WO2023177954A1 - Combination therapy for treatment of cancer, methods and systems of delivery thereof - Google Patents

Abstract

Compositions and methods for treating malignant tumors with a combination of IAP antagonist and TNFα. TNFα is delivered locally via microphages/T cells transduced with viral vectors encoding a tumor targeting molecule, such as a chimeric antigen receptor, and TNFα. The tumor targeting molecule allows the transduced macrophages or T cells to bind specifically to targeted tumor cells and release TNFα in the vicinity of the tumor cells. TNFα released from the transduced cells works with lAPa to induce apoptosis of the targeted tumor cells.

Description

DOCKET NO: 1134-102 PCT

TITLE

COMBINATION THERAPY FOR TREATMENT OF CANCER, METHODS AND SYSTEMS OF DELIVERY THEREOF

[0001] This application claims priority from U.S. Provisional Patent App. No. 63/269,562, filed March 18, 2022, which is incorporated herein by reference.

[0002] This invention was made with government support under W81XWH-22-1- 0049 awarded by the U.S. Department of Defense Breast Cancer Research Program. The government has certain rights in the invention.

FIELD

[0003] This application relates generally to the field of treatment of cancer and delivery of anti-tumor agents to tumor sites.

BACKGROUND

[0004] Cancer is a major public health problem worldwide and is the second leading cause of death, the greatest number of which are from cancers of the lung, colorectum and prostate in men and breast in women. In 2022, 1,918,030 new cancer cases and 609,360 cancer deaths are projected to occur in the United States, including approximately 350 deaths per day from lung cancer, the leading cause of cancer death.

[0005] Most of solid cancers can be surgically removed. The greatest challenging issue for the cancers are their recurrence and metastasis post-surgery to other organs, causing about 90% of cancer deaths. For decades, radiation therapy (RT) and chemotherapy (CT) are used before and/or after surgery' in order to prevent the recurrence and metastasis of the cancer. However, only a small proportion (5-10%) of patients benefit from chemotherapy or radiation therapy. One reason for the poor therapeutic outcome could be that many patients already have micro-metastases when their primary cancers are diagnosed. In recent years, therapeutic advance has been achieved for some cancers that are caused by a single gene mutation, for example, epidermal growth factor receptor tyrosine kinase inhibitors that are targeted against the most common NSCLC (non-smaH cell lung cancer) driver mutations. Immunotherapy targeting programmed cell death protein- 1/programmed death ligand- 1 immune checkpoint have been approved by the US FDA to treat melanoma. These targeted therapies against a single gene or immune molecule cannot be used in most cancers since only small number of cancers are caused by a single gene mutation and the immune molecules play a role in small number of patients. Elimination of the mortality related to cancers still depend on the development and application of a generalized approach in most cancers.

SUMMARY

[0006] One aspect of the present application relates an expression vector that comprises: (1) a first nucleotide sequence encoding a chimeric protein comprising a membrane anchoring domain and a cell targeting domain, and (2) a second nucleotide sequence encoding TNFa. In some embodiments, the expression vector further comprises a regulatory sequence operably linked to at least one of the first and the second nucleotide sequence.

[0007] Another aspect of the present application relates to a cell comprising (1) a chimeric protein on a cell surface, wherein the chimeric protein comprises a cell membrane anchoring domain and a cell targeting domain, and (2) an expression cassette that expresses TNF. In some embodiment, the cell is a macrophage, a T cell, a nature killer cell, a monocyte or a dendritic cell.

[0008] Another aspect of the present application relates to a method for treating tumor or cancer in a subject. The method comprises the steps of administering to the subject an effective amount of the chimeric protein/TNFa expressing cells of the present application, and administering to the subject an effective amount of an IAP antagonist.

[0009] Another aspect of the present application relates to a method for treating a tumor in a subject. The method comprises the steps of administering to the subject an effective amount of TNFa; and administering to the subject an effective amount of an IAP antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Fig. 1 shows SM-164 combined with TNFa eliminates metastases in bone and lung from MDA-MB-231 cells in mice. Panel (A): Scheme showing that MDA-MB-23 l^lucl cells were inoculated into the left cardiac ventricle of 7-wk-old female athymic nude mice. After 2 weeks, mice with established bone metastases, determined by BLI, were randomly divided into 4 groups, 7-8 mice per group, which were treated with vehicle (Veh), 3 mg/ml SM-164 (SM), SM+ 0.2 pg TNFa or standard chemotherapy (SCT) including adriamycin, cytoxan and zoledronate. Panel (B): Tumor growth in the legs was evaluated by bioluminescence imaging (BLI) signal intensity, calculated on individual legs, after 1 and 2 weeks of treatment. *p<0.05 and **p<0.01. ANOVA+/Dunnett test based on logarithm transformed intensity. The horizontal line at 10⁵ indicates the mean value in the mice at baseline. Panel (C): Mice were euthanized on day 29 and the total numbers and percentage of tibiae and femora (bones) with metastases (Mets) (*p<0.05, non-parameter analysis). Panel (D): tumor burden was determined on H&E-stained sections (*p<0.05, **p<0.01, ANOVA+/Dunnett test). Panel (E): The number of mice with lung metastases was evaluated (*p<0.05, non-parameter analysis).

[0011] Fig. 2 shows IL-4- polarized Macs produce TNFa to trigger SM-164-induced apoptosis of MDA-231 cells. Panel (A): GFP⁺ MDA-231 cells were cultured alone or together with WT mouse bone marrow (BM) cells treated with M-CSF plus PBS or 10 ng/ml IL-4 for 3 days. 3 nM SM-164 plus PBS or TNFR:Fc were added for the last 16 hrs. # of GFP⁺ (live) MDA-231 cells. Panel (B): % of AnV⁺PI'^/+ apoptotic cells in GFP⁺ population were analyzed by flow cytometry based on total cell#. Dead MDA231 cells lost GFP. ANOVA+/Dunnett test.

[0012] Fig. 3 shows Mucl is highly expressed on a variety of human breast cancer cells. Human breast cancer cell lines, MDA-MB-231, MDA-MB-436, MCF7 and BT474, were stained with PE-conjugated anti-Mucl antibody to analyze Mucl expression by flow cytometry.

[0013] Fig. 4 shows the cloning of a chimeric receptor/ligand and hTNFa into an expression vector. Panel (A) shows the chimeric receptor/ligand cDNA is cloned to an expression vector. Panel (B) shows hTNFa cDNA is cloned to an expression vector. Panel (C) shows both cDNAs of hTNFa and chimeric receptor/ligand are cloned to the same expression vector.

[0014] Fig. 5 shows the construction of a chimeric receptor/ligand to recognize tumor cells. Panel (A) shows a chimeric receptor-antibody recognizes tumor cell surface antigen. Panel (B) shows a chimeric receptor-ligand recognizes tumor cell surface receptors.

[0015] Fig. 6 shows generation of chimeric receptor/ligand macrophages expressing hTNFa.

[0016] Fig. 7 shows generation of a chimeric receptor/ligand T cells expressing hTNFa.

[0017] Fig. 8 shows construction of M-Macs^TNh. Panel (A) Structure scheme for human CD33 protein. Panel (B) Chimeric CD33-Mucl scFv was designed by linking the DNA of Mucl monoclonal antibody scFv to a segment (transmembrane and cytoplasmic domain) of human CD33 DNA. Panel (C) The synthesized cDNA of CD33-scFv and hTNFa was inserted into a pLV-Duet lentiviral vector, respectively. The control vectors were constructed by inserting CD33-scFv, CD33 or hTNFa to pLV-mCherry. Panel (D) 2xl0⁵ Macs, infected with pLV-Duet^CD33'^scFv'^cheny or pLV-Duet^CD33'^cheny-lentivirus, were injected via tail vein into athymic nude mice, which had intratibial injections of IxlO⁵ GFP⁺MDA- MB-231 cells 5 days earlier. After 3 days, fluorescence images were taken to trace both cells in frozen sections of tibiae. Macs carrying CD33-scFv (mCherry⁺M) binding to MDA-MB- 231 cells (GFP⁺MDA231) and merged as yellow (dark arrows in lower panel); in contrast, Macs carrying CD33 alone (white arrows in upper panel) were distributed in the BM without binding to GFP⁺ cancer cells.

[0018] Fig. 9 shows M-Macs™^ combined with SM-164 kill MDA231 cells. M- Macs^TNF, Macs^scFv, Macs^CD33 and Macs^{I NF} were generated by infecting the cultured Macs with pLV-Duet^CD33'^{scFv hTNF}, pLV-Duet^CD33'^scFv, pLV-Duet^CD33 and pLV-Duet^hTNF lentivirus, respectively, as in Fig.4C. Each type of these engineered Macs were co-cultured with 2xl0⁵ GFP⁺ MDA-231 cells for 24 hrs. 3nM SM-164 was added over-night, including a dish without Macs (-ve control) and one with Macs+TNFa as +ve control). Panel (A) % of GFP⁺ cells were analyzed by flow. Panel (B) The total# of GFP⁺ cells (live) was calculated.

[0019] Fig. 10 shows working model for lAPa + M-Macs™¹) M-Macs^{I NF} recognize and bind to TNBC (1) and produce TNFa (2) which synergize with an lAPa that degrades IAP proteins (3) to induce cancer cell apoptosis (4) by triggering the release of cytochrome-c (C) from mitochondria (4).

[0020] Fig. 11 shows scheme illustrating the experimental procedures that M- Macs^TNF combined with SM-164 prevent metastases following surgical removal of orthotopically implanted patient-derived xenograft (PDX) of triple-negative breast cancer (TNBC).

[0021] Fig. 12 shows injected Macs survive after 7 days in vivo. C57B16 mouse Macs^scFv'^cheny, generated as Fig. 9, were injected via tail vein into recipient C57B16 mice. After 14 days, the mice were killed to trace the injected cells in bone (A), lung (B) and brain (C) using anti-cherry H4C. Tr.B=trabecular bone, C.B=cortical bone.

[0022] Fig. 13 shows scheme illustrating the experimental procedures that M- Macs^{I NF} combined with SM-164 treat advanced metastases in lung, bone and brain from

TNBC PDX. [0023] Fig. 14 shows M-Macs™^ in combination with SM-164 effectively treat human Her2+ breast cancer in a mouse model. Panel (A) A diagraph illustrating the procedure using M-Macs™^ compared with an lAPa to treat Her2⁺ breast cancer. Panel (B) Cancer BLI signal in mammary fat pads at baseline (two weeks after cancer injection) showing no difference among groups. Panel (C) Cancer BLI signal in mammary fat pads after two weeks of treatment. Panel (D) Cancer BLI signal change in each same mammary fat pad after treatment vs. its respective baseline. 4 mice per group with 7-11 mammary fat pads with BLI signal higher than 10⁷ photons/s/cm²/sr.

[0024] Fig. 15 shows M-Macs™^ in combination with SM-164 has better effect than SM-164 alone to reduce or eliminate human Her2⁺ breast cancer in a mouse model. The mice, as in Fig.l, were euthanized after two weeks of treatment. The tumors in mammary fat pads were collected to measure weight. Only those tumors with basal BLI signal higher than 10⁸ photons/s/cm²/sr were used for statistical analysis.

[0025] Fig. 16 shows that M-Macs™^ produce hTNF in culture medium. hTNF concentration was determined by ELISA. Control: MDA-MB-231 breast cancer cell culture medium. M-Macs™^: 6 culture medium samples from two independent experiments.

[0026] FIG. 17 shows the effectiveness of M-Macs^TNh combined with an IAP antagonist in treating cancer in a patient-derived xenograft (PDX) model. 50 ul of minced tumor tissue from NSG mouse bearing PDX model of breast cancer (Jax Lab #TM00090) was injected into subcutaneously on the left trunk of a NSG mouse. After two weeks when the tumor grow to about 0.5 cm, the mice were randomly divided into 3 groups, 8-9 mice per group. Each mouse in group 3 was injected subcutaneously with 3xl0⁵ of M-Mac^TNh, generated as in Fig.14-17. After 3 days, the mice were begun to treat with vehicle or 3 mg/kg of SM-164, twice a day for 10 days. The mice were euthanized and the tumors were collected to scale their weights.

DETAILED DESCRIPTION

[0027] Reference will be made in detail to certain aspects and exemplary embodiments of the application, illustrating examples in the accompanying structures and figures. The aspects of the application will be described in conjunction with the exemplary embodiments, including methods, materials and examples, such description is non-limiting and the scope of the application is intended to encompass all equivalents, alternatives, and modifications, either generally known, or incorporated here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. One of skill in the art will recognize many techniques and materials similar or equivalent to those described here, which could be used in the practice of the aspects and embodiments of the present application. The described aspects and embodiments of the application are not limited to the methods and materials described.

[0028] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0029] Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will 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. It is also understood that when a value is disclosed that "less than or equal to "the value," greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value " 10" is disclosed the "less than or equal to 10" as

[0030] This application discloses a new method to target a variety of malignant tumors to deliver TNFa using adoptive cells. When used to treat tumors by general delivery routes to the body, TNFa can induce serious side effects, including systemic shock and inflammatory reactions, due to its cytotoxic, cytostatic, and immunomodulatory effects and these have prevented its therapeutic administration to humans. Thus, it is important to develop a novel targeted approach to deliver TNFa locally to treat the malignancies specifically in combination with an lAPa.

[0031] Unexpectedly, combined treatment of an lAPa, SM-164, and TNFa completely eliminated all lung metastasis and half of bone metastasis from human MDA- MB-231 cells in mice in vivo, as described herein. In contrast, neither SM-164 alone nor SCT had an effect to eliminate the established bone and lung metastasis. Therefore, an lAPa in combination with TNFa is an approach to treat malignant tumors, particularly, for those with metastases. I. Definitions

[0032] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0033] The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

[0034] A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0035] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0036] The term "nucleotide sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0037] The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0038] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0039] As used herein, the term "regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0040] An “expression cassette” is a distinct component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell. In each successful transformation, the expression cassette directs the cell's machinery to make RNA and protein(s). Some expression cassettes are designed for modular cloning of protein-encoding sequences so that the same cassette can easily be altered to make different proteins.

[0041] A "chimeric protein" is a protein having non-naturally occurring amino acid sequences fused together.

[0042] A “membrane anchoring domain” is a transmembrane protein domain which spans the cell membrane when in situ naturally. The membrane anchoring domain functions to anchor proteins to the cell membrane. The membrane anchoring domain may also include intracellular protein domains.

[0043] A “cell targeting domain” is a protein domain which enables the specific recognition of a distinct cell type by virtue of binding with cell surface receptors/antigens that distinguish that cell type. For example, the cell targeting domain may be portions of an antibody which recognizes a cell surface antigen or may be a ligand (for example cytokine or chemokine) which recognizes a cell surface receptor.

[0044] As used herein, the term "effective amount" is an amount necessary or sufficient to achieve the desired biological effect or the selected result, and such amount can be determined by one of ordinary skill in the art as a matter of routine experimentation.

[0045] The term "antigen" or " Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present application includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0046] “Apoptosis” is caspase-mediated programmed cell death under physiological or pathological stimuli. Inhibitor of apoptosis proteins (LAP), a family of E3 ubiquitin ligases including the most widely studied cIAPl, cIAP2 and XIAP, cause the ubiquitin degradation of caspases 3, 7 and 9, to block caspase-induced apoptosis. Under the intrinsic or extrinsic signal stimulation, endogenous Second Mitochondria-Derived Activator of Caspase (SMAC) induces the degradation of IAP proteins, resulting in caspase activation and the subsequent apoptosis. IAP proteins are frequently over-expressed in human tumors to promote cancer cell survival. [0047] The terms "IAP antagonist (IAPa)" and "IAP inhibitor" are used interchangeably and refers to a substance which interferes with, or inhibits, a physiological action or biological activity of IAP, either directly or indirectly. Examples of IAP antagonist include, but are not limited to, IAP degrading compounds (IAPDC) and second mitochondria- derived activator of caspase (SM AC) mimetics. Examples of IAPa include, but are not limited to, GDC-0917 (CUDC-427), GDC-0152, LCL161, AT-406 (Xevinapant, Debiol 143), HGS1029, Birinapant (TL32711, NSC-756502), SM-164, SM-122, SM-1387, BV6, MV1, TQB-3728, APG-1387, BI-891065, T-3256336, ASTX660 (tolinapant), LBW- 242, NVP-LBW242, AT-IAP

[0048] The term "SMAC mimetic" refers to a small-molecule inhibitor for therapeutic inhibition of IAP which small-molecule inhibitor mimics the N-terminal four-amino acid stretch of the endogenous SMAC sequence and is at least partly comprised of non-peptidic elements. The N-terminal sequence of endogenous SMAC is Al a- Vai -Pro-lie (A VP I) and is required for binding to IAP.

II. Expression vector expressing a chimeric receptor/ligand and TNFa

[0049] One aspect of the present application relates to an expression vector comprising: (1) a first nucleotide sequence encoding a chimeric protein comprising a membrane anchoring domain and a cell targeting domain; (2) a second nucleotide sequence encoding TNFa; and (3) a regulatory sequence operably linked to at least one of the first and the second nucleotide sequence. The expression vector can be plasmid vectors or viral vectors. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a retrovirus vector. In certain embodiments, the viral vector is a lentivirus vector.

Membrane anchoring domain

[0050] The membrane anchoring domain is a transmembrane domain that anchors the chimeric protein on cell surface in an orientation that exposes the cell targeting domain on the surface of a cell. In certain embodiments, the membrane anchoring domain comprises the transmembrane domain of a myeloid cell receptor selected from the group consisting of CDl la (ITGAL); CDl lb (ITGAM); CDl lc (ITGAX); CD14; CD16 (FCGR3A); CD18 (ITGB2); CD33 (Siglec-3); CD36 (SCARB3); CD40; CD45 (PTPRC); CD63; CD64 (FCGR1A); CD66b (CEACAM8); CD68 (SCARD1); CD80 (B7-1); CD85; CD86 (B7-2); CD115(IFNgR); CD163; CD169 (SIGLEC1); CD192 (CCR2); CD195 (CCR5); CD200R (CD200R1); CD204 (MSR1); CDC206 (MRC1); CD209 (DC-SIGN); CD282 (TLR2); CD284 (TLR4); EMR1 (ADGRE1); Ly6C (Ly6cl); Marco (MARCO); MER (MERTK); CCRL1 (CXC3CR1); Tim4 (TIMD4); and IL-1R (ILR1). In some related embodiments, the membrane anchoring domain further comprises the intracellular domain of a myeloid cell receptor selected from the group consisting of CD1 la (ITGAL); CD1 lb (ITGAM); CD11c (ITGAX); CD14; CD16 (FCGR3A); CD18 (ITGB2); CD33 (Siglec-3); CD36 (SCARB3); CD40; CD45 (PTPRC); CD63; CD64 (FCGR1A); CD66b (CEACAM8); CD68 (SCARD1); CD80 (B7-1); CD85; CD86 (B7-2); CD115 (IFNgR); CD163; CD169 (SIGLEC1); CD192 (CCR2); CD 195 (CCR5); CD200R (CD200R1); CD204 (MSR1); CDC206 (MRC1); CD209 (DC-SIGN); CD282 (TLR2); CD284 (TLR4); EMR1 (ADGRE1); Ly6C (Ly6cl); Marco (MARCO); MER (MERTK); CCRL1 (CXC3CR1); Tim4 (TIMD4); and IL-1R (ILR1)

[0051] In certain embodiments, the membrane anchoring domain comprises the transmembrane domain of a T cell receptor selected from the group consisting of CD3e, CD4, CD5, CD8a, CD8b, CD25, CD27 (TNFRS7); CD28, CD30 (TNFRSF8), CD39 (FR4), CD62L, CD73 (GARP), CD80 (B7-1), CD86 (B7-2), CD103, CD134 (GITR), CD152 (CTLA-4), CD153 (CD30L), CD192 (CCD2), CD196 (CCR6), CD223, and CD137 (4-1BB). In some related embodiments, the membrane anchoring domain further comprises the intracellular domain of a T cell receptor selected from the group consisting of CD3e, CD4, CD5, CD8a, CD8b, CD25, CD27 (TNFRS7); CD28, CD30 (TNFRSF8), CD39 (FR4), CD62L, CD73 (GARP), CD80 (B7-1), CD86 (B7-2), CD103, CD134 (GITR), CD152 (CTLA-4), CD153 (CD30L), CD192 (CCD2), CD196 (CCR6), CD223, and CD137 (4-1BB).

[0052] In certain embodiments, the chimeric protein nucleotide sequence encodes a membrane anchoring region that comprises a CD66b transmembrane and intracellular region: I P NITTKNSGSYACHTTNSATGRNRTTVRMetITVSDALVQGSSPGLSARATVSIMetIGVL ARVALI (SEQ ID NO:1).

[0053] In certain embodiments, the chimeric protein nucleotide sequence encodes a membrane anchoring domain that comprises a CD33 transmembrane and intracellular region: GAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKL HGPTETSSCSGAAPTVEMetDEELHYASLNFHMetNPSKDTSTEYSEVRTQ (SEQ ID NO:2).

[0054] In certain embodiments, the chimeric protein nucleotide sequence encodes a membrane anchoring domain that comprises a CD28 transmembrane and intracellular region: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRS (SEQ ID NO:3). Cell targeting domain

[0055] The cell targeting domain binds specifically to a target cell, such as a tumor cell. In certain embodiments, the cell targeting domain is a scFV light chain and/or heavy chain against/targeting an antigen on a cell surface, such as a tumor antigen on the surface of a tumor cell. In some embodiments, the cell targeting domain specifically binds to a protein selected from the group consisting of alphafetoprotein (AFP) (hepatocellular carcinoma, germ cell tumors), CD66e (CEACAM5) (colorectal cancer), CA-125 (Mucin 16) (ovarian cancer), Mucin-1 (Muc-1) (cancers from epithelial cells, as well as B-cell lymphoma and multiple myeloma); Her-2 (human epidermal growth factor receptor 2) (breast cancer); mesothelin (MSLN) (malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma); PSCA (prostate stem cell antigen) (prostate, bladder, pancreatic, placenta, colon, kidney, and stomach cancers); Claudin 18.2 (gastric cancer, esophageal adenocarcinoma (EAC)); EpCAM (colon, pancreatic, prostate, gastric, liver); Nectin4/FAP (Nectin4-positive advanced malignant solid tumor); Lewis Y (gastrointestinal carcinomas, cancers originated epithelial cells); Glypican-3 (hepatocellular carcinoma); CD171 (neuroectodermal tumors, ovarian serous carcinoma, malignant mesothelioma, and testicular embryonal carcinoma); PSMA (prostate); CD20 (melanoma); ephrin type-A receptor 2 (EPHA2) (osteosarcoma, glioma); CD80/86 (lung), c-MET (breast, hepatocellular); DLL-3 (small cell lung cancer (SCLC)); gplOO (melanoma); MAGE-A1/3/4 (lung); LMP1 (nasopharyngeal); .

[0056] In certain embodiments, the chimeric protein nucleotide sequence encodes a cell targeting domain that is Mucl scFV light chain: MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPG NTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQG TTLTVSS (SEQ ID. NO:4). In certain embodiments, the chimeric protein nucleotide sequence encodes a cell targeting domain that is Mucl scFV heavy chain: SDIVMetTQSPSSLTVTAGEKVTMetlCKSSQSLLNSGDQKNYLTWYQQKPGQPPKLLI FWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLE LK (SEQ ID. NO:5)

[0057] In certain embodiments, the chimeric protein nucleotide sequence encodes a cell targeting domain that comprises Her2 scFV light chain: MetDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIK (SEQ ID. NO:6) [0058] In certain embodiments, the chimeric protein nucleotide sequence encodes a cell targeting domain that comprises Her2 scFV heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG QGTLVTVSS (SEQ ID. NO:7).

[0059] In certain embodiments, the cell targeting domain comprises a ligand, such as a cytokine or chemokine, which can bind a receptor on tumor surface. The receptor on tumor surface include but not limited to EGFR (lung, liver, stomach cancer); VEGFR2 (melanoma, brain); EGFRIII (glioblastoma and brain tumor); IL-13Ra2 (glioblastoma); DR5 (hepatoma) and FR-a (ovarian)

[0060] In certain embodiments, the chimeric protein nucleotide sequence encodes a cell targeting domain that comprises a sequence from human EGF: MetNSDSECPLSHDGYCLHDGVMetYIEALDKYACNCVVGYIGERCQYRDLKWWEL R (SEQ ID NO:8), which recognizes and bind to EGFR on tumor surface.

Linker and hinge regions

[0061] In certain embodiments, the chimeric protein nucleotide sequence encodes a linker region between scFV light chain and heavy chain sequences: GGGGSGGGGSGGGG (SEQ ID. NO:9)

[0062] In certain embodiments, the chimeric protein nucleotide sequence encodes a hinge region between the cell targeting domain and the membrane anchoring domain: ESKYGPPCPSCP (SEQ. ID. NO: 10).

[0063] In certain embodiments, the expression vector encodes Chimeric CD66b- Mucl scFV: MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPG NTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQG TTLTVSSGGGGSGGGGSGGGGSDIVMetTQSPSSLTVTAGEKVTMetICKSSQSLLNSG DQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA VYYCQNDYSYPLTFGAGTKLELKESKYGPPCPSCPIPNITTKNSGSYACHTTNSATGR NRTTVRMetITVSDALVQG SSPGLSARATVSIMetIGVLARVALI (SEQ ID NO:11).

[0064] In certain embodiments, the expression vector encodes Chimeric CD33-Mucl scFV: MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPG NTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQG TTLTVSSGGGGSGGGGSGGGGSDIVMetTQSPSSLTVTAGEKVTMetICKSSQSLLNSG DQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA VYYCQND YS YPLTFGAGTKLELKESK YGPPCPSCPGAIGG AG VI ALL ALC LC LIFFIV KTHRRKAART AVGRNDTHPTTGS ASPKHQKKSKLHGPTETS SC SGAAPTVEMDEEL HYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 12).

[0065] In certain embodiments, the expression vector encodes Chimeric CD33-Her2 scFV: Met DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGG GG SGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG DGFYAMDYWGQGTLVTVSSESKYGPPCPSCPGAIGGAGVTALLALCLCLIFFIVKTH RRKAART AVGRNDTHPTTGS ASPKHQKKSKLHGPTETS SC SGAAPTVEMetDEELHY ASLNFHGMetNPSKDTSTEYSEVRTQ (SEQ ID NO: 13).

[0066] In certain embodiments, the expression vector encodes Chimeric CD33-hEGF: MetNSDSECPLSHDGYCLHDGVCMetYIEALDKYACNCVVGYIGERCQYRDLKWWE LRE SKYGPPCPSCPGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSA SP KHQKKSKLHGPTETSSCSGAAPTVEMetDEELHYASLNFHGMetNPSKDTSTEYSEVR TQ (SEQ ID NO: 14)

[0067] In certain embodiments, the expression vector encodes Chimeric CD28-Mucl scFV: MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSP GNTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQ GTTLTVSSGGGGSGGGGSGGGGSDIVMetTQSPSSLTVTAGEKVTMetICKSSQSLLNS GDQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDL AVYYCQNDYSYPLTFGAGTKLELKESKYGPPCPSCPFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:15)

[0068] In certain embodiments, the expression vector encodes Chimeric CD28-hEGF:

MetNSDSECPLSHDGYCLHDGVCMetYIEALDKYACNCVVGYIGERCQYRDLKWWE

LR ESKYGPPCPSCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR

RPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 16).

TNFa sequence

[0069] In some embodiments, the TNFa coding sequence is located in an expression cassette that is different from the expression cassette for the chimeric protein. In other embodiments, the TNFa coding sequence is located in the same expression cassette as the chimeric protein. In certain embodiments, the nucleotide sequence encoding TNFa is human TNFa-1 full length: MetSTESMetIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGAT TLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQ WL NRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSY Q TKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAE SG QVYFGIIAL (SEQ. ID. NO: 17) and its possible variants or its truncated form.

[0070] In certain embodiments, the nucleotide sequence encoding TNFa is human TNFa-2 soluble form:

MetVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSE GLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAK PWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ. ID. NO: 18) and its possible variants or its truncated form.

[0071] Nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the chimeric protein, by deriving the chimeric protein nucleotide sequence from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the chimeric protein nucleotide sequence of interest can be produced synthetically, rather than cloned.

[0072] In some embodiments, the coding sequences of the chimeric antigen receptor and TNFa can be cloned into the same expression vector, such as a lentiviral/retroviral vector (Fig. 1, Panel C). In some embodiments, the coding sequence of the chimeric antigen receptor and the coding sequence of TNFa are cloned into two different expression vectors, such as two different lentiviral/retroviral vectors (Fig. 1, Panel A, Panel B). When the coding sequence of chimeric antigen receptor and TNFa are cloned to two lentiviral/retroviral vectors independently, the packaged virus will be combined equally to infect target cells, such as macrophage or T cells.

Expression vectors

[0073] The expression vectors can be any vector suitable for replication and integration eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The 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. 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, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[0074] A number of viral based systems have been developed for chimeric protein nucleotide sequence transfer into mammalian cells. For example, retroviruses provide a convenient platform for chimeric protein nucleotide sequence delivery systems. A selected chimeric protein nucleotide sequence 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. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0075] Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term chimeric protein nucleotide sequence transfer since they allow long-term, stable integration of a chimeric protein nucleotide sequence and its propagation in daughter cells. Lenti viral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In brief summary, the expression of natural or synthetic nucleic acids encoding chimeric protein is typically achieved by operably linking a nucleic acid encoding the chimeric protein or portions thereof to a promoter, and incorporating the construct into an expression vector.

[0076] Methods of introducing and expressing chimeric protein nucleotide sequences 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). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0077] 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 chimeric protein nucleotide sequence into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus , adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat, Nos. 5,350,674 and 5,585,362. 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 (eg. an artificial membrane vesicle).

[0078] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). 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.

III. Tumor-targeting, TNFq-expressing Cells

[0079] Another aspect of the application relates to a cell comprising (1) a chimeric protein on a cell surface, wherein the chimeric protein comprises a cell membrane anchoring domain and a cell targeting domain and (2) an expression cassette that expresses TNFa.

[0080] In certain embodiments, the cell is a macrophage, monocyte, granulocyte, or dendritic cell.

[0081] In certain embodiments, a cell is transduced with the expression vector as described herein.

[0082] In certain embodiments, the cell is a macrophage, or a T cell, or a nature killer cell or other subset of immune cell.

[0083] When macrophages (monocyte/dendritic cells) are used as a tool to deliver TNFa, a segment of a macrophage/monocyte/dendritic cell receptor containing cytoplasmic and transmembrane domain will be used to link a monoclonal antibody scFv against a tumor antigen or a ligand (cytokine/chemokine/hormone) that can bind to a tumor surface marker, like Mucin 1, as in Fig. 3, through a hinge. When T cells are used as a tool to deliver TNFa, a segment of a T cell receptor containing cytoplastic and transmembrane domain will be used to link a monoclonal antibody scFv or a ligand, as described herein, through a hinge.

[0084] In addition, the cytoplasmic domain of the T cell receptor may be mutated to block its downstream signaling in order to avoid the activation and cytokine releases of the adoptive T cells since the adoptive T cell is mainly serves as a tool for targeted delivery of TNFa to cancer cells, which is different from classical Car T cells that kill cancer cells by releasing cytokines.

[0085] A source of T cells is obtained from a subject. T 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. In certain embodiments of the present application, any number of T cell lines available in the art, may be used. In certain embodiments of the present application, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.

[0086] The blood will be drawn from a patient to expand macrophage (monocyte/dendritic cells) or T cells, which will be engineered to over-express a chimeric antigen receptor that recognizes a tumor surface marker and a secreted TNFa. The tumor surface marker can be an antigen or receptor, which are described herein.

[0087] The adoptive macrophage or T cells expressing TNFa can be locally or systemically given back to the patient and then an lAPa can be given orally, intravenously or by another route.

IV. Methods for treating cancer using a combination therapy of TNFo/TNFq- expressing cells and IAP antagonists

[0088] Another aspect of the present application relates to a method for treating a tumor or cancer in a subject. The method comprises the step of administering to the subject an effective amount of the cells expressing TNFa as described herein and administering to the subject an effective amount of an IAP antagonist. In some embodiments, the IAP antagonist is administered for a period of 1-3 months after cell administration. In further embodiments, a first dose of the IAP antagonist is given within 14 days of cell administration.

[0089] Another aspect of the present application relates to a method for treating a tumor or cancer in a subject. The method comprises the steps of administering to the subject an effective amount of TNFa; and administering to the subject an effective amount of an IAP antagonist. In some embodiments, the IAP antagonist is administered prior to, concurrently with, or after, the administration of the TNFa. In some embodiments, the TNFa is administered systemically. In some embodiments, the TNFa is administered intravenously, intramuscularly or subcutaneously. In some embodiments, the TNFa is administered directly into the tumor/cancer tissue or mass.

[0090] Cancers that may be treated with the methods of the present application include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors, The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the CARs of the application include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included, hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemnia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0091] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovionia, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochronocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tuinor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a gliona (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealona, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[0092] In some embodiments, wherein the tumor is a malignant tumor selected from, but not limited to, acute myelogenous leukemia; adult T-cell lymphoma; advanced solid tumor; cutaneous T-cell lymphoma; lymphoma; peripheral T-cell lymphoma; T-cell lymphoma; advanced solid tumor, breast tumor; lymphoma; solid tumor squamous cell carcinoma; head and neck tumor; hematological neoplasm; hepatitis B virus infection; metastatic colorectal cancer; metastatic pancreas cancer; peritoneal tumor; hepatocellular carcinoma; germ cell tumors; colorectal cancer; ovarian cancer; cancers from epithelial cells, as well as B-cell lymphoma and multiple myeloma; malignant mesothelioma; pancreatic, ovarian or lung adenocarcinoma; prostate, bladder, pancreatic, placenta, colon, kidney or stomach cancers; gastric cancer; esophageal adenocarcinoma (EAC); liver cancer; nectin-4- positive advanced malignant solid tumor; gastrointestinal carcinomas; cancers originated epithelial cells; hepatocellular carcinoma; neurpectodermal humors, ovarian serous carcinoma, malignant mesothelioma, and testicular embryonal carcinoma; melanoma, osteosarcoma, glioma; small cell lung cancer (SCLC); nasopharyngeal; glioblastoma and brain tumor. In particular embodiments, the tumor is breast tumor.

[0093] In some embodiments, the IAP antagonist is selected from the group consisting of: GDC-0917 (CUDC-427), GDC-0152, LCL161, AT-406 (Xevinapant, Debiol 143), HGS1029, Birinapant (TL32711, NSC-756502), SM-164, SM-122, SM-1387, BV6, MV1 , TQB-3728, APG-1387, BI-891065, T-3256336, ASTX660 (tolinapant), LBW- 242, NVP-LBW242, AT-IAP, inhibitor of apoptosis protein 1 inhibitor; inhibitor of apoptosis protein 2 inhibitor; X-linked inhibitor of apoptosis protein inhibitor; kidney inhibitor of apoptosis protein inhibitor, XIAP antagonists, SMAC mimetics, proline mimetics, second mitochondria-derived activator of caspases mimetics.

[0094] One of ordinary skill will understand that the IAP antagonist used in the methods described herein may be delivered in forms including, but not limited to, an oral formulation, small molecule therapeutic, peptidomimetic, capsule formulation, tablet formulation, infusion, intravenous formulation. One of ordinary skill will understand that the particular formulation or method of delivery of the IAP antagonist is not limiting on the methods described herein.

[0100] The present application is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference.

EXAMPLES

Example 1. SM-164, given in combination with TNFa, but not alone effectively treats advanced TNBC metastasis in bone and lung in a mouse model:

[0101] The presence of TNFa in the body can explain why lAPa alone inhibits tumor growth and eliminates early metastatic TNBCs in bone and lung in mice. However, like standard chemotherapy (SCT), which includes Adriamycin (ADR, 3 mg/kg), Cytoxan (CYT, 10 mg/kg) and Zoledronic acid (ZA, 0.1 mg/kg), SM-164 alone was less effective to treat advanced bone and lung metastasis from MDA-MB-231 cells (Fig. 1).

[0102] In contrast, SM-164 plus TNFa (0.2 pg) eliminated all established metastasis in lung and half metastasis in bone from MDA-MB-231 cells in mice but SM-164 alone or SCT did not (Fig. 1). The tumor burden in the remaining half bone, although they were not eliminated, in the mice treated with SM-164+TNFa was significantly lower than those mice treated with SM-164 alone or SCT. However, TNFa can induce serious side effects in humans, including systemic shock and inflammatory reactions.

Example 2. Macrophages produce TNFa to trigger SM-164 induction of breast cancer apoptosis:

[0103] Macrophages (Macs) are the main sources of TNFa and among the most abundant support cells in a tumor microenvironment. Macs are classified as inflammatory (Ml) and anti-inflammatory (M2) Macs, which are linked to Thl- and Th2 type immune responses, respectively. Although deleterious inflammation is a primary feature of BC, tumor-associated Mac (TAM) mainly exhibits an M2 phenotype. IL-4 is traditionally considered a factor that polarizes M2s.

[0104] It was found that IL-4 alone did not trigger SM-164 induction of MDA-MB- 231 apoptosis (Fig. 2, panels A and B). However, after the cancer cells had been preincubated with mouse bone marrow cells with IL-4 in the presence of M-CSF, a factor that is essential for Mac survival and proliferation, SM-164 significantly increased apoptosis of the cancer cells and decreased the total number of GFP+ cancer cells (Fig. 2, panels A and B). Importantly, addition of a TNF receptor IgG:Fc fusion protein (TNFR:Fc) blocked apoptosis induced by IL-4-polarized Macs and SM-164 (Fig. 2, panels A and B), suggesting that IL-4- polarized Macs produce TNFa to trigger SM-164 induction of BC apoptosis.

Example 3. Mucl-directed engineered Macs guide an lAPa to eliminate metastatic BC specifically

[0105] Mucinl (Mucl) is a glycoprotein with extensive O-linked glycosylation in its extracellular domain. It is expressed on the surface of epithelial cells in many tissues. Mucl overexpression is often associated with tumorigenesis and metastases, including BC, pancreatic and lung cancer.

[0106] An engineered CAR T cell targeting a Mucl glycopeptide epitope, widely expressed by adenocarcinomas, using scFv has also been developed, and this has been shown to eliminate Mucl -expressing tumors in mouse models of leukemia and pancreatic cancer. It was confirmed that TNBC MDA-MB-231 cells highly express Mucl protein on their surface (Fig. 3).

[0107] CD33 is a transmembrane receptor in myeloid cells, mediating cell-cell interactions to maintain immune cells in a resting state. cDNA of Mucl monoclonal antibody scFv was molecularly linked to a cDNA of human CD33 segment with a transmembrane/cytoplasmic domain, which was called chimeric CD33-scFv. The cDNA of chimeric CD33-scFv and hTNFa were respectively inserted into a duet lentiviral vector, modified based on pLV-mCherry (Addgene), to construct pLV-Duet^CD33'^{scFv hTNF} plasmid. Cultured Macs from a patient/animal will be infected with pLV-Duet^CD33'^{scFv hTNF} plasmid to generate engineered Macs expressing Mucl Ab scFv on their surface and secreted TNFa, which are called Mucl-directed Macs expressing TNFa (M-Macs™¹). Administered M- Macs^{I NF} will trace and bind to metastatic TNBC cells and produce TNFa locally to kill the metastatic cancer specifically and effectively in combination with an lAPa.

Example 4. Cultured Macs expressing scFv linked to CD33 specifically recognized and attached to MDA-MB-231 cells

[0108] MacsCD33-scFv-mCherry and MacsCD33-mCherry generated by infecting cultured Macs with pLV-Duet-CD33-scFv-mCherry and the control pLV-Duet-CD33- mCherry lentivirus, respectively, intravenously injected into female NSG mice that had been injected with GFP+ MDA-MB-231 cells in the tibial marrow cavity 5 days earlier. After 3 days, the mice were euthanized and their bones were processed as frozen sections to study the interactions of Macs expressing Mucl scFv linked to CD33 (MacsCD33-scFv-mCherry) with the cancer cells using fluorescent microscopy.

[0109] Compared to Macs^CD33'^mCheny that did not co-localize with MDA-MB-231 cells, most of Macs^CD33'^scFv'^mCheny bound GFP+ MDA-MB-231 cells to become yellow (Fig. 8, panel D), confirming that cultured Macs expressing scFv linked to CD33 specifically recognized and attached to MDA-MB-231 cells.

Example 5. M-Macs^TNF expressing both scFv and TNFa together with SM-164 effectively killed MDA-MB-231 cells

[0110] Of note, Macs expressing chimeric CD33-scFv alone (Macs^CD33'^scFv'^mCheny) did not induce more apoptosis of MDA-MB-231 cells than the control Macs expressing CD33 (Fig. 9, panels A and B) in the presence of SM-164 in vitro. However, M-Macs™¹^ expressing both scFv and TNFa together with SM-164 effectively killed MDA-MB-231 cells (Fig. 9, panels A and B), the remaining number of GFP+ (live) MDA-MB-231 cells in M-Macs^{I NF} dishes being reduced to that in cultures treated with TNFa -/+ Macs in the presence of SM- 164 (Fig. 9, panels A and B).

Example 6. A model of how M-Macs^TNF enable lAPa to eliminate TNBC metastasis

[OHl] Macs can be generated from patients with TNBC and engineered to carry Mucl monoclonal antibody scFv on their surface and produce secreted TNFa, and transfer these engineered Macs back to patients to target the metastatic TNBC, enabling an lAPa to kill the cancer cells specifically and effectively. A working model illustrating how M- Macs^TNh enable lAPa to eliminate TNBC metastasis is illustrated in Fig. 10.

Example 7. Transferred M-Macs^TNF in combination with an lAPa will effectively prevent metastases following surgical removal of orthotopic PDX of TNBC

[0112] TNBC is the most aggressive subtype BC with poor prognosis. Particularly, it does not respond to the targeted therapies against hormone (estrogen or progesterone) or Her2. There is an unmet need to develop a targeted therapy for TNBC to improve survival.

[0113] The efficacy of M-Macs^TNh in combination with SM-164 to prevent the recurrence and metastasis using a commercial PDX model of TNBC from Jax lab, which has 30 available PDXs from 30 different TNBC patients, is tested. As an alternative, freshly resected TNBC samples from patients are used.

[0114] T cells are the important source of TNFa, and lAPa can augment human and mouse T cell responses and cytokine production to physiologically relevant stimuli. Thus, immune deficiency in NSG mice could limit the efficacy of lAPa to treat cancer. Humanized hCD34+ NSG (Hu-NSG) mice containing mature T cells and B cells are established. The treatments begin at the 8th day after surgical resection of orthotopically implanted PDX, mimicking an actual treatment procedure.

[0115] Experimental procedures, outlined in Fig. 11.

Establishment of Hu-NSG mice

[0116] 3 -wk-old female NSG mice (NOD-scid IL2Rgammanull, Jax Lab) will be total body irradiated with 200 cGy.

[0117] 1 * 105 enriched CD34+ hematopoietic stem cell (HSC) from human umbilical cord blood (UCB) from a single donor are injected intravenously into each mouse 24 hours after irradiation.

[0118] The engraftment levels of human CD45+ cells and human immune cell populations, including CD45+, CD3+, and CD4+, CD8+ T cells, B cells, NK cells, MDSCs, and other lineage-negative cells are determined in the peripheral blood by flow cytometry.

Mice with over 25% human CD45+ cells in the peripheral blood are considered Hu-NSG mice.

[0119] Each PDX tissue is implanted to the mammary fat pads of 12 Hu-NSG mice from the same HSC donor. Each group has duplicate mice to ensure enough number of mice with successful engraftment of HSC and the cancer from PDX samples.

[0120] The breast tumors are surgically resected when they are 1 cm, by which time about 50% of the mice develop lung metastasis from orthotopically implanted stable line of xenograft. The mice are randomly divided into 6 groups as shown in Fig. 11.

[0121] Freshly collected human peripheral blood or Commercial human CD14+ monocytes are used to culture Macs by M-CSF because they will be generated from patient's blood in future. The cultured Macs will be infected with pLV-DuetCD33-scFv+hTNF lentiviral plasmid to generate M-Macs™^ as in Fig. 8, panel D. This will take about 7 days.

[0122] Adjuvant therapy begins at day 8 after tumor resection, which mimics an actual care procedure. 2xl0⁵ M-Macs^TNh will be injected into mice in groups 3) and 4) via tail vein. The injected Macs are detected in bone, lung and brain after 7 days (Fig. 12). One dose of M-Macs^TNh is sufficient for therapeutic purposes because the lifespan of Macs can be months.

[0123] Vehicle, SM-164 (3 mg/kg), SM-164 + TNF 0.2pg twice daily and SCT one cycle each week, as in Fig. 1, a lOOpl volume will be I.P. injected into the mice for 3 weeks, as shown in Fig.11.

[0124] PDXs, CD34+ HSCs from 8 donors enable analysis whether their diversity affects the therapeutic response.

End-point evaluation:

[0125] Survival rate: body weight is measured weekly to monitor the health status. The mice is euthanized when they are dehydrated and/or paralyzed.

[0126] Digital X-ray on lower and upper extremities and spine is performed to investigate osteolytic bone lesion followed by histological examination, as in Fig. l.

[0127] The mammary fat pad, lymph nodes, lung, brain, liver and kidney is used to evaluate the visible tumor nodes on their surface followed by histological examination for tumor recurrence and metastasis.

[0128] IAP and Mucl protein levels and molecular sub-typing of TNBC in PDX tissues are tested by immunostaining and TNF receptor genetic variant is tested by PCR to analyze if they are related to the therapeutic response. [0129] Levels of TNF and other cytokines in the plasma are tested by ELISA and cytokine array to analyze if M-MacsTNF induce cytokine production and stimulate tumor growth;

[0130] % and classification of active T cells, Thl, Th2, Thl7 and cytotoxic T cells, as well as B cells in blood are analyzed by flow cytometry.

[0131] The interaction between M-Macs ^Nh and metastatic BCs is tested via double immunostaining of anti-Mucl and anti-Ku80/XRCC5, specifically expressed on human cells.

[0132] These tests demonstrate SM-164 in combination with M-Macs™¹', which target the metastatic TNBC and secrete TNF, is better than SCT and SM-164 alone to improve survival of mice with TNBC by preventing cancer recurrence and metastasis.

[0133] The mean or the median of the 3 repeats from each PDX for every treatment is used to reduce variation within a group. For the calculation of the survival rate, total number of mice (3x8=24) for each treatment is used.

[0134] 50% of the mice with 1 cm breast tumors from orthotopic PDX have pathologically detected lung metastasis. Some of the remaining mice have undetectable micro-metastasis. Therefore, SM-164 alone prolongs the survival of the mice by killing early micro metastasis in lung, bone and other organs, and its effect is better than SCT in the PDX model, like the model from MDA-MB-231 cells.

[0135] Since combined SM-164 with TNF a, but not SM-164 alone eliminates advanced metastasis from MDA-MB-231 cells (Fig. l), SM-164 in combination with either M-Macs^TNh or TNFa significantly increases the survival compared to SM-164 alone, vehicle or SCT in a PDX model. SM-164 combined with M-Macs™^ has a better effect than its combination with TNFa because M-Macs™^ will directly target and kill the metastatic cancer when SM-164 is given. M-Macs^TNh alone has a complex effect on the progression of the metastasis because TNFa can kill the cancer cells but it also induces inflammation and elevates cIAP protein to promote cancer growth.

[0136] High levels of IAP proteins in PDX samples enable them to grow faster while low levels of IAPS enable them to respond better to SM- l 64/M-Macs^TNh therapy. Intact TNF receptor on the surface of TNBC cells is required for TNF /IAPa to induce their apoptosis.

[0137] Thl and Th2 produce TNFa or polarize Macs to produce TNFa, which promote cancer growth by stimulating the expression of IAP proteins, but could improve cancer response to M-Macs^TNF/IAPa therapy. In addition, M-Macs^TNF/IAPa activates cytotoxic T cells to repress the cancer growth. [0138] Macrophages are terminal differentiated cells and their life span can be months. The therapeutic period of M-Macs ^Nh with an lAPa is about 3-4 weeks. M-Macs™^ are possibly alive and produce TNFa for a short period after completion of cancer therapy. Even if M-Macs™^ induce chronic inflammation, it will disappear soon. Bisphosphonates (BP), such as ZA, accelerate the reduction of inflammation. BPs are used to treat osteoporosis and bone metastasis, because they inhibit osteoclasts, induce apoptosis of Macs and impair Mac polarization. The use of a BP can also avoid the concern that an lAPa could induce osteoporosis and secondarily increase bone metastasis by stimulating osteoclast formation through activating NIK71. An alternative is to treat the mice with a TNFa blocker to mitigate inflammation.

[0139] Unlike CAT-T cells, which kill cancer cells by releasing cytokines, M- Macs^TNh alone do not kill cancer cells. They function as a tool to recognize and attach to the metastatic BC and solely express TNFa locally, enabling SM-164 or other lAPa to kill cancer cells specifically and effectively. CD33 in myeloid cells maintain immune cells at a resting state. The extracellular domain has been replaced by anti-Mucl scFv and thus the chimeric CD33 in M-Macs™^ does not induce immune repression. The cytoplasmic signal peptide of CD33 has been deleted. Thus M-Macs^TNh will not be activated or inhibited via CD33 signaling. M-Macs™^ do not cause cytokine release syndrome.

[0140] TNF -related apoptosis inducing ligand (TRAIL) has been reported to synergize with lAPa to kill cancer cells. The efficacy of Trail+SM-164 to eliminate metastatic BC is tested.

[0141] M-Macs^TNh target Mucl on normal epithelial cells in some tissues, but they are enriched on BC with overexpression of Mucl, as shown in Fig.3. Even if M-Macs^TNh are off-target, this will not harm normal cells even in the presence of SM-164 because normal cells express low levels of IAP proteins and their proliferation and survival do not depend on IAP proteins. No side effects have been observed in SM-164-treated mice, including normal WT mice that were treated with SM-164 for 8 months. The dose of SM-164 to induce B cell toxic death in the presence of TNFa is 300-1000-fold higher than the dose that induces BC cell apoptosis in vitro (data not shown), consistent with its low toxicity. Histological analysis of internal organs and chemical analysis for the parameters of liver and kidney function enables further monitoring if the engineered macrophage/IAPa induces any side-effects.

[0142] Among the various IAP antagonists tested, SM-164 is the strongest one to kill the BC cells and degrade cIAP proteins. However, other IAP antagonists, in particular those with confirmed biosafety through clinical trials, can be used as long as their concentration in vivo can reach the threshold that kills the cancer cells in combination with TNF.

Example 8. Transferred M-Macs^TNF in combination with an lAPa effectively treat the established metastases in brain, lung and bone from PDXs of TNBC

[0143] Except for local lymph node and lung metastasis, the orthotopically implanted PDX seldom spreads to other distant organs such as liver, bone and brain. This is probably caused by the fact that the period of the studies is relatively short in animals while the spreading of most BCs in humans take years. The cell suspension from the stable PDX to the mice is inoculated via tail vein, marrow cavity and intracarotid artery injection to force tumor residence and growth in lung, bone and brain, aiming to investigate the efficacy of M- Macs^TNh in combination with an lAPa to eliminate advanced TNBC metastasis.

Experimental procedures, outlined in Fig. 13.

[0144] Each PDX tissue is meshed to cell suspension, which is incubated with lentivirus carrying luciferase (luci) overnight to label the cells with luci, following the published protocol.

[0145] IxlO⁵ of the cell suspension is injected to Hu-NSG mice via 1) tail vein, 2) tibial bone marrow cavity and 3) intracarotid artery, 12 mice each site from each PDX, duplicate for each group, as described above.

[0146] Generate M-Macs™^ as described above.

[0147] After 2 weeks of cancer injections, the mice with established cancer in lung, bone or brain, identified by BLI as in Fig. 5, are randomly divided into 6 groups, as in Fig. 13.

[0148] 2xl0⁵ M-Macs^TNh is injected into mice in group 3) and 4) via tail vein.

[0149] The design in Fig. 13 is followed to treat the mice for 3 weeks as described above.

End-point evaluation:

[0150] Monitoring the dynamic cancer growth and metastasis by BLI as in Fig.l as well as body weight weekly.

[0151] Euthanizing the mice at day 35. Evaluation of the tumor burden in lung, bone and brain by histology.

[0152] Serum level of human TNFa and double immunostaining of anti-Mucl and anti-Ku80, as in described above. [0153] SM-164 in combination with M-Macs^{I Nh} is better than SCT or SM-164 alone to treat advanced metastasis in lung, bone and brain from TNBC. The mean or the median of the 3 repeats from each PDX for every treatment is used to reduce variation within a group.

[0154] SCT or SM-164 alone slightly inhibits the tumor growth in lung, bone and brain from the stable lines of PDX, but will not eliminate them, like from the MDA-MB-231 cells (Fig. 1). In contrast, SM-164 plus M-Macs^TNh or TNFa eliminate at least half of the tumors in lung, bone and brain, like the MDA-MB-231 cells (Fig. 1). They will also inhibit the progression of the remaining tumors in lung, bone and brain. SM-164 combined with M- Macs^TNh has a better effect than its combination with TNF to eliminate the established cancers for the reasons discussed above. Administration of M-Macs^TNh alone promotes the progression of the implanted cancers in lung, bone and brain as discussed above.

[0155] Although SM-164 is the strongest lAPa that kills BC cells, it is not known if it can go through the blood-brain barrier to kill brain cancer. A control lAPa, GDC-0152 that can enter into the brain to kill glioblastomas, provides proof of concept evidence that an lAPa in combination with M-Macs^TNh is an approach to treat advanced brain metastasis from TNBC.

[0156] SM-164 combined with M-Macs™^ also impacts survival after the stable lines of PDX grow to an advanced stage in lung, bone and brain. The metastatic BC can spread from one organ to another.

[0157] Statistical Plan and Data Analysis: Descriptive statistics are presented by means and standard deviations for continuous variables. When data distributions are skewed, median and interquartile range are used instead. In addition, frequencies are presented for categorical variables. Comparisons between two groups are analyzed using independent sample t test and those among 3 or more groups using one-way analysis of variance followed by Dunnett' s post-hoc multiple comparisons. When data distributions are not normal, Krusal Wallas is used to compare medians instead. Chi-square test is used for comparisons of frequency of bone, brain and lung metastasis in mice. Time-to-event data is analyzed using the Kaplan-Meier estimation. All analyses are performed at two tailed 0.05 significance level.

[0158] The power analysis was performed based on the ANOVA test. According to preliminary data, the means of tumor volume in the long bone in vehicle, SM-164, SCT and BV6 were 6.55, 0.28, 1.19 and 4.45 (mm²) with 3.78, 0.71, 1.58 and 2.37 (mm²) SD, respectively. Seven subjects (initially 8 to ensure enough subjects at the end) are needed in each group to achieve at least 85% power at 0.05 two tailed significance level. Example 9. M-Macs^TNF in combination with an lAPa can be used for targeted therapy of a variety of BCs

[0159] Mucl is expressed on the surface of epithelial cells in many tissues and its overexpression is often associated with tumorigenesis and metastases, including BC, pancreatic and lung cancer. Mucl is not only highly expressed on the surface of MDA-MB- 231 TNBC cells but also on TNBC MDA-MB-436, ER⁺ MCF7 and Her2⁺ BT474 human BC (Fig.3). Thus, M-Macs™^ in combination with an lAPa can be used for targeted therapy of a variety of BCs and many other cancers originated from epithelial cells.

Example 10. Cancer in mice treated with SM-164 + M-Macs^TNF and with SM-164 + TNFa was reduced to unmeasurable level

[0160] As illustrated in Fig.14A, 2xl0⁵ of BT474 human breast cancer cells was injected to a mammary fat pad, 4 sites each mouse, in 3-month-old NSG mice that had been injected with a 17-(3 estradiol pellet one day before. After 2 weeks, the mice were scanned with IVIS Spectrum imaging (PerkinElmer imaging system) to monitor cancer growth. The mice with established cancer (BLI signal higher than 10⁷ in at least one mammary in the mouse) were divided into 4 groups, 1) vehicle; 2) SM-164; 3) SM-164 + M-Macs^TNh and 4) SM-164 + TNF (0.2pg), through stratified sampling based on the highest BLI intensity in each mouse: >10⁹, 10⁸-10⁹ and 10⁷-10⁸ photons/s/cm²/sr. The cancer BLI signal, 4 mice each group, did not have difference among groups at baseline (Fig. 14, panel B). At day 17, IxlO⁵ of M-Macs^TNh was injected to a mammary fat pat in group 3. From day 21, vehicle, SM-164 (group 2 &3) and SM-164 + TNF (group) were IP injected to the mice, 2 times/day, for 2 weeks, as in Fig.l4A.

[0161] Generation of engineered macrophage. 15 ml of peripheral blood drawn from a healthy adult subject donor was incubated with 4-fold volume of NH4C1 buffer (Stemcell Tec, Cat# 07850) to lyse the red blood cells. The peripheral blood mononuclear cells (PBMCs) was suspended in monocyte culture medium (alpha-MEM containing 10% FBS, 1% non-essential amino acid and 1% L-glutamine), and cultured with 5 ng/ml of both human M-CSF and GM-CSF in four 100-mm culture dishes (lx 10⁷ cells/dish). After 5 days of culture, the cells in each dish were incubated with 4 ml of culture medium plus 1 ml of pLV- chimeric CD66-Mucl scFv and 1ml of pLV-hTNF lentiviral supernatant, 3 ug/ml of polybrene, 5 ng/ml of human M-CSF and GM-CSF for 2 days. The medium with viral particle was removed and continue to culture with M-CSF and GM-CSF for additional 5 days. The culture medium were collected for the testing human TNF concentration by ELISA (Fig.16) and the macrophages (M-Macs™¹) attached to the dishes were harvested and injected to the mice bearing cancer, as described above.

[0162] After 2 weeks of treatment, the mice were scanned with IVIS again, which shows that BLI signal in mice treated with SM-164 alone was significantly lower than those treated with vehicle, and the BLI signal was further decreased in mice with addition of either M-Macs^TNh or TNFa compared to SM-164 alone (Fig.14, panel C). Compared to baseline, the cancer BLI signal was increased in vehicle treated mice while it was reduced in mice treated with SM-164 alone or its combination with either M-Macs™^ or TNFa (Fig. 14, panel D upper panel). The BLI signal was increased 5 folds (median) in mice treated with vehicle while it was decreased by 0.66 fold in mice treated with SM-164 alone after 2-weeks of treatment (Fig. 14, panel D lower panel).

[0163] Expectedly, addition of M-Macs^TNh and TNFa in mice treated with SM-164 further decreased the cancer signal by 0.1 and 0.09 fold to their respective baseline signal, and the fold change of BLI signal in mice with addition of M-Macs^TNh after treatment did not have difference compared to those with TNFa (Fig.14, panel D lower panel). Of note is that the cancer BLI signal in 5/11 of mammary fat pads in mice treated with SM-164 + M- Macs^TNh and 3/7 in those with SM-164 + TNFa were reduced to unmeasurable level (under 10⁷). In contrast, none of the cancer BLI signal was reduced to unmeasurable level in mice treated with SM-164 alone.

[0164] The mice were euthanized at day 38 to collect the tumors and measure their weight. It was found that the cancer with BLI signal less than 10⁸ photons/s/cm2/sr at baseline was invisible or too small in mice treated with vehicle by the end. Thus, only those cancers with basal BLI signal higher than 10⁸ photons/s/cm²/sr in a mammary fat pad were used to evaluate tumor size. As expected and consistent with the BLI signal data (Fig. 14), tumor weight in mice treated with SM-164 alone was significantly lower than those treated with vehicle, and it was further decreased in mice treated with SM-164 in combination with M-Macs^TNh (Fig. 15), in which 4/11 of the tumors disappeared after treatment.

[0165] Tumor weight in mice treated with SM-164 + TNFa, although 3/7 of tumor disappeared, did not show statistical difference compared to those treated with SM-164 alone (Fig.15) probably because initial number of tumors with BLI signal higher than 10⁸ photons/s/cm²/sr was smaller and tumor size (initial BLI signal) had bigger variance in the group of SM-164 + TNFa. [0166] In summary, one time injection of IxlO⁵ of M-Macs ^Nh is as effective as or is better than daily twice injection of 0.2pg TNF a in treating or eliminating established Her2+ human breast cancer in animal model when they are combined with an IAP antagonist.

[0167] Fig. 16 shows M-Macs™^ secret hTNF. The culture medium of engineered macrophages (M-Macs^TNF), as in Figs.14 and 15, were used to test hTNF concentration by ELISA (Invitrogen). Control = culture medium from MDA-MB-231 breast cancer cells. M- Macs™^ 6 samples from two independent experiments.

[0168] A traditional cancer cell line xenograft model does not reflect cancer diversity because there are no supporting cells. Thus, the treatment results of a cell line in animals do not work in clinical trials in some cases. Patient-derived xenografts (PDX) can be developed to overcome these shortfalls. Human cancer tissue freshly collected during surgery or biopsy is directly engrafted into immunocompromised mice. The tumor of the PDX model can be expanded via serial transplantation in mice while maintaining the histology and architecture of the original tumors. Thus, using the PDX model to test the effect of the adoptive cells in combination with an IAP antagonist in treating cancer would reflect their real effects in humans.

[0169] 50 pl of minced tumor tissue from an NSG mouse bearing PDX model of breast cancer (Jax Lab #TM00090) was injected subcutaneously into the left trunk of an NSG mouse. After two weeks, when the tumors were about 0.5 cm in size, representing the rapid growth and advanced stage, the mice were randomly divided into 3 groups, 8-9 mice per group. Each mouse in group 3 was injected subcutaneously with 3xl0⁵ of M-Mac ^Nh, generated as in Fig.14-17. After 3 days, the mice were treated with vehicle or 3 mg/kg of SM- 164 twice a day for 10 days. The mice were euthanized, and the tumors were collected to scale their weights. The results indicate that SM-164 alone did not change the tumor size and weight compared to the vehicle (Fig.17). This is consistent with the facts that an IAP antagonist alone, which effectively degrades IAP proteins, does not kill cancer and TNF level is too low in vivo and is not efficient to trigger the cancer apoptosis when IAP proteins are degraded by an IAP antagonist. In contrast, the adoptive macrophage (M-Mac™¹) combined with SM-164 markedly and significantly reduced the tumor size and weight compared to either vehicle or SM-164 alone (Fig.17). These finding further confirm that the adoptive cells that target cancer to produce TNF in combination with an IAP antagonist is a practical approach to treat advanced stage cancer. [0170] Herein incorporated by reference is the sequence listing .xml file named 1134- 102. xml created February 17, 2023, which size is 26 KB.

[0171] While various embodiments have been described above, it should be understood that such disclosures have been presented by way of example only and are not limiting. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary embodiments but should be defined only in accordance with the following claims and their equivalents.

[0172] The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Appendix chimeric protein a membrane anchoring region that comprises a CD66b transmembrane and intracellular region

chimeric protein a membrane anchoring domain that comprises a CD33 transmembrane and intracellular region:

GAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKL HGPTETSSCSGAAPTVEMetDEELHYASLNFHMetNPSKDTSTEYSEVRTQ (SEQ ID

NO:2) chimeric protein a membrane anchoring domain that comprises a CD28 transmembrane and intracellular region:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP

YAPPRDFAAYRS (SEQ ID NO:3) chimeric protein a cell targeting domain that is Mucl scFV light chain:

MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPG NTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQG TTLTVSS (SEQ ID. NO:4) chimeric protein a cell targeting domain that is Mucl scFV heavy chain:

SDIVMetTQSPSSLTVTAGEKVTMetlCKSSQSLLNSGDQKNYLTWYQQKPGQPPKLLI FWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLE LK (SEQ ID. NO:5) chimeric protein a cell targeting domain that comprises Her2 scFV light chain:

MetDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIK (SEQ

ID. NO:6) chimeric protein a cell targeting domain that comprises Her2 scFV heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG QGTLVTVSS (SEQ ID. NO:7) chimeric protein a cell targeting domain that comprises a sequence from human EGF: MetNSDSECPLSHDGYCLHDGVMetYIEALDKYACNCVVGYIGERCQYRDLKWWEL R (SEQ ID NO:8) chimeric protein a linker region between scFV light chain and heavy chain sequences: GGGGSGGGGSGGGG (SEQ ID. NO:9) chimeric protein a hinge region between the cell targeting domain and the membrane anchoring domain: ESKYGPPCPSCP (SEQ. ID. NO: 10)

Chimeric CD66b-Mucl scFV:

MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPG NTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQG TTLTVSSGGGGSGGGGSGGGGSDIVMetTQSPSSLTVTAGEKVTMetICKSSQSLLNSG DQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA VYYCQNDYSYPLTFGAGTKLELKESKYGPPCPSCPIPNITTKNSGSYACHTTNSATGR NRTTVRMetITVSDALVQG SSPGLSARATVSIMetIGVLARVALI (SEQ ID NO:11)

Chimeric CD33-Mucl scFV:

MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPG NTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQG TTLTVSSGGGGSGGGGSGGGGSDIVMetTQSPSSLTVTAGEKVTMetICKSSQSLLNSG DQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA VYYCQNDYSYPLTFGAGTKLELKESKYGPPCPSCPGAIGGAGVTALLALCLCLIFFIV K I HRRK AAR1 A VGRND THPI TGS ASPKHQKKSKLHGPI El S SC SG AAPI VEM DEEL

HYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 12)

Chimeric CD33-Her2 scFV: Met

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS

ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGG GG

SGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG DGFYAMDYWGQGTLVTVSSESKYGPPCPSCPGAIGGAGVTALLALCLCLIFFIVKTH RRKAARTAVGRNDTHPTTGS ASPKHQKKSKLHGPTETS SC SGAAPTVEMetDEELHY ASLNFHGMetNPSKDTSTEYSEVRTQ (SEQ ID NO: 13)

Chimeric CD33-hEGF:

MetNSDSECPLSHDGYCLHDGVCMetYIEALDKYACNCVVGYIGERCQYRDLKWWE LRE

SKYGPPCPSCPGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSA SP

KHQKKSKLHGPTETSSCSGAAPTVEMetDEELHYASLNFHGMetNPSKDTSTEYSEVR TQ (SEQ ID NO: 14)

Chimeric CD28-Mucl scFV:

MetQVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSP GNTDIKYNDKFKGKATLTVDRSSSTAYMetQLNSLTSEDSAVYFCKTSTFFFDYWGQ GTTLTVSSGGGGSGGGGSGGGGSDIVMetTQSPSSLTVTAGEKVTMetICKSSQSLLNS GDQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDL AVYYCQNDYSYPLTFGAGTKLELKESKYGPPCPSCPFWVLVVVGGVLACYSLLVTV

AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 15)

Chimeric CD28-hEGF :

MetNSDSECPLSHDGYCLHDGVCMetYIEALDKYACNCVVGYIGERCQYRDLKWWE LR

ESKYGPPCPSCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 16) human TNFa-l full length:

MetSTESMetIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGAT

TLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQ WL

NRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSY

Q

TI<VNLLSAII<SPCQRETPEGAEAI<PWYEPIYLGGVFQLEI<GDRLSAEINRPDYLDFAE

SG QVYFGIIAL (SEQ. ID. NO: 17) human TNFa-2 soluble form:

MetVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSE

GLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAK

PWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ. ID. NO: 18)

Claims

WHAT IS CLAIMED IS:

1. An expression vector comprising:

(1) a first nucleotide sequence encoding a chimeric protein comprising a membrane anchoring domain and a cell targeting domain;

(2) a second nucleotide sequence encoding TNFa; and

(3) a regulatory sequence operably linked to at least one of the first and the second nucleotide sequence.

2. The expression vector of claim 1, wherein the vector is a viral vector.

3. The expression vector of claim 2, wherein the viral vector is a retrovirus vector.

4. The expression vector of claims 1 to 3, wherein the viral vector is a lentivirus vector.

5. The expression vector of any one of claims 1-4, wherein the membrane anchoring domain comprises a transmembrane domain of a myeloid cell receptor selected from the group consisting of CDl la, CDl lb, CDl lc, CD14; CD16, CD18, CD33, CD36, CD40; CD45, CD63, CD64, CD66b, CD68, CD80, CD85, CD86, CD115, CD163; CD169, CD192, CD 195, CD200R, CD204, CD206, CD209, CD282, CD284, EMR1, Ly6C, Marco, MER, CCRL1, Tim4 and IL-1R.

6. The expression vector of any one of claims 1-4, wherein the membrane anchoring domain comprises a transmembrane domain of a T cell receptor selected from the group consisting of CD3e, CD4, CD5, CD8a, CD8b, CD25, CD27, CD28, CD30, CD39, CD62L, CD73, CD80, CD86, CD103, CD134, CD152, CD153, CD192, CD196, CD223 and CD137.

7. The expression vector of any one of claims 1-4, wherein the membrane anchoring domain comprises SEQ ID NO: 1, 2 or 3.

8. The expression vector of any one of claims 1-7, wherein the cell targeting domain comprises a cell targeting sequence selected from a protein in the group consisting of alphafetoprotein, CD66e, CA-125, Mucin-1; Her-2, mesothelin, PSCA (prostate stem cell antigen), Claudin 18.2, EpCAM, Nectin4/FAP, Lewis Y, Glypican-3, CD171, PSMA, CD20, ephrin type-A receptor 2 (EPHA2), CD80/86, c-MET, DLL-3, gplOO, MAGE-A1/3/4, LMP1, EGFR, VEGFR2, EGFRIII, IL-13Ra2, DR5 and FR-a.

9. The expression vector of any one of claims 1-7, wherein the cell targeting domain comprises SEQ ID NO:4, 5, 6, 7 or 8.

10. The expression vector of any one of claims 1-4, wherein the chimeric protein comprises SEQ ID NO: 11, 12, 13, 14, 15 or 16.

I L A cell transduced with the expression vector of any one of claims 1-10.

12. The cell of claim 11, wherein the cell is a macrophage or monocyte or granulocyte or dendritic cell, or a T cell, or a nature killer cell.

13. A cell comprising: a chimeric protein on a cell surface, wherein the chimeric protein comprises a cell membrane anchoring domain and a cell targeting domain; and an expression cassette that expresses TNFa.

14. The cell of claim 13, wherein the cell is a macrophage or monocyte or granulocyte or dendritic cell.

15. The cell of claim 13, wherein the cell is a T-cell.

16. A method for treating tumor in a subject, comprising the steps of: administering to the subject an effective amount of the cells of any one of claims 11- 15; and administering to the subject an effective amount of an IAP antagonist.

17. The method of claim 16, wherein the IAP antagonist is administered for a period of 1-3 months after cell administration, wherein a first dose of the IAP antagonist is giving within 14 days of cell administration.

18. The method of claim 16 or 17, wherein the tumor is a malignant tumor selected from the group consisting of hepatocellular carcinoma, colorectal cancer, ovarian cancer, B- cell lymphoma and multiple myeloma, breast cancer, mesothelioma, pancreatic cancer, lung cancer, prostate cancer, melanoma, osteosarcoma, glioma and brain tumor.

19. The method of claim 17, wherein the tumor is breast cancer.

20. The method of any one of claims 16-19, wherein the IAP antagonist is selected from the group consisting of BI-5, BI-891065, T-3256336, LBW-242, NVP-LBW242, ASTX-660 (tolinapant), AT-IAP, TQB-3728, GT-13065, IGM-9427, NSC-756502, birinapant (TL32711), LCL161, CUDC-427, GDC-0152, BV6, MV1, APG-1387, AT-406 (Xevinapant, Debiol 143), SM-1387, XIAP antagonists, second mitochondria-derived activator of caspases mimetics, proline mimetics and SMAC mimetics.

21. A method for treating a tumor in a subject, comprising the steps of: administering to the subject an effective amount of TNFa; and administering to the subject an effective amount of an IAP antagonist.

22. The method of claim 21, wherein the IAP antagonist is administered prior to, concurrently with, or after, the administration of the TNFa.

23. The method of claim 21 or 22, wherein the tumor is a malignant tumor selected from the group consisting of hepatocellular carcinoma, colorectal cancer, ovarian cancer, B-cell lymphoma and multiple myeloma, breast cancer, mesothelioma, pancreatic cancer, lung cancer, prostate cancer, melanoma, osteosarcoma, glioma and brain tumor.

24. The method of claim 23, wherein the tumor is breast cancer.

25. The method of any one of claims 21-24, wherein the IAP antagonist is selected from the group consisting of HGS1029, BI-891065, T-3256336, LBW-242, NVP-LBW242, ASTX660 (tolinapant), AT-IAP, TQB-3728, Birinapant (TL32711, NSC-756502), LCL161, CUDC-427 (GDC-0917), GDC-0152, BV6, MV L APG-1387, AT-406 (Xevinapant, Debiol 143), SM-164, SM-122, SM-1387, XIAP antagonists, second mitochondria-derived activator of caspases mimetics, proline mimetics and SMAC mimetics.