Lilrb3 modulators and methods of use thereof

Abstract

The present disclosure provides for a chimeric antigen receptor (CAR) polypeptide comprising a LILRB3 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region, and methods of use thereof. Further provided herein are antibodies that specifically bind to LILRB3 and methods of use thereof.

Description

[0001] LILRB3 MODULATORS AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 609,092 filed on December 12, 2023, the disclosure of which is hereby expressly incorporated by reference herein in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The sequence listing submitted on December 12, 2024, as an .XML file entitled “10063- 091 WO1_ST26” created on December 11 , 2024, and having a file size of 242,282 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0006] BACKGROUND

[0007] Acute myeloid leukemia (AML) is the most common acute leukemia, accounting for an estimated 19,940 new cases and 11,180 deaths in the US in 2019. AML is characterized by heterogeneous neoplastic proliferation of immature myeloid cells, leading to an accumulation of malignant leukemic cells in bone marrow, peripheral blood, and tissue. These malignant immature myeloid cells replace normal bone marrow cells, leading to multilineage cytopenias and clinical symptoms, such as fatigue, weakness, and predisposition to infection due to impaired leukocyte function. In medically fit adults, first- line medical therapy is often intensive induction chemotherapy, consisting of cytarabine (AraC) and an anthracycline, which may be followed by consolidation chemotherapy and / or hematopoietic stem cell transplantation. The rate of complete remission in patients under 60 is approximately 60- 85%, with 5-year disease-free survival at 30%. In patients over 60, complete remission is markedly decreased at 40-55%, and 5-year disease-free survival is also drastically lower at 5-10%. Despite intensive therapy, relapsed / refractory disease occurs in 10-40% of patients. Therefore, a major challenge in AML treatment is treating residual disease, especially AML cells residing in the bone marrow, where it is difficult to achieve a sufficiently high concentration of chemotherapeutic agents. Currently, there are limited reliable treatment modalities for controlling residual disease. One potential treatment modality is chimeric antigen receptor (CAR) T-cells, which although commonly used clinically in B-cell malignancies, are not well developed in the context of myeloid malignancies due to difficulty in identifying efficacious, clinically tolerable, myeloid- specific targets.

[0008] The compositions and methods disclosed herein address these and other needs. SUMMARY

[0009] In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to chimeric antigen receptors and methods relating thereto.

[0010] Thus, in one example, a chimeric antigen receptor polypeptide is provided, including a LILRB3 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region.

[0011] In a further example, an isolated nucleic acid sequence encoding a recombinant polypeptide of any of the CAR polypeptides disclosed herein is provided.

[0012] Additionally, a vector comprising the isolated nucleic acid sequence disclosed herein is provided.

[0013] Further provided herein is a cell comprising the vector disclosed herein.

[0014] Also provided herein is a method of treating leukemia in a subject in need thereof, including administering to the subject a therapeutically effective amount of the chimeric antigen receptor polypeptide disclosed herein.

[0015] Previously, there has been no effective method or modality for AML treatment, especially in patients with relapsed tumor or AML metastases in the bone or other metastatic sites. The LILRB3 targeting CAR-T and anti-LILRB3 antibodies disclosed herein efficiently eliminate the AML and tumor cells expressing the LILRB3 receptor. Also demonstrated herein is that antagonist and agonist antibodies can induce the AML cholesterol metabolism pathway that promotes the tumor invasion and drug resistance.

[0016] LILRB3 as a target contributes to AML tumor killing and is part of a pathway that promotes tumor differentiation and increases its sensitivity to tumor killing. This tumor killing ability can have a synergistic effect when utilized with chemotherapy or immune therapy to prevent the drug resistance or tumor refractory to chemotherapy.

[0017] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0019] Figure 1A - Figure 1C show LILRB3 expressed in human AML and predicts overall survival. (A) LILRB 1-4 expression in PBMCs from 14 AML patients, gated on viable CD33+ cells. (B) Overall survival of TCGA AML patients with low LILRB3 expression (log2 RSEM normalized count less than 6.881) compared to intermediate (between 6.881 and 9.215) and high LILRB3 expression (greater than 9.215) with a p-value of 0.048 (logrank Mantel-Cox test), adapted from UCSC Xena Browser24. (C) Log2 expression of LILRB3 in various AML types compared to normal hematopoietic cells and myeloid cells from Bloodspot25.

[0020] Figure 2A - Figure 2K show an example LILRB3 blockade modulating macrophage differentiation and inhibiting AML proliferation. (A) LILRB3 expression of U937 in the presence or absence of IFNy (50 ng / ml) or AraC (50 nM) treatment assessed by flow cytometry. Representative dot plots from repeated experiments are shown. (B) The mean expression of LILRB3 on U937 cells in the presence or absence of IFNy or AraC treatment. (C) IFNy production from human PBMC cocultured with untreated U937 vs U937 cells pretreated with 500nM AraC, IFNy was measured in the coculture supernatant after 60 hours via ELISA, n=3 wells per PBMC donor, per condition. (D) Supernatant from PBMC and U937 cells cultured as described in D, was supplemented to culture of U937 cells with or without anti-IFNy antibody, LILRB3 expression was determined via flow cytometry after 24hrs. (E) LILRB3 expression in CD34+ Human AML patient blast cells in the presence or absence of IFNy (lOOng / ml) or poly(LC) (lOOug / ml), percentages added manually for clarity. (F) The binding affinities and specificity of two anti- LILRB3 clones were assessed by Octet. (G) Measurement of TNFa secretion (pg / ml) from monocyte differentiation assay by ELISA. (H) CD163, CD86, CD206, and HLA-DR expression in MCSF treated monocytes in M2 polarized conditions treated with anti-LILRB3 antagonist antibody. (I) The effect of anti-LILRB3 on U937 proliferation in the presence or absence of various concentrations of IFNy pretreatment. (J) The effect of two anti-LILRB3 clones on U937 proliferation in the presence of IFNy pretreatment (50 ng / ml). (K) The effect of two anti-LILRB3 clones on HL-60 proliferation in the presence of IFNy pretreatment (50 ng / ml). Error bars indicate the mean of standard deviation (SD). Comparisons performed using one-way ANOVA with Tukey adjusted P values for multiple comparisons (B, D, J, K) or student t-test (C,G) or multiple T tests with Holm Sidak correction for multiple comparisons (I); *P<0.05; **P<0.01 ; ***P<0.001, ****P<0.0001. Figure 3 A - Figure 3D show an example LILRB3 blockade promoting differentiation- related gene expression and surface markers. Relative PU.l , C / EBPa, C / EBPp, IRF1 , IRF8 gene expression in (A) U937 cells pretreated with IFNy and treated with anti-LILRB3 or IgG control and in (B) U937 cells pretreated with AraC and treated with antagonist LILRB3 or IgG control. Relative C / EBPa, C / EBPp, C / EBPs, IRF4, IRF8, CMYC, JUNB, PU.l and RUNX1 gene expression in (C) U937 cells with overexpressed LILRB3 receptor and treated with antagonist or agonist anti-LILRB3 or IgG control. (D) U937 cells pretreated with IFNy and treated with antagonist or agonist anti-LILRB3 or IgG control. Comparisons performed via one-way ANOVA with Tukey adjusted P values for multiple comparisons; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0021] Figure 4 A - Figure 4D show an assessment of LILRB3 downstream signaling by reverse phase protein array (RPPA) and western blot. (A) RPPA heatmap of differentially expressed proteins in U937 cells pretreated with IFNy and treated with antagonist LILRB3 or IgG control. (B) Western blot of U937 cells pretreated with IFNy and treated with antagonist LILRB3 or IgG control. Cells were stimulated with LPS for 0, 10, 30 minutes (C) Relative protein expression of phosphorylated pRb, phospho-p38, and phospho-Akt in U937 cells treated with anti-LILRB3 or IgG control at 30 minute time point. (D) Western blot of THP-1 LILRB3 expressing cells treated with antagonist anti-LILRB3 or IgG control. Cells were stimulated with LPS for 0, 10, 30 minutes Comparisons via student t-test; *P<0.05; **P<0.01; ***P<0.001.

[0022] Figure 5A - Figure 5G show an RNAseq analysis of U937 cells treated with aLILRB3 antagonist and agonist antibody. Experiments performed with antagonist antibody (A) RNA-seq heatmap of differentially expressed genes of IFNy pretreated U937 cells treated with IgG control vs Antagonist LILRB3 antibody (cutoff: 0.5 > log2 > -0.5, p-value < 0.05, adjusted p-value < 0.05). (B) Canonical pathway analysis generated by Ingenuity Pathway Analysis software (p-value < 0.05). (C) Upstream Regulator Analysis of differentially expressed gene sets. Negative z-scores represent predicted inhibition, and positive z-scores represent predicted activation of upstream regulators. (D) Gene Set Enrichment Analysis of RNA-seq differentially expressed genes of U937 cells treated with LILRB3 antibody compared to IgG control. (E-F) Experiments performed with agonist antibody. (E) RNA-seq heatmap of differentially expressed genes of IFNy pretreated U937 cells treated with IgG control vs Agonist LILRB3 antibody (cutoff: 0.5 > log2 > -0.5, p-value < 0.05, adjusted p-value < 0.05). (F) Volcano plot of differentially expressed genes of IgG control vs Agonist LILRB3. Horizontal dotted line represents adjusted P value (FDR) = 0.1, vertical dotted line represents log2FC =0.5. (G) Pathway analysis generated by IPA. Figure 6A - Figure 6M show example LILRB3-CAR T-cells demonstrating AML directed cytotoxicity in vitro and decreasing LILRB3-transduced MV3-1 1 leukemic burden in vivo. Tn vitro cytotoxicity of LILRB3-CAR T-cells against primary AML cells at (A), LILRB3 transduced MV4-11 cells (B), and LILRB3 Transduced THP-1 cells (C) at 7 hours. (D) Pictorial depiction of experimental design (created with BioRender.com) (E) Bioluminescence images of mice implanted with LILRB3 transduced MV4-11 cells on day -5. Mice treated with either PBS vehicle control (n=3), control T-cells (n=5), or LILRB3-CAR T-cells (n=7). CAR-T and control T cells derived from two separate human donors; the two leftmost panels of each column derived from donor 1 with the remaining panels derived from donor 2. Tumor burden was followed by serial bioluminescence imaging on days 0, 7, 14, 21, 28, and 96. (F) Quantification of flux with each group represented as the mean + standard deviation. Groups compared via repeated measures ANOVA with Geisser-Greenhouse correction and Tukey method for correction for multiple comparisons. (G) Quantification of flux with each mouse represented individually. (H) Kaplan- Meier survival curve of mice. Groups compared with log rank test. (I) In vitro cytotoxicity of AML patient derived CAR T cells against LILRB3 transduced MV4-11 cells at 24 hrs. Surface expression of the T cell activation marker CD25 (J) and degranulation marker CD107a (K) of AML patient derived CAR T cells cocultured with LILRB3 transduced MV4-11 cells at an E:T of 1:1. (L-M) In vivo demonstration of autologous AML CAR T cell treatment. (L) Peripheral blood hCD33+ cell concentration prior to T cell engraftment. (M) Bone marrow leukemic burden 10 days following T cell engraftment. *P<0.05,**P<0.01 ; ***P<0.001 ; ****P<0.0001.

[0023] Figure 7A - Figure 7L show example murine LILRB3-CAR T-cells demonstrating AML directed cytotoxicity in vitro with limited hematologic and metabolic toxicity in vivo. (A) In vitro cytotoxicity of murine LILRB3-CAR T-cells against LILRB3 transduced C1498 cells at 24 hrs. (B-K) Complete blood count (CBC) and metabolic panel (CMP) from LILRB3 transgenic mice treated with 5x106 murine LILRB3-CAR T-cells (n=4 CBC, n=5 CMP) vs control T cells (n=5) vs PBS (n=3). Blood was collected on Day 9 following T-cell injection. One outlier removed from PBS treated monocyte group using Iglewicz and Hoaglin's robust test for outliers (cutoff Z>2). (L) Weekly body weights of LILRB3 transgenic mice treated with 2 doses of 5x106 murine LILRB3- CAR T-cells (n=10) vs control T cells (n=6) vs at PBS (n=4) at days 0 and 15. Groups compared using one way ANOVA, with Dunnet’s correction for multiple comparisons. *P<0.05.

[0024] Figure 8A - Figure 8G show LILRB3 expression in human AML patients and overall survival. (A) Gating Strategy for Figure 1A. PE channel represents the specific LILRB being probed for in each subsample, LILRB3 in this instance. (B) LILRB3 mRNA expression by FAB subtype from LAML TCGA project. (C) Survival by FAB subtype in TCGA cohort. (D) Survival of M4 / M5 patients stratified by LILRB3 expression. (E-F) LILRB3 surface expression on CD45+ / CD38- / CD34+ gated human hematopoietic stem / progenitor cells isolated from the peripheral blood of a healthy human donor, (E) Representative gating, (F) quantification of LILRB3+ cells, n=3 donors. Groups compared with paired T test. (G) Normalized LILRB 3 protein expression on AML samples compared with HD Lin- cells, each bar represents 1 patient; adapted from Kramer. *P<0.05,**P<0.01.

[0025] Figure 9A - Figure 9J show LILRB3 expression and the effect of an example LILRB3 blockade on macrophages differentiation. (A) Gating strategy for Fig. 9A. (B) LILRB3 induction in U937 cells with 50nM LPS. US:unstained (C) LILRB3 surface expression on U937 following stimulation removal (IFNy / AraC), in serum free media (D) LILRB3 induction in HL60 cells with IFNg. (E) IFNg production from untreated U937 vs U937 cells treated with 500nM AraC, IFNg was measured in the coculture supernatant after 60 hours via ELISA, n=3 wells per PBMC donor, per condition. (F) LILRB3 expression in a second human AML patient blast cells in the presence or absence of IFNy (lOOng / ml) or PolylC (lOOug / ml) (F) Gating strategy for Fig. 2E / S. Fig. 9D. (H-I) LILRB3 expression on CD34+ AML blast cell subpopulation compared with CD34- subpopulation. Two patients and two gating strategies are shown for representation purposes. (J) HLA-DR CD163 and CD206 surface expression MFI in IgG vs LILRB3 antagonist antibody. Groups compared via paired T test. *P<0.05.

[0026] Figure 10A - Figure 10F show anti-LILRB3 inhibiting AML proliferation. (A) Tumor sizes of subcutaneous transplanted U937 cells in NSG-SGM3 mice treated with anti-LILRB3_l antibody (n=4) or isotype-matched antibody (IgGl) (n=5). (B) Tumor sizes of subcutaneous transplanted U937 cells in NSG-SGM3 mice treated with anti-L!LRB3_2 antibody (n=6) or isotype matched antibody (IgGl) (n=5). (C) Corresponding tumor mass at experiment endpoint from (B) (n=5 IgG, n=6 anti-LILRB3). Left panel each group is represented as mean ± standard deviation. Right panel each mouse is represented individually. (D) Tumor sizes of subcutaneous transplanted U937 cells in NSG-SGM3 mice treated with anti-LILRB3_3 antibody (n=13) or isotype matched antibody (IgGl) (n= 15). Left panel each group is represented as mean ± standard deviation. Right panel each mouse is represented individually. (E) Corresponding tumor mass at experiment endpoint from, (D) (n=12 IgG, n=10 anti-LILRB3. (F) Tumor sizes of subcutaneous transplanted Cl 498 mouse leukemia cells in transgenic LILRB3 mice with C57BL / 6J background treated with anti-LILRB3_l antibody (n=4) or isotype matched antibody (IgGl) (n=3). Figures A,B,D,F left panel represents group the mean ± standard deviation, right panel, each mouse is represented individually. Figures C,E indicators of 75thpercentile, median and 25thpercentile are overlaid over dots representing each mouse. Groups compared via repeated measures ANOVA with Geisser-Greenhouse correction (A,B,D,F) or student t-test (C,E); *P<0.05,**P<0.01 ; ***P<0.001.

[0027] Figure 11A - Figure 11B show example LILRB3 blockade promoting differentiation related gene expression and surface markers. (A) Differentiation related genes in Control vs. IFNy treated HL-60, IgG vs Anti-LILRB3 antibody. (B) CD 14 / CD 16 staining in HL-60 cells treated with Ctrl vs IFNy and IgG vs Anti-LILRB3 antibody.

[0028] Figure 12A - Figure 12B show an example assessment of LILRB3 downstream signaling by reverse phase protein array (RPPA) and western blot. (A) Western blot of U937 cells pretreated with or without IFNy and treated with anti-LILRB3 or IgG control. Cells were stimulated with LPS for 0, 10, 30 minutes. Samples were blotted for total and phospho-mTOR. (B) Western blot of THP-1 LILRB3 expressing cells treated with anti-LILRB3 or IgG control. Samples were blotted for total and phospho-mTOR.

[0029] Figure 13A - Figure 13F show RNAseq analysis of U937 cells treated with aLILRB3 antagonist antibody. Heatmaps of differentially expressed genes in IgG treated versus anti- LILRB3 treated U937 cells that match genes found in the GSEA gene sets.

[0030] Figure 14 shows an example LILRB3 blockade promoting a cell maturation in leukemia cells.

[0031] Figure 15A - Figure 15H show example LILRB3 CAR T-cells decrease LILRB3 transduced MV4-11 tumor burden in-vivo. (A) Pictorial depiction of human CAR T construct transduced into donor PBMC for in-vitro and in-vivo CAR T experiments. Made in SnapGene. (B) CD69 activation marker upregulation in CAR jurkat cells cultured alone vs cocultured with THP1 vs cocultured with LILRB3 transduced THP-1. (C) 24 hr cytotoxicity of mCherry labelled MV411 LILRB3 cells via flow cytometry. (D) 24 hr cytotoxicity of mCherry labelled U937 LILRB3 cells. (E) Control T cell and CAR T proliferation in response to LILRB3 protein stimulation. (F) human CD3 (hCD3) positive cells at day 10 following CAR T cell engraftment in AML engrafted mice. (G) Pictorial depiction murine CAR T construct transduced into donor PBMC for in-vitro and in-vivo CAR T experiments. Made in SnapGene. (H) IFNy release from murine control T cell and CAR T-cells cocultured with C1498 LILRB3 cells. (D) multiple T tests with Holm Sidak correction for multiple comparisons. (E) one-way ANOVA with Tukey adjusted P values for multiple comparisons *P<0.05,**P<0.01; ***P<0.001; ****P<0.0001.

[0032] Figure 16A - Figure 16C show LILRB expression in the brain and other organs. (A) LILRB 1-5 expression by q-PCR in brain organoid tissue derived from human iPSC and healthy donor human PBMC. (B) LILRB 1-5 expression in iPSC derived human brain organoids. (C) Gene expression of LILRB3 in various human cell types.

[0033] Figure 17 shows LILRB3 CAR T-cells decreasing LILRB3 transduced MV4-11 tumor burden in-vivo. Pictorial depiction of human CAR T construct transduced into donor PBMC for in-vitro and in-vivo CAR T experiments. Made in SnapGene.

[0034] Figure 18 shows SDS-PAGE result of chimeric and PTM removal antibodies under nonreducing and reducing conditions. Lane M is a marker. Lane 1 is U529DFD220 Chimeric PTM remove I +chimeric YL with 99% purity. Lane 2 is U529DFD220 Chimeric PTM remove2+chimeric VL with 99% purity. Lane 3 is U529DFD220 Chimeric VH+VL with 99% purity. Lane 4 is Human IgG. Lane 5 is U529DFD220 Chimeric PTM remove 1 +chimeric VL with 97% purity. Lane 6 is U529DFD220 Chimeric PTM remove2+chimeric YL with 98% purity. Lane 7 is U529DFD220Chimeric VH+VL with 99% purity.

[0035] Figure 19A - Figure 19C show sensor-grams of HMRI Antigen A to selected clones: (a) chimeric VH+VL; (b) chimeric PTM remove 1 ; (c) chimeric PTM remove 2.

[0036] Figure 20 shows sensor-grams of HMRI Antigen A to selected clones.

[0037] Figure 21 A - Figure 21 C show SDS-PAGE result of humanized antibody under nonreducing and reducing conditions, (a) Lane M is the marker. Lane 1 is U529DFD220 U529DFD220-VH(lgG4 G56A)-BM1 +VL-BM2 with 99% purity. Lane 2 is U529DFD220 U529DFD220-VH(lgG4 G56A)-GRAFTED+VL-BM2 with 99% purity. Lane 3 is U529DFD220 U529DFD220-VH(lgG4 G56A)-GRAFTED+VL-BM3 with 99% purity. Lane 4 is human IgG. (b) Lane M is a marker. Lane 1 is U529DFD220 U529DFD220-VH(lgG4 G56A)-BM1 +VL-BM2 with 90% purity. Lane 2 is U529DFD220 U529DFD220 -VH(lgG4 G56A)-GRAFTED+VL-BM2 with 90% purity. Lane 3 is U529DFD220 U529DFD220-VH(lgG4 G56A)-GRAFTED+VL-BM3 with 99% purity. Lane 4 is Human IgG. (c) Lane M is a marker. Lane 1 is U529DFD220 Chimeric - VH(lgG4 G56A)+VL with 99%. Lane 2 is U529DFD220 Chimeric- VH(lgG4 G56A)+VL with 99% purity. Lane 3 is Human IgG.

[0038] Figure 22A - Figure 22D show sensor-grams of HMRI Antigen A to antibodies.

[0039] Figure 23 shows sensor grams of HMRI Antigen A to selected clones.

[0040] Figure 24A - Figure 24H show that human PBMCs were treated with anti-LILRB3 antibodies overnight and then cells were stimulated with LPS for 6 hours, supernatant were analyzed for the human TNF-alpha by ELISA. The human PBMCs were treated with anti-LILRB3 antibodies overnight and then cells were stimulated with anti-human CD3 for 72 hours, supernatant were analyzed for the human IFN-gamma by ELISA. DETAILED DESCRIPTION

[0041] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0042] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0043] As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0044] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0045] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0046] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0047] Definitions

[0048] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0049] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a disorder”, includes, but is not limited to, two or more such compounds, compositions, or disorders, and the like.

[0050] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0051] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’ , less than y’ , and Tess than z’ . Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0052] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0053] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1 % by weight or less, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition.

[0054] “Reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control (e.g., an untreated tumor).

[0055] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0056] The term “amino acid sequence” refers to a list of abbreviations, letters, characters, or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0057] The term “antibody” refers to natural or synthetic antibodies that selectively bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.

[0058] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecule is a fusion protein.

[0059] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides, or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0060] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLAST? is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLAST? identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values, and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code. The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide, or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5 ’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0061] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0062] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0063] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0064] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0065] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally, a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0066] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g., immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g., an antibody specifically binds to an antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M-1(e.g., 106M-1, 107M-1, 108M-1, 109M-1, I O10M-1, 1011M-1, and 1012M-1or more) with that second molecule.

[0067] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0068] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0069] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0070] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0071] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (e.g., a degenerate variant), substitutions within the wobble position of each codon (e.g. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0072] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, virus, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). Compositions

[0073] Chimeric Antigen Receptor ( CAR ) Polypeptides

[0074] Provided herein is a chimeric antigen receptor (CAR) polypeptide comprising a LILRB3 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory signaling region.

[0075] Chimeric antigen receptors (CARs) are receptor proteins that have been engineered to give T cells the ability to target a specific antigen. The receptors are chimeric in that they combine both antigen binding and T cell activating functions into a single receptor.

[0076] Leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3) is a protein that in humans is encoded by the LILRB3 gene. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is found in a gene cluster at chromosomal region 19ql 3.4. The encoded protein belongs to the subfamily B class of LIR receptors, which have two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The receptor is expressed on immune cells.

[0077] The disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain is responsible for antigen recognition. It also generally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane domain (TD), is as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell. The endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. For example, the endodomain can contain an intracellular signaling domain (ISD) and optionally a co-stimulatory signaling region (CSR).

[0078] Antigen binding domain refers to the region of an antibody that binds to antigens. It can comprise one constant and one variable domain of each of the heavy chain and light chain.

[0079] A transmembrane domain is a membrane-spanning protein domain. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (e.g., comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, or CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.

[0080] A hinge domain is a flexible amino acid stretch that is present in some immunoglobulins. The hinge domain provides segmental flexibility and can allow for crosslinking of two antigens or binding of two antigenic determinants on the same antigen molecule. The hinge sequence may be positioned between the antigen recognition moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0081] An intracellular signaling domain communicates via protein-protein interactions against effector proteins, which in turn pass a signal to the destination.

[0082] The co-stimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or their ligands, that is required for an efficient response of lymphocytes to an antigen.

[0083] In some examples, the LILRB3 antigen binding domain is a single-chain variable fragment (scFv) of an antibody that specifically binds to LILRB3. A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins, connected with a short linker peptide.

[0084] In further examples, the co-stimulatory signaling region comprises a cytoplasmic domain of costimulatory molecule 4-1BB. 4-1BB is a co-stimulatory glycoprotein receptor that is part of the tumor necrosis factor superfamily. It is an inducible cell surface receptor that is expressed in the presence of activating stimuli and functions in cell signaling during T cell activation and proliferation.

[0085] In certain examples, the intracellular signaling domain comprises a CD3 zeta (CD3Q signaling domain. Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. However, in preferred embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3Q. T-cell surface glycoprotein CD3 zeta (CD3Q chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene.

[0086] In specific examples, the CAR polypeptide is defined by the formula: SP-LILRB3-HG- TM-CSR-ISD; or SP-LILRB3-HG-TM-ISD-CSR wherein “SP” represents a signal peptide; wherein “LILRB3” represents an LILRB 3 -binding region; wherein “HG” represents an optional hinge domain; wherein “CSR” represents a co-stimulatory signaling region; wherein “ISD” represents an intracellular signaling domain; and wherein represents an optional bivalent linker.

[0087] A signal peptide is a short amino acid sequence that controls protein secretion and translocation.

[0088] A bivalent linker is a single chemical entity composed of two pharmacophores covalently linked by a spacer of variable size. The bivalent linker can be any molecule suitable to link a compound or nucleic acid to a polynucleotide sequence. Methods and compositions for conjugating biomolecules, such as polynucleotides, are disclosed in G.T. Hermanon, Bioconjugate Techniques (2nded.), Academic Press (2008), which is incorporated by reference in its entirety for the teaching of these techniques. In some cases, the bivalent linker comprises one or more amino acids. However, it can also comprise a peptide bond directly linking the disclosed domains.

[0089] In some examples, the CAR polypeptide is defined by the formula: SP-(VL-VH)n-HG- TM-CSR-ISD; or SP-(VH-VL)n-HG-TM-CSR-ISD; SP-(VL-VH)n-HG-TM-ISD-CSR; or SP- (VH-VL)n-HG-TM-CSR-ISD wherein “SP” represents a signal peptide; wherein “VL” represents a light chain variable region; “wherein VH” represents a heavy chain variable region; wherein “n” is > 1; wherein ‘HG” represents an optional hinge domain; wherein “CSR” represents a costimulatory signaling region; wherein “ISD” represents an intracellular signaling domain; and wherein represents an optional bivalent linker.

[0090] A heavy chain variable region (VH) is the large polypeptide subunit of an antibody. A light chain variable region (VL) is the small polypeptide subunit of an antibody.

[0091] In some examples, the scFv comprises a heavy chain variable region (VH) with a CDR1 comprising a sequence with at least 60% identity to SEQ ID NOS: 10, 16, or 22, a CDR2 comprising a sequence with at least 60% identity to SEQ ID NOS: 11, 17, or 23, and a CDR3 comprising a sequence with at least 60% identity to SEQ ID NOS: 12, 18, or 24.

[0092] In certain examples, the scFv nucleic acid sequence comprises a heavy chain variable region (VH) with a CDR1 comprising a sequence with at least 60% identity to SEQ ID NOS: 37, 43, or 49, a CDR2 comprising a sequence with at least 60% identity to SEQ ID NOS: 38, 44, or 50, and a CDR3 comprising a sequence with at least 60% identity to SEQ ID NOS: 39, 45, or 51 .

[0093] In certain examples, the scFv comprises a light chain variable region (VL) with a CDR1 comprising a sequence with at least 60% identity to SEQ ID NOS: 13, 19, or 25, a CDR2 comprising a sequence with at least 60% identity to SEQ ID NOS: 14, 20, or 26, and a CDR3 comprising a sequence with at least 60% identity to SEQ ID NOS: 15, 21, or 27.

[0094] In further examples, the scFv nucleic acid sequence comprises a light chain variable region (VL) with a CDR1 comprising a sequence with at least 60% identity to SEQ ID NOS: 40, 46, or 52, a CDR2 comprising a sequence with at least 60% identity to SEQ ID NOS: 41, 47, or 53, and a CDR3 comprising a sequence with at least 60% identity to SEQ ID NOS: 42, 48, or 54.

[0095] As used herein, at least 60% identity includes at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity. In further examples, at least 60% identity includes at least 62%, at least 64%, at least 66%, at least 68%, at least 72%, at least 74%, at least 76%, at least 78%, at least 82%, at least 84%, at least 86%, at least 88%, at least 92%, at least 94%, at least 96%, or at least 98%.

[0096] In further examples, the scFv comprises a VL with a CDR1, CDR2, and CDR3, and a VH with a CDR1, CDR2, and CDR3, wherein: VL CDR1 is SEQ ID NO: 13; VL CDR2 is SEQ ID NO: 14; VL CDR3 is SEQ ID NO: 15; VH CDR1 is SEQ ID NO: 10; VH CDR2 is SEQ ID NO: 11; and VH CDR3 is SEQ ID NO: 12; or VL CDR1 is SEQ ID NO: 19; VL CDR2 is SEQ ID NO: 20; VL CDR3 is SEQ ID NO: 21; VH CDR1 is SEQ ID NO: 16; VH CDR2 is SEQ ID NO: 17; and VH CDR3 is SEQ ID NO: 18; or VL CDR1 is SEQ ID NO: 25; VL CDR2 is SEQ ID NO: 26; VL CDR3 is SEQ ID NO: 27; VH CDR1 is SEQ ID NO: 22; VH CDR2 is SEQ ID NO: 23; and VH CDR3 is SEQ ID NO: 24.

[0097] In some examples, VL CDR1 is SEQ ID NO: 13; VL CDR2 is SEQ ID NO: 14; VL CDR3 is SEQ ID NO: 15; VH CDR1 is SEQ ID NO: 10; VH CDR2 is SEQ ID NO: 11; and VH CDR3 is SEQ ID NO: 12.

[0098] In further examples, VL CDR1 is SEQ ID NO: 19; VL CDR2 is SEQ ID NO: 20; VL CDR3 is SEQ ID NO: 21; VH CDR1 is SEQ ID NO: 16; VH CDR2 is SEQ ID NO: 17; and VH CDR3 is SEQ ID NO: 18.

[0099] In specific examples, VL CDR1 is SEQ ID NO: 25; VL CDR2 is SEQ ID NO: 26; VL CDR3 is SEQ ID NO: 27; VH CDR1 is SEQ ID NO: 22; VH CDR2 is SEQ ID NO: 23; and VH CDR3 is SEQ ID NO: 24.

[0100] A complementarity-determining region (CDR) is a part of the variable chains in immunoglobulins (antibodies) and T cell receptors. CDRs are the most variable part of the molecules and are therefore important to the diversity of antigen specificities generated by lymphocytes. There are three CDRs (CDR1 , CDR2, and CDR3) arranged non-consecutively on the amino acid sequence of a variable domain of an antigen receptor. In some examples, antigen receptors are composed of two variable domains (on two different polypeptide chains: the heavy chain variable region and the light chain variable region) and therefore there are six CDRs for each antigen receptor.

[0101] In some examples, the scFv comprises a heavy chain variable region (VH) comprising a sequence with at least 60% identity to SEQ ID NOS: 4, 6, or 8 and a light chain variable region (VL) comprising a sequence with at least 60% identity to SEQ ID NOS: 5, 7, or 9.

[0102] In certain examples, the scFv nucleic acid sequence comprises a heavy chain variable region (VH) comprising a sequence with at least 60% identity to SEQ ID NOS: 31, 33, or 35 and a light chain variable region (VL) comprising a sequence with at least 60% identity to SEQ ID NOS: 32, 34, or 36.

[0103] In further examples, the scFv comprises a sequence with at least 60% identity to SEQ ID NOS : 1 -3 or a fragment thereof.

[0104] In some examples, the scFv nucleic acid sequence comprises a sequence with at least 60% identity to SEQ ID NOS: 28-30 or a fragment thereof.

[0105] The CAR polypeptide can include antagonist and / or agonist antibodies. Further, antagonist and agonist antibodies can induce the AML cholesterol metabolism pathway that promotes the tumor invasion and drug resistance. In some examples, the binding affinity of the antagonist is modified so as to change the antagonist into an agonist, and vice versa to modify an agonist into an antagonist. See Yu et al., Reducing affinity as a strategy to boost immunomodulatory antibody agonism. Nature 2023 February 16, vol. 614: 539-547; Yu et al., Isotype switching converts anti- CD40 antagonism to agonism to elicit potent antitumor activity. CellPress 2020 June 8, Cancer Cell 37: 850-866.

[0106] Isolated Nucleic Acids, Vectors, and Cells

[0107] Also provided herein is an isolated nucleic acid sequence encoding the recombinant polypeptide disclosed herein. Further disclosed is a vector comprising the isolated nucleic acid sequence as disclosed herein. Also disclosed herein is a cell comprising the vector as disclosed herein. In some examples, the cell reduced tumor activity when the antigen binding domain of the CAR polypeptide binds to LILRB3.

[0108] Nucleic acid sequences encoding the disclosed CARs, and regions thereof, can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0109] Expression of nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0110] The disclosed nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0111] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. In some examples, the polynucleotide vectors are lentiviral or retroviral vectors.

[0112] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.

[0113] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. The promoter can alternatively be an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0114] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.

[0115] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibioticresistance genes.

[0116] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0117] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well- known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0118] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.

[0119] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0120] In the case where a non- viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).

[0121] Also disclosed are immune effector cells that are engineered to express the disclosed

[0122] CARs. These cells are preferably obtained from the subject to be treated (e.g., are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0123] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes.

[0124] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0125] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen- presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI , TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response. Cytotoxic T cells (Tc cells, or CTLs) destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0126] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0127] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Tregcells have been described — naturally occurring Tregcells and adaptive Trcgcells.

[0128] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id.

[0129] Recombinant Antibodies

[0130] Also provided herein is a recombinant antibody, wherein the antibody comprises a light chain variable region (VL) with a CDR1, CDR2, and CDR3, and a heavy chain variable region (VH) that comprises a CDR1, CDR2, and CDR3, further wherein the VL CDR1 comprises a sequence with at least 60% identity to SEQ ID NOS: 72, 78, 84, 90, 96, 102, or 108 and / or the VH CDR1 comprises a sequence with at least 60% identity to SEQ ID NOS: 69, 75, 81 , 87, 93, 99, or 105.

[0131] In some examples, the VL CDR2 comprises a sequence with at least 60% identity to SEQ ID NOS: 73, 79, 85, 91, 97, 103, or 109 and / or the VH CDR2 comprises a sequence with at least 60% identity to SEQ ID NOS: 70, 76, 82, 88, 94, 100, or 106.

[0132] In further examples, the VL CDR3 comprises a sequence with at least 60% identity to SEQ ID NOS: 74, 80, 86, 92, 98, 104, or 110 and / or the VH CDR3 comprises a sequence with at least 60% identity to SEQ ID NOS: 71, 77, 83, 89, 85, 101, or 107. In certain examples, the VL comprises a sequence with at least 60% identity to SEQ ID NOS: 56, 58, 60, 62, 64, 66, or 68 and / or the VH comprises a sequence with at least 60% identity to SEQ ID NOS: 55, 57, 59, 61, 63, 65, or 67.

[0133] In specific examples, VL CDR1 is SEQ ID NO: 72; VL CDR2 is SEQ ID NO: 73; VL CDR3 is SEQ ID NO: 74; VH CDR1 is SEQ ID NO: 69; VH CDR2 is SEQ ID NO: 70; and VH CDR3 is SEQ ID NO: 71 ; or VL CDR1 is SEQ ID NO: 78; VL CDR2 is SEQ ID NO: 79; VL CDR3 is SEQ ID NO: 80; VH CDR1 is SEQ ID NO: 75; VH CDR2 is SEQ ID NO: 76; and VH CDR3 is SEQ ID NO: 77; or VL CDR1 is SEQ ID NO: 84; VL CDR2 is SEQ ID NO: 85; VL CDR3 is SEQ ID NO: 86; VH CDR1 is SEQ ID NO: 81; VH CDR2 is SEQ ID NO: 82; and VH CDR3 is SEQ ID NO: 83; or VL CDR1 is SEQ ID NO: 90; VL CDR2 is SEQ ID NO: 91; VL CDR3 is SEQ ID NO: 92; VH CDR1 is SEQ ID NO: 87; VH CDR2 is SEQ ID NO: 88; and VH CDR3 is SEQ ID NO: 89; or VL CDR1 is SEQ ID NO: 96; VL CDR2 is SEQ ID NO: 97; VL CDR3 is SEQ ID NO: 98; VH CDR1 is SEQ ID NO: 93; VH CDR2 is SEQ ID NO: 94; and VH CDR3 is SEQ ID NO: 95; or VL CDR1 is SEQ ID NO: 102; VL CDR2 is SEQ ID NO: 103; VL CDR3 is SEQ ID NO: 104; VH CDR1 is SEQ ID NO: 99; VH CDR2 is SEQ ID NO: 100; and VH CDR3 is SEQ ID NO: 101; or VL CDR1 is SEQ ID NO: 108; VL CDR2 is SEQ ID NO: 109; VL CDR3 is SEQ ID NO: 110; VH CDR1 is SEQ ID NO: 105; VH CDR2 is SEQ ID NO: 106; and VH CDR3 is SEQ ID NO: 107.

[0134] In some examples, VL CDR1 is SEQ ID NO: 72; VL CDR2 is SEQ ID NO: 73; VL CDR3 is SEQ ID NO: 74; VH CDR1 is SEQ ID NO: 69; VH CDR2 is SEQ ID NO: 70; and VH CDR3 is SEQ ID NO: 71.

[0135] In further examples, VL CDR1 is SEQ ID NO: 78; VL CDR2 is SEQ ID NO: 79; VL CDR3 is SEQ ID NO: 80; VH CDR1 is SEQ ID NO: 75; VH CDR2 is SEQ ID NO: 76; and VH CDR3 is SEQ ID NO: 77.

[0136] In specific examples, VL CDR1 is SEQ ID NO: 84; VL CDR2 is SEQ ID NO: 85; VL CDR3 is SEQ ID NO: 86; VH CDR1 is SEQ ID NO: 81; VH CDR2 is SEQ ID NO: 82; and VH CDR3 is SEQ ID NO: 83.

[0137] In certain examples, VL CDR1 is SEQ ID NO: 90; VL CDR2 is SEQ ID NO: 91 ; VL CDR3 is SEQ ID NO: 92; VH CDR1 is SEQ ID NO: 87; VH CDR2 is SEQ ID NO: 88; and VH CDR3 is SEQ ID NO: 89.

[0138] In some examples, VL CDR1 is SEQ ID NO: 96; VL CDR2 is SEQ ID NO: 97; VL CDR3 is SEQ ID NO: 98; VH CDR1 is SEQ ID NO: 93; VH CDR2 is SEQ ID NO: 94; and VH CDR3 is SEQ ID NO: 95. In further examples, VL CDR1 is SEQ ID NO: 102; VL CDR2 is SEQ ID NO: 103; VL CDR3 is SEQ ID NO: 104; VH CDR1 is SEQ ID NO: 99; VH CDR2 is SEQ ID NO: 100; and VH CDR3 is SEQ ID NO: 101.

[0139] In specific examples, VL CDR1 is SEQ ID NO: 108; VL CDR2 is SEQ ID NO: 109; VL CDR3 is SEQ ID NO: 110; VH CDR1 is SEQ ID NO: 105; VH CDR2 is SEQ ID NO: 106; and VH CDR3 is SEQ ID NO: 107.

[0140] A recombinant antibody is a monoclonal antibody produced in vitro with genetic manipulation using synthetic genes. Heavy chain variable regions, light chain variable regions, and complementarity determining regions have the same meaning herein as discussed above.

[0141] In some examples, the antibody and / or polypeptide provided herein promotes and controls the lipid metabolism, particularly cholesterol biosynthesis, which aids in treating AML, myeloma, and other cancer tumor metastases and invasion. In further examples, LILRB3 agonist promotes and modulates the lipid metabolism and mTOR pathway, and controls tumor invasion. In certain examples, an antagonist controls lipid accumulation and cholesterol synthesis. In specific examples, an agonist controls and / or reduces inflammation (i.e., promote phagocytosis and reduce inflammation cytokines). In some examples, the agonists used are those with a high affinity.

[0142] Methods

[0143] Immune effector cells expressing the disclosed CARs can elicit an anti-tumor immune response. The anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response.

[0144] Adoptive transfer of immune effector cells expressing chimeric antigen receptors is a promising anti-cancer therapeutic. Following the collection of a patient’ s immune effector cells, the cells may be genetically engineered to express the disclosed CARs, then infused back into the patient.

[0145] The disclosed CAR-modified immune effector cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components, cytokines, or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are in some embodiments formulated for intravenous administration. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0146] When “an immunologically effective amount”, “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104to 109cells / kg body weight, such as 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0147] In certain embodiments, it may be desired to administer activated T cells to a subject and then subsequently re-draw blood (or have an apheresis performed), activate T cells therefrom according to the disclosed methods, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw / multiple reinfusion protocol may serve to select out certain populations of T cells.

[0148] The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (IV) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by IV injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection. In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as 0KT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

[0149] Methods of Treating Leukemia

[0150] The present disclosure, in one aspect, provides for a method of treating leukemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimeric antigen receptor polypeptide as disclosed herein.

[0151] The present disclosure, in one aspect, provides for a method of treating leukemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant antibody as disclosed herein.

[0152] In some examples, the leukemia comprises acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or myelodysplastic syndrome (MDS). In some examples, the leukemia comprises acute myeloid leukemia (AML).

[0153] Leukemia is a cancer of the body’s blood-forming tissues, including the bone marrow and the lymphatic system. In subjects with leukemia, the bone marrow produces an excessive amount of abnormal white blood cells, which do not function properly. Symptoms of leukemia include fever or chills, persistent fatigue / weakness, frequent or severe infections, losing weight without trying, swollen lymph nodes, enlarged liver or spleen, easy bleeding or bruising, recurrent nosebleeds, tine red spots in your skin (petechiae), excess sweating, especially at night, and / or bone pain or tenderness. Leukemia is believed to occur when blood cells acquire mutations in their genetic material, thereby resulting in the blood cells continuing to grow and divide. This blood cell production becomes out of control and the abnormal cells crowd out healthy blood cells in the bone marrow, leading to fewer healthy white blood cells, red blood cells, and platelets, causing the signs and symptoms of leukemia.

[0154] Leukemia is classified by how fast it progresses: acute leukemia and chronic leukemia. Acute leukemia worsens quickly and therefore requires aggressive and timely treatment. Chronic leukemia can produce no early symptoms and can go unnoticed or undiagnosed for years. A second type of classification is by the type of white blood cell affected: lymphocytic leukemia and myelogenous leukemia. Lymphocytic leukemia affects the lymphoid cells (lymphocytes), which form lymphoid or lymphatic tissue. Myelogenous leukemia affects the myeloid cells. Myeloid cells give rise to red blood cells, white blood cells, and platelet-producing cells.

[0155] Major types of acute leukemia include acute lymphocytic leukemia (ALL) and acute myeloid leukemia (acute myelogenous leukemia; AML). ALL is the most common type of leukemia in young children. ALL can also occur in adults. AML occurs in children and adults but is the most common type of acute leukemia in adults. Major types of chronic leukemia include chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). CLL is the most common chronic adult leukemia. CML mainly affects adults. A person with CML may have few or no symptoms for months or years before entering a phase in which the leukemia cells grow more quickly. Other types of leukemia include hairy cell leukemia, myelodysplastic syndromes, or myeloproliferative disorders.

[0156] In AML, the myeloid stem cells generally become a type of immature white blood cells called myeloblasts (or myeloid blasts). The myeloblasts in AML are abnormal and do not become healthy white blood cells, but rather become abnormal white blood cells, red blood cells, and / or platelets (also cells leukemia cells or blasts). Leukemia cells can build up in the bone marrow and blood, thereby creating less space for healthy white blood cells, red blood cells, and platelets. Leukemia cells can spread outside the blood to other parts of the body, including the central nervous system, such as brain and spinal cord, skin and / or gums. In some examples, leukemia cells form a solid tumor called a myeloid sarcoma, also called an extramedullary myeloid tumor, granulocytic sarcoma, or chloroma.

[0157] There are subtypes of AML which are based on the maturity of the cancer cells at the time of diagnosis and how different those cells are from normal cells. Furthermore, AML is described as untreated, remission, refractory, or recurrent. Untreated AML indicated that the disease is newly diagnosed. At least 20% of the cells in the bond marrow are blasts (leukemia cells) and there are certain gene changes. The complete blood count is abnormal and there are signs or symptoms of leukemia. AML in remission indicates that the complete blood count is normal, less than 5% of the cells in the bone marrow are blasts, and there are no signs or symptoms of leukemia in the brain, spinal cord, or elsewhere in the body. Refractory AML refers to the disease after treatment with chemotherapy wherein patients with untreated AML will not go into remission. Recurrent AML is cancer that has recurred after remission. AML can come back in the blood or bone marrow.

[0158] In further examples, the method further comprises administering a targeted therapy. In certain examples, the targeted therapy comprises gemtuzumab ozogamicin, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, or any combination thereof.

[0159] In certain examples, the targeted therapy comprises Arsenic Trioxide, Azacitidine, Daunorubicin Hydrochloride, Cyclophosphamide, Cytarabine, Daunorubicin Hydrochloride, Dexamethasone, Doxorubicin Hydrochloride, Idarubicin Hydrochloride, Mitoxantrone Hydrochloride, Pemigatinib, Prednisone, Quizartinib Dihydrochloride, Olutasidenib, Rituximab, Thioguanine, Tisagenlecleucel, Venetoclax, Vincristine Sulfate, or any combination thereof.

[0160] In specific examples, the method further comprises administering chemotherapy, radiation therapy, stem cell transplantation, or any combination thereof.

[0161] Chemotherapy is a cancer treatment that utilizes drugs to stop the growth of cancer cells, whether that is by killing the cells or by stopping them from dividing. Chemotherapy can be taken by the mouth or injected into a vein or muscle, in which case the drugs enter the bloodstream and can reach cancer cells throughout the body. In some examples chemotherapy can be placed directly into the cerebrospinal fluid, an organ, or a body cavity, in which the drugs mainly impact the cancer cells in those areas. In further examples, intrathecal chemotherapy can be used to treat adult AML that spread to the brain and spinal cord. Combination chemotherapy is another option in which treatment utilizes more than one anticancer drug. The way in which chemotherapy is administered depends on the subtype of AML being treated and whether leukemia cells have spread to the brain and spinal cord.

[0162] Radiation therapy is a cancer treatment that utilizes high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing. External radiation therapy uses a machine outside the body to send radiation toward the area of the body with cancer. Total body irradiation is another type of radiation therapy wherein radiation is sent toward the whole body.

[0163] Stem cell transplantation is a treatment in which stem cells (immature blood cells) are removed from the blood or bone marrow of the patient, or donor, and frozen and sorted. After the patient completes chemotherapy and / or radiation, the stored stem cells are thawed and given back to the patient through an infusion. The reinfused stem cells then grow into (and restore) the body’s blood cells.

[0164] Method of Reducing Tumor Activity Also disclosed herein is a method of reducing tumor activity in a subject with leukemia, the method comprising administering to the subject a therapeutically effective amount of the chimeric antigen receptor polypeptide as disclosed herein.

[0165] Further disclosed herein is a method of reducing tumor activity in a subject with leukemia, the method comprising administering to the subject a therapeutically effective amount of a recombinant antibody as disclosed herein.

[0166] In some examples, the leukemia comprises acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or myelodysplastic syndrome (MDS). In some examples, the leukemia comprises acute myeloid leukemia (AML).

[0167] In further examples, the method further comprises administering a targeted therapy. In certain examples, the targeted therapy comprises gemtuzumab ozogamicin, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, or any combination thereof.

[0168] In specific examples, the method further comprises administering chemotherapy, radiation therapy, stem cell transplantation, or any combination thereof.

[0169] Table 1. CAR-T Therapy Amino Acid Sequences

[0170] Table 2. CAR-T Nucleic Acid Sequences

[0171] Table 3. Antagonist Antibody Amino Acid Sequences

[0172] Table 4. Antagonist Antibody Nucleic Acid Sequences

[0173] Table 5. Additional Antagonist Antibody Sequences

[0174] Table 6. 55 C Mouse Sequences A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0175] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0176] EXAMPLES

[0177] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

[0178] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0179] Example 1: LILRB3 modulates acute myeloid leukemia progression and acts as an effective target for CAR T-cell therapy

[0180] LILRB3 regulates differentiation and proliferation in acute myeloid leukemia and can be targeted with monoclonal antibodies and CAR T cells to suppress leukemia growth. Identifying novel cell surface receptors that regulate leukemia cell differentiation and can be targeted to inhibit cellular proliferation is crucial to improve current treatment modalities in acute myeloid leukemia (AML), especially for relapsed or chemotherapy -refractory leukemia. Leukocyte immunoglobulin- like receptor type B (LILRB) is an immunomodulatory receptor originally found to be expressed in myeloid cells. LILRB receptors can be induced under inflammatory stimuli and chemotherapy treatment conditions. Blockade of LILRB 3 inhibited leukemia cell proliferation and leukemia progression. Additionally, treatment with LILRB3 blocking antibodies upregulated myeloid lineage differentiation transcription factors, including PU.l, C / EBP family, and IRF, whereas phosphorylation of proliferation regulators, e.g., AKT, cyclin DI , and retinoblastoma protein (Rb), was decreased. Conversely, transcriptomic analysis showed LILRB3 activation by agonist antibodies may enhance leukemia survival through upregulation of cholesterol metabolism, which has been shown to promote leukemia cell survival. Moreover, LTLRB3- targeted CAR T cells exhibited potent antitumor effects both in vitro and in vivo. Taken together, the results suggest that LILRB3 is a potentially potent target for multiple treatment modalities in AML.

[0181] Leukocyte immunoglobulin-like receptors B (LILRB) are a family of transmembrane receptors containing intracellular immunoreceptor tyrosine-based inhibition motifs (ITIMs), which act as an immune checkpoint, playing an important role in controlling T-cell activation, macrophage function, and immunologic inflammatory responses, such as TNF-a secretion. Compared to other members of the LILRB family, L1LRB3 was more myeloid-specific and therefore represented an attractive target for AML.

[0182] The type II interferon IFNy is a pleotropic cytokine with a role in cancer immunobiology. In AML, IFNy is produced by leukemic cells as well as immune cell subsets, contributing to the immunophenotype of the AML tumor microenvironment. Interest in therapeutic IFNy continues, with an ongoing clinical trial examining exogenous IFNy treatment to induce graft-vs-leukemia effect in AML / MDS patients (NCT04628338).

[0183] IFNy as well as AraC, induced LILRB3 expression in AML cell lines and primary AML cells. LILRB 3 was a mediator in AML proliferation and phenotype determination, with antagonist antibodies inhibiting proliferation and promoting a less proliferative and more mature, quiescent phenotype. LILRB 3 specific CAR T-cells are potently cytotoxic to primary AML cells and AML cell lines in vitro. In vivo, LILRB3 specific CAR T-cell therapy effectively reduced leukemic burden and improved mortality with lasting remissions in mice.

[0184] Methods

[0185] Cell lines and in vitro assays

[0186] AML cell lines, U937, HL-60, THP-1, MV411, C1498 purchased from the American Type Culture Collection (ATCC, Manassas, VA), were cultured in RPMI 1640 (Coming 10043CV) supplemented with 10% FBS, 0.1 mM non-essential amino acids (Coming 25-025-CIR), and 100 nM penicillin / streptomycin (Gibco 15140122). Short tandem repeat cell line authentication was performed by ATCC prior to delivery. Cell lines are maintained Mycoplasma contamination free with routine testing performed via PCR (abm cat. G238). The LILRB3 transduced cell lines were generated by retroviral transduction of THP-1 cells with LILRB 3 using a custom pMX-puro- LILRB3 plasmid (pMX- puro: Cell Biolabs RTV-012 with inserted full-length LILRB3 plasmid). For the PBMC-U937 coculture assays, 5xl04U937 were cultured in a 96-well round bottom plate in RPMI-1640 media supplemented with cytarabine at a cytotoxic concentration of 500 nM for 48 hours. The culture media was then removed and 2x10shuman PBMC were added for an E:T ratio of 1 :0.25. Supernatants were collected after 60 hours and IFNy was measured by ELISA (ELISA MAX™ Deluxe Set Human IFNy), n=3 wells per donor per condition. This assay was repeated with two healthy human PBMC donors. Using a third healthy donor, 50 p L of cell-free supernatant was removed from each well and diluted 1 : 1 in 50 pL of RPMI containing 5xl04U937 cells. 10 pg / mL of IFNy blocking antibody (InVivoMAb anti-human IFNy, clone B27) was added to one group of wells. After 24 hours cells were harvested for flow cytometry as described below.

[0187] For cell proliferation assays, AML cells were pretreated with 50 ng / ml IFNy followed by treatment with 5 pg / mL LILRB3 antibodies for 72 hours. Cells were pulsed with 1 DCi tritiated thymidine (Perkinelmer NET027X005MC) for the last 16 hours of culture. For quantitative-PCR (q-PCR), cells were treated for 72 hours in the presence of anti-LILRB3 (5 pg / mL) or isotype-matched Ig.

[0188] Flow cytometric analysis

[0189] In all flow assays, dead cells were excluded by fixable viability dye (Invitrogen Cat:65- 0863-14). Commercially available antibodies were used for all assays, LILRB3 (PE, Biolegend Cat: 337704), CD3 (APC, Biolegend Cat:317318), CD19(PE, Biolegend cat:302208), CD25 (AlexaFluor 647, Biolegend Cat: 302618, CD33 (PE-Cy7, Biolegend Cat: 366618), CD34 (APC, Invitrogen Cat: 17- 0349-42, PE, Biolegend Cat:343506), CD38(FITC, Biolegend Cat: 303504, PE-cy7, ebioscience 25- 0389-71), CD45 (FITC, Biolegend Cat:304006, APC, Biolegend Cat:368512), CD69 (PE, 310906), CD86 (FITC, Biolegend Cat:374204), CD107a (APC, Biolegend Cat: 328620) CD163 (APC, Biolegend Cat:33610), CD206 (APC-Cy7, Biolegend Cat: 321120) , HLA-DR (PE, Biolegend Cat:307606).

[0190] Clonal hybridoma cells were cultured in ClonaCelLHY Medium A (StemCell Technologies Cat:03801) followed by adaptation to serum-free conditions using Hybridoma-SFM (ThermoFisher Scientific Cat: 12045-076). Antibody-containing supernatants were harvested by centrifugation (800xg, 10 mins), then concentrated using Amicon Ultra- 15 centrifugal filter concentrators with a nominal molecular weight limit of 100 kDa. Concentrated supernatants were then purified using Nab Protein A / G Spin Kit (Thermo Fisher Scientific) per manufacturer’s instructions. Purified monoclonal antibodies were desalted using Zeba™ Spin Desalting Columns, 7K MWCO (Thermo Fisher Scientific) followed by concentration using centrifugal filters (Thermo Fisher Scientific), if needed. LPS contamination was assessed in all purified antibody preparations using the Pierce Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific). Agonist and antagonist antibodies were classified using functional assays on human PBMC, where agonist decreased TNF-a production, IFNy production, and CD3 stimulated T cell proliferation compared to IgG control, whereas the antagonist antibodies increased each of these indicators.

[0191] Biolayer Interferometry Binding Assay

[0192] Real-time binding affinities of LILRB3-His (SinoBiological) and L1LRB3 antibodies were determined through the use of biolayer interferometry on an Octet Red system (Fortebio, Menlo Park, CA, USA). Anti-LILRB3 antibody (10 pg / mL) was coupled to kinetics grade Protein G / mlgG high binding biosensors (Fortebio). Sensors coated with LILRB3 antibodies were allowed to bind to LILRB3-His in PBS with 0.1% (v / v) Tween-20 and 10% DMSO at increasing concentrations. Binding kinetics were calculated using the Octet Red software package, which determines the best fit for the observed binding curves and calculates the association rate constants. LILRB3-His was allowed to dissociate by incubation of the sensors in PBS with 0.1% Tween-20 and 10% DMSO. Best fit dissociation curves were determined, and dissociation rate constants were calculated. Binding affinities (kD) were calculated as the kinetic dissociation rate constant divided by the kinetic association rate constant.

[0193] Generation and differentiation of human monocyte-derived macrophages

[0194] Buffy coats from healthy donors were purchased from the New York Blood Center and Gulf Coast Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated by a LymphoPrep (StemCell #07851). CD33+ monocytes were purified from PBMCs using CD33+ magnetic beads (Miltenyi Biotech, Cat: 130-045-501). CD14 was not used to purify monocytes but was used later to assess macrophage differentiation. Purified CD33+ monocytes were treated with anti-LILRB3 or isotype control (1 pg / mL) in the presence of M-CSF (50 ng / ml) (PeproTech: SOO- 25) for 5 days. After 5-day culture, immature macrophages were obtained for flow cytometric analyses or further stimulated with LPS (50 ng / ml) for 16-24 hours. The supernatants were collected and TNF-a concentrations in supernatants were assessed by ELISA (eBioscience).

[0195] Western blot

[0196] U937 and LILRB3-transduced THP-1 cells were treated with anti-LILRB3 or isotype control Ig (1 pg / mL) for 24 h followed by stimulation with LPS (50 ng / ml), for 0, 10, 30 min. The cells were lysed using a cell lysis reagent (Sigma- Aldrich). Protein samples were separated on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked using a 4% skim milk solution, then incubated with an appropriate antibody followed by a secondary antibody conjugated to horseradish peroxidase. Antibodies for total p38, phospho-p38 (p- p38), total Akt, p-Akt, total Retinoblastoma (Rb), phospho-Rb (Ser807 / 811), total mTOR, and phospho-mTOR were purchased from Cell Signaling Technology, Inc. (Beverly, MA) and the antibody for GAPDH was purchased from Santa Cruz Biotechnology. The immunoreactive bands were visualized using the ECL system (Thermo Scientific).

[0197] Generation of anti-LILRB3 hybridomas

[0198] LILRB3-specific antibodies were produced and screened. Briefly, LILRB3 monoclonal antibodies were generated by immunizing mice with LILRB3 cDNA followed by boosting with LILRB3 recombinant proteins. Commercially available anti-LILRB3 was purchased from Biolegend (MKT5.1,Cat:337704). Outgrowing fused hybridoma clones were expanded and culture supernatants were collected. LILRB3 binding activity was assessed using a LILRB3 transduced 2B4 T-cell hybridoma cell line. LILRB3+ 2B4 cells were incubated with LILRB3 antibody followed by staining with polyclonal goat anti-mlgG secondary antibody (Biolegend Cat:405307) and flow cytometric analysis. LILRB3 binding was confirmed in multiple cell lines transduced with L1LRB3 including HT29 and HCT116.

[0199] In-vivo leukemia proliferation

[0200] All animal experiments were conducted in accordance with institutional 1ACUC guidelines. 3xl06U937 or IxlO6C1498 LILRB3 cells were subcutaneously implanted into NSG- SGM3 mice (Jackson Laboratories, Cat:013062) or C57BL / 6 LILRB3 transgenic mice, respectively. C57BL / 6 LILRB3 transgenic mice were generated by pronuclear injection of BAC DNA. Before injection, U937 cells were pretreated with 50 ng / ml IFNy to stimulate LILRB3 expression. After tumor was established (average tumor 3-5mm in diameter), mice were treated with anti-LILRB3 or isotype-matched control Ig (100 Dg / mouse) via intraperitoneal injection followed by subsequent injections repeated every 3 days. Tumor size was measured on the same day as injections and every 3 days, thereafter. Tumors were dissected for further analyses following the 8th injection or when tumor sizes grew to larger than 20 mm in diameter. Tumor volumes were calculated via the formula: volume (mm3) = (length x width2) / 2.

[0201] Reverse -phase protein array (RPPA)

[0202] U937 cells (1 .5 x 106cells) were pretreated with or without IFNy for 48 hours followed by overnight treatment with anti-LILRB3 (1 pg / mL) or isotype- matched Ig control. Cells were lysed for 20 minutes in lysis buffer: 1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCh, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 10% glycerol, containing freshly added protease and phosphatase inhibitors from Roche Applied Science (Cat: 05056489001 and 04906837001). Cell lysates were centrifuged at 14,000 RPM for 20 minutes and supernatants were collected. The lysate was mixed with 4 x SDS buffer without bromophenol blue and boiled for 5 minutes at 100°C. RPPA was performed at the MD Anderson Functional Proteomics RPPA core facility. Heatmaps of differentially expressed genes were created using Heatmapper. Expression values are z-score normalized, and high, intermediate, and low expressions are shown as red, black, and green, respectively.

[0203] RNA sequencing and analysis

[0204] RNA sample quality and quantity were assessed using Nanodrop, agarose gel electrophoresis, and Agilent 2100. cDNA library preparation was performed using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on the Illumina Hiseq X Ten at 150bp paired end reads with 20M read depth. All samples had Q30 > 90%. Both library preparation and sequencing were performed by Novogene (Sacramento, California). Differential gene analysis was performed using the HISAT2-Cufflinks workflow. Volcano plots were generated using the EnhancedVolcano R package. Pathway analysis and upstream regulator analysis were performed using Ingenuity Pathway Analysis (IPA) software (Qiagen).

[0205] Generation of the retroviral vector encoding LILRB3-CAR

[0206] The retroviral vector encoding the LILRB3-CAR was based on a LILRB 3 -specific hybridoma cell line generated in this manuscript. A LILRB3-specific single chain variable fragment (scFv) was derived from this hybridoma and subcloned into a SFG retroviral vector encoding a 4- IBB. zeta CAR, a 2A sequence, and truncated CD19 (tCD19). The 4-lBB.zeta CAR consisted of an immunoglobulin heavy-chain leader peptide, the LILRB3-specific scFv, a human IgGl hinge, a CD8a transmembrane domain, and a 4-lBB.zeta signaling domain. CAR cloning was verified by sequencing. RD114 pseudotyped retroviral particles were generated by transient transfection of 293T cells.

[0207] Generation of LILRB3-CAR T- cells

[0208] Human PBMC from healthy donors were stimulated with CD3 / CD28 on day 1 and cultivated in RPMI medium supplemented with IL-7 (10 ng / ml) and IL- 15 (5 ng / ml) on day 2. On day 3, the primary T cells were transduced with retroviral particles encoding the LILRB3-CAR and tCD19. On day 5, the transduction efficiency was assessed by CD19 expression of transduced T cells.

[0209] Functional evaluation of CAR T-cells in vitro and in vivo

[0210] CAR T-cells were cocultured with primary AML cells (AML 8), LILRB 3 -transduced MV4-11, and LILRB3-transduced THP-1 and their cytolytic activity was evaluated using LDH release assays (CytoTox 96 Non-Radioactive Cytotoxicity Assay). Cytolytic activity was measured at the following E:T ratios: 0.1 :1, 0.2:1, 1: 1, 5:1, 10:1. For 7-hour cytotoxicity studies CAR T-cells were cocultured with primary AML cells (AML 8), LILRB3- transduced MV4-11 , and LILRB3-transduced THP-1 and their cytolytic activity was evaluated using LDH release assays (CytoTox 96 Non-Radioactive Cytotoxicity Assay). Cytolytic activity was measured at the following E:T ratios: 0.1:1, 0.2:1, 1:1, 5:1, 10:1. Percent of cell lysis was calculated using the formula: (Experimental LDH Release (OD450) - Effector Spontaneous Release (OD450) - Target Spontaneous Release (OD450) ) / (Target Maximum LDH Release (OD450) - Target Spontaneous Release (OD450)). Maximum LDH release was determined by the addition of the provided lysis solution. 24-hour cytotoxicity was performed using fluorescently labelled target cells. Target cells were imaged using Incucyte live cell imaging or flow cytometry as indicated. Percent of cell killing was determined using the following equation: %cytotoxicity= 100 - ((test RFU / no T cell average RFU)*100), where RFU was relative fluorescent units.

[0211] For the mv411 xenograft in vivo study, NSG-SGM3 mice were injected intravenously (iv) with IxlO6firefly luciferase expressing LILRB 3 -transduced MV4-11 cells. On day 5 post tumor cell injection, mice were randomized to receive a single iv dose of 1) PBS vehicle control, 2) 5xl06untransduced T- cells (Control T-cells), or 3) 5xl06LILRB3-CAR T-cells. In vivo experiments were repeated using two different T cell donors. Control T-cells and CAR T-cells were donor- matched. Tumor growth was followed by serial bioluminescence imaging.

[0212] For the human PDX in vivo model, 2xl07AML8 PBMC were injected into NSG-SGM3 mice via tail vein 24 hours following busulfan myelo-conditioning at 30 mg / kg. Engraftment of human PBMC was verified at day 30 and mice were grouped by peripheral blood human CD33+ cell percentage and randomized to receive 5xl06CAR T vs control T-cells. 10 days after T cell engraftment mice were sacrificed and bone marrow was analyzed via flow cytometry for AML cell content as a percentage of total viable bone marrow cells.

[0213] In vivo hematology and serum chemistry toxicity studies

[0214] To evaluate the toxicity of LILRB3 specific CAR T-cell therapy, an LILRB3 transgenic autologous murine CAR T-cell model was employed. CAR T-cells were generated using splenocytes from LILRB3 transgenic mice transduced with the CAR construct depicted in Figure 15G. Mice were injected 5xl06CAR T vs control T vs PBS via tail vein injection and sacrificed 9 days subsequently for determination of complete blood counts and serum chemistry. Complete blood counts and chemistry panels were performed by the MD Anderson DVMS Veterinary Pathology Core on Advia 2120i (Siemens) and Integra 400 (Roche) instruments, respectively.

[0215] Statistical analysis Data are presented as the mean ± SD unless otherwise specified. Biological replicates are represented as individual data points within each group. In-vitro data were compared using a two- tailed Student's / -test for tests of two samples and ANOVA for samples of three or more samples. ANOVA P values were adjusted for multiple comparisons using Tukey’s correction; student t-test P values were adjusted for multiple comparisons using Holm-Sidak correction. Repeated measures data (in-vivo time course experiments) were compared using repeated measures ANOVA with Geisser-Greenhouse correction. Percent data was transformed prior to analysis using the square root transformation to meet normality and heteroscedasticity assumptions of ANOVA. All analyses were conducted using Prism or SPSS. Data were considered statistically significant at a value of p < 0.05.

[0216] Study Approval

[0217] Human samples were obtained under the approved IRB protocol Pro00007175:1 at Houston Methodist. Human samples were obtained anonymously, without access to clinical data. All animal procedures were approved by the IACUC of Houston Methodist Research Institute.

[0218] Results

[0219] L1LRB in human AML

[0220] To determine the translational potential of the LILRB protein family as targets for AML treatment, LILRB expression in PBMCs from AML patients was investigated. CD33+ myeloid cells from 14 AML patients were screened for LILRB 1-4 surface expression via flow cytometry. LILRB 3 exhibited the highest surface expression levels among LILRB members in this patient population. Notably, wide variation of LILRB expression was observed, suggesting that LILRB expression was patient dependent (Figure 1A, Table 14, Figure 15 A; Figure 17). Moreover, LILRB3 expression varies across FAB subtype, with enriched expression predominant in the M4 and M5 subtypes (Figure 15B). Elevated LILRB3 expression was correlated with increased mortality in AML — analysis of TCGA data demonstrates significantly decreased survival for patients in the high and intermediate tertiles of LILRB3 expression compared with the patients in the lowest LILRB 3 expression tertile (Figure IB).

[0221] Table 14. LILRB expression in patients.

[0222] LILRB3 expression has increased specificity for AML vs healthy human hematopoietic progenitor cells. Analysis of aggregated microarray datasets demonstrates that AML cells have increased expression of LILRB3 than healthy donor derived normal hematopoietic progenitor cells, but decreased expression than mature monocytes and PMN (Figure 1C). Similarly, analysis of proteomic datasets demonstrates increased LILRB3 protein expression on AML samples compared with lineage (Lin-) cells from healthy donors (Figure 9G). Consistently, a low percentage (7.9 ± 1.3%) of CD45+ / CD38- / CD34+ gated human hematopoietic stem / progenitor cells isolated from the peripheral blood of healthy human donors demonstrated surface LILRB3 expression (Figure 8E - Figure 8F).

[0223] LILRB3 expression in leukemia cells and generation of functional LILRB3 antibodies

[0224] Since IFNy-related gene expression profiles can be used to predict both chemoresistance and response to immunotherapy in refractory AML, chemotherapy induced IFNy can enhance the expression of the LILRB family of proteins. Additionally, chemotherapy drugs commonly used in AML were screened for the ability to modulate LILRB3 expression. The U937 cell line, a well- documented AML model, was a neoplastic histiocytic cell line which has native LILRB3 expression. Treatment with IFNy, AraC and LPS induced upregulation of LILRB3 expression in U937 cells (Figure 2A - Figure 2B, Figure 9A - Figure 9B), with increased expression of LILRB3 persisting in vitro after withdrawal of IFNy / AraC in serum free conditions (Figure 19C). Another AML cell line, HL60, similarly exhibited LILRB3 upregulation following IFNy exposure (Figure 9D). Notably, in vivo AraC may upregulate LILRB3 through multiple mechanisms, both acting directly on the AML cells and through the induction of IFNy response in PBMC exposed to AraC treated AML cells. Coculture of human PBMC with AraC pretreated U937 cells increased PBMC IFNy production when compared to coculture with untreated U937 cells (Figure 2C). Low levels of IFNy were present in the supernatant of AraC treated U937 cells in the absence of PBMC, suggesting the majority of IFNy production was derived from PBMC and not U937 cells (Figure 9E). Moreover, supplementation of U937 cells with conditioned media derived from the PBMC / AraC-U937 coculture increased LILRB3 expression on U937 cells, compared to conditioned media derived from the coculture of PBMC / untreated-U937 cells (Figure 2D). This upregulation of LILRB3 was attenuated with the addition of anti-IFNy antibody. LILRB3 upregulation was also observed in CD34+ gated primary AML patient samples treated with IFNy and poly(LC) (polyinosinic:polycytidylic acid), a potent inducer of interferons (Figure 2E, Figure 9F, Figure 9G).

[0225] To investigate the role and biological significance of LILRB3 in AML, monoclonal antibodies targeting LILRB3 (aLILRB3) were generated. Antibodies generated from hybridoma production were screened and characterized using both binding and functional assays in normal human myeloid cells. As high homology exists between the LILRB family members, bio-layer interferometry was performed to demonstrate antibody specificity and affinity to LILRB 3 (Figure 2F). In M2 skewed monocyte-derived macrophages (MDM), treatment with two distinct anti- LILRB3 antibodies (aLILRB3_l, aLILRB3_2) increased secreted TNF-a (Figure 2G), while simultaneously decreasing the surface expression of the M2 markers (CD 163, CD206) and increasing the surface expression of the Ml markers (HLA-DR, CD86) (Figure 2H, Figure 9J). Taken together, these results demonstrate functional antagonism of LILRB3. Agonist antibody activity was designated to clones which inhibit TNF-a production and T-cell proliferation in PBMC and skew MDM to M2 phenotype (data not shown).

[0226] Inhibition of in vitro and in vivo leukemia cell proliferation by anti-LILRB3 antagonist antibodies

[0227] The direct effect of antagonist anti-LILRB3 antibodies on cell proliferation in the leukemia cell lines was further evaluated. IFNy treatment sensitized U937 cells to anti-LILRB3 antibody treatment, in a dose-dependent manner, resulting in decreased proliferation (Figure 21). Treatment with IFNy (50 ng / ml) in combination with different anti-LILRB3 antibodies (aLILRB3_l, aLILRB3_2) (5 pg / mL) significantly inhibited proliferation of both U937 and HL-60 cells after 72 hours incubation when compared to IgG control -treated cells (Figure 2J, Figure 2K).

[0228] To assess the efficacy of anti-LILRB3 proliferation inhibition in vivo, U937 cells were subcutaneously implanted in NSG-SGM3 mice. Following tumor development, mice were treated with either antagonist anti-LILRB3 antibody or control IgG via intraperitoneal injection every three days. Multiple antagonist LILRB3 clones inhibited proliferation in vivo, with marked reduction in tumor volume and mass in anti-LILRB3 treated mice compared to IgG controls (Figure 10A - Figure 10F). Furthermore, this was repeated using a syngeneic leukemia model, where the broad immunostimulatory effects of LILRB3 blockade may enhance the direct AML cell acting effects in reducing tumor burden. The C1498 cell line was transduced with LILRB3 and subcutaneously implanted in LILRB3 transgenic C57BL / 6 mice to avoid LILRB3 mediated rejection of C1498 cells. Consistent with the xenograft results, antagonist anti-LILRB3 antibody diminished tumor growth with tumor regression observed in multiple mice (Figure 10F).

[0229] LILRB3 blockade promotes differentiation-related gene expression and surface markers

[0230] As the natural ligand for LILRB3 was not well established, agonist and antagonist antibodies were used to elucidate the downstream effects of LILRB3 signaling in AML. Since antagonist anti-LILRB3 antibodies exerted a regulatory effect on macrophage differentiation (Figure 2H) and AML cell proliferation (Figure 21 - Figure 2K, Figure 10A - Figure 10F), their effect on signature myeloid differentiation transcription factors in U937 and HL-60 cells was assessed. Pre-incubation with either IFNy or AraC followed by antagonist anti-LILRB3 treatment resulted in higher transcription of myeloid differentiation genes, such as PU.l, CCAAT / enhancer- binding proteins (CEBP-a, CEBP-P), and interferon regulatory factors (IRF1, IRF8) in U937 cells (Figure 3A, Figure 3B) and PU.l, CEBP-P, IRF1 and IRF5 in HL-60 cells (Figure 11 A). Furthermore, HL-60 cells treated with antagonist anti-LILRB3 antibody had increased CD 14 expression compared to IgG control. Combination with IFNy treatment further increased CD 14 expression (Figure 1 IB).

[0231] To contrast the effects of functional agonism and antagonism of LILRB3 signaling in AML, the expression of AML phenotype-contributing genes was assessed in LILRB3 transduced (Figure 3C) and IFNy pretreated U937 cells (Figure 3D) treated with either nonspecific IgG, antagonist anti-LILRB3 or agonist anti-LILRB3 antibodies. Antagonist anti-LILRB3 antibody treatment increased expression of CEBP genes, IRF genes, JUNB, and PU.l, and decreased sternness-related genes RUNX1 and CMYC. The agonist, conversely, decreased CEBPs and IRF4 / 8 and increased CMYC and RUNX1 gene expression in both models of induced LILRB3 expression in U937 cells.

[0232] Assessment of signaling associated with LILRB3 antagonism by reverse-phase protein array (RPPA), western blot, and RNA sequencing

[0233] Antagonism of LILRB3 alters the phosphoproteomic landscape of U937 cells. U937 cells treated with antagonist anti-LILRB3 antibody exhibited decreased phosphor-Rb, increased PTEN, and decreased cyclin D3 expression when compared to control IgG via reverse-phase protein array analysis (RPPA) (Figure 4A). Western blot analysis of LPS- stimulated U937 cells treated with antagonist anti-LILRB3 antibody reproduced the trends observed in RPPA, with decreased phosphor-Rb and pAkt as compared to the control IgG antibody (Figure 4B, Figure 4C). Similar results were observed using the THP-1 cell line transduced with LILRB3 (Figure 4D). Interestingly, levels of phospho-p38 were increased in both cell lines following anti-LILRB3 antibody treatment, indicating the activation of stress pathways (Figure 4B - Figure 4D).

[0234] To assess gene expression profile changes, RNA sequencing was performed on IFNy pretreated U937 cells treated with either IgG control or antagonist anti-LILRB3 antibody. 810 significant differentially expressed genes were represented in the heatmap (Figure 5A). Pathway analysis with ingenuity pathway analysis (IP A) revealed downregulation of genes involved in eIF2 signaling and mTOR signaling (Figure 5B). Inhibition of mTOR signaling was also observed at the protein level via western blot (Figure 12A - Figure 12B). Interestingly, upstream regulator analysis predicted a downregulation of MYC and Rb activity (Figure 5C). Gene Set Enrichment Analysis (GSEA) recapitulated the results observed with pathway analysis and upstream regulator analysis. Antagonist anti-LILRB3 antibody treatment decreased mTOR signaling-related genes and decreased hallmark cMyc signature genes when compared to IgG treatment in U937 cells while also reducing the expression of leukemia sternness signature genes (Figure 5D, Figure 13 A - Figure 13F).

[0235] Alternatively, agonist anti-LILRB3 antibodies were used to determine the effect of stimulation of LILRB3 signaling in leukemia cells. RNA sequencing of U937 cells pretreated with IFNy and either IgG control or agonist anti-LILRB3 antibody revealed 904 significant differentially expressed genes (Figure 5E, Figure 5F). Notable downregulated genes include inflammatory chemokine CXCL10 and CXCL11. Genes upregulated by agonist LILRB3 antibody include IL10, CD163, and MMP9, which together may promote an immunosuppressed microenvironment supporting tumor invasion. Pathway enrichment analysis using IPA predicted an increase in cholesterol biosynthesis and regulation of SREBP signaling (Figure 5G). Additional upregulated pathways consistent with ITIM agonism were also observed including T cell exhaustion signaling and the Th2 pathway.

[0236] LILRB 3 -specific CAR T-cells demonstrate antileukemic activity against cell lines and patient derived samples in vitro and in vivo

[0237] To confirm the in vivo efficacy of LILRB3-CAR T-cells, firefly luciferase-expressing LILRB3- transduced MV4-11 cells were implanted into NSG-SGM3 mice. Five days later, mice were randomized into groups that received PBS (n=3), healthy donor derived control T-cells (n=5), or healthy donor derived LILRB3-CAR T-cells (n=7) (Figure 6D). LILRB3-CAR T-cells produced a marked reduction in leukemic burden and improved survival compared to control groups (Figure 6E - Figure 6H). Furthermore, all mice treated with LILRB3-CAR T-cells showed lasting remission with undetectable tumor burden by luminescent imaging on day 96 and survival eclipsing 100 days post tumor inoculation.

[0238] Additionally, autologously generated CAR T cells derived from an AML patient sample (AML8) were evaluated in vitro and in vivo. AML patient derived T cells demonstrated increased in vitro killing of LILRB 3 -transduced MV4-11 and U937 cells across E:T ratios compared with control T cells (Figure 61, Figure 15C, Figure 8D) with increased surface expression of the activation marker CD25 and the degranulation markers CD 107a (Figure 6J - Figure 6K). AML patient derived LILRB 3 CAR T cells also show increased proliferation at baseline, which was further induced through exposure to LILRB 3 protein (Figure 15E) compared with untransduced T cell controls.

[0239] In vivo efficacy of AML patient derived LILRB3 CAR T cells was demonstrated through an autologous PDX model. AML PBMC were injected into NSG-SGM3 mice via tail vein 24 hours following busulfan myeloconditioning. Engraftment of human PBMC was verified at day 30 and mice were grouped by peripheral blood human CD33+ cell percentage and randomized to receive 5xl06CAR T vs control T-cells (Figure 6L). 10 days post T cell engraftment, CAR T-cell treated mice had significantly decreased AML burden compared with control T-cell treated mice. 3 of 4 mice were without measurable disease in the bone marrow following treatment (Figure 6M), with CAR T persistence observed in splenocytes at day 10 (Figure 15F).

[0240] Murine LILRB3-specific CAR T-cells demonstrate in vitro AML cytolysis with a favorable safety profile in vivo

[0241] To evaluate the toxicity of LILRB3 specific CAR T-cell therapy, an LILRB3 transgenic autologous murine CAR T-cell model was employed. CAR T-cells were generated using splenocytes from LILRB3 transgenic mice transduced with the CAR construct depicted in Figure 15G. Functional activity of the murine CAR T-cells were confirmed with LILRB 3 transduced C1498 murine AML cell killing (Figure 7A) and corresponding IFNy release (Figure 15H). For toxicity studies LILRB3 transgenic naive mice were adoptively transferred with 5x106CAR T vs control T vs PBS and sacrificed 9 days subsequently for determination of complete blood counts and serum chemistry (Figure 7B - Figure 7K). LILRB3 specific CAR treated mice demonstrated mild monocytopenia; however, no other major hematologic or metabolic disturbance was observed. An additional cohort of LILRB3 transgenic mice was treated with 2 doses of CAR T vs control T-cells vs PBS and followed for weight change, no significant weight loss or change in body condition was observed in any group (Figure 7L).

[0242] Discussion

[0243] Although knowledge of the role of LILRB3 in AML has expanded recently with the demonstration that LILRB3 acts through TRAF2 and cFLIP to promote AML cell survival, much remains to be known about LILRB3 signaling in leukemia. In AML patients, increased expression was associated with increased mortality (Figure 1). Notably, LILRB3 was consistently enriched in the moderately poor prognosis M4 / M5 subtypes while LILRB3 expression decreased in the better prognosis M3 subtype. This expression distribution may contribute to the difference observed in survival due to LILRB3 expression, as when the population was subset for the M4 / M5 subtypes only, there was no statistical difference in survival (Figure 15D). Moreover, LILRB3 directed therapies may be preferentially suited for AML patients with the M4 / M5 subtypes, who currently do not have subtype specific therapies available.

[0244] Inflammatory cytokines, such as IFNy and chemotherapy increase LILRB3 expression (Figures 2A-2K), potentially through the alteration of the epigenetic landscape of these cells, promoting transcriptional activators or inactivating suppressors of LILRBs. Robust interferon secretion from human PBMC exposed to AraC pretreated AML cells was demonstrated. Therefore, AraC may be able to induce LILRB3 via multiple mechanisms in vivo, through both direct action on the tumor cells and interferon induction from stimulated immune cells in the tumor microenvironment. Interestingly, IFNy signaling in multiple cancers induces a multigenic program resistant to immune checkpoint therapies, and persistent IFN signaling results in increased resistance to radiotherapy and immune checkpoint blockade therapies including anti-CTLA4 and anti-PD-Ll possibly due to induction of LILRB proteins.

[0245] Normal myeloid cells, such as monocytes and macrophages, also express the LILRB family of proteins. Blockade of LILRB3 with antibodies on M-CSF cultured MDM promoted TNF-a secretion and reduced CD 163 surface expression (Figures 2A-2K). CD 163 is a scavenger receptor found in tumor-infiltrating M2-like anti-inflammatory macrophages and is correlated with decreased overall survival in multiple cancer types. This suggests that the antagonist antibodies are functional and can promote a pro-inflammatory phenotype (Ml -like) in macrophages. These observations are consistent with the increased inflammatory responses observed in MDSC populations in PIRB knockout mice.

[0246] LILRB3 blockade via anti-LILRB3 antagonist antibodies decreased cell proliferation in both U937 and HL-60 cells in vitro and also inhibited tumor growth in vivo (Figure 21 - Figure 2K, Figure 10A - Figure 10F).

[0247] The results suggest that treatment with antagonist anti-LILRB3 promotes a prodifferentiation transcriptome in AML cells. Quantitative PCR showed that U937 cells pretreated with IFNy or AraC subsequently treated with antagonist anti-LILRB3 antibodies had increased expression of PU.l, C / EBP, and IRF family of genes as compared to control IgG-treated cells (Figure 3 A - Figure 3D). PU. l, C / EBPa, C / EBPp, IRF1 and IRF8 are transcription factors that regulate myeloid and granulocytic differentiation and maturation of progenitor cells. C / EBPa and IRF8 play roles in the differentiation of macrophages and increased IRF8 expression has been shown to inhibit AML growth. PU.l, a transcription factor that plays a role in myeloid cell differentiation, was often downregulated in AML by several oncogenic products such as RUNX1- ETO, FLT3-ITD, and PML-RARa. Transgenic mice with decreased PU. l expression in hematopoietic stem cells and myeloid progenitors compared to wild-type mice develop an aggressive form of AML. C / EBPa was involved in the maturation of myeloid progenitors into granulocytic / myeloid progenitors and further granulocytic differentiation in myeloid cells. In mouse models of myeloid leukemia, C / EBP-P promotes neutrophilic differentiation and improves overall survival. Interestingly, all-trans retinoic acid (ATRA) promotes differentiation in acute promyelocytic leukemia ( APL) cells by increasing PU.1 and C / EBPP expression. Despite inducing complete remission in a high proportion of patients with acute promyelocytic leukemia, clinical relapses can occur with continued ATRA treatment due to acquired resistance. Therefore, further evaluation of the combination of ATRA and anti-LILRB3 blockade may be worthwhile. Additionally, functional antagonism of LILRB3 promoted an anti-proliferative phosphoproteomic state. RPPA analysis revealed that U937 cells treated with antagonist anti-LILRB3 antibody had decreased phospho-Rbl protein levels compared to control-treated cells (Figure 4A). This result was confirmed by western blot, and an increase in phospho-p38 in anti-LILRB3 antibody-treated leukemia cells compared IgG-treated controls at 30 minutes was observed following stimulation (Figure 4B - Figure 4D). p38 was upstream of pRb and an increase in phospho-p38 results in decreased phospho-pRb. Rb contributes to cell proliferation and cell cycle progression. In its hypo- phosphorylated form, pRbl remains bound to the E2F transcription factor and prevents progression through the Gl / S phase resulting in decreased proliferation.

[0248] Cumulatively these data suggest LILRB3 blockade provides a multifaceted mechanism for leukemia differentiation. The proposed model was that anti-LILRB3-mediated blockade increases p38 signaling, decreases phospho-pRb, and promotes expression of the differentiation-related transcription factors PU. 1, C / EBP, IRF1, resulting in decreased cell proliferation and enhanced cell differentiation (Figure 14). This model was supported by pathway analysis of differentially expressed genes observed through RNA sequencing (Figure 5A - Figure 5D). Although these cellular changes could potentially induce alterations or arrest in the cell cycle, and therefore enhance the antiproliferative effects of chemotherapy, this has yet to be directly demonstrated. Antagonist anti-LILRB3 treated U937 cells showed significant inhibition in gene sets related to eIF2, eIF4, and mTOR pathways which may be related to decreased Akt phosphorylation (Figure 4A - Figure 4D; Figure 5A - Figure 5G). Notably, members of the eIF4 transcription factor family are increased in M4 / M5 AML subtypes and clinical improvement through inhibition of eIF4E has been demonstrated in these patients; the role for eiF2 remains less clear. mTOR activation has been found to promote leukemia progression and decreased eIF2 and mTOR may potentially synergize to decrease overall protein synthesis and therefore inhibit cell growth and proliferation. Upstream regulator analysis demonstrated decreased c-M YC and RB 1 activity (Figure 5 A - Figure 5G). The transcription factor cMyc was activated in leukemia and was a known oncogene that inhibits leukemia cell differentiation, promotes drug resistance, and promotes cancer sternness in AML. Gene set enrichment analysis showed a significant decrease in mTOR signaling and cMyc signature genes when U937 cells are treated with LILRB3 antagonist antibody vs IgG control (Figure 5D). These results suggest that the AKT / mTOR / cMyc axis was involved in decreased proliferation and increased differentiation of U937 cells induced by LILRB3 blockade.

[0249] Additionally, the laboratory has generated multiple functional LILRB3 specific agonist antibodies, which are tools in determining the effects of LILRB3 signaling on leukemia cells as a high- affinity natural ligand for LILRB3 was unknown. As opposed to LILRB3 antagonism, functional agonism of LILRB3 increased the expression of genes that promote an aggressive AML phenotype (Figure 3C, Figure 3D). RNAseq of U937 cells treated with LILRB3 agonist exhibited significantly increased CD 163, IL- 10, MMP1, and MMP9 gene expression and decreased the expression of the inflammatory genes CXCL10 and CXCL11. Moreover, pathway analysis of RNAseq results identified other AML promoting transcriptional changes induced by agonist LILRB3 antibodies, including the upregulation of cholesterol biosynthesis and metabolism in U937 cells (Figure 5A - Figure 5G). High intracellular cholesterol levels have been shown to promote leukemia cell survival, and there was evidence that acute cholesterol response increases chemotherapy resistance; whereas, cholesterol-modulating drugs, such as statins, can kill AML cells and sensitize them to chemotherapies. Further elucidation of the impact of LILRB3 signaling in AML metabolism represents a potentially therapeutically relevant future direction. Cumulatively, these data suggests that LILRB agonism promotes progression, immune invasion, and proliferation of AML.

[0250] In addition to exploring the regulation LILRB 3 expression and the phenotype changes secondary to LILRB3 downstream signaling, LILRB3 was an effective target for CAR T-cell therapy. The inductive effect of AraC and IFNy on LILRB 3 expression greatly enhances the potential for L1LRB3 as a target for CAR T therapy. L1LRB3 induction from AraC may increase the usefulness of an LILRB3 specific CAR T cell in the consolidation setting or as a bridging therapy prior to stem cell transplant. Interestingly, a cytotoxic positive feedback loop was potentially formed, where IFNy produced from CAR T-cells enhances LILRB 3 expression on AML cells, which in turn enhances the cytotoxic potential of LILRB3 specific CAR T-cells. Although, excessive IFNy signaling may the increased risk of development of cytokine release syndrome and macrophage activation syndrome. These toxicides could potentially be abrogated through antibody mediated blockade of IFNy, or CAR T cell depletion as needed.

[0251] LILRB3-CAR T-cells demonstrated potent AML cell killing both in vitro and in vivo (Figures 6A-6M). Importantly, LILRB3-CAR T-cells were effective at killing primary patient AML cells in vitro and in vivo without inducing major toxicity in LILRB3 expressing transgenic mice and therefore demonstrate potential for clinical translation (Figure 6 A - Figure 6M; Figure 7A - 7L). Moreover, low relative expression of LILRB3 in brain organoids and human brain tissue may portend reduced on-target, off-tumor neurologic toxicity compared with other members of the LILRB family (Figure 16A - Figure 16C).

[0252] LILRB 3 specific CAR T cells may also have the potential to target highly malignant AML blast cells. Surface expression of CD34, an AML blast marker, was associated with chemoresistance and worsened prognosis. Similar to HSCs from healthy donors (Figure 1C, Figure 8E - Figure 8G), native LILRB3 expression was decreased in the most immature FAB subtypes (Figure 15B) and also CD34+ cells compared to CD34- cells in the patient cohort (Figure 9G, Figure 9H). However, stimulation with IFNy and poly(LC) drastically increased LILRB3 expression in CD34+ cells (Figure 2E, Figure 9F, Figure 9G), therefore potentially allowing for the targeting of these AML blasts with LILRB 3 targeted CAR T-cell therapy. Additionally, the use of LILRB3 scFv as a component of a split or bi-specific CAR may increase the ability to target AML blasts or increase the myeloid specificity of AML CAR products currently in development. LILRB3 expression was enriched in AML cells compared to immature progenitor and stem- like cells but decreased as compared to normal mature myeloid cells such as monocytes. Similar to the AML cell line U937, primary AML cells expressed a higher level of LILRB3 upon treatment of IFNy or poly(LC). LILRB3 signaling promotes a proliferative aggressive state in AML through a wide variety of transcriptomic and phosphoproteomic mechanisms which can be inhibited with antagonist anti- LILRB3 antibodies. Finally, LILRB3 represents a promising target for CAR T-cells as LILRB3 specific CAR T-cells killed AML cells in vitro and produced lasting remission in vivo.

[0253] Example 2: Back Mutation Antibody Humanization

[0254] The aim of this project was to humanize a mouse monoclonal antibody (mAb) using CDR grafting plus back mutation method without sacrificing the binding affinity of the parental (chimeric) antibody.

[0255] Materials

[0256] • Amino acid sequences of parental antibody provided by client;

[0257] » HMRI Antigen A protein prepared by client;

[0258] • pcDNA3.4 expression vector and Expi293F cell prepared by GenScript;

[0259] • Biological safety cabinet (Thermo Scientific, Model. 1384);

[0260] • Orbital shaker (Shanghai Zhichu Instrument Co., Ltd, Model: ZCZY-BS8);

[0261] " FreeStyle 293 medium (lifetechnologies, Cat. No.12338-018);

[0262] • 125-ml shake flask (Corning, Cat. No. 431143);

[0263] • 500-ml shake flask (Coming, Cat. No. 431145);

[0264] • Protein-A resin (GenScript, Cat. No. L00210);

[0265] • Binding buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0;

[0266] • Elution buffer: 0.1 M Glycine- HCI, pH 3.2;

[0267] • Neutralization buffer: 1 M Tris-HCI, pH 9.0;

[0268] • Biacore T200 (GE Healthcare);

[0269] • Series S Sensor Chip CMS (GE Healthcare, Cat. No.: BR-1005-30);

[0270] • Series S Sensor Chip Protein A (GE Healthcare, Cat. No.:29-1275-55);

[0271] • HBS-EP: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH 7.4;

[0272] » NHS: 100 mM N-hydroxy succinimide in H2O;

[0273] » EDC: 400 mM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide in H2O;

[0274] « Ethanolamine: 1 M ethanolamine hydrochloride, adjusted to pH 8.5 with NaOH Methods

[0275] Construction and production of chimeric and PTM removal antibodies

[0276] The DNA sequences encoding the chimeric & PTM removal antibodies heavy and light chains were synthesized and inserted into pcDNA3.4 vector to construct expression plasmids of full-length IgGs. Expression of chimeric & PTM removal antibodies were conducted in Expi293F cell culture and the supernatants were purified with protein A affinity column. The purified antibody was buffer-exchanged into PBS using dialysis bag. The concentration and purity of the purified protein were determined by 00280 and SOS-PAGE, respectively. The NG amino acids on the heavy chain CDR2 region was mutated to QG and NA, named PTM remove 1 and remove 2, respectively.

[0277] Binding confirmation of chimeric and PTM removal antibodies

[0278] The affinity of chimeric & PTM removal antibodies to antigen HMRI Antigen A were determined using a Surface Plasmon Resonance (SPR) biosensor, Biacore T200. Antibody was immobilized on the sensor chip through Fc capture method. HMRI Antigen A protein was used as the analyte. The data of dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over ka.

[0279] Antibody humanization by CDR grafting plus back mutation

[0280] The structure of parental antibody was modelled by computer-aided homology modelling program. Humanized antibodies were designed using CDR grafting. Briefly, the CORs of parental antibody were grafted into the human acceptors to obtain humanized light chains and humanized heavy chains for each parental antibody. Four heavy chains and 4 light chains were paired with each other for affinity ranking experiment.

[0281] Production and affinity ranking of humanized antibodies

[0282] The designed plasmids of heavy chain and light chain were sent for 10 mL transfection following GenScript's standard operating procedures (SOP). For affinity ranking, Antibodies was immobilized on the sensor chip through Fe capture method. Antigen was used as the analyte. The surface was regenerated before the injection of another antibody. The process was repeated until all antibodies were analyzed. The off-rates of antibodies were obtained from fitting the experimental data locally to 1: 1 interaction model using the Biacore T200 evaluation software. The antibodies were ranked by their dissociation rate constants (off-rates, kd). Based on the ranking result, the top 3 clones were selected.

[0283] Construction and production of selected humanized IgGs The top 3 binders were selected to express in Expi293F cell culture. The recombinant IgGs secreted to the medium were purified using protein A affinity chromatography following GenScript's SOP. The purified antibody was buffer exchanged into PBS using dialysis bag. The concentration and purity of the purified protein were determined by 00280 and SOS -PAGE, respectively. The selected heavy and light chains sequences were shown in Appendix I.

[0284] Affinity measurement of purified humanized IgGs

[0285] The affinity of purified antibody binding to HMRI Antigen A was individually determined using a Surface Plasmon Resonance (SPR) biosensor, Biacore T200 (GE Healthcare). Antibodies were immobilized on the sensor chip through Fe capture method. HMRI Antigen A was used as the analyte. The data of dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over ka.

[0286] Results

[0287] Chimeric and PTM removal antibodies production

[0288] Chimeric & PTM removal antibodies were expressed and purified according to GenScript’s SOP respectively. The purified IgG migrated as ~ 150 kDa band in SOS-PAGE under non-reducing condition,- 50 kDa and- 25 kOa bands under reducing condition. Evaluating by the SOS-PAGE result, the purity of IgGs were> 95% (Figure 18).

[0289] Binding confirmation of chimeric and PTM removal antibodies

[0290] The results indicated that chimeric & PTM removal antibodies can bind to the HMRI Antigen A. The affinity and kinetics of HMRI Antigen A to Chimeric IgG was summarized in Table 6 and Table 7, and the sensor-grams were shown in Figure 19A - Figure 19C.

[0291] Table 7. Details parameters of affinity validation. Flow rale

[0292] Ta

[0293] Affinity ranking of humanized antibodies

[0294] The affinity of HMRJ Antigen A to humanized Abs supernatant was summarized in

[0295] Table 8 and Table 9. The sensor-grams were shown in Figure 20. Table 9. Details parameters of affinity validation.

[0296] Table 10. Binding kinetics of HMRI Antigen A to selected clones. Production of purified IgGs Three selected humanized antibodies were expressed and purified according to GenScript’s SOP. Evaluating from the SDS-PAGE, the purity of humanized IgGs were >90% (Figure 19A - Figure 19C; Figure 21 A - Figure 21C).

[0297] Affinity measurement of purified IgGs The affinity and kinetic antibodies to HMR1 Antigen A was summarized in Table 10 and

[0298] Table 11 , and the sensor-grams were shown in Figure 22A - Figure 22D.

[0299] Table 11. Details parameters of affinity validation.

[0300] Table 12. Binding kinetics of HMR1 Antigen A to antibodies Summary

[0301] In this example, mouse monoclonal antibody (mAb) was successfully humanized. Four heavy chains and four humanized light chains were designed, synthesized, and inserted into expression vector. The humanized antibodies were expressed, and then used for affinity ranking test. Finally, three humanized antibodies with similar binding affinity to Chimeric (VH-G56A) antibodies were purified for the delivery. Example 3: Affinity Ranking

[0302] Overview

[0303] Affinity to HMRI Antigen A to select clones was measured using Biacore T200. Materials

[0304] Ligand: selected clones

[0305] Analyte: HMRI Antigen A

[0306] Methods

[0307] Table 13. Details parameters of validity validation. Results

[0308] All the data were processed using the Biacore T200 Evaluation software version 3.1. Flow cell 1 and buffer injection were used as double reference for Response Units subtraction. The binding kinetic data was shown in Table 8, and the binding sensor-grams were shown in Figure 23.

[0309] Table 14. Binding kinetics of HMRI Antigen A to selected clones.

[0310] Other advantages which are obvious, and which are inherent to the invention, will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

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Claims

CLAIMSWhat is claimed is:

1. A chimeric antigen receptor (CAR) polypeptide comprising a LILRB3 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region.

2. The polypeptide of claim 1 , wherein the LILRB3 antigen binding domain is a single-chain variable fragment (scFv) of an antibody that specifically binds to LILRB3.

3. The polypeptide of any one of claims 1-2, wherein the co-stimulatory signaling region comprises a cytoplasmic domain of costimulatory molecule 4-1BB.

4. The polypeptide of any one of claims 1-3, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

5. The polypeptide of any one of claims 1-4, wherein the CAR polypeptide is defined by the formula:SP-LILBR3-HG-TM-CSR-ISD; orSP-L1LRB3-HG-TM-1SD-CSR wherein “SP” represents a signal peptide; wherein “LILRB3” represents an LILRB3 -binding region; wherein “HG” represents an optional hinge domain; wherein “CSR” represents a co-stimulatory signaling region; wherein “ISD” represents an intracellular signaling domain; and wherein represents an optional bivalent linker.

6. The polypeptide of any one of claims 1-5, wherein the CAR polypeptide is defined by the formula:SP-(VL-VH)n-HG-TM-CSR-ISD; or SP-(VH-VL)n-HG-TM-CSR-ISD; SP-(VL-VH)n- HG-TM-ISD-CSR; or SP-(VH-VL)n-HG-TM-CSR-ISD wherein “SP” represents a signal peptide; wherein “VL” represents a light chain variable region;wherein “VH” represents a heavy chain variable region; wherein “n” is > 1 ; wherein “HG” represents an optional hinge domain; wherein “CSR” represents a co-stimulatory signaling region; wherein “ISD” represents an intracellular signaling domain; and whereinrepresents an optional bivalent linker.

7. The polypeptide of any one of claims 2-6, wherein the scFv comprises a heavy chain variable region (VH) with a CDR1 comprising a sequence with at least 60% identity to SEQ ID NOS: 10, 16, or 22, a CDR2 comprising a sequence with at least 60% identity to SEQ ID NOS: 11, 17, or 23, and a CDR3 comprising a sequence with at least 60% identity to SEQ ID NOS: 12, 18, or 24.

8. The polypeptide of any one of claims 2-7, wherein the scFv comprises a light chain variable region (VL) with a CDR1 comprising a sequence with at least 60% identity to SEQ ID NOS: 13, 19, or 25, a CDR2 comprising a sequence with at least 60% identity to SEQ ID NOS: 14, 20, or 26, and a CDR3 comprising a sequence with at least 60% identity to SEQ ID NOS: 15, 21, or 27.

9. The polypeptide of any one of claims 2-6, wherein the scFv comprises a heavy chain variable region (VH) comprising a sequence with at least 60% identity to SEQ ID NOS: 4, 6, 8 and a light chain variable region (VL) comprising a sequence with at least 60% identity to SEQ ID NOS: 5, 7, 9.

10. The polypeptide of any one of claims 2-6, wherein the scFv comprises a sequence with at least 60% identity to SEQ ID NOS: 1-3 or a fragment thereof.

11. An isolated nucleic acid encoding the polypeptide of any one of claims 1-10.

12. A vector comprising the isolated nucleic acid of claim 11.

13. A cell comprising the vector of claim 12.

14. The cell of claim 13, wherein the cell reduces tumor activity when the antigen bindingdomain of the CAR polypeptide binds to LILRB3.

15. A method of treating leukemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimeric antigen receptor polypeptide of any one of claims 1-10.

16. The method of claim 15, wherein the leukemia comprises acute myeloid leukemia (AML).

17. The method of any one of claims 15-16, further comprising administering a targeted therapy.

18. The method of claim 17, wherein the targeted therapy comprises gemtuzumab ozogamicin, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, or any combination thereof.

19. The method of any one of claims 15-18, wherein the method further comprises administering chemotherapy, radiation therapy, stem cell transplantation, or any combination thereof.

20. A recombinant antibody, wherein the antibody comprises a light chain variable region (VL) with a CDR1, CDR2, and CDR3, and a heavy chain variable region (VH) that comprises a CDR1, CDR2, and CDR3, further wherein the VL CDR1 comprises a sequence with at least 60% identity to SEQ ID NOS: 72, 78, 84, 90, 96, 102, or 108 and / or the VH CDR1 comprises a sequence with at least 60% identity to SEQ ID NOS: 69, 75, 81, 87, 93, 99, or 105.

21. The recombinant antibody of claim 20, wherein the VL CDR2 comprises a sequence with at least 60% identity to SEQ ID NOS: 73, 79, 85, 91, 97, 103, or 109 and / or the VH CDR2 comprises a sequence with at least 60% identity to SEQ ID NOS: 70, 76, 82, 88, 94, 100, or 106.

22. The recombinant antibody of any one of claims 20-21, wherein the VL CDR3 comprises a sequence with at least 60% identity to SEQ ID NOS: 74, 80, 86, 92, 98, 104, or 110 and / or the VH CDR3 comprises a sequence with at least 60% identity to SEQ ID NOS: 71 , 77, 83, 89, 85, 101, or 107.

23. The recombinant antibody of any one of claims 20-22, wherein the VL comprises a sequence with at least 60% identity to SEQ ID NOS: 56, 58, 60, 62, 64, 66, or 68 and / or the VH comprises a sequence with at least 60% identity to SEQ ID NOS: 55, 57, 59, 61, 63, 65, or 67.

24. The recombinant antibody of any one of claims 20-23, wherein:VL CDR1 is SEQ ID NO: 72;VL CDR2 is SEQ ID NO: 73;VL CDR3 is SEQ ID NO: 74;VH CDR1 is SEQ ID NO: 69;VH CDR2 is SEQ ID NO: 70; andVH CDR3 is SEQ ID NO: 71 ; orVL CDR1 is SEQ ID NO: 78;VL CDR2 is SEQ ID NO: 79;VL CDR3 is SEQ ID NO: 80;VH CDR1 is SEQ ID NO: 75;VH CDR2 is SEQ ID NO: 76; andVH CDR3 is SEQ ID NO: 77; orVL CDR1 is SEQ ID NO: 84;VL CDR2 is SEQ ID NO: 85;VL CDR3 is SEQ ID NO: 86;VH CDR1 is SEQ ID NO: 81 ;VH CDR2 is SEQ ID NO: 82; andVH CDR3 is SEQ ID NO: 83; orVL CDR1 is SEQ ID NO: 90;VL CDR2 is SEQ ID NO: 91 ;VL CDR3 is SEQ ID NO: 92;VH CDR1 is SEQ ID NO: 87;VH CDR2 is SEQ ID NO: 88; andVH CDR3 is SEQ ID NO: 89; orVL CDR1 is SEQ ID NO: 96;VL CDR2 is SEQ ID NO: 97;VL CDR3 is SEQ ID NO: 98;VH CDR1 is SEQ ID NO: 93;VH CDR2 is SEQ ID NO: 94; andVH CDR3 is SEQ ID NO: 95; orVL CDR1 is SEQ ID NO: 102;VL CDR2 is SEQ ID NO: 103;VL CDR3 is SEQ ID NO: 104;VH CDR1 is SEQ ID NO: 99;VH CDR2 is SEQ ID NO: 100; andVH CDR3 is SEQ ID NO: 101; orVL CDR1 is SEQ ID NO: 108;VL CDR2 is SEQ ID NO: 109;VL CDR3 is SEQ ID NO: 110;VH CDR1 is SEQ ID NO: 105;VH CDR2 is SEQ ID NO: 106; andVH CDR3 is SEQ ID NO: 107.

25. A method of treating leukemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant antibody of any one of claims 20-24.

26. The method of claim 25, wherein the leukemia comprises acute myeloid leukemia (AML).

27. The method of any one of claims 25-26, further comprising administering a targeted therapy.

28. The method of claim 27, wherein the targeted therapy comprises gemtuzumab ozogamicin, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, or any combination thereof.

9. The method of any one of claims 25-28, wherein the method further comprises administering chemotherapy, radiation therapy, stem cell transplantation, or any combination thereof.

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