Methods of treating neogenetic diseases
阅读说明：本技术 治疗新生性疾病的方法 (Methods of treating neogenetic diseases ) 是由 朱正仑 于 2018-03-28 设计创作，主要内容包括：治疗癌症的方法,所述方法包括：提供经修饰的巨噬细胞或单核细胞,其含有编码Hom-1多肽或其含有Hom-1同源框域的片段的外源核酸序列,其中所述经修饰的巨噬细胞或单核细胞表达Hom-1多肽或其片段；并且对患有癌症的受试者施用所述经修饰的巨噬细胞或单核细胞。(A method of treating cancer, the method comprising: providing a modified macrophage or monocyte comprising an exogenous nucleic acid sequence encoding a Hom-1 polypeptide or a Hom-1 homeobox domain-containing fragment thereof, wherein the modified macrophage or monocyte expresses a Hom-1 polypeptide or fragment thereof; and administering the modified macrophage or monocyte to a subject having cancer.)
1. A method of treating cancer, the method comprising:
providing a modified macrophage or monocyte comprising an exogenous nucleic acid sequence encoding a Hom-1 polypeptide or a Hom-1 homeobox domain-containing fragment thereof, wherein the modified macrophage or monocyte expresses the Hom-1 polypeptide or fragment thereof; and is
Administering the modified macrophage or monocyte to a subject having cancer.
2. The method of claim 2, wherein the modified macrophage or monocyte is produced by introducing the exogenous nucleic acid into a macrophage or monocyte derived from the subject or another subject.
3. The method of claim 1, wherein the exogenous nucleic acid is an mRNA molecule.
4. The method of claim 3, wherein the mRNA molecule is a chemically modified mRNA molecule.
5. The method of claim 1, wherein the modified macrophage exhibits the M1 phenotype.
6. The method of claim 1, wherein the exogenous nucleic acid sequence is operably linked to a heterologous or endogenous promoter.
7. The method of claim 6, wherein the promoter is a constitutive promoter.
8. The method of claim 6, wherein the promoter is an inducible promoter.
9. The method of any one of claims 1 to 8, further comprising administering an immunomodulator to the subject.
10. The method of claim 9, wherein the immunomodulator is selected from the group consisting of: CAR-T cells, immune checkpoint inhibitors and antibodies against tumor specific antigens, tumor associated antigens or neoantigens (neoantigen).
11. The method of claim 10, wherein the neoantigen is CK 20.
12. The method of any one of claims 1-11, further comprising detecting a lower level of Hom-1 expression in tumor-associated macrophages of the subject prior to the administering step as compared to a control.
13. The method of claim 1, wherein the cancer is selected from the group consisting of: leukemia, sarcoma, osteosarcoma, lymphoma, melanoma, glioma, glioblastoma, pheochromocytoma, liver cancer, ovarian cancer, skin cancer, testicular cancer, stomach cancer, pancreatic cancer, kidney cancer, breast cancer, prostate cancer, colorectal cancer, head and neck cancer, brain cancer, esophageal cancer, bladder cancer, adrenal cortex cancer, lung cancer, bronchial cancer, thyroid cancer, endometrial cancer, nasopharyngeal cancer, cervical cancer, liver cancer, metastatic cancer, and cancer of unknown primary site.
14. A method of treating cancer, the method comprising:
contacting a macrophage or monocyte with one or more agents that induce the expression of Hom-1, such that the expression level of endogenous Hom-1 in the macrophage or monocyte is greater than prior to the contacting step; and is
Administering the macrophage or monocyte and the immunomodulator so contacted to a subject having cancer.
15. The method of claim 14, wherein the immunomodulator is selected from the group consisting of: CAR-T cells, immune checkpoint inhibitors and antibodies against tumor-specific antigens, tumor-associated antigens or neoantigens.
16. The method of claim 15, wherein the neoantigen is CK 20.
17. The method of claim 14, wherein the macrophages or monocytes are autologous or allogeneic to the subject.
18. The method of claim 14, wherein the contacting step and the applying step are repeated at least once.
19. A method of treating cancer, the method comprising:
contacting a macrophage or monocyte with an agent that induces expression of the M1 gene or an agent that inhibits expression of the M2 gene, thereby producing a macrophage that exhibits the M1 phenotype; and is
Administering the macrophages and immunomodulators so produced to a subject having cancer.
20. The method of claim 19, wherein the macrophages or monocytes are autologous or allogeneic to the subject.
21. The method of claim 19, wherein the immunomodulator is selected from the group consisting of: CAR-T cells, immune checkpoint inhibitors and antibodies against tumor-specific antigens, tumor-associated antigens or neoantigens.
22. The method of claim 20, wherein the neoantigen is CK 20.
Background
Macrophages are the performers of innate and adaptive immunity and have been recognized as a key component of tumors and their microenvironment. Extensive studies have shown a role for tumor-associated macrophages (TAMs) in the growth, invasion and metastasis of almost all tumors. TAMs are derived from circulating monocytes and exhibit a wide range of phenotypes ranging from an M1-like phenotype in the early stages of selected tumors to an M2-like phenotype in most advanced tumors. Consistent with its role in promoting tumorigenesis, M2-like TAMs have a characteristic phenotype with elevated expression of IL-10, IL4, MMPs, VEGF, but reduced expression of cytotoxic iNO and ROI and proinflammatory cytokines involved in tumoricidal activity. TAMs, in addition to their intrinsic function in promoting tumorigenesis, contribute to the suppression of anti-tumor immunity through a balance in alternating T cell responses and tumor microenvironment. The functional plasticity of TAMs is well recognized. It has been proposed that TAMs can function as attractive targets for anti-tumor therapy by converting the pro-tumor M2-like TAMs to an anti-tumor M1-like phenotype. Over the past few years, the potential functions of a variety of extracellular and intrinsic factors in the conversion of TAMs into tumoricidal cells have been explored. However, the lack of understanding of how intracellular factors control the plasticity of TAMs limits the effectiveness and potential applications of targeted TAMs in tumor therapy.
Tumor-specific and tumor-associated antigens have been extensively studied as targets for cancer immunotherapy. Targeting these antigens can potentially minimize off-target toxicity with increased efficacy. On the other hand, antibodies against these antigens are often less effective alone.
Summary of The Invention
In one aspect, provided herein are methods of treating cancer. The method comprises providing a modified macrophage or monocyte comprising an exogenous nucleic acid sequence encoding a Hom-1 polypeptide or a fragment thereof comprising a Hom-1 homeobox domain, wherein the modified macrophage or monocyte expresses a Hom-1 polypeptide or fragment thereof; administering the modified macrophage or monocyte to a subject having cancer. The modified macrophages exhibited the M1 phenotype. In some embodiments, the nucleic acid sequence is an mRNA molecule. In some embodiments, the exogenous nucleic acid sequence is operably linked to a heterologous or endogenous promoter. The method may further comprise administering an immunomodulator to the subject.
In another aspect, described herein is a method of treating cancer, the method comprising contacting a macrophage or monocyte with one or more agents that induce the expression of Hom-1, whereby the expression level of endogenous Hom-1 in the macrophage or monocyte is higher than prior to the contacting step; and administering the macrophage or monocyte and the immunomodulator so contacted to the subject having the cancer.
Also described herein are methods of treating cancer comprising contacting a macrophage or monocyte with an agent that induces expression of the M1 gene or an agent that inhibits expression of the M2 gene, thereby producing a macrophage that exhibits the M1 phenotype; administering the macrophages and immunomodulators so produced to a subject having cancer.
In any of the methods, the immunomodulator may be selected from the group consisting of: CAR-T cells, immune checkpoint inhibitors and antibodies against tumor-specific antigens, tumor-associated antigens or neoantigens. In some embodiments, the neoantigen is CK 20.
Any of the methods disclosed herein can further comprise, prior to the administering step, detecting a lower level of Hom-1 expression in tumor-associated macrophages of the subject as compared to a control.
The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages of the embodiments will be apparent from the description and from the claims.
Detailed Description
It was unexpectedly found that Hom-1 expression in TAMs is significantly reduced compared to macrophages isolated from normal tissues. It was further found, also unexpectedly, that increasing the expression of Hom-1 in TAMs transformed them into M1-like macrophages with tumoricidal activity. In addition, it has been shown that co-administration of a TAM expressing Hom-1 and an antibody against the neoantigen is surprisingly effective for inhibiting tumor growth in vivo. Thus, Hom-1-modulated TAMs can be used as a novel form of cancer therapy, either alone or in combination with immune modulators (e.g., CAR-T cells, immune checkpoint inhibitors, or antibodies against tumor-specific, tumor-associated, or neoantigens (e.g., CK20 for colorectal cancer, ME1 for lung cancer, and CDC27 for melanoma).
The human homeobox transcription factor Hom-1 is a canonical antagonist of Wnt signaling. Disclosed herein are the nucleic acid sequence of Hom-1 (SEQ ID NO:1) and the amino acid sequence encoded thereby (SEQ ID NO: 2). Positions 91-151 within SEQ ID NO 2 encompass the homeobox domain.
Described herein are methods of treating cancer in a subject by administering to the subject macrophages that exhibit anti-tumor activity. As used herein, unless otherwise specified, the terms "macrophage exhibiting anti-tumor activity", "M1-like macrophage" and "macrophage exhibiting the M1 phenotype" are used interchangeably.
M1-like macrophages can be produced by: (1) increasing expression of Hom-1 in macrophages or monocytes; (2) increasing the expression of one or more M1 genes in macrophages or monocytes; and/or (3) inhibiting the expression of one or more M2 genes in macrophages or monocytes.
Since it was shown that Hom-1 expression is both necessary and sufficient for monocyte differentiation into macrophages, monocytes (e.g., monocytes derived from the peripheral blood of a subject) can be used in this method. In other words, increasing the expression of Hom-1 in monocytes may promote their differentiation into macrophages.
Macrophages or monocytes may be induced ex vivo to express higher levels of endogenous Hom-1. Various agents or therapeutic methods can be used to induce Hom-1 expression, such as LPS, Cholera Toxin (CTX), chemotherapeutic agents, radiation, cytokines (e.g., GM-CSF), phorbol 12-myristate 13-acetate (PMA), and antibodies or RNAi against inhibitors of Hom-1 expression.
Modified macrophages or monocytes exhibiting the M1 phenotype may be produced by introducing into the macrophages or monocytes an exogenous mRNA molecule (e.g., a synthetic mRNA molecule) comprising a sequence encoding a Hom-1 polypeptide or fragment thereof comprising a Hom-1 homeobox domain. Thus, expression of Hom-1 in macrophages or monocytes is transiently increased to induce the M1 phenotype. The modified macrophages or monocytes may then be administered to a cancer patient. The mRNA molecule may be a chemically modified mRNA to promote mRNA stability and/or translation efficiency.
Macrophages or monocytes that have been genetically modified to express elevated levels of Hom-1 may also be used to treat a subject with cancer. For example, a genetically modified macrophage or monocyte can contain a nucleic acid sequence encoding a Hom-1 polypeptide or a fragment thereof that comprises a Hom-1 homeobox domain. The nucleic acid sequence is operably linked to a promoter, and the modified macrophage or monocyte expresses the Hom-1 polypeptide or a fragment thereof. Such modified macrophages or modified monocytes (which will differentiate into macrophages) express sufficiently high levels of Hom-1 to exhibit anti-tumor activity and/or an M1 phenotype.
Genetically modified macrophages or monocytes may also be produced by introducing additional copies of the Hom-1 gene into the macrophages or monocytes. For example, an expression construct comprising a Hom-1 nucleic acid sequence (encoding a Hom-1 polypeptide or a fragment thereof comprising a Hom-1 homeobox domain) operably linked to an endogenous Hom-1 promoter can be introduced into a macrophage or monocyte.
The expression level of endogenous Hom-1 in macrophages or monocytes can also be increased by genetically modifying the regulatory elements for Hom-1 expression. For example, one or more negative transcriptional or translational regulatory elements of Hom-1 can be modified, deleted, or replaced to increase Hom-1 transcript and/or protein levels. Genome editing techniques using CRIPR, TALENs or ZFNs, or other techniques known in the art, can be used to alter the regulatory elements of Hom-1 expression.
The Hom-1 polypeptide or a fragment thereof comprising the Hom-1 homeobox domain can also be introduced into macrophages or monocytes by direct peptide delivery.
Methods known in the art can be used to genetically modify macrophages and monocytes. For example, an exogenous expression construct for expression of Hom-1 can be introduced (e.g., stably or transiently transfected) into macrophages or monocytes. In one embodiment, the Hom-1 nucleic acid sequence is operably linked to a heterologous (i.e., non-Hom-1 promoter) constitutive or inducible promoter. In one embodiment, the Hom-1 nucleic acid sequence is operably linked to an endogenous promoter.
M1-like tumoricidal macrophages can also be produced by inducing M1 gene expression in macrophages or monocytes. Agents that can induce the M1 gene include, but are not limited to, LPS, CTX, PMA, GM-SCF, INF γ, and chemotherapeutic agents. Macrophages or monocytes may also be genetically modified to express elevated levels of the M1 gene. The M1 genes include IL1b, IL6, IL12, IL23, TNF α, iNO, CD40, CD80, CD86, CD68, TLR4, TLR2, IL-1R, MHCII, CCL15, CCL20, CXCL9, CXCL1 and SOCS 3.
Inhibition of the expression of the M2 gene in macrophages or monocytes may also produce M1-like tumoricidal macrophages. Agents that inhibit the M2 gene include anti-IL 4 agents (e.g., antibodies or RNAi agents), anti-IL 13 agents (e.g., antibodies or RNAi agents), antibodies to the M2 protein, and RNAi agents that target the M2 gene. M2 genes include ARG1, MMP9, CCL18, VEGF, IL10, IL4, TGFb, CD163, CD206, CD68, TLR8, TLR1, MHCII, TGM2, DcoyR, IL-1RII, Ym1/2, MMR/CD206, and SR.
Allogeneic or autophagic macrophages or monocytes may be used to produce M1-like macrophages. If heterologous macrophages or monocytes are used, HLA matching may be performed to avoid or minimize host responses. HLA-mismatched macrophages or monocytes may also be used. Autologous macrophages or monocytes may be obtained from a cancer patient using methods known in the art.
Immunomodulators enhance, inhibit or modulate one or more components of the immune system. Such modulators include CAR-T cells, immune checkpoint inhibitors or antibodies against tumor specific antigens, tumor associated antigens or neoantigens (e.g., CK20 for colorectal cancer, ME1 for lung cancer and CDC27 for melanoma).
The resulting M1-like macrophages and/or immunomodulators can be administered to a subject by infusion or injection (e.g., by intravenous, intrathecal, intramuscular, intracavity, intratracheal, intraperitoneal, intracranial, subcutaneous or another type of intraluminal route), transdermal administration, or other routes known in the art. In one example, macrophages, monocytes and/or immunomodulators can be injected directly into or around the site or tissue where the tumor is found (e.g., the liver or pancreas).
The subject may be treated with M1-like macrophages as often as necessary (e.g., every 1 to 30 days) and multiple times (e.g., 1-30 times) to treat the cancer. The M1-like macrophages described herein may also be used in combination therapy with other cancer treatments (e.g., radiation, chemotherapy, and small molecule drugs).
The data below show that Hom-1 expression converts TAMs into tumoricidal cells independent of tumor type. Thus, M1-like macrophages as described herein can be used to treat any cancer, particularly cancers associated with TAMs that express low levels of Hom-1. Prior to treating a subject with M1-like macrophages as described above, it can be useful to determine whether the level of Hom-1 expression in the subject's TAM is lower than the level of Hom-1 expression found in a control group (e.g., macrophages found in the subject's normal tissue).
Examples of cancers that can be treated with M1-like macrophages include, but are not limited to, carcinomas and sarcomas, such as leukemias, sarcomas, osteosarcomas, lymphomas, melanomas, gliomas, glioblastoma, pheochromocytoma, liver cancer, ovarian cancer, skin cancer, testicular cancer, stomach cancer, pancreatic cancer, kidney cancer, breast cancer, prostate cancer, colorectal cancer, head and neck cancer, brain cancer, esophageal cancer, bladder cancer, adrenal cortex cancer, lung cancer, bronchial cancer, thyroid cancer, endometrial cancer, nasopharyngeal cancer, cervical cancer, liver cancer, metastatic cancer, and cancers of unknown primary sites.
Detection of a lower level of expression of the Hom-1 or M1 gene or a higher level of expression of the M2 gene in macrophages found in the microenvironment of a tissue region as compared to the level of a control (e.g., the corresponding level in macrophages of normal tissue) indicates that the tissue region is or is at risk of becoming cancer. Accordingly, methods for identifying whether a suspect tissue region is cancer or is at risk of becoming cancer are also contemplated herein.
Further described herein are methods of identifying candidate compounds for treating cancer. A test cell (i.e., macrophage or monocyte) can be contacted with a test compound and the expression level of: (i) hom-1, (ii) a reporter gene operably linked to the Hom-1 promoter, (iii) an M1 gene, (iv) a reporter gene operably linked to the M1 promoter, (v) an M2 gene, or (vi) a reporter gene operably linked to the M2 promoter. A test compound that increases the expression level of any one of (i) - (iv) and/or decreases the expression level of (v) or (vi) as compared to a control (e.g., the corresponding level in a test cell not contacted with the test compound) is a candidate compound for treating cancer.
In one screening method, a test compound is added to a co-culture containing a test cell and a cancer sample. A test compound is a candidate compound for treating cancer if it (i) increases the level of expression of Hom-1, a reporter gene operably linked to the Hom-1 promoter, M1 gene, a reporter gene operably linked to the M1 promoter in a test cell, (ii) inhibits a significant decrease in the level of expression of Hom-1, a reporter gene operably linked to the Hom-1 promoter, M1 gene, or a reporter gene operably linked to the M1 gene in a test cell, or (iii) decreases the level of expression of the M2 gene or a reporter gene operably linked to the M2 promoter in a test cell, as compared to a control.
In a co-culture system, it can also be determined whether the test compound has an inhibitory effect on the cancer sample. A test compound that inhibits a cancer sample (e.g., inhibits growth of cancer cells, kills cancer cells, or reduces the size of a cancer sample) as compared to a control is a candidate compound for treating cancer. The test cell and the cancer sample may be in direct contact with each other. Alternatively, the test cell and the cancer sample are not in direct contact (e.g., using a transwell insert). The cancer sample can be a sample containing cancer cells, e.g., a cancer tissue sample, cancer cells isolated from a cancer tissue sample, or cells of a cancer cell line. The cancer tissue sample may be obtained from a surgical dissected specimen from a cancer patient. Such cancer tissue samples may comprise TAMs.
The screening method can also be performed with a cancer tissue sample in the absence of test cells. The cancer tissue sample can be contacted with the test compound. After a period of time, the TAM can be isolated from the cancer tissue sample and the level of expression of the Hom-1, M1 gene or M2 gene in the TAM can be determined. Alternatively, or in addition, the expression level of Hom-1 in the tissue sample can be determined. A test compound is a candidate compound for treating cancer if it (i) increases the expression level of the Hom-1 or M1 gene, (ii) inhibits a significant decrease in the expression level of the Hom-1 or M1 gene, and/or (iii) decreases the expression level of the M2 gene, as compared to a control. A test compound that inhibits a cancer tissue sample (e.g., reduces the size of the sample) as compared to a control is also considered a candidate compound for treating cancer.
Test compounds to be screened (e.g., proteins, peptides, peptide mimetics, peptoids, antibodies, RNAi, small molecules, or other drugs) can be obtained using methods known in the art.
In any of the methods described herein, the expression level of the Hom-1, M1 gene, or M2 gene can be determined at the mRNA level or the protein level. Promoter activity can also be measured. Methods for measuring mRNA levels, protein levels, and promoter activity are well known in the art.
In any of the above screening methods, the test cell may be a macrophage or monocyte. The macrophage can be a M1 macrophage, a M2 macrophage, a tumor-associated macrophage, a tissue macrophage, or a monocyte-derived macrophage. The test cell may also be a monocyte.
The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are incorporated by reference in their entirety.
Examples
We identified a role for human homeobox protein Hom-1 in the functional polarization of human macrophages. We found that Hom-1 promotes M1 activation of human macrophages and is required for M1 activation of human macrophages, but is not required for expression of key genes involved in M2 activation.
Using primary TAMs isolated from cancer, we found a significant reduction in Hom-1 expression in TAMs compared to macrophages isolated from normal control tissues. We show that the expression profile of HAM-1 in TAM correlates with the TAM phenotype. In addition, ectopic expression of Hom-1 converts TAM to an M1-like phenotype. Both in vitro and in vivo data show that Hom-1 confers tumoricidal activity to TAMs. Our studies have shown together that Hom-1 converts TAMs into tumoricidal cells. We show that the TAM expressing Hom-1 (exhibiting the M1 phenotype) exerts a strong inhibitory effect on the growth of a variety of cancers, suggesting the role of Hom-1 regulated TAM as a novel form of treatment for cancer.
Hom-1 expression is reduced in TAM
In advanced tumors, TAM showed a pro-tumor M2-like phenotype. See Bronte and Murray (2015), NatMed 21, 117-. The plasticity of TAMs is well understood and a variety of cytokines are associated with polarization of TAMs towards the M2 phenotype. See Noy and Pollard (2014), Immunity 41, 49-61. In contrast, the transcriptional mechanisms that control TAM polarization remain largely unknown.
TAMs were isolated from tumor tissue and macrophages were isolated from normal mucosa 15cm from the tumor site using surgical specimens discarded from colon cancer resection, as described below. As previously described, FACS analysis showed that TAMs expressed significantly higher levels of cell surface markers associated with the M2 phenotype (e.g., CD68, CD163, CD206) compared to macrophages isolated from normal control mucosa. See Zhang et al (2013), Eur J Cancer 49, 3320-. We found no significant difference in expression of the non-distinctive macrophage marker CD33 in TAM and control macrophages.
To determine whether Hom-1 can play a role in TAMs, we quantified Hom-1 expression in TAMs by qRT-PCR and found that Hom-1 expression was significantly reduced in TAMs compared to its expression in control macrophages.
VentX modulates TAM plasticity and polarizes TAM towards the M1 phenotype
Previous studies indicate that LPS can induce TAM to show the M1 phenotype. See Zhang et al. To determine whether Hom-1 plays a role in TAM plasticity, we examined the expression of Hom-1 in LPS-exposed TAM. We found that Hom-1 expression was significantly elevated in TAM after stimulation with LPS. In parallel with the elevated Hom-1 expression and in agreement with previous findings, LPS stimulation of TAMs resulted in increased secretion of inflammatory cytokines and cytotoxic iNO.
To determine whether Hom-1 plays a regulatory role in TAM plasticity, we examined the effect of Hom-1 knockdown on TAM phenotype. Treatment of TAM with anti-Hom-1 Morpholino (MO) resulted in about an 80% reduction in Hom-1 expression. Consistent with the role of Hom-1 as a key regulator of TAM plasticity, we found that Hom-1MO terminates LPS-induced secretion of inflammatory cytokines and cytotoxic iNO in TAM. CD206 is the mannose receptor and M2 cell surface marker, which is highly expressed in TAMs. Reflecting the plasticity of TAMs, exposure to LPS of TAMs resulted in a significant reduction in the CD68+ CD206+ population and an increase in the number of CD68+ CD 206-cells. Hom-1MO abolished both of these effects of LPS on TAM (p < 0.01).
The correlation between the level of Hom-1 expression and the TAM phenotype prompted us to further explore the idea that Hom-1 controls TAM plasticity. TAM was isolated and transfected with plasmids encoding GPF-Hom-1 or control GFP. TAM transfected with GFP-Hom-1 showed a characteristic M1 morphology, with elongated/fibroblast-like cell shape, compared to TAM transfected with control GFP. FACS analysis showed a significant increase in surface expression of the M1 markers CD40, CD80 and CD86 in TAM transfected with GFP-Hom-1. In addition, in TAM transfected with GFP-Hom-1, secretion of the proinflammatory cytokines TNF alpha, IL-1 beta and IL-12 was significantly increased, while secretion of the M2 cytokine IL-10 was significantly decreased. Consistently, gene expression analysis showed a significant increase in M1 genes such as IL-1 β, IL-6, TNF- α, and iNO in TAM transfected with GFP-Hom-1, while M2 genes such as CCL18, MMP9, VEGFA, and Arg1 in TAM transfected with GFP-Hom-1. Together, our data suggest that Hom-1 modulates TAM plasticity and promotes M1 polarization of TAM.
Hom-1 conversion of TAM into tumor suppressor cells
Our finding that Hom-1 promotes M1 polarization of TAMs prompted us to explore whether Hom-1 modified TAMs could exert tumor suppression. Freshly isolated TAM from colon cancer was transfected with plasmids encoding GPF-Hom-1 or control GFP. The modified TAMs were then co-cultured with tumor or normal tissue of the same patient using a rotating well culture system. Notably, after 7-10 days of co-culture, tumor volume was significantly reduced (about 70%) (p <0.01) during incubation with GFP-Hom-1 modified TAMs, while there was no significant change in tumor size in incubation with GFP-transfected TAMs or TAMs alone. To determine whether the reduction in tumor volume was associated with a reduction in cancer cells, we performed tissue sectioning, H & E staining and immunohistochemistry on colon cancer cells using the CK20 antibody. We found CK20 positive tumor cells to be present in nests (nest), cords (cord) and patches of tumors incubated with TAMs or GFP-modified TAMs. However, CK20 positive tumor cells disappeared during incubation with TAM transfected with GFP-Hom-1. The specificity of the tumoricidal effect of Hom-1 modified TAMs is demonstrated by the following findings: the Hom-1 modified TAM exerts minimal effects on the volume and morphology of normal colonic mucosa during incubation with either GFP or GFP-Hom-1 transfected TAM.
Hom-1 promotes the tumoricidal function of TAM in vivo
Our findings that Hom-1 converts TAMs into tumoricidal cells in vitro have prompted us to explore the potential role of Hom-1-regulated TAMs in tumorigenesis in vivo. Colon cancer was cut into pieces of about 0.5cm and surgically inoculated into the subcutaneous space on the ventral side of NSG mice. One week later, TAM transfected with MO-Hom-1 (Hom-1 inhibitory) or GFP-Hom-1 (Hom-1 expressive) was injected via the tail vein of mice. Tumors were formed in mice injected with TAM transfected with MO-Hom-1, but not in mice injected with TAM transfected with GFP-Hom-1, 8 weeks after xenografting.
Furthermore, we evaluated the effect of anti-CK 20 antibody in combination with TAM transfected with MO-Hom-1 in the same mouse model. Mice were administered antibodies alone or in combination with MO-Hom-1 transfected TAM after tumor formation. Tumors in mice administered only antibody continue to grow. On the other hand, tumors in mice administered with the antibody and MO-Hom-1 transfected TAM stopped growing or grew at a much slower rate than mice treated with the antibody alone.
We also found that TAMs or monocytes can be induced to exhibit the M1 phenotype by culturing them in M1 differentiation medium. These M1 differentiated TAMs/monocytes can be infused into NSG mice and inhibit cancer growth in vivo. The effect of M1 differentiated TAM on tumor growth was abolished by inhibiting the expression of Hom-1 in these TAMs or monocytes.
Effect of Hom-1 ectopic expression in TAM on various cancer types
TAMs have been implicated in carcinogenesis of essentially all tumors. After our study of TAMs in colon cancer cells, we extended the study to other tumor types.
Surgical categories of lung, melanoma, esophageal, gastric, and pancreatic cancers were obtained and TAMs were isolated as described above. Macrophages from corresponding normal tissues of the same patient were obtained. Real-time RT-PCR was used to quantify the expression of Hom-1 in TAM and tissue macrophages. Hom-1 expression was lower in TAM of all these tumors compared to that in corresponding macrophages from distant normal tissues.
To determine whether Hom-1 can transform these TAMs into tumoricidal cells, GFP or GFP-Hom-1 was transfected into the TAM. After 48 hours of transfection, GFP positive cells were sorted out and co-cultured with individual tumors. Tumor volume decreased in all tumors during co-culture with GFP-Hom-1 transfected TAM, but not with control GFP transfected TAM. Our results show that Hom-1 can convert TAMs into tumoricidal cells independent of the tumor type.
Collecting colon tissue samples
Cancer and normal tissues were obtained from surgically excised specimens from patients in pathology laboratories. Approximately 5-10 grams of tissue was collected from normal mucosa at 15cm from each tumor mass or tumor mass. Patient blood samples were also collected.
Preparation of intraepithelial lymphocytes
Lamina Propria Monocytes (LPMCs) were isolated using the previously described technique (Kamada N et al, 2008; Pignata C et al, 1990) with modifications. In short, will be dissectedFresh mucosa and tumor masses of (2) were used in 10cm dishes without Ca2+And does not contain Mg2+Is washed with Hank's Balanced Salt Solution (HBSS) (life technologies) containing 2% Fetal Bovine Serum (FBS) and 1mMD Dithiothreitol (DTT) (Sigma-Aldrich) to remove mucus. The mucosa and tumor were cut into 0.5cm pieces with a razor blade and incubated in 6-well plates with 5mL HBSS containing 1mM EDTA (Sigma-Aldrich) for 1 hour at 37 ℃ before passing through grey-mesh (gray-mesh) (100 microns). The flow-through contained intraepithelial lymphocytes and epithelial cells and was analyzed by flow cytometry.
Isolation of macrophages from tumor mass and normal mucosa
Subsequently, the mucosa and tumor were treated with HBSS (Ca-containing) containing 2% FBS, 1.5mg/mL collagenase D (Roche), 0.1mg/mL DNase I2+And Mg2+) Incubated at 37 ℃ for 1 hour. The digested tissue was passed through a grey mesh (70 micron) filter. The flow-through was collected and resuspended in a 40% Percoll solution (Pharmacia) and then layered over 60% Percoll and centrifuged at 2000rpm for 30 minutes without braking. The LPMC at the interface was collected. EasySep without CD16 depletionTMHuman monocyte/macrophage enrichment kit (StemCell Technologies) normal mucosal macrophages and TAMs were purified from LPMC according to the manufacturer's instructions. Cells isolated by these techniques are typically more than 98% viable by Propidium Iodide (PI) staining. The purity of intestinal macrophages is over 95%.
Preparation of macrophages from peripheral blood
Peripheral Blood Mononuclear Cells (PBMCs) from healthy adult donors at the bushigen women hospital (Brigham and Womenhospital) were isolated by Ficoll density gradient centrifugation. EasySep without CD16 depletionTMHuman monocyte enrichment kit human monocytes were purified from PBMCs according to the manufacturer's instructions. Purified cells were cultured in complete RPMI medium containing 10ng/mL M-CSF (PeproTech). After enrichment, monocytes were cultured in complete RPMI medium containing M-CSF for 5 days and the cells were used in a co-culture system.
FACS analysis
After immunolabeling the cells with fluorochrome-conjugated antibodies, TAM and other lymphocytes were phenotyped using flow cytometry. The following antibodies were used: PE-conjugated anti-CD 3(OKT3), CD25(BC96), CD14(61D3), CD68(eBio Y182A), CD163 (ebigh 161), -CD206, FITC-conjugated anti-CD 4(RPA-T4), CD33(HIM3-4), APC-conjugated anti-CD 8(OKT8), CD4(OKT4) (eBioscience, Inc). Intracellular staining was performed for Foxp3(236A/E7), IFN-. gamma., perforin and granzyme B with PE conjugated antibodies according to the protocol provided by the manufacturer. Isotope control labeling was performed in parallel. The antibody was diluted according to the supplier's recommendations. Labeled cells were collected on a FACScan flow cytometer using Cell-Quest software (BDbiosciences) and analyzed by FlowJo software. Results are expressed as percentage of positive cells.
Organotypic co-culture of tumors and macrophages
The transwell inserts (0.4 μm pore size, Costar, Corning) were placed in 12-well polystyrene tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). The mucosa and tumor mass were weighed and washed with 1xPBS buffer plus antibiotics, then cut into 0.5cm pieces. Approximately 50mg of tissue was seeded in the upper compartment of a 12-well rotating well and filled with 0.5 mlpmlr pmi 1640 complete medium. Mixing 5x 105Was added to the lower compartment at a density of 50 ten thousand cells/well, with no direct cell-tissue contact, and was filled with 2mL of PRMI complete medium. The plates were incubated at 37 ℃ with 5% CO2The following incubation was performed. 0.5mL of medium was collected for cytokine analysis and fresh medium was added every three days. After co-culturing for two weeks, lower compartment macrophages were collected and total RNA was isolated by TRIzol reagent (Ambion). Tumor and normal mucosa were monitored twice weekly with calipers according to the formula (length x width)2) The tumor volume was calculated 2. Photographs of the tissues were taken with a 3 megapixel CMOS camera on a Leica EZ4D stereomicroscope or a digital camera on an iPhone.
Immunohistochemistry
Tumor or normal tissues were fixed in formalin (Fisher Scientific Company, Kalamazoo, MI). CK20 staining (Dako, Carpinteria, CA, clone Ks20.8, 1:50) and hematoxylin/eosin (H & E) staining were performed. CK20 staining was performed on a Leica Bone III staining platform for 20 minutes online using Epitope Retrieval 2 and using the Bone Polymer Refine detection kit. Microscopy was performed using Nikon Eclipse Ti fluorescence microscopy. Images were captured using a color camera at 40 x original magnification using NIS Elements imaging software (Nikon). The brightness and contrast of the representative image are adjusted equally between the groups.
Hom-1 overexpression
GFP-Hom-1 was transfected into blood macrophages and TAM by lipofectamine 2000(Life Technologies) according to the manufacturing protocol. 48 hours after transfection, cells were filtered through a 70um filter for cell sorting. GFP positive cells were sorted under sterile conditions by BD FACSAria II in BakerBio-Protect Hood. After sorting, cells were cultured in RPMI 1640 complete medium. Hom-1 knockdown
Colonic TAMs or human primary monocytes were transfected with Morpholino (MO) antisense oligonucleotides using a human monocyte nuclear transfection reagent (Nucleofector) kit (Lonza, walker, MD) according to the manufacturer's instructions. Briefly, 5X 106The individual cells were resuspended with 2.5nmol of Hom-1MO oligonucleotide or standard control MO oligonucleotide in 100. mu.l of nuclear transfection reagent solution and electroporated with a nuclear transfection reagent II device (Lonza). The cells were then immediately removed from the device and incubated overnight with 1ml of pre-warmed human monocyte nuclear transfection reagent medium containing 2mM glutamine and 10% FBS. The cells were then resuspended in complete RPMI medium and treated with appropriate cytokines to induce differentiation into macrophages. All MO oligonucleotides were ordered from Gene Tools (Philograph, OR).
Cytokine measurement
The levels of IL-1 β, IL-10, TNF- α and IL-12p70 in supernatants of LPS-treated blood macrophages from E.coli or LPS-treated TAM were quantified using an ELISA kit obtained from eBiosciences. The analysis was performed according to the manufacturer's instructions.
Quantitative RT-PCR
Isolation of Total RNA Using TRIzol reagent, andthe amount of RNA was measured by NanoDrop 2000(Thermo Scientific). Equal amounts of RNA were used for first strand cDNA synthesis using the SuperScript III first Strand Synthesis System (Life Technologies) according to the manufacturer's protocol. To amplify the Hom-1 cDNA by conventional PCR, we used the AccuPrimeTaq DNA polymerase system (Life Technologies) according to the manufacturer's instructions. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. GAPDH was used as an internal control. We performed quantitative measurements of Hom-1 and other gene cDNAs on a LightCycler (480Real-Time PCRSystem; Roche) using SYBR Green. Then using the comparative Ct method (Δ Δ C)tMethod) the relative expression profile of the mRNA is calculated.
Arginase activity and NO assay
Arginase activity in cell lysates was quantified by measuring urea production using the QuantiChrom arginase assay kit (DARG-200; BioAssays Systems). Nitrite concentration in the culture supernatant was determined using a Griess kit (Molecular Probes).
Statistical analysis
Statistical analysis was performed using Student's test.
Other embodiments
All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the described embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt them to various usages and conditions. Accordingly, other embodiments are within the claims.
Sequence listing
<110> Zhu Zhen Lun
<120> method for treating a novel disease
<130>218008-0007PCT
<150>US 62/477,754
<151>2017-03-28
<150>US 62/516,401
<151>2017-06-07
<160>2
<170>PatentIn version 3.5
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Met Arg Leu Ser Ser Ser Pro Pro Arg Gly Pro Gln Gln
1 5 10
ctc tcc agc ttt ggc tcc gtg gac tgg ctc tcc cag agc agc tgc tca 98
Leu Ser Ser Phe Gly Ser Val Asp Trp Leu Ser Gln Ser Ser Cys Ser
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ggg ccg acc cac acc ccc agg cct gcc gac ttc tcc ctg ggg agc ctc 146
Gly Pro Thr His Thr Pro Arg Pro Ala Asp Phe Ser Leu Gly Ser Leu
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cct ggc cca ggc cag aca tcc ggc gcc cgg gag ccc cct cag gcc gtc 194
Pro Gly Pro Gly Gln Thr Ser Gly Ala Arg Glu Pro Pro Gln Ala Val
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agc atc aag gag gcc gcc ggg tcc tca aat ctg cct gcg ccg gag agg 242
Ser Ile Lys Glu Ala Ala Gly Ser Ser Asn Leu Pro Ala Pro Glu Arg
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acc atg gcc ggg ttg agt aag gag cca aat acc ttg cgg gcc ccc cgt 290
Thr Met Ala Gly Leu Ser Lys Glu Pro Asn Thr Leu Arg Ala Pro Arg
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gtc cgc aca gcc ttc acc atg gag cag gtc cgc acc ttg gag ggc gtc 338
Val Arg Thr Ala Phe Thr Met Glu Gln Val Arg Thr Leu Glu Gly Val
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ttc cag cac cac cag tac ctg agc cct ctg gag cgg aag agg ctg gcc 386
Phe Gln His His Gln Tyr Leu Ser Pro Leu Glu Arg Lys Arg Leu Ala
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agg gag atg cag ctc tca gag gtc cag ata aaa acc tgg ttt cag aat 434
Arg Glu Met Gln Leu Ser Glu Val Gln Ile Lys Thr Trp Phe Gln Asn
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cgc cgc atg aaa cac aaa cgg caa atg cag gac ccc cag ctg cac agc 482
Arg Arg Met Lys His Lys Arg Gln Met Gln Asp Pro Gln Leu His Ser
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ccc ttc tcg ggg tct ctc cat gcg ccc cca gct ttc tac tca acg tct 530
Pro Phe Ser Gly Ser Leu His Ala Pro Pro Ala Phe Tyr Ser Thr Ser
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tct ggc ctt gcc aat ggc ctg cag ctg ctg tgc cct tgg gca ccc ctg 578
Ser Gly Leu Ala Asn Gly Leu Gln Leu Leu Cys Pro Trp Ala Pro Leu
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tcc ggg ccc cag gct ctg atg ctg ccc cct ggc tcc ttc tgg ggt ctc 626
Ser Gly Pro Gln Ala Leu Met Leu Pro Pro Gly Ser Phe Trp Gly Leu
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tgc caa gtg gca caa gag gcc ctg gca tct gcg gga gct tcc tgc tgc 674
Cys Gln Val Ala Gln Glu Ala Leu Ala Ser Ala Gly Ala Ser Cys Cys
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Gly Gln Pro Leu Ala Ser His Pro Pro Thr Pro Gly Arg Pro Ser Leu
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Gly Pro Ala Leu Ser Thr Gly Pro Arg Gly Leu Cys Ala Met Pro Gln
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Thr Gly Asp Ala Phe
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cttgctgatc gcacctggct cctacctgga ggactcagtt gttctgttta catcctggtg 878
gcacctctca ccctgaccca cacaaaggtt ctggagatta ctggagaata tatataaata 938
tatatatgta cgtatatatg taaatacaca tatacgtata tataaatata tatatacata 998
tgtgtgtgta tatatatata tatttttttt tttttttttt tttttgagac ggagtgttgc 1058
tctgtcaccc aggctggagt gcaatgacgc aatctcggct cactgcaacc tccgcctcct 1118
gggttcaagc gattctccag cctcagcctc ccgagtagct gggattacag acacccgcca 1178
ccacgcccgg ctaatttttt ctatttttag tagaaatggg gtttcaccat gttagccagg 1238
ctggtctcaa actcctgacc ctgtgatccg cccgcctcgg cctcccaaag tgctgggatt 1298
acaggcatga gccactgcac ccggccctga gaatatattt attaaagcca cctcttcact 1358
gaaagttacc gaaagagtcg gtttaggaag gaaacgaagg gtcagtgaac agagtcaaat 1418
gcagaagtgg gcttgtcatg ggtagggctt tcggcgtacg ataaaaggat catttgtttt 1478
ttaaaagggg ttggaaaaac tggttttcca gttggaaaca gtaaaggttg taagctttgt 1538
gtgtacaaaa gaaaacaggg aatgcaggtg tgtttatagc gttgtggttc aagtccctct 1598
taacaagaac tccaaagctg gaaagcagga gggaacaaag gtgaacatga aggcgaggat 1658
gctggggccc tgcagtgcgc tctaggctgt gcgtgagccg ggactgtacc cacagcttgc 1718
tgagggctgc tcttcttggg ccagggaaag cagggcagcc gggacctgcg gctgtgcctg 1778
gactgaagct gtcccgcagg tccccaccct ccaacacgtg ctcacctgtc cccctcctcg 1838
cagcagcctc gggacaaaac aatgactcaa ggacagcact tctcgcagaa ggtctggaag 1898
tgcccagaat gggaggcacg gaagcccctc ccggggagga ctcccgcgtt gatggaccgt 1958
tcttggtgca gactcctgac tgcgtgcatg aaacctgaga caagtgcaat tccttccatg 2018
tcgccccaga gtgcccagga ggcaggcagt gcggggtgcc caggcagacg ggttcagcct 2078
gcagaactgg aggcgacctg tgaaacccac ccgggcaccc caacaggaac agaagcgtgg 2138
tcctgcggct gcgtccccag cgagtttcac tttccccttg ctcgtttctc ccttgttgta 2198
agtgtttaca actggcatgt gcttttaaac gtcaggtaag aggggaacag ctgctgtaca 2258
tcgtcctggc gagtgacaat gtgacagaag cctgggcgag gccctcggag ggcagcagct 2318
ggacaggggc tactgggttt ggcctggaca gcactgattt gtggatgtgg atgggggcac 2378
gttgtccgtg ataaaagtac aagtgcccct cacaaaaaaa aaaaaaaaaa 2428
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Met Arg Leu Ser Ser Ser Pro Pro Arg Gly Pro Gln Gln Leu Ser Ser
1 5 10 15
Phe Gly Ser Val Asp Trp Leu Ser Gln Ser Ser Cys Ser Gly Pro Thr
20 25 30
His Thr Pro Arg Pro Ala Asp Phe Ser Leu Gly Ser Leu Pro Gly Pro
35 40 45
Gly Gln Thr Ser Gly Ala Arg Glu Pro Pro Gln Ala Val Ser Ile Lys
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Glu Ala Ala Gly Ser Ser Asn Leu Pro Ala Pro Glu Arg Thr Met Ala
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Gly Leu Ser Lys Glu Pro Asn Thr Leu Arg Ala Pro Arg Val Arg Thr
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Ala Phe Thr Met Glu Gln Val Arg Thr Leu Glu Gly Val Phe Gln His
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His Gln Tyr Leu Ser Pro Leu Glu Arg Lys Arg Leu Ala Arg Glu Met
115 120 125
Gln Leu Ser Glu Val Gln Ile Lys Thr Trp Phe Gln Asn Arg Arg Met
130 135 140
Lys His Lys Arg Gln Met Gln Asp Pro Gln Leu His Ser Pro Phe Ser
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Gly Ser Leu His Ala Pro Pro Ala Phe Tyr Ser Thr Ser Ser Gly Leu
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Ala Asn Gly Leu Gln Leu Leu Cys Pro Trp Ala Pro Leu Ser Gly Pro
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Gln Ala Leu Met Leu Pro Pro Gly Ser Phe Trp Gly Leu Cys Gln Val
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Ala Gln Glu Ala Leu Ala Ser Ala Gly Ala Ser Cys Cys Gly Gln Pro
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Leu Ala Ser His Pro Pro Thr Pro Gly Arg Pro Ser Leu Gly Pro Ala
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Leu Ser Thr Gly Pro Arg Gly Leu Cys Ala Met Pro Gln Thr Gly Asp
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Ala Phe