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1.
Article in English | MEDLINE | ID: mdl-38829958

ABSTRACT

INTRODUCTION: Recent studies have identified a critical role for stromal-immune cell interactions in immunity and immune tolerance. Transcriptomic profiling has implicated stromal cells in immune-mediated disorders including the two common forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC). Stromal-immune interactions may edify inflammatory state and the development of IBD-related complications such as fibrosis; yet the lack of protein markers has hampered studying stromal-immune perturbation. METHODS: In this study, we designed a 40-color spectral flow cytometry assay to characterize hematopoietic and nonhematopoietic cells in intestinal biopsies and matched blood samples from patients with CD or UC. RESULTS: We identified circulating stromal-like cells that are significantly more abundant in IBD blood samples than in healthy controls. Those cells expressed podoplanin (PDPN), a commonly used marker for fibroblasts, and they were associated with activated and memory T and B cells, and altered NK cell, monocyte, and macrophage populations. PDPN+ cells in the blood correlated with PDPN+ cells in the colon. Principal component analysis distinctly separated healthy blood samples from IBD blood samples, with stromal-like cells and B cell subtypes dominating the IBD signature; Pearson correlation detected an association between PDPN+ stromal-like cells and B cell populations in IBD blood and gut biopsies. DISCUSSION: These observations suggest that PDPN+ cells in the blood may serve as a biomarker of IBD. Understanding the relationship between stromal cells and immune cells in the intestine and the blood may provide a window into disease pathogenesis and insight into therapeutic targets for IBD.

2.
Gastro Hep Adv ; 2(8): 1103-1119, 2023.
Article in English | MEDLINE | ID: mdl-38098742

ABSTRACT

Cancer immunotherapy has become an indispensable mode of treatment for a multitude of solid tumor cancers. Colorectal cancer (CRC) has been one of the many cancer types to benefit from immunotherapy, especially in advanced disease where standard treatment fails to prevent recurrence or results in poor survival. The efficacy of immunotherapy in CRC has not been without challenge, as early clinical trials observed dismal responses in unselected CRC patients treated with checkpoint inhibitors. Many studies and clinical trials have since refined immunotherapies available for CRC, solidifying immunotherapy as a powerful asset for CRC treatment. This review article examines CRC immunotherapies, from their foundation, through emerging avenues for improvement, to future directions.

5.
Adv Sci (Weinh) ; 10(23): e2300548, 2023 08.
Article in English | MEDLINE | ID: mdl-37271874

ABSTRACT

Pancreatic ductal adenocarcinoma (PDA) is a clinically challenging disease with limited treatment options. Despite a small percentage of cases with defective mismatch DNA repair (dMMR), PDA is included in the most immune-resistant cancer types that are poorly responsive to immune checkpoint blockade (ICB) therapy. To facilitate drug discovery combating this immunosuppressive tumor type, a high-throughput drug screen platform is established with the newly developed T cell-incorporated pancreatic tumor organoid model. Tumor-specific T cells are included in the pancreatic tumor organoids by two-step cell packaging, fully recapitulating immune infiltration in the immunosuppressive tumor microenvironment (TME). The organoids are generated with key components in the original tumor, including epithelial, vascular endothelial, fibroblast and macrophage cells, and then packaged with T cells into their outside layer mimicking a physical barrier and enabling T cell infiltration and cytotoxicity studies. In the PDA organoid-based screen, epigenetic inhibitors ITF2357 and I-BET151 are identified, which in combination with anti-PD-1 based therapy show considerably greater anti-tumor effect. The combinatorial treatment turns the TME from immunosuppressive to immunoactive, up-regulates the MHC-I antigen processing and presentation, and enhances the effector T cell activity. The standardized PDA organoid model has shown great promise to accelerate drug discovery for the immunosuppressive cancer.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , T-Lymphocytes , Pancreatic Neoplasms/drug therapy , Carcinoma, Pancreatic Ductal/drug therapy , Immunotherapy , Organoids/pathology , Tumor Microenvironment , Pancreatic Neoplasms
6.
Nat Biomed Eng ; 5(11): 1320-1335, 2021 11.
Article in English | MEDLINE | ID: mdl-34725507

ABSTRACT

In breast cancer, genetic heterogeneity, the lack of actionable targets and immune evasion all contribute to the limited clinical response rates to immune checkpoint blockade therapy. Here, we report a high-throughput screen based on the functional interaction of mouse- or patient-derived breast tumour organoids and tumour-specific cytotoxic T cells for the identification of epigenetic inhibitors that promote antigen presentation and potentiate T-cell-mediated cytotoxicity. We show that the epigenetic inhibitors GSK-LSD1, CUDC-101 and BML-210, identified by the screen, display antitumour activities in orthotopic mammary tumours in mice, that they upregulate antigen presentation mediated by the major histocompatibility complex class I on breast tumour cells and that treatment with BML-210 substantially sensitized breast tumours to the inhibitor of the checkpoint programmed death-1. Standardized measurements of tumour-cell killing activity facilitated by tumour-organoid-T-cell screens may help with the identification of candidate immunotherapeutics for a range of cancers.


Subject(s)
Antigen Presentation , Breast Neoplasms , Animals , CD8-Positive T-Lymphocytes , Epigenesis, Genetic , Female , Humans , Mice , Organoids
7.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: mdl-33830945

ABSTRACT

One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.


Subject(s)
Antigen Presentation/drug effects , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Immune Checkpoint Inhibitors/pharmacology , Immunity, Cellular/drug effects , Immunotherapy , Lactones/pharmacology , Neoplasms, Experimental/therapy , Sesquiterpenes/pharmacology , Animals , Antigens, Neoplasm/genetics , HCT116 Cells , Humans , Immune Checkpoint Inhibitors/pharmacokinetics , Immunity, Cellular/genetics , Lactones/pharmacokinetics , Mice , Mice, Inbred BALB C , Mice, Transgenic , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasms, Experimental/genetics , Neoplasms, Experimental/immunology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , Sesquiterpenes/pharmacokinetics
8.
Sci Transl Med ; 13(580)2021 02 10.
Article in English | MEDLINE | ID: mdl-33568521

ABSTRACT

The clinical challenge for treating HER2 (human epidermal growth factor receptor 2)-low breast cancer is the paucity of actionable drug targets. HER2-targeted therapy often has poor clinical efficacy for this disease due to the low level of HER2 protein on the cancer cell surface. We analyzed breast cancer genomics in the search for potential drug targets. Heterozygous loss of chromosome 17p is one of the most frequent genomic events in breast cancer, and 17p loss involves a massive deletion of genes including the tumor suppressor TP53 Our analyses revealed that 17p loss leads to global gene expression changes and reduced tumor infiltration and cytotoxicity of T cells, resulting in immune evasion during breast tumor progression. The 17p deletion region also includes POLR2A, a gene encoding the catalytic subunit of RNA polymerase II that is essential for cell survival. Therefore, breast cancer cells with heterozygous loss of 17p are extremely sensitive to the inhibition of POLR2A via a specific small-molecule inhibitor, α-amanitin. Here, we demonstrate that α-amanitin-conjugated trastuzumab (T-Ama) potentiated the HER2-targeted therapy and exhibited superior efficacy in treating HER2-low breast cancer with 17p loss. Moreover, treatment with T-Ama induced immunogenic cell death in breast cancer cells and, thereby, delivered greater efficacy in combination with immune checkpoint blockade therapy in preclinical HER2-low breast cancer models. Collectively, 17p loss not only drives breast tumorigenesis but also confers therapeutic vulnerabilities that may be used to develop targeted precision immunotherapy.


Subject(s)
Breast Neoplasms , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line, Tumor , Female , Humans , Immunotherapy , Receptor, ErbB-2/genetics , Trastuzumab
9.
J Clin Invest ; 131(1)2021 01 04.
Article in English | MEDLINE | ID: mdl-32990678

ABSTRACT

Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost CD8+ T cell-mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made toward harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8+ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy.


Subject(s)
Antigen Presentation , Antigens, Neoplasm/immunology , Breast Neoplasms/immunology , Myelin and Lymphocyte-Associated Proteolipid Proteins/immunology , Neoplasm Proteins/immunology , Tumor Escape , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Female , Histocompatibility Antigens Class I/immunology , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Inbred BALB C , Mice, Nude
10.
JCI Insight ; 5(9)2020 05 07.
Article in English | MEDLINE | ID: mdl-32376804

ABSTRACT

Immune checkpoint blockade immunotherapy delivers promising clinical results in colorectal cancer (CRC). However, only a fraction of cancer patients develop durable responses. The tumor microenvironment (TME) negatively impacts tumor immunity and subsequently clinical outcomes. Therefore, there is a need to identify other checkpoint targets associated with the TME. Early-onset factors secreted by stromal cells as well as tumor cells often help recruit immune cells to the TME, among which are alarmins such as IL-33. The only known receptor for IL-33 is stimulation 2 (ST2). Here we demonstrated that high ST2 expression is associated with poor survival and is correlated with low CD8+ T cell cytotoxicity in CRC patients. ST2 is particularly expressed in tumor-associated macrophages (TAMs). In preclinical models of CRC, we demonstrated that ST2-expressing TAMs (ST2+ TAMs) were recruited into the tumor via CXCR3 expression and exacerbated the immunosuppressive TME; and that combination of ST2 depletion using ST2-KO mice with anti-programmed death 1 treatment resulted in profound growth inhibition of CRC. Finally, using the IL-33trap fusion protein, we suppressed CRC tumor growth and decreased tumor-infiltrating ST2+ TAMs. Together, our findings suggest that ST2 could serve as a potential checkpoint target for CRC immunotherapy.


Subject(s)
Colorectal Neoplasms/immunology , Interleukin-1 Receptor-Like 1 Protein/immunology , Interleukin-33/immunology , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/immunology , Animals , Cell Line, Tumor , Databases, Genetic , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Tumor-Associated Macrophages/cytology
11.
Nat Nanotechnol ; 15(4): 342, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31953520

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

12.
Nat Nanotechnol ; 14(5): 496, 2019 May.
Article in English | MEDLINE | ID: mdl-30837690

ABSTRACT

The Supplementary Information originally published with this Article was an older version, in which 'IFN-γ' was misspelled 'INF-γ' in Supplementary Fig. 9 and the ß-Actin blot in Supplementary Fig. 13 was the wrong image. The Supplementary Information has now been replaced.

13.
Nat Nanotechnol ; 14(4): 388-397, 2019 04.
Article in English | MEDLINE | ID: mdl-30804480

ABSTRACT

TP53 is the most frequently mutated or deleted gene in triple negative breast cancer (TNBC). Both the loss of TP53 and the lack of targeted therapy are significantly correlated with poor clinical outcomes, making TNBC the only type of breast cancer that has no approved targeted therapies. Through in silico analysis, we identified POLR2A in the TP53-neighbouring region as a collateral vulnerability target in TNBC tumours, suggesting that its inhibition via small interfering RNA (siRNA) may be an amenable approach for TNBC targeted treatment. To enhance bioavailability and improve endo/lysosomal escape of siRNA, we designed pH-activated nanoparticles for augmented cytosolic delivery of POLR2A siRNA (siPol2). Suppression of POLR2A expression with the siPol2-laden nanoparticles leads to enhanced growth reduction of tumours characterized by hemizygous POLR2A loss. These results demonstrate the potential of the pH-responsive nanoparticle and the precise POLR2A targeted therapy in TNBC harbouring the common TP53 genomic alteration.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/enzymology , Animals , Cell Line, Tumor , Cell Proliferation , Endosomes/metabolism , Female , Gene Deletion , Humans , Hydrogen-Ion Concentration , Lysosomes/metabolism , Mice, Inbred C57BL , Mice, Nude , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Triple Negative Breast Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism
14.
Nat Commun ; 9(1): 4718, 2018 11 09.
Article in English | MEDLINE | ID: mdl-30413718

ABSTRACT

Chromosome 17q23 amplification occurs in ~11% of human breast cancers. Enriched in HER2+ breast cancers, the 17q23 amplification is significantly correlated with poor clinical outcomes. In addition to the previously identified oncogene WIP1, we uncover an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing 17q23 amplicons. The 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 selectively inhibits the proliferation, survival and tumorigenic potential of the HER2+ breast cancer cells harboring 17q23 amplification. To overcome the resistance of trastuzumab-based therapies in vivo, we develop pH-sensitive nanoparticles for specific co-delivery of the WIP1 and miR-21 inhibitors into HER2+ breast tumors, leading to a profound reduction of tumor growth. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the trastuzumab-resistant HER2+ breast cancers.


Subject(s)
Breast Neoplasms/genetics , Chromosomes, Human, Pair 17/genetics , Drug Resistance, Neoplasm/genetics , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DEAD-box RNA Helicases/metabolism , Drug Delivery Systems , Drug Resistance, Neoplasm/drug effects , Female , Gene Amplification/drug effects , Humans , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Nanoparticles/chemistry , Protein Phosphatase 2C/genetics , Protein Phosphatase 2C/metabolism , Receptor, ErbB-2/metabolism , Trastuzumab/pharmacology , Trastuzumab/therapeutic use
15.
Nat Commun ; 9(1): 4394, 2018 10 22.
Article in English | MEDLINE | ID: mdl-30349055

ABSTRACT

Heterozygous deletion of chromosome 17p (17p) is one of the most frequent genomic events in human cancers. Beyond the tumor suppressor TP53, the POLR2A gene encoding the catalytic subunit of RNA polymerase II (RNAP2) is also included in a ~20-megabase deletion region of 17p in 63% of metastatic castration-resistant prostate cancer (CRPC). Using a focused CRISPR-Cas9 screen, we discovered that heterozygous loss of 17p confers a selective dependence of CRPC cells on the ubiquitin E3 ligase Ring-Box 1 (RBX1). RBX1 activates POLR2A by the K63-linked ubiquitination and thus elevates the RNAP2-mediated mRNA synthesis. Combined inhibition of RNAP2 and RBX1 profoundly suppress the growth of CRPC in a synergistic manner, which potentiates the therapeutic effectivity of the RNAP2 inhibitor, α-amanitin-based antibody drug conjugate (ADC). Given the limited therapeutic options for CRPC, our findings identify RBX1 as a potentially therapeutic target for treating human CRPC harboring heterozygous deletion of 17p.


Subject(s)
Chromosomes, Human, Pair 17/metabolism , Prostatic Neoplasms, Castration-Resistant/metabolism , RNA Polymerase II/metabolism , Apoptosis/genetics , Apoptosis/physiology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Chromosomes, Human, Pair 17/genetics , Humans , Immunoblotting , Immunohistochemistry , Immunoprecipitation , Male , Prostatic Neoplasms, Castration-Resistant/genetics , RNA Polymerase II/genetics , Sequence Deletion/genetics , Sequence Deletion/physiology , Transcription, Genetic/genetics , Transcription, Genetic/physiology , Ubiquitin-Protein Ligases
16.
ACS Nano ; 12(10): 9815-9829, 2018 10 23.
Article in English | MEDLINE | ID: mdl-30256609

ABSTRACT

In vitro transcribed mRNA constitutes a versatile platform to encode antigens and to evoke CD8 T-cell responses. Systemic delivery of mRNA packaged into cationic liposomes (lipoplexes) has proven particularly powerful in achieving effective antitumor immunity in animal models. Yet, T-cell responses to mRNA lipoplexes critically depend on the induction of type I interferons (IFN), potent pro-inflammatory cytokines, which inflict dose-limiting toxicities. Here, we explored an advanced hybrid lipid polymer shell mRNA nanoparticle (lipopolyplex) endowed with a trimannose sugar tree as an alternative delivery vehicle for systemic mRNA vaccination. Like mRNA lipoplexes, mRNA lipopolyplexes were extremely effective in conferring antitumor T-cell immunity upon systemic administration. Conversely to mRNA lipoplexes, mRNA lipopolyplexes did not rely on type I IFN for effective T-cell immunity. This differential mode of action of mRNA lipopolyplexes enabled the incorporation of N1 methyl pseudouridine nucleoside modified mRNA to reduce inflammatory responses without hampering T-cell immunity. This feature was attributed to mRNA lipopolyplexes, as the incorporation of thus modified mRNA into lipoplexes resulted in strongly weakened T-cell immunity. Taken together, we have identified lipopolyplexes containing N1 methyl pseudouridine nucleoside modified mRNA as potent yet low-inflammatory alternatives to the mRNA lipoplexes currently explored in early phase clinical trials.


Subject(s)
Inflammation/immunology , Lipids/immunology , RNA, Messenger/immunology , T-Lymphocytes/immunology , Animals , Dendritic Cells/immunology , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Particle Size , Polymers/chemistry , Surface Properties , Tumor Cells, Cultured
17.
World J Gastroenterol ; 24(34): 3834-3848, 2018 Sep 14.
Article in English | MEDLINE | ID: mdl-30228778

ABSTRACT

Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors.


Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Colorectal Neoplasms/drug therapy , Immunoconjugates/pharmacology , Tumor Escape/drug effects , Alpha-Amanitin/pharmacology , Alpha-Amanitin/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/mortality , Costimulatory and Inhibitory T-Cell Receptors/antagonists & inhibitors , Costimulatory and Inhibitory T-Cell Receptors/immunology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/immunology , Humans , Immunoconjugates/therapeutic use , Immunotherapy/methods , Microsatellite Instability/drug effects , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleic Acid Synthesis Inhibitors/therapeutic use , RNA Polymerase II/antagonists & inhibitors , Treatment Outcome , Tumor Escape/genetics , Tumor Escape/immunology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Tumor Suppressor Protein p53/genetics
18.
J Clin Invest ; 128(7): 2951-2965, 2018 07 02.
Article in English | MEDLINE | ID: mdl-29649003

ABSTRACT

A synthetic lethality-based strategy has been developed to identify therapeutic targets in cancer harboring tumor-suppressor gene mutations, as exemplified by the effectiveness of poly ADP-ribose polymerase (PARP) inhibitors in BRCA1/2-mutated tumors. However, many synthetic lethal interactors are less reliable due to the fact that such genes usually do not perform fundamental or indispensable functions in the cell. Here, we developed an approach to identifying the "essential lethality" arising from these mutated/deleted essential genes, which are largely tolerated in cancer cells due to genetic redundancy. We uncovered the cohesion subunit SA1 as a putative synthetic-essential target in cancers carrying inactivating mutations of its paralog, SA2. In SA2-deficient Ewing sarcoma and bladder cancer, further depletion of SA1 profoundly and specifically suppressed cancer cell proliferation, survival, and tumorigenic potential. Mechanistically, inhibition of SA1 in the SA2-mutated cells led to premature chromatid separation, dramatic extension of mitotic duration, and consequently, lethal failure of cell division. More importantly, depletion of SA1 rendered those SA2-mutated cells more susceptible to DNA damage, especially double-strand breaks (DSBs), due to reduced functionality of DNA repair. Furthermore, inhibition of SA1 sensitized the SA2-deficient cancer cells to PARP inhibitors in vitro and in vivo, providing a potential therapeutic strategy for patients with SA2-deficient tumors.


Subject(s)
Antigens, Nuclear/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Nuclear Proteins/genetics , Animals , Antigens, Nuclear/chemistry , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/chemistry , DNA Breaks, Double-Stranded , Female , Gene Knockdown Techniques , Genes, Essential , Humans , Mice , Mice, Nude , Neoplasms/pathology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Phthalazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/genetics , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/genetics , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Xenograft Model Antitumor Assays , Cohesins
19.
N Biotechnol ; 45: 69-79, 2018 Oct 25.
Article in English | MEDLINE | ID: mdl-29574274

ABSTRACT

Single-domain antibody fragments (sdAbs) are the smallest functional antigen-binding fragments, derived from heavy chain-only camelid antibodies. When designed as radiolabeled monomeric probes for imaging and therapy of cancer, their fast and specific targeting results in high tumor-to-background ratios early after injection. However, their moderate absolute uptake into tumors might not always be sufficient to treat cancerous lesions. We have evaluated the pharmacokinetics of seven constructs derived from a CD20-targeting monomeric sdAb (αCD20). The constructs differed in affinity or avidity towards CD20 (dimeric αCD20-αCD20 and αCD20 fused to a non-targeting control sdAb, referred to as αCD20-ctrl) and blood half-lives (αCD20 fused to an albumin-targeting sdAb (αAlb) = αCD20-αAlb). The constructs were radiolabeled with 111In (imaging) and 177Lu (therapy) using the bifunctional chelator CHX-A"-DTPA and evaluated in vitro and in vivo. In mice, tumor uptake of 177Lu-DTPA-αCD20 decreased from 4.82 ±â€¯1.80 to 0.13 ±â€¯0.05% IA/g over 72 h. Due to its rapid blood clearance, tumor-to-blood (T/B) ratios of >100 were obtained within 24 h. Although in vitro internalization indicated that dimeric 177Lu-DTPA-αCD20-αCD20 was superior in terms of total cell-associated radioactivity, this was not confirmed in vivo. Blood clearance was slower and absolute tumor uptake became significantly higher for αCD20-αAlb. Blood levels of 177Lu-DTPA-αCD20-αAlb decreased from 68.30 ±â€¯10.53 to 3.58 ±â€¯0.66% IA/g over 120 h, while tumor uptake increased from 6.21 ±â€¯0.94 to 24.90 ±â€¯2.83% IA/g, resulting in lower T/B ratios. Taken together, these results indicate that the increased size of dimeric αCD20-αCD20 or the fusion of monomeric αCD20 to an albumin-targeting moiety (αAlb) counterbalance their improved tumor targeting capacity compared to monomeric αCD20.


Subject(s)
Antigens, CD20/immunology , Lutetium/chemistry , Radioisotopes/chemistry , Single-Domain Antibodies/immunology , Animals , Antigen-Antibody Reactions , Cell Line, Tumor , Dimerization , Humans , Mice , Single-Domain Antibodies/chemistry
20.
Int J Cancer ; 143(3): 686-698, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29464699

ABSTRACT

Improved understanding of cancer immunology has provided insight into the phenomenon of frequent tumor recurrence after initially successful immunotherapy. A delicate balance exists between the capacity of the immune system to control tumor growth and various resistance mechanisms that arise to avoid or even counteract the host's immune system. These resistance mechanisms include but are not limited to (i) adaptive expression of inhibitory checkpoint molecules in response to the proinflammatory environment and (ii) amplification of cancer stem cells, a small fraction of tumor cells possessing the capacity for self-renewal and mediating treatment resistance and formation of metastases after long periods of clinical remission. Several individual therapeutic agents have so far been developed to revert T-cell exhaustion or disrupt the cross-talk between cancer stem cells and the tumor-promoting microenvironment. Here, we demonstrate that a three-arm combination therapy-consisting of an mRNA-based vaccine to induce antigen-specific T-cell responses, monoclonal antibodies blocking inhibitory checkpoint molecules (PD-1, TIM-3, LAG-3), and antibodies targeting IL-6 and TGF-ß-improves the therapeutic outcome in subcutaneous TC-1 tumors and significantly prolongs survival of treated mice. Our findings point to a need for a rational development of multidimensional anticancer therapies, aiming at the induction of tumor-specific immunity and simultaneously targeting multiple resistance mechanisms.


Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Interleukin-6/antagonists & inhibitors , Neoplasms/genetics , Neoplasms/metabolism , RNA, Messenger/genetics , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Antineoplastic Agents, Immunological/therapeutic use , Cell Line, Tumor , Disease Models, Animal , Gene Expression , Humans , Immunotherapy , Interleukin-6/metabolism , Melanoma, Experimental , Mice , Neoplasms/pathology , Neoplasms/therapy , Recurrence , SOXB1 Transcription Factors/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism
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