Synthetic lilrb4-binding proteins, manufacture, and uses thereof

Abstract

The present disclosure provides, among other things, synthetic LILRB4-binding proteins, compositions containing such binding proteins, and methods of making and using such binding proteins.

Description

Attorney Docket No.: AIP-010WOSYNTHETIC LILRB4-BINDING PROTEINS, MANUFACTURE, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of and priority to U.S. Provisional Patent Application No.63 / 566,178, filed March 15, 2024, the entire contents of which are hereby incorporated by reference for all purposes. SEQUENCE LISTING

[0002] The present specification makes reference to a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on February 20, 2025, is named AIP-010WO_SL.xml and is 135,875 bytes in size. FIELD

[0003] The disclosure relates generally to synthetic leukocyte immunoglobulin-like receptor B 4 (LILRB4) binding proteins, their manufacture, and their use in treatment of various disorders, such as cancer. BACKGROUND

[0004] LILRB4 (also known as ILT3) is an immune checkpoint molecule expressed in myeloid antigen presenting cells (APCs), such as monocytes and dendritic cells. High expression of LILRB4 in APCs suppress immune response and lead to immune tolerance. Elevated levels of LILRB4 correlate with poor prognosis of various cancer types, including acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and non- small-cell lung cancer (NSCLC), evidencing its role in regulation of immune activation and making it a potential target for cancer immunotherapy. See Yang et al. (2022) BLOOD SCI. 4(2): 49, and Abdallah et al. (2021) FRONT. IMMUNOL.12: 717998. Other pathologies in which LILRB4 has been implicated include B cell-related malignancies, like systemic lupus erythematosus, and allergies. See Boonpiyathad et al. (2019) ALLERGY.74(5): 976, and Inui et al. (2016) INT. IMM.28(12): 597.

[0005] The downstream activity of LILRB4 is believed to involve the deactivation of several protein kinases by suppressing phosphorylation and activation of tyrosine-protein kinase Syk through the phosphatase activity of SHP-1. See Lu et al. (2009) JBC.284(50): 34839, and Park et al. (2016) SCI. REP.6: 35085. In monocytes, co-ligation of LILRB4 with Page 1 of 66       Attorney Docket No.: AIP-010WOFc-gamma receptor I (FcγRI) is implicated in this signaling pathway by suppressing cytokine production and immune activation. Id.

[0006] Even though the potential for LILRB4 to act both as a biomarker and target of various diseases has been demonstrated, knowledge on the interacting ligands for its function are scarce beyond the recently-identified ones (CD166, APOE, and CNTFR). See Deng et al. (2021) ANTIBODY THERAPEUTICS.4(1): 16. Consequently, the present understanding of the mechanism and involved pathways of LILRB4 is limited. As such, current therapies that target LILRB4 are limited to antibodies and antibody-drug conjugates that specifically bind to LILRB4 in order to enable proper activation of immune cells. See Paavola et al. (2021) CANCER IMMUNOL. RES.9(11): 1283, Deng et al. (2019) CANCER IMMUNOL. RES.7(8): 1244, Anami et al. (2020) MOL. CANCER THER.19(11): 2330, and Condurso et al. (2022) JOURNAL FOR IMMUNOTHERAPY OF CANCER.10: 483.

[0007] Accordingly, despite the developments made to date, there remains a need for other therapeutics that target tumor-associated antigens such as LILRB4. SUMMARY

[0008] The disclosure is based, in part, upon the development of synthetic LILRB4- binding proteins. LILRB4, an immune checkpoint molecule, is expressed on various cancer types, including acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and non-small-cell lung cancer (NSCLC). Currently, there is a long felt and unmet need for improvements in compositions and methods for targeting cancer cells, e.g., myeloid cancer cells, using agents that bind cancer markers such as LILRB4. As provided herein, LILRB4-binding proteins with improved safety, selectivity, efficacy, potency, manufacturability, scalability, and stability can meet such needs.

[0009] In one aspect, provided are synthetic LILRB4-binding proteins which comprise (a) an amino acid sequence from 35 to 100 amino acid residues in length; (b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for LILRB4 stronger than 1 µM; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to LILRB4-upon cooling to room temperature after thermal denaturation at 95 °C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to LILRB4-after incubation for 16 hours at 37 °C of incubation in PBS relative to the protein under the same conditions prior to the incubation; and / or (iii) Page 2 of 66       Attorney Docket No.: AIP-010WOretains at least 90% binding affinity to LILRB4 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0010] In another aspect, provided are synthetic LILRB4-binding proteins which comprise (a) an amino acid sequence from 35 to 100 amino acid residues in length; (b) a net negative charge in PBS; (c) a binding affinity for LILRB4 stronger than 1 µM; (d) at least one alpha helix; (e) at least three beta strands; and (f) at least three amino acid loops, wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first beta strand to a terminal amino acid (e.g., a N-terminal amino acid) of a first alpha helix; a second loop having a second amino acid sequence connects a second terminal amino acid (e.g., an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta strand; and a third loop having a second amino acid sequence connects a second terminal amino acid (e.g., an C-terminal amino acid) of the second beta strand to a terminal amino acid (e.g., an N- terminal amino acid) of a third beta strand.

[0011] In certain embodiments, provided synthetic LILRB4-binding proteins of any of the foregoing aspects comprise one or more of the following features: (a) free of tryptophan residues; (b) free of methionine residues; (c) free of cysteine residues; (d) does not comprise an unpaired cysteine residue when cysteine residues are present in the protein; (e) free of N-linked glycosylation sites; (f) free of L-X-R-R sequences, wherein X represents any amino acid residue; and (g) soluble up to at least 0.5 mM in PBS at 4 °C for one month.

[0012] In some embodiments, provided synthetic LILRB4-binding protein are characterized by a binding affinity for LILRB4 that is between 1 μM to about 0.001 nΜ; about 1 μM to about 0.01 nM, about 1 μM to about 0.75 nM; about 1 μM to about 0.5 nM; about 1 μM to about 0.25 nM; about 1 μM to about 1 nΜ; about 0.75 μM to about 1 nM, about 0.5 μM to about 1 nM; about 0.25 μM to about 1 nΜ; about 0.10 μM to about 1 nΜ; about 75 nΜ to about 1 nΜ; about 50 nΜ to about 1 nΜ; about 25 nΜ to about 1 nΜ; about 10 nΜ to about 1 nΜ; and about 5 nΜ to about 1 nΜ. In some embodiments, the binding affinity for LILRB4 is stronger than about 1 μM, about 0.75 μM, about 0.5 μM, about 0.25 μM, about 0.1 μM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM. Page 3 of 66       Attorney Docket No.: AIP-010WO

[0013] In some embodiments, the N-terminus of the first beta strand is preceded by one or more N-terminal amino acid residues and / or the C-terminus of the third beta strand is followed by one or more C-terminal amino acid residues.

[0014] In certain embodiments, the synthetic LILRB4-binding proteins comprise from 35 to 95 amino acid residues in length, from 35 to 90 amino acid residues in length, from 35 to 85 amino acid residues in length, from 35 to 80 amino acid residues in length, from 35 to 75 amino acid residues in length, from 35 to 70 amino acid residues in length, from 35 to 65 amino acid residues in length, from 35 to 60 amino acid residues in length, from 35 to 55 amino acid residues in length, from 35 to 50 amino acid residues in length, from 35 to 45 amino acid residues in length, from 35 to 40 amino acid residues in length, from 40 to 95 amino acid residues in length, from 40 to 90 amino acid residues in length, from 40 to 85 amino acid residues in length, from 40 to 80 amino acid residues in length, from 40 to 75 amino acid residues in length, from 40 to 70 amino acid residues in length, from 40 to 65 amino acid residues in length, from 40 to 60 amino acid residues in length, from 40 to 55 amino acid residues in length, from 40 to 50 amino acid residues in length, from 40 to 45 amino acid residues in length, from 45 to 95 amino acid residues in length, from 45 to 90 amino acid residues in length, from 45 to 85 amino acid residues in length, from 45 to 80 amino acid residues in length, from 45 to 75 amino acid residues in length, from 45 to 70 amino acid residues in length, from 45 to 65 amino acid residues in length, from 45 to 60 amino acid residues in length, from 45 to 55 amino acid residues in length, from 45 to 50 amino acid residues in length, from 50 to 95 amino acid residues in length, from 50 to 90 amino acid residues in length, from 50 to 85 amino acid residues in length, from 50 to 80 amino acid residues in length, from 50 to 75 amino acid residues in length, from 50 to 70 amino acid residues in length, from 50 to 65 amino acid residues in length, from 50 to 60 amino acid residues in length, or from 50 to 55 amino acid residues in length.

[0015] For example, provided synthetic LILRB4-binding proteins may comprise 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

[0016] In certain embodiments, within the provided synthetic LILRB4-binding proteins, (a) the first alpha helix contains at least two hydrophobic amino acid residues, wherein, optionally, one or more of the at least two hydrophobic amino acid residues is not solvent accessible; (b) the second beta strand contains at least one hydrophobic amino acid residue, wherein, optionally, the at least one hydrophobic amino acid residue is not solvent accessible; (c) the third beta strand contains at least one hydrophobic amino acid residue, Page 4 of 66       Attorney Docket No.: AIP-010WOwherein, optionally, one or more of the at least one hydrophobic amino acid residue is not solvent accessible; (d) the first, second, and / or third beta strand contains at least one or two solvent accessible amino acid residues; (e) the first alpha helix contains at least one or two solvent accessible amino acid residues; (f) the first and / or second and / or third loop contains at least one hydrophobic amino acid residue; or (g) the binding protein comprises any combination of elements selected from (a), (b), (c), (d), (e), and (f).

[0017] In some embodiments, within the provided synthetic LILRB4-binding proteins, (a) the first, second, and / or third beta strand each contains at least two hydrophobic amino acid residues; (b) the first, second, and third beta strand each contains at least one solvent accessible amino acid residue; (c) the first, second, and third beta strand each contains at least two hydrophobic and one solvent accessible amino acid residue; (d) the first alpha helix contains at least two hydrophobic amino acid residues; (e) the first alpha helix contains at least one solvent accessible amino acid residue; (f) the first alpha helix contains at least two hydrophobic and one solvent accessible amino acid residues; (g) the first alpha helix sheet contains at least four solvent accessible amino acid residues; and / or (h) the first, second, and / or third loop contains at least one hydrophobic amino acid residue.

[0018] In one aspect, provided are synthetic LILRB4-binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula I), wherein D1, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and L1, L2, and L3 are loops 1, 2 and 3, respectively; wherein D1, D2, D3, and D4 independently comprise any of the following combinations: (a) D1 comprises the amino acid sequence ITV, (b) D2 comprises an amino acid sequence of LLX9ASVVAYQIX18X19X20(SEQ ID NO: 8), wherein X9 is E or V; X18 is D or Q; X19 is H, R, or S; and X20 is A, D, or E; (c) D3 comprises an amino acid sequence of X25VX27IX29YD (SEQ ID NO: 9), wherein X25is A, R or Y; X27is E, Q, or S; and X29 is H, R, or Y; and (d)D4 comprises the amino acid sequence RYYIVTTE (SEQ ID NO: 10).

[0019] In certain embodiments, L1 comprises the amino acid sequence DS; L2 comprises the amino acid sequence NPNV (SEQ ID NO: 11); and / or L3 comprises the amino acid sequence EETH (SEQ ID NO: 12).

[0020] In one aspect, provided are synthetic LILRB4-binding proteins comprising an amino acid sequence of: Page 5 of 66       Attorney Docket No.: AIP-010WOITVDSLLX9ASVVAYQIX18X19X20NPNVX25VX27IX29YDEETHRYYIVTTE (SEQ ID NO: 7), wherein X9is E or V; X18is D or Q; X19is H, R, or S; X20is A, D, or E; X25is A, R or Y; X27 is E, Q, or S; and X29 is H, R, or Y.

[0021] In some embodiments, provided synthetic LILRB4-binding proteins comprise an N-terminal amino acid which is N-terminal to D1, for example, a P (proline).

[0022] In some embodiments, provided synthetic LILRB4-binding proteins comprise a C-terminal amino acid which is C-terminal to D4, for example, a T (threonine).

[0023] In certain embodiments, the provided synthetic LILRB4-binding proteins comprise an amino acid sequence selected from the group consisting of: ITVDSLLVASVVAYQIQSDNPNVRVEIRYDEETHRYYIVTTE (SEQ ID NO: 13), ITVDSLLVASVVAYQIQSANPNVRVEIRYDEETHRYYIVTTE (SEQ ID NO: 14), ITVDSLLVASVVAYQIDHDNPNVRVEIRYDEETHRYYIVTTE (SEQ ID NO: 15), ITVDSLLEASVVAYQIQRENPNVYVSIYYDEETHRYYIVTTE (SEQ ID NO: 16), ITVDSLLEASVVAYQIQRENPNVAVQIHYDEETHRYYIVTTE (SEQ ID NO: 17), and ITVDSLLEASVVAYQIQRENPNVYVQIHYDEETHRYYIVTTE (SEQ ID NO: 18).

[0024] In certain embodiments, the provided synthetic LILRB4-binding proteins comprise an amino acid sequence selected from the group consisting of: PITVDSLLVASVVAYQIQSDNPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 1), PITVDSLLVASVVAYQIQSANPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 2), PITVDSLLVASVVAYQIDHDNPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 3), PITVDSLLEASVVAYQIQRENPNVYVSIYYDEETHRYYIVTTET (SEQ ID NO: 4), PITVDSLLEASVVAYQIQRENPNVAVQIHYDEETHRYYIVTTET (SEQ ID NO: 5), and PITVDSLLEASVVAYQIQRENPNVYVQIHYDEETHRYYIVTTET (SEQ ID NO: 6).

[0025] In some embodiments, the provided synthetic LILRB4-binding proteins have a binding affinity for LILRB4 stronger than 1 µM.

[0026] In some embodiments, the synthetic LILRB4-binding proteins have a binding affinity for LILRB4 that is between 1 μM to about 0.001 nΜ; about 1 μM to about 0.01 nM; about 1 μM to about 0.75 nM; about 1 μM to about 0.5 nM; about 1 μM to about 0.25 nM; about 1 µM to about 0.01 nM; about 1 μM to about 1 nΜ; about 0.5 μM to about 1 nM; about 0.25 μM to about 1 nΜ; about 0.10 μM to about 1 nΜ; about 0.10 μM to about 0.01 nΜ; about 75 nΜ to about 1 nΜ; about 50 nΜ to about 1 nΜ; about 25 nΜ to about 1 nΜ; about 10 nΜ to about 1 nΜ; and about 5 nΜ to about 1 nΜ. Page 6 of 66       Attorney Docket No.: AIP-010WO

[0027] For example, the synthetic LILRB4-binding proteins may have a binding affinity for LILRB4 that is stronger than about 1 μM, about 0.75 μM, about 0.5 μM, about 0.25 μM, about 0.1 μM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0028] In some embodiments, the synthetic LILRB4-binding proteins have an amino acid sequence having at least 70 (e.g., 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9) percent identity to that of any one of SEQ ID NOs: 1-6.

[0029] In some embodiments, the synthetic LILRB4-binding proteins comprise a paratope defined by: a leucine, optionally substituted with glycine, isoleucine, or valine at position 6; a serine, optionally substituted with isoleucine, threonine, or valine amino at position 10; a valine, optionally substituted with alanine, isoleucine, or leucine at position 11; a tyrosine, optionally substituted with alanine, histidine, isoleucine, or valine at position 14; and an isoleucine, optionally substituted with leucine or valine at position 27, wherein the position numbers correspond to those of SEQ ID NO: 7 from N-terminus to C-terminus.

[0030] In one aspect, provided are fusion proteins comprising a synthetic LILRB4- binding protein as disclosed herein. In certain embodiments, provided fusion proteins comprising at least a first effector molecule which may, in some embodiments, be linked to the synthetic LILRB4-binding protein through a linker. In some embodiments, the fusion proteins comprise a second effector molecule which may, in some embodiments, be linked to the first effector molecule or to the synthetic LILRB4- binding protein through a linker. The linker may be, for example, an amino acid linker, such as an amino acid linker which comprises glycine and serine amino acid residues. In some embodiments, the amino acid linker is a (GGS)nlinker. In some of the provided fusion proteins, the LILRB4-binding protein is at a terminus of the fusion protein, for example, at the N-terminus or at the C-terminus of the fusion protein. The first and / or second effector molecule may be, for example, a binding protein capable of binding a target other than LILRB4. In some embodiments, the first and / or second effector molecule is a synthetic binding protein. The first and / or second effector molecule may, in some embodiments, comprise a detectable label, optionally a fluorescent label. Page 7 of 66       Attorney Docket No.: AIP-010WO

[0031] In one aspect, provided are conjugates comprising a synthetic LILRB4-binding proteins or fusion protein as disclosed herein conjugated to a chelating moiety.

[0032] In some embodiments, the chelating moiety is complexed with a metal, such as a radionuclide. In some embodiments, the chelating moiety is DOTA (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid).

[0033] In one aspect, provided are pharmaceutical compositions comprising a synthetic LILRB4-binding protein, a fusion protein, or a conjugate as disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, provided pharmaceutical compositions are formulated for administration by a systemic route, e.g., intravenous administration.

[0034] In one aspect, provided are methods comprising a step of contacting a LILRB4- expressing cell with a synthetic LILRB4-binding protein, a fusion protein, a conjugate, or a pharmaceutical composition as disclosed herein. In certain embodiments, the LILRB4- expressing cell is a cancer cell. In some embodiments, the step of contacting occurs inside a subject, e.g., a human subject.

[0035] In another aspect, provided are methods of treating, ameliorating, or preventing a disease or condition associated with LILRB4 comprising a step of administering to a subject an effective amount of a synthetic binding protein, a fusion protein, a conjugate, or a pharmaceutical composition as disclosed herein. In some embodiments, the subject is a human. In some embodiments, the subject is diagnosed with or at risk of having tumor and / or one or more cancer-related disorders. For example, the tumor may be a myeloid tumor and / or the cancer-related disorder is a myeloid cancer-related disorder. For example, the subject may be diagnosed with or at risk of having acute myeloid leukemia (AML). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG.1 is a schematic illustration of a synthetic LILRB4-binding protein binding to LILRB4 on a cancer cell.

[0037] FIG.2 is a graph of circular dichroism (CD) spectra showing the stability profile of a synthetic LILRB4-binding protein (Reference Miniprotein 1 (SEQ ID NO: 1)) over a wavelength range of 200-260 nm during heating and cooling measured at 5 °C intervals between temperatures of 25 °C – 95 °C (not all data points shown for clarity). Page 8 of 66       Attorney Docket No.: AIP-010WO

[0038] FIG.3 depicts a three-dimensional (3D) representation and a secondary structure representation of an exemplary LILRB4- binding protein, Reference Miniprotein 1, along with a portion of its amino acid sequence (SEQ ID NO: 13), with domains D1, D2, D3, and D4 connected with loops L1, L2, and L3, indicated as shown.

[0039] FIGs.4A-D are graphs depicting results from in vitro binding assays of a synthetic LILRB4-binding protein (Reference Miniprotein 1 (SEQ ID NO: 1)) (FIGs.4A and FIGs.4C) or a commercial LILRB4 antibody (FIGs.4B and 4D) to AML cancer cell lines MV-4-11 (FIGs.4A and 4B) and OCI-AML-3 (FIGs.4C and 4D). Plotted on the x-axis is the fluorescence intensity, with increased intensity indicating binding to cells; cell counts are plotted on the y-axis. For the assay, LILRB4 binding synthetic proteins were genetically fused to a FLAG® tag. Cell binding was detected using an anti-FLAG® tag antibody.

[0040] FIGs.5A-5E are binding curves reflecting binding of Reference Miniprotein 1 to LILRB1 (FIG.5A), LILRB2 (FIG.5B), LILRB3 (FIG.5C), LILRB4 (FIG.5D), or LILRB5 (FIG.5E). DETAILED DESCRIPTION

[0041] The disclosure provides, among other things, synthetic LILRB4-binding proteins, methods of making such proteins, and methods of using such proteins to treat a disease or condition, e.g., cancer. It is contemplated that LILRB4-binding proteins provided herein have the ability to target cancer cells, e.g., selectively and with binding affinities suitable for use in therapeutic and / or diagnostic applications. I. DEFINITIONS

[0042] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g., polypeptide and polynucleotide chemistry and synthesis, molecular and cellular biology, protein biology and biochemistry, immunology, etc. as described herein are those well- known and commonly used in the art.

[0043] As used herein, the singular forms “a,” “an” and “the” include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g., a synthetic LILRB4-binding protein includes a single binding protein, a plurality of synthetic binding proteins, etc. Page 9 of 66       Attorney Docket No.: AIP-010WO

[0044] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0045] Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations.

[0046] As used herein, the phrases “solvent accessible residue” and “solvent accessible amino acid” refer to an amino acid that, when disposed in a folded molecule (e.g., in its a tertiary conformation) and in a solvent, is characterized in that the amino acid is at least partially accessible or exposed to the solvent. Solvent accessible amino acid residues can be determined using a variety of approaches including, e.g., Rosetta software suite, Neighbor Count, and Neighbor vector algorithms (Durham et. al. (2009) J. MOL. MODEL.15(9): 1093- 108).

[0047] As used herein, the phrase “conservative substitution” refers to a substitution with a structurally and / or functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) and Threonine (T); 2) Aspartic Acid (D) and Glutamic Acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W). Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix). In certain embodiments, a binding protein of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions relative to a reference amino acid sequence.

[0048] As used herein, the phrase “corresponding to” designates a position / identity of an amino acid residue or a nucleic acid in a polymeric molecule such as an amino acid residue in an amino acid sequence or a nucleic acid in a nucleic acid sequence. It is understood by Page 10 of 66       Attorney Docket No.: AIP-010WOthe skilled artisan that such amino acid residues or nucleic acids in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that, for example, an amino acid in a first polymer “corresponding to” position seven in the reference amino acid, for example, need not actually be the seventh amino acid in the first polymer. Those of ordinary skill in the art are aware of methodology to identify “corresponding” amino acid residues or nucleic acids between two molecules (e.g., a polymer and a reference polymer), including, such as, commercially available algorithms, databases, or other information given context regarding particular polymers.

[0049] As used herein, the term “domain” refers to a region or segment of a given synthetic binding protein disclosed herein, and can include one or more structural features (e.g., amino acid, primary, structure or secondary structure features) and / or one or more functional features (e.g., binding properties). In the context of secondary structure, a “structural domain” can be an uninterrupted linear sequence that adopts a single type of secondary structure, for example, ten continuous amino acid residues that are all part of the same alpha helix structure or beta sheet or strand. In the context of binding, a “binding domain” can be a discontinuous portion of the overall amino acid sequence that facilitates chemical interactions with a target molecule or indirectly stabilizes such interactions.

[0050] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g., a synthetic LILRB4-binding protein disclosed herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0051] As used herein, the term “effector” refers to a molecule or molecular entity that confers one or more particular characteristics on itself or another molecule or molecular entity with or to which it is associated. For example, an effector may include a synthetic binding protein (e.g., a miniprotein, or something other than a miniprotein that is associated with a synthetic LILRB4-binding protein disclosed herein (e.g., via a covalent linkage), such as a detectable label (e.g., visualizable or otherwise measurable such as by fluorescence or radiolabel detection), small molecule, nanoparticle (e.g., a lipid nanoparticle, a polymer nanoparticle, etc.), polynucleotide (e.g., an aptamer, an siRNA, an shRNA, an oligonucleotide, etc.), a radionuclide or a chelating moiety complexed with a radionuclide, etc. An effector may be a synthetic binding protein (e.g., a monovalent synthetic binding protein linked to a LILRB4-binding protein disclosed herein to create a bivalent synthetic Page 11 of 66       Attorney Docket No.: AIP-010WOprotein where one or both of the proteins causes a change, e.g., in a cellular function, e.g., in a disease state, etc.).

[0052] As used herein, the term “loop” refers to (i) a structure (e.g., polypeptide) that connects two structural domains (e.g., a loop may be disposed between two alpha helices, between an alpha helix and a beta sheet or strand, or between two beta sheets or strands in a given synthetic LILRB4-binding protein) and / or (ii) a structure (e.g., peptide) present at the N- and / or C-terminal end of a given monovalent synthetic binding protein.

[0053] As used herein, the term “linker” refers to a structure (e.g., a polypeptide linker, or a chemical crosslinker (e.g., a homobifunctional or a heterobifunctional cross linking agent) between two molecules (e.g., two synthetic LILRB4-binding proteins disclosed herein) or between, e.g., a synthetic LILRB4-binding protein and an effector, wherein each of the entities that are linked is covalently linked to one another.

[0054] As used herein, the terms / phrases “synthetic binding protein,” “synthetic miniprotein,” and “miniprotein” are used interchangeably, and refer to a polypeptide between about 35 to about 100 amino acid residues in length, e.g., from about 40 to 100 amino acid residues, from about 40 to about 90 amino acid residues, from about 40 to 80 amino acid residues, from about 40 to about 70 amino acid residues, from about 40 to 60 amino acid residues, from about 40 to about 50 amino acid residues in length, or 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acid residues in length, that are capable of binding to a given target, e.g., LILRB4, with a desired binding affinity (e.g., stronger than 1 µM).

[0055] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences (e.g., a polypeptide) have the same amino acid or nucleotide at the same positions in an alignment. The percent identity between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. It is contemplated that a reference sequence can be an amino acid sequence corresponding to an entire LILRB4-binding protein or a portion thereof. A reference sequence may be an amino acid sequence that corresponds to a particular domain or domains (e.g., an alpha helix, a loop region) or a combination of domains (e.g., a combination of a loop and an alpha helix). Alignment for purposes of determining percent Page 12 of 66       Attorney Docket No.: AIP-010WOsequence identity (e.g., amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, MUSCLE, or BioPython software. For a discussion of basic issues in searching sequence databases, see Altschul et al. (1994) NATURE GENETICS 6:119-129, which is incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0056] As used herein, the term “synthetic” refers to a molecule that is (i) not naturally occurring, (ii) not present in nature, (iii) does not comprise entirely natural components, or (iv) a combination of any one of (i), (ii) and (iii). For example, a synthetic peptide does not exist naturally, is produced or otherwise modified by human intervention, such as techniques including recombinant or cell-free synthesis, and / or the peptide may comprise one or more non-naturally occurring amino acid residues.

[0057] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0058] As used herein, the phrase “pharmaceutically acceptable carrier” as used herein refers to an agent (e.g., excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded.2020). Page 13 of 66       Attorney Docket No.: AIP-010WO

[0059] As used herein, the term “epitope” refers to a region of a protein that is specifically recognized by a binding partner, such as an antibody or another binding protein. The epitope may generally span a portion of the protein. Often, proteins may have multiple such regions where binding partners can attach. Epitopes typically fall into two classes: continuous epitopes (also known as linear epitopes), which are epitopes defined by linear sequences of consecutive amino acid residues, and discontinuous epitopes (also known as conformational epitopes), which are epitopes defined by discontinuous amino acid residues that are brought together into spatial proximity when a protein is in its folded state.

[0060] As used herein, the term "paratope" refers to the specific region of a binding molecule (e.g., binding protein) that recognizes and binds an epitope of a target molecule. The paratope typically comprises 5-20 amino acid residues that are solvent accessible and in close proximity in three-dimensional space.

[0061] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0062] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0063] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present Page 14 of 66       Attorney Docket No.: AIP-010WOdisclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0064] Throughout the text, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0065] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described and depicted herein.

[0066] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0067] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0068] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context. Page 15 of 66       Attorney Docket No.: AIP-010WO

[0069] It should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0070] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc. II. LILRB4

[0071] Provided herein are synthetic binding proteins (also referred to as miniproteins) that bind LILRB4 (also known as ILT3). LILRB4 is an immune checkpoint molecule expressed in myeloid antigen presenting cells (APCs), such as monocytes and dendritic cells. High expression of LILRB4 in APCs suppress immune response and lead to immune tolerance. Elevated levels of LILRB4 correlate with poor prognosis of various cancer types, including acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and non-small-cell lung cancer (NSCLC).

[0072] LILRB4 has an extracellular segment (“ectodomain”) which consists of two immunoglobulin-like structural domains, D1 and D2, each of which consists of antiparallel fragments. The D1–D2 outer domain of LILRB4 adopts a blunt interdomain angle of 107°, which is stabilized by hydrophobic interactions. In the D2 structural domain, LILRB4 appears to have two new 3(10) helix regions.

[0073] LILRB4’s intracellular segment is made up of three receptor tyrosine-based inhibitory groups (ITIMs). ITIM is commonly found on receptor molecules on the surface of some immune cells, and its primary role in signaling is to inhibit or negatively regulate immune responses. When the ITIM receptor binds to its ligand, the tyrosine in the ITIM region may be phosphorylated, triggering a series of signaling events that ultimately lead to an inhibitory response in immune cells. III. LILRB4-BINDING PROTEINS

[0074] The disclosure provides synthetic LILRB4-binding proteins, and methods of identifying, making, characterizing, formulating, and using such LILRB4-binding proteins. In some embodiments, synthetic LILRB4-binding proteins have advantages over small molecule and large molecule (e.g., biologics) based therapeutics. For example, small molecule therapeutics may suffer from off-target activities and / or may have long half-lives, Page 16 of 66       Attorney Docket No.: AIP-010WOwhich can risk negative impact on one more organs or organ systems, such as kidneys. Similarly, large molecules such as biologics are expensive to produce, are challenging to produce in uniform batches of drug substance (including, especially, e.g., at commercially- scalable amounts), and can be challenging to formulate, transport, store, and administer to subjects.

[0075] Synthetic LILRB4-binding proteins disclosed herein avoid certain such disadvantages as they have high binding specificity to LILRB4, can be engineered to have desired pharmacodynamic and pharmacokinetic properties (e.g., a desirable circulating half- life in plasma), reduced immunogenicity (e.g., do not elicit an immune response against them), are chemically and thermally stable, and are resistant to protease degradation (e.g., via cleavage at L-X-R-R sites, wherein X represents any amino acid residue), deamination and post translational modification (e.g., glycosylation, e.g., N-linked glycosylation, e.g., glycosylation at N-X-S / T sites, wherein X represents any amino acid residue), and are stable in different redox environments.

[0076] A synthetic LILRB4-binding protein described herein can have mM-level solubility. In some embodiments, the LILRB4-binding protein has a solubility greater than 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL or 10 mg / mL in an aqueous solution. The LILRB4-binding proteins described herein are designed to specifically bind LILRB4. Synthetic LILRB4 miniproteins as provided herein can, in some embodiments, bind with an affinity at least at a level of a reference LILRB4 antagonist (e.g., an LILRB4 antagonist antibody such as IO-202 (Immune-Onc)).

[0077] In one aspect, provided are LILRB4-binding proteins comprising: (a) an amino acid sequence from 35 to 100 amino acid residues in length; (b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for LILRB4- stronger than 1 µM; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to LILRB4- upon cooling to room temperature after thermal denaturation at 95 °C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to LILRB4-after incubation for 16 hours at 37 °C of incubation in PBS relative to the protein under the same conditions prior to the incubation; and / or (iii) retains at least 90% binding affinity to LILRB4 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation. Page 17 of 66       Attorney Docket No.: AIP-010WO

[0078] In one aspect, provided are synthetic LILRB4-binding proteins comprising: (a) an amino acid sequence from 35 to 100 amino acid residues in length; (b) a net negative charge in PBS; (c) a binding affinity for LILRB4 stronger than 1 µM; (d) at least one alpha helix; (e) at least three beta strands; and (e) at least three amino acid loops, wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first beta strand to a terminal amino acid (e.g., a N-terminal amino acid) of a first alpha helix; a second loop having a second amino acid sequence connects a second terminal amino acid (e.g., an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta strand; and a third loop having a second amino acid sequence connects a second terminal amino acid (e.g., an C-terminal amino acid) of the second beta strand to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta strand.

[0079] Synthetic LILRB4-binding proteins of the present disclosure may include naturally-occurring or non-naturally occurring amino acids. It is understood that certain amino acids may have and / or take on different characteristics (e.g., hydrophobic, hydrophilic, neutral, etc.) depending upon the context (e.g., macro and / or micro-environment including, but not limited to surrounding amino acids, environmental conditions such as solvent type, pH, etc.). Various terms and phrases may be used herein to describe, identify, and / or characterize amino acids. In addition, a single amino acid, at any given time, may have more than one characteristic or identity. Depending upon the context, a hydrophobic amino acid may be selected from alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, tryptophan, tyrosine, lysine, and arginine. A hydrophilic amino acid may be an amino acid selected from cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tryptophan, and tyrosine. A charged amino acid may be an amino acid selected from arginine, histidine, lysine, aspartic acid (aspartate), and glutamic acid (glutamate). A positively-charged amino acid may be an amino acid selected from arginine, histidine, and lysine. A negatively-charged amino acid may be an amino acid selected from aspartic acid (aspartate) and glutamic acid (glutamate). An uncharged or neutral amino acid may be selected from alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Synthetic LILRB4-binding proteins of present disclosure have a primary structure comprising certain key features. For example, synthetic LILRB4-binding proteins can have amino acid sequences that include various combinations Page 18 of 66       Attorney Docket No.: AIP-010WOof hydrophobic and solvent accessible amino acids organized into certain domains. Primary structures (i.e., amino acid sequences) of the synthetic LILRB4-binding proteins will have a combination of one or more types (e.g., hydrophobic, e.g., solvent accessible) amino acids.

[0080] In each of the foregoing aspects, the synthetic LILRB4-binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein; (e) free of N-linked glycosylation sites (e.g., N-X-S / T, wherein X represents any amino acid residue); (f) free of protease cleavage sites (e.g., L-X-R-R sequences, wherein X represents any amino acid residue); and (g) soluble up to at least 0.5 mM in PBS at 4 °C for one month.

[0081] Preferably, the LILRB4 binding sites are designed to be free of glycosylation sites (e.g., free of N-linked glycosylation sites), for example, free of peptide sequences that are substrates for glycosylation (e.g., N-X-S / T (wherein X represents any amino acid residue), which can be a substrate for an oligosaccharyltransferase (OST) complex). Alternatively or additionally, the LILRB4 binding sites are designed to be free of protease cleavage sites, for example, free of peptide sequences that are substrates for proteases (e.g., L-X-R-R (wherein X represents any amino acid residue), which can be a substrate for a Kexin / KEX2 protease). Similarly, the LILRB4-binding proteins may also be designed to be free of other protease cleavage sites for other proteolytic enzymes such as trypsin, chymotrypsin, elastase, subtilisin, etc. Some synthetic LILRB4-binding proteins may be designed to avoid cleavage by certain other enzymes, including depending upon linkers and fusion protein partners.

[0082] By way of non-limiting example, a structural arrangement in a synthetic LILRB4- binding protein may be depicted as N- En1Ln2Hn3Ln4En5Ln6En7-C, where H is an alpha helix, E is a beta strand, L is a loop, each of n1-n5 represents an integer indicating the number of amino acids in that structural domain, N and C represent N-terminal and C-terminals, respectively. As disclosed herein, certain LILRB4-binding proteins can be represented according to a formula (Formula I): D1-L1-D2-L2-D3-L3-D4. In some embodiments, D2 corresponds to an alpha helical domain, and D1, D3, and D4 each correspond to a beta strand domain; and L1, L2, and L3 are each, independently, loops. The components of Formula I can correspond to the aforementioned structural arrangement as follows: D2 to Hn3 and each of D1, D3, and D4 to each of En1, En5, and En7. The number of E amino acids does not have to be the same across beta strands, for example, n1, n5, and n7 may be, but do not have to be, the same numbers. For example, an exemplary formula of a synthetic LILRB4-binding Page 19 of 66       Attorney Docket No.: AIP-010WOprotein may comprise N—C as depicted pictorially below denoting the amino acids in a beta strand domain (E), a helix domain (H), or a loop domain (L):

[0083] N-EEELLHHHHHHHHHHHHHHLLLLEEEEEEELLLLEEEEEEEE-C.

[0084] A person skilled in the art can determine which amino acids of a given sequence constitute a loop, beta strand or sheet, or a helix. See, e.g., Mirdita, et al. (2022) NAT. METHODS (19): 679-682.

[0085] Any given H domain (e.g., Hn3) in an alpha helix that is part of a synthetic LILRB4-binding protein may independently contain between about 4 amino acids and about 20 amino acids in length. An H domain may independently comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids in length. Any given E domain (e.g., En1, En5, or En7) in a beta strand that is part of a synthetic LILRB4-binding protein may independently contain between about 3 and about 15 amino acids in length. An E domain may independently comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids in length.

[0086] In a LILRB4-binding protein with more than one alpha helical structure, each alpha helix may comprise the same number of amino acids in each of its H domains or different numbers of amino acids in length in reference to the primary structure of each helical region. That is, in some synthetic LILRB4-binding proteins having more than one alpha helix, each H domain in the binding protein is the same length. In some synthetic LILRB4-binding proteins having one or more alpha helix, one or more H domains has a different length relative to other H domains in the binding protein. In some embodiments, an H domain has zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine or more conserved amino acids (e.g., relative to other LILRB4-binding proteins).

[0087] In a LILRB4-binding protein with more than one beta strand, each beta strand may comprise the same number of amino acids in each of its E domains or different numbers of amino acids in length in reference to the primary structure of each sheet region. That is, in some synthetic LILRB4-binding proteins having more than one beta strand, each E domain in the binding protein is the same length. In some synthetic LILRB4-binding proteins having one or more beta strands, one or more E domains has a different length relative to other E domains in the binding protein. In some embodiments, an E domain has zero, one, two, Page 20 of 66       Attorney Docket No.: AIP-010WOthree, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more conserved amino acids (e.g., relative to other LILRB4-binding proteins).

[0088] In an exemplary LILRB4-binding protein disclosed herein, the binding protein comprises at least one alpha helix, at least three beta strands, and at least three loops (a first loop, a second loop, and a third), wherein the first loop has a first amino acid sequence that connects a terminal amino acid (e.g., a C-terminal amino acid) of a first beta strand to a terminal amino acid (e.g., a N-terminal amino acid) of a first alpha helix, a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., an C- terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta strand; and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g., an C-terminal amino acid) of the second beta strand to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta strand.

[0089] A synthetic LILRB4-binding protein may have one of several consensus sequence structures. Consensus sequences will generally have certain “fixed” amino acid positions as well as those that can be varied, such as by changing to another amino acid. Sometimes changing amino acids at certain positions can alter the function of the synthetic binding protein by increasing or decreasing affinity for the target (i.e., LILRB4). However, all the binding proteins disclosed herein, although having different primary structures have a minimal “threshold” binding affinity to LILRB4. In some embodiments, a threshold binding affinity may be greater than about 10 µM, about 1 µM, about 100 nM, about 10 nM, or about 1 nM.

[0090] In one aspect, provided are LILRB4-binding proteins comprising an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula I) wherein D1, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and L1, L2, and L3 are loops 1, 2 and 3, respectively.

[0091] Table 1 provides exemplary sequences for D1, D2, D3, D4, L1, L2, and L3. TABLE 1A. Exemplary Miniprotein Domain Consensus Sequences Domain SEQ ID NO: Consensus SequencePage 21 of 66       Attorney Docket No.: AIP-010WOD2 8 LLX9ASVVAYQIX18X19X20whereinTABLE 1B. Exemplary Miniprotein Variable Substitutions Domain Consensus Amino Acid residue / Amino Acid Residues sequence or Consensus position

[0092] In some embodiments, the synthetic LILRB4-binding protein comprises an amino acid sequence of ITVDSLLX9ASVVAYQIX18X19X20NPNVX25VX27IX29YDEETHRYYIVTTE (SEQ ID NO: Page 22 of 66       Attorney Docket No.: AIP-010WO7), wherein X9 is E or V; X18 is D or Q; X19 is H, R, or S; X20 is A, D, or E; X25 is A, R or Y; X27is E, Q, or S; and X29is H, R, or Y

[0093] For example, below are examples of full sequences of Reference Miniproteins 1-6: PITVDSLLVASVVAYQIQSDNPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 1), PITVDSLLVASVVAYQIQSANPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 2), PITVDSLLVASVVAYQIDHDNPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 3) PITVDSLLEASVVAYQIQRENPNVYVSIYYDEETHRYYIVTTET (SEQ ID NO: 4) PITVDSLLEASVVAYQIQRENPNVAVQIHYDEETHRYYIVTTET (SEQ ID NO: 5) PITVDSLLEASVVAYQIQRENPNVYVQIHYDEETHRYYIVTTET (SEQ ID NO: 6) IV. LOOPS

[0094] Any of a variety of loops of various lengths can be used between domains (e.g., between D1 and D2, between D2 and D3, and / or between D3 and D4) of a synthetic LILRB4-binding protein disclosed herein. Each loop may be of a same or different length. For example, each loop may independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more amino acid residues or more in length. In some embodiments, each loop is independently between at least 2, 3, 4, 5, or 6 amino acids in length. Tables 2A, 2B, and 2C provide non-limiting examples of sequences and lengths for loops L1, L2, and L3, respectively, which, when used, can produce LILRB4 miniproteins (e.g., such as those disclosed herein, e.g., in Tables 2A-2C) that can still strongly bind to LILRB4 (e.g., as compared to LILRB4 miniproteins with “parental loops” such as disclosed in L1 (amino acid sequence “DS”), L2 (NPNV, SEQ ID NOs: 11 or 26), and L3 (ETTH, SEQ ID NOs: 12 or 83) of SEQ ID NO: 1, respectively)). TABLE 2A. Exemplary sequences and lengths for L1 SEQ L1 sequence Loop Total ID length miniprotein NO: length      Attorney Docket No.: AIP-010WOTABLE 2B. Exemplary sequences and lengths for L2 SEQ L2 se uence Loo Total    Attorney Docket No.: AIP-010WOSEQ L3 sequence Loop Total ID length miniprotein NO: length    Attorney Docket No.: AIP-010WO     Attorney Docket No.: AIP-010WO35to 95 amino acid residues in length, from 35 to 90 amino acid residues in length, from 35 to 85 amino acid residues in length, from 35 to 80 amino acid residues in length, from 35 to 75 amino acid residues in length, from 35 to 70 amino acid residues in length, from 35 to 65 amino acid residues in length, from 35 to 60 amino acid residues in length, from 35 to 55 amino acid residues in length, from 35 to 50 amino acid residues in length, from 35 to 45 amino acid residues in length, from 35 to 40 amino acid residues in length, from 40 to 95 amino acid residues in length, from 40 to 90 amino acid residues in length, from 40 to 85 amino acid residues in length, from 40 to 80 amino acid residues in length, from 40 to 75 Page 27 of 66       Attorney Docket No.: AIP-010WOamino acid residues in length, from 40 to 70 amino acid residues in length, from 40 to 65 amino acid residues in length, from 40 to 60 amino acid residues in length, from 40 to 55 amino acid residues in length, from 40 to 50 amino acid residues in length, from 40 to 45 amino acid residues in length, from 45 to 95 amino acid residues in length, from 45 to 90 amino acid residues in length, from 45 to 85 amino acid residues in length, from 45 to 80 amino acid residues in length, from 45 to 75 amino acid residues in length, from 45 to 70 amino acid residues in length, from 45 to 65 amino acid residues in length, from 45 to 60 amino acid residues in length, from 45 to 55 amino acid residues in length, from 45 to 50 amino acid residues in length, from 50 to 95 amino acid residues in length, from 50 to 90 amino acid residues in length, from 50 to 85 amino acid residues in length, from 50 to 80 amino acid residues in length, from 50 to 75 amino acid residues in length, from 50 to 70 amino acid residues in length, from 50 to 65 amino acid residues in length, from 50 to 60 amino acid residues in length, or from 50 to 55 amino acid residues in length.

[0096] For example, provided synthetic LILRB4-binding proteins may comprise 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues. V. SYNTHESIS OF LILRB4-BINDING PROTEINS

[0097] Synthetic LILRB4-binding proteins described herein may be produced by methods known to those of ordinary skill in the art. Methods may include, for example, biological approaches, such as recombinant approaches and / or chemical approaches, such as solid phase and / or liquid phase chemical synthesis, etc., or combinations thereof.

[0098] With regard to recombinant approaches, a variety of methodologies can be implemented to produce the binding proteins disclosed herein. For example, DNA molecules encoding the binding proteins can be synthesized chemically and / or cloned / produced using recombinant DNA methodologies. The resulting DNA molecules encoding binding proteins of interest can be ligated to other nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct (i.e., expression vector). Thereafter, the resulting expression vectors are introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include E. coli cells, Bacillus subtilis cells, Pichia Pastoris cells, Saccharomyces cerevisiae cells, Kluyveromyces lactis cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). The transformed host cells can be grown Page 28 of 66       Attorney Docket No.: AIP-010WOunder conditions that permit the host cells to express the genes that encode the binding proteins.

[0099] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., T7, lac, Trp or Tac, and, in some contexts, a prokaryotic signal sequence or fusion to a protein such as, e.g., Trx, MBP, SUMO, or OsmY. The expressed protein may be secreted. The expressed protein can be harvested after disruption of the cells by French press or sonication (e.g., in the presence of 4-6 M urea). Alternatively, or in addition, the binding proteins can be harvested and purified or isolated from cell extracts using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags. Protease cleavage with SUMO Protease (Ulp), thrombin, enterokinase, TEV protease, 3C protease may be used to cleave affinity tags and fusion proteins from the miniprotein binder. The protein may be further purified with reverse phase HPLC using a C- 18 column and eluted in a solvent gradient (e.g., gradient of acetonitrile). The protein may then be lyophilized to remove solvent and may be resuspended in phosphate buffered saline. Purification by reverse phase HPLC may be used to remove endotoxin from samples expressed in E. coli.

[0100] If the engineered gene is expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers. The vector may also optionally contain fusion domains which can be used to facilitate expression and secretion. Vectors may also optionally contain enzyme (e.g., protease) cleavage sites. The gene construct can be introduced into eukaryotic host cells using conventional transfection (e.g., for mammalian) and transformation (e.g., for yeast).

[0101] In addition, the synthetic binding proteins may be produced in cell-free systems. For example, chemical synthesis such as organic chemical synthesis using liquid and / or solid phase chemical processes may be used. Such processes and tools for performing such processes, such as various automatic synthesizers, are well known to those of ordinary skill in the art and such tools are widely commercially available. More specifically, methods of chemically synthesizing polypeptides are well known in the art and include, but are not limited to, solid-phase peptide synthesis, liquid-phase peptide synthesis, and organic Page 29 of 66       Attorney Docket No.: AIP-010WOsynthesis methods. In some synthetic approaches, an amino group of one amino acid (or amino acid derivative) is linked to a carboxyl group of another amino acid (or amino acid derivative) that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group attacks the activated carboxyl group, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the α-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) may be blocked (“protected”) from participating in the chemical reaction. Thus, only particular active groups react such that the desired product is formed. Blocking groups useful for this purpose include, without limitation, tertbutoxycarbonyl groups (t-Boc) and benzoyloxycarbonyl groups to protect amine groups; and simple esters (such as methyl and ethyl groups) and benzyl esters to protect carboxyl groups. Blocking groups can typically be subsequently removed with a treatment that leaves peptide bonds intact (for example, treatment with dilute acid). This process of protecting reacting groups that should not react, coupling to form a peptide bond, and deprotecting reactive groups may be repeated. A peptide may be synthesized by sequentially adding amino acid residues to a growing peptide chain.

[0102] Both liquid-phase and solid phase peptide synthesis methods can be used to make the binding proteins described herein. In solid-phase peptide synthesis, the growing peptide chain is typically linked to an insoluble matrix (such as, for example, polystyrene beads) by linking the carboxyterminal amino acid to the matrix. At the end of synthesis, the peptide can be released from the matrix using a cleaving reagent that does not disrupt peptide bonds, such as hydrofluoric acid (HF). Protecting groups are also typically removed at this time. Automated, high throughput, and / or parallel peptide synthesis methods may also be used in accordance with the disclosure. For more information about peptide synthesis methods, see, e.g., Merrifield (1969) ADV. ENZYMOL. RELAT. AREAS MOL. BIOL., 32: 221-96; Fridkin et al. (1974) ANN. REV. BIOCHEM.43(0): 419-43; Merrifield (1997) METH. ENZYMOL.289: 3-13; Sabatino et al. (2009) CURR. OPIN. DRUG DISCOV. DEVEL., 11(6): 762-70.

[0103] Once synthesized, the binding proteins can be purified using standard approaches including, for example, chromatographic (e.g., reverse phase HPLC) and affinity binding approaches. The resulting binding proteins can then be characterized using a variety of chemical, biological and biophysical approaches, e.g., as described herein. Page 30 of 66       Attorney Docket No.: AIP-010WOVI. CHARACTERIZATION OF LILRB4-BINDING PROTEINS A. Biophysical characterization

[0104] The synthetic binding proteins described herein may be characterized using a variety of approaches to determine, e.g., secondary and tertiary conformation, binding affinity, binding selectivity, stability (e.g., thermostability, chemical stability, propensity to degrade, etc.), solubility, etc.

[0105] For example, protein conformation may be measured via circular dichroism spectroscopy, infrared spectroscopy, NMR, X-ray crystallography, cryo-electron microscopy and AlphaFold (alphafold.ebi.ac.uk / ). Binding affinity and / or selectivity may be determined using assays such as flow cytometric analyses using, e.g., yeast or mammalian cells, biolayer interferometry and / or surface plasmon resonance measurements, each of which will be able to determine different types and specificities of binding.

[0106] Binding affinity and / or avidity can be determined by measuring the equilibrium dissociation constant (KD) of a synthetic LILRB4-binding protein to a target. In some embodiments, the binding affinity (KD) of synthetic LILRB4-binding proteins in the range of 10-5M or less, or ranging down to 10-10M or lower, (e.g., about 10-6,10-7, 10-8, 10-9, 10-10M or less).

[0107] In some embodiments, the synthetic LILRB4-binding protein comprises a binding affinity characterized by a dissociation constant ranging from about 1 pM to 1 ^M. In some embodiments, the binding affinity is between about 0.001 nM to about 1 ^M; about 0.01 nM to about 1 µM; about 0.1 nM to about 1 µM; about 1 nM to about 1 ^M; about 1 nM to about 0.5 ^M; about 1 nM to about 0.25 ^M; about 1 nM to about 0.10 ^M; about 1 nM to about 75 nM; about 1 nM to about 50 nM; about 1 nM to about 25 nM; about 1 nM to about 10 nM; and about 1 nM to about 5 nM. In some embodiments, the binding affinity is stronger than about 1 ^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 ^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM to about 0.01 nM, and about 0.01 nM to about 0.001 nM. One of ordinary skill in the art will readily know how to calculate equilibrium dissociation constants using measured Ka(1 / sec) and Kd(1 / secM) of the synthetic LILRB4-binding proteins. Page 31 of 66       Attorney Docket No.: AIP-010WO

[0108] Other analytical techniques (some of which are also used for synthesis and purification) include, without limitation, HPLC, LCMS, quantitative thin layer chromatography and others known to those of skill in the art. Stability can be measured using assays that expose binding proteins to elevated temperatures (e.g., 37 °C, e.g., 95 °C, etc.) and / or chemical denaturants (e.g., urea, and guanidine hydrochloride) and then observe whether the protein refolds into its pre-exposure structure / conformation and / or regains binding activity to a given target molecule. Degradation can be evaluated using techniques such as reverse phase HPLC or gel electrophoresis to monitor resistance of a synthetic binding protein to degradation. B. Biochemical characterization

[0109] The biological activity of the binding proteins can be determined via in vitro and in vivo assays (see, e.g., Example 4). Such assays can be used to determine, e.g., whether a binding protein binds LILRB4 on cells and has antagonistic (or agonistic) properties. For example, the suitable assays can be performed to determine whether a synthetic LILRB4- binding protein disclosed herein can, e.g., partially or completely inhibit downstream LILRB4-mediated signaling.

[0110] Various assays may be used to evaluate efficacy of synthetic LILRB4-binding proteins to determine their ability to antagonize LILRB4-mediated signaling. Certain assays can measure the antagonism of LILRB4 signaling by its natural ligands (e.g., ALCAM, APOE, fibronectin, or galectin-8), e.g., by assessing effects on T cell activity, tumor development, and / or signaling via the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling axis.

[0111] Assays can be conducted with various LILRB4-expressing cells, such as LILRB4- expressing cancer cells (e.g., blood cancer cells such as acute myeloid leukemia (AML), multiple myeloma (MM), or mantle cell lymphoma (MCL) cells). Assays can be conducted on established cell lines or on patient-derived (e.g., AML patient-derived) cells. Non-limiting examples of suitable cells for such assays include MV-4-11, OCI-AML3, and Jeko-1 cells.

[0112] In some embodiments, a concentration of the synthetic LILRB4-binding protein of about 105-10-12M (e.g., about 10-5, about 10-6, about 10-7, about 10-8, about 10-9, about 10-10, about 10-11, or about 10-12) may be enough to target cancer cells (as measured, e.g., by detectable reporter) through binding of a synthetic LILRB4-binding protein to LILRB4 in accordance with the present disclosure. Page 32 of 66       Attorney Docket No.: AIP-010WO

[0113] Characterization assays may also be conducted in vivo. For example, synthetic LILRB4-binding proteins fused to other effector proteins may be tested for efficacy in controlling and reducing the growth of LILRB4+ tumors in mice treated with a LILRB4- targeting protein. VII. LILRB4-BINDING PROTEIN CONJUGATES

[0114] It is contemplated that the synthetic binding proteins may be engineered to modify certain desired properties (e.g., binding affinity, binding avidity, or pharmacokinetic or pharmacodynamic properties). This can be achieved by conjugating (e.g., chemical conjugation or via a fusion protein) a synthetic binding protein to an effector molecule or to a chelating moiety which can complex with an effector molecule (e.g., a metal such as a radionuclide). For example, the synthetic binding protein can be conjugated to a second binding molecule, e.g., a second synthetic binding molecule, which can be the same or different from the first synthetic binding protein, or an antibody or antibody fragment) or a molecule that directly (e.g., bovine serum albumin (BSA), murine serum albumin (MSA), or human serum albumin (HSA)) or indirectly (e.g., an engineered binding site for BSA, MSA, or HSA) enhances the PK or PD properties of the binding molecule. By way of non-limiting examples, half-life can be extended by a variety of approaches known to those of skill in the art including use of polyethylene glycol (PEG), fusion proteins (e.g., Fc fusions, albumin fusions), engineered Fc binding such as engineered binding to neonatal Fc receptor (FcRn), antibody conjugation (e.g., to an antibody or fragment thereof), e.g., other protein engineering approaches that change the stability and / or clearance of a protein from an organism. It is contemplated that certain half-life extension approaches such as PEGylation may reduce clearance and prolong circulation. Fusions, such as to Fc or albumin, and / or addition of binding domains such as engineered HSA domains may prolong half-life due to circulatory half-life properties of the molecules (e.g., longer circulatory half-life via binding to albumin in the serum). For instance, a serum albumin binding domain may facilitate longer half-life via binding to albumin in circulating blood. One mechanism that extension could occur may include via recycling of a synthetic LILRB4-binding protein by binding to neonatal Fc receptor (FcRn), which could thereby extend the serum half-life of the synthetic LILRB4-binding protein.

[0115] It is contemplated that a variety of effector molecules can be used to modify the properties of the synthetic LILRB4-binding protein disclosed herein. Furthermore, it is Page 33 of 66       Attorney Docket No.: AIP-010WOcontemplated that the effector may be a cytotoxic molecule or detectable label (e.g., radiolabel or fluorescent tag) which can be used in a detection assay, e.g., a diagnostic assay.

[0116] It is contemplated that the effector molecule can be chemically conjugated to the synthetic binding protein or can be incorporated into the synthetic binding protein as a fusion protein. The chemical conjugation can be accomplished by including a conjugation site into the synthetic binding protein, e.g., via inclusion of a derivatizable amino acid (e.g., a lysine or cysteine amino acid). The conjugation site can then be used to link the effector molecule to the binding protein, either directly or indirectly (e.g., via a linker, such as homobifunctional or a heterobifunctional cross-linking agent). By way of non-limiting example, linkers may be or include hydrazone, PEG, bifunctional 4-(4-acetylphenoxy) butanoic acid moiety, maleimidocaproyl, maleimidomethyl cyclohexane-1-carboxylate, maleimidocaproyl group with a tetrapeptide portion consisting of the amino acid sequence, glycine-glycine-phenylalanine-glycine, and / or maleimidocaproyl group with PEG. Exemplary homo- and heterobifunctional cross-linking agents can also include, for example, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), sulfo-NHS (N- hydroxysulfo succinimide), NHS (N-hydroxysuccinimide), dimethyl pimelimidate dihydrochloride, suberic acid bis(N-hydroxysuccinimide ester), DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DSP (dithiobis(succinimidyl propionate)), sulfo- SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SPDP ((succinimidyl 3-(2-pyridyldithio)propionate), N-succinimdyl oxycarbonylethyl methanethio sulfonate, 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, 6-Maleimidocaproic acid sulfo-N-succinimidyl ester, maleimidoacetic acid N-hydroxysuccinimide ester, N- succinimidyl iodoacetate, and 4-(4-maleimidophynyl) butyric acid N-hydroxysuccinimide ester.

[0117] In some embodiments, provided synthetic binding proteins are conjugated to a chelating moiety, e.g., a chelating moiety which can complex with a metal such as a radionuclide or a non-metal radiolabel. Non-limiting examples of chelating moieties include DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α″, α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10- tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2′,2″-(10-(2,6- Page 34 of 66       Attorney Docket No.: AIP-010WOdioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7- triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA (1,4,7,10,13,16- hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13- pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaacetic acid), H4Octapa (N,N′-bis(6- carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diacetic acid), H2Dedpa (1,2-[[6-(carboxy)- pyridin-2-yl]-methylamino]ethane), H6phospa (N,N′-(methylenephosphonate)-N,N′-[6- (methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine- N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinones), and porphyrin.

[0118] In some embodiments, the chelating moiety is DOTA.

[0119] In some embodiments, the chelating moiety has the structure: orPage 35 of 66       Attorney Docket No.: AIP-010WOwherein R2is optionally substituted hydrogen or —CO2H.

[0121] Persons skilled in the art may determine the appropriate chelating moiety depending on the particular situation, e.g., depending on the metal (e.g., radionuclide) intended to be complexed with the chelating moiety.

[0122] In some embodiments, the chelating moiety is complexed with a radionuclide.

[0123] In some embodiments, the radionuclide is useful in diagnostic or imaging applications. Examples of such radionuclides, include, but are not limited to, gamma emitters, such as62Cu,64Cu,67Ga,68Ga,86Y,89Zr, and111In.

[0124] In some embodiments, the radionuclide is useful in therapeutic applications, such as beta or alpha emitters.

[0125] In certain embodiments, the radionuclide is an alpha-emitting radionuclide. As one of ordinary skill in the art would appreciate, the chelator can chelate an alpha-emitting radionuclide through one or more coordinate bonds. In some embodiments, the alpha- emitting radionuclide is selected from the group consisting of211At,212Bi,213Bi,225Ac,223Ra,212Pb,227Th,149Tb, and255Fm. In some embodiments, the alpha-emitting radionuclide is225Ac.225Ac (Actinium-225) is an alpha-emitting radioisotope with a 10-day half-life that is of particular interest for medical applications.225Ac decays in a series of steps that ultimately emits 4 alpha particles before reaching a stable isotope,209Bi, thereby increasing the potency.

[0126] In some embodiments, the radionuclide is a beta and / or gamma-emitting radionuclide, such as a radionuclide selected from the group consisting of32P,47Sc,67Cu,77As,89Sr,90Y,99Tc,105Rh,109Pd,111Ag,131I,153Sm,159Gd,165Dy,166Ho,169Er,177Lu,186Re,188Re,194Ir,198Au, and199Au.

[0127] In some embodiments, the radioisotope is177Lu (Lutetium-177), which emits both gamma-irradiation suitable for imaging and medium-energy beta-irradiation suitable for radiotherapy.

[0128] Linkers may be used to link two or more synthetic LILRB4-binding proteins to one another, and may also be used to link one or more synthetic LILRB4-binding proteins to one or more effectors. A linker may be a peptide linker or a chemical linker. Linkers may be covalently bound (e.g., to an amino acid) to a synthetic LILRB4-binding protein and covalently bound to a second agent (e.g., an effector, e.g., a second binding protein). The Page 36 of 66       Attorney Docket No.: AIP-010WOcomposition and / or length of the linker may be designed with a particular functionality in mind, but preferably is non-immunogenic. A linker may contain one or more glycine amino acids and / or one or more serine amino acids. Exemplary linkers can comprise one or multiples of (Gly2Ser)n, (Gly3Ser)n, or (Gly4Ser)n, where n can be 1, 2, 3 etc.

[0129] It is contemplated that the synthetic LILRB4-binding proteins disclosed herein can monovalent or multivalent. Multivalent proteins may include, but are not limited to bivalent and trivalent formats. A multivalent molecule may include two, three, four, or more monovalent synthetic LILRB4-binding proteins, wherein at least one linker connects a C- terminal amino acid of a first monovalent binding protein to an N-terminal amino acid of a second monovalent synthetic binding protein, such that the first and the second monovalent synthetic LILRB4-binding proteins are linked together. For example, in some embodiments, a multivalent (e.g., bivalent) miniprotein may comprise two miniproteins, each independently between about 35 and 100 amino acids and associated (e.g., linked, conjugated) with one another. In some embodiments, multivalent molecules may be fused and / or combined to another molecule, such as an effector molecule, e.g., a half-life extender (e.g., a site or a protein that binds to serum albumin to extend serum half life, etc.).

[0130] In certain embodiments, the disclosure provides a multivalent (e.g., bivalent) protein comprising a multiple synthetic LILRB4-binding proteins disclosed herein. The multivalent protein can comprise a first synthetic LILRB4-binding protein and a second synthetic LILRB4-binding protein linked together through at least one linker. A linker (e.g., a glycine and serine containing linker (e.g., GGS), can connect a C-terminal amino acid of the first synthetic LILRB4-binding protein to an N-terminal amino acid of the second synthetic LILRB4-binding protein. Depending upon the circumstances, the multivalent binding protein can have a binding affinity stronger than the binding affinity of each synthetic LILRB4-binding protein alone. In certain embodiments, the multivalent protein comprises a synthetic LILRB4-binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in SEQ ID NO: 1-6 or 13- 18, or as otherwise described herein. VIII. PHARMACEUTICAL COMPOSITIONS

[0131] Once produced, a synthetic LILRB4-binding protein disclosed herein can be formulated into a pharmaceutical composition. Page 37 of 66       Attorney Docket No.: AIP-010WO

[0132] For therapeutic use, a synthetic LILRB4-binding protein disclosed herein is combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc.) used in formulating and preparing pharmaceutical compositions are known and / or readily accessible to those of skill in the art. Depending upon the circumstances, a carrier can include a liquid (e.g., a sterile liquid) or a solid. A carrier may be selected from or comprise water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically a carrier is approved by United States Food and Drug Administration and meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other International Pharmacopoeia. Suitable formulations for use in the present disclosure are found in see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded.2020). For a brief review of methods for drug delivery, see, e.g., Langer (1990) SCIENCE 249:1527-1533. The resulting pharmaceutical compositions are suitable for administration to a subject (e.g., an animal, e.g., a mammal, e.g., a human).

[0133] A pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or Page 38 of 66       Attorney Docket No.: AIP-010WOhydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded.2020)).

[0134] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(−)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0135] Depending upon the circumstances, a pharmaceutical composition may contain nanoparticles, or lipid droplets, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED.1: 10-29).

[0136] Pharmaceutical compositions containing a synthetic LILRB4-binding protein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the synthetic peptide is administered by subcutaneous administration.

[0137] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded.2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene Page 39 of 66       Attorney Docket No.: AIP-010WOglycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0138] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyethoxylated castor oil or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0139] Pharmaceutical formulations preferably are sterile. Formulations can be sterilized, for example, by methods appropriate to retain activity and stability of the synthetic LILRB4- binding protein included therein. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0140] Depending upon the drug substance and formulation, the resulting dosage forms can be stable for extended periods of time, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more, when the dosage form is a liquid or solid. The formulations can be stable at room temperature or higher. It is contemplated that the dosage form is stable at ambient conditions in PBS. Alternatively the dosage form is frozen (e.g., a liquid or a lyophilizate) and stable under appropriate temperatures such as, e.g., -20 °C, -80 °C).

[0141] Depending upon the circumstances, the dosage forms can be formulated as a unit dose, which can include, for example, about 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 g, 1.5 g, 2.5 g, 5 g, or 10 g of the drug substance.

[0142] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein are generally administered by parenteral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion. In certain embodiments, it is contemplated that the synthetic constructs disclosed herein can be administered by systemic administration. Page 40 of 66       Attorney Docket No.: AIP-010WO

[0143] Generally, a therapeutically effective amount of active component, for example, a synthetic LILRB4-binding protein disclosed herein, is in the range of 0.1 mg / kg to 1000 mg / kg, e.g., 1 mg / kg to 100 mg / kg, e.g., 10 mg / kg to 500 mg / kg, e.g., 500 mg / kg to 1000 mg / kg. In certain embodiments, the effective amount is in the range of 15 to 50 mg / kg. In certain embodiments, the effective amount is 15 mg / kg. In certain embodiments, the effective amount is 30 mg / kg. In certain embodiments, the effective amount is 50 mg / kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the synthetic peptide, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. IX. METHODS OF USE AND TREATMENT

[0144] The LILRB4-binding proteins described herein can be used in a variety of different contexts, including therapeutic and / or diagnostic contexts. For example, the binding proteins can be used in a method of targeting LILRB4, such as a method which comprises contacting a cell that expresses LILRB4 on its cell surface with a composition comprising the synthetic LILRB4-binding protein disclosed herein. Additionally or alternatively, the LILRB4-binding proteins described herein can be used to modulate LILRB4 activity. For example, such a method may comprise contacting a cell that expresses LILRB4 on its cell surface with a composition comprising the synthetic LILRB4-binding protein disclosed herein. Additionally or alternatively, the LILRB4-binding proteins described herein can be used to detect LILRB4-positive cells, e.g., cancer cells such as myeloid cancer cells (e.g., acute myeloid leukemia (AML) cancer cells). For example, such a method may comprise contacting a cell suspected of expressing LILRB4 on its cell surface with a composition comprising the synthetic LILRB4-binding protein conjugate comprising a detectable label, as disclosed herein.

[0145] In each method, the LILRB4-binding protein or the LILRB4-binding protein conjugate further comprises an effector molecule. In some embodiments of each of the Page 41 of 66       Attorney Docket No.: AIP-010WOforegoing methods, the synthetic LILRB4-binding protein or the LILRB4-binding protein conjugate inhibits or reduces LILRB4 activity in the presence of a LILRB4 ligand relative to LILRB4 activity in the presence of the LILRB4 ligand but in the absence of the synthetic LILRB4-binding protein or the LILRB4-binding protein conjugate.

[0146] The LILRB4-binding proteins may be used in treatment of a disease, disorder, or condition mediated by LILRB4.

[0147] In certain embodiments, the disclosure provides a method of decreasing LILRB4- mediated activity in a subject in need thereof. The method comprises administering to the subject an effective amount of the pharmaceutical composition comprising a synthetic LILRB4 binding protein or LILRB4-binding protein conjugate disclosed herein. As such, the compositions described herein can be used in treating cancer in a subject in need thereof.

[0148] Exemplary diseases, disorders, or conditions that may be treated with the LILRB4- binding proteins disclosed herein include cancer.

[0149] It is contemplated that therapy can be accomplished using a synthetic LILRB4- binding protein (or LILRB4-binding protein conjugate) alone, as a monotherapy, or as part of a combination therapy. The combination therapy may include one or more additional agents or therapeutic approaches known to those of skill in the art for treating cancer, and may have been previously used, be already ongoing, or added to a treatment for a subject in need thereof.

[0150] A subject may be evaluated, e.g., by a healthcare provider, before, during, and / or after treatment with a composition provided herein. Depending on the outcome of the evaluation, a treatment may be continued or ceased, treatment frequency or dosage may change, or the patient may be treated with a different synthetic LILRB4-binding protein. Subjects may be administered a composition comprising the synthetic LILRB4-binding protein for a discrete period of time according to dosage paradigms described herein, including, optionally, until the disease, disorder, or condition is treated.

[0151] Subjects that can be treated include those suspected as having, having, or at risk of having cancer. In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is a liquid tumor. For example, in some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), myeloma (e.g., multiple myeloma), lymphoma (e.g., mantle cell lymphoma)), and multiple myeloma (MM). In some embodiments, the cancer is a solid tumor. Page 42 of 66       Attorney Docket No.: AIP-010WO

[0152] The methods described herein may include a step of selecting a treatment for a subject in need thereof. The method includes (a) identifying (e.g., diagnosing) the subject with a condition, and (b) selecting a synthetic LILRB4-binding protein or LILRB4-binding protein conjugate as described herein, to treat the subject. Synthetic LILRB4-binding proteins or LILRB4-binding protein conjugate administered in an effective amount to a subject in need thereof may result in one or more of (a) reduced cancer cell proliferation, (b) increased tumor cell death, (c) improved immune cell function, (d) reduced incidence of cancer remission (e) reduced pain, or (f) increased quality of life and any combinations of any of the above.

[0153] Certain diseases can be treated by modulating, e.g., reducing, LILRB4-mediated signaling.

[0154] LILRB4-mediated activity may be evaluated by detecting levels of one or more downstream molecules. In certain embodiments, such molecules are expressed at higher levels in a subject having or suspected as having a disease, e.g., cancer.

[0155] The present disclosure provides methods of treating a subject in need thereof by administering an effective amount of the synthetic LILRB4-binding protein or LILRB4- binding protein conjugate to the subject. The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The phrase administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially Page 43 of 66       Attorney Docket No.: AIP-010WOadditive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0156] In certain embodiments, a method or composition described herein, is administered in combination with one or more additional therapies, e.g., surgery, radiation therapy, or administration of another therapeutic preparation. In certain embodiments, the additional therapy may include an anti-cancer agent. Exemplary anti-cancer agents include, for example, small molecules and biologic immunomodulators. EXAMPLES EXAMPLE 1: INITIAL SCREENING

[0157] This Example describes an initial in vitro screen of synthetic miniproteins using a library screen to identify miniproteins capable of binding LILRB4.

[0158] The library was designed in silico and members were screened for certain characteristics, such as selectivity and specificity of binding to LILRB4 at particular concentrations of LILRB4 (e.g., 10 μM). Once identified, certain synthetic binding proteins were synthesized for use in downstream screening and discovery processes.

[0159] The miniproteins with higher Next Generation Sequencing (NGS) counts were expressed and purified in soluble form from E. coli and further characterized.

[0160] A set of synthetic binding proteins containing six unique proteins (having SEQ ID NOs: 1-6) were individually tested for LILRB4 binding by surface plasmon resonance (SPR). Initially, miniproteins encoded by each of these sequences were expressed and purified from E. coli to use in binding assays, including SPR assays. For all proteins that showed specific binding to LILRB4 (as determined by measurable binding to the LILRB4 ectodomain protein and not to a streptavidin-coated chip surface), binding affinities were determined. In parallel with binding affinity testing, each of the six proteins was individually tested for the ability to bind to LILRB4 in the cell context by using tumor cell lines that express LILRB4 on its surface.

[0161] One protein (Reference Miniprotein 1 with amino acid sequence of SEQ ID NO: 1) was selected for further analysis, e.g., as described in Example 2. FIG.3 depicts a three- dimensional (3D) representation and a secondary structure representation of Reference Miniprotein 1 along with a portion of its amino acid sequence (SEQ ID NO: 13) displayed with domains (D1, D2, D3, and D4), connected with loops (L1, L2, and L3), indicated as Page 44 of 66       Attorney Docket No.: AIP-010WOshown. Note that the N-terminal proline and C-terminal threonine of Reference Miniprotein 1 (SEQ ID NO: 1) are not shown in FIG.3.

[0162] Reference Miniprotein 1 was confirmed to bind to the LILRB4 ectodomain by SPR (ka 7.5 x 105s-1M-1; kd 6.38 x 10-04s-1; KD 8.52 x 10-10M) and was folded and thermostable as shown by circular dichroism spectroscopy from about 25oC to about 95oC (FIG.2). Table 3 summarizes the results for Reference Miniproteins 1-6. TABLE 3. Binding Characteristics of Exemplary Reference Miniproteins SEQ ID NO: / Ka (1 / Ms) Kd (1 / s) KD (M) ReferenceEXAMPLE 2: CHARACTERIZATION OF MINIPROTEIN VARIANTS WITH DIFFERING LOOP LENGTHS

[0163] This Example describes the characterization of Reference Miniprotein 1 (identified as described in Example 1) in which the lengths of loops 1, 2, and / or 3 vary compared to that in Reference Miniprotein 1.

[0164] The binding characteristics (as determined by surface plasmon resonance) of the variants are summarized in Tables 4A, 4B, and 4C below. Each variant had the sequence of Reference Protein 1 (SEQ ID NO: 1) except that the sequence of the indicated loop (i.e., parental loop, e.g., L1, L2, or L3 with sequences of: L1 (DS), L2 (NPNV, SEQ ID NO: 11 or 26); and L3 (EETH; SEQ ID NO: 12 or 83), respectively) was substituted with a sequence (e.g., L1 sequence, L2 sequence, or L3 sequence) shown in each of the respective Tables, 4A-4C. Page 45 of 66       Attorney Docket No.: AIP-010WOTABLE 4A. Binding Characteristics of Exemplary Variants of Reference Proteins – L1 substitutions ) 0 7 7 8 9999TABLE 4B. Binding Characteristics of Exemplary Variants of Reference Proteins – L2 substitutions L2 sequence SEQ Length Binding properties (by SPR) ID ) 1008 110808 080708 060908  Attorney Docket No.: AIP-010WO) 08050907081107070710080808 070707 07 07 08 09 07 08 08 08 08 08  Attorney Docket No.: AIP-010WO) 07 08 07 07 08 09 09 07 07 07 08 07 07 09 07 09 080707 07    Attorney Docket No.: AIP-010WOTABLE 4C. Binding Characteristics of Exemplary Variants of Reference Proteins – L3 substitutions ) 1008 090908 08100909080908080810080909 0807 09 07 09 10 08 08   Attorney Docket No.: AIP-010WO07 08 07 07 09 07 09 08 08 07 08 07 08 10 07 07 08 08 09 07 08 08    Attorney Docket No.: AIP-010WO08 08 07 08 07 07 08

[0165] Thus, the present Example demonstrates that the loops within synthetic LILRB4- binding miniproteins can be lengthened, and the total size of the LILRB4-binding miniprotein can be lengthened up to a length of at least 100 without abolishing binding to LILRB4. EXAMPLE 3: VARIANT MINIPROTEIN STABILITY

[0166] This Example describes in vitro chemical stability of various Reference Miniproteins (SEQ ID NOs: 1-6) using biophysical characterization after exposure to exemplary chemical denaturants.

[0167] Each of these LILRB4 miniproteins developed and characterized in Example 1 were exposed to 4 M urea. Measurements of whether these proteins had properly folded three-dimensional structure after heating to 95℃ and then returning to 25℃ as measured by circular dichroism (CD) spectroscopy were determined. FIG.2 shows that exemplary Reference Miniprotein 1 (SEQ ID NO: 1) was thermally stable at least from 25℃ - 75℃. In addition, Reference Miniproteins 2-6 (corresponding to SEQ ID NOs: 2-6) were thermally stable, retaining their conformations across a variety of conditions and maintaining binding across a range of temperatures (as shown by proper refolding after heating to 95℃ and then cooling to 25℃). Reference Miniproteins 1-6 (corresponding to SEQ ID NOs: 1-6) all showed chemical stability in 4 M urea because after exposure to urea and subsequent dilution into PBS, the Reference Miniproteins each folded into their expected, respective, molecular structures (as prior to urea exposure) at 25℃ as measured by CD spectroscopy. Page 51 of 66       Attorney Docket No.: AIP-010WOEXAMPLE 4: IN VITRO BINDING ACTIVITY OF SYNTHETIC LILRB4-BINDING MINIPROTEINS

[0168] This Example demonstrates the ability of LILRB4 miniproteins to bind to tumor cells that express LILRB4 on their surface.

[0169] A FLAG®-tagged version of an exemplary synthetic LILRB4-binding protein (Reference Miniprotein 1) was generated (FLAG®-p16-LILRB4-binding protein; p16 is an amino acid linker). MV-4-11 or OC-AML3 cells, both of which express LILRB4, were incubated with the FLAG®-tagged LILRB4-binding protein. Cells were then incubated with a fluorescently labeled anti-FLAG® tag antibody. Cells were then analyzed by flow cytometry for staining, with increased mean fluorescence intensity (MFI) indicative of binding between the LILRB4-binding protein and the LILRB4-expressing cells.

[0170] As shown in FIGs.4A and 4C, the synthetic LILRB4-binding protein bound to both MV-4-11 and OCI-AML3 cells (evidenced by the right-most peak in each of these figures, corresponding to higher MFI).

[0171] As controls, some cells were incubated with a commercial LILRB4 antibody instead of the FLAG®-tagged synthetic LILRB4-binding protein (see FIGs.4B and 4D), and some cells were incubated with anti-FLAG® antibody without prior incubation with FLAG®-tagged LILRB4-binding protein (see left-most peak in FIG.4A and 4C).

[0172] Thus, this Example demonstrates that a synthetic LILRB4-binding protein of the present disclosure binds to LILRB4 expressed on cells. EXAMPLE 5: IN VITRO BINDING ACTIVITY OF SYNTHETIC LILRB4-BINDING MINIPROTEINS AGAINST LILRB FAMILY MEMBERS

[0173] This Example demonstrates the binding specificity of a LILRB4 miniprotein disclosed herein for LILRB4.

[0174] The leukocyte immunoglobulin-like receptor (LILRB) protein family includes five proteins having high sequence homology with one another. Example canonical sequences of each protein, which each may have more than one isoform, are provided below.

[0175] LILRB1 - Uniprot ID Q8NHL6-1 MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYR EKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIK PTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGP Page 52 of 66       Attorney Docket No.: AIP-010WOVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGS DAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAP SDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRST YQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPT STSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQ RKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQ AVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSL TLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 138) LILRB2 - Uniprot ID Q8N423 MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYR EKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKP TLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPV SPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVMAPGESLTLQCVSD VGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPS DPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIH EYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPIS TPGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQGKHWTSTQR KADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAAASEAPQD VTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH (SEQ ID NO: 139)

[0176] LILRB3 - Uniprot ID O75022-1 MTPALTALLCLGLSLGPRTRVQAGPFPKPTLWAEPGSVISWGSPVTIWCQGSQEAQEYRLHK EGSPEPLDRNNPLEPKNKARFSIPSMTEHHAGRYRCHYYSSAGWSEPSDPLEMVMTGAYSKP TLSALPSPVVASGGNMTLRCGSQKGYHHFVLMKEGEHQLPRTLDSQQLHSRGFQALFPVGPV TPSHRWRFTCYYYYTNTPWVWSHPSDPLEILPSGVSRKPSLLTLQGPVLAPGQSLTLQCGSD VGYNRFVLYKEGERDFLQRPGQQPQAGLSQANFTLGPVSPSNGGQYRCYGAHNLSSEWSAPS DPLNILMAGQIYDTVSLSAQPGPTVASGENVTLLCQSWWQFDTFLLTKEGAAHPPLRLRSMY GAHKYQAEFPMSPVTSAHAGTYRCYGSYSSNPHLLSHPSEPLELVVSGHSGGSSLPPTGPPS TPGLGRYLEVLIGVSVAFVLLLFLLLFLLLRRQRHSKHRTSDQRKTDFQRPAGAAETEPKDR GLLRRSSPAADVQEENLYAAVKDTQSEDRVELDSQSPHDEDPQAVTYAPVKHSSPRREMASP PSSLSGEFLDTKDRQVEEDRQMDTEAAASEASQDVTYAQLHSLTLRRKATEPPPSQEGEPPA EPSIYATLAIH (SEQ ID NO: 140) Page 53 of 66       Attorney Docket No.: AIP-010WO

[0177] LILRB4 - Uniprot ID: Q8NHJ6-1 MIPTFTALLCLGLSLGPRTHMQAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDK EESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPVGWSQPSDPLELVMTGAYSKP TLSALPSPLVTSGKSVTLLCQSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPVT SVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSPTRSVSTAAGPEDQPLMPTGS VPHSGLRRHWEVLIGVLVVSILLLSLLLFLLLQHWRQGKHRTLAQRQADFQRPPGAAEPEPK DGGLQRRSSPAADVQGENFCAAVKNTQPEDGVEMDTRQSPHDEDPQAVTYAKVKHSRPRREM ASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTLRQKATEPPPSQEGA SPAEPSVYATLAIH (SEQ ID NO: 141)

[0178] LILRB5 - Uniprot ID O75023-1 MTLTLSVLICLGLSVGPRTCVQAGTLPKPTLWAEPASVIARGKPVTLWCQGPLETEEYRLDK EGLPWARKRQNPLEPGAKAKFHIPSTVYDSAGRYRCYYETPAGWSEPSDPLELVATGFYAEP TLLALPSPVVASGGNVTLQCDTLDGLLTFVLVEEEQKLPRTLYSQKLPKGPSQALFPVGPVT PSCRWRFRCYYYYRKNPQVWSNPSDLLEILVPGVSRKPSLLIPQGSVVARGGSLTLQCRSDV GYDIFVLYKEGEHDLVQGSGQQPQAGLSQANFTLGPVSRSHGGQYRCYGAHNLSPRWSAPSD PLDILIAGLIPDIPALSVQPGPKVASGENVTLLCQSWHQIDTFFLTKEGAAHPPLCLKSKYQ SYRHQAEFSMSPVTSAQGGTYRCYSAIRSYPYLLSSPSYPQELVVSGPSGDPSLSPTGSTPT PGPEDQPLTPTGLDPQSGLGRHLGVVTGVSVAFVLLLFLLLFLLLRHRHQSKHRTSAHFYRP AGAAGPEPKDQGLQKRASPVADIQEEILNAAVKDTQPKDGVEMDAPAAASEAPQDVTYAQLH SLTLRREATEPPPSQEREPPAEPSIYAPLAIH (SEQ ID NO: 142)

[0179] To determine whether Reference Miniprotein 1 (having the sequence of SEQ ID NO: 1) binds specifically to LILRB4, surface plasmon resonance (SPR) was used to examine binding of Reference Miniprotein 1 to each of the LILRB family members. Biotinylated LILRB proteins were purchased from Acro and immobilized on a SPR streptavidin chip for subsequent use in SPR experiments. As shown in FIGs.5A-5E, Reference Miniprotein 1 bound to LILRB4 (FIG.5D) but not to other LILRB family members (FIGs.5A-5C, 5E). EXAMPLE 6: IN VITRO BINDING ACTIVITY OF DOTA CONJUGATED SYNTHETIC LILRB4-BINDING MINIPROTEINS

[0180] Reference Miniprotein 1 (having the amino acid sequence of SEQ ID NO: 1) was chosen for installing a lysine residue to enable site specific conjugation with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which enables loading the binder with metals such as radionuclides. In order to do so, a glycine and serine linker containing a Page 54 of 66       Attorney Docket No.: AIP-010WOsingle lysine residue was added to the C-terminus of the sequence (Compound 1). Two unique DOTA reagents were selected for conjugation. The first was p-SCN-Bn-DOTA from Macrocyclics (Catalog B-205) and the second was DOTA-NHS-ester from Macrocyclics (Catalog B-280). Conjugation was performed in aqueous buffer using standard laboratory techniques. Conjugated compounds were further purified by size exclusion chromatography. Site specific labeling was verified by mass spectrometry.

[0181] Table 5 presents the Ka, Kd, and calculated KDvalues obtained from these experiments. Binding to LILRB4 by the two conjugate compounds (Compounds 2 and 3) and by the unconjugated compound (Compound 1) were determined by SPR.

[0182] As shown in Table 5, both conjugated compounds bound to LILRB4 with KDvalues in the subnanomolar range, with one conjugate (Compound 2) demonstrating binding with a KDvalue of less than 15 pM. Therefore, these results demonstrate the feasibility of conjugating Reference Miniprotein 1 while retaining effective binding to LILRB4. Table 5. Binding characteristics of Exemplary DOTA conjugated miniproteins Compound Sequence Conjugation Binding properties (by SPR) ) 12 11 10OTHER EMBODIMENTS

[0183] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that Page 55 of 66       Attorney Docket No.: AIP-010WOcome within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein. Page 56 of 66

Claims

Attorney Docket No.: AIP-010WOWHAT IS CLAIMED IS:

1. A synthetic LILRB4-binding protein, the binding protein comprising: (a) an amino acid sequence from 35 to 100 amino acid residues in length; (b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for LILRB4 stronger than 1 µM; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to LILRB4 upon cooling to room temperature after thermal denaturation at 95 °C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to LILRB4 after incubation for 16 hours at 37 °C of incubation in PBS relative to the protein under the same conditions prior to the incubation; and / or (iii) retains at least 90% binding affinity to LILRB4 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

2. A synthetic LILRB4-binding protein, the binding protein comprising: (a) an amino acid sequence from 35 to 100 amino acid residues in length; (b) a net negative charge in PBS; (c) a binding affinity for LILRB4 stronger than 1 µM; (d) at least one alpha helix; (e) at least three beta strands; and (f) at least three amino acid loops, wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first beta strand to a terminal amino acid (e.g., a N-terminal amino acid) of a first alpha helix; a second loop having a second amino acid sequence connects a second terminal amino acid (e.g., an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta strand; and a third loop having a second amino acid sequence connects a second terminal amino acid (e.g., an C-terminal amino acid) of the second beta strand to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta strand. Page 57 of 66       Attorney Docket No.: AIP-010WO3. The synthetic LILRB4-binding protein of claim 1 or 2, wherein the binding protein comprises one or more of the following features: (a) free of tryptophan residues; (b) free of methionine residues; (c) free of cysteine residues; (d) does not comprise an unpaired cysteine residue when cysteine residues are present in the protein; (e) free of N-linked glycosylation sites; (f) free of L-X-R-R sequences, wherein X represents any amino acid residue; and (g) soluble up to at least 0.5 mM in PBS at 4 °C for one month.

4. The synthetic LILRB4-binding protein of any one of claims 1-3, wherein the binding affinity for LILRB4 is between 1 μM to about 0.001 nΜ; about 1 μM to about 0.01 nM, about 1 μM to about 0.75 nM; about 1 μM to about 0.5 nM; about 2.5 μM to about 0.25 nM; about 1 μM to about 1 nΜ; about 0.75 μM to about 1 nM, about 0.5 μM to about 1 nM; about 0.25 μM to about 1 nΜ; about 0.10 μM to about 1 nΜ; about 75 nΜ to about 1 nΜ; about 50 nΜ to about 1 nΜ; about 25 nΜ to about 1 nΜ; about 10 nΜ to about 1 nΜ; and about 5 nΜ to about 1 nΜ.

5. The synthetic LILRB4-binding protein of any one of claims 1-4, wherein the binding affinity for LILRB4 is stronger than about 1 μM, about 0.75 μM, about 0.5 μM, about 0.25 μM, about 0.1 μM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

6. The synthetic LILRB4-binding protein of any one of claims 2-5, wherein the N- terminus of the first beta strand is preceded by one or more N-terminal amino acid residues.

7. The synthetic LILRB4-binding protein of any one of claims 2-6, wherein the C- terminus of the third beta strand is followed by one or more C-terminal amino acid residues.

8. The synthetic LILRB4-binding protein of any one of claims 1-7, wherein the protein comprises from 35 to 95 amino acid residues in length, from 35 to 90 amino acid residues in length, from 35 to 85 amino acid residues in length, from 35 to 80 amino acid residues in Page 58 of 66       Attorney Docket No.: AIP-010WOlength, from 35 to 75 amino acid residues in length, from 35 to 70 amino acid residues in length, from 35 to 65 amino acid residues in length, from 35 to 60 amino acid residues in length, from 35 to 55 amino acid residues in length, from 35 to 50 amino acid residues in length, from 35 to 45 amino acid residues in length, from 35 to 40 amino acid residues in length, from 40 to 95 amino acid residues in length, from 40 to 90 amino acid residues in length, from 40 to 85 amino acid residues in length, from 40 to 80 amino acid residues in length, from 40 to 75 amino acid residues in length, from 40 to 70 amino acid residues in length, from 40 to 65 amino acid residues in length, from 40 to 60 amino acid residues in length, from 40 to 55 amino acid residues in length, from 40 to 50 amino acid residues in length, from 40 to 45 amino acid residues in length, from 45 to 95 amino acid residues in length, from 45 to 90 amino acid residues in length, from 45 to 85 amino acid residues in length, from 45 to 80 amino acid residues in length, from 45 to 75 amino acid residues in length, from 45 to 70 amino acid residues in length, from 45 to 65 amino acid residues in length, from 45 to 60 amino acid residues in length, from 45 to 55 amino acid residues in length, from 45 to 50 amino acid residues in length, from 50 to 95 amino acid residues in length, from 50 to 90 amino acid residues in length, from 50 to 85 amino acid residues in length, from 50 to 80 amino acid residues in length, from 50 to 75 amino acid residues in length, from 50 to 70 amino acid residues in length, from 50 to 65 amino acid residues in length, from 50 to 60 amino acid residues in length, or from 50 to 55 amino acid residues in length.

9. The synthetic LILRB4-binding protein of any one of claims 1-8 wherein the protein comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

10. The synthetic LILRB4-binding protein of any of claims 2-9, wherein (a) the first alpha helix contains at least two hydrophobic amino acid residues, wherein, optionally, one or more of the at least two hydrophobic amino acid residues is not solvent accessible; (b) the second beta strand contains at least one hydrophobic amino acid residue, wherein, optionally, the at least one hydrophobic amino acid residue is not solvent accessible; (c) the third beta strand contains at least one hydrophobic amino acid residue, wherein, optionally, one or more of the at least one hydrophobic amino acid residue is not solvent accessible; Page 59 of 66       Attorney Docket No.: AIP-010WO(d) the first, second, and / or third beta strand contains at least one or two solvent accessible amino acid residues; (e) the first alpha helix contains at least one or two solvent accessible amino acid residues; (f) the first and / or second and / or third loop contains at least one hydrophobic amino acid residue; or (g) the binding protein comprises any combination of elements selected from (a), (b), (c), (d), (e), and (f).

11. The synthetic LILRB4-binding protein of any one of claims 1-10, wherein: (a) the first, second, and / or third beta strand each contains at least two hydrophobic amino acid residues; (b) the first, second, and third beta strand each contains at least one solvent accessible amino acid residue; (c) the first, second, and third beta strand each contains at least two hydrophobic and one solvent accessible amino acid residue; (d) the first alpha helix contains at least two hydrophobic amino acid residues; (e) the first alpha helix contains at least one solvent accessible amino acid residue; (f) the first alpha helix contains at least two hydrophobic and one solvent accessible amino acid residues; (g) the first alpha helix sheet contains at least four solvent accessible amino acid residues; and / or (h) the first, second, and / or third loop contains at least one hydrophobic amino acid residue.

12. A synthetic LILRB4- binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula I), wherein Page 60 of 66       Attorney Docket No.: AIP-010WOD1, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and L1, L2, and L3 are loops 1, 2 and 3, respectively; wherein D1, D2, D3, and D4 independently comprise any of the following combinations: (a) D1 comprises the amino acid sequence ITV, (b) D2 comprises an amino acid sequence of LLX9ASVVAYQIX18X19X20 (SEQ ID NO: 8), wherein X9is E or V; X18 is D or Q; X19 is H, R, or S; and X20 is A, D, or E; (c) D3 comprises an amino acid sequence of X25VX27IX29YD (SEQ ID NO: 9), wherein X25 is A, R or Y; X27 is E, Q, or S; and X29 is H, R, or Y; and (d) D4 comprises the amino acid sequence RYYIVTTE (SEQ ID NO: 10).

13. The synthetic LILRB4-binding peptide of claim 12, wherein L1 comprises the amino acid sequence DS; L2 comprises the amino acid sequence NPNV (SEQ ID NO: 11); and / or L3 comprises the amino acid sequence EETH (SEQ ID NO: 12).

14. A synthetic LILRB4-binding protein comprising an amino acid sequence of ITVDSLLX9ASVVAYQIX18X19X20NPNVX25VX27IX29YDEETHRYYIVTTE (SEQ ID NO: 7), wherein X9 is E or V; X18 is D or Q; X19 is H, R, or S; Page 61 of 66       Attorney Docket No.: AIP-010WOX20 is A, D, or E; X25 is A, R or Y; X27is E, Q, or S; and X29is H, R, or Y.

15. The synthetic LILRB4-binding protein of any one of claims 12-13, comprising an N- terminal amino acid which is N-terminal to D1.

16. The synthetic LILRB4-binding protein of claim 15, wherein the N-terminal amino acid is P.

17. The synthetic LILRB4-binding protein of any one of claims 12-13 and 15-16, comprising a C-terminal amino acid which is C-terminal to D4.

18. The synthetic LILRB4-binding protein of claim 17, wherein the C-terminal amino acid is T.

19. The synthetic LILRB4-binding protein of any one of claims 12-18, comprising an amino acid sequence selected from the group consisting of: ITVDSLLVASVVAYQIQSDNPNVRVEIRYDEETHRYYIVTTE (SEQ ID NO: 13), ITVDSLLVASVVAYQIQSANPNVRVEIRYDEETHRYYIVTTE (SEQ ID NO: 14), ITVDSLLVASVVAYQIDHDNPNVRVEIRYDEETHRYYIVTTE (SEQ ID NO: 15), ITVDSLLEASVVAYQIQRENPNVYVSIYYDEETHRYYIVTTE (SEQ ID NO: 16), ITVDSLLEASVVAYQIQRENPNVAVQIHYDEETHRYYIVTTE (SEQ ID NO: 17), and ITVDSLLEASVVAYQIQRENPNVYVQIHYDEETHRYYIVTTE (SEQ ID NO: 18).

20. The synthetic LILRB4-binding protein of claim 19, comprising an amino acid sequence selected from the group consisting of: PITVDSLLVASVVAYQIQSDNPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 1), PITVDSLLVASVVAYQIQSANPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 2), PITVDSLLVASVVAYQIDHDNPNVRVEIRYDEETHRYYIVTTET (SEQ ID NO: 3), PITVDSLLEASVVAYQIQRENPNVYVSIYYDEETHRYYIVTTET (SEQ ID NO: 4), PITVDSLLEASVVAYQIQRENPNVAVQIHYDEETHRYYIVTTET (SEQ ID NO: 5), and PITVDSLLEASVVAYQIQRENPNVYVQIHYDEETHRYYIVTTET (SEQ ID NO: 6). Page 62 of 66       Attorney Docket No.: AIP-010WO21. The synthetic LILRB4-binding protein of any one of claims 12-20, having a binding affinity for LILRB4 stronger than 1 µM.

22. The synthetic LILRB4-binding protein of any one of claims 12-21, having a binding affinity for LILRB4 that is between 1 μM to about 0.001 nΜ; about 1 μM to about 0.01 nM; about 1 μM to about 0.75 nM; about 1 μM to about 0.5 nM; about 1 μM to about 0.25 nM; about 1 µM to about 0.01 nM; about 1 μM to about 1 nΜ; about 0.5 μM to about 1 nM; about 0.25 μM to about 1 nΜ; about 0.10 μM to about 1 nΜ; about 0.10 μM to about 0.01 nΜ; about 75 nΜ to about 1 nΜ; about 50 nΜ to about 1 nΜ; about 25 nΜ to about 1 nΜ; about 10 nΜ to about 1 nΜ; and about 5 nΜ to about 1 nΜ.

23. The synthetic LILRB4-binding protein of any one of claims 12-21, having a binding affinity for LILRB4 that is stronger than about 1 μM, about 0.75 μM, about 0.5 μM, about 0.25 μM, about 0.1 μM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

24. The synthetic LILRB4-binding protein of any one of claims 12-23, having an amino acid sequence having at least 70 (e.g., 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9) percent identity to that of any one of SEQ ID NOs: 1-6.

25. The synthetic LILRB4-binding protein of any one of claims 1-24, comprising a paratope defined by: a leucine, optionally substituted with glycine, isoleucine, or valine at position 6; a serine, optionally substituted with isoleucine, threonine, or valine amino at position 10; a valine, optionally substituted with alanine, isoleucine, or leucine at position 11; a tyrosine, optionally substituted with alanine, histidine, isoleucine, or valine at position 14; and an isoleucine, optionally substituted with leucine or valine at position 27, wherein the position numbers correspond to those of SEQ ID NO: 7 from N-terminus to C-terminus. Page 63 of 66       Attorney Docket No.: AIP-010WO26. A fusion protein comprising the synthetic LILRB4-binding protein of any one of claims 1-25.

27. The fusion protein of claim 26, comprising at least a first effector molecule.

28. The fusion protein of claim 27, wherein the first effector molecule and the LILRB4- binding protein are linked through a linker.

29. The fusion protein of claim 27 or 28, further comprising a second effector molecule.

30. The fusion protein of claim 29, wherein the second effector molecule and the first effector molecule are linked through a linker.

31. The fusion protein of claim 28 or 30, wherein the linker is an amino acid linker.

32. The fusion protein of claim 31, wherein the amino acid linker comprises glycine and serine amino acid residues.

33. The fusion protein of claim 32, wherein the amino acid linker is a (GGS)n linker.

34. The fusion protein of any one of claims 26-33, wherein the LILRB4-binding protein is at a terminus of the fusion protein.

35. The fusion protein of claim 34, wherein the LILRB4-binding protein is at the N- terminus of the fusion protein.

36. The fusion protein of claim 34, wherein the LILRB4-binding protein is at the C- terminus of the fusion protein.

37. The fusion protein of any one of claims 27-36, wherein the first and / or second effector molecule is a binding protein capable of binding a target other than LILRB4.

38. The fusion protein of any one of claims 27-37, wherein the first and / or second effector molecule is a synthetic binding protein.

39. The fusion protein of any one of claims 27-38, wherein the first and / or second effector molecule comprises a detectable label, optionally wherein the detectable label is a fluorescent label.

40. A conjugate comprising the synthetic LILRB4-binding protein of any one of claims 1- 25 or the fusion protein of any one of claims 26-39 conjugated to a chelating moiety.

41. The conjugate of claim 40, wherein the chelating moiety is complexed with a metal.

42. The conjugate of claim 41, wherein the metal is a radionuclide. Page 64 of 66       Attorney Docket No.: AIP-010WO43. The conjugate of any one of claims 40-42, wherein the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

44. A pharmaceutical composition comprising the synthetic LILRB4-binding protein of any one of claims 1-25, the fusion protein of any one of claims 26-39, or the conjugate of any one of claims 40-43 and a pharmaceutically acceptable carrier.

45. The pharmaceutical composition of claim 44, formulated for administration by a systemic route.

46. The pharmaceutical composition of claim 45, wherein the systemic route is intravenous administration.

47. A method comprising a step of contacting a LILRB4-expressing cell with a synthetic LILRB4-binding protein of any one of claims 1-25, a fusion protein of any one of claims 26- 39, the conjugate of any one of claims 40-43, or the pharmaceutical composition of any one of claims 44-46.

48. The method of claim 47, wherein the LILRB4-expressing cell is a cancer cell.

49. The method of claim 47, wherein the step of contacting occurs inside a subject.

50. The method of claim 49, wherein the subject is a human.

51. A method of treating, ameliorating, or preventing a disease or condition associated with LILRB4 comprising a step of administering to a subject an effective amount of the synthetic binding protein of any one of claims 1-25, the fusion protein of any one of claims 26-39, the conjugate of any one of claims 40-43, or the pharmaceutical composition of any one of claims 44-46.

52. The method of claim 51, wherein the subject is a human.

53. The method of claim 51 or 52, wherein the subject is diagnosed with or at risk of having tumor and / or one or more cancer-related disorders.

54. The method of claim 53, wherein the tumor is a myeloid tumor and / or the cancer- related disorder is a myeloid cancer-related disorder.

55. The method of claim 54, wherein the subject is diagnosed with or at risk of having acute myeloid leukemia (AML). Page 65 of 66