and the slides mounted with VectaMount™ (Vector Laboratories; Cat. No. H- 5000).

[00205] As shown in Figure 1, a group of hybridomas (listed in Table 4) can recognize LILRB2 expressed in HEK293 cells.

[00206] Table 4. Hybridomas Isolated for Screening

[00207] Specificity assessment by ELISA

[00208] The inventors then assessed the binding specificities of the anti-LILRB2 antibodies (recombinant and/or hybridoma supernatant) to synthetic peptides from LILRs by ELISA. Based on a multiple sequence alignment, peptides from each LILR as listed in Table 5 were assessed. Each peptide with a C-terminal lysine-biotin moiety was synthesized using standard methods at Elim Biopharmaceuticals and purified by HPLC to >95% and characterized by mass spectrometry prior to use.

[00209] Table 5. LILR peptides for ELISA

[00210] Peptides were coated at 10 pg/mL in sodium bicarbonate, pH 9.6 overnight at 4°C on a 96-well MaxiSorp plate, washed 3x with PBS/Tween and blocked with 1% BSA in PBS. After washing 3x with PBS/Tween the mouse antibodies were incubated in PBS/Tween + 0.5% BSA for 2 hours. After washing 6x with PBS/Tween, the mouse antibodies were bound using a 1 :10,000 dilution of goat anti-mouse IgG-HRP conjugation in PBS/Tween + 0.5% BSA. Samples were washed 6x with PBS/Tween and detected with 3, 3’, 5,5’- Tetram ethylbenzidine (TMB).

[00211] As shown in Table 6, a group of clones, including 16B9, 29F12 and 37B2, demonstrated binding specificity to LILRB2.

[00212] Table 6. Specificity of anti-LILRB2 antibodies by ELISA [00213] Specificity assessment by IHC

[00214] The inventors then assessed the specificity of anti-LILRB2 hybridoma clones in the IHC assay using HEK293 cells expressing LILRs. In short, HEK293 cells cultured on microscope slide coverslips were transiently transfected with LILRB2, LILRB1, LILRB3 or LILRA6. Non-transfected cells were used as negative control. The specificity of 16B9, 29F12, 49G11, 32G8, and 37B2 were tested using IHC staining with 1 :5 diluted supernatant from each hybridoma clone. A more detailed description of the method is provided below.

[00215] HEK293 cells were seeded on 20 x 20 millimeter (mm) microscope slide coverslips placed in 6-well plates and cultured overnight to reach 80% confluency. DNA plasmids (5 pg) coding full length LILRB2 protein were transfected using Lipofectamine™ 3000 following manufacturer’s instruction. Transfected HEK293 cells were cultured for 4 days in a 5% CO ₂ incubator. The transiently transfected HEK293 cells were stained by IHC using VECTASTAIN Elite ABC HRP Kit according to the manual. Briefly, the coverslips were fixed in 4% paraformaldehyde in phosphate-buff ered saline (PBS) for 10 minutes, washed with PBS, incubated in BLOXALL Blocking Solution for 10 minutes, washed with PBS, blocked with 2.5% normal horse serum for 20 minutes, blocked with Avidin/Biotin Blocking Kit, incubated with diluted primary antibody overnight at 4°C, washed 3 times with PBS, incubated with diluted biotinylated anti -mouse secondary antibody for 1 hour at room temperature, washed 3 times with PBS, incubated with VECTASTAIN ABC buffer for 30 minutes, washed 3 times with PBS, and then developed with 3,3’ Di aminobenzidine (DAB) for 3 minutes. After wash with PBS 3 times, the coverslips were mounted on microscope slides with VectaMount™.

[00216] As shown in Figure 2, clones 16B9, 29F12, 49G11, and 37B2 demonstrated specific binding to LILRB2.

EXAMPLE 3

[00217] As the results in Example 2 showed that clones 16B9 and 37B2 demonstrated high binding specificity to LILRB2, the inventors further characterized the properties of these two clones.

[00218] Titration of recombinant 16B9 and 37B2 antibodies in the IHC assay

[00219] The inventors titrated the recombinant anti-LILRB2 antibodies derived from clones 16B9 and 37B2 using FFPE sections of HEK293 or HEK293 LILRB2 cell pellets. The methods of IHC assay using FFPE sections of HEK293 or HEK293 LILRB2 cell pellets have been described in Example 2 supra. As shown in Figure 3, recombinant 16B9 and 37B2 antibodies bind to LILRB2 in the IHC assay at the concentrations of 10 to 0.15625 pg/mL.

[00220] Specificity of recombinant 16B9 and 37B2 antibodies

[00221] The inventors then evaluated the cross-reactivity of recombinant 16B9 and 37B2 antibodies to the peptides from different LILR members using ELISA and bio-layer interferometry (BLI).

[00222] The method of ELISA has been described in Example 2 supra. As shown in Figure 4B, recombinant 16B9 specifically binds to LILRB2.

[00223] To assess the binding specificity of recombinant 16B9 and 37B2 antibodies in a BLI assay, 16B9 and 37B2 binding kinetics and affinity measurements to the biotinylated LILRB2 peptide (Biotin-RB2) were performed on a Gator BLI platform (GatorBio) using a built-in kinetic assay setup program. The basic parameters including data acquisition (Frequency: 5 Hz), shaker setting (tilt @ 30OC), and pre-wet & pre-mix setting (Time: 300sec with shaker A & B speed: 1000 rpm), are used throughout the experiments. Biotin- RB2 was immobilized onto SA (Streptavidin) probes and incubated with varying concentrations of 16B9 or 37B2 (0 to 67 nM) in solution. The experiment included 5 steps:

[00224] a. Pre-wet, regeneration/neutralization (three times), & baseline probe procedure (300 sec, 30 sec, & 120 sec @ 1000 rpm)

[00225] b. Biotin-RB2 (6.7ug/mL) loading onto SA probes (300 sec @ 1000 rpm)

[00226] c. Equilibrate the loading baseline with K Buffer (300 sec @ 1000 rpm)

[00227] d. Association of 16B9 or 37B2 with immobilized Biotin-RB2 for the measurement of Kon (300 sec @ 1000 rpm)

[00228] e. Dissociation of 16B9 or 37B2 with immobilized Biotin-RB2 for the measurement of Kais (500 sec @ 1000 rpm)

[00229] Baseline and dissociation steps were conducted in K-buffer, and background subtraction was performed to correct for biosensor drifting. Background wavelength shifts were measured from reference biosensors that were loaded with 16B9 or 37B2 but did not go through interactions with the target peptide. Binding affinity constants were determined using a 1 : 1 fitting model (Global Fit) with Gator’s data analysis software 1.6.1.1203, and the K _D was calculated using the ratio K _dis /K _on . [00230] Screening 16B9 and 37B2 against the biotinylated immunogen peptides performed with similar assay procedure as described above except a fixed concentration of 16B9 and 37B2 (67 nM) was used to generate their binding sensorgrams.

[00231] As shown in Figure 4C and Figure 4D, both recombinant 16B9 and 37B2 antibodies demonstrated binding specificity to LILRB2.

[00232] Detection of endogenous LILRB2 in human normal tissue by IHC using recombinant 16B9 and 37B2

[00233] The recombinant 16B9 and 37B2 antibodies were used to detect endogenous LILRB2 in human normal tissues by IHC staining of FFPE sections of lung, spleen or bone marrow from healthy human donors.

[00234] FFPE tissue sections mounted on microscope slides were deparaffinized twice in xylene baths for 10 minutes and then sequentially rehydrated in 100%, 95%, 70% and 50% ethanol baths for 5 minutes each. After rinsing in tap water for 5 minutes, the sections were subjected to antigen-unmasking in a 2100-Retriever using Target Retrieval Solution, Citrate pH 6. Sections were rinsed in tap water for 5 minutes, then blocked and stained using ImmPRESS® HRP Horse Anti -Mouse IgG PLUS Polymer Kit or ImmPRESS® HRP Horse Anti-Rabbit IgG PLUS Polymer according to the manufacturer’s manual. Briefly, the sections were incubated in BLOXALL Blocking Solution for 10 minutes, washed in PBS, blocked with 2.5% horse serum for 20 minutes, then incubated with diluted primary antibodies (anti-LILRB2, anti-CDl lb or anti-CD163) for 30 minutes at room temperature. The sections were then washed in PBS, incubated with ImmPRESS Horse Reagent, washed in PBS. The signal was developed using ImmPACT DAB Eqv working solution. The cell nuclei were counterstained with hematoxylin for 30 seconds. The sections were then rinsed in tap water for 5 minutes and dehydrated in 50%, 70%, 95%, 100% ethanol baths and twice in xylene baths for 5 minutes each. The sections were air-dried, and the slides mounted with VectaMount™.

[00235] As shown in Figure 5, Figure 6, and Figure 7, the recombinant 16B9 and 37B2 antibodies detected endogenous LILRB2 in human normal or tumor tissues by IHC or immunofluorescence staining, at a concentration 0.3125-2.5 pg/mL (16B9 antibody) and 0.625 -2.5 pg/mL (37B2 antibody). The staining pattern of the myeloid cell markers CDl lb or CD163 was also analyzed to compare with the staining pattern of 16B9 or 37B2, [00236] Recombinant 16B9 antibody was used to detect endogenous LILRB2 in human normal tissues by immunofluorescence staining. FFPE tissue sections mounted on microscope slides were deparaffinized twice in xylene baths for 10 minutes and then sequentially rehydrated in 100%, 95%, 70% and 50% ethanol baths for 5 minutes each. After rinsing in tap water for 5 minutes, the sections were subjected to antigen-unmasking in 2100- Retriever using Target Retrieval Solution, Citrate pH 6. The slides were rinsed in tap water for 5 minutes, permeabilized with 0.4% Triton X-100 for 10 minutes, washed with PBS, and block with 5% goat and donkey serum in PBS for 1-2 hours, incubated with diluted primary antibodies (anti-LILRB2 or anti-CDl lb or anti-CD163) overnight at 4°C, washed with PBS 3 times, incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 and Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1 : 1000 dilution for 1 hour. The sections were washed 3 times with PBS and stained with DAPI for 1 minute, rinsed with PBS and mount the slides with ProLong™ Gold Antifade Mountant.

[00237] As shown in Figure 6 and Figures 7 A-B, FFPE tissue sections of human normal and tumor tissues were stained with recombinant 16B9 antibody, with the same staining pattern as the myeloid cell markers CDl lb (Figure 6A and Figure 6B) or CD163 (Figure 7A and Figure 7B).

[00238] Identification of the critical LILRB2 amino acids for binding to 16B9 or 37B2 antibodies

[00239] The inventors then identified amino acid residues of LILRB2 that are critical for the binding of 16B9 and 37B2 antibodies using BLI analysis of captured biotinylated peptides. The methods of BLI analysis have been described supra. As shown in Figure 8E and Figure 8F, an isoleucine residue is critical for the binding of 16B9 and 37B2 antibodies to LILRB2. Binding to this isoleucine is important for the specificity of 16B9 and 37B2 to LILRB2 over LILRA6 and LILRB3 which have closely related peptides.

[00240] 16B9 antibody detects LILRB2 in FFPE samples pre-treated with therapeutic anti-LILRB2 antibody

[00241] The inventors also tested whether 16B9 recombinant antibody can detect LILRB2 by IHC staining in FFPE sections prepared from samples that have been exposed to, or treated with, a therapeutic antibody (e.g. antagonist or agonist antibody) targeting LILRB2. Cultured HEK293 LILRB2 cells were detached from tissue culture dishes, resuspended in culture medium, and then treated, or left untreated, with 20 pg/mL anti-LILRB2 therapeutic antibody in suspension for 1 hour at 37°C. The cells were pelleted by centrifugation, washed in PBS and processed into FFPE sections. The sections were stained by IHC using 16B9 recombinant antibody at different concentrations. The nuclei were counterstained with hematoxylin. As shown in Figure 9, 16B9 recombinant antibody can detect LILRB2 by IHC staining in HEK293 LILRB2 FFPE samples that have been pre-treated with a therapeutic anti-LILRB2 antibody.

EXAMPLE 4

[00242] This example illustrates the epitope mapping and LILRB2 peptide specificity of 16B9 antibody.

[00243] The contributions of specific residues in the peptide used to generate the 16B9 mAb were tested by an alanine scanning binding ELISA. The importance of the wildtype residue was quantified by an increase in the ELISA EC ₅₀ value. Over 10-fold increases in the ELISA EC ₅₀ values occurred at amino acid substitutions PAA, TAA, GAA, PAA and IAA in peptides #4-8, with the TAA, GAA and IAA substitutions in peptides #5, #6 and #8 showing the largest effects.

[00244] The specificity of 16B9 antibody binding to the LILRB2 peptide sequence was tested by making analogous peptides for all other LILR receptors and determining the ELISA binding EC ₅₀ values (Table 7). Binding to LILRA6 and LILRB3 peptides was measured but at levels 2.8 x 10 ⁶ -fold below LILRB2. Therefore, it was possible to dilute the primary antibody in a range to detect LILRB2 and not other family members.

[00245] Table 7. Alanine scanning binding ELISA of 16B9 antibody.

[00246] For the alanine scanning ELISA, high protein binding, 96-well ELISA plates were coated with lOOuL/well of 4ug/mL Streptavidin in Phosphate Buffered Saline, pH 7.4 (PBS) overnight at 4-8°C. After washing with PBS, 0.05% Tween-20 (PBST), N-terminal biotinylated peptides were captured in the SA-coated wells using lOOuL/well of lOug/mL peptide in (PBS) overnight at 4-8°C. After blocking with 1% BSA/PBS for Ih and washing in PBST, the 16B9 antibody from lOug/ml with 1 :3 dilutions in 0.5%BSA/PBST was allowed to bind for 1 hour at ambient temperature, washed in PBST and the secondary antibody peroxidase-AffiniPure Goat Anti-Mouse IgG, Fey Fragment Specific (min X Hu, Bov, Hrs Sr Prot) was added for 1 hour at ambient temperature. After a final washing in PBST the plates were developed with a TMB (3,3',5,5'-Tetramethylbenzidine) substrate.

[00247] For the LILR family peptide ELISA, high protein binding, 96-well ELISA plates were coated with lOOuL/well of lOug/ml peptide in sodium bicarbonate, pH9.6 overnight at 4-8°C. After washing in PBST and blocking Ih in 1%BSA/PBS, the 16B9 antibody from lOug/ml with 1 :3 dilutions in 0.5%BSA/PBST was allowed to bind for 2 hours at ambient temperature. After washing in PBST the secondary antibody peroxidase- AffiniPure Goat Anti-Mouse IgG, Fey Fragment Specific (min X Hu, Bov, Hrs Sr Prot) was added for 1 hour at ambient temperature. After a final washing in PBST the plates were developed with a TMB substrate.

EXAMPLE 5

[00248] This example illustrates the paratope optimization of the 16B9 antibody.

[00249] As illustrated in Examples 2 and 3, the 16B9 antibody is a specific IHC reagent in tissues important to LILRB2 therapeutic applications. Off-target IHC staining to unknown cytoplasmic antigens were observed in some normal human tissues such as heart and testes. The inventors explored whether the paratope of 16B9 could be edited to decrease this off-target staining while maintaining sensitivity and specificity to LILRB2. The mouse 16B9 antibody was converted to a scFv format and tested to show it retained its binding properties. Next a systematic “NNK walk” across each of the heavy chain CDRs was designed into an oligonucleotide pool (Table 8) used to generate a scFv library. In this way, each CDR residue is tested one at a time as either wildtype or any of the other 20 amino acids in the context of the otherwise wildtype sequence. As shown in Example 4, the 16B9 binding is strongly dependent on the PTGPI motif. This motif can be identified by the NCBI Blastp tool in other proteins, such as titin. Therefore, using ribosome display, the inventors compared binding of the 16B9 scFv NNK walk library to peptide #1 (wildtype), peptide #5 (TAA) and peptide #8 (IAA), with the hope to select for changes in the heavy chain CDRs that would de-emphasize the importance of the contacts with threonine and isoleucine in PTGPI and recruit or strengthen alternative contacts within the 16 amino acid long peptide. Binding was quantified statistically by the enrichment of sequences in NGS data in each selected library after three rounds of ELISA binding. A logo plot of a selection of the NGS sequences is shown in Figure 10, with the top 100 sequences obtained for peptide #1 selection listed in SEQ ID NOs: 78-183, the top 100 sequences obtained for peptide #5 selection listed in SEQ ID NOs: 184-346, and the top 100 sequences obtained for peptide #8 selection listed in SEQ ID NOs: 347-539. Ten variants representing single amino acid CDR changes enriched in NGS data after binding both peptide #5 and #8 but not peptide #1 were made as recombinant IgG (Table 9). After initial binding confirmations, four variants with different changes in peptide binding specificity (Figure 11) and representing substitutions in either CDR1, CDR2 or CDR3 were selected for H4C testing (Figures 12 and 13). Although each antibody variant showed evidence of paratope shifting by lowering or eliminating binding to LILRA6 and LILRB3 peptides, the H4C signal differential between the unknown off-target cytoplasmic protein and on-target LILRB2 membrane protein worsened than the original 16B9 antibody. Therefore, we determined that the PTGPI motif is essential to the quality of on-target LILRB2 IHC binding.

EXAMPLE 6

[00250] This example illustrates the staining and scoring of human cancer samples based on LILRB2, CD3 and CD 163 IHC.

[00251] CD163, CD3 and LILRB2 were stained with respective IHC antibodies in 195 evaluable samples from 19 different human cancer indications. Figure 14A shows the number of samples in each LILRB2 IHC reactivity score (0-5) in macrophages across solid tumor types. Figure 14B shows the average score and correlation between LILRB2, CD 163 and CD3 IHC staining across tumor types. Figure 14C shows the ranking of tumor types stained by IHC based on reactivity scores. Each tissue was evaluated by CRO’s board- certified pathologist separately for LILRB2, CD163, and CD3 reactivity. Positively staining immune cells were assessed and scored within tumor and tumor-induced stroma (TIS). The number of reactive cells for each biomarker in each sample was counted at 20X magnification over multiple fields and the cell counts were converted to IHC reactivity score of 0-5 as defined here: 5: >100 reactive cells; 4: 51-100 reactive cells; 3: 26-50 reactive cells; 2: 11-25 reactive cells; 1 : 1-10 reactive cells; 0: <1 reactive cells.

[00252] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Table 8. Oligonucleotide pool for NKKwalk

Table 9. Recombinant IgG sequences derived from variants enriched in NGS data after binding both peptide #5 and #8 but not peptide #1