RESUMEN
Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Antineoplásicos/inmunología , Inmunoconjugados/inmunología , Oligopéptidos/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Femenino , Proteínas Ligadas a GPI/inmunología , Células HEK293 , Humanos , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacología , Ratones SCID , Ratas Sprague-Dawley , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.
Asunto(s)
alfa-Globulinas/química , Antineoplásicos/farmacología , Inmunoconjugados/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Haplorrinos , Humanos , Inmunoconjugados/química , Ratones , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.
Asunto(s)
Antineoplásicos Inmunológicos/química , Cisteína/química , Disulfuros/química , Inmunoconjugados/química , Maleimidas/química , Trastuzumab/química , Animales , Antineoplásicos Inmunológicos/sangre , Cisteína/sangre , Cisteína/genética , Disulfuros/sangre , Estabilidad de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoconjugados/sangre , Maleimidas/sangre , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligopéptidos/sangre , Oligopéptidos/química , Agregado de Proteínas , Estabilidad Proteica , Ratas , Trastuzumab/sangre , Trastuzumab/genéticaRESUMEN
Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/antagonistas & inhibidores , Inmunoconjugados/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/inmunología , Células HEK293 , Humanos , Inmunoconjugados/inmunología , Inmunoconjugados/farmacocinética , Células MCF-7 , Macaca fascicularis , Ratones Desnudos , Ratones SCID , Ratas Sprague-Dawley , Receptores de Esteroides/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genéticaRESUMEN
A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.
Asunto(s)
Anticuerpos/administración & dosificación , Benzodiazepinas/administración & dosificación , Inmunoconjugados/administración & dosificación , Neoplasias/tratamiento farmacológico , Pirroles/administración & dosificación , Animales , Anticuerpos/química , Anticuerpos/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/inmunología , Benzodiazepinas/química , Benzodiazepinas/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Disulfuros/química , Disulfuros/inmunología , Humanos , Inmunoconjugados/química , Ratones , Neoplasias/inmunología , Neoplasias/patología , Pirroles/química , Pirroles/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody drug conjugates (ADCs) constitute a family of cancer therapeutics designed to preferentially direct a cytotoxic drug to cells expressing a cell-surface antigen recognized by an antibody. The antibody and drug are linked through chemistries that enable release of the cytotoxic drug or drug adduct upon internalization and digestion of the ADC by the cell. Over 40 distinct ADCs, targeting an array of antigens and utilizing a variety of drugs and linkers, are undergoing clinical evaluation. This review primarily covers ADCs that have advanced to clinical investigation with a particular emphasis on how the individual targets, linker chemistries, and appended drugs influence their behavior.
Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Tecnología Farmacéutica/métodos , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/química , Ensayos Clínicos Fase I como Asunto , Liberación de Fármacos , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Inmunoconjugados/químicaRESUMEN
Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inmunotoxinas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Receptores Acoplados a Proteínas G/inmunología , Animales , Antineoplásicos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Estudios de Factibilidad , Femenino , Regulación Neoplásica de la Expresión Génica , Genes APC , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Concentración 50 Inhibidora , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Antibody-drug conjugates (ADC) selectively deliver a cytotoxic drug to cells expressing an accessible antigenic target. Here, we have appended monomethyl auristatin E (MMAE) to an antibody recognizing the SLC34A2 gene product NaPi2b, the type II sodium-phosphate cotransporter, which is highly expressed on tumor surfaces of the lung, ovary, and thyroid as well as on normal lung pneumocytes. This study evaluated its efficacy and safety in preclinical studies. EXPERIMENTAL DESIGN: The efficacy of anti-NaPi2b ADC was evaluated in mouse ovarian and non-small cell lung cancer (NSCLC) tumor xenograft models, and its toxicity was assessed in rats and cynomolgus monkeys. RESULTS: We show here that an anti-NaPi2b ADC is effective in mouse ovarian and NSCLC tumor xenograft models and well-tolerated in rats and cynomolgus monkeys at levels in excess of therapeutic doses. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue target expression. The nonproliferative nature of normal pneumocytes, together with the antiproliferative mechanism of MMAE, likely mitigates the potential liability of this normal tissue expression. CONCLUSIONS: Overall, our preclinical results suggest that the ADC targeting NaPi2b provides an effective new therapy for the treatment of NSCLC and ovarian cancer and is currently undergoing clinical developments.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Macaca fascicularis , Masculino , Ratones , Oligopéptidos/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Ratas , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We are interested in identifying mechanisms of resistance to the current generation of antibody-drug conjugates (ADC) and developing ADCs that can overcome this resistance. EXPERIMENTAL DESIGN: Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor monomethyl auristatin E (MMAE) attached to the antibody by the protease-cleavable linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). Early clinical trial data suggest that these ADCs have promising efficacy for the treatment of non-Hodgkin lymphoma (NHL); however, some patients do not respond or become resistant to the ADCs. Anthracyclines are very effective in NHL, but ADCs containing the anthracycline doxorubicin were not clinically efficacious probably due to the low drug potency and inadequate linker technology. The anthracycline analogue PNU-159682 is thousands of times more cytotoxic than doxorubicin, so we used it to develop a new class of ADCs. We used the same MC-vc-PAB linker and antibody in pinatuzumab vedotin but replaced the MMAE with a derivative of PNU-159682 to make anti-CD22-NMS249 and tested it for in vivo efficacy in xenograft tumors resistant to MMAE-based ADCs. RESULTS: We derived cell lines from in vivo xenograft tumors that were made resistant to anti-CD22-vc-MMAE and anti-CD79b-vc-MMAE. We identified P-gp (ABCB1/MDR1) as the major driver of resistance to the vc-MMAE-based conjugates. Anti-CD22-NMS249 was at least as effective as anti-CD22-vc-MMAE in xenograft models of the parental cell lines and maintained its efficacy in the resistant cell lines. CONCLUSIONS: These studies provide proof of concept for an anthracycline-based ADC that could be used to treat B-cell malignancies that are resistant to vc-MMAE conjugates.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/análogos & derivados , Inmunoconjugados/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Aminobenzoatos/farmacología , Animales , Antraciclinas/administración & dosificación , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos ICR , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligopéptidos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Oligopéptidos/uso terapéutico , Animales , Antineoplásicos/química , Western Blotting , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunoconjugados/química , Inmunohistoquímica , Ratones , Ratones SCID , Oligopéptidos/química , Ratas , Ratas Sprague-Dawley , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Chemotherapies are limited by a narrow therapeutic index resulting in suboptimal exposure of the tumor to the drug and acquired tumor resistance. One approach to overcome this is through antibody-drug conjugates (ADC) that facilitate greater potency via target-specific delivery of highly potent cytotoxic agents. EXPERIMENTAL DESIGN: In this study, we used a bioinformatics approach to identify the lymphocyte antigen 6 complex locus E (LY6E), an IFN-inducible glycosylphosphatidylinositol (GPI)-linked cell membrane protein as a promising ADC target. We developed a monoclonal anti-LY6E antibody and characterized in situ LY6E expression in over 750 cancer specimens and normal tissues. Target-dependent anti-LY6E ADC killing was investigated both in vitro and in vivo using patient-derived xenograft models. RESULTS: Using in silico approaches, we found that LY6E was significantly overexpressed and amplified in a wide array of different human solid tumors. IHC analysis revealed high LY6E protein expression in a number of tumor types, such as breast, lung, gastric, ovarian, pancreatic, kidney and head/neck carcinomas. Characterization of the endocytic pathways for LY6E revealed that the LY6E-specific antibody is internalized into cells leading to lysosomal accumulation. Consistent with this, a LY6E-specific ADC inhibited in vitro cell proliferation and produced durable tumor regression in vivo in clinically relevant LY6E-expressing xenograft models. CONCLUSIONS: Our results identify LY6E as a highly promising molecular ADC target for a variety of solid tumor types with current unmet medical need.
Asunto(s)
Antígenos de Neoplasias/farmacología , Antígenos de Superficie/inmunología , Antineoplásicos/farmacología , Inmunoconjugados/farmacología , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Citometría de Flujo , Proteínas Ligadas a GPI/inmunología , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/síntesis química , Inmunoconjugados/farmacología , Maitansina/análogos & derivados , Ingeniería de Proteínas , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , TrastuzumabRESUMEN
Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, ß-catenin independent pathway and suppress a canonical Wnt/ß-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Evolución Molecular , Modelos Moleculares , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Transducción de Señal/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Catálisis , Células HEK293 , Humanos , Immunoblotting , Luciferasas , Datos de Secuencia Molecular , Fosforilación , Estructura Terciaria de Proteína , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/química , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Therapies targeting the mitogen-activated protein (MAP) kinase pathway in melanoma have produced significant clinical responses; however, duration of response is limited by acquisition of drug resistance. Rational drug combinations may improve outcomes in this setting. We assessed the therapeutic combination of an antibody-drug conjugate (ADC) targeting the endothelin B receptor (EDNRB) with small-molecule inhibitors of the MAP kinase signaling pathway in melanoma. Cell lines and tumor models containing either mutant BRAF or NRAS, or wild-type for both, were exposed to small-molecule inhibitors of BRAF and MEK. Expression of EDNRB was analyzed and the therapeutic impact of combining the anti-EDNRB ADC with the BRAF and MEK inhibitors was assessed. Increased expression of EDNRB in response to inhibition of BRAF and/or MEK was observed and augmented the antitumor activity of the ADC. Enhanced target expression and ADC antitumor activity were realized irrespective of the response of the tumor model to the BRAF or MEK inhibitors alone and could be achieved in melanoma with mutant NRAS, BRAF, or neither mutation. Cells that acquired resistance to BRAF inhibition through long-term culture retained drug-induced elevated levels of EDNRB expression. Expression of EDNRB was not enhanced in normal human melanocytes by inhibition of BRAF and the combination of the ADC with MAPK inhibitors was well-tolerated in mice. The anti-EDNRB ADC combines well with BRAF and MEK inhibitors and could have therapeutic use in the majority of human melanoma cases.
Asunto(s)
Antagonistas de los Receptores de la Endotelina B/administración & dosificación , Melanoma/tratamiento farmacológico , Melanoma/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Receptor de Endotelina B/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors--target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0-8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Antineoplásicos/uso terapéutico , Especificidad de Anticuerpos , Sistemas de Liberación de Medicamentos , Inmunoconjugados/uso terapéutico , Maitansina/análogos & derivados , Neoplasias/tratamiento farmacológico , Ado-Trastuzumab Emtansina , Brentuximab Vedotina , Humanos , Maitansina/uso terapéutico , Trastuzumab , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.
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Anticuerpos/inmunología , Inmunización , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Plásmidos , Vacunas de ADN , Animales , Línea Celular , ADN Complementario/inmunología , ADN Complementario/farmacología , Humanos , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Plásmidos/inmunología , Plásmidos/farmacología , Estructura Secundaria de Proteína , Vacunas de ADN/inmunología , Vacunas de ADN/farmacologíaRESUMEN
Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased ß-catenin-mediated signaling. However, continued requirement of Wnt/ß-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/ß-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/ß-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting ß-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/ß-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that ß-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/ß-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic.
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Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Genes APC , Mutación , Piperidinas/farmacología , Sulfonas/farmacología , Tanquirasas/antagonistas & inhibidores , Triazoles/farmacología , Animales , Proteína Axina/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Ratones , Esferoides Celulares/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/fisiologíaRESUMEN
Receptor-interacting protein kinase 4 (RIPK4) is required for epidermal differentiation and is mutated in Bartsocas-Papas syndrome. RIPK4 binds to protein kinase C, but its signaling mechanisms are largely unknown. Ectopic RIPK4, but not catalytically inactive or Bartsocas-Papas RIPK4 mutants, induced accumulation of cytosolic ß-catenin and a transcriptional program similar to that caused by Wnt3a. In Xenopus embryos, Ripk4 synergized with coexpressed Xwnt8, whereas Ripk4 morpholinos or catalytic inactive Ripk4 antagonized Wnt signaling. RIPK4 interacted constitutively with the adaptor protein DVL2 and, after Wnt3a stimulation, with the co-receptor LRP6. Phosphorylation of DVL2 by RIPK4 favored canonical Wnt signaling. Wnt-dependent growth of xenografted human tumor cells was suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may contribute to the growth of certain tumor types.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Vía de Señalización Wnt , Proteínas de Xenopus/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Citosol/metabolismo , Proteínas Dishevelled , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Trasplante de Neoplasias , Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Trasplante Heterólogo , Proteína Wnt3A/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMEN
The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3ß to foster phosphorylation of the Wnt effector ß-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced ß-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.