RESUMO
We investigated relationships among chimeric TCR (cTCR) expression density, target Ag density, and cTCR triggering to predict lysis of target cells by cTCR(+) CD8(+) T human cells as a function of Ag density. Triggering of cTCR and canonical TCR by Ag could be quantified by the same mathematical equation, but cTCR represented a special case in which serial triggering was abrogated. The magnitude of target lysis could be predicted as a function of cTCR triggering, and the predicted minimum cTCR density required for maximal target lysis by CD20-specific cTCR was experimentally tested. cTCR density below approximately 20,000 cTCR/cell impaired target lysis, but increasing cTCR expression above this density did not improve target lysis or Ag sensitivity. cTCR downmodulation to densities below this critical minimum by interaction with Ag-expressing targets limited the sequential lysis of targets in a manner that could be predicted based on the number of cTCRs remaining. In contrast, acute inhibition of lysis of primary, intended targets (e.g., leukemic B cells) due to the presence of an excess of secondary targets (e.g., normal B cells) was dependent on the Ag density of the secondary target but occurred at Ag densities insufficient to promote significant cTCR downmodulation, suggesting a role for functional exhaustion rather than insufficient cTCR density. This suggests increasing cTCR density above a critical threshold may enhance sequential lysis of intended targets in isolation, but will not overcome the functional exhaustion of cTCR(+) T cells encountered in the presence of secondary targets with high Ag density.
Assuntos
Citotoxicidade Imunológica/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Modelos Imunológicos , Proteínas Mutantes Quiméricas/antagonistas & inibidores , Proteínas Mutantes Quiméricas/genética , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Antígenos CD20/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Relação Dose-Resposta Imunológica , Humanos , Células Jurkat , Modelos Lineares , Proteínas Mutantes Quiméricas/biossíntese , Valor Preditivo dos Testes , Receptores de Antígenos de Linfócitos T/biossíntese , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologiaRESUMO
Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.
Assuntos
Compostos Heterocíclicos com 1 Anel , Rim/metabolismo , Neoplasias/metabolismo , Polietilenoglicóis , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Compostos de Sulfidrila/química , Animais , Sítios de Ligação , Radioisótopos de Cobre/química , Radioisótopos de Cobre/farmacocinética , Feminino , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Rim/diagnóstico por imagem , Camundongos , Camundongos Nus , Estrutura Molecular , Transplante de Neoplasias , Neoplasias/diagnóstico por imagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
UNLABELLED: The CD20 cell surface antigen is expressed at high levels by over 90% of B-cell non-Hodgkin lymphomas (NHL) and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific PET imaging of NHL, we sought to develop PET agents targeting CD20. METHODS: Two recombinant anti-CD20 rituximab fragments, a minibody (scFv-C(H)3 dimer; 80 kDa) and a modified scFv-Fc fragment (105 kDa), designed to clear rapidly, were generated. Both fragments were radiolabeled with (124)I, and the minibody was additionally labeled with (64)Cu (radiometal) after conjugation to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). The radioiodinated fragments and the radiometal-labeled minibody were evaluated in mice as small-animal PET imaging agents for the in vivo imaging of human CD20-expressing lymphomas. RESULTS: Rapid and specific localization to CD20-positive tumors was observed with the radioiodinated fragments. However, the tumor uptake levels and blood activities differed, resulting in different levels of contrast in the images. The better candidate was the minibody, with superior uptake (2-fold higher than that obtained with scFv-Fc) in CD20-positive tumors and low uptake in CD20-negative tumors. Ratios of CD20-positive tumors to CD20-negative tumors at 21 h were 7.0 +/- 3.1 (mean +/- SD) and 3.9 +/- 0.7 for the minibody and scFv-Fc, respectively. The ratio achieved with the (64)Cu-DOTA-minibody at 19 h was about 5-fold lower because of higher residual background activity in CD20-negative tumors. CONCLUSION: A radioiodinated minibody and a radioiodinated scFv-Fc fragment produced excellent, high-contrast images in vivo. These new immunoPET agents may prove useful for imaging CD20-positive lymphomas in preclinical models and in humans with NHL.
Assuntos
Anticorpos Monoclonais , Linfoma de Células B/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Tomografia por Emissão de Pósitrons/veterinária , Compostos Radiofarmacêuticos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos de Imunoglobulinas/imunologia , Linfoma de Células B/imunologia , Camundongos , Camundongos Endogâmicos C3H , Compostos Radiofarmacêuticos/imunologia , Reprodutibilidade dos Testes , Rituximab , Sensibilidade e EspecificidadeRESUMO
Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignancies. We studied the behavior of three anti-carcinoembryonic antigen (CEA) single-chain Fv-Fc (scFv-Fc) variants (I253A, H310A, and H310A/H435Q; Kabat numbering system) that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2beta, 27.7 h) achieved the highest tumor uptake (44.6% ID/g at 24 h), whereas the radiometal-labeled H310A/H435Q fragment with the most rapid elimination (T1/2beta, 7.05 h) reached a maximum of 28.0% ID/g at 12 h postinjection. The H310A protein was characterized by both intermediate serum half-life and tumor uptake. The 111In-based biodistribution studies showed that all three fragments were eliminated primarily through the liver, and hepatic radiometal activity correlated with the rate of fragment clearance. The 111In-labeled H310A/H435Q protein exhibited the highest liver uptake (23.5% ID/g at 24 h). Metabolism of the 125I-labeled scFv-Fc proteins resulted in low normal organ activity. Finally, the 125I/111In biodistribution data allowed for dose estimations, which suggest the 131I-labeled scFv-Fc H310A/H435Q as a promising candidate for radioimmunotherapy.
Assuntos
Antígeno Carcinoembrionário/imunologia , Imunoconjugados/farmacocinética , Fragmentos de Imunoglobulinas/metabolismo , Radioisótopos de Índio/farmacocinética , Radioisótopos do Iodo/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Feminino , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Fragmentos de Imunoglobulinas/imunologia , Marcação por Isótopo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Distribuição Tecidual , Transplante HeterólogoRESUMO
Increasing interest in the use of radiolabeled antibodies for cancer imaging and therapy drives the need for more efficient production of the antibody conjugates. Here, we illustrate a method for rapid and efficient production of radiolabeled antibody conjugates using vacuum diafiltration guided by mathematical modeling. We apply this technique to the production of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated antibodies at the milligram and gram production scale and achieve radiolabeling efficiencies >95% using In-111. Using vacuum diafiltration, antibody-chelate conjugation and purification can be accomplished within the same vessel, and the entire process can be completed in <24 h. Vacuum diafiltration also offers safer and gentler processing conditions by eliminating the need to keep the retentate vessel under positive pressure through applied gas pressure or shear-inducing restriction points in the retentate flow path. Experimental data and mathematical model calculations suggest there exists a weak binding affinity (approximately 10(4)M(-1)) between the charged chelate molecules (e.g., DOTA) and the antibodies that slows the removal of excess chelate during purification. By analyzing the radiolabeling efficiency as a function of the number of diavolumes, we demonstrate the importance of balancing the removal of free chelate with the introduction of metal contaminants from the diafiltration buffer and also illustrate how to optimize radiolabeling of antibody conjugates under a variety of operating conditions. This methodology is applicable to the production of antibody conjugates in general.
Assuntos
Anticorpos Monoclonais , Filtração/métodos , Compostos Heterocíclicos com 1 Anel/química , Imunoconjugados/química , Marcação por Isótopo/métodos , Radioimunoterapia/métodos , Compostos Radiofarmacêuticos/química , Animais , Quelantes/química , Humanos , Modelos Teóricos , VácuoRESUMO
The goal of this study was to characterize the relationship between tumor uptake of 64Cu-DOTA-trastuzumab as measured by PET/CT and standard, immunohistochemistry (IHC)-based, histopathologic classification of human epidermal growth factor receptor 2 (HER2) status in women with metastatic breast cancer (MBC). Methods: Women with biopsy-confirmed MBC and not given trastuzumab for 2 mo or more underwent complete staging, including 18F-FDG PET/CT. Patients were classified as HER2-positive (HER2+) or -negative (HER2-) based on fluorescence in situ hybridization (FISH)-supplemented immunohistochemistry of biopsied tumor tissue. Eighteen patients underwent 64Cu-DOTA-trastuzumab injection, preceded in 16 cases by trastuzumab infusion (45 mg). PET/CT was performed 21-25 (day 1) and 47-49 (day 2) h after 64Cu-DOTA-trastuzumab injection. Radiolabel uptake in prominent lesions was measured as SUVmax Average intrapatient SUVmax (
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Compostos Organometálicos/metabolismo , Adulto , Idoso , Transporte Biológico , Neoplasias da Mama/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Receptor ErbB-2/metabolismo , TrastuzumabRESUMO
Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was approximately 12 days. Additionally, (124)I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.
Assuntos
Antígeno Carcinoembrionário/imunologia , Imunoconjugados/farmacocinética , Fragmentos de Imunoglobulinas/metabolismo , Radioisótopos do Iodo/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Animais , Antígeno Carcinoembrionário/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mieloma Múltiplo/metabolismo , Tomografia por Emissão de Pósitrons , Distribuição Tecidual , Transplante HeterólogoRESUMO
We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 +/- 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 +/- 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 +/- 4.0% ID/g). A minibody derived from a second anti-p185(HER2) antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 +/- 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 +/- 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C(H)2-C(H)3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 +/- 2.4% ID/g) and reduced kidney uptake (13.1 +/- 1.5% ID/g). Thus, by manipulating the size and format of anti-p185(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.
Assuntos
Imunoconjugados/farmacocinética , Fragmentos de Imunoglobulinas/metabolismo , Neoplasias/diagnóstico por imagem , Receptor ErbB-2/imunologia , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Antígeno Carcinoembrionário/imunologia , Radioisótopos de Cobre/farmacocinética , Feminino , Humanos , Imunoconjugados/química , Fragmentos de Imunoglobulinas/química , Radioisótopos de Índio/química , Radioisótopos de Índio/farmacocinética , Rim/diagnóstico por imagem , Rim/imunologia , Rim/metabolismo , Camundongos , Camundongos Nus , Neoplasias/imunologia , Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Distribuição TecidualRESUMO
PURPOSE/OBJECTIVE: Previous radioimmunotherapy (RIT) clinical trials at this institution with (90)Y-labeled cT84.66 anti-CEA (carcinoembryonic antigen) evaluated the antibody conjugated to diethylenetriaminepentaacetic acid (DTPA). The aim of this phase I therapy trial was to evaluate cT84.66 conjugated to the macrocyclic chelate (90)Y-DOTA and labeled with (90)Y in a comparable patient population. EXPERIMENTAL DESIGN: Patients with metastatic CEA-producing cancers were entered in this trial. If antibody targeting to tumor was observed after the administration of (111)In-DTPA cT84.66, the patient then received the therapy infusion of (90)Y-DOTA-cT84.66 1 week later. Serial nuclear scans, blood and urine collections, and computed tomography (CT) scans were performed to assess antibody biodistribution, pharmacokinetics, toxicities, and antitumor effects. RESULTS: Thirteen (13) patients were treated in this study. Dose-limiting hematologic toxicity was experienced at initial starting activity levels of 12 and 8 mCi/m(2). Subsequent patients received systemic Ca-DTPA at 125 mg/m(2) every 12 hours for 3 days post-therapy to allow for a dose escalation to 16 mCi/m(2), where hematologic toxicity was observed with an associated maximum tolerated dose (MTD) of 13.4 mCi/m(2). Tumor doses ranged from 4.4 to 569 cGy/mCi, which translated to 97-12,500 cGy after a single infusion of (90)Y-DOTA-cT84.66. Human anti-chimeric antibody (HACA) response developed in 8 of 13 patients and prevented additional therapy in 4 patients. CONCLUSIONS: This study demonstrates the feasibility of using (90)Y-DOTA-cT84.66 for antibody-guided radiation therapy. Immunogenicity of the DOTA-conjugated cT84.66 antibody was not appreciably greater than that observed with (90)Y-DTPA-cT84.66 in previous trials. Dose-limiting hematopoietic toxicity with (90)Y-DOTA-cT84.66 decreased with Ca-DTPA infusions post-therapy and appears to be comparable to previously published results for (90)Y-DTPA-cT84.66. The highest antibody uptake and tumor doses were to small nodal lesions, which supports the predictions from preclinical and clinical data that RIT may be best applied in the minimal tumor burden setting.
Assuntos
Antígeno Carcinoembrionário/imunologia , Imunotoxinas/uso terapêutico , Neoplasias/imunologia , Neoplasias/radioterapia , Radioimunoterapia/métodos , Radioisótopos de Ítrio/uso terapêutico , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Antígeno Carcinoembrionário/biossíntese , Feminino , Compostos Heterocíclicos com 1 Anel/efeitos adversos , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Humanos , Imunoglobulina G/imunologia , Imunotoxinas/efeitos adversos , Imunotoxinas/farmacocinética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/patologia , Radioimunoterapia/efeitos adversos , Radioisótopos de Ítrio/efeitos adversos , Radioisótopos de Ítrio/farmacocinéticaRESUMO
PURPOSE: Targeted systemic radiation therapy using radiolabeled antibodies results in tumor doses sufficient to produce significant objective responses in the radiosensitive hematological malignancies. Although comparable doses to tumor are achieved with radioimmunotherapy (RIT) in solid tumors, results have been modest primarily because of their relative lack of radiosensitivity. For solid tumors, as with external beam radiotherapy, RIT should have a more important clinical role if combined with other systemic, potentially radiation-enhancing chemotherapy agents and if used as consolidative therapy in the minimal tumor burden setting. The primary objective of this trial was to evaluate the feasibility and toxicities of systemic 90Y-chimeric T84.66 (cT84.66) anti-carcinoembryonic antigen RIT in combination with continuous infusion 5-fluorouracil (5-FU). EXPERIMENTAL DESIGN: Patients with chemotherapy-refractory metastatic colorectal cancer were entered. The study was designed for each patient to receive 90Y-cT84.66 anti-carcinoembryonic antigen at 16.6 mCi/m2 as an i.v. bolus infusion combined with 5-FU delivered as a 5-day continuous infusion initiated 4 h before antibody infusion. Cohorts of patients were entered at 5-FU dose levels of 700, 800, 900, and 1000 mg/m2/day. Upon reaching the highest planned dose level of 5-FU, a final cohort received 90Y-cT84.66 at 20.6 mCi/m2 and 5-FU at 1000 mg/m2/day. For all patients, Ca-diethylenetriaminepentaacetic acid at 125 mg/m2 every 12 h was administered for the first 72 h after 90Y-cT84.66. Patients were eligible to receive up to three cycles of 90Y-cT84.66/5-FU every 6 weeks. RESULTS: Twenty-one patients were treated on this study. All had been heavily pretreated with 19 having previously received 5-FU and 16 having failed two to four chemotherapy regimens. A maximum-tolerated dose of 16.6 mCi/m2 90Y-cT84.66 combined with 1000 mg/m2/day 5-FU was reached. These dose levels are comparable with maximum-tolerated dose levels of each agent alone. Thirteen patients received one cycle and 8 patients two cycles of therapy. Hematopoietic toxicity was dose-limiting and reversible. RIT did not appear to increase nonhematopoietic toxicities associated with 5-FU. Two of 19 patients assayed developed a human anti-chimeric antibody immune response after the first cycle of therapy, which is significantly less than that observed in a previous trial evaluating 90Y-cT84.66 alone. No objective responses were observed. However, 11 patients with progressive disease entering the study demonstrated radiological stable disease of 3-8 months duration and 1 patient demonstrated a mixed response. CONCLUSIONS: Results from this trial are encouraging and demonstrate the feasibility and possible advantages of combining continuous infusion 5-FU with 90Y-cT84.66 RIT. The addition of 5-FU does not appear to significantly enhance hematological toxicities of the radiolabeled antibody. In addition, 5-FU reduces the development of human anti-chimeric antibody response, permitting multicycle therapy in a larger number of patients. Future efforts should continue to focus on integrating radiation therapy delivered by radiolabeled antibodies into established 5-FU regimens.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/terapia , Fluoruracila/uso terapêutico , Radioimunoterapia , Proteínas Recombinantes de Fusão/uso terapêutico , Radioisótopos de Ítrio/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/secundário , Terapia Combinada , Estudos de Viabilidade , Humanos , Dose Máxima TolerávelRESUMO
PURPOSE: The chimeric T84.66 (cT84.66) minibody is a novel engineered antibody construct (V(L)-linker-V(H)-C(H)3; 80 kDa) that demonstrates bivalent and high affinity (4 x 10(10) m(-1)) binding to carcinoembryonic antigen (CEA). The variable regions (V(L) and V(H)) assemble to form the antigen-combining sites, and the protein forms dimers through self-association of the C(H)3 domains. In animal models, the minibody demonstrated high tumor uptake, approaching that of some intact antibodies, substantially faster clearance than intact chimeric T84.66, and superior tumor-to-blood ratios compared with the cT84.66 F(ab')(2) fragment, making it attractive for further evaluation as an imaging and therapy agent. The purpose of this pilot clinical study was to determine whether (123)I-cT84.66 minibody demonstrated tumor targeting and was well tolerated as well as to begin to evaluate its biodistribution, pharmacokinetics, and immunogenicity in patients with colorectal cancer. EXPERIMENTAL DESIGN: Ten patients with biopsy-proven colorectal cancer (6 newly diagnosed, 1 pelvic recurrence, 3 limited metastatic disease) were entered on this study. Each received 5-10 mCi (1 mg) of (123)I-labeled minibody i.v. followed by serial nuclear scans and blood and urine sampling over the next 48-72 h. Surgery was performed immediately after the last nuclear scan. RESULTS: Tumor imaging was observed with (123)I-labeled minibody in seven of the eight patients who did not receive neoadjuvant therapy before surgery. Two patients received neoadjuvant radiation and chemotherapy, which significantly reduced tumor size before surgery and minibody infusion. At surgery, no tumor was detected in one patient and only a 2-mm focus was seen in the second patient. (123)I-labeled minibody tumor targeting was not seen in either of these pretreated patients. Mean serum residence time of the minibody was 29.8 h (range, 10.9-65.4 h). No drug-related adverse reactions were observed. All 10 patients were evaluated for immune responses to the minibody, with no significant responses observed. CONCLUSION: This pilot study represents one of the first clinical efforts to evaluate an engineered intermediate-molecular-mass radiolabeled antibody construct directed against CEA. cT84.66 minibody demonstrates tumor targeting to colorectal cancer and a faster clearance in comparison with intact antibodies, making it appropriate for further evaluation as an imaging and therapy agent. The mean residence time of the minibody in patients is longer than predicted from murine models. We therefore plan to further evaluate its biodistribution and pharmacokinetic properties with minibody labeled with a longer-lived radionuclide, such as (111)In.
Assuntos
Anticorpos/química , Anticorpos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/terapia , Fragmentos de Imunoglobulinas/uso terapêutico , Imunoterapia/métodos , Radioisótopos do Iodo/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Cromatografia Líquida de Alta Pressão , Dimerização , Feminino , Humanos , Cinética , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Projetos Piloto , Estrutura Terciária de Proteína , Radiometria , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Natural killer (NK) cells play a vital antitumor role as part of the innate immune system. Efficacy of adoptive transfer of NK cells depends on their ability to recognize and target tumors. We investigated whether low dose focused ultrasound with microbubbles (ldbFUS) could facilitate the targeting and accumulation of NK cells in a mouse xenograft of human colorectal adenocarcinoma (carcinoembryonic antigen (CEA)-expressing LS-174T implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice) in the presence of an anti-CEA immunocytokine (ICK), hT84.66/M5A-IL-2 (M5A-IL-2). Human NK cells were labeled with an FDA-approved ultra-small superparamagnetic iron oxide particle, ferumoxytol. Simultaneous with the intravenous injection of microbubbles, focused ultrasound was applied to the tumor. In vivo longitudinal magnetic resonance imaging (MRI) identified enhanced accumulation of NK cells in the ensonified tumor, which was validated by endpoint histology. Significant accumulation of NK cells was observed up to 24 hrs at the tumor site when ensonified with 0.50 MPa peak acoustic pressure ldbFUS, whereas tumors treated with at 0.25 MPa showed no detectable NK cell accumulation. These clinically translatable results show that ldbFUS of the tumor mass can potentiate tumor homing of NK cells that can be evaluated non-invasively using MRI.
Assuntos
Adenocarcinoma/terapia , Neoplasias Colorretais/terapia , Ondas de Choque de Alta Energia/uso terapêutico , Microbolhas/uso terapêutico , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Xenoenxertos , Humanos , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos NOD , Transplante de NeoplasiasRESUMO
An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.
Assuntos
Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/uso terapêutico , Neoplasias Mamárias Experimentais/terapia , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Dados de Sequência MolecularRESUMO
An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disulfide-bonded dimer. This cysteine-modified diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys-diabody could be chemically modified using a thiol-specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site-specifically. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.
Assuntos
Antígeno Carcinoembrionário/química , Dissulfetos , Animais , Anticorpos/química , Sequência de Bases , Ligação Competitiva , Cisteína/química , Dimerização , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Neoplasias/metabolismo , Peptídeos/química , Estrutura Terciária de Proteína , Compostos de Sulfidrila , Fatores de Tempo , Tomografia Computadorizada de EmissãoRESUMO
Chimeric T84.66 (cT84.66) is a monoclonal antibody (mAb) of high specificity and affinity for the tumor-associated carcinoembryonic antigen (CEA). Radiolabeled cT84.66 has demonstrated utility in the clinic as a reagent for the radioimmunoscintigraphy and radioimmunotherapy of CEA-positive colorectal and breast malignancies. To extend the therapeutic efficacy of T84.66, humanization by complementary determining region (CDR) grafting was employed. CDR grafting is a well-established technique, though often a series of framework back-mutations is required to restore high affinity. Recently, the crystal structure of the T84.66 diabody (scFv dimer) derived from the murine T84.66 mAb was determined, facilitating the humanization process by the availability of crystal structure data for both the graft donor and graft acceptor. A search of the Protein Data Bank revealed close structural similarity (r.m.s.d. of 1.07 A) between the Fv of T84.66 and the Fv of 4D5v8, a humanized anti-p185HER2 antibody marketed as Herceptin (Trastuzumab). This resulted in two humanized versions of the T84.66 M5A and M5B mAbs that differed only in the number of murine residues present in the C-terminal half of CDR-H2. Biochemical analysis and animal biodistribution studies were conducted to evaluate the humanized mAbs. The M5A, M5B and cT84.66 mAbs showed sub-nanomolar affinity for CEA and as radiolabeled mAbs exhibited specific tumor localization in tumor bearing mice. The T84.66 M5A mAb was selected for clinical development due to a slightly higher tumor uptake and a larger content of human residues, and was renamed hT84.66. A limited-scale production and animal imaging study have demonstrated hT84.66's ability to support clinical trials. Planned clinical trials will determine the effective utilization of this structure-based approach in the development of a promising new therapeutic.
Assuntos
Anticorpos Monoclonais/imunologia , Antígeno Carcinoembrionário/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Radioisótopos de Índio , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
UNLABELLED: Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C(H)3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with (124)I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. METHODS: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of (124)I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or >90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9-3.1 MBq (53-85 microCi) of (124)I-minibody or with 3.1 MBq (85 microCi) of (124)I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using (18)F-FDG 2 d before imaging with (124)I-minibody. RESULTS: PET images using (124)I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using (124)I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the (124)I-diabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the (124)I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (<3 mm in diameter) could be imaged, and (124)I-anti-CEA minibodies, compared with (18)F-FDG, demonstrated highly specific localization. CONCLUSION: (124)I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.
Assuntos
Anticorpos Monoclonais , Antígenos/metabolismo , Antígeno Carcinoembrionário/metabolismo , Neoplasias Colorretais/diagnóstico por imagem , Glioma/diagnóstico por imagem , Radioisótopos do Iodo , Tomografia Computadorizada de Emissão/métodos , Animais , Anticorpos Monoclonais/farmacocinética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Imunoconjugados/farmacocinética , Radioisótopos do Iodo/farmacocinética , Marcação por Isótopo/métodos , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Transplante de Neoplasias , Engenharia de Proteínas/métodos , Controle de Qualidade , Radioimunoensaio/métodos , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Transplante HeterólogoRESUMO
UNLABELLED: Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. METHODS: Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). RESULTS: Liver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from (64)Cu-DOTA-trastuzumab was similar to (18)F-FDG. CONCLUSION: (64)Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a (64)Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. (64)Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.
Assuntos
Anticorpos Monoclonais Humanizados , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Radioisótopos de Cobre , Compostos Organometálicos , Tomografia por Emissão de Pósitrons , Receptor ErbB-2/metabolismo , Adulto , Idoso , Neoplasias da Mama/metabolismo , Feminino , Humanos , Fígado/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Radiometria , Reprodutibilidade dos Testes , Fatores de Tempo , Tomografia Computadorizada por Raios X , TrastuzumabRESUMO
PURPOSE: Targeted nanotherapies are being developed to improve tumor drug delivery and enhance therapeutic response. Techniques that can predict response will facilitate clinical translation and may help define optimal treatment strategies. We evaluated the efficacy of diffusion-weighted magnetic resonance imaging to monitor early response to CRLX101 (a cyclodextrin-based polymer particle containing the DNA topoisomerase I inhibitor camptothecin) nanotherapy (formerly IT-101), and explored its potential as a therapeutic response predictor using a mechanistic model of tumor cell proliferation. EXPERIMENTAL DESIGN: Diffusion MRI was serially conducted following CRLX101 administration in a mouse lymphoma model. Apparent diffusion coefficients (ADCs) extracted from the data were used as treatment response biomarkers. Animals treated with irinotecan (CPT-11) and saline were imaged for comparison. ADC data were also input into a mathematical model of tumor growth. Histological analysis using cleaved-caspase 3, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, Ki-67, and hematoxylin and eosin (H&E) were conducted on tumor samples for correlation with imaging results. RESULTS: CRLX101-treated tumors at day 2, 4, and 7 posttreatment exhibited changes in mean ADC = 16 ± 9%, 24 ± 10%, 49 ± 17%, and size (TV) = -5 ± 3%, -30 ± 4%, and -45 ± 13%, respectively. Both parameters were statistically greater than controls [p(ADC) ≤ 0.02, and p(TV) ≤ 0.01 at day 4 and 7], and noticeably greater than CPT-11-treated tumors (ADC = 5 ± 5%, 14 ± 7%, and 18 ± 6%; TV = -15 ± 5%, -22 ± 13%, and -26 ± 8%). Model-derived parameters for cell proliferation obtained using ADC data distinguished CRLX101-treated tumors from controls (P = 0.02). CONCLUSIONS: Temporal changes in ADC specified early CRLX101 treatment response and could be used to model image-derived cell proliferation rates following treatment. Comparisons of targeted and nontargeted treatments highlight the utility of noninvasive imaging and modeling to evaluate, monitor, and predict responses to targeted nanotherapeutics.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/administração & dosagem , Ciclodextrinas/administração & dosagem , Imagem de Difusão por Ressonância Magnética , Linfoma/patologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Linfoma/tratamento farmacológico , Camundongos , Camundongos Nus , Modelos Biológicos , Nanomedicina , Resultado do Tratamento , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Noninvasive methods are needed to explore the heterogeneous tumor microenvironment and its modulation by therapy. Hybrid PET/MRI systems are being developed for small-animal and clinical use. The advantage of these integrated systems depends on their ability to provide MR images that are spatially coincident with simultaneously acquired PET images, allowing combined functional MRI and PET studies of intratissue heterogeneity. Although much effort has been devoted to developing this new technology, the issue of quantitative and spatial fidelity of PET images from hybrid PET/MRI systems to the tissues imaged has received little attention. Here, we evaluated the ability of a first-generation, small-animal MRI-compatible PET scanner to accurately depict heterogeneous patterns of radiotracer uptake in tumors. METHODS: Quantitative imaging characteristics of the MRI-compatible PET (PET/MRI) scanner were evaluated with phantoms using calibration coefficients derived from a mouse-sized linearity phantom. PET performance was compared with a commercial small-animal PET system and autoradiography in tumor-bearing mice. Pixel and structure-based similarity metrics were used to evaluate image concordance among modalities. Feasibility of simultaneous PET/MRI functional imaging of tumors was explored by following (64)Cu-labeled antibody uptake in relation to diffusion MRI using cooccurrence matrix analysis. RESULTS: The PET/MRI scanner showed stable and linear response. Activity concentration recovery values (measured and true activity concentration) calculated for 4-mm-diameter rods within linearity and uniform activity rod phantoms were near unity (0.97 ± 0.06 and 1.03 ± 0.03, respectively). Intratumoral uptake patterns for both (18)F-FDG and a (64)Cu-antibody acquired using the PET/MRI scanner and small-animal PET were highly correlated with autoradiography (r > 0.99) and with each other (r = 0.97 ± 0.01). On the basis of these data, we performed a preliminary study comparing diffusion MRI and radiolabeled antibody uptake patterns over time and visualized movement of antibodies from the vascular space into the tumor mass. CONCLUSION: The MRI-compatible PET scanner provided tumor images that were quantitatively accurate and spatially concordant with autoradiography and the small-animal PET examination. Cooccurrence matrix approaches enabled effective analysis of multimodal image sets. These observations confirm the ability of the current simultaneous PET/MRI system to provide accurate observations of intratumoral function and serve as a benchmark for future evaluations of hybrid instrumentation.
Assuntos
Imageamento por Ressonância Magnética/instrumentação , Imageamento por Ressonância Magnética/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Tomografia por Emissão de Pósitrons/instrumentação , Tomografia por Emissão de Pósitrons/métodos , Algoritmos , Animais , Anticorpos , Autorradiografia , Calibragem , Radioisótopos de Cobre , Fluordesoxiglucose F18 , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos C57BL , Imagens de Fantasmas , Compostos Radiofarmacêuticos , Reprodutibilidade dos TestesRESUMO
This case study describes early phase purification process development for a recombinant anticancer minibody produced in mammalian cell culture. The minibody did not bind to protein A. Cation-exchange, anion-exchange, hydrophobic-interaction, and hydroxyapatite (eluted by phosphate gradient) chromatographic methods were scouted, but the minibody coeluted with BSA to a substantial degree on each. Hydroxyapatite eluted with a sodium chloride gradient separated BSA and also removed a dimeric contaminant, but BSA consumed so much binding capacity that this proved impractical as a capture tool. Capto MMC media proved capable of supporting adequate capture and significant dimer removal, although both loading and elution selectivity varied dramatically with the amount of supernatant applied to the column. An anion-exchange step was included to fortify overall virus and DNA removal. These results illustrate the value of multimodal chromatography methods when affinity chromatography methods are lacking and conventional alternatives prove inadequate.