Prediction, diagnosis, and treatment of multiple myeloma

Abstract

The present disclosure provides improved compositions and methods for detecting, diagnosing, prognosing, monitoring, and treating hematological disorders including multiple myeloma in a subject. In particular, the disclosure provides methods for detecting IL4I1 in subjects to reliably diagnose, predict survival, or monitor multiple myeloma in the subject and methods for inhibiting IL4I1 to treat multiple myeloma in the subject.

Description

PREDICTION, DIAGNOSIS, AND TREATMENT OF MULTIPLEMYELOMACROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority and benefits of U.S. Provisional Patent Application No. 63 / 641,934, filed May 2, 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (IMBC_012_01WO_SeqList_ST26.xml; Size: 38,430 bytes; and Date of Creation: May 2, 2025) are herein incorporated by reference in its entirety.FIELD

[0003] The compositions and methods of the present disclosure relate generally to detection of biomarkers for the diagnosis, prognosis, treatment, and monitoring of cancer. In particular, the present disclosure relates to compositions and methods for detection of interleukin 4 induced gene 1 (IL4I1) for the diagnosis, prognosis, treatment, and monitoring of multiple myeloma.BACKGROUND

[0004] Multiple myeloma (MM) is the second most common hematologic malignancy. Alkylating agents such as melphalan and cyclophosphamide and other drugs, including vinca alkaloids, nitrosoureas, and anthracy clines, are only marginally effective in treating MM when combined with steroids and do not improve survival compared to the combination of oral melphalan and prednisone. Nor has the commonly used combination of vincristine, doxorubicin, and dexamethasone improved median survival compared to other treatment regimens in randomized clinical trials.

[0005] Proteasome inhibitors (Pis) such as bortezomib or carfilzomib, immunomodulatory agents such as thalidomide, pomalidomide or lenalidomide, pegylated liposomal doxorubicin, and arsenic trioxide reduce myeloma cell growth in laboratory studies and have been used clinically to treat MM patients. Initially, Pis were used alone, but these drugs have now been combined with each other or with chemotherapy or glucocorticosteroids. High-dosechemotherapy followed by autologous hematopoietic support also represents another treatment option for MM patients. Despite the many advances that have improved patients’ outcomes, MM remains incurable.SUMMARY

[0006] In one aspect, provided herein is a composition comprising an inhibitor of interleukin-4 induced gene 1 (IL4I1) and optionally a pharmaceutically acceptable excipient, wherein the IL4I1 inhibitor inhibits IL4I1 expression or function.

[0007] In some embodiments of the compositions of the disclosure, the IL4I1 inhibitor comprises a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense oligonucleotide (ASO), or a ribozyme that inhibits IL4I1 gene expression. In certain embodiments, the IL4I1 inhibitor comprises an siRNA. In particular embodiments, the siRNA comprises: an antisense strand comprising CUUUAAUGCGAGGUCCUCGtg (SEQ ID NO: 3) and a sense strand comprising CGAGGACCUCGCAUUAAAGtt (SEQ ID NO: 4); an antisense strand comprising CUCUCAACCAGGCCCUCAA[dT][dT] (SEQ ID NO: 5) and a sense strand comprising UUGAGGGCCUGGUUGAGAG[dT][dT] (SEQ ID NO: 6); an antisense strand comprising CCAAGUUCACCCAGUACGA[dT][dT] (SEQ ID NO: 7) and a sense strand comprising UCGUACUGGGUGAACUUGG[dT][dT] (SEQ ID NO: 8); an antisense strand comprising GUCCCUUAUGGCCGCAUCU[dT][dT] (SEQ ID NO: 9) and a sense strand comprising AGAUGCGGCCAUAAGGGAC[dT][dT] (SEQ ID NO: 10); or an antisense strand comprising CAUUGAAGGCGGCCACUCA[dT][dT] (SEQ ID NO: 11) and a sense strand comprising UGAGUGGCCGCCUUCAAUG[dT][dT] (SEQ ID NO: 12); and the antisense strand comprises a sequence that is a reverse complement of at least a region of a IL4I1 mRNA transcript.

[0008] In some embodiments of the compositions of the disclosure, the IL4I1 inhibitor comprises an antibody or an antigen-binding fragment thereof that binds IL4I1.

[0009] In some embodiments of the compositions of the disclosure, the IL4I1 inhibitor comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof,each of n and m is independently 0, 1, 2 or 3, each of R1and R2is independently halogen, C1-6 alkyl, OCi-6 alkyl or C1-6 haloalkyl;R3is H, C1-6 alkyl or C3-6 cycloalkyl; andR4is H or oxo (=0).

[0010] In some embodiments of the compositions of the disclosure, the IL4I1 inhibitor comprises a compound of formula (II), or a pharmaceutically acceptable salt thereof,each of n and m is independently 0, 1, 2 or 3, each of R1and R2is independently halogen, Ci-6 alkyl, OCi-6 alkyl or Ci-6 haloalkyl;R3is H, Ci-6 alkyl, OCi-6 alkyl or C3-6 cycloalkyl; andR5is heterocyclyl, NH2, NH(CI-6 alkyl) or N(CI-6 alkyl)2.

[0011] In some embodiments of the compositions of the disclosure comprising an IL4I1 inhibitor comprising a compound of formula (I) or (II), n is 1. In certain embodiments, R1is halogen, C1-3 alkyl, OC1-3 alkyl or C1-3 haloalkyl. In certain embodiments, R1is halogen or C1-3 haloalkyl. In certain embodiments, R1is F or CF3. In certain embodiments, R1is F. In certain embodiments, R2is halogen or C1-3 haloalkyl. In certain embodiments, R2is F, Cl, or CF3 In certain embodiments, R3is H or C1-4 alkyl. In certain embodiments, R3is H, CH3, CH2CH3 or CH(CH3)2. In certain embodiments, R3is H. In certain embodiments, R4is H. In certain embodiments, R5is 5-6 membered N-heterocyclyl or N(Ci-6 alkyl)2.In certain embodiments,

[0012] In some embodiments of the compositions of the disclosure comprising an IL4I1 inhibitor comprising a compound of formula (I) or (II), the compound of formula (I) or formula (II) binds to the oxidase catalytic site of IL4I1.

[0013] In some embodiments of the compositions of the disclosure comprising an IL4I1 inhibitor, the IL4I1 inhibitor comprises any one of the compounds in Table A, or a pharmaceutically acceptable salt thereof.

[0014] In some embodiments of the compositions of the disclosure comprising an IL4I1 inhibitor, the IL4I1 inhibitor binds to the oxidase catalytic site of IL4I1.

[0015] In another aspect, provided herein is a method of synthesizing a urea derivative compound, wherein the urea derivative compound binds to the oxidase catalytic site of IL4I1, comprising: a. contacting Compound 1 with TFA to form a mixture;,(Compound 1) b. adding NaBH(OAc)s after step (a); c. adding Compound 2 after step (b);(Compound 2) d. warming up the mixture from step (c) slowly to room temperature; e. adding the mixture from step (d) into ice-cold water to form a crude solid; f. extracting the crude solid with Hex / EA (5: 1), thereby forming a filtrate; g. concentrating the filtrate, thereby forming Compound 3 as a solid;(Compound 3) h. dissolving Compound 3 from step (g) in iPrOH to form a solution; i. adding NH4CI to the solution of step (h); j . adding Fe powder to the solution of step (i) to form a mixture; k. diluting the mixture from step (j) with EtOAc and H2O; l. filtering the diluted mixture from step (k) through celite;m. separating and concentrating the organic phase from the filtrate from step (1) to form a residue; n. dissolving the residue in EtOAc and drying over MgSCU to form a crude solid; o. mixing the crude solid with Hex / MTBE to form a mixture; p. filtering the mixture from step (o), thereby forming Compound 4 as a crude solid;(Compound 4) q. mixing Compound 4 from step (p) with CDI in THF to form a mixture; r. concentrating the mixture from step (q) and adding EtOAc to form a slurry; s. filtering the slurry from step (r) to produce a solid and a mother liquid; and t. drying the solid from step (s) to produce said urea derivative compound(urea derivative compound).

[0016] In some embodiments of a method of synthesizing a urea derivative compound of the disclosure, the method further comprises: u. washing the mother liquid from step (s) with 2N HC1; v. isolating the organic phase from step (u); w. drying the organic phase from step (v) and concentrating to form a semi solid; x. adding ACN to the semi solid of step (w) to produce a mixture; y. filtering the mixture from step (x), thereby forming a solid; and z. drying the solid of step (y) to produce said urea derivative compound.

[0017] In another aspect, provided herein is a method of diagnosing multiple myeloma (MM) in a subject, comprising: a. detecting an amount of IL-4 Induced 1 (IL4I1) polypeptide or IL4I1 gene expression in a biological sample obtained from the subject; and b. comparing the amount of IL4I1 polypeptide or gene expression detected in step (a) to a predetermined cut-off value or to an amount detected in a control biological sample,where an increased amount of IL4I1 polypeptide or gene expression of (a) as compared to the predetermined cut-off value or amount in the control biological sample of (b) indicates the presence of MM.

[0018] In certain embodiments, the method further comprises administering to the subject a treatment for MM if step (b) indicates MM is present, wherein the treatment for MM comprises the composition of any one of claims 1-23.

[0019] In another aspect, provided herein is a method of prognosing the survival of a subject having or suspected of having MM, comprising: a. detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject; and b. comparing the amount of IL4I1 polypeptide or gene expression detected in step (a) to a median value of levels of IL4I1 polypeptide or gene expression in a population of subjects being treated for MM, where an increased amount of IL4I1 polypeptide or gene expression of (a) as compared to the median value of IL4I1 polypeptide or gene expression of (b) indicates a reduced chance of survival for the subject having MM, and wherein a decreased amount of IL4I1 polypeptide or gene expression of (a) as compared to the median value of IL4I1 polypeptide or gene expression of (b) indicates an increased chance of survival for the subject having MM.

[0020] In certain embodiments of a method of prognosing the survival of a subject, the subject has been diagnosed with MM. In particular embodiments, the subject has received treatment for MM. In some embodiments of a method of prognosing the survival of a subject, a subject with an increased amount of IL4I1 polypeptide or gene expression as compared to the median value of IL4I1 polypeptide or gene expression has a 10%, 30%, 50%, or 70% reduction in the chance of survival compared to the survival of a subject having the median level of IL4I1 polypeptide or gene expression.

[0021] In certain embodiments of a method of prognosing the survival of a subject, the method further comprises removing the subject having MM from a treatment or a maintenance therapy of MM if step (b) indicates an increased chance of survival for the subject.

[0022] In another aspect, provided herein is a method of monitoring the progression or response of a subject diagnosed with MM to treatment of MM, comprising: a. detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject at a first time point;b. detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject at a second time point or following treatment; and c. comparing the amount detected in step (a) to the amount detected in step (b), where an increased amount of IL4I1 polypeptide or gene expression of (b) as compared to the amount of IL4I1 polypeptide or a gene expression of (a) indicates that said MM is progressing, and wherein a decreased amount of IL4I1 polypeptide or a gene expression of (b) as compared to the amount of IL4I1 polypeptide or a gene expression of (a) indicates that said MM is entering remission or responding to treatment. In some embodiments of a method of monitoring the progression or response of a subject diagnosed with MM, the method further comprises removing the subject from a treatment or a maintenance therapy of MM if step (b) indicates said MM is entering remission or responding to treatment.

[0023] In some embodiments of the methods of diagnosing, prognosing, or monitoring of the disclosure, the biological sample is a serum sample, a plasma sample, or a sample comprising RNA isolated from a subject’s bone marrow mononuclear cells or peripheral blood mononuclear cells. In certain embodiments, the biological sample is supernatant obtained from a cell culture of the subject’s bone marrow mononuclear cells. In certain embodiments, the biological sample is supernatant obtained from a cell culture of the subject’s peripheral blood mononuclear cells.

[0024] In some embodiments of the methods of diagnosing, prognosing, or monitoring of the disclosure, the amount of IL4I1 polypeptide is detected by immunohistochemistry, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, flow cytometry, or strip assay. In certain embodiments, the amount of IL4I1 polypeptide is detected by ELISA. In certain embodiments, the amount of IL4I1 polypeptide is detected by lateral flow assay.

[0025] In some embodiments of the methods of diagnosing, prognosing, or monitoring of the disclosure, the amount of IL4I1 gene expression is detected by Northern blotting, qPCR, RT- PCR, microarray, or RNA-seq.

[0026] Also provided herein is a kit for detecting, diagnosing, predicting survival, staging, or monitoring MM in a subject, comprising a reagent suitable for determining a level of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject. In some embodiments of a kit of the disclosure, the biological sample is a serum sample, a plasma sample, or a sample comprising RNA isolated from a subject’s bone marrow mononuclear cellsor peripheral blood mononuclear cells. In certain embodiments, the biological sample is supernatant obtained from a cell culture of the subject’s bone marrow mononuclear cells. In certain embodiments, the biological sample is supernatant obtained from a cell culture of the subject’s peripheral blood mononuclear cells.

[0027] In some embodiments of a kit of the disclosure, the reagent suitable for determining a level of IL4I1 polypeptide or gene expression comprises an antibody or antigenbinding fragment thereof specific for IL4I1. In certain embodiments, the kit comprises an ELISA assay. In certain embodiments, the kit comprises a lateral flow assay.

[0028] In some embodiments of a kit of the disclosure, the reagent suitable for determining a level of IL4I1 polypeptide or gene expression comprises one or more oligonucleotides or primers that specifically hybridize to the IL4I1 gene or a segment thereof, or an mRNA transcript thereof.

[0029] In another aspect, disclosed herein is a method of treating a subject having, at risk of having, or suspected of having MM, comprising administering to the subject a composition of the disclosure. In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the subject has been diagnosed with having MM using a method of diagnosing of the disclosure or a kit of the disclosure.

[0030] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule selected from the group consisting of a proteasome inhibitor, thalidomide or a derivative thereof, an anti-CD38 antibody, an anti-SLAMF7 antibody, a steroid, a chemotherapeutic agent, a bispecific T cell engager, a histone deacetylase inhibitor, a selective inhibitor of nuclear export, an antibiotic, and a BCMA-directed CAR T cell, wherein the molecule is administered before, simultaneously with, or after administering the composition, optionally wherein the molecule and the IL4I1 inhibitor are present in the subject during an overlapping time period. In certain embodiments, the molecule is administered before administering the composition. In certain embodiments, the molecule is administered simultaneously with administering the composition. In certain embodiments, the molecule is administered after administering the composition.

[0031] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is a proteasome inhibitor selected from the group consisting of bortezomib, carfilzomib, oprozomib, ixazomib citrate, marizomib, delanzomib, and syringolin A.

[0032] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is thalidomide, or a derivative thereof selected from the group consisting of thalidomide, lenalidomide, pomalidomide, linomide, CC-1088, CDC-501, and CDC-801.

[0033] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is an anti-CD38 antibody. In certain embodiments, the anti-CD38 antibody is Isatuximab or Daratumumab.

[0034] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is an anti-SLAMF7 antibody. In certain embodiments, the anti-SLAMF7 antibody is Elotuzumab.

[0035] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule administered to the subject is a steroid, optionally wherein the steroid is a glucocorticoid. In certain embodiments, the glucocorticoid is selected from the group consisting of hydroxy corti sone, cortisone, desoxy corticosterone, fludrocortisone, betamethasone, dexamethasone, prednisolone, prednisone, methylprednisolone, methylprednisone, paramethasone, triamcinolone, flumethasone, fluocinolone, fluocinonide, fluprednisolone, halcinonide, flurandrenolide, meprednisone, and medrysone.

[0036] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is a chemotherapeutic agent selected from the group consisting of melphalan, cyclophosphamide, bendamustine, doxorubicin, and pegylated liposomal doxorubicin.

[0037] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is a bispecific T cell engager. In certain embodiments, the bispecific T cell engager is talquetamab, teclistamab, or elranatamab.

[0038] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule administered to the subject is a histone deacetylase inhibitor, optionally wherein the histone deacetylase inhibitor is panobinostat.

[0039] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is a selective inhibitor of nuclear export, optionally wherein the selective inhibitor of nuclear export is selinexor.

[0040] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is an antibiotic, optionally wherein the antibiotic is clarithromycin.

[0041] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, the method comprises further administering to the subject a molecule, where the molecule is a BCMA-directed CAR T cell. In certain embodiments, the BCMA- directed CAR T cell is ide-cel or cilta-cel.

[0042] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, a composition is administered through parenteral administration. In certain embodiments, the parenteral administration is subcutaneous, intramuscular, intravenous, or intrathecal administration.

[0043] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, a composition is administered through intraosseous infusion.

[0044] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, a composition is administered through intratumoral injection.

[0045] In some embodiments of a method of treating a subject having, at risk of having, or suspected of having MM, a composition is administered through enteral administration. In certain embodiments, the enteral administration route is oral.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1A and FIG. IB provide graphs showing serum IL4I1 levels in MM patients, as measured by ELISA. FIG. 1A shows a comparison of IL4I1 levels between normal (n=50) and all MM patients (n=182). FIG. IB shows a comparison of IL4I1 levels between normal, frontline, and relapsed / refractory multiple myeloma (RRMM) patients. The data were analyzed using a nonparametric unpaired t test and are presented as the mean ± SEM.

[0047] FIGs. 2A-D provide Kaplan-Meier curves showing the correlation of serum IL4I1 levels with survival in MM patients. FIG. 2A shows a comparison of progression-free survival (PFS) between patients with IL4I1 below the median (Hl - lower half) and those with IL4I1 above the median (H2 - upper half). FIG. 2B shows a comparison of PFS between patients withthe highest IL4I1 quartile (>Q1) and the remaining 75% of patients (<Q 1). FIG. 2C shows a comparison of overall survival (OS) between patients with IL4I1 below the median (Hl - lower half) and those with IL4I1 above the median (H2 - upper half). FIG. 2D shows a comparison of OS between patients with the highest IL4I1 quartile (>Q1) and the remaining 75% of patients (<Q1).

[0048] FIGs. 3A-D provide Kaplan-Meier curves showing the correlation of serum IL4I1 levels with survival in RRMM (n=76) and frontline MM (n=106) patients. FIG. 3A shows a comparison of PFS and FIG. 3B shows a comparison of OS between RRMM patients with the lowest IL4I1 quartile (QI) and those with the highest IL4I1 quartile (Q4). FIG. 3C shows a comparison of PFS and FIG. 3D shows a comparison of OS between frontline patients with the lowest IL4I1 quartile (QI) and those with the highest IL4I1 quartile (Q4).

[0049] FIG. 4 provides a graph showing serum IL4I1 levels for 50 MM patients in CR compared to that for 50 MM patients with PD.

[0050] FIGs. 5A-5L provide representative graphs of serum IL4I1 levels, as well as levels of other serum markers, in RRMM patients over the course of the disease, including at baseline (T1 : immediately prior to treatment), in the middle (T2 and T3 : at their best response), and at the end (T4). Graphs providing data for a RRMM patient with partial response (PR) to treatment and PD at T4 show IL4I1 levels (FIG. 5A), IL4I1 and sBCMA level (FIG. 5B), IL4I1 and M- protein levels (FIG. 5C), and IL4I1 and sFLC levels (FIG. 5D). Graphs providing data for a RRMM patient in CR to treatment, with disease remaining in CR at T4 show IL4I1 levels (FIG. 5E), IL4I1 and sBCMA levels (FIG. 5F), IL4I1 and M-protein levels (FIG. 5G), and IL4I1 and sFLC levels (FIG. 5H). FIGs. 5I-5L provide graphs showing IL4I1 levels in 4 RRMM patients with standard disease.

[0051] FIGs. 6A-C provide graphs showing serum BCMA levels in MM patients. FIG. 6A shows BCMA levels for patients in the highest quartile (>Q4, n=46) for serum IL4I1 levels compared to that for the remainder 75% of patients (<Q4, n=138). FIG. 6B shows BCMA levels for patients in the lowest quartile (<Q1, n=45) for serum IL4I1 levels compared to that for the remainder 75% of patients (>Q1, n=138). FIG. 6C shows BCMA levels for patients in the lowest quartile QI (n=45) for serum IL4I1 levels compared to that in the highest quartile Q4 (n=45).

[0052] FIG. 7A provides a bar chart showing the IL4I1 levels in the supernatant of bone marrow mononuclear cells (BMMCs) from MM patients in CR or with PD. FIG. 7B providesillustrative images showing immunohistochemistry staining of IL4I1 in bone marrow cells of MM patients in CR or with PD.

[0053] FIG. 8 provides images showing immunohistochemistry staining of IL4I1 in bone marrow cells of MM patients assigned a clinical status of complete response (CR) or progressive disease (PD).

[0054] FIG. 9 provides a bar chart showing the relative gene expression levels of IL4I1, as detected by ELISA, in cell culture supernatants from 24 hour or 48 hour cultures of bone marrow cells of MM patients assigned a clinical status of CR or PD.

[0055] FIG. 10A provides a bar chart showing the plasma mouse IL4I1 levels in SCID mice bearing human MM xenografts. FIG. 10B provides a bar chart showing the levels of human IL4I1 in mouse plasma from mice bearing human MM xenografts. The data were analyzed using a two-way ANOVA test and are presented as the mean ± SEM. A p-value of less than 0.05 was considered statistically significant. ****p<0.0001.

[0056] FIGs. 11A-B provide graphs showing mouse and human IL4I1 levels in plasma of SCID mice bearing LAG-K2 patient-derived xenografts (PDXs), as determined by ELISA. FIG. 11 A shows the measurement of human-specific IL4I1 in the blood plasma of control (n=3) and tumor-bearing (n=3) SCID mice. FIG. 11B shows the correlation between plasma IL4I1 levels and LAG-k2 tumor growth. The data were analyzed using an unpaired t test and are presented as the mean ± SEM.

[0057] FIGs. 12A-F provide graphs showing the effect of the JAK inhibitors pacritinib, fedratinib, and ruxolitinib on IL4I1 (FIGs. 12A, C, and E) and AHR (FIGs. 12B, D, and F) gene expression in BMMCs from two MM patients (FIGs. 12A-D) and the U266 MM cell line (FIGs. 12E-F)

[0058] FIGs. 13A-B provide graphs showing qRT-PCR analysis of IL4I1 expression after transfection with siRNA targeting IL4I1 in BMMCs from a patient with PD (FIG. 13A) and U266 cell line (FIG. 13B).

[0059] FIGs. 14A-C provide graphs showing, at the indicated time points, the viability of BMMCs from a MM patient with PD (FIG. 14A), BMMCs from a MM patient with CR (FIG. 14B), and the U266 cell line (FIG. 14C), following IL4I1 inhibition by siRNA at the indicated concentrations. All experiments were performed in triplicates from each sample, and data are presented as mean ± SEM.

[0060] FIG. 15A provides a schematic diagram outlining the experimental and treatment plan to determine the effects of IL4I1 inhibition on MM progression in the LAGX-1 PDX SCIDmouse model. FIG. 15B provides a graph showing plasma IL4I1 levels on day 16 (the day following the final siRNA injection) in sham and siRNA-treated LAGX-1 PDXs SCID mice. FIG. 15C provides a plot comparing the tumor growth rates in siRNA-treated and untreated groups (n=5 / group). FIG. 15D provides a graph showing the final tumor volume, calculated as length x width2x 0.5, on day 16 post-siRNA treatment. The data were analyzed using a nonparametric unpaired t test and are presented as the mean ± SEM.

[0061] FIGs. 16A-B provide graphs showing qRT-PCR analysis of IL4I1 expression after transfection with siRNA targeting IL4I1 in the U266 cell line (FIG. 16A) and BMMCs (FIG.16B). The data were analyzed using a nonparametric unpaired t test and are presented as the mean ± SEM.

[0062] FIGs. 17A-C provide graphs showing the effect of IL4I1 inhibition by siRNA targeting IL4I1 on LAG-K2 PDXS in SCID mice. FIG. 17A provides a schematic diagram illustrating the treatment plan. FIG. 17B provides a plot comparing tumor growth rates in the siRNA combo-treated and untreated groups (n=8 / group). FIG. 17C provides a graph showing final tumor volume on day 34 post-siRNA treatment. Data in FIGs. 17B-C were analyzed using a nonparametric unpaired t-test and are presented as the mean ± SEM.

[0063] FIG. 18 provides a schematic showing the synthesis of Compound 301 as a urea derivative, which acts as an IL4I1 inhibitor, through a three-step process.

[0064] FIG. 19 shows the purity analysis of Compound 301 using high-performance liquid chromatography (HPLC).

[0065] FIG. 20 shows liquid chromatography-mass spectrometry (LSMS) analysis of Compound 301.

[0066] FIG. 21 shows structural analysis of Compound 301 using nuclear magnetic resonance (NMR) spectroscopy.

[0067] FIGs. 22A-D provide graphs showing the percentage of viable cells, as determined by MTT assay, after ex vivo or in vitro treatment of PD BMMCs (FIG. 22A), CR BMMCs (FIG. 22B), the MMES cell line (FIG. 22C), and LAGX-1 tumor xenograft single cells (FIG. 22D), with Compound 301 at the indicated concentrations and times. All experiments were performed in triplicates for each sample. Two-way ANOVA was used for statistical analysis, and data are presented as mean ± SEM.

[0068] FIG. 23A provides a schematic illustrating the IL4I1 enzymatic reaction, which releases H2O2 as a byproduct from the degradation of phenylalanine. FIGs. 23B-C providemeasurements of IL4I1 L-amino acid oxidase (LAAO) activity as determined by H2O2 release from BMMCs (FIG. 23B) and the MM.1 S cell line (FIG. 23C).

[0069] FIG. 24 provides a graph showing IL4L1 enzymatic activity at various concentrations of Compound 301, an IL4L1 inhibitor.

[0070] FIG. 25A-E provide graphs and images showing the effect of Compound 301, an IL-411 inhibitor, on LAG-K2 PDXS in SCID mice. FIG. 25A provides a schematic diagram illustrating the treatment plan. FIG. 25B provides a plot comparing tumor growth rates between the Compound 301-treated and placebo groups (n=8-9 / group). FIG. 25C provides a graph showing final tumor volume on day 30 post-treatment. FIG. 25D provides a graph showing final tumor weights. FIG. 25E provides an image showing tumor growth in SCID mice. Data in FIGs. 25C-D were analyzed using a nonparametric unpaired t-test and are presented as the mean ± SEM.

[0071] FIG. 26A-G provide graphs showing body and organ weights of SCID mice bearing LAG-K2 PDXS treated with Compound 301. FIG. 26A provides a graph showing body weights recorded throughout the study. FIG. 26B provides a graph showing final body weights at day 30. FIGs. 26C-H provide graphs showing liver weight (FIG. 26C), lung weight (FIG. 26D), kidney weight (FIG. 26E), spleen weight (FIG. 26F), heart weight (FIG. 26G), and brain weight (FIG. 26H) at the end of the study on day 30.

[0072] FIGs. 27A-C provide graphs showing the effect of Compound 301, on LAG-K2 PDXs in SCID mice. FIG. 27A provides a schematic diagram illustrating the treatment plan. FIG. 27B provides a plot of tumor growth rates between the Compound 301-treated and placebo groups (n=2 / group). FIG. 27C provides a graph showing final tumor volume, calculated as length x width2x 0.5, on day 15 post-treatment. FIG. 27D provides a plot showing tumor weights at the end of the study on day 15. FIG. 27E provides images showing tumor growth in treated and control SCID mice. Data are presented as the mean ± SEM.

[0073] FIGs. 27F-M provide graphs showing body and organ weights of SCID mice bearing LAG-K2 PDXS treated with Compound 301. FIG. 27F provides a graph showing body weights recorded throughout the study. FIG. 27G provides a graph showing final body weights at day 30. FIGs. 27H-M provide graphs showing liver weight (FIG. 27H), lung weight (FIG. 271), kidney weight (FIG. 27 J), spleen weight (FIG. 27K), heart weight (FIG. 27L), and brain weight (FIG. 27M) at the end of the study on day 15.

[0074] FIGs. 28A-D provide graphs and images showing the effects of Compound 301, on MM1.S cell line-derived human MM tumors in SCID mice. FIG. 28A provides a plotcomparing tumor growth rates between the Compound 301 -treated and placebo groups (n=3- 4 / group). FIG. 28B provides a graph showing final tumor volume, calculated as length x width2x 0.5, on day 9 post-treatment. FIG. 28C provides images showing tumor growth in treated and control SCID mice. FIG. 28D provides images showing tumor growth in treated and control SCID mice. Data are presented as the mean ± SEM.

[0075] FIGs. 28E-L provide graphs showing body and organ weights of SCID mice bearing LAG-K2 PDXS treated with Compound 301. FIG. 28E provides a graph showing body weights recorded throughout the study. FIG. 28F provides a graph showing final body weights at day 15. FIG. 28G-L provide graphs showing liver weight (FIG. 28G), lung weight (FIG. 28H), kidney weight (FIG. 281), spleen weight (FIG. 28J), heart weight (FIG. 28K), and brain weight (FIG. 28L) at the end of the study on day 15.

[0076] FIGs. 29A-L provide sets of four graphs showing changes in serum IL4I1 levels during the course of the disease at baseline (T1 : immediately prior to treatment), middle (T2 and T3 : at their best response), and end (T4), in RRMM patients with PD (FIGs. 29A-F) or CR (FIGs. 29G-L) at T4, with each set of four showing (i) serum IL4I1 levels during the course of the disease, (ii) IL4I1 and sBCMA levels, (iii) IL4I1 and M-protein levels, (iv) IL4I1, and sFLC levels.

[0077] FIG. 30 provides representative images showing intracellular staining of IL4I1 protein in BMMCs from MM patients.

[0078] FIGs. 31A-E provide chemical structures of urea derivative Compound 301 and its analogs.

[0079] FIGs. 32A-R provide schematics for the synthesis of Compound 301 and its analogs.

[0080] FIGs. 33A-C provide graphs showing the percentage of viable LAGk- l cells following treatment with Compound 301 and its analogs at increasing concentrations.

[0081] FIG. 34 provides a graph showing IC30 values of Compound 301 and its analogs on ex vivo cultures of LAG I xenografts.

[0082] FIGs. 35A-E provide graphs showing IL4L1 enzymatic activity at various concentrations of Compound 301 and its analogs.

[0083] FIG. 36 provides a graph showing the IC50 values of Compound 301 and its analogs for inhibiting IL4L1 enzymatic activity.

[0084] FIG. 37A provides flow cytometry dot plots showing analysis of CD 138+ cells and FIG. 37B provides graphs showing the percentage of viable cells after treatment of bonemarrow cells from a myeloma patient with various concentrations of Compound 301 and its analogs.

[0085] FIG. 38 provides graphs showing the percentage of viable LAGX-1 cells after treatment with Compound 315 or standard clinical drugs.DETAILED DESCRIPTIONOverview

[0086] The present disclosure provides methods for diagnosing, prognosing, and monitoring certain types of cancers (e.g., MM) by determining IL4I1 polypeptide or gene (e.g., mRNA transcript) expression levels. The present disclosure further provides methods for treating cancer (e.g., MM) comprising inhibiting the production, activity, expression, or function of IL4Il.

[0087] Interleukin-4 induced gene 1 (IL4I1), which may also be referred to as interleukin- 4 induced 1, is an enzyme involved in amino acid metabolism that is overexpressed in certain cancer cells but not in normal cells, making it a promising target for cancer treatment. Specifically, it is an L-amino acid oxidase. A variety of immune cells, including B cells, macrophages, and dendritic cells, express IL4I1. Overexpression of IL4I1 in tumor cells has led to enhanced tumor cell growth and suppression of anti-tumor immunity through activation of the aryl hydroxylase receptor (AHR). This protein has been found to have immunosuppressive properties, which can lead to uncontrolled tumor growth. By depleting phenylalanine, IL4I1 inhibits the proliferation and activation of T cells. IL4I1 interferes with T cell receptor (TCR) signaling, alters the differentiation of naive T cells, and restricts the proliferation of effector T cells. The expression of this protein in tumors influences the tumor microenvironment in a way that hinders the antitumor cytotoxic T cell response, thereby aiding cancer immune evasion. Tumor associated macrophages which are tumor stimulatory express IL4I1. This protein also drives M2 macrophage polarization, and these cells possess both tumor stimulatory and immune inhibitory features. These types of macrophages also express high amounts of checkpoint inhibitor proteins on their cell surface that suppress T cell responses that eliminate tumor cells. Importantly, IL4I1 signals through the Janus kinase (JAK) pathway. Activation of this pathway stimulates production of checkpoint proteins, resulting in impaired anti-cancer immunity. JAK inhibitors have been shown to block the production of checkpoint inhibitor proteins, allowing immune-based killing of cancer cells to be restored. The checkpoint inhibitor proteins also stimulate IL4I1 activity, thereby orchestrating tumor cell escape from immune baseddestruction. Elevated levels of IL4I1 expression have been observed in certain hematological cancers, including acute myeloid leukemia and chronic lymphocytic leukemia. Several studies have explored the relationship between IL4I1 expression and clinical outcomes in cancer patients. High levels of IL4I1 have been associated with a poor prognosis in various cancers, including breast cancer and melanoma. However, IL4I1 has not been associated with MM and serum levels of IL4I1 have not previously been investigated in any disease.

[0088] The present inventor has surprisingly discovered that IL4I1 polypeptide or gene (e.g., mRNA transcript) expression levels in a biological sample can be reliably used as prognosis for progression-free survival and / or overall survival of a subject having or suspected of having multiple myeloma (MM). In addition, IL4I1 polypeptide or gene expression (e.g., mRNA transcript) levels in a biological sample can be reliably used to predict time to progression to MM in a subject. Furthermore, the present inventor has surprisingly found that IL4I1 polypeptide or gene expression (e.g., mRNA transcript) levels in a biological sample can be reliably used for diagnosing MM and monitoring progression of MM or response to treatment of MM in a subject. Importantly, the present inventor found that inhibition of IL4I1 slows tumor progression, demonstrating the utility of IL4I1 inhibition in treating MM.Diagnosis, Prognosis, and Monitoring of MM

[0089] The present disclosure provides methods of detection and / or diagnosis of multiple myeloma in subjects, methods of prognosis of survival in subjects diagnosed with multiple myeloma, and methods of monitoring a response of the disease to treatment, based upon the level of IL4I1 polypeptide or gene expression (e.g., mRNA transcript) in a biological sample obtained from a subject, including, e.g., a subject's blood, plasma, serum, bone marrow, or tissue.

[0090] In some embodiments, the biological sample includes, without limitation, cells in culture, cell supernatants, cell lysates, blood, serum, plasma, urine, cerebral spinal fluid, biological fluid, and tissue samples. In certain embodiments, the biological sample is supernatant obtained from a culture of the subject's cells. In certain embodiments, the cells are the subject's bone marrow mononuclear cells. In certain embodiments, the cells are the subject's peripheral blood mononuclear cells. In certain embodiments, the biological sample is serum. In certain embodiments, the biological sample is plasma.

[0091] In some embodiments, a method of diagnosing multiple myeloma (MM), comprises:

[0092] (a) detecting an amount of IL-4 Induced gene 1 (IL4I1) polypeptide or IL4I1 gene expression in a biological sample obtained from a subject; and

[0093] (b) comparing the amount of IL4I1 polypeptide or gene expression detected in step(a) to a predetermined cut-off value or to an amount detected in a control biological sample, wherein an increased amount of IL4I1 polypeptide or gene expression in the biological sample of (a) as compared to the predetermined cut-off value or amount in the control biological sample of (b) indicates the presence of MM.

[0094] In some embodiments, a method of prognosing the survival of a subject having or suspected of having multiple myeloma (MM) comprises:

[0095] (a) detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from a subject; and

[0096] (b) comparing the amount of IL4I1 polypeptide or gene expression detected in step(a) to a median value of levels of IL4I1 polypeptide or gene expression in a population of subjects being treated for MM, wherein an increased amount of IL4I1 polypeptide or gene expression in the biological sample of (a) as compared to the median value or corresponding amount of IL4I1 polypeptide or gene expression in the treated population of (b) indicates a reduced chance of survival for the subject having MM.

[0097] In certain embodiments of the method of prognosing the survival of a subject having or suspected of having MM, the subject has been diagnosed with MM. In certain embodiments, the subject has received treatment for MM. In certain embodiments, wherein the subject has an increased amount of IL4I1 polypeptide or gene expression as compared to the median value, the subject has a 10%, 30%, 50%, or 70% reduction in the chance of survival compared to the survival of a subject having the median level or less than the median value of IL4I1 polypeptide or gene expression.

[0098] In some embodiments, a method of monitoring the progression or response to treatment of multiple myeloma (MM), comprises:

[0099] (a) detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from a patient diagnosed with MM at a first timepoint;

[0100] (b) detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the patient at a second time point or following treatment; and

[0101] (c) comparing the amount detected in step (a) to the amount detected in step (b), wherein an increased amount of IL4I1 polypeptide or a gene expression in the biological sample of (b) as compared to the amount of IL4I1 polypeptide or a gene expression in the biologicalsample of (b) indicates that said multiple myeloma is progressing, and wherein a decreased amount of IL4I1 polypeptide or gene expression in the biological sample of (b) as compared to the amount in the biological sample of (a) indicates that said MM is entering remission or responding to treatment.

[0102] In some embodiments, the IL4I1 polypeptide is a soluble IL4I1 polypeptide. In certain embodiments, the IL4I1 polypeptide is a fragment thereof of the full-length IL4I1 polypeptide. A variety of methods of determining IL4I1 polypeptide levels are known and available in the art. In some embodiments, these involve the use of an IL4I1 binding agent, such as an IL4I1 specific antibody. There are a variety of assay formats known as ordinary skill in the art and suitable for using a binding agent to detect polypeptide markers in a sample. Nonlimiting examples of such assay formats include immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay. In certain embodiments, the IL4I1 polypeptide or a fragment thereof is detected using an ELISA assay. In certain embodiments, the IL4I1 polypeptide or a fragment thereof is detected using a lateral flow assay. In some embodiments, the detection is performed using an antibody specific to the IL4I1 polypeptide or a fragment thereof. In certain embodiments, the antibody specific for IL4I1 polypeptide or a fragment thereof is a monoclonal antibody. In further embodiments, the antibody specific for IL4I1 polypeptide or a fragment thereof is a polyclonal antibody.

[0103] In some embodiments, the methods of determining the level of IL4I1 comprises detecting enzymatic activity of IL4I1 in the biological sample. In certain embodiments, detecting the enzymatic activity of IL4I1 comprises the detection of the amount and / or concentration of IL4I1 metabolites in the biological sample. Non -limiting examples of methods for detecting the enzymatic activity of IL4I1 include the chromatography, NMR, metabolite sensors, antibodies, ELISAs, enzymatic assays, colorimetric assays, or fluorescence assays.

[0104] In some embodiments, the IL4I1 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 16, or 17. In other embodiments, the IL4I1 polypeptide comprises an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 85%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 1, 16, or 17. In certainembodiments, the IL4I1 polypeptide comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 1, 16, or 17. In certain embodiments, the IL4I1 polypeptide comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 1, 16, or 17. In certain embodiments, the IL4I1 polypeptide comprises an amino acid sequence having at least about 75% sequence identity with SEQ ID NO: 1, 16, or 17.

[0105] In some embodiments, the level of IL4I1 gene expression refers to the level of mRNA expression of the gene encoding IL4I1. The level of mRNA expression can be determined by measuring the amounts of IL4I1 mRNA.

[0106] In some embodiments, IL4I1 gene expression, e.g., mRNA levels, is detected using genetic tools known in the arts, such as northern blotting, PCR, microarrays, or RNA-seq. In some embodiments, IL4I1 gene expression is detected using PCR. In certain embodiments, the detection is performed using qPCR, including RT-PCR and qRT-PCR. In some embodiments, detection involves measuring IL4I1 mRNA levels present in the biological sample. In some embodiments, detection is performed using one or more primers specific for IL4I1. In some embodiments, the IL4I1 gene detected is the human IL4I1 gene, a variant, or an allele thereof. In some embodiments, the IL4I1 gene expression is relative IL4I1 gene expression. Relative IL4I1 gene expression in a biological sample can be determined by comparison to one or more control genes, such as one or more housekeeping genes, in said biological sample, or a control sample, such as a corresponding biological sample derived from a healthy subject, a group of healthy subjects, a patient, or a patient group.

[0107] In some embodiments, the IL4I1 gene or a variant or an allele thereof comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the IL4I1 gene comprises an nucleic acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 85%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 2. In certain embodiments, the IL4I1 gene comprises an nucleic acid sequence having at least about 90% sequence identity with SEQ ID NO: 2. In certain embodiments, the IL4I1 gene comprises an nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 2. In certain embodiments, the IL4I1 gene comprises an nucleic acid sequence having at least about 75% sequence identity with SEQ ID NO: 2.

[0108] In some embodiments, the IL41 cDNA sequence or a variant or an allele thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 13-15. In some embodiments, the IL41 cDNA sequence comprises an nucleic acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 85%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity with any one of SEQ ID NOs: 13-15. In certain embodiments, the IL41 cDNA sequence comprises a nucleic acid sequence having at least about 90% sequence identity with any one of SEQ ID NOs: 13-15. In certain embodiments, the IL41 cDNA sequence comprises an nucleic acid sequence having at least about 80% sequence identity with any one of SEQ ID NOs: 13-15. In certain embodiments, the IL41 cDNA sequence comprises a nucleic acid sequence having at least about 75% sequence identity with any one of SEQ ID NOs: 13-15. In some embodiments, the IL41 mRNA sequence or a variant or an allele thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 13-15 or an antisense sequence thereof, wherein T are replaced with U.

[0109] In some embodiments, the diagnosis, prognosis, or monitoring of multiple myeloma involves determining the absolute amount of a IL4I1 polypeptide or a fragment thereof in a biological sample of a subject. In certain embodiments, the levels of IL4I1 polypeptide or a fragment thereof in a biological sample of a patient suffering from MM is greater than about 1 ng / mL, greater than about 2 ng / mL, greater than about 3 ng / mL, greater than about 4 ng / mL, greater than about 5 ng / mL, or greater than about 8 ng / mL. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in a biological sample of a patient of greater than about 2 ng / mL indicates a reduced chance of PFS for the subject. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in a biological sample of the subject of greater than about 3 ng / mL indicates a reduced chance of PFS for the subject. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in a biological sample of the subject of greater than about 4 ng / mL indicates a reduced chance of PFS for the subject. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in a biological sample of the subject of greater than about 5 ng / mL indicates a reduced chance of PFS for the subject. In certain embodiments, the biological sample is a serum sample, or plasma sample, or supernatant obtained from a culture of the subject's bone marrow mononuclear cells or peripheral blood mononuclear cells.

[0110] In certain embodiments, subjects with IL4I1 polypeptide levels greater than about 6 ng / mL in a biological sample have reduced PFS to MM compared to subjects with IL4I1 polypeptide levels lower than about 3 ng / mL. In other embodiments, subjects with IL4I1 polypeptide levels greater than about 3 ng / mL in a biological sample have reduced PFS to MM compared to subjects with IL4I1 polypeptide levels lower than about 3 ng / mL. Therefore, in certain embodiments, IL4I1 polypeptide levels greater than about 3 ng / mL correlate with a shorter PFS among subjects having or suspected of having MM. In certain embodiments, IL4I1 polypeptide levels lower than about 3 ng / mL correlate with a longer PFS among subjects having or suspected of having MM. In certain embodiments, the biological sample is a serum sample, or plasma sample, or supernatant obtained from a culture of the subject's bone marrow mononuclear cells or peripheral blood mononuclear cells. In certain embodiments, the biological sample is a serum sample, or plasma sample, or supernatant obtained from a culture of the subject's bone marrow mononuclear cells or peripheral blood mononuclear cells.[OHl] In some embodiments, a method of prognosis predicting time to progression (TTP) to MM in a subject is provided. In certain embodiments, the levels of serum IL4I1 polypeptide levels or a fragment thereof in biological samples obtained from patients suffering from MM are greater than about 1 ng / mL, greater than about 3 ng / mL, greater than about 4 ng / mL, greater than about 5 ng / mL, greater than about 6 ng / mL, or greater than about 10 ng / mL. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in a biological sample of the subject of greater than about 10 ng / mL, about 6 ng / mL, or about 3 ng / mL correlates with a reduced TTP of MM. In certain embodiments, the biological sample is a serum sample, or plasma sample, or supernatant obtained from a culture of the subject's bone marrow mononuclear cells or peripheral blood mononuclear cells.

[0112] In some embodiments, a method of prognosis for the OS of a subject having or suspected of having MM is provided. In certain embodiments, the levels of serum IL4I1 polypeptide levels or a fragment thereof in a biological sample obtained from patients suffering from MM is greater than about 1 ng / mL, greater than about 3 ng / mL, greater than 6ng / mL, or greater than about 10 ng / mL. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of greater than about 1 ng / mL indicates a shorter overall survival (OS) for the subject. In other embodiments, an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of greater than about 3 ng / mL indicates a shorter OS for the subject. In certain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of greater than about 6 ng / mL indicates a shorter OS for the subject. Incertain embodiments, an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of greater than about 10 ng / mL indicates a shorter OS for the subject. In certain embodiments, the biological sample is a serum sample, or plasma sample, or supernatant obtained from a culture of the subject's bone marrow mononuclear cells or peripheral blood mononuclear cells.

[0113] In some embodiments, a method of monitoring disease progression or response to treatment of MM is provided. In certain embodiments, an amount of IL4I1 or a fragment thereof in a biological sample is obtained from a subject diagnosed with MM at a time point following treatment, wherein an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of greater than about 2 ng / mL, about 3 ng / mL, greater than about 4 ng / mL, greater than about 5 ng / mL, greater than about 6 ng / mL, greater than about 7 ng / mL, greater than about 8 ng / mL, greater than about 9 ng / mL, or greater than about 10 ng / mL indicates that the cancer will progress sooner. In other embodiments, an amount of IL4I1 or a fragment thereof in a biological sample is obtained from a subject diagnosed with MM at a time point following treatment, wherein an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of lower than about 6 ng / mL, lower than about 5 ng / mL, lower than about 4 ng / mL, lower than about 3 ng / mL, lower than about 2 ng / mL, or lower than about 1 ng / mL indicates that the cancer is more likely to enter remission or respond to treatment. In certain embodiments, an amount of IL4I1 or a fragment thereof in a biological sample is obtained from a subject diagnosed with MM at a time point following treatment, wherein an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of lower than about 6 ng / mL indicates that the cancer is more likely to enter remission or respond to treatment. In other embodiments, an amount of IL4I1 or a fragment thereof in a biological sample is obtained from a subject diagnosed with MM at a time point following treatment, wherein an amount of IL4I1 polypeptide or a fragment thereof in the biological sample of lower than about 3 ng / mL indicates that the cancer is more likely to enter remission or respond to treatment.

[0114] In some embodiments, the diagnosis, prognosis, or monitoring of MM involves determining the relative amount of a IL4I1 polypeptide or a fragment thereof in a biological sample of a subject having or suspected of having MM as compared to the amount of a IL4I1 polypeptide or a fragment thereof, or IL4I1 gene expression, in a biological sample of a normal control subject or a patient in CR. Thus, in certain embodiments, the diagnosis, prognosis, or monitoring of MM is monitored by the presence of an increased amount or a decreased amount of IL4I1 polypeptide expression level in a subject having or suspected of having MM relative tothat in a biological sample of a normal control subject or a patient in CR, or relative to a median value of IL4I1 polypeptide expression level in a population of normal control subjects or patients in CR. In some embodiments, an increased amount of the levels of IL4I1 polypeptide or a fragment thereof refers to at least about 2 -fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels of IL4I1 as compared to those in a normal control subject or a patient in CR. In general, a disease state is monitored by the presence of at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels of IL4I1 as compared to those in a normal control subject or a patient in CR or to a median value of IL4I1 polypeptide expression level in a population of normal control subjects or patients in CR. In some embodiments, an increased amount or higher levels of IL4I1 polypeptide or a fragment thereof compared to corresponding levels in healthy individuals or a patient in CR correlate with a reduced TTP and shorter PFS. In other embodiments, an increased amount or higher levels of IL4I1 polypeptide or a fragment thereof compared to corresponding levels in healthy individuals or a patient in CR correlate with a shorter OS among subjects having or suspected of having MM. Therefore, in certain embodiments, an increased amount or higher levels of IL4I1 polypeptide or a fragment thereof compared to corresponding levels in healthy individuals or a patient in CR are predictive of a shorter PFS, reduced TTP, and / or shorter OS in subjects having or suspected of having MM.

[0115] In some embodiments, very high levels of IL4I1 polypeptide or a fragment thereof compared to corresponding levels in healthy individuals or a patient in CR also correlate with a shorter PFS, reduced TTP, and / or shorter OS in subjects having or suspected of having a solid tumor cancer. In certain embodiments, the levels of IL4I1 polypeptide or a fragment thereof in biological samples of subjects having or suspected of having MM is higher than about 6 ng / mL. In certain embodiments, the levels of IL4I1 polypeptide or a fragment thereof in biological samples of subjects having or suspected of having MM is higher than about 10 ng / mL.

[0116] In some embodiments, the diagnosis, prognosis, or monitoring of MM involves determining the relative amount of a IL4I1 polypeptide or a fragment thereof in a biological sample of a subject having or suspected of having MM as compared to a predetermined cut-off value. Thus, in certain embodiments, the diagnosis, prognosis, or monitoring of MM is monitored by the presence of “an increased amount” or “a decreased amount” of IL4I1 polypeptide expression compared to a predetermined cut-off value. In certain embodiments, an increased amount of IL4I1 polypeptide expression in a sample refers to a sample generating asignal that is statistically stronger than the predetermined cut-off value, whereby the sample is considered positive for a disease condition (e.g., MM). In certain embodiments, the sample generates a signal that is up to about two standard deviations, up to about three standard deviations, up to about five standard deviations, up to about 10 standard deviations, up to about 20 standard deviations, up to about 30 standard deviations, up to about 40 standard deviations, up to about 50 standard deviations, up to about 60 standard deviations, up to about 70 standard deviations, up to about 80 standard deviations, up to about 90 standard deviations, or up to about 100 standard deviations above the predetermined cut-off. Accordingly, an increased amount of IL4I1 polypeptide expression may refer to up to about two standard deviations, up to about three standard deviations, up to about five standard deviations, up to about 10 standard deviations, up to about 20 standard deviations, up to about 30 standard deviations, up to about 40 standard deviations, up to about 50 standard deviations, up to about 60 standard deviations, up to about 70 standard deviations, up to about 80 standard deviations, up to about 90 standard deviations, or up to about 100 standard deviations above the predetermined cut-off.

[0117] In certain embodiments, an increased amount of IL4I1 polypeptide expression in a sample refers to a sample generating a signal that is at least about 2 -fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels above the predetermined cut-off. Accordingly, an increased amount of IL4I1 polypeptide expression may refer to at least about 2 -fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels above the predetermined cut-off.

[0118] In certain embodiments, an increased amount of IL4I1 polypeptide expression in a sample refers to a sample generating a signal that is at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% greater than the predetermined cut-off. Accordingly, an increased amount of IL4I1 polypeptide expression may refer to at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, or higher levels above the predetermined cut-off.

[0119] In other embodiments, the sample generates a signal that is up to about two standard deviations, up to about three standard deviations, up to about five standard deviations, up to about 10 standard deviations, up to about 20 standard deviations, up to about 30 standarddeviations, up to about 40 standard deviations, up to about 50 standard deviations, up to about 60 standard deviations, up to about 70 standard deviations, up to about 80 standard deviations, up to about 90 standard deviations, or up to about 100 standard deviations below the predetermined cut-off. Accordingly, a decreased amount of IL4I1 polypeptide expression may refer to up to about two standard deviations, up to about three standard deviations, up to about five standard deviations, up to about 10 standard deviations, up to about 20 standard deviations, up to about 30 standard deviations, up to about 40 standard deviations, up to about 50 standard deviations, up to about 60 standard deviations, up to about 70 standard deviations, up to about 80 standard deviations, up to about 90 standard deviations, or up to about 100 standard deviations below the predetermined cut-off.

[0120] In certain embodiments, a decreased amount of IL4I1 polypeptide expression in a sample refers to a sample generating a signal that is at least about 2 -fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or lower levels below the predetermined cut-off. Accordingly, a decreased amount of IL4I1 polypeptide expression may refer to at least about 2 -fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or lower levels below the predetermined cut-off.

[0121] In certain embodiments, a decreased amount of IL4I1 polypeptide expression in a sample refers to a sample generating a signal that is at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% below the predetermined cut-off. Accordingly, a decreased amount of IL4I1 polypeptide expression may refer to at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% below the predetermined cut-off.

[0122] In some embodiments, higher levels of IL4I1 polypeptide or a fragment thereof compared to a predetermined cut-off value correlate with a reduced TTP and shorter PFS among subjects having or suspected of having MM. In other embodiments, higher levels of IL4I1 polypeptide or a fragment thereof compared to a predetermined cutoff value correlate with a shorter OS in subjects having or suspected of having MM. Therefore, in certain embodiments, higher levels of IL4I1 polypeptide or a fragment thereof compared to a predetermined cut-off value are predictive of a shorter PFS, reduced TTP, and / or shorter OS in subjects having or suspected of having MM.

[0123] In some embodiments, very high levels of IL4I1 polypeptide or a fragment thereof compared to a predetermined cut-off value also correlate with a shorter PFS, reduced TTP, and / or shorter OS in subjects having or suspected of having MM. In certain embodiments, the levels of IL4I1 polypeptide or a fragment thereof in biological samples of subjects having or suspected of having MM is higher than about 6 ng / mL. In certain embodiments, the levels of IL4I1 polypeptide or a fragment thereof in biological samples of subjects having or suspected of having MM is higher than about 10 ng / mL.

[0124] In some embodiments, a predetermined cut-off value is a median value of the expression levels of a IL4I1 polypeptide or a fragment thereof in a population of normal control subjects. In some embodiments, a predetermined cut-off value is a median value of the expression levels of a IL4I1 polypeptide or a fragment thereof in a population of patients being treated for MM. In some embodiments, a predetermined cut-off value is a median value of the expression levels of a IL4I1 polypeptide or a fragment thereof in a population of MM patients in CR. In some embodiments, a predetermined cut-off value is a median value of the expression levels of a IL4I1 polypeptide or a fragment thereof in a population of MM patients in PD.

[0125] In some embodiments, the diagnosis, prognosis, or monitoring of MM involves determining the relative IL4I1 gene expression in a biological sample of a subject having or suspected of having MM as compared to the relative IL4I1 gene expression in a biological sample of a normal control subject or a patient in CR. Thus, in certain embodiments, the diagnosis, prognosis, or monitoring of MM is monitored by the presence of “an increased amount” or “a decreased amount” of IL4I1 gene expression compared to a predetermined cut-off value, an amount in a control biological sample, or a median value of IL4I1 gene expression in a population of subjects being treated for MM (e.g. a population of patients in CR).

[0126] In some embodiments, an increased amount of IL4I1 gene expression refers to at least about 1-fold, at least about 2 -fold, at least about 3 -fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels of IL4I1 gene expression as compared to a predetermined cut-off value, an amount in a control biological sample (e.g. a normal control subject or a patient in CR), or a median value of IL4I1 gene expression in a population of normal control subjects or patients in CR. In some embodiments, an increased amount of IL4I1 gene expression refers to at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, or higher levels of IL4I1 gene expression as compared to a predeterminedcut-off value, an amount in a control biological sample (e.g. a normal control subject or a patient in CR), or a median value of IL4I1 gene expression in a population of normal control subjects or patients in CR. In particular embodiments, IL4I1 gene expression is determined by mRNA levels.

[0127] In some embodiments, a decreased amount of IL4I1 gene expression refers to at least about 2 -fold, at least about 3-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels of IL4I1 gene expression as compared to a predetermined cut-off value, an amount in a control biological sample (e.g. a normal control subject or a patient in CR), or a median value of IL4I1 gene expression in a population of normal control subjects or patients in CR. In some embodiments, a decreased amount of IL4I1 gene expression refers to at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, or lower levels of IL4I1 gene expression as compared to a predetermined cut-off value, an amount in a control biological sample (e.g., a normal control subject or a patient in CR), or a median value of IL4I1 gene expression in a population of normal control subjects or patients in CR.

[0128] In general, a disease state is monitored by the presence of at least about 1-fold, at least about 2-fold, at least about 3 -fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or higher levels of IL4I1 gene expression as compared to those in a normal control subject or a patient in CR. In some embodiments, higher levels of IL4I1 gene expression compared to corresponding levels in healthy individuals or a patient in CR correlate with a reduced TTP and shorter PFS. In other embodiments, higher levels of IL4I1 gene expression compared to corresponding levels in healthy individuals or a patient in CR correlate with a shorter OS in subjects having or suspected of having MM. Therefore, in certain embodiments, higher levels of IL4I1 gene expression compared to corresponding levels in healthy individuals or a patient in CR are predictive of a shorter PFS, reduced TTP, and / or shorter OS in subjects having or suspected of having MM.MM-related diseases

[0129] The methods for diagnosis, prognosis, or monitoring of MM of the present disclosure can be applied to diseases and conditions related to MM. Non-limiting examples of MM-related diseases and conditions include monoclonal gammopathy of undeterminedsignificance (MGUS), smoldering (S)MM, Waldenstrom’s macroglobulinemia (WM), and primary amyloidosis (PA). In some embodiments, serum IL4I1 levels or relative IL4I1 gene expression can be used for diagnosis of MM-related diseases and conditions. In some embodiments, serum IL4I1 levels or relative IL4I1 gene expression can be used to determine PFS and OS among patients with WM and PA.

[0130] Patients with MGUS harbor the same monoclonal protein present among patients with MM but its levels are lower and BM plasma cell involvement is lower. Although MGUS patients do not require treatment for MM, approximately 1% of them / year develop active MM requiring treatment. Similarly, patients with SMM meet the criteria for having MM but do not require treatment for MM, but they have a higher risk of developing active MM than those with MGUS. It has been previously shown that serum BCMA levels predict the chances of both of these types of patients developing active myeloma requiring treatment. In some embodiments, baseline levels of serum IL4I1 are used to predict the risk of developing active MM among these patients. In some embodiments, the combination of serum IL4I1 and BCMA levels are used for diagnosis, prognosis, or monitoring of MM or MM-related diseases and conditions. In certain embodiments, the combination of serum IL4I1 and BCMA levels are used to predict transformation to active MM among individuals with MGUS or SMM. In some embodiments, the combination of serum BCMA and IL4I1 levels are used as predictors of outcome for MM, WM, and PA patients. Methods for diagnosis, prognosis, and monitoring of MM and MM- related diseases by determining BCMA levels are described in U.S. Patent No. 10,126,301, which is incorporated herein in its entirety by reference.Detection Systems and Kits

[0131] In various embodiments, the present disclosure provides detection systems and kits for MM. A detection system or kit of the present disclosure may be used for diagnosis, prognosis, or monitoring of MM patients in a biological sample, e.g., serum, of a subject. In some embodiments, the diagnostic kit could include the method for the detection of an antigenantibody reaction in addition to the material. In certain embodiments, the detection method comprises flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assays, or strip assay. The reactivity of the antigen recognition material could be confirmed using device detecting an enzyme reaction, fluorescence, luminescence, or radiation. In some embodiments, the diagnosis, prognosis, ormonitoring of MM can be made with a flow cytometry kit, an immunohistochemistry kit, an ELISA kit, or a lateral flow or strip kit including an anti-IL4Il antibody or an antigen binding fragment thereof.

[0132] In some embodiments, a kit or system may comprise one or more or all of the following components: 1) one or more standards comprised of IL4I1; 2) a binding agent, such as an antibody or a plurality of antibodies, that are specific for IL4I1; 3) written instructions; 4) diluents for samples and the standards; 5) a wash buffer; 6) color reagents; 7) stop solution; and 8) a carrier, such as an antibody carrier, for example, a lateral flow device, or a microplate with bound antibody, or polystyrene beads.

[0133] In some embodiments, the detection system or kit used to diagnose, prognose, or monitor MM is a quantitative ELISA that determines the concentration or concentrations of IL4I1 in accordance with methods embodied by the present disclosure.

[0134] In other embodiments, the diagnosis, prognosis, or monitoring of MM may be carried out using a lateral flow assay. Such lateral flow assays have the potential to be a cost- effective, fast, simple, and sensitive method, for instance for on-site screening assays. The lateral flow assay comprises a carrier that allows a lateral flow to occur wherein either the sample or the detection reagent is displaced form one location on the carrier to another. There are many formats of lateral flow assays suitable for use in a method embodied by the present disclosure, and the skilled person will readily know how to select and optimize a particular format. An example of a lateral flow test strip of the present disclosure comprises, for example, the following components: sample pad; an absorbent pad onto which the test sample is applied; a conjugate or reagent pad that contains antibodies specific to the target analyte (IL4I1) and conjugated to colored particles (usually colloidal gold particles, or latex microspheres); a reaction membrane, typically a hydrophobic nitrocellulose or cellulose acetate membrane onto which anti-target analyte antibodies are immobilized in a line across the membrane as a capture zone or test line (a control zone may also be present, containing antibodies specific for the conjugate antibodies); and a wick or waste reservoir, a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it.

[0135] A detection system in kit form can include, for example, an amount sufficient for at least one assay and an antibody composition or monoclonal antibody composition that binds to the IL4I1 serological biomarker for MM as a packaged reagent. Instructions for use of the packaged reagent are also typically included.

[0136] In various embodiments, the present disclosure provides detection systems and kits for MM. A detection system or kit of the present disclosure may be used for diagnosis, prognosis, or monitoring of MM patients in a biological sample, e.g., bone marrow mononuclear cells, of a subject. In some embodiments, the biological sample is RNA (e.g., mRNA) isolated from bone marrow mononuclear cells or peripheral blood mononuclear cells. The diagnostic / detection kit could include the method for the detection of IL4I1 gene expression. In certain embodiments, the detection method comprises northern blotting, PCR, microarrays, or RNA-seq. In some embodiments, the detection system and kits of the present disclosure comprises one or more oligonucleotide or primer that specifically hybridizes to the IL4I1 gene.

[0137] As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits an antibody composition or monoclonal antibody composition. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide, or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed.

[0138] Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent / sample admixtures, temperature, buffer conditions and the like.

[0139] In particular embodiments, a detection system of the present invention further includes a label or indicating means capable of signaling the formation of a complex containing a polypeptide or antibody molecule of the present invention.

[0140] "Complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

[0141] As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents such labels are themselves well-known in clinicaldiagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and / or systems.

[0142] The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. In certain embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-azino-di-(3-ethyl- benzthiazoline-G-sulfonic acid) (ABTS). Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124 I, 125 I, 128 I, 132 I and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups.Compositions

[0143] The present disclosure further provides compositions for the treatment of MM, characterized by a modulation, such as a decrease in the activity (e.g., enzymatic activity) and / or expression of IL4I1. In some embodiments, the compositions described herein comprises an inhibitor of IL4I1. An inhibitor of IL4I1 may inhibit: production of IL4I1 polypeptide, activity (e.g., enzymatic activity) of IL4I1 protein, expression of IL4I1 transcripts, and / or function of IL4I1 protein. Functions of IL4I1 include, but are not limited to, catalyzing the oxidative deamination of L-amino acids, activation of aryl hydrocarbon receptor (AHR), inhibition of the proliferation and activation of T cells, driving M2 macrophage polarization, and activation of the Janus kinase (JAK) pathway. In some embodiments of the IL4I1 inhibitors described herein that inhibit IL4I1 expression or function, “inhibit” refers to at least a partial reduction in the production of a IL4I1 polypeptide, at least a partial reduction in the enzymatic activity of IL4I1 protein, at least a partial reduction in the expression of IL4I1 transcripts, at least a partial reduction in the expression of IL4I1 transcripts and / or at least a partial reduction in a function of IL4I1, such as catalyzing the oxidative deamination of L-amino acids. In particular embodiments, the at least partial reduction is a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.IL4I1 inhibitors

[0144] IL4I1 is a secreted L-amino acid oxidase (LAAO) and acts as an enzyme that converts phenylalanine to phenylpyruvate, hydrogen peroxide, and ammonia. In some embodiments, the compositions described herein for the treatment of MM comprises an inhibitor of IL4I1 enzymatic activity. In certain embodiments, the compositions described herein for the treatment of MM comprises an inhibitor of IL4I1 that reduces the ability of IL4I1 to convert phenylalanine to phenylpyruvate, hydrogen peroxide, and ammonia. Disclosed herein are urea derivative compounds that bind to the oxidase catalytic site of IL4I1 and inhibit the enzymatic activity of IL4I1. In some embodiments, the urea derivative compounds are a compound of formula (I), (la), (II) or a pharmaceutically acceptable salt thereof. In embodiments, the urea derivative compounds are any one of the compounds of Table A.

[0145] In embodiments, the IL4I1 inhibitor of the present disclosure comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof,

[0146] wherein

[0147] each of n and m is 0, 1, 2 or 3;

[0148] each of R1and R2is independently halogen, Ci-6 alkyl, OCi-6 alkyl or Ci-6 haloalkyl;

[0149] R3is H, Ci-6 alkyl or C3-6 cycloalkyl; and

[0150] R4is H or oxo (=0).

[0151] In embodiments, the IL4I1 inhibitor of the present disclosure comprises asalt thereof, wherein the definition of R1, R2, R3, and m are as defined in formula (I).

[0152] In embodiments, the IL4I1 inhibitor of the present disclosure comprises a compound of formula (II), or a pharmaceutically acceptable salt thereof,

[0154] each of n and m is 0, 1, 2 or 3;

[0155] each of R1and R2is independently halogen, Ci-6 alkyl, OCi-6 alkyl or Ci-6 haloalkyl;

[0156] R3is H, Ci-6 alkyl or C3-6 cycloalkyl; and

[0157] R5is heterocyclyl, NH2, NH(CI-6 alkyl) or N(CI-6 alkyl)2.

[0158] In embodiments of the compound of formula (I), (II) or a pharmaceutically acceptable salt thereof, n is 1, 2 or 3. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 2. In embodiments, n is 3.

[0159] In embodiments of the compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof, m is 1, 2 or 3. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3.

[0160] In embodiments of the compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof, R1is halogen, C1-3 alkyl, OC1-3 alkyl or C1-3 haloalkyl. In embodiments, R1is F, Cl, CH2, CH2CH3, OCH3, CHF, CHF2or CF3. In embodiments, R1is F or CF3. In embodiments, R1is F.

[0161] In embodiments of the compound of formula (I), (II), or a pharmaceutically acceptable salt thereof, n is i and R1is F.

[0162] In embodiments of the compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof, R2is halogen, C1-3 alkyl, OC1-3 alkyl or C1-3 haloalkyl. In embodiments, R2is halogen or C1-3 haloalkyl. In embodiments, R2is F, Cl, CHF, CHF2 or CF3. In embodiments, R2is F, Cl, or CF3.

[0163] In embodiments of the compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof, R3is H or C1-4 alkyl. In embodiments, R3is H, CH3, CH2CH3 or CH(CH3)2. In embodiments, R3is H.

[0164] In embodiments of the compound of formula (I), or a pharmaceutically acceptable salt thereof, R4is H. In embodiments of the compound of formula (I), or a pharmaceutically acceptable salt thereof, R4is oxo (=0).

[0165] In embodiments of the compound of formula (II), or a pharmaceutically acceptable salt thereof, R5is N-heterocyclyl, NH2, NH(CI-6 alkyl) or N(CI-6 alkyl)2. In embodiments, R5isN-heterocyclyl or N(Ci-6 alkyl)2. In embodiments, R5is 5-6 membered N-heterocyclyl or N(Ci-6 alkyl)2. In embodiments, R5is 5-6 membered N-heterocyclyl or N(Ci-3 alkyl)2. In embodiments,

[0166] In embodiments, provided herein is a compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof.

[0167] In embodiments, provided herein is a compound set forth in Table A, or a pharmaceutically acceptable salt thereof.

[0168] In embodiments, the IL4I1 inhibitor used in the present disclosure comprises a compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof. In embodiments, the IL4I1 inhibitor used in the present disclosure is a compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof.

[0169] In embodiments, the IL4I1 inhibitor used in the present disclosure comprises a compound in Table A, or a pharmaceutically acceptable salt thereof. In embodiments, the IL4I1 inhibitor used in the present disclosure is a compound in Table A, or a pharmaceutically acceptable salt thereof. In embodiments, the IL4I1 inhibitor used in the present disclosure comprises a compound shown in any one of FIGs. 31A-E, or a pharmaceutically acceptable salt thereof.

[0170] In embodiments, provided herein is a product obtainable by any of the processes or examples disclosed herein.

[0171] In embodiments, provided herein is a process of synthesizing a compound according to the figures and procedures of Example 10. In embodiments, provided herein a process of synthesizing a compound according to the procedures shown in FIGs. 32A-R.

[0172] In embodiments, provided herein is one or more compounds synthesized according to the figures and procedures of Example 10. In embodiments, provided herein is one or more compounds synthesized according to the procedures shown in FIGs. 32A-R.Table A: Exemplary structures of IL4I1 inhibitors

[0173] Also disclosed are methods of synthesizing a urea derivative compound that is useful as an IL4I1 inhibitor. For example, a urea derivative compound, referred herein as “Compound 301,” which binds to the oxidase catalytic site of IL4I1, was designed and synthesized as shown in the schema below.

[0174] Three step synthesis schema for Compound 301

[0175] Step 1 : Compound 1 (30 g, 232 mmol, 1 equiv) was added to 10V TFA slowly under cooling. NaBH(OAc)3 (98.6 g, 464 mmol, 2 equiv) was added portionwisely while maintaining internal temperature under 10 C. After stirring at 0°C for 30 min, compound 2 (39.3 g, 232 mmol, 1 equiv) was added portion-wisely. The reaction was allowed to warm up slowly to RT and stirred for 20h. The reaction mixture was slowly added into ice-water (15 V) with stirring, and the crude solid was filtered. The crude solid was dried and extracted with Hex / EA (5: 1) two times. Filtrate was combined and concentrated to give a compound 3 as a solid (36 g).

[0176] Step 2: Compound 3 from previous step (50 g, 177.3 mmol, 1 equiv) was dissolved in iPrOH (5 V). NH4C1 (47.4 g, 886.5 mmol, 5 equiv) in water (3 V) was added. The reaction mixture was heated to 75C internally. Fe powder (29.79 g, 531.9 mmol, 3 equiv) was added portionwisely. The reaction was heated at 8O0C internally for 2h. High-performance liquid chromatography (HPLC) showed slowing down of conversion. To the reaction was added extra 2 equiv Fe powder and was heated at 8O0C for another 2h. HPLC showed completion of reaction. The reaction mixture was diluted with 5 V EtOAc and 2V H2O and then was filtered through celite. The organic phase was separated and concentrated on a rotavap, and the residue was redissolved in EtOAc, dried over MgSO4, and concentrated on a rotavap. The crude solid was then slurried in Hex / MTBE (15: 1, 4V) and filtered to give a crude compound 4 as a solid (37 g).

[0177] Step 3: Compound 4 (102 g, 404.8 mmol, 1 equiv) from the previous step was mixed with CDI (85.2 g, 526.2 mmol, 1.3 equiv) in THF (10 V). The reaction was heated for 3 h at 65 C. The reaction mixture was concentrated and added EtOAc to a slurry, which was stirred for 30 min. The solid was filtered and dried in vacuum oven overnight (crop 1, 67 g >99A%). The mother liquor was washed with 2N HC1 x 2 (3 equiv in total), and then organic phase was dried over MgSO4 and concentrated to a semi solid, to which was added ACN to a slurry, and filtered to get a solid, which was dried in vacuum oven overnight (crop 2, 25 g, >98A%).

[0178] In some embodiments, the compositions described herein comprise a compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions described herein comprise a compound in Table A, or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions described herein comprise compound 301, compound 302, compound 303, compound 304, compound 305, compound 306, compound 307, compound 309, compound 315, compound 317, compound 318, compound 319, compound 320, compound 321, compound 322, compound 323, compound 324, compound 325, or a pharmaceutically acceptable salt thereof.

[0179] In some embodiments, the compositions described herein comprise a urea derivative compound. In certain embodiments, the urea derivative comprises the structure ofCompound 301.Compound 301

[0180] In some embodiments, the urea derivative is a compound of a compound of formula (I), (la), (II), or a pharmaceutically acceptable salt thereof, optionally wherein the urea derivative is synthesized according to the procedures shown in FIGs 32A-R.

[0181] In some embodiments, the urea derivative is compound 301, compound 302, compound 303, compound 304, compound 305, compound 306, compound 307, compound 309, compound 315, compound 317, compound 318, compound 319, compound 320, compound 321, compound 322, compound 323, compound 324, compound 325, or a pharmaceutically acceptable salt thereof, optionally wherein the urea derivative is synthesized according to the procedures shown in FIGs. 32A-R.

[0182] In some embodiments, the urea derivative is Compound 301, optionally wherein the urea derivative is synthesized by the three-step synthesis schema presented above. siRNA

[0183] In some embodiments, the composition for the treatment of MM comprises an inhibitor of IL4I1 which inhibits IL4I1 gene expression. In some embodiments, the inhibition of IL4I1 gene expression is achieved by decreasing production of IL4I1 mRNA transcripts. In some embodiments, a composition comprising an inhibitor of IL4I1 which inhibits IL4I1 gene expression comprises a small interfering RNA (siRNA) oligo, micro-RNA (miRNA), antisense oligonucleotide (ASO), or ribozyme that inhibits IL4I1 gene expression. In some embodiments, the compositions described herein comprise a siRNA oligo. An siRNA oligo that inhibits IL4I1 gene expression as described herein comprises an antisense strand comprising a sequence that is a reverse complement of at least a region of a IL4I1 mRNA transcript. In certain embodiments, the sequence is at least or about 12, at least or about 14, at least or aboutl6, at least or about 18, or at least or about 20 nucleotides in length. In some embodiments, the IL4I1 mRNA transcript comprises a sequence encoded by any one of SEQ ID NOs: 13-15, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 85%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity with any one of SEQ ID NOs: 13-15. In some embodiments, a siRNA oligo that inhibits IL4I1 gene expression as described herein targets at least a region of a IL4I1 mRNA transcript encoded by any one of SEQ ID NOs: 13-15, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 85%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 98%, or at least about 99% sequence identity with any one of SEQ ID NOs: 13-15.

[0184] In some embodiments, a composition described herein comprises a siRNA oligo. In certain embodiments, the composition comprises a siRNA oligo that targets at least a region of a IL4I1 mRNA transcript encoded by any one of SEQ ID NOs: 13-15, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 85%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity with any one of SEQ ID NOs: 13-15. In certain embodiments, the composition comprises one or more of the following siRNA oligos: a) an antisense strand comprising CUUUAAUGCGAGGUCCUCGtg (SEQ ID NO:3) and a sense strand comprising CGAGGACCUCGCAUUAAAGtt (SEQ ID NO:4); b) an antisense strand comprising CUCUCAACCAGGCCCUCAA[dT][dT] (SEQ ID NO: 5) and a sense strand comprising UUGAGGGCCUGGUUGAGAG[dT][dT] (SEQ ID NO: 6); c) an antisense strand comprising CCAAGUUCACCCAGUACGA[dT][dT] (SEQ ID NO: 7) and a sense strand comprising UCGUACUGGGUGAACUUGG[dT][dT] (SEQ ID NO: 8); d) an antisense strand comprising GUCCCUUAUGGCCGCAUCU[dT][dT] (SEQ ID NO: 9) and a sense strand comprising AGAUGCGGCCAUAAGGGAC[dT][dT] (SEQ ID NO: 10); or e) an antisense strand comprising CAUUGAAGGCGGCCACUCA[dT][dT] (SEQ ID NO: 11) and a sense strand comprising UGAGUGGCCGCCUUCAAUG[dT][dT] (SEQ ID NO: 12).

[0185] It will be appreciated that [dT][dT] in SEQ ID NOs: 3-12 above indicate an optional deoxythymidine dinucleotide 3’ overhang, which may confer increased nuclease resistance and reduce degradation of the siRNA oligos. Other optional 3’ or 5’ overhangs include “tg” and “tf ’ overhangs, which may facilitate guiding the siRNA duplex to DICER. Therefore, in some embodiments, the siRNA oligos described herein do not comprise [dT][dT] overhangs or “tg” and “tf ’ overhangs.

[0186] The compositions described herein may further comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except as far as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of the present disclosure.

[0187] When administered to a subject, the compositions described herein may inhibit IL4I1 gene expression or protein production. In some embodiments, the compositions are administered to human subjects. In a further embodiment, the compositions are administered to a subject who is a patient.

[0188] The phrase “active ingredient” generally refers to the IL4I1 inhibitors (e.g., urea derivative compounds), siRNA oligos, miRNAs, antisense oligonucleotides, or ribozymes to be delivered as described herein. Formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0189] A composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0190] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treatedand further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0191] Pharmaceutically acceptable excipients used in the manufacture of the compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0192] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, and / or suspensions. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0193] In some embodiments, the compositions described herein are administered through parenteral injections. In certain embodiments, the parenteral route of injection is intradermal (IM), subcutaneous (SQ), intramuscular (IM), or intravenous (IV). In some embodiments, the route of administration is intratumor. In some embodiments, the compositions described herein are administered directly into the bone marrow of a subject. In certain embodiments, the compositions is administered by intraosseous infusion.

[0194] In some embodiments, the compositions described herein are administered through enteral administration. In certain embodiments, the enteral administration route is oral administration or sublingual administration. In certain embodiments, the IL4I1 inhibitors and pharmaceutical compositions comprising the same described herein are administered orally.Methods of treatment

[0195] The present disclosure further provides methods for treating a subject having or suspected of having MM. MM is a B cell malignancy of mature plasma cell morphology. Theseplasma cells proliferate in BM and may invade adjacent bone and sometimes the blood. Types of multiple myeloma that may be treated as disclosed herein include, but are not limited to, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non- secretory myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0196] In some embodiments, the methods described herein comprise administering to the subject an inhibitor of IL4I1 or a composition described herein. An inhibitor of IL4I1 may be a polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In some embodiments of the methods for treating a subject with MM, the method comprises administering to the subject an inhibitor of IL4I1. In certain embodiments, the inhibitor of IL4I1 comprises or consists of Compound 1. In certain embodiments, the inhibitor of IL4I1 comprises siRNA targeting a nucleic acid sequence encoding IL4I1 or a complementary sequence thereof. In some embodiments, the method comprises administering a polypeptide that specifically binds IL4I1. In some embodiments, the method comprises administering an anti-IL4Il antibody. In some embodiments, the method comprises administering a composition described herein. In some embodiments, the term “an IL4I1 inhibitor” and the term “an inhibitor of IL4H” are used interchangeably.

[0197] In some embodiments of the methods described herein, a composition described herein is administered to a subject to treat relapsed / refractory MM. In some embodiments, a composition described herein is administered to a subject to treat overt MM, SMM, plasma cell leukemia, non-secretory myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, or extramedullary plasmacytoma. In certain embodiments, compositions described herein are administered to a subject with relapsed / refractory MM that has been unsuccessfully treated with one, two, three, or more treatments. In particular embodiments, compositions described herein are administered to a subject with relapsed / refractory MM that has been unsuccessfully treated with one, two, three, or more treatments, including at least one Pi and / or an immunomodulatory drug (IMiD). In some embodiments, the subject's MM is refractory to three treatment regimens. In certain embodiments, the subject's MM is refractory to three treatment regimens, including at least one Pi and an IMiD. In some embodiments, the subject's MM is double-refractory to one or more treatment regimens.

[0198] Examples of Pis to which a subject's MM may be refractory include, but are not limited to, bortezomib and carfilzomib. Examples of IMiDs to which a subject's MM may be refractory include, but are not limited to thalidomide, lenalidomide, and pomalidomide.

[0199] Examples of other treatments to which a subject’s MM may be refractory include, but are not limited to, dexamethasone, and antibody -based therapies selected from the group consisting of elotuzumab, daratumumab, MOR03087, isatuximab, bevacizumab, cetuximab, siltuximab, tocilizumab, elsilimomab, azintrel, rituximab, tositumomab, milatuzumab, lucatumumab, dacetuzumab, figitumumab, dalotuzumab, AVE 1642, tabalumab, pembrolizumab, pidilizumab, and nivolumab.

[0200] In some embodiments, a subject's MM is refractory to treatment with daratumumab. In some embodiments, a subject's MM is refractory to treatment with an IMiD, a Pi, and dexamethasone.

[0201] The present disclosure provides methods for treating MM in a subject comprising administering a therapeutically effective amount of an inhibitor of IL4I1 to the subject. Also provided are methods for treating MM in a subject comprising administering a therapeutically effective amount of a compound of formula (I), (la), (II) or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the methods of the present disclosure for treating MM in a subject comprises administering a therapeutically effective amount of a compound of Table A. In some embodiments, the methods of the present disclosure for treating MM in a subject comprises administering a therapeutically effective amount of a compound of Table A , or a pharmaceutically acceptable salt thereof to the subject.

[0202] In some embodiments, the inhibitor of IL4I1 of any of the methods herein comprises a compound of formula (I), (la), (II) or a pharmaceutically acceptable salt thereof. In certain embodiments, the inhibitor of IL4I1 comprises any one of the compounds of Table A, or a pharmaceutically acceptable salt thereof. In certain embodiments, the inhibitor of IL4I1 comprises Compound 301, or a pharmaceutically acceptable salt thereof.

[0203] In some embodiments of the methods for treating a subject with MM, treatment comprises determining the presence of MM in the subject using a method of diagnosing MM or a kit described herein and administering to the subject an inhibitor of IL4I1, a polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or the compositions described herein. In some embodiments of the methods for treating a subject with MM, the method comprises administering to the subject an inhibitor of IL4I1. In some embodiments, the method comprises administering a polypeptide that specifically binds IL4I1. In some embodiments, the method comprises administering an anti-IL4Il antibody. In some embodiments, the method comprises administering the compositions described herein.

[0204] The methods of treatment of the present disclosure can be used in combination with other therapies for treatments of MM patients. In some embodiments of the treatment of MM of the present disclosure, the treatment further comprises administering to the subject a Pi, an IMiDs such as thalidomide or a derivative thereof, an anti-CD38 antibody, an anti-SLAMF7 antibody, a steroid, a chemotherapeutic agent, a bispecific T cell engager, a histone deacetylase inhibitor, a selective inhibitor of nuclear export, an antibiotic, or a BCMA-directed chimeric antigen receptor (CAR) T cell, before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein.

[0205] In some embodiments, the treatment further comprises administering to the subject a Pi before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In some embodiments, a Pi refers to a substance which directly or indirectly inhibits the 20S and / or 26S proteasome or an activity thereof. In particular embodiments, proteasome inhibition is specific, i.e., the Pi inhibits proteasome activity at a concentration that is lower than the concentration of the inhibitor required to produce another, unrelated biological effect. In certain embodiments, the Pi is selected from the group consisting of: bortezomib, carfilzomib, oprozomib, ixazomib citrate, marizomib, delanzomib, syringolin, ONX 0912, CEP-18770, and MLN9708.

[0206] In some embodiments, the treatment further comprises administering to the subject thalidomide or a derivative thereof before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, thalidomide or the derivative thereof is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, linomide, CC-1088, CDC- 501, and CDC-801.

[0207] In some embodiments, the treatment further comprises administering to the subject an anti-CD38 antibody before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the anti-CD38 antibody is isatuximab or daratumumab.

[0208] In some embodiments, the treatment further comprises administering to the subject an anti-SLAMF7 antibody before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the anti-SLAMF7 antibody is elotuzumab.

[0209] In some embodiments, the treatment further comprises administering to the subject a steroid before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the steroid is a glucocorticoid. In certain embodiments, the glucocorticoid is selected from the group consisting of hydroxy corti sone, cortisone, desoxy corticosterone, dexamethasone, fludrocortisone, betamethasone, dexamethasone, prednisolone, prednisone, methylprednisolone, methylprednisone, paramethasone, triamcinolone, flumethasone, fluocinolone, fluocinonide, fluprednisolone, halcinonide, flurandrenolide, meprednisone, and medrysone.

[0210] In some embodiments, the treatment further comprises administering to the subject a chemotherapeutic agent before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the chemotherapeutic agent is selected from the group consisting of melphalan, cyclophosphamide, bendamustine, doxorubicin, and pegylated liposomal doxorubicin.

[0211] In some embodiments, the treatment further comprises administering to the subject a bispecific T cell engager before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the bispecific T cell engager is talquetamab, teclistamab, or elranatamab.

[0212] In some embodiments, the treatment further comprises administering to the subject a histone deacetylase inhibitor before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the histone deacetylase inhibitor is panobinostat.

[0213] In some embodiments, the treatment further comprises administering to the subject a selective inhibitor of nuclear export, before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the selective inhibitor of nuclear export is selinexor.

[0214] In some embodiments, the treatment further comprises administering to the subject an antibiotic before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the antibiotic is clarithromycin.-M-

[0215] In some embodiments, the treatment further comprises administering to the subject a BCMA-directed CAR T-cell before, simultaneously, or after administering the inhibitor of IL4I1, the polypeptide that specifically binds IL4I1, an anti-IL4Il antibody, or a composition described herein. In certain embodiments, the BCMA-directed CAR T cell is ide-cel or cilta-cel.

[0216] In some embodiments, the compositions described herein, or other therapies used in combination with said compositions, are administered through parenteral injections. In certain embodiments, the parenteral route of injection is intradermal (IM), subcutaneous (SQ), intramuscular (IM), and intravenous (IV), intraosseous infusion, or intratumoral. In some embodiments, the compositions described herein, or combinations thereof, are administered directly into the bone marrow of a subject. In certain embodiments, the compositions is administered by intraosseous infusion.Definitions

[0217] The terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise.

[0218] The term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0219] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0220] The terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0221] Numerical ranges are provided for certain quantities in the present application. It is to be understood that these ranges comprise all subranges therein. Thus, the range “from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

[0222] Unless otherwise indicated, the term "sequence identity" and related terms and expressions used in the context of describing nucleic acid or amino acid sequences refer to a sequence that has at least 20% sequence identity to a reference sequence. Examples include at least: 20%, 25%. 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence (subject sequence) to which test sequences (query sequence) are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well- known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0223] Depending on the algorithm, the calculated percent sequence identity may differ. For example, there are at least three ways in which to calculate a percent sequence identity.% Query sequence identity: = (Number of alignment identities) / (Length of Query sequence);% Subject sequence identity: = (Number of alignment identities) / (Length of Subject sequence);%Alignment sequence identity = (Number of alignment identities) / (Length of Alignment)

[0224] Accordingly, when the term “sequence identity” is used herein, it can include any of the above non-limiting methodologies provided above to calculate.

[0225] The terms “treatment” or “treating” used to refer to treatment or treating of a subject indicate any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In some embodiments, “treatment” or “treating” includes a partial remission. In some embodiments, “treatment” or “treating” includes a complete remission.

[0226] An “effective amount” means the amount of compound or pharmaceutical formulation, that when administered to a patient for treating a state, disorder or condition is sufficient to effect such treatment.

[0227] The term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical formulation that is sufficient to result in a desired clinical benefit after administration to a patient in need thereof. A “therapeutically effective amount,” in some embodiments, is a dose or amount of a compound or pharmaceutical formulation that is sufficient to result in prophylaxis after administration to a patient in need thereof.

[0228] The terms “administer” or “administration” refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a formulation of the invention) into a patient, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.

[0229] As used herein, the terms "promoting," "enhancing," "stimulating," or "increasing" generally refer to the ability of compositions contemplated herein to produce or cause a greater physiological response (i.e., measurable downstream effect), as compared to the response caused by either vehicle or a control molecule / composition. One such measurable physiological response includes, without limitation, increased cell killing and / or tumor reduction, increasedsurvival, increased treatment efficacy compared to normal, untreated, or control -treated subjects. The physiological response may be increased by at least 5%, 10%, 20%, 30%>, 40%>, 50%>, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or greater compared to the response measured in normal, untreated, or control -treated subjects. An "increased" or "enhanced" response or property is typically "statistically significant" , and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) that produced by normal, untreated, or control -treated subjects.

[0230] As used herein, the term "statistically significant" generally refers to a study result where the P value of the data analysis is less than 0.05, unless otherwise stated.

[0231] As used herein, the terms "correlated with" or "associated with" refer to the levels of IL4I1 in a biological sample of a subject that has a statistically significant correlation with a physiologic state, e.g., disease status or extent of the disease, response to treatment, and survival. The strength of the correlation between IL4I1 levels and the presence or absence of a particular physiologic state may be determined by a statistical test of significance. Methods for determining the strength of a correlation between the expression level of a differentially- expressed gene and a particular physiologic state by assigning a statistical score to the correlation are reviewed in Holloway et al. (2002) Nature Genetics Suppl. 32:481-89, Churchill (2002) Nature Genetics Suppl. 32:490-95, Quackenbush (2002) Nature Genetics Suppl. 32: 496- 501; Slonim (2002) Nature Genetics Suppl. 32:502-08; and Chuaqui et al. (2002) Nature Genetics Suppl. 32:509-514; each of which is herein incorporated by reference in its entirety.

[0232] As used herein, a "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. Non-limiting examples of a biological sample include blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived there from and the progeny thereof. In some embodiments, said samples may be manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses a clinical sample, cells in culture, cell supernatants, cell lysates, serum, plasma, urine, cerebral spinal fluid, biological fluid, and tissue samples. The sample may be pretreated as necessary by dilution in an appropriate buffer solution or concentrated, if desired. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, preferably at physiological pH can be used. Biological samples can be derivedfrom patients using well known techniques such as venipuncture, lumbar puncture, fluid sample such as saliva or urine, or tissue biopsy and the like. In some embodiments, the biological sample is serum. In some embodiments, the biological sample is plasma. In some embodiments, the biological sample is bone marrow cells. In some embodiments, the biological sample is bone marrow mononuclear cells. In some embodiments, the biological sample is peripheral blood mononuclear cells. In some embodiments, the biological sample is supernatant from a cell culture comprising bone marrow mononuclear cells. In some embodiments, the biological sample is supernatant from a cell culture comprising peripheral mononuclear cells. In some embodiments, the biological sample comprises RNA (e.g,. mRNA) isolated from bone marrow mononuclear cells. In some embodiments, the biological sample comprises mRNA (e.g., mRNA) isolated from peripheral blood mononuclear cells.

[0233] As used herein, “control” or “control sample” refers to, for example, a biological sample from a healthy subject or group of healthy subjects, a prior sample from the same patient, or a different patient or a patient group. In certain embodiments, a control or control sample is from the same patient or group of patients as the sample where IL4I1 is detected but taken from the patient or group of patients at a previous time point.

[0234] The terms "polypeptide" and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. In various embodiments, IL4I1 polypeptides are contemplated for use within diagnostic, prognostic, or monitoring compositions and methods disclosed herein. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0235] As used herein, the term "IL4I1 polypeptide" is intended to generically refer to both the wild-type and variant IL4I1 polypeptides, unless specifically denoted otherwise. IL4I1 polypeptides are encoded by the IL4I1 gene. As it is commonly used in the art, the term "gene" is intended to refer to the genomic region encompassing 5' untranslated region(s) (UTR), exons, introns, and 3' UTR. Individual segments may be specifically referred to, e.g., promoter, coding region, etc. Combinations of such segments that provide for a complete IL4I1 protein may be referred to generically as a protein coding sequence. The "IL4I1 polypeptide" encompasses an amino acid sequence encoded by an open reading frame (ORF) of a known IL4I1polynucleotide, including the full-length native polypeptide and fragments thereof, particularly biologically active fragments and / or fragments corresponding to functional domains, e.g., a region or domain having biological activity, etc.; antigenic fragments thereof, and including fusions of the subject polypeptides to other proteins or parts thereof.

[0236] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides may have any three- dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single- and double-stranded and triple helical molecules."Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double- stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.

[0237] The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art. Nucleic acids may be naturally occurring, e.g., DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars, or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'- S-5'-0-phosphorothioate, 3'-CH2-5'-0-phosphonate and 3'-NH-5'-0-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.

[0238] A "subject," "subject in need of treatment," "subject in need thereof," "individual," or "patient" as used herein, includes any animal that exhibits a symptom of a disease, disorder, or condition that can be diagnosed and / or treated with methods and / or compositionscontemplated herein. In some embodiments, the disease, disorder, or condition relates to a hematological malignancy, e.g., multiple myeloma. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals (such as horses, cows, sheep, pigs), and domestic animals or pets (such as a cat or dog). In some embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human primate. In preferred embodiments, the subject is a human.

[0239] The term "relapse" refers to the diagnosis of return, or signs and symptoms of return, of a cancer after a period of improvement or remission.

[0240] "Remission," also known as "clinical remission," includes both partial and complete remission (CR). In partial remission, some, but not all, signs and symptoms of cancer have disappeared. In CR, all signs and symptoms of cancer have mostly disappeared, although cancer still may be in the body.

[0241] "Refractory" refers to a cancer that is resistant to, or non-responsive to, therapy with a particular therapeutic agent. A cancer can be refractory from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period or during a subsequent treatment period.

[0242] As used herein, the term "cell viability" refers to whether a cell is living or dead. Cell viability measurements may be used to evaluate the death or life of cells. Cell viability tests can be used to determine the effectiveness of agents to promote cell survival or induce cell death. Non-limiting examples of methods to determine cell viability include methods that use (1) nucleic acid binding dyes which are cell-impermeant to detect a compromised plasma membrane, (2) amine-reactive viability dyes which are cell-impermeant and dimly fluorescent when bound to extracellular amine proteins on live cells but undergo a significant increase in fluorescence when these dyes enter dead cells and react with intracellular amine proteins; or (3) enzyme activity viability substrates that react with cellular enzymes in live cells or react with cellular enzymes released from damaged cells and that produce a fluorescent or colorimetric readout.

[0243] Cell proliferation MTS assay is an assay that depends on conversion of the MTS tetrazolium compound to a colored formazan product in metabolically active cells; it is therefore an assay of viable cell number. A decline in conversion of the tetrazolium compound relates to an increase in cell killing as long as cell disappearance by apoptosis in untreated cell populations is absent or negligible (as with most healthy primary cell populations).

[0244] Oxo” refers to the =0 substituent.

[0245] Halogen refers to F, Cl, Br, and I.

[0246] “Alkyl” or “alkyl group” refers to a fully saturated, straight, or branched hydrocarbon chain radical having from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 12 are included. An alkyl comprising up to 12 carbon atoms is a C1-C12 alkyl, an alkyl comprising up to 10 carbon atoms is a Cl -CIO alkyl, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl. A C1-C5 alkyl includes C5 alkyls, C4 alkyls, C3 alkyls, C2 alkyls and Cl alkyl (i.e., methyl). A C1-C6 alkyl includes all moi eties described above for C1-C5 alkyls but also includes C6 alkyls. A Cl -CIO alkyl includes all moi eties described above for C1-C5 alkyls and C1-C6 alkyls, but also includes C7, C8, C9 and CIO alkyls. Similarly, a C1-C12 alkyl includes all the foregoing moieties, but also includes Cl 1 and C12 alkyls. Non-limiting examples of C1-C12 alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n- hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0247] “Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical consisting solely of carbon and hydrogen atoms, which can include fused, bridged, or spiro ring systems, having from three to twenty carbon atoms, e.g., having from three to 10 carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group can be optionally substituted.

[0248] “Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, tri chloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3 -bromo-2 -fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group can be optionally substituted.

[0249] “Heterocyclyl” “heterocyclic ring” or “heterocycle” refers to a stable 3- to 20-membered non-aromatic, saturated or partially unsaturated ring radical which consists of two to twelve carbon ring atoms and from one to six heteroatoms as ring atoms selected fromnitrogen, oxygen or sulfur, at least one non-aromatic, saturated or partially unsaturated ring containing at least one heteroatom as a ring atom. Unless stated otherwise specifically in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused, bridged, or spiro ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quatemized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. In embodiments where “L” is heterocyclyl, the heterocyclyl radical is a diradical. Unless stated otherwise specifically in the specification, a heterocyclyl group can be optionally substituted.

[0250] “N-heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. Unless stated otherwise specifically in the specification, a N-heterocyclyl group can be optionally substituted.

[0251] In this specification, unless stated otherwise, the term “pharmaceutically acceptable” is used to characterize a moiety (e.g., a salt, dosage form, or excipient) as being appropriate for use in accordance with sound medical judgment. In general, a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.

[0252] The term “pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically acceptable salts include those obtained by reacting the active compound functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc. Those skilled in the art will further recognize that acid addition salts may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.

[0253] As used herein, the symbol “ I ” or “ (hereinafter can be referred to as “a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example,XY— ! —XY“ ! ” or “ « ” indicates that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity can be specified by inference.ExamplesMaterials and MethodsCollection of clinical samples

[0254] Peripheral blood and BM samples were collected from MM, smoldering (S) MM and healthy age- and sex- matched subjects. The study was approved by the Institutional Review Board (Western IRB BIO-101) and informed consent was obtained in accordance with the Declaration of Helsinki. Patients were defined as having SMM, MM patients with active disease according to the International Myeloma Working Group (IMWG) and treated patients as showing PD or responsive disease (partial response (PR), very good (VG) PR, or CR) according to the IMWG criteria. Serum was obtained from these patients and analyzed for serum IL4I1 levels using two different comparisons: 1) IL4I1 serum levels were compared between age- and sex-matched healthy controls, untreated SMM patients and those with active MM before starting the treatment including newly diagnosed and RRMM patients, and 2) levels among MM patients with PD compared with those in complete remission (CR) from their treatment. The IMWG Uniform Response Criteria was used to determine the patient’s clinical status. Progress! on -free survival (PFS) was defined as the time from treatment start to disease progression or death from any cause, while overall survival (OS) was the time from MM diagnosis for frontline patients and time from starting new treatment for those with RRMM to death or last follow-up.Preparation of patents' bone marrow mononuclear cells (BMMCs)

[0255] Fresh BM aspirates were collected from MM patients with either CR or PD (5 patients / group). Aspirates were diluted with 0.9% NaCl (1 :5), were filtrated (100 pM), and BMMCs were isolated using density gradient centrifugation using Ficoll (Cambrex; 800 g, 20min, without brake). BMMCs were washed three times with PBS (800 g), counted, and used for the experiments.U266 cell line

[0256] The human MM cell line U266 was obtained from American Type Culture Collection (Rockville, MD, USA). The cell line was maintained in RPMI 1640 (Omega Scientific, Tarzana, CA, USA) supplemented with 10% fetal bovine serum, 2 mmol / 1 1- glutamine, and essential amino acids in an atmosphere of 5% carbon dioxide at 37°C.Assessment of serum IL4I1 and B-cell maturation antigen (BCMA) using enzyme-linked immunosorbent assays (ELISAs)

[0257] Serum BCMA levels were determined as previously published in multiple papers by our group (Ghermezi et al. Haematologica. 2017; Jew et al. Targeted Oncology 2023).

[0258] The quantification of IL4I1 levels in human serum was conducted using a human specific ELISA as per the instructions provided by the manufacturer (BIOMATIK, DE, USA, Cat. No. EKU05341). The serum samples were diluted at a ratio of 1 :500 in a diluent, and a volume of 100 pl from this diluted mixture was utilized for the ELISA. IL4I1 levels were represented as the mean of triplicate samples for each specimen, measured in ng / ml. To measure production of IL4I1 by BMMCs, these cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol / liter L-Glutamine, 1% penicillin / streptomycin (2 x 105in 300 pl) and incubated at 37°C in 5% CO2 incubator. After a period of 24-72 hours, the supernatant was collected and analyzed for IL4I1 levels using the human IL4I1 ELISA kit.

[0259] To assess the plasma levels of IL4I1 in mice, blood samples were obtained either from the cheek vein or directly from the heart (during euthanasia) of both healthy and tumor- afflicted mice. Following centrifugation, plasma was isolated and examined for IL4I1 concentrations. A mouse-specific ELISA kit (MyBioresource # MBS1605213) was employed for measuring mouse IL4I1 levels.Immunohistochemistry of Cytospin slides

[0260] To analyze cellular IL4I1, BMMCs were incubated with Brefeldin A (BFA) solution, an inhibitor of protein secretion, overnight in a complete medium (RPMU640, enriched with 10% fetal bovine serum, 2 mM / L L-glutamine, and 1% penicillin / streptomycin). The culture was maintained at 37°C in a 5% CO2 incubator. The cells were collected, fixed with 2% paraformaldehyde, and rinsed twice with PBS before being suspended in a 500 pl solution (105 cells / ml). These cells were then transferred to a silane-coated glass slide (Millipore- Sigma,USA) using a Cytospin (Cytospin 4, Thermo Fischer, USA) at 1000 RPM for 5 minutes. After inactivation of endogenous peroxidase with 3% H2O2 for 5 min and blocking with blocking with 3% BSA for 30 min, the samples were incubated with an anti-IL4Il primary antibody overnight at 4°C (rabbit polyclonal, 1 :200 dilution; ThermoFisher Scientific # PA5-115418). This was followed by a 2-hour incubation at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1 :200 dilution; SeraCare # 5220-0283). The binding of the primary antibody was visualized using 3-amino-9-ethylcarbazole (AEC) substrate solution (Vector Laboratories, CA, USA #SK- 4200). Finally, the sections were counterstained with Mayer’s hematoxylin and mounted using Epredia™ Aqua-Mount Mounting Medium (Epredia #13800). IL4I-1 positive cells were counted with QuPath version 0.5.1 using brightfield image analysis with OD image detection and cytoplasm max OD intensity threshold (Bankhead, P. et al. Reports 2017)Measurement of IL4I1 production in patient-derived xenograft in SCID mice

[0261] Male CB17-SCID mice, aged four-six weeks, were procured from Charles River (Wilmington, MA) for all human MM xenograft studies. These mice were housed in a specific pathogen-free zone within our animal resources facility. All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee. In brief, SCID mice were anesthetized with isoflurane before surgery. A fresh MM tumor, called LAG-K2, measuring 20 to 40 mm3 was implanted into the superficial gluteal muscle of the left hind limb. The tumors were allowed to grow for the specified time points. Tumor sizes were measured, and volumes (mm3) were calculated using the formula: length x width2x 0.5. Blood samples were taken before tumor implantation, at the midpoint (days 15 and 30 postimplantation), and at the end of the study (day 42 post-implantation). These samples were analyzed for human IL4I1 and mouse IL4I1 in mouse plasma using ELISA kits as previously described.Gene expression analysis using qRT-PCR

[0262] BMMCs from patients were cultured overnight. Following harvesting, total RNA was extracted from these cells using the Invitrogen PureLink RNA mini kit (#12183018A). The concentrations of RNA were determined using a nanodrop spectrometer (ThermoSci entific). For each sample, approximately 1 pg of RNA was used for the synthesis of complementary DNA (cDNA) using the RT Kit (Applied Biosystems #4440040). A qPCR reaction was then set up using the TaqMan Universal Master Mix (ThermoScientific) and primers specific to the gene of interest (Assays: Hs00169233_ml AHR FAM, Hs00541746_ml IL4I1 FAM, HsOl 101944_slACTBL2 VIC), following the standard protocol. After 50 cycles of DNA amplification on a thermal cycler (AB Applied Biosystems: StepOnePlus), gene expression analysis was performed. The data are presented as 2AACT.Designing of small interfering RNA (siRNA) for blocking IL4I1 expression

[0263] To block expression of IL4I1 expression, we designed and synthesized the siRNAs presented in Table 4. All sequences were constructed in 5' to 3' directions. siRNA transfection and assessment of cell proliferation in vitro

[0264] BMMCs from MM patients or the MM cell line U266 were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM / L L-Glutamine, and 1% penicillin / streptomycin at 37°C in 5% CO2 incubator. For the survival assay, cells were seeded in 96-well plates at a density of 1 X 105 cells / 100 pl per well. After an overnight incubation, cells were transfected with 5-40 nM siRNA using a lipofectamine RNAi MAX reagent (Invitrogen #13778-075) as per the manufacturer’s instructions. Cell viability was then assessed at 24, 48 and 72 hours using an MTS (3 (4,5 dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay. Also known as the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay, the MTS assay is a homogeneous, colorimetric method used to determine the number of viable cells in proliferation, cytotoxicity, or chemosensitivity assays. Following the addition of 50 pl MTS reagent to each well, the plate was incubated for 1-2 hours. The absorbance of each well was then recorded at 490 nM using a 96-well plate reader. The absorbance was converted into a numeric value that could be normalized to control (non-transfected) cells. Relative IL4I1 gene expression was determined after exposure to the siRNA construct using qRT PCR.Effects of siRNA IL4I1 treatment on MM patient derived xenograft growth in SCID mice

[0265] Male CB17 SCID mice, aged four weeks, were procured from Charles River (Wilmington, MA) for all human MM xenograft studies. These mice were housed in a specific pathogen-free zone within our animal resources facility. All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee. In brief, SCID mice were anesthetized with isoflurane before surgery. A fresh MM tumor (LAGA- I or LAGK-2) measuring 20 to 40 mm3 was implanted into the superficial gluteal muscle of the left hind limb. The tumors were allowed to grow until a visible and measurable tumor was observed in the mouse which took approximately 3 weeks. Tumor sizes were measured, and tumor volumes (mm3) were calculated as follows: length x width 2 x 0.5. Whentumors reached approximately 200 mm3, mice were randomly divided into two groups. A vehicle or anti-IL4I-l siRNA-seql alone or in combination with other four siRNA constructs (siRNA-seq2-5) were injected directly into the tumor. The In vivo-jetPEI transfection reagent (Polyplus Transfection, Illkirch, France) was used as a delivery agent to administer IL4I-1 siRNA intratumorally every 3rd day (2 pg siRNA in 2 pl jetPEI diluted in 50 pL 5% glucose per dose). Tumor sizes were measured every 3rd day. After two weeks, the tumors were harvested, processed for protein and RNA extraction, and stored at -80 °C, or immediately fixed in 10% formalin overnight at 4°C and processed for paraffin embedding and histology analysis.Designing and synthesis of the IL4I1 enzymatic inhibitor compound Compound 301

[0266] A urea derivative compound to bind with oxidase catalytic site of IL4I1 enzyme was designed, called “Compound 301”, and synthesized following 3 steps protocol as described below.

[0267] Three step synthetic schema for Compound 301

[0268] Step l :

[0269] Compound 1 (30 g, 232 mmol, 1 equiv) was added to 10V TFA slowly under cooling. NaBH(OAc)3 (98.6 g, 464 mmol, 2 equiv) was added portionwisely while maintaining internal temperature under 10 C. After stirring at 0°C for 30 min, compound 2 (39.3 g, 232 mmol, 1 equiv) was added portion-wisely. The reaction was allowed to warm up slowly to RT and stirred for 20h. The reaction mixture was slowly added into ice-water (15 V) with stirring, and the crude solid was filtered. The crude solid was dried and extracted with Hex / EA (5:1) two times. Filtrate was combined and concentrated to give a compound 3 as a solid (36 g).1 2 3

[0270] Step 2:

[0271] Compound 3 from previous step (50 g, 177.3 mmol, 1 equiv) was dissolved in iPrOH (5V). NH4C1 (47.4 g, 886.5 mmol, 5 equiv) in water (3V) was added. The reactionmixture was heated to 75C internally. Fe powder (29.79 g, 531.9 mmol, 3 equiv) was added portionwisely. The reaction was heated at 8O0C internally for 2h. High-performance liquid chromatography (HPLC) showed slowing down of conversion. To the reaction was added extra 2 equiv Fe powder and was heated at 8O0C for another 2h. HPLC showed completion of reaction. The reaction mixture was diluted with 5 V EtOAc and 2V H2O and then was filtered through celite. The organic phase was separated and concentrated on a rotavap, and the residue was redissolved in EtOAc, dried over MgSO4, and concentrated on a rotavap. The crude solid was then slurried in Hex / MTBE (15: 1, 4V) and filtered to give a crude compound 4 as a solid

[0272] Step 3:

[0273] Compound 4 (102 g, 404.8 mmol, 1 equiv) from the previous step was mixed with CDI (85.2 g, 526.2 mmol, 1.3 equiv) in THF (10 V). The reaction was heated for 3 h at 65 C.The reaction mixture was concentrated and added EtOAc to a slurry, which was stirred for 30 min. The solid was filtered and dried in vacuum oven overnight (crop 1, 67 g >99A%). The mother liquor was washed with 2N HC1 x 2 (3 equiv in total), and then organic phase was dried over MgSO4 and concentrated to a semi solid, to which was added ACN to a slurry, and filtered to get a solid, which was dried in vacuum oven overnight (crop 2, 25 g, >98A%).Measurement of IL4I1 L-amino acid oxidase (LAAO) activity

[0274] A cell lytic method for suspension culture was used to measure LAAO activity.Cells were cultured at the desired density in 80 pl of RPMI media (MM1.S: 3 x 105 or BMMCs: 1 x 106 cells) in a white 96-well plate with 20 mM phenylalanine (Millipore-Sigma) as the IL4I1 substrate and increasing concentrations of Compound 301. The release of H2O2 was thenmeasured using the ROS-Glo H2O2 Assay Kit (Promega). Specifically, after incubating the plate in a CO2 incubator for 20 hours, 20 pl of H2O2 substrate solution (from the kit) was added and gently mixed by pipetting, bringing the final volume to 100 pl per well. The plate was incubated for another 4 hours to observe the effects of Compound 301 over a total of 24 hours. Finally, 100 pl of ROS-Glo detection solution was added, and the plate's chemiluminescence was immediately recorded at 25°C using a fluorescent plate reader.

[0275] A chemical method was used to evaluate IL4I1 enzymatic kinetics. Serial dilutions of Compound 301 (0, 0.025, 0.5, 1, 2, 3, 4, and 5 pM) were incubated with 0.1 pg of human recombinant IL4I1 (R&D #5684-AO), 1 mM phenylalanine (enzyme substrate; Sigma, Catalog #P2126), 1 unit / ml horseradish peroxidase (HRP; Sigma, Catalog #P8375), and 50 pM Amplex Ultra Red (AUR; Molecular Probes, Catalog #A36006) in a total of 100 pl reaction buffer (50 mM Sodium Phosphate, pH 7.0) in a black 96-well plate. The plate was read at excitation and emission wavelengths of 544 nm and 590 nm, respectively, in kinetic mode for 5 minutes using a fluorescent plate reader (BMG LAB TECH). Specific enzyme activity (pmol / min / pg) was calculated using the formula: Adjusted Vmax [Relative Fluorescence Unit (RFU) / min)] x Conversion Factor (pmol / RFU) / amount of enzyme (pg). To calculate the conversion factor, a fluorescent standard was prepared by incubating 50 pM AUR, 1 unit / ml HRP, 1 mM phenylalanine, and a range of hydrogen peroxide (0-500 pmol, Sigma, Catalog #H1009) in Assay Buffer. The resulting amounts of oxidized AUR were used to determine the conversion factor.Treatment of human MM xenografts with Compound 301 drug

[0276] Compound 301 stock was prepared in DMSO at a concentration of 400 mg / ml. Patient-derived xenografts LAGK-2 or LAGk- l were implanted and grown in SCID mice as described previously for the IL4I1 siRNA treatment studies. For cell line-derived xenografts, 1 x 107 MM1.S cells were injected subcutaneously into SCID mice. When the tumors reached a measurable volume (-150 mm3), the mice were randomly divided into two groups and administered orally either 25 mg / kg of Compound 301 in DMSO or vehicle (50 pl volume / dose, twice daily, via gavage). Tumor sizes were measured daily, and tumor volumes (mm3) were calculated as follows: length x width2x 0.5. After 2-4 weeks, depending on tumor growth to a permissible volume without ulceration, the mice were sacrificed, and tumors along with all other organs were harvested for weight measurement and further analysis.Statistical Analysis

[0277] The relationships between IL4I1, baseline characteristics, and patient survival (PFS and OS) were analyzed using Kaplan-Meier analysis and log-rank tests. Statistical differences between groups were determined using a two-tailed Student’s t-test. For multiple group comparisons, one-way or two-way ANOVA with Bonferroni’s correction was used. Univariate analysis was performed with Cox proportional hazard models. A p-value of less than 0.05 was considered statistically significant and data were presented as mean ± SD or SEM. GraphPad Prism 8 software was utilized for graph creation and statistical analysis.Example 1: Baseline serum IL4I1 levels are elevated in MM patients and correlate with clinical outcomes

[0278] Serum IL4I1 levels were determined for a total of 182 MM patients (106 newly diagnosed and 76 RRMM) prior to starting treatment, along with 50 premalignant SMM patients. To establish normal IL4I1 levels in humans, serum IL4I1 levels were determined for 50 healthy individuals (without MM). The demographic and clinical characteristics of the MM patients are detailed in Table 1 below.Table 1 : MM patient characteristics

[0279] The median age was 65.5 years for the MM group, with males comprising 56% of the patient population. ELISA was used to determine the serum levels of IL4I1 as described inMaterials and Methods section above. As shown in FIG. 1A, as the disease progressed, baseline serum IL4I1 levels significantly increased, with a median of 1.16 ng / ml in healthy individuals, 2.717 ng / ml in SMM patients, and 4.621 ng / ml in active MM patients. As shown in FIG. IB, further analysis of the MM cohort revealed that the median IL4I1 level was significantly higher (p < 0.0001) in RRMM patients (5.92 ng / ml) compared to newly diagnosed MM patients.

[0280] The relationship between baseline serum IL4I1 levels and patient survival was investigated next. As shown in FIG. 2A, patients with IL4I1 levels above the median had a significantly shorter PFS of 7.8 months compared to 22.77 months for those below the median (p < 0.0001). When patients were divided into quartiles based on their IL4I1 levels, those in the highest quartile (> 8.34 ng / mL; n = 45) had a significantly shorter median PFS of 4.64 months, compared to 21.85 months for the other three quartiles, as shown in FIG. 2B (p < 0.0001; n=137). Also, as shown in FIG. 2C, patients with IL4I1 levels above the median had a shorter OS of 50.83 months, versus 81.48 months for those below the median (p = 0.009). Lastly, as shown in FIG. 2D, the OS for patients in the highest quartile was also significantly shorter at 21.21 months, compared to 70.44 months for those in the lower three quartiles (p = 0.0022).

[0281] Given baseline serum IL4I1 levels were significantly different between newly diagnosed and RRMM patients, newly diagnosed and RRMM patients were separately analyzed in terms of serum baseline IL4I1 levels and their survival. In the RRMM cohort, those in the lowest quartile (n=19) with a median IL4I1 level of 2.55 ng / ml showed a PFS 8.745 months as compared to 2.04 months among those in the highest quartile with a median IL4I1 level of 9.4 ng / ml, as shown in FIG. 3A (p = 0.02; n=19). Similarly, the OS was also significantly longer in the lowest quartile than the highest quartile, as shown in FIG. 3B (32.94 months vs. 11.24 months; p = 0.019). Among frontline patients, those in the lowest quartile (n=26) with a median serum IL4I1 level of 1.281 ng / ml had a 65.08 months PFS as compared to 27.1 months in the highest quartile (n=26) with the median IL4I1 level of 6.526 ng / ml, as shown in FIG. 3C (p = 0.14). The OS for the lowest and highest quartiles in the frontline setting was 81.4 months compared to undefined, as shown in FIG. 3D (p = 0.625). The differences in PFS and OS were not statistically significant for frontline patients, possibly due to the fact that a majority of frontline patients responded to treatment, resulting in a low number showing PD or death. Collectively, these findings demonstrate that baseline IL4I1 levels in previously untreated patients could influence the length of their response to treatment and OS.Example 2: Serum IL4I1 levels correlate with a newly diagnosed patient’s current clinical status

[0282] To identify the potential link between IL4I1 and the clinical status of the patient’s MM, IL4I1 expression was assessed in two distinct patient groups: those who were in CR and those with PD according to the IMWG Uniform Response Criteria. Each group consisted of 50 patients, and their demographics characteristics are shown in Table 2 below.Table 2: Demographic Characteristics of MM Patients for the PD vs CR Comparison Studies

[0283] The median ages of the CR and PD groups were 69 and 70 years, respectively, with 61% males (58% in the PD group and 64% in the CR group). As shown in FIG. 4, the serum IL4I1 level in the PD group was significantly higher (median 6.62 ng / ml) compared to those in CR (median 3.00 ng / ml; p<0.0001).Example 3: Serum IL4I1 levels correlates with changes in RRMM patients’ clinical status and other conventional serum MM biomarkers

[0284] To evaluate whether serum IL4I1 levels can monitor the clinical status of MM patients, 14 RRMM patients were tracked longitudinally from the start of treatment (Tl), through their best response (T2 and T3), to the end of treatment (T4). At T4, the disease progressed in half of the patients (n=7) and remained stable in the other half (n=7). Serum IL4I1 levels were measured from Tl to T4 in all patients. As shown in FIGs. 5A-5H and FIGs. 29A- 29L, the levels of IL4I1 decreased and stayed low in patients without disease progression, while it increased again among those with PD. In contrast to patients who responded to therapy, the level of serum IL4I1 was consistently higher in non-responder RRMM patients who did not show a response to therapy. In other words, there were no significant changes in serum IL4I1 levels when patients had standard disease during treatment, as shown in FIGs. 5I-5L. These studies show that changes in serum IL4I1 levels can reflect the clinical status of the disease.

[0285] Given that sBCMA levels have been shown to predict the clinical status and outcomes of MM patients, with higher levels indicating worse outcomes, correlation between baseline IL4I1 levels and sBCMA levels among MM patients was investigated. As shown in FIGs. 6A-6C, the changes in IL4I1 levels across quartiles parallel the changes in sBCMAlevels. For example, MM patients in the highest quartile (n=45) with a median IL4I1 level of 8.342 ng / ml had significantly higher sBCMA levels (1152 ng / ml) compared to the remaining three quartiles (n=137) with a median IL4I1 level of 3 ng / ml, as shown in FIG. 6A (418 ng / ml; p<0.0001). Similarly, IL4I1 levels correlated with sBCMA and other conventional serum MM markers, such as serum monoclonal (M) protein and serum free light chain (sFLC), in RRMM patients over the course of their disease (T1-T4), as shown in FIGs. 5A-5H, 6A-6C, and 29A- 29L. These results showed that changes in sBCMA, M protein, and sFLC closely matched changes in serum IL4I1 levels during their course of disease. This data demonstrate a strong association between IL4I1 levels and current MM disease status, supporting the use of IL4I1 as a biomarker for monitoring disease progression.Example 4: Bone marrow from patients in PD shows higher supernatant levels and protein and gene expression of IL4I1 than those in CR

[0286] To measure production of IL4I1 by BMMCs from MM patients in both CR and PD, these cells were cultured in complete medium (RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol / liter L-Glutamine, and 1% penicillin / streptomycin) and incubated at 37°C in 5% CO2 incubator. After 24-48 hours, the supernatant was collected and analyzed for IL4I1 levels using the above ELISA kit. As shown in FIG. 7A, the supernatant from PD patients showed 6-7 times more IL4I1 (1.7 and 2.4 ng / ml at 24 and 48 hours, respectively) compared to the amount in CR patients (0.22 and 0.38 ng / ml, respectively).

[0287] To determine whether BMMCs from MM patients in CR showed different amounts of IL4I1 protein, IL4I1 staining was evaluated on slides using Cytospin following immunohistochemical staining (H4C). BMMCs were cultured in complete medium in the presence of Brefeldin A, an inhibitor of protein secretion, for 12 hours. The culture was maintained at 37°C in a 5% CO2 incubator. The cells were collected, fixed with 2% paraformaldehyde, and rinsed twice with PBS before being suspended in a 500 pl solution (105cells / ml). These cells were then transferred to a silane-coated glass slide (Millipore-Sigma, USA) using a Cytospin (Cytospin 4, Thermo Fischer, USA) at 1000 RPM for 5 minutes. After inactivation of endogenous peroxidase with 3% H2O2 for 5 min and blocking with blocking with 3% BSA for 30 min, the samples were incubated with an anti-IL4Il primary antibody overnight at 4°C (rabbit polyclonal, 1 :200 dilution; ThermoFisher Scientific # PA5-115418). This was followed by a 2-hour incubation at room temperature with horseradish peroxidase (HRP)- conjugated goat anti-rabbit IgG (1 :200 dilution; SeraCare # 5220-0283). The binding of theprimary antibody was visualized using 3-amino-9-ethylcarbazole (AEC) substrate solution (Vector Laboratories, CA, USA #SK- 4200). Finally, the sections were counterstained with Mayer’s hematoxylin and mounted using Epredia™ Aqua-Mount Mounting Medium (Epredia #13800). IL4I1 -positive cells were counted with QuPath version 0.5.1 using brightfield image analysis with OD image detection and cytoplasm max OD intensity threshold (Bankhead, P. et al. Reports 2017).

[0288] As shown in FIG. 7B and FIG. 8, IL4I1 staining of BMMCs by IHC revealed that 100% of BM samples from PD patients were positive for IL4I1 protein expression. In contrast, all CR patient samples did not show detectable IL4I1 protein.Table 3: Percentage of IL4I1 positive cells in PD patients’ BMMCs

[0289] Specifically, the proportion of IL4I1 -positive cells in PD patient samples ranged from 11% to 68% with a median of 44% IL4I1 -positive cells (Table 3). Further IHC staining and flow cytometric analysis are performed to identify the specific BM cell types producing IL4I1 in these patients.

[0290] Additionally, qRT-PCR analysis was used to determine transcript levels of IL4I1 in CR versus PD BMMCs. As shown in FIG. 9, PD BMMCs expressed 40-90 times more IL4I1 mRNA than CR BMMCs. Together, the data demonstrate a positive correlation between the clinical status of the MM patient and IL4I1 production from their BM.Example 5: IL4I1 is directly released by multiple myeloma tumor cells

[0291] To determine if serum IL4I1 detected in human MM serum is released exclusively from MM tissues and not from other normal tissues and to assess its association with MM growth, human IL4I1 concentrations were measured in the plasma of SCID mice with patient- derived MM xenografts. Six- to eight-week-old male severe combined immunodeficient (SCID) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in a specificpathogen- free area in our animal resources facility. All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee. Animals were anesthetized with ketamine, xylazine, and isoflurane prior to surgery and were euthanized when tumors reached 2 cm in diameter.

[0292] To establish tumor xenografts, BM biopsies were obtained from MM patients. Biopsy tissue was surgically implanted in the hind limbs of anesthetized SCID mice and passaged through succeeding generations. Myeloma tumors (LAGK-1 A or LAGK-2 or LAGk-1) were excised from an anesthetized donor mouse, sectioned into 20 to 40 mm pieces, and surgically implanted into the left superficial gluteal muscle of anesthetized naive SCID mice. Recipient mice received weekly injections of anti-asialo GM1 rabbit serum (Wako Bioproducts, Richmond, VA) to further suppress immune activity.

[0293] To assess the plasma levels of IL4I1 in mice, blood samples were obtained either from the cheek vein or directly from the heart (during euthanasia) of both healthy and tumor- afflicted mice. Following centrifugation, plasma was isolated and examined for IL4I1 concentrations. Depending on the species specificity required, either a human-specific ELISA kit was used for human IL4I1 or a mouse-specific ELISA kit (MyBioresource # MB SI 605213) was employed for mouse IL4I1.

[0294] As shown in FIG. 10A, mouse IL4I1 levels remained consistent regardless of whether the mice had MM xenografts. However, human IL4I1 levels were found to rise to high levels in the plasma of tumor-bearing mice and found at only background levels in mice without MM in all 3 PDX models (FIG. 10B and HA).

[0295] In addition, plasma human IL4I1 levels closely correlated with tumor growth, as shown in FIG. 11B. Together, the data demonstrate that the human MM tumor cells are the source of the increased IL4I1 production and secretion of this protein into the blood.Example 6: Treatment with JAK inhibitors reduces IL4I1 and AHR gene expression

[0296] JAK inhibitors have previously been shown to reduce gene expression of the checkpoint inhibitor proteins PDL-1 and B7 / H3, and checkpoint inhibitor proteins have been shown to induce IL4I1 expression in some tumor types but not MM. Fresh MM BMMCs and the MM cell line U266 were exposed to the JAK inhibitors pacritinib, fedratinib and ruxolitinib to determine whether JAK inhibitors can reduce expression of IL4I1 and its downstream effector AHR. As shown in FIGs. 12A-12D, both pacritinib and fedratinib markedly reduced both IL4I1 and AHR gene expression in BMMCs from two MM patients (BM3570 and BM3819), as measured by qPCR analysis, whereas ruxolitinib had similar but less dramatic effects. Followingtreatment with these three JAK inhibitors, U266 cells also showed some reduction in IL4I1 gene expression whereas AHR gene expression became undetectable, as shown in FIGs. 12E-12F. These results demonstrate the ability of JAK inhibitors to reduce IL4I1 and AHR expression in MM BM. Without being held to theory, the effects are likely attributable to the ability of these kinase inhibitors to block checkpoint inhibitor protein expression, which has been previously demonstrated in MM. These studies provide support for the ability of JAK inhibitors to reduce IL4I1 and its downstream effector AHR expression in MM BM.Example 7: Blocking IL4I1 reduces human MM BMMC proliferation

[0297] To inhibit IL4I1 expression, anti-IL4Il siRNA oligos were created and synthesized. All sequences are constructed in 5' to 3' directions. Exemplary siRNA oligos are shown in Table 4.Table 4: Exemplary anti-IL4Il siRNA oligos

[0298] MM BMMCs or U266 MM cells were seeded in 96-well plates at a density of 1 x 105cells / 100 pL per well. After an overnight incubation, cells were transfected with 5-40 nM siRNA using a lipof ectamine RNAi MAX reagent (Invitrogen #13778-075) as per the manufacturer’s instructions. An ex vivo culture of BMMCs from a patient with PD was transfected with increasing concentrations of anti-IL4Il siRNA sequence 1 (siRNA-seql), ranging from 5 to 20 nM.

[0299] qRT-PCR analysis for IL4I1 gene expression at intervals of 24-72 hours demonstrated a concentration-dependent silencing of IL4I1 gene expression, with reducedexpression of approximately 45% at 20 nM after 48 hours, as shown in FIG. 13 A. Using U266 MM cells, the maximum inhibition of IL4I1 by siRNA-seql occurred at 40 nM after 48 hours, resulting in more than 50% gene silencing, as shown in FIG. 13B. Both 20 and 40 nM concentrations were subsequently used in further experiments.

[0300] To evaluate the impact of blocking IL4I1 expression on MM cell proliferation, PD and CR BMMCs were transfected with 20 nM and 40nM siRNA-seql and cell proliferation was measured using an MTS assay at 24, 48, and 72 hours. The survival rate of PD BMMCs significantly decreased with siRNA at both 20 nM and 40 nM: 92%, 68.5%, and 58.6% for 20 nM at 24, 48, and 72 hours respectively, and 80.6%, 44.5%, and 38.2% for 40 nM at 24, 48, and 72 hours, respectively. Compared to the non-transfected controls, there was a 41.56% and 61.78% reduction in viability after 72 hours, as shown in FIG. 14A.

[0301] A similar experiment was conducted using CR BMMCs as a control and using the highest concentration of siRNA-seql (40 nM). Importantly, the effect of IL4I1 blocking on CR BMMCs was minimal after 72 hours of culture, with more than 80% cell survival compared to only 38% with PD BMMCs, as shown in FIG. 14B. These results show that IL4I1 blocking is selective for MM cells, demonstrating higher antiproliferative effects on PD BMMCs with >50% malignant plasma cells than in BMMCs derived from a patient in CR. These experiments were replicated with U266 cells, an established aggressive MM cell line. Similar to PD BMMCs, transfection of U266 cells with siRNA-seql significantly reduced the proliferation of the cells, with approximately a 50% reduction at 72 hours, as shown in FIG. 14C. These in vitro data demonstrate that siRNA-mediated inhibition of IL4I1 results in dose- and timedependent killing of myeloma cells.

[0302] Together, these data demonstrate that inhibiting IL4I1 is effective in reducing human myeloma cell viability and has potential as a treatment for this B-cell malignancy.Example 8: IL4I1 blocking reduces growth of MM patient-derived xenograft in SCID mice

[0303] The MM patient-derived xenograft LAGk-1 in SCID mice was utilized to examine the anti-MM effect of siRNA-seql in vivo. Tumors were intramuscularly implanted as outlined in Materials and Methods above. Once the mice developed palpable tumors, they were divided into two groups. For blocking IL4I1, one group received intratumoral treatment with siRNA- seql for two weeks (2 pg / injection, every 3rd day), while the other group received placebo. The experimental plan is depicted in FIG. 15A. Tumor volumes were recorded twice a week. To verify the blocking of IL4I1, blood was collected from the mice intracardially during euthanasia the day after the final intratumor siRNA injection (on day 16) and plasma samples wereanalyzed for IL4I1 levels. Treatment with siRNA-seql significantly reduced the levels of IL4I1 in the plasma, indicating the efficiency of siRNA-seql in diminishing IL4I1 expression, as shown in FIG. 15B. Additionally, treatment with siRNA-seql significantly reduced the growth of LAG I tumors compared to the control group, as shown in FIG. 15C. The final tumor volume was 3,501 mm3in the siRNA treated group versus 5,673 mm3in the control group, as shown in FIG. 15D (p<0.001).

[0304] To achieve maximum IL4I1 silencing, a combination of five different siRNA constructs (seql-5) targeting the IL4I1 gene was assessed. Following transfection, a 70-80% inhibition of IL4I1 gene expression was achieved in the U266 MM cell line and BMMCs, as shown in FIGs. 16A-B). This siRNA combination was then assessed in another MM patient- derived xenograft (PDX) model, LAGK-2. AS depicted in FIG. 17A, when tumors became visible after implantation in the hind limbs of SCID mice, the IL4I1 siRNA combination (seql- 5) was injected intratumorally (0.4 pg of each siRNA, totaling 2 pg per injection, every third day), and tumor growth was monitored until 34 days post-treatment. The growth of LAGK-2 xenografts was reduced throughout the study, with a final tumor volume of 2,216.78 mm3in the siRNA-treated group compared to 4,258.76 mm3in the sham group at day 34, as shown in FIGs. 17B-C. Collectively, the in vivo data from both LAGX- I and LAGK-2 xenograft models demonstrate that blocking IL4I1 is a promising strategy for treating MM.In conclusion, both in vitro and in vivo data demonstrate that IL4I1 blocking is a promising strategy for treating MM. Further preclinical studies with escalating doses and different schedules of administration will be conducted to determine the optimal dose and schedule of siRNAs to maximize the anti -MM effect of blocking IL4I1 expression.Example 9: A novel IL4I1 inhibitor suppresses MM growth

[0305] A chemical compound, Compound 301, was synthesized and developed as a small molecule drug to inhibit IL4I1 enzymatic activity, as shown in FIG. 18. The synthesis involved a three-step process to create a urea derivative that binds IL4I1 and blocks its catalytic activity. At each step, the intermediate compounds were verified for yield and desired structure using 1H NMR and MS analysis. The detailed protocol is described in the Materials and Methods section above. The final compound was thoroughly characterized for purity and structural configuration using HPLC, Liquid chromatography-mass spectrometry (LCMS), and proton nuclear magnetic resonance (1H NMR), as shown in FIGs. 19-21). These analyses confirmed the compound's high purity of >99% (FIG. 19), desired molecular weight of 279 (FIG. 20), and structural configuration (FIG. 21). Finally, the solubility of Compound 301 was evaluated in varioussolvents, including DMSO, Tween-80, and various ratios (v / v) of the solvents in water and PBS. Table 5 below provides the results of the solubility analysis. The compound demonstrated solubility up to 404 mg / ml in DMSO.Table 5: Solubility analysis of Compound 301 in different solvent and pH,

[0306] The effect of Compound 301 on BMMCs was examined in a concentration and time dependent manner. Specifically, BMMCs from either PD or CR patients were cultured ex vivo and treated with increasing concentrations of Compound 301, and cell proliferation was determined at 24, 48, and 72 hours. At 10, 20 and 40 pM concentrations, a significantly higher cell death (20-40%) was observed in PD BMMCs (FIG. 22A), while no significant effect was found in CR BMMCs (FIG. 22B), indicating that Compound 301 had a selective effect on BMMCs with a higher percentage of plasma cells. A similar effect of Compound 301 was observed on the MM1.S cell line and LAGX-1 xenograft single cell cultures, with a 30 to 40 percent reduction in cell proliferation at 40 pM concentration, as shown in FIGs. 22C and 22D.

[0307] To verify whether Compound 301 blocks IL4I1 enzymatic activity, the inventor developed a cellular assay to detect hydrogen peroxide (H2O2) release, a byproduct of ZL4H's catalytic activity. FIG. 23A depicts the chemical reaction catalyzed by IL4I1. We cultured IxlO6cells, either BMMCs or the MM1.S cell line, with the IL4I1 substrate L-phenylalanine and treated them with increasing concentrations of Compound 301. The H2O2 released into the supernatant was measured using the Promega ROS-Glo H2O2 Assay. In both BMMCs and MM1.S cells, Compound 301 treatment significantly reduced H2O2 production in a concentration-dependent manner, as shown in FIGs. 23B and 23C.

[0308] A chemical method using human recombinant IL4I1 was used to assess the effectiveness of the Compound 301 compound in inhibiting enzyme kinetics, as described in the methods section above. Consistent with the cellular lytic assay, the enzyme kinetic assay demonstrated that Compound 301 effectively blocked enzyme activity, with an IC50 value of 0.0887 pM. as shown in FIG. 24. These results indicate that Compound 301 specifically inhibits ILdll's catalytic activity.

[0309] To evaluate the anti -MM activity of Compound 301 in vivo, PDX LAGK-2 cells were implanted in SCID mice. Once the mice developed palpable tumors, they were randomized and treated orally with 25 mg / kg of Compound 301 twice daily via oral gavage, while the control group received a placebo. The experimental plan is illustrated in FIG. 25A. Treatment with Compound 301 significantly reduced the growth of LAGK-2 tumors compared to the control group, with final tumor volumes of 549.1 mm3in the Compound 301 treated group versus 1,968 mm3in the control group, as shown in FIGs. 25B-D (n=8-9 / group, p < 0.0001). At the end of the study, weight measurements were recorded for the tumors and organs from the mice. Consistent with the tumor volumes, the tumor weight was significantly lower in the Compound 301 treated group compared to the control group, as shown in FIG. 25E. The body weights of the animals, which were monitored throughout the study, showed no significant difference between the Compound 301 -treated and placebo groups. As shown in FIG. 26A and 26B, the average final body weight was 22.7 grams in the control group and 19.6 grams in the Compound 301-treated group (n=5-6 / group). Additionally, there were no differences in the weights of organs, including the liver, lung, kidney, spleen, heart, and brain, as shown in FIGs. 26C-26H (n=5-6 / group).

[0310] To ensure rigor, the study was repeated with two additional MM models: LAGk- l PDX and MM1.S cell line-derived xenografts. Both of these xenografts grow faster compared to LAGK-2 tumors, and the study was conducted for approximately two weeks from the start ofdrug treatment. LAGX- I PDX was implanted, grown, and treated similarly to LAGK-2 tumors, as illustrated in FIG. 27A. Consistent with the results for LAGK-2, Compound 301 treatment significantly suppressed the growth of LAGX- I tumors compared to the control group, with final tumor volumes of 1,053.7 mm3in the Compound 301 treated group versus 1,898.5 mm3in the control group (n=7 / group, p < 0.001, FIG. 27B-D). Additionally, there was no significant difference in body and organ weights at the end of the study (FIG. 27F-M).

[0311] To establish MM1.S cell line-derived xenografts, 10 million MM1.S cells were injected subcutaneously into SCID mice, leading to tumor growth within 10 days. The mice were then treated with Compound 301 for 16 days, similar to the PDX tumors, as depicted in FIG. 28A. Consistent with the PDX tumors, Compound 301 treatment significantly reduced the growth of MM1.S tumors compared to the placebo group (tumor volume: 745 mm3versus 2,105 mm3) (n=12-14 / group, p < 0.01, FIG. 28B-D). Again, the body weights and organ weights of Compound 301 treated mice were comparable to those of the placebo group (FIG. 28E-28L). Thus, preclinical data from BMMCs ex vivo and in vivo from patient-derived xenografts and MM cell line-derived xenografts suggest the potential use of Compound 301 for MM treatment, with no adverse effects on body weight and organ weights. Further preclinical studies with escalating doses and varied time courses will be necessary to determine the optimal dose to maximize Compound 301's anti -MM effect.Example 10: Synthesis and testing anti-myeloma effect of 18 new Compound 301 analogs.

[0312] As depicted in FIGs. 31A -E, seventeen analogs of the Compound 301 were synthesized alongside Compound 301 using the chemical reactions shown in FIGs. 32A-R. At each step, the structure and molecular weight of the desired intermediate and final compounds were verified using NMR and LC-MS analysis. The detailed protocol, including the steps for converting one chemical compound to another, their reaction conditions, and the generation of Compound 301 and Compound 301 analogs, is described in Table 6. In Table 6, RT denotes Room Temperature; NaBH(OAc)3 denotes Sodium triacetoxyborohydride; TFA denotes Trifluoroacetic acid; CDI denotes Carbonyldiimidazole; Pd / c denotes palladium on carbon; Mel denotes methyl iodide; DMF denotes dimethylformamide, THF denotes Tetrahydrofuran, eq denotes Equivalent; and DIPEA denotes N,N-Diisopropylethylamine.Table 6: Chemical synthesis of Compound 301 analogs. Structural chemical reactions for all compounds have been shown in Figure 32,

[0313] To determine the effectiveness of these analog compounds, ex vivo single cell cultures of LAGX- I xenograft were treated in a concentration-dependent manner for 72 hours, and cell death was determined using the MTS assay. In addition to Compound 301, several other compounds were found to be highly effective for anti-myeloma effects, with IC30 values of 24.22 pM, 27.3 pM, 30.62 pM, 33.31 pM, 34.26 pM, 36.16 pM, 40.59 pM, and 42 pM for Compound 315, Compound 301, Compound 307, Compound 305, Compound 304, Compound 302, Compound 306, and Compound 322 compounds, respectively, as shown in FIGs. 33A-C and FIG. 34. Other compounds were less effective than Compound 301, including Compound 309, Compound 325, Compound 324, Compound 321, Compound 303, Compound 320, Compound 319, Compound 317, and Compound 323.

[0314] As described in the methods above and in FIG. 24, a chemical method using human recombinant IL4I1 was employed to assess the effectiveness of the analogs of Compound 301 in inhibiting IL4I1 enzymatic activity. Consistent with their anti-myeloma effects, many of these analogs are highly effective in blocking IL4I1 enzyme activity, as shown in FIGs. 35A-E. For example, the IC50 values for Compound 301, Compound 315, Compound 306, Compound 322, Compound 305, and Compound 307 were 0.057 nM, 0.058 nM, 0.067 nM, 0.070 nM, 0.080 nM, and 0.110 nM, respectively, as shown in FIG. 36.

[0315] Next, the effectiveness of these six analogs was analyzed on myeloma patient bone marrow containing approximately 15% plasma cells (FIG. 37A). Similar to the xenograft experiments, these compounds were effective in reducing the growth of patient tumor cells ex vivo. Notably, Compound 315, Compound 306, and Compound 307 were 1.5 times more effective than Compound 301. For instance, IC30 values for Compound 315, Compound 307, and Compound 306 were 17-18 pM, compared to 25 pM for Compound 301. Importantly, these three selected compounds were significantly more effective than some well-known anti-myeloma clinical drugs, with IC30 values of 35.58 pM for the immunomodulatory agents lenalidomide and 78.52 pM for pomalidomide on this myeloma patient’s bone marrow (FIG. 37B). IC30 values for Compound 301 and six selected analogs are provided in Table 7 below.Table 7: IC30 of Compound 301 and analogs

[0316] For further comparison, the effect of Compound 315 was evaluated alongside standard anti -myeloma clinical drugs on LAG I PDX cells ex vivo. Compound 315 was significantly better at suppressing LAGX-1 cell growth, with an IC30 value of 24.22 pM compared to 75.37 pM, 63.17 pM, 44.89 pM, and 9.59 mM for lenalidomide, pomalidomide, dexamethasone, and cyclophosphamide, respectively (FIG. 38). These in vitro and ex vivo studies from myeloma patient bone marrow and xenograft studies demonstrate that some IL4I1 blockers, including Compound 301, Compound 307, and Compound 315 are highly effective for myeloma suppression and show anti-myeloma effects at lower concentrations than standard antimyeloma drugs.

[0317] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0318] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference wasspecifi cally and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0319] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSI / We claim:

1. A composition comprising an inhibitor of interleukin-4 induced gene 1 (IL4I1) and optionally a pharmaceutically acceptable excipient, wherein the IL4I1 inhibitor inhibits IL4I1 expression or function.

2. The composition of claim 1, wherein the IL4I1 inhibitor comprises a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense oligonucleotide (ASO), or a ribozyme that inhibits IL4I1 gene expression .

3. The composition of claim 1 or 2, wherein the IL4I1 inhibitor comprises an siRNA.

4. The composition of claim 3, wherein the siRNA comprises: a. an antisense strand comprising CUUUAAUGCGAGGUCCUCGtg (SEQ ID NO:3) and a sense strand comprising CGAGGACCUCGCAUUAAAGtt (SEQ ID NO:4); b. an antisense strand comprising CUCUCAACCAGGCCCUCAA[dT][dT] (SEQ ID NO: 5) and a sense strand comprising UUGAGGGCCUGGUUGAGAG[dT][dT] (SEQ ID NO: 6); c. an antisense strand comprising CCAAGUUCACCCAGUACGA[dT][dT] (SEQID NO: 7) and a sense strand comprisingUCGUACUGGGUGAACUUGG[dT][dT] (SEQ ID NO: 8); d. an antisense strand comprising GUCCCUUAUGGCCGCAUCU[dT][dT] (SEQID NO: 9) and a sense strand comprisingAGAUGCGGCCAUAAGGGAC[dT][dT] (SEQ ID NO: 10); or e. an antisense strand comprising CAUUGAAGGCGGCCACUCA[dT][dT] (SEQID NO: 11) and a sense strand comprisingUGAGUGGCCGCCUUCAAUG[dT][dT] (SEQ ID NO: 12); and wherein the antisense strand comprises a sequence that is a reverse complement of at least a region of a IL4I1 mRNA transcript.

5. The composition of claim 1, wherein the IL4I1 inhibitor comprises an antibody or an antigen-binding fragment thereof that binds IL4I1.

6. The composition of claim 1, wherein the IL4I1 inhibitor comprises a compound of formula(I), or a pharmaceutically acceptable salt thereof,each of n and m is independently 0, 1, 2 or 3, each of R1and R2is independently halogen, Ci-6 alkyl, OCi-6 alkyl or Ci-6 haloalkyl;R3is H, Ci-6 alkyl or C3-6 cycloalkyl; andR4is H or oxo (=0).

7. The composition of claim 1, wherein the IL4I1 inhibitor comprises a compound of formula(II), or a pharmaceutically acceptable salt thereof,each of n and m is independently 0, 1, 2 or 3, each of R1and R2is independently halogen, C1-6 alkyl, OC1-6 alkyl or C1-6 haloalkyl;R3is H, C1-6 alkyl, OC1-6 alkyl or C3-6 cycloalkyl; andR5is heterocyclyl, NH2, NH(CI-6 alkyl) or N(CI-6 alkyl)2.

8. The composition of claim 6 or 7, wherein n is 1.

9. The composition of any one of claims 6-8, wherein R1is halogen, C1-3 alkyl, OC1-3 alkyl or C1-3 haloalkyl.

10. The composition of claim 9, wherein R1is halogen or C1-3 haloalkyl.

11. The composition of claim 10, wherein R1is F or CF3.

12. The composition of claim 11, wherein R1is F.

13. The composition of any one of claims 6-12, wherein R2is halogen or C1-3 haloalkyl.

14. The composition of claim 13, wherein R2is F, Cl, or CF3.

15. The composition of any one of claims 6-14, wherein R3is H or C1-4 alkyl.

16. The composition of claim 15, wherein R3is H, CH3, CH2CH3 or CH(CH3)2.

17. The composition of claim 16, wherein R3is H.

18. The composition of any one of claims 6 or 8-14, wherein R4is H.

19. The composition of any one of claims 7 or 8-17, wherein R5is 5-6 membered N- heterocyclyl or N(Ci-6 alkyl)2.

20. The composition of claim 19, wherein R is21. The composition of any one of claims 6 - 20, wherein the compound of formula (I) or formula (II) binds to the oxidase catalytic site of IL4I1.

22. The composition of claim 1, wherein the IL4I1 inhibitor comprises any one of the compounds in Table A, or a pharmaceutically acceptable salt thereof.

23. The composition of any one of claims 1-22, wherein the IL4I1 inhibitor binds to the oxidase catalytic site of IL4I1.

24. A method of synthesizing a urea derivative compound, wherein the urea derivative compound binds to the oxidase catalytic site of IL4I1, comprising: a. contacting Compound 1 with TFA to form a mixture;(Compound 1)b. adding NaBH(OAc)s after step (a); c. adding Compound 2 after step (b);(Compound 2) d. warming up the mixture from step (c) slowly to room temperature; e. adding the mixture from step (d) into ice-cold water to form a crude solid; f. extracting the crude solid with HexZEA (5: 1), thereby forming a filtrate; g. concentrating the filtrate, thereby forming Compound 3 as a solid;(Compound 3) h. dissolving Compound 3 from step (g) in iPrOH to form a solution; i. adding NH4CI to the solution of step (h); j . adding Fe powder to the solution of step (i) to form a mixture; k. diluting the mixture from step (j) with EtOAc and H2O; l. filtering the diluted mixture from step (k) through celite; m. separating and concentrating the organic phase from the filtrate from step (1) to form a residue; n. dissolving the residue in EtOAc and drying over MgSO4 to form a crude solid; o. mixing the crude solid with Hex / MTBE to form a mixture; p. filtering the mixture from step (o), thereby forming Compound 4 as a crude solid;(Compound 4) q. mixing Compound 4 from step (p) with CDI in THF to form a mixture; r. concentrating the mixture from step (q) and adding EtOAc to form a slurry; s. filtering the slurry from step (r) to produce a solid and a mother liquid; and t. drying the solid from step (s) to produce said urea derivative compound(urea derivative compound).

25. The method of claim 24, further comprising: u. washing the mother liquid from step (s) with 2N HC1; v. isolating the organic phase from step (u); w. drying the organic phase from step (v) and concentrating to form a semi solid; x. adding ACN to the semi solid of step (w) to produce a mixture; y. filtering the mixture from step (x), thereby forming a solid; and z. drying the solid of step (y) to produce said urea derivative compound.

26. A method of diagnosing multiple myeloma (MM) in a subject, comprising: a. detecting an amount of IL-4 Induced 1 (IL4I1) polypeptide or IL4I1 gene expression in a biological sample obtained from the subject; and b. comparing the amount of IL4I1 polypeptide or gene expression detected in step (a) to a predetermined cut-off value or to an amount detected in a control biological sample, wherein an increased amount of IL4I1 polypeptide or gene expression of (a) as compared to the predetermined cut-off value or amount in the control biological sample of (b) indicates the presence of MM.

27. The method of claim 26, further comprising administering to the subject a treatment for MM if step (b) indicates MM is present, wherein the treatment for MM comprises the composition of any one of claims 1-23.

28. A method of prognosing the survival of a subject having or suspected of having MM, comprising: a. detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject; and b. comparing the amount of IL4I1 polypeptide or gene expression detected in step (a) to a median value of levels of IL4I1 polypeptide or gene expression in a population of subjects being treated for MM,wherein an increased amount of IL4I1 polypeptide or gene expression of (a) as compared to the median value of IL4I1 polypeptide or gene expression of (b) indicates a reduced chance of survival for the subject having MM, and wherein a decreased amount of IL4I1 polypeptide or gene expression of (a) as compared to the median value of IL4I1 polypeptide or gene expression of (b) indicates an increased chance of survival for the subject having MM.

29. The method of claim 28, wherein the subject has been diagnosed with MM.

30. The method of claim 28 or 29, wherein the subject has received treatment for MM.

31. The method of any one of claims 28-30, wherein the subject with an increased amount of IL4I1 polypeptide or gene expression as compared to the median value of IL4I1 polypeptide or gene expression has a 10%, 30%, 50%, or 70% reduction in the chance of survival compared to the survival of a subject having the median level of IL4I1 polypeptide or gene expression.

32. The method of any one of claims 28-30, further comprising removing the subject having MM from a treatment or a maintenance therapy of MM if step (b) indicates an increased chance of survival for the subject.

33. A method of monitoring the progression or response of a subject diagnosed with MM to treatment of MM, comprising: a. detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject at a first time point; b. detecting an amount of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject at a second time point or following treatment; c. comparing the amount detected in step (a) to the amount detected in step (b), wherein an increased amount of IL4I1 polypeptide or gene expression of (b) as compared to the amount of IL4I1 polypeptide or a gene expression of (a) indicates that said MM is progressing, and wherein a decreased amount of IL4I1 polypeptide or a gene expression of (b) as compared to the amount of IL4I1 polypeptide or a gene expression of (a) indicates that said MM is entering remission or responding to treatment.

34. The method of claim 33, further comprising removing the subject from a treatment or a maintenance therapy of MM if step (b) indicates said MM is entering remission or responding to treatment.

35. The method of any one of claims 26-34, wherein the biological sample is a serum sample, a plasma sample, or a sample comprising RNA isolated from a subject’s bone marrow mononuclear cells or peripheral blood mononuclear cells.

36. The method of any one of claims 26-34, wherein the biological sample is supernatant obtained from a cell culture of the subject’s bone marrow mononuclear cells.

37. The method of any one of claims 26-34, wherein the biological sample is supernatant obtained from a cell culture of the subject’s peripheral blood mononuclear cells.

38. The method of any one of claims 26-37, wherein the amount of IL4I1 polypeptide is detected by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, flow cytometry, or strip assay.

39. The method of claim 38, wherein the amount of IL4I1 polypeptide is detected by ELISA.

40. The method of claim 38, wherein the amount of IL4I1 polypeptide is detected by lateral flow assay.

41. The method of any one of claims 26-37, wherein the amount of IL4I1 gene expression is detected by Northern blotting, qPCR, RT-PCR, microarray, or RNA-seq.

42. A kit for detecting, diagnosing, predicting survival, staging, or monitoring MM in a subject, comprising a reagent suitable for determining a level of IL4I1 polypeptide or gene expression in a biological sample obtained from the subject.

43. The kit of claim 42, wherein the biological sample is a serum sample, a plasma sample, or a sample comprising RNA isolated from a subject’s bone marrow mononuclear cells or peripheral blood mononuclear cells.

44. The kit of claim 42, wherein the biological sample is supernatant obtained from a cell culture of the subject’s bone marrow mononuclear cells.

45. The kit of claim 42, wherein the biological sample is supernatant obtained from a cell culture of the subject’s peripheral blood mononuclear cells.

46. The kit of any one of claims 42-45, wherein the reagent comprises an antibody or antigenbinding fragment thereof specific for IL4I1.

47. The kit of any one of claims 42-46, wherein the kit comprises an ELISA assay.

48. The kit of any one of claims 42-46, wherein the kit comprises a lateral flow assay.

49. The kit of any one of claims 42-45, wherein the reagent comprises one or more oligonucleotides or primers that specifically hybridize to the IL4I1 gene or a segment thereof, or an mRNA transcript thereof.

50. A method of treating a subject having, at risk of having, or suspected of having MM, comprising administering to the subject the composition of any one of claims 1-23.

51. The method of claim 50, wherein the subject has been diagnosed with having MM using the method of claim 26 or the kit of any one of claims 42-49.

52. The method of claim 50 or 51, further comprising administering to the subject a molecule selected from the group consisting of a proteasome inhibitor, thalidomide or a derivative thereof, an anti-CD38 antibody, an anti-SLAMF7 antibody, a steroid, a chemotherapeutic agent, a bispecific T cell engager, a histone deacetylase inhibitor, a selective inhibitor of nuclear export, an antibiotic, and a BCMA-directed CAR T cell, wherein the molecule is administered before, simultaneously with, or after administering the composition, optionally wherein the molecule and the IL4I1 inhibitor are present in the subject during an overlapping time period.

53. The method of claim 52, wherein the molecule is administered before administering the composition.

54. The method of claim 52, wherein the molecule is administered simultaneously with administering the composition.

55. The method of claim 53, wherein the molecule is administered after administering the composition.-go-56. The method of any one of claims 52-55, wherein the molecule is a proteasome inhibitor selected from the group consisting of bortezomib, carfilzomib, oprozomib, ixazomib citrate, marizomib, delanzomib, and syringolin A.

57. The method of any one of claims 52-55, wherein the molecule is thalidomide, or a derivative thereof selected from the group consisting of thalidomide, lenalidomide, pomalidomide, linomide, CC-1088, CDC-501, and CDC-801.

58. The method of any one of claims 52-55, wherein the molecule is an anti-CD38 antibody.

59. The method of claim 58, wherein the anti-CD38 antibody is Isatuximab or Daratumumab.

60. The method of any one of claims 52-55, wherein the molecule is an anti-SLAMF7 antibody.

61. The method of claim 60, wherein the anti-SLAMF7 antibody is Elotuzumab.

62. The method of any one of claims 52-55, wherein the molecule administered to the subject is a steroid, optionally wherein the steroid is a glucocorticoid.

63. The method of claim 62, wherein the glucocorticoid is selected from the group consisting of hydroxy corti sone, cortisone, desoxycorticosterone, fludrocortisone, betamethasone, dexamethasone, prednisolone, prednisone, methylprednisolone, methylprednisone, paramethasone, triamcinolone, flumethasone, fluocinolone, fluocinonide, fluprednisolone, halcinonide, flurandrenolide, meprednisone, and medrysone.

64. The method of any one of claims 52-55, wherein the molecule is a chemotherapeutic agent selected from the group consisting of melphalan, cyclophosphamide, bendamustine, doxorubicin, and pegylated liposomal doxorubicin.

65. The method of any one of claims 52-55, wherein the molecule is a bispecific T cell engager.

66. The method of claim 65, wherein the bispecific T cell engager is talquetamab, teclistamab, or elranatamab.

67. The method of any one of claims 52-55, wherein the molecule administered to the subject is a histone deacetylase inhibitor, optionally wherein the histone deacetylase inhibitor is panobinostat.

68. The method of any one of claims 52-55, wherein the molecule is a selective inhibitor of nuclear export, optionally wherein the selective inhibitor of nuclear export is selinexor.

69. The method of any one of claims 52-55, wherein the molecule is an antibiotic, optionally wherein the antibiotic is clarithromycin.

70. The method of any one of claims 52-55, wherein the molecule is a BCMA-directed CAR T cell.

71. The method of claim 70, wherein the BCMA-directed CAR T cell is ide-cel or cilta-cel.

72. The method of any one of claims 50-71, wherein the composition is administered through parenteral administration.

73. The method of claim 72, wherein the parenteral administration is subcutaneous, intramuscular, intravenous, or intrathecal administration.

74. The method of any one of claims 50-71, wherein the composition is administered through intraosseous infusion.

75. The method of any one of claims 50-71, wherein the composition is administered through intratumoral injection.

76. The method of any one of claims 50-71, wherein the composition is administered through enteral administration.

77. The method of claim 76, wherein the enteral administration route is oral.

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