RESUMO
In the 40-year history of biopharmaceuticals, there have been a few cases where the final products contained residual host cell protein (HCP) impurities at levels high enough to be of concern. This article summarizes the industry experience in these cases where HCP impurities have been presented in public forums and/or published. Regulatory guidance on HCP impurities is limited to advising that products be as pure as practical, with no specified numerical limit because the risk associated with HCP exposure often depends on the clinical setting (route of administration, dose, indication, patient population) and the particular impurity. While the overall safety and purity track record of the industry is excellent, these examples illustrate several important lessons learned about the kinds of HCPs that co-purify with products (e.g., product homologs, and HCPs that react with product), and the kinds of clinical consequences of HCP impurities (e.g., direct biological activity, immunogenicity, adjuvant). The literature on industry experience with HCP impurities is scattered, and this review draws in to one reference documented examples where the data have been presented in meetings, patents, product inserts, or press releases, in addition to peer-reviewed journal articles. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:828-837, 2018.
Assuntos
Produtos Biológicos/análise , Proteínas/análise , Animais , Células CHO , Cromatografia Líquida , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Humanos , Espectrometria de Massas em TandemRESUMO
During expression of biotherapeutic proteins, complex mixtures of additional proteins are also produced by normal expression machinery of the host cell (termed "host cell proteins," or HCP). HCPs pose a potential impact to patient safety and product efficacy, and therefore must be well-characterized and the ability of the process to clear these proteins must be demonstrated. Due to the complexity of HCP, the method(s) used for monitoring must be demonstrated to provide sufficient information about relevant proteins. The most commonly used analytical method for monitoring HCP is an enzyme-linked immunosorbent assay (ELISA). To ensure development of a suitable HCP ELISA, careful selection of critical reagents (anti-HCP antibodies and analytical standard) is crucial. During a recent major update to the manufacturing process of a biotherapeutic, we re-evaluated the suitability of the existing HCP ELISA for monitoring the HCP population in the updated process. In the evaluation, we compared a process-specific ELISA to a platform ELISA. Despite qualitative differences in the HCP profiles in 2D PAGE, LC-MS/MS showed that the HCP populations in the two analytical standards were similar. The process-specific HCP antibody had adequate HCP coverage, but was more sensitive to a few dominant proteins that were present in the upstream purification process. The platform HCP antibody had very broad coverage and additionally, could detect the majority of potential HCP impurities from this process. Furthermore, the platform HCP antibody was not biased toward a few dominant proteins and was more sensitive in the downstream purification process. Due to its broad HCP coverage and sensitivity, we conclude that our platform HCP ELISA method is superior to the process-specific HCP ELISA method.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Proteínas , Animais , Anticorpos/metabolismo , Células CHO , Cromatografia Líquida/métodos , Cricetinae , Cricetulus , Eletroforese em Gel Bidimensional , Proteínas/análise , Proteínas/química , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from â¼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (â¼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.
Assuntos
Proliferação de Células , Proteoma/análise , Animais , Células CHO , Sobrevivência Celular , Cromatografia Líquida , Cricetulus , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , L-Lactato Desidrogenase/análise , Lisofosfolipase/análise , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multi-product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information.
Assuntos
Anticorpos/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas/análise , Proteínas/química , Proteínas Recombinantes/metabolismo , Animais , Biotecnologia , Células CHO , Cricetinae , Cricetulus , Eletroforese em Gel Bidimensional , Proteínas/isolamento & purificação , Projetos de PesquisaRESUMO
Resins containing immobilized Staphylococcal Protein A (PA) are widely used in the commercial purification of recombinant human monoclonal antibody (rhuMAb IgG) biotherapeutics. Therefore, a sensitive assay for leached PA is needed to ensure that PA is not present at unacceptable levels as an impurity in the final product. PA impurities are measured by an ELISA using chicken anti-PA antibodies. However, PA in the presence of IgG product forms a PA/IgG complex that interferes in the assay. In this report a multi-product PA ELISA is described, wherein the PA/IgG complex is dissociated by heating in the presence of detergents and chelators prior to the ELISA. The dissociation facilitates the accessibility of the anti-PA antibodies to bind to PA in the immunoassay. Heat is provided by a novel microwave technology which allows brief heating time and high sample throughput using a microtiter plate for sample heating. Thus, broadly applicable dissociation conditions, suitable for all 21 rhMab IgGs tested to date were identified. This approach streamlines the measurement of leached PA, allows higher sample testing throughput, facilitates application across multiple products, and facilitates assay automation. Data comparing in-process samples tested with both the former product-specific ELISA and this new multi-product assay are shown.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/análise , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/análise , Complexo Antígeno-Anticorpo/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Proteína Estafilocócica A/imunologiaRESUMO
BACKGROUND: Prolonged cold ischemia is thought to exacerbate ischemia-reperfusion injury and graft coronary artery disease (GCAD). We investigated the effect of varying lengths of cold ischemia on inflammation and apoptosis during ischemia-reperfusion injury and correlated this with the degree of GCAD in rat cardiac allografts. METHODS: PVG rat (RT1c) hearts subjected to 30, 60, 90, 120, or 150 minutes of cold ischemia were heterotopically transplanted into ACI rats (RT1a). Grafts were procured after 4 hours of reperfusion and analyzed for superoxide generation, myeloperoxidase activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) production, cardiomyocyte apoptosis, and caspase-2, -3, -8, -9 activities. Additional transplanted animals received cyclosporine A (7.5 mg/kg/day) for 10 days as chronic rejection models. Indices of GCAD were determined at 90 days. RESULTS: A direct linear correlation was found between cold ischemic time, ischemia-reperfusion injury, and GCAD. Superoxide generation, myeloperoxidase activity, TNF-alpha, IL-1beta, MCP-1/CCL2 production, cardiomyocyte apoptosis, and caspase-2, -3, -8, and -9 activities increased with ischemic time, peaking at 120 minutes and plateauing at 150 minutes. GCAD, assessed by the percentage of luminal narrowing, the intima/media ratio, and the percentage of diseased vessels, worsened with increased ischemic time, peaking at 120 minutes and plateauing at 150 minutes. All tested variables in both the acute and chronic phases were significantly increased with 120-minute ischemia compared with 30-minute ischemia. CONCLUSIONS: These data indicate that the degree of cardiomyocyte apoptosis and inflammatory response in cardiac allografts during ischemia-reperfusion injury depends on the duration of cold ischemia. More important, that prolonged cold ischemia correlates with increased GCAD.
Assuntos
Isquemia Fria/efeitos adversos , Doença das Coronárias/etiologia , Transplante de Coração/efeitos adversos , Traumatismo por Reperfusão Miocárdica/patologia , Animais , Apoptose , Caspases/metabolismo , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Transplante de Coração/imunologia , Marcação In Situ das Extremidades Cortadas , Interleucina-1/metabolismo , Masculino , Traumatismo por Reperfusão Miocárdica/etiologia , Miócitos Cardíacos , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transplante Heterotópico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Graft coronary artery disease (GCAD) is the leading cause of death after the first year of heart transplantation. The reduced bioavailability of endothelium-derived nitric oxide (NO) may play a role in endothelial vasodilator dysfunction and the structural changes that are characteristic of GCAD. A potential contributor to endothelial pathobiology is asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor. We hypothesized that lowering ADMA concentrations by dimethylarginine dimethylaminohydrolase (DDAH) overexpression in the recipient might suppress GCAD and long-term immune responses in murine cardiac allografts. METHODS AND RESULTS: In one series, donor hearts of C-H-2(bm12)KhEg (H-2(bm12)) wild-type (WT) mice were heterotopically transplanted into C57BL/6 (H-2b) transgenic mice overexpressing human DDAH-I or WT littermates and procured after 4 hours of reperfusion (WT and DDAH-I recipients, n=6 each). In a second series, donor hearts were transplanted into DDAH-I-transgenic or WT mice and procured 30 days after transplantation (n=7 each). In DDAH-I recipients, plasma ADMA concentrations were lower, in association with reduced myocardial generation of superoxide anion (WT versus DDAH-I, 465.7+/-79.8 versus 173.4+/-32.3 micromol.L(-1).mg(-1).h(-1); P=0.02), inflammatory cytokines, adhesion molecules, and chemokines. GCAD was markedly reduced in cardiac allografts of DDAH-I-transgenic recipients as assessed by luminal narrowing (WT versus DDAH, 79+/-2% versus 33+/-7%; P<0.01), intima-media ratio (WT versus DDAH, 1.1+/-0.1 versus 0.5+/-0.1; P<0.01), and the percentage of diseased vessels (WT versus DDAH, 100+/-0% versus 62+/-10%; P<0.01). CONCLUSIONS: Overexpression of DDAH-I attenuated oxidative stress, inflammatory cytokines, and GCAD in murine cardiac allografts. The effect of DDAH overexpression may be mediated by its reduction of plasma and tissue ADMA concentrations.
Assuntos
Amidoidrolases/metabolismo , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/prevenção & controle , Transplante de Coração/efeitos adversos , Amidoidrolases/farmacologia , Animais , Arginina/análogos & derivados , Arginina/antagonistas & inibidores , Arginina/sangue , Arginina/metabolismo , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Feminino , Coração/efeitos dos fármacos , Coração/fisiopatologia , Transplante de Coração/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocardite/prevenção & controle , Miocárdio/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Nitritos/metabolismo , Concentração Osmolar , Período Pós-Operatório , Gravidez , Superóxidos/antagonistas & inibidores , Fatores de Tempo , Transplante Heterotópico , Transplante Homólogo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND: We investigated the feasibility of bioluminescence imaging (BLI) for the in vivo assessment of cardiac allograft viability and visualization of passenger leukocytes during the course of acute rejection. METHODS AND RESULTS: Hearts of FVB (H-2q) luciferase-green fluorescent protein transgenic mice (beta-actin promoter) or FVB luciferase transgenic mice (CD5 promoter) were heterotopically transplanted into either BALB/c (H-2d) or FVB recipients. Light intensity emitting from the recipient animals was measured daily by in vivo BLI until 12 days after transplantation. Graft beating score (0 to 4) was assessed by daily abdominal palpation until 12 days after transplantation. Inflammatory cell infiltration (CD45 stain) and structural changes of green fluorescent protein-positive cardiomyocytes were followed by immunohistochemistry. All cardiac allografts were acutely rejected by 12 days after transplantation. The intensity of light emitting from cardiac allografts declined 4 days after transplantation and correlated with graft beating scores (R2=0.91, P=0.02). Immunohistochemistry confirmed these results by showing an increase of CD45+ inflammatory cell infiltration and destruction of green fluorescent protein-positive cardiomyocytes in the cardiac allografts during acute rejection. In vivo BLI visualized migration and proliferation of CD5+ passenger leukocytes in both syngeneic and allogeneic recipients. In the allograft recipients, light signal from CD5+ passenger leukocytes peaked at 6 hours and diminished by 12 hours, whereas in the syngeneic recipients, the signal remained high until 10 days after transplantation. CONCLUSIONS: BLI is a useful modality for the quantitative assessment of in vivo cardiac graft viability and tracking of passenger leukocytes in vivo during the course of acute rejection.
Assuntos
Genes Reporter , Rejeição de Enxerto/patologia , Transplante de Coração/imunologia , Luminescência , Subpopulações de Linfócitos T/transplante , Transplante Homólogo/patologia , Animais , Antígenos CD5/análise , Divisão Celular , Linhagem da Célula , Estudos de Viabilidade , Feminino , Fibroblastos/transplante , Luciferina de Vaga-Lumes , Rejeição de Enxerto/imunologia , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia de Fluorescência , Miócitos Cardíacos/química , Miócitos Cardíacos/patologia , Subpopulações de Linfócitos T/imunologia , Transplante Heterotópico , Transplante Homólogo/imunologia , Transplante IsogênicoRESUMO
BACKGROUND: We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection. METHODS AND RESULTS: In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1 x 10(6) cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation. CONCLUSIONS: In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.
Assuntos
Diferenciação Celular/imunologia , Transplante de Tecido Fetal/imunologia , Rejeição de Enxerto/imunologia , Neoplasias Cardíacas/etiologia , Isquemia Miocárdica/cirurgia , Miocárdio/imunologia , Células-Tronco Pluripotentes/transplante , Complicações Pós-Operatórias/etiologia , Transplante de Células-Tronco , Teratoma/etiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular/imunologia , Linhagem Celular/transplante , Células Dendríticas/imunologia , Feminino , Granulócitos/imunologia , Neoplasias Cardíacas/patologia , Transplante de Coração/imunologia , Injeções Intramusculares , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/imunologia , Complicações Pós-Operatórias/patologia , Transplante de Células-Tronco/efeitos adversos , Subpopulações de Linfócitos T/imunologia , Teratoma/patologia , Transplante Heterotópico/imunologia , Transplante Homólogo/imunologia , Transplante Isogênico/imunologiaRESUMO
OBJECTIVE: Transplanted hearts subjected to prolonged ischemia develop ischemia-reperfusion injury and graft coronary artery disease. To determine the effect of delta-protein kinase C and -protein kinase C on ischemia-reperfusion injury and the resulting graft coronary artery disease induced by prolonged ischemia, we used a delta-protein kinase C-selective inhibitor peptide and an -protein kinase C-selective activator peptide after 30 or 120 minutes of ischemia. METHODS: Hearts of piebald viral glaxo (PVG) rats were heterotopically transplanted into allogeneic August Copenhagen Irish (ACI) rats. After cardioplegic arrest of the donor heart, -protein kinase C activator was injected antegrade into the coronary arteries. Hearts were procured and bathed in -protein kinase C activator, and before reperfusion, delta-protein kinase C inhibitor was injected into the recipient inferior vena cava. Controls were treated with saline. To analyze ischemia-reperfusion injury, grafts were procured at 4 hours after transplantation and analyzed for superoxide generation; myeloperoxidase activity; tumor necrosis factor alpha, interleukin 1beta, and monocyte/macrophage chemoattractant protein 1 production; and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase 2, 3, 8, and 9 activity. To analyze graft coronary artery disease, another set of animals underwent equal ischemic times and treatment strategies and then after 90 days were analyzed for graft coronary artery disease indexes. RESULTS: All measures of ischemia-reperfusion injury and graft coronary artery disease after 120 minutes of ischemia in the saline-treated group were significantly increased relative to those observed after 30 minutes of ischemia. It is important to note that all ischemia-reperfusion injury parameters and graft coronary artery disease indexes decreased significantly in the protein kinase C regulator-treated group in comparison to saline-treated controls; additionally, these values were equivalent to those in saline-treated controls with 30 minutes of ischemia. CONCLUSIONS: Combined treatment with -protein kinase C activator and delta-protein kinase C inhibitor reduces ischemia-reperfusion injury and decreases the resulting graft coronary artery disease induced by prolonged ischemia.
Assuntos
Doença das Coronárias/prevenção & controle , Modelos Animais de Doenças , Transplante de Coração/efeitos adversos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Oligopeptídeos/uso terapêutico , Proteína Quinase C , Animais , Apoptose , Caspases/análise , Caspases/metabolismo , Doença das Coronárias/diagnóstico , Doença das Coronárias/etiologia , Doença das Coronárias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Rejeição de Enxerto , Marcação In Situ das Extremidades Cortadas , Inflamação , Masculino , Traumatismo por Reperfusão Miocárdica/diagnóstico , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/química , Peroxidase/análise , Peroxidase/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Proteína Quinase C-épsilon , Ratos , Ratos Endogâmicos , Índice de Gravidade de Doença , Superóxidos/análise , Superóxidos/metabolismo , Fatores de Tempo , Transplante HeterotópicoRESUMO
After cardiac transplantation, graft damage occurs secondary to ischemia-reperfusion injury and acute rejection. This damage ultimately leads to the development of graft coronary artery disease (GCAD), which limits long-term graft survival. Apoptosis is directly involved in graft injury, contributing to the development of GCAD. To assess the role of the antiapoptotic factor Bcl-2 in the process of GCAD, we transplanted hearts from FVB transgenic mice overexpressing human Bcl-2 under the control of alpha-myosin heavy chain promoter into allogenic C57BL/6 mice. Bcl-2 overexpression led to reduced cytochrome c-mediated caspase-9-dependent cardiomyocyte apoptosis and local inflammation (neutrophil infiltration and proinflammatory cytokine production) in cardiac allografts during ischemia-reperfusion injury and also led to reduced immune responses (inflammatory cell infiltration, production of T(H)1 cytokines and chemokines, and expression of adhesion molecules) during acute and chronic rejection without affecting host CD4(+) and CD8(+) cell responses in the spleen. Thus, local Bcl-2 expression directly contributes to the modulation of local immune responses in allograft rejection, resulting in attenuated GCAD. In conclusion, our findings suggest that the modulation of Bcl-2 expression by pharmacologic up-regulation or gene transfer may be of clinical benefit in the short- and long-term function of cardiac allografts.