ABSTRACT
KRAS mutations drive a quarter of cancer mortality, and most are undruggable. Several inhibitors of the MAPK pathway are FDA approved but poorly tolerated at the doses needed to adequately extinguish RAS/RAF/MAPK signaling in the tumor cell. We found that oncogenic KRAS signaling induced ferrous iron (Fe2+) accumulation early in and throughout mutant KRAS-mediated transformation. We converted an FDA-approved MEK inhibitor into a ferrous iron-activatable drug conjugate (FeADC) and achieved potent MAPK blockade in tumor cells while sparing normal tissues. This innovation allowed sustainable, effective treatment of tumor-bearing animals, with tumor-selective drug activation, producing superior systemic tolerability. Ferrous iron accumulation is an exploitable feature of KRAS transformation, and FeADCs hold promise for improving the treatment of KRAS-driven solid tumors.
Subject(s)
Neoplasms , Proto-Oncogene Proteins p21(ras) , Animals , Cell Line, Tumor , Iron/pharmacology , Mutation/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Signal TransductionABSTRACT
αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.
Subject(s)
Immunity , Immunotherapy , Integrins/metabolism , Models, Biological , Neoplasms/immunology , Neoplasms/therapy , T-Lymphocytes/immunology , Animals , Antibodies, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , CTLA-4 Antigen/immunology , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Granzymes/metabolism , Interferon-gamma/metabolism , Lymphocyte Depletion , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Neoplasms/genetics , Neoplasms/pathology , Signal Transduction , Smad3 Protein/metabolism , Survival Analysis , T-Lymphocytes, Cytotoxic/immunology , Transforming Growth Factor beta/metabolism , Tumor Microenvironment/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolismABSTRACT
Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861.
Subject(s)
Antigens, CD19/immunology , Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Receptors, Chimeric Antigen/immunology , Humans , Immunotherapy, Adoptive , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , ProteomicsABSTRACT
PURPOSE: New therapies have changed the outlook for patients with multiple myeloma, but novel agents are needed for patients who are refractory or relapsed on currently approved drug classes. Novel targets other than CD38 and BCMA are needed for new immunotherapy development, as resistance to daratumumab and emerging anti-BCMA approaches appears inevitable. One potential target of interest in myeloma is ICAM1. Naked anti-ICAM1 antibodies were active in preclinical models of myeloma and safe in patients, but showed limited clinical efficacy. Here, we sought to achieve improved targeting of multiple myeloma with an anti-ICAM1 antibody-drug conjugate (ADC). EXPERIMENTAL DESIGN: Our anti-ICAM1 human mAb was conjugated to an auristatin derivative, and tested against multiple myeloma cell lines in vitro, orthotopic xenografts in vivo, and patient samples ex vivo. The expression of ICAM1 was also measured by quantitative flow cytometry in patients spanning from diagnosis to the daratumumab-refractory state. RESULTS: The anti-ICAM1 ADC displayed potent anti-myeloma cytotoxicity in vitro and in vivo. In addition, we have verified that ICAM1 is highly expressed on myeloma cells and shown that its expression is further accentuated by the presence of bone marrow microenvironmental factors. In primary samples, ICAM1 is differentially overexpressed on multiple myeloma cells compared with normal cells, including daratumumab-refractory patients with decreased CD38. In addition, ICAM1-ADC showed selective cytotoxicity in multiple myeloma primary samples. CONCLUSIONS: We propose that anti-ICAM1 ADC should be further studied for toxicity, and if safe, tested for clinical efficacy in patients with relapsed or refractory multiple myeloma.
Subject(s)
Antibodies, Monoclonal/pharmacology , Immunoconjugates/pharmacology , Intercellular Adhesion Molecule-1/genetics , Multiple Myeloma/drug therapy , ADP-ribosyl Cyclase 1/antagonists & inhibitors , ADP-ribosyl Cyclase 1/immunology , Adult , Aged , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Anti-Idiotypic/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Flow Cytometry , Heterografts , Humans , Immunoconjugates/immunology , Intercellular Adhesion Molecule-1/immunology , Male , Mice , Middle Aged , Multiple Myeloma/genetics , Multiple Myeloma/immunology , Multiple Myeloma/pathologyABSTRACT
A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins-MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.
ABSTRACT
Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.
Subject(s)
Adenocarcinoma/metabolism , Antibodies, Monoclonal/pharmacology , Antigens, Neoplasm/metabolism , Membrane Cofactor Protein/metabolism , Neuroendocrine Tumors/metabolism , Prostate/metabolism , Prostatic Neoplasms/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/immunology , Androstenes/pharmacology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/immunology , Antibodies, Neoplasm/pharmacology , Antibody Affinity , Antigens, Neoplasm/immunology , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , Benzamides , Cell Line, Tumor , Female , Humans , Macaca fascicularis , Male , Membrane Cofactor Protein/genetics , Membrane Cofactor Protein/immunology , Neuroendocrine Tumors/drug therapy , Neuroendocrine Tumors/immunology , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathology , Recombinant Fusion Proteins , Signal Transduction/drug effects , Therapeutics , Tumor Microenvironment , Xenograft Model Antitumor AssaysABSTRACT
The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Activation of JAK/STAT signaling is thought to be a central component of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor Food and Drug Administration (FDA) approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Herein, we validated in vitro, in stromal-responsive human myeloma cell lines, and in vivo, in orthotopic disseminated xenograft models of myeloma, that tofacitinib showed efficacy in myeloma models. Furthermore, tofacitinib strongly synergized with venetoclax in coculture with bone marrow stromal cells but not in monoculture. Surprisingly, we found that ruxolitinib, an FDA approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. Transcriptome analysis and unbiased phosphoproteomics revealed that bone marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib reverses the growth-promoting effects of the tumor microenvironment. As tofacitinib is already FDA approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.
Subject(s)
Bone Marrow/drug effects , Bone Marrow/pathology , Drug Repositioning , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Tumor Microenvironment/drug effects , Animals , Cell Communication , Disease Models, Animal , Humans , Janus Kinases/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Multiple Myeloma/metabolism , Phosphoproteins/metabolism , Piperidines/administration & dosage , Plasma Cells/metabolism , Plasma Cells/pathology , Protein Kinase Inhibitors/administration & dosage , Proteome , Proteomics/methods , Pyrimidines/administration & dosage , Pyrroles/administration & dosage , STAT Transcription Factors/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neoplasm/pharmacology , Immunoconjugates/pharmacology , Membrane Cofactor Protein/antagonists & inhibitors , Multiple Myeloma/drug therapy , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/immunology , Cell Line, Tumor , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 1/immunology , Gene Dosage/immunology , Humans , Immunoconjugates/immunology , Membrane Cofactor Protein/genetics , Membrane Cofactor Protein/immunology , Mice , Multiple Myeloma/genetics , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Xenograft Model Antitumor AssaysABSTRACT
Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site-specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, (111)In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1-dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.
Subject(s)
Biomarkers, Tumor/metabolism , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/metabolism , Epithelial Cells/metabolism , Optical Imaging/methods , Proteinase Inhibitory Proteins, Secretory/metabolism , Serine Endopeptidases/metabolism , Antibodies/immunology , Humans , Indium Radioisotopes/metabolism , Mass Spectrometry , Serine Endopeptidases/immunologyABSTRACT
The cell surface protease membrane-type serine protease-1 (MT-SP1), also known as matriptase, is often upregulated in epithelial cancers. We hypothesized that dysregulation of MT-SP1 with regard to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1), a situation that increases proteolytic activity, might be exploited for imaging purposes to differentiate malignant from normal tissue. In this study, we show that MT-SP1 is active on cancer cells and that its activity may be targeted in vivo for tumor detection. A proteolytic activity assay with several MT-SP1-positive human cancer cell lines showed that MT-SP1 antibodies that inhibit recombinant enzyme activity in vitro also bind and inhibit the full-length enzyme expressed on cells. In contrast, in the same assay, MT-SP1-negative cancer cell lines were inactive. Fluorescence microscopy confirmed the cell surface localization of labeled antibodies bound to MT-SP1-positive cells. To evaluate in vivo targeting capability, 0.7 to 2 nmoles of fluorescently labeled antibodies were administered to mice bearing tumors that were positive or negative for MT-SP1. Antibodies localized to MT-SP1-positive tumors (n = 3), permitting visualization of MT-SP1 activity, whereas MT-SP1-negative tumors (n = 2) were not visualized. Our findings define MT-SP1 activity as a useful biomarker to visualize epithelial cancers using a noninvasive antibody-based method.