RESUMO
Integrin beta-6, a component of the heterodimeric adhesion receptor alpha-v/beta-6, is overexpressed in numerous solid tumors. Its expression has been shown by multiple investigators to be a negative prognostic indicator in diverse cancers including colorectal, non-small cell lung, gastric, and cervical. We developed SGN-B6A as an antibody-drug conjugate (ADC) directed to integrin beta-6 to deliver the clinically validated payload monomethyl auristatin E (MMAE) to cancer cells. The antibody component of SGN-B6A is specific for integrin beta-6 and does not bind other alpha-v family members. In preclinical studies, this ADC has demonstrated activity in vivo in models derived from non-small cell lung, pancreatic, pharyngeal, and bladder carcinomas spanning a range of antigen expression levels. In nonclinical toxicology studies in cynomolgus monkeys, doses of up to 5 mg/kg weekly for four doses or 6 mg/kg every 3 weeks for two doses were tolerated. Hematologic toxicities typical of MMAE ADCs were dose limiting, and no significant target-mediated toxicity was observed. A phase I first-in-human study is in progress to evaluate the safety and antitumor activity of SGN-B6A in a variety of solid tumors known to express integrin beta-6 (NCT04389632).
Assuntos
Antineoplásicos , Carcinoma , Imunoconjugados , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Prognóstico , Integrinas , Linhagem Celular TumoralRESUMO
SGN-CD228A is an investigational antibody-drug conjugate (ADC) directed to melanotransferrin (CD228, MELTF, MFI2, p97), a cell-surface protein first identified in melanoma. SGN-CD228A consists of a humanized antibody, hL49, with high specificity and affinity for CD228 that is stably conjugated to 8 molecules of the clinically validated microtubule-disrupting agent monomethyl auristatin E (MMAE) via a novel glucuronide linker. We performed comprehensive IHC studies, which corroborated published RNA sequencing data and confirmed low CD228 expression in normal tissues and high expression in several cancers, including melanoma, squamous non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), colorectal cancer, and pancreatic cancer. SGN-CD228A was efficiently internalized in various tumor cell types, and its cytotoxic activity was dependent on CD228 expression and internalization and intrinsic sensitivity to the MMAE payload. Compared with the valine-citrulline dipeptide linker, the novel glucuronide linker increased the cellular retention of MMAE in vitro and conferred improved antitumor activity against melanoma cell lines in vitro and in vivo. In addition, SGN-CD228A was active across melanoma, TNBC, and NSCLC cell line- and patient-derived xenograft models with heterogeneous antigen expression. In vivo, CD228 expression was important for response to SGN-CD228A but was not well correlated across all tumor types, suggesting that other factors associated with ADC activity are important. Overall, SGN-CD228A is a CD228-directed, investigational ADC that employs innovative technology and has compelling preclinical antitumor activity. SGN-CD228A is investigated in a Phase I clinical trial (NCT04042480) in patients with advanced solid tumors.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Imunoconjugados , Neoplasias Pulmonares , Melanoma , Neoplasias de Mama Triplo Negativas , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Glucuronídeos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To provide a better understanding of the pharmacokinetics-pharmacodynamics relationships of antibody-based drugs, we analyzed several chimeric and humanized monoclonal antibodies or antibody-drug conjugates (ADC) for PK and efficacy among four strains of mice. Notably, antibodies and ADCs displayed a dose-dependent drug disposition profile in the plasma of NSG mice. The increased clearance rate in NSG mice resulted in the reduction of antitumor activity of ADCs. Furthermore, we identified that the abnormal clearance was mediated by Fc-FcγR interaction by comparing antibodies that lack FcγR binding capacity. We also found a high percentage of FcγR-expressing macrophages in the bone marrow, spleen, and liver of NSG mice, which may be responsible for the abnormal distribution of antibodies. Overall, these findings suggest that preclinical evaluation of efficacy and pharmacokinetics of antibodies and ADCs need to consider mouse strain-induced variations.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Imunoconjugados/metabolismo , Antígeno Ki-1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células Mieloides/imunologia , Células Mieloides/metabolismo , Glutamato de Sódio/farmacologia , Resultado do Tratamento , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR.
Assuntos
Imunoconjugados/farmacologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Humanos , Imunoconjugados/imunologia , Leucemia Mieloide Aguda/imunologia , Camundongos , Camundongos SCID , Células THP-1 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The primary mechanism of antibody-drug conjugates (ADC) is targeted delivery of a cytotoxic payload to tumor cells via cancer-associated membrane receptors. However, the tumor microenvironment likely plays a role in ADC penetration, distribution, and processing and thus impacts the overall antitumor activity. Here, we report on the potential contribution of Fc-FcγR interactions between ADCs and tumor-associated macrophages (TAM) to the preclinical antitumor activities of ADCs. In the CD30+ L-428 Hodgkin lymphoma model, anti-CD30-vcMMAE and a non-binding control (hIgG-vcMMAE) demonstrated similar antitumor activity as well as similar payload release in the tumors. IHC analysis revealed L-428 tumors contained highly abundant TAMs, which were confirmed to bind ADCs by IHC and flow cytometry. The infiltration of TAMs was further found to correlate with the antitumor activity of the non-binding hIgG-vcMMAE in five additional xenograft models. hIgG1V1-vcMMAE, bearing a mutation in the Fc region which ablates Fc gamma receptor (FcγR) binding, lost antitumor activity in three TAM-high xenograft models, suggesting Fc-FcγR interactions modulate the TAM-ADC interaction. Our results suggest that TAMs can contribute to ADC processing through FcγR interaction in preclinical tumor models and may represent an important additional mechanism for drug release from ADCs. Correlative studies in clinical trials will further shed light on whether TAMs play a role in patients' response to ADC therapies. Mol Cancer Ther; 16(7); 1347-54. ©2017 AACR.
Assuntos
Doença de Hodgkin/tratamento farmacológico , Imunoconjugados/administração & dosagem , Receptores de IgG/imunologia , Microambiente Tumoral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Imunoconjugados/imunologia , Macrófagos/imunologia , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this article, we describe a novel antibody-drug conjugate (ADC; SGN-LIV1A), targeting the zinc transporter LIV-1 (SLC39A6) for the treatment of metastatic breast cancer. LIV-1 was previously known to be expressed by estrogen receptor-positive breast cancers. In this study, we show that LIV-1 expression is maintained after hormonal therapy in primary and metastatic sites and is also upregulated in triple-negative breast cancers. In addition to breast cancer, other indications showing LIV-1 expression include melanoma, prostate, ovarian, and uterine cancer. SGN-LIV1A consists of a humanized antibody conjugated through a proteolytically cleavable linker to monomethyl auristatin E, a potent microtubule-disrupting agent. When bound to surface-expressed LIV-1 on immortalized cell lines, this ADC is internalized and traffics to the lysozome. SGN-LIV1A displays specific in vitro cytotoxic activity against LIV-1-expressing cancer cells. In vitro results are recapitulated in vivo where antitumor activity is demonstrated in tumor models of breast and cervical cancer lineages. These results support the clinical evaluation of SGN-LIV1A as a novel therapeutic agent for patients with LIV-1-expressing cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Imunoconjugados/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Afinidade de Anticorpos , Antineoplásicos/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Imunoconjugados/administração & dosagem , Imunofenotipagem , Lisossomos/metabolismo , Células MCF-7 , Microtúbulos/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.
Assuntos
Anticorpos Monoclonais Humanizados/química , Benzodiazepinas/química , Resistencia a Medicamentos Antineoplásicos , Imunoconjugados/química , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/química , Animais , Apoptose , Ciclo Celular , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/genética , Dimerização , Desenho de Fármacos , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/imunologia , CamundongosRESUMO
The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDP-fucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.
Assuntos
Anticorpos Monoclonais/metabolismo , Vacinas Anticâncer/farmacologia , Fucose/farmacologia , Fucosiltransferases/antagonistas & inibidores , Guanosina Difosfato Fucose/metabolismo , Polissacarídeos/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cromatografia Líquida , Cricetinae , Cricetulus , Desenho de Fármacos , Feminino , Fucose/química , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neutrófilos/metabolismoRESUMO
An anti-CD70 antibody conjugated to monomethylauristatin F (MMAF) via a valine-citrulline dipeptide containing linker has been shown previously to have potent antitumor activity in renal cell cancer xenograft studies. Here, we generated a panel of humanized anti-CD70 antibody IgG variants and conjugated them to MMAF to study the effect of isotype (IgG1, IgG2, and IgG4) and Fcgamma receptor binding on antibody-drug conjugate properties. All IgG variants bound CD70+ 786-O cells with an apparent affinity of approximately 1 nmol/L, and drug conjugation did not impair antigen binding. The parent anti-CD70 IgG1 bound to human FcgammaRI and FcgammaRIIIA V158 and mouse FcgammaRIV and this binding was not impaired by drug conjugation. In contrast, binding to these Fcgamma receptors was greatly reduced or abolished in the variant, IgG1v1, containing the previously described mutations, E233P:L234V:L235A. All conjugates had potent cytotoxic activity against six different antigen-positive cancer cell lines in vitro with IC50 values of 30 to 540 pmol/L. The IgGv1 conjugate with MMAF displayed improved antitumor activity compared with other conjugates in 786-O and UMRC3 models of renal cell cancer and in the DBTRG05-MG glioblastoma model. All conjugates were tolerated to > or =40 mg/kg in mice. Thus, the IgG1v1 MMAF conjugate has an increased therapeutic index compared with the parent IgG1 conjugate. The improved antitumor activity of the IgG1v1 auristatin conjugates may relate to increased exposure as suggested by pharmacokinetic analysis. The strategy used here for enhancing the therapeutic index of antibody-drug conjugates is independent of the antigen-binding variable domains and potentially applicable to other antibodies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Ligante CD27/imunologia , Imunoconjugados/uso terapêutico , Engenharia de Proteínas , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Imunoglobulina G/imunologia , Camundongos , Camundongos Nus , Modelos Moleculares , Receptores de IgG/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anti-CD30 diabodies were engineered with two cysteine mutations for site-specific drug conjugation in each chain of these homodimeric antibody fragments. Diabodies were conjugated with approximately 4 equivalents of the anti-tubulin drugs, monomethyl auristatin E or F, via a protease-cleavable dipeptide linker, to create the conjugates, diabody-vcE4 and diabody-vcF4, respectively. Diabody conjugation had only minor (<3-fold) effects on antigen binding. Diabody-vcF4 was potently cytotoxic against the antigen-positive cell lines, Karpas-299 (34 pmol/L IC(50)) and L540cy (22 pmol/L IC(50)), and was 8- and 21-fold more active than diabody-vcE4 against these cell lines, respectively. Clearance of diabody-vcF4 (99-134 mL/d/kg) was 5-fold slower than for the nonconjugated diabody in naive severe combined immunodeficient mice. Diabody-vcF4 had potent and dose-dependent antitumor activity against established Karpas-299 xenografts and gave durable complete responses at well-tolerated doses. Biodistribution experiments with diabody-[(3)H]-vcF4 (0.72-7.2 mg/kg) in tumor-bearing mice showed a dose-dependent increase in total auristatin accumulation in tumors (< or =520 nmol/L) and decrease in relative auristatin accumulation (< or =8.1 %ID/g), with peak localization at 4 to 24 h after dosing. Diabody-vcF4 had approximately 4-fold lower cytotoxic activity than the corresponding IgG1-vcF4 conjugate in vitro. A similar potency difference was observed in vivo despite 25- to 34-fold faster clearance of diabody-vcF4 than IgG1-vcF4. This may reflect that dose-escalated diabody-vcF4 can surpass IgG1-vcF4 in auristatin delivery to tumors, albeit with higher auristatin exposure to some organs including kidney and liver. Diabody-drug conjugates can have potent antitumor activity at well-tolerated doses and warrant further optimization for cancer therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Antígeno Ki-1/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Feminino , Imunoglobulina G/imunologia , Antígeno Ki-1/imunologia , Camundongos , Camundongos SCIDRESUMO
The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.
Assuntos
Anticorpos Monoclonais/imunologia , Antineoplásicos/farmacocinética , Imunoconjugados/farmacocinética , Oligopeptídeos/farmacocinética , Animais , Anticorpos Monoclonais/química , Antineoplásicos/química , Sequência de Bases , Sítios de Ligação , Cisteína/química , Dissulfetos/química , Dissulfetos/metabolismo , Imunoconjugados/imunologia , Antígeno Ki-1/imunologia , Ligantes , Dose Máxima Tolerável , Camundongos , Oligopeptídeos/química , Engenharia de Proteínas , Serina/química , Solventes/química , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.
Assuntos
Padronização Corporal , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Fatores de Transcrição/metabolismo , Vespas , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Hibridização In Situ , Proteínas de Insetos/genética , Dados de Sequência Molecular , RNA Mensageiro Estocado/metabolismo , Alinhamento de Sequência , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Vespas/anatomia & histologia , Vespas/embriologiaRESUMO
Expression array data for >3000 individual clones from two suppression subtractive hybridization libraries revealed 147 genes overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Of these 147 genes, 30 genes have previously unknown cancer association and 65 genes have been associated with cancers other than NSCLC. The identification of 52 genes previously associated with NSCLC by different methodologies supports the validity of the strategy used here. Of the 147 genes, 19 have no prior named Unigene cluster designation, and are designated herein as L1 to L19. Quantitative real-time PCR and cancer profiling arrays were used as independent validation tools to confirm tumor overexpression for five of the 'L' genes in tumor cell lines and patient samples from NSCLC and other cancers. Follow-up studies for candidate NSCLC-associated genes can be useful in providing valuable insight into the etiology of lung cancer as well as providing potentially interesting diagnostic or therapeutic targets for further investigation.