RESUMO
BACKGROUND: One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 ("A2") that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH). METHODS: A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2- HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking. RESULTS: A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the 'on' and 'off' cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2- tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity. CONCLUSIONS: We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.
Assuntos
Neoplasias Pulmonares , Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T , Complexo Ferro-Dextran/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Antígenos HLA-ARESUMO
Ewing's and osteogenic sarcoma are two of the leading causes of cancer deaths in children and adolescents. Recent data suggest that sarcomas may depend on the insulin-like growth factor type 1 (IGF-1) receptor (IGF1R) and/or the insulin receptor (INSR) to drive tumor growth, survival, and resistance to mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We evaluated the therapeutic value of ganitumab (AMG 479; C(6472)H(10028)N(1728)O(2020)S(42)), an anti-IGF1R, fully human monoclonal antibody, alone and in combination with rapamycin (mTORC1 inhibitor) in Ewing's (SK-ES-1 and A673) and osteogenic (SJSA-1) sarcoma models. IGF1R was activated by IGF-1 but not by insulin in each sarcoma model. INSR was also activated by IGF-1 in the SJSA-1 and SK-ES-1 models, but not in the A673 model where insulin was the preferred INSR ligand. Ganitumab significantly inhibited the growth of SJSA-1 and SK-ES-1 xenografts; inhibition was associated with decreased IGF1R and Akt phosphorylation, reduced total IGF1R and bromodeoxyuridine detection, and increased caspase-3 expression. Ganitumab inhibited rapamycin-induced IGF1R, Akt, and glycogen synthase kinase-3ß hyperphosphorylation in each sarcoma model. However, ganitumab in combination with rapamycin also resulted in a marked increase in INSR expression and activity in the SJSA-1 and A673 models. The in vivo efficacy of ganitumab in the two ganitumab-sensitive models (SJSA-1 and SK-ES-1) was significantly enhanced in combination with rapamycin. Our results support studying ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas and suggest that INSR signaling is an important mechanism of resistance to IGF1R blockade.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Sarcoma de Ewing/tratamento farmacológico , Sirolimo/uso terapêutico , Animais , Antibióticos Antineoplásicos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Nus , Osteossarcoma/metabolismo , Fosforilação/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/metabolismo , Sarcoma de Ewing/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-beta) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-beta1 on breast cancer cells in the presence or absence of overexpressed HER-2. METHODS: Cell proliferation assays were used to determine the effect of TGF-beta on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-beta1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-beta signaling pathway was assessed using TGF-beta1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. RESULTS: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-beta1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-beta in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-beta induced pro-invasive and pro-metastatic gene signature. CONCLUSION: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-beta. In contrast, HER-2 and TGF-beta signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.
Assuntos
Neoplasias da Mama/genética , Receptor ErbB-2/biossíntese , Fator de Crescimento Transformador beta/fisiologia , Northern Blotting , Western Blotting , Neoplasias da Mama/patologia , Proliferação de Células , Progressão da Doença , Células Epiteliais , Feminino , Perfilação da Expressão Gênica , Engenharia Genética , Humanos , Glândulas Mamárias Humanas/citologia , Mesoderma , Invasividade Neoplásica , Transdução de Sinais , Fator de Crescimento Transformador beta1RESUMO
Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding assays showed that ganitumab recognized murine IGF1R with sub-nanomolar affinity (KD=0.22ânM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon carcinoma cells and tumors. Addition of ganitumab to 5-fluorouracil resulted in enhanced inhibition of tumor growth in the CT26 model. Pharmacological intervention with ganitumab in naïve nude mice resulted in a number of physiological changes described previously in animals with targeted deletions of Igf1 and Igf1r, including inhibition of weight gain, reduced glucose tolerance and significant increase in serum levels of GH, IGF1 and IGFBP3. Flow cytometric analysis identified GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These findings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly regulates serum IGF1 levels through Gh signaling.
Assuntos
Anticorpos Monoclonais/farmacologia , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Cinética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Receptor IGF Tipo 1/química , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer. EXPERIMENTAL DESIGN: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I-, IGF-II-, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo. RESULTS: In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho-IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I-, IGF-II-, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo. CONCLUSIONS: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho-IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947-58. ©2014 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados , Western Blotting , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Paclitaxel/administração & dosagem , Receptor IGF Tipo 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling. METHODOLOGY/PRINCIPAL FINDINGS: A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists. CONCLUSIONS/SIGNIFICANCE: The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos , Sítios de Ligação/genética , Proliferação de Células , Mapeamento de Epitopos , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Fosforilação , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismoRESUMO
In preclinical models, both dietary fat reduction and insulin-like growth factor I receptor (IGF-1R) blockade individually inhibit prostate cancer xenograft growth. We hypothesized that a low-fat diet combined with IGF-1R blockade would cause additive inhibition of prostate cancer growth and offset possible untoward metabolic effects of IGF-1R blockade antibody therapy. Fifty severe combined immunodeficient mice were injected with 22Rv1 cells subcutaneously. Ten days postinjection, the animals were randomized to four groups: (i) high-fat diet + saline (HF); (ii) high-fat diet + IGF-1R blocking antibody, ganitumab (HF/Ab); (iii) low-fat diet + saline (LF); and (iv) low-fat diet + ganitumab (LF/Ab). After 19 days of treatment, the animals were euthanized, serum was collected, and tumors were weighed. Tumor Ki67, Akt and extracellular signal-regulated kinase (ERK) activation, serum insulin, IGF-I and TNF-α were measured. In vitro, ganitumab treatment inhibited growth and induced apoptosis in several prostate cancer cell lines. In vivo, tumor weights and volumes were unaffected by the different treatments. The LF/Ab therapy significantly reduced proliferation (Ki67) and ERK activation in tumors. The HF/Ab group had significantly higher serum insulin levels than the HF group. However, LF/Ab combination significantly reduced serum insulin back to normal levels as well as normalizing serum TNF-α level. Whereas the combination of low-fat diet and IGF-1R blockade did not have additive inhibitory effects on tumor weight, it led to reduced tumor cell proliferation and a reduction in serum insulin and TNF-α levels.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Dieta com Restrição de Gorduras , Neoplasias da Próstata/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic carcinoma is a leading cause of cancer deaths, and recent clinical trials of a number of oncology therapeutics have not substantially improved clinical outcomes. We have evaluated the therapeutic potential of AMG 479, a fully human monoclonal antibody against insulin-like growth factor (IGF) type I receptor (IGF-IR), in two IGF-IR-expressing pancreatic carcinoma cell lines, BxPC-3 and MiaPaCa2, which also differentially express insulin receptor (INSR). AMG 479 bound to IGF-IR (K(D) 0.33 nmol/L) and blocked IGF-I and IGF-II binding (IC(50) < 0.6 nmol/L) without cross-reacting to INSR. AMG 479 completely inhibited ligand-induced (IGF-I, IGF-II, and insulin) activation of IGF-IR homodimers and IGF-IR/INSR hybrids (but not INSR homodimers) leading to reduced cellular viability in serum-deprived cultures. AMG 479 inhibited >80% of basal IGF-IR activity in BxPC-3 and MiaPaCa2 xenografts and prevented IGF-IR and IGF-IR/INSR hybrid activation following challenge with supraphysiologic concentrations of IGF-I. As a single agent, AMG 479 inhibited (â¼ 80%) the growth of pancreatic carcinoma xenografts, and long-term treatment was associated with reduced IGF-IR signaling activity and expression. Efficacy seemed to be the result of two distinct biological effects: proapoptotic in BxPC-3 and antimitogenic in MiaPaCa2. The combination of AMG 479 with gemcitabine resulted in additive inhibitory activity both in vitro and in vivo. These results indicate that AMG 479 is a clinical candidate, both as a single agent and in combination with gemcitabine, for the treatment of patients with pancreatic carcinoma
Assuntos
Anticorpos Monoclonais/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Animais , Anticorpos Monoclonais Humanizados , Antimetabólitos Antineoplásicos/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologia , Ligação Proteica/efeitos dos fármacos , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Increasing evidence continues to emerge supporting the early hypothesis that BRCA1 might be involved in transcriptional processes. BRCA1 physically associates with more than 15 different proteins involved in transcription and is paradoxically involved in both transcriptional activation and repression. However, the underlying mechanism by which BRCA1 affects the gene expression of various genes remains speculative. In this study, we provide evidence that BRCA1 protein complexes interact with specific DNA sequences. We provide data showing that the upstream stimulatory factor 2 (USF2) physically associates with BRCA1 and is a component of this DNA-binding complex. Interestingly, these DNA-binding complexes are downregulated in breast cancer cell lines containing wild-type BRCA1, providing a critical link between modulations of BRCA1 function in sporadic breast cancers that do not involve germline BRCA1 mutations. The functional specificity of BRCA1 tumor suppression for breast and ovarian tissues is supported by our experiments, which demonstrate that BRCA1 DNA-binding complexes are modulated by serum and estrogen. Finally, functional analysis indicates that missense mutations in BRCA1 that lead to subsequent cancer susceptibility may result in improper gene activation. In summary, these findings establish a role for endogenous BRCA1 protein complexes in transcription via a defined DNA-binding sequence and indicate that one function of BRCA1 is to co-regulate the expression of genes involved in various cellular processes.