Unlabeled Antibody Research Articles

Synthesis and biological investigations of a ZnPc-antiCEA bioconjugate for imaging of colorectal cancer.

Two zinc(II) phthalocyanines (ZnPcs) were conjugated with a monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA), using an in situ activated carboxylic acid on the ZnPcs. The bioconjugate with the highest ZnPc/MAb ratio of 3 was investigated in vitro for its ability to target and fluorescently label human colorectal HT-29 cells. The ZnPc-CEA MAb 2 was observed to efficiently target HT-29 cells, about 37 times more than unconjugated ZnPc. Furthermore, in the presence of a 4-fold excess of unlabelled anti-CEA antibody, the fluorescence signal of 2 was reduced by ~90% showing that the targeting is CEA-mediated. These studies further confirm the high specificity of Pc-antibody conjugates for antigens over-expressed on tumor cells and warrant further investigations of these immunoconjugates and their derivatives for imaging of colorectal cancer.

Safety and Efficacy of 188-Rhenium-Labeled Antibody to Melanin in Patients with Metastatic Melanoma

There is a need for effective “broad spectrum” therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of 188Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi 188Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of 188Re-6D2 was escalated to 54 mCi. SPECT/CT revealed 188Re-6D2 uptake in melanoma metastases. The mean effective half-life of 188Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that 188Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

Antibody repertoire profiling using bacterial display identifies reactivity signatures of celiac disease.

A general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (n = 45) or healthy subjects (n = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not with those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation. Individual peptides were dissimilar with known CD-specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and the classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.

Moving Radioimmunotherapy Forward for Follicular Lymphoma

The treatment of follicular non-Hodgkin lymphoma (NHL), which is the second most common NHL in Western countries, continues to evolve. In this issue of Journal of Clinical Oncology, two important reports deal with the upfront use of radioimmunotherapy (RIT) in follicular lymphoma (FL). Both of these studies enrolled FL patients who were considered to be in need of therapy. The report by Scholz et al 1 used RIT as a single-agent without any chemotherapy, whereas the second study by Press et al 2 was a phase III randomized trial of the standard six cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) versus six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) with RIT as consolidation (Southwest Oncology Group [SWOG] S0016). The results of these studies have implications for current care and the design of future studies but, as is typical for FL studies, leave us with nagging unanswered questions. RIT is a unique therapeutic modality that uses an antibody carrier to target high-energy, short–path-length radionuclides to tumor sites with little effect on other solid organs. If pretherapy guidelines are followed, the only toxicity is reversible myelosuppression. The known sensitivity of FL to conventional radiation therapy and the usual widespread nature of FL make this type of NHL an attractive target for RIT. Indeed, early randomized studies of unlabeled antibody versus the radiolabeled version of essentially the same antibody in relapsed indolent NHL demonstrated the advantage of the radioimmunoconjugate (RIC) with respect to the overall response rate (ORR) and complete response (CR) rates. 3,4

Combination Therapy Targeting Two Different Antigens with Anti-CD22 Radioimmunotherapy and Anti-CD20 Immunotherapy in Non-Hodgkin Lymphoma (NHL): Phase I Results

AbstractAbstract 3680 Background:Current NHL radioimmunotherapy is administered with unlabeled antibody targeting the same B-cell antigen. This decreases sequestration of the radiolabeled antibody by normal B-lymphocytes, but potentially blocks access of the radiolabeled antibody to the tumor sites. In clinical studies, yttrium-90 radiolabeled anti-CD22 epratuzumab (90Y-epratuzumab) has demonstrated therapeutic activity with acceptable toxicity when given weekly × 2 at doses up to 20 mCi/m2 (Morschhauser et al., J Clin Oncol. 2010;28:3709–16), while separate studies showed that a 200-mg/m2 dose of anti-CD20 veltuzumab given weekly × 4 readily depletes peripheral blood B-cells and is also therapeutically active (Morschhauser et al., J Clin Oncol, 2009;27:3346–53). In mice bearing human B-cell lymphoma xenografts, this non-competing combination showed improved therapeutic responses over either agent alone (Mattes et al., Clin Cancer Res. 2008;14:6154–60). As such, a multicenter, phase I/II study was undertaken to evaluate combination therapy with 90Y-epratuzumab and veltuzumab. Methods:Adult patients with aggressive NHL who failed ≥ 1 prior standard regimen (excluding autologous SCT) were eligible if they had measurable CT lesions, ECOG 0–1 performance status, <25% bone marrow involvement, hemoglobin ≥ 10 g/dL, ANC ≥ 1.5 × 109/L, platelets ≥ 100 × 109/L and were >4 weeks beyond last treatment. All patients received a 4-week treatment regimen (weekly 200 mg/m2 veltuzumab, 3–5 mCi 111In-epratuzumab on week 2, 90Y-epratuzumab on weeks 3 and 4). Using NCI CTCAE v4.0 toxicity criteria, dose-limiting toxicity (DLT) was defined as Grade 4 hemoglobin, platelet or neutrophil levels >7 days or not recovered to Grade 1 by 12 weeks, or any non-hematologic Grade 3 or 4 events. Starting at 15 mCi/m2, a standard 3+3 dose escalation determined the MTD of 90Y-epratuzumab. Treatment response utilized CT-based 2007 IWG revised criteria. Serial 111In imaging and serum samples were used to assess biodistribution, tumor targeting and determine normal organ radiation dosimetry. ELISA-based assays were used to measure veltuzumab pharmacokinetics and any human anti-veltuzumab antibodies (HAHA). Results:Fifteen patients (10 male/5 female, 56 – 84 years old) with diffuse large B-cell lymphoma (DLBCL N=7), transformed follicular lymphoma (TFL, N=4) or mantle cell lymphoma (MCL, N=4) have now been enrolled. They had 3 median prior treatments (1 – 7) [RCHOP (N=15); bendamustine plus rituximab (N=5); ICE, lenalidomide, bortezomib (N=2 each); others (1 each)] and had low (N=3), low-intermediate (N=3), high-intermediate (N=7), or high (N=2) IPI risk scores. Treatment was well-tolerated with no infusion reactions. 111In-imaging showed normal biodistribution, with acceptable normal organ dosimetry for the scheduled 90Y dose, and tumor targeting occasionally visualized. There was no evidence of HAHA, and the serum clearance of veltuzumab and 111In-epratzumab in combination was comparable to that reported previously for these agents alone. The 90Y dose was repeatedly de-escalated from 15 (N=3) to 12 (N=3), 9 (N=6), and currently 6 (N=3) mCi/m2 due to 7 hematologic DLTs at ≥ 9 mCi/m2in 6 patients involving Grade 3-thrombocytopenia (N=5) or neutropenia (N=2). Clinically, there were 3 SAEs (pneumonia, non-infectious pneumonitis, fracture from fall), but no other major infections or bleeding. Of 11 patients who have now had treatment response assessments, 5 (45%) had objective responses, including one DLCBL patient with a complete response (CR) continuing 12 months later and 4 patients (1 DLBCL, 3 TFL) with partial responses (PRs), with one continuing at 4 weeks and 3 relapsed at 3 – 6 months. Four other patients (1 DLBCL, 3 MCL) achieved stable disease (SD) continuing up to 6 months as best response, resulting in a disease control rate of 82% (CR+PR+SD). Conclusions:Combination treatment was well-tolerated, with the PK behavior of the two agents substantially unchanged, and with initial evidence of therapeutic activity (including one durable CR) in these difficult-to-treat patients. Hematologic toxicity necessitated lowering the 90Y dose, most likely due to the prior aggressive chemotherapies or depletion of the normal sequestering B-cell pool by veltuzumab preceding 90Y-epratzumab. The trial is continuing to determine an acceptable 90Y dose and define the safety and efficacy profile of this combination approach. Disclosures:Sharkey:Immunomedics: Employment. Rojo:Immunomedics: Employment. Wegener:Immunomedics: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy

In spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium((188)Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy. Mice bearing A2058 melanoma xenografts were treated with either 1.5 mCi (188)Re-6D2 antibody, saline, unlabeled 6D2 antibody or (188)Re-labeled non-specific IgM. On Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P<0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers--chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells. These results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5+ and JARID1B+cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers.

Molecular Biology Techniques Q&amp;A

Molecular Biology Techniques Q&amp;A

Differential Effects of Predosing on Tumor and Tissue Uptake of an111In-Labeled Anti-TENB2 Antibody–Drug Conjugate

TENB2, also known as tomoregulin or transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is a transmembrane proteoglycan overexpressed in human prostate tumors. This protein is a promising target for antimitotic monomethyl auristatin E (MMAE)-based antibody-drug conjugate (ADC) therapy. Nonlinear pharmacokinetics in normal mice suggested that antigen expression in normal tissues may contribute to targeted mediated disposition. We evaluated a predosing strategy with unconjugated antibody to block ADC uptake in target-expressing tissues in a mouse model while striving to preserve tumor uptake and efficacy. Unconjugated, unlabeled antibody was preadministered to mice bearing the TENB2-expressing human prostate explant model, LuCaP 77, followed by a single administration of (111)In-labeled anti-TENB2-MMAE for biodistribution and SPECT/CT studies. A tumor-growth-inhibition study was conducted to determine the pharmacodynamic consequences of predosing. Preadministration of anti-TENB2 at 1 mg/kg significantly increased blood exposure of the radiolabeled ADC and reduced intestinal, hepatic, and splenic uptake while not affecting tumor accretion. Similar tumor-to-heart ratios were measured by SPECT/CT at 24 h with and without the predose. Consistent with this, the preadministration of 0.75 mg/kg did not interfere with efficacy in a tumor-growth study dosed at 0.75 mg or 2.5 mg of ADC per kilogram. Overall, the potential to mask peripheral, nontumor antigen uptake while preserving tumor uptake and efficacy could ameliorate toxicity and may significantly affect future dosing strategies for ADCs.

Sustained levels of antibodies against Aβ in amyloid-rich regions of the CNS following intravenous dosing in human APP transgenic mice

Passive immunization with anti-Aβ antibodies leads to the reduction of AD-like neuropathology in transgenic mice. Previously we showed that anti-Aβ antibodies enter the brain and bind to amyloid plaques. Now using 125I-labeled 3D6, the mouse parent antibody of the clinical candidate bapineuzumab, we further characterized the pharmacokinetic profile of this antibody in the brain and serum. Our studies demonstrated that following a single intravenous injection, the labeled antibody accumulates and persists in plaque rich regions of the brain in transgenic PDAPP mice. Accumulation was specific to amyloid since it did not occur in non-transgenic animals lacking human APP, could not be measured in transgenic animals prior to plaque deposition, and correlated with the level of plaque burden in aging transgenic mice. After a single intravenous injection, CNS levels of 125I‐labeled 3D6 continued to increase for 14days even as serum levels of the antibody declined. The calculated half-life of antibody in the circulation was 6days, while antibody levels in the CNS remained stable for nearly a month. When given at supra-therapeutic levels, unlabeled antibody did not compete with tracer levels of labeled antibody for accumulation in the CNS, indicating that the binding capacity of plaques was very high. Our results demonstrate that even when administered in the periphery at very low (tracer) doses, 3D6 and bapineuzumab cross the blood brain barrier to accumulate in plaque rich regions of the brain. CNS clearance is markedly slower than in the serum and correlates with binding to deposited amyloid in a transgenic model of Alzheimer's disease.

Improvement of the efficiency and simplification of ELISA tests for rapid and ultrasensitive detection of okadaic acid in shellfish

Okadaic acid (OA) is a marine toxin ingested by shellfish. The consumption of contaminated seafood causes diarrheic shellfish poisoning, responsible for gastrointestinal troubles in humans. The development of fast, reliable and sensitive methods for OA analysis is an evident necessity in order to guarantee the seafood safety and to protect human health.In this work, competitive indirect immunoassays were developed for the toxin detection. Indirect and direct labeling were used and compared. With indirect labeling, the unlabeled anti-OA antibody binds to the toxin and a labeled secondary antibody is used for the signal generation. Alternatively, using the direct labeling, the label is attached via a covalent bond to the anti-OA antibody. In this work, peroxidase was conjugated to the anti-OA antibody through a periodate activation. The peroxidase–antibody conjugate is used for both toxin recognition and signal generation. In the last case, ELISA test is simplified and the colorimetric competitive indirect immunoassay based on direct labeling is more sensitive to OA than the test based on indirect labeling. In optimized conditions, the IC50 value and detection limit were respectively 0.066 μg L−1 and 0.0061 μg L−1. Then, the colorimetric immunoassay based on direct labeling was validated with certified reference mussel samples, demonstrating the efficiency of the approach.

Abstract 1574: Quantitation of HLA-A2-MAGE3271-279 complexes on the DC surface improves anticancer vaccine designs

Abstract Background: Dendritic cells (DC) loaded with tumor antigen (TA)-derived peptides are used as vehicles for anti-cancer vaccines. HLA class-I restricted short synthetic peptides are commonly used to load DC for therapy. However, randomized clinical trials have failed to confirm the efficacy of DC-based therapies. We hypothesized that new methods for the quantification of peptide loading of DC are needed to improve the overall response rate to anti-tumor vaccines. Methods: An unlabeled monoclonal antibody (Ab), 12B6 Ab, recognizing HLA-A2-MAGE3271-279 complexes was generated by S. Ferrone. Monocyte-derived DC were generated from HLA-A2+ buffy coats of healthy donors in media containing IL-4 and GM-CSF. DC harvested on day 5 of culture were matured by further 48-h exposure to LPS or Interferon-α (IFN-γ). The HLA-A2+ cell line (T2) deficient for LMP2, TAP1 and TAP2 was maintained in a complete RPMI medium. Peptides were synthesized at the University of Pittsburgh Peptide Synthesis facility. An HLA-A2+ CTL clone specific for MAGE3271-279 was used in ELISPOT assays. Cells were stained with 12B6 Ab followed by a PE-labeled secondary Ab. Flow cytometry was used to measure mean fluorescence intensity (MFI) of the HLA class-I-peptide complexes and the loading kinetics. HLA class-I-peptide complexes were dissociated by treatment of cells with citric acid, pH 3.0. Results: The 12B6 Ab reacted with HLA-A2+DC or T2 cells loaded with MAGE3271-279 but not with HLA-A2− DC loaded with MAGE3271-279 or cells loaded with irrelevant peptides. The MFI obtained for the complexes positively correlated with the concentration of MAGE3271-279 used for loading. Maximum levels of HLA-A2-MAGE3271-279 were detectable on DC after 30min loading, while optimal loading of T2 cells required 4h incubation. The levels of detectable complexes correlated with the total expression levels of HLA-A2 molecules on the DC surface. However, donors with comparable levels HLA-A2 expression showed different levels HLA-A2-MAGE3271-279 expression after loading with the same peptide concentration. The MFI for HLA-A2-MAGE3271-279 on DC, but not on T2 cells, was increased when peptide loading was performed after low pH treatment. This treatment did not affect expression of co-stimulatory molecules on DC. Importantly, the peptide-specific stimulation of CTL generated with mDC loaded after low pH treatment resulted in a significantly increased percentage of IFN-γ secreting T cells. Conclusion: The 12B6 Ab is specific for HLA-A2-MAGE3271-279 complexes and allows for their quantification on the surface of DC. Loading of DC was increased by the removal of HLA class-I-self antigen complexes prior to the addition of the TA-derived exogenous peptide. The treated DC showed a higher density of HLA-A2-MAGE3271-279 by flow cytometry and were more effective in priming MAGE3271-279 specific CTL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1574. doi:1538-7445.AM2012-1574

Maximizing tumour exposure to anti‐neuropilin‐1 antibody requires saturation of non‐tumour tissue antigenic sinks in mice

Neuropilin-1 (NRP1) is a VEGF receptor that is widely expressed in normal tissues and is involved in tumour angiogenesis. MNRP1685A is a rodent and primate cross-binding human monoclonal antibody against NRP1 that exhibits inhibition of tumour growth in NPR1-expressing preclinical models. However, widespread NRP1 expression in normal tissues may affect MNRP1685A tumour uptake. The objective of this study was to assess MNRP1685A biodistribution in tumour-bearing mice to understand the relationships between dose, non-tumour tissue uptake and tumour uptake. Non-tumour-bearing mice were given unlabelled MNRP1685A at 10 mg·kg(-1) . Tumour-bearing mice were given (111) In-labelled MNRP1685A along with increasing amounts of unlabelled antibody. Blood and tissues were collected from all animals to determine drug concentration (unlabelled) or radioactivity level (radiolabelled). Some animals were imaged using single photon emission computed tomography - X-ray computed tomography. MNRP1685A displayed faster serum clearance than pertuzumab, indicating that target binding affected MNRP1685A clearance. I.v. administration of (111) In-labelled MNRP1685A to tumour-bearing mice yielded minimal radioactivity in the plasma and tumour, but high levels in the lungs and liver. Co-administration of unlabelled MNRP1685A with the radiolabelled antibody was able to competitively block lungs and liver radioactivity uptake in a dose-dependent manner while augmenting plasma and tumour radioactivity levels. These results indicate that saturation of non-tumour tissue uptake is required in order to achieve tumour uptake and acceptable exposure to antibody. Utilization of a rodent and primate cross-binding antibody allows for translation of these results to clinical settings.

Treatment with Unlabeled mAb BR96 After Radioimmunotherapy with 177Lu-DOTA-BR96 in a Syngeneic Rat Colon Carcinoma Model

Metastatic disease after successful treatment of the primary tumor continues to be a therapeutic challenge. Enhancement of therapeutic effects by the administration of unlabeled monoclonal antibodies (mAbs) after radioimmunotherapy (RIT) may provide a means of preventing or delaying the development of metastatic disease. In the present study, Brown Norway rats with syngeneic grafted colon carcinomas were administered the minimal effective therapeutic dose of 400 MBq/kg lutetium-177 ((177)Lu)-DOTA-BR96. After 2 weeks, half of the animals were given 15 mg/kg unlabeled mAb BR96 as consolidation therapy. Treatment response and toxicity were monitored 100 days after the treatment with unlabeled BR96. The treatment with unlabeled mAb after RIT resulted in a complete response (CR) in 19 of 19 animals, while RIT alone resulted in a CR in 17 of 19 animals. The additional treatment did not affect the number of animals with metastatic disease or the time to clinical symptoms of metastases. RIT resulted in reversible myelotoxicity. The unlabeled mAb BR96 did not cause any additional toxicity, making it possible to repeat the consolidation therapy.

Predictors of long-term outcome from intraperitoneal radioimmunotherapy for ovarian cancer.

Data was analyzed from 92 patients > 5 years after intraperitoneal (IP) radionuclide therapy (RIT) with (90)Y- or (177)Lu-CC49 to determine prognostic factors. Patients had CC49 antibody-reactive ovarian cancer confined to the abdominal cavity after primary debulking and chemotherapy. The first 27 patients received IP (177)Lu-CC49 alone; the remainder received Interferon (IFN), to increase the expression of the tumor-associated glycoprotein-72 (TAG-72) antigen, +/- IP paclitaxel (25-100 mg/m(2)) 2 days before RIT. Factors assessed by univariate (and some multivariate) analysis included age, race, body size, interval between initial diagnosis and RIT, interval between 2nd look surgery and RIT, (90)Y versus (177)Lu, MBq dose, paclitaxel dose, grade of tumor, extent of initial surgery, size of disease deposits prior to RIT, intensity of TAG reactivity, the addition of unlabeled antibody, and the development of human anti-mouse antibody and/or serum sickness after murine antibody. A statistically significant improvement in progression-free survival (p ≤ 0.05) was noted for less bulky disease and younger age. Administration of paclitaxel plus IFN, an immune response, and use of (90)Y showed a favorable nonsignificant trend. Dose escalation of radionuclide did not change risk of progression; thus, this therapy may have therapeutic efficacy at modest dose levels.

Unlabeled multi tumor marker detection system based on bioinitiated light addressable potentiometric sensor

Multi biomarkers' assays are of great significance in clinical diagnosis. A label-free multi tumor markers' parallel detection system was proposed based on a light addressable potentiometric sensor (LAPS). Arrayed LAPS chips with basic structure of Si(3)N(4)-SiO(2)-Si were prepared on silicon wafers, and the label-free parallel detection system for this component was developed with user friendly controlling interfaces. Then the l-3,4-dihydroxyphenyl-alanine (L-Dopa) hydrochloric solution was used to initiate the surface of LAPS. The L-Dopa immobilization state was investigated by the theoretical calculation. L-Dopa initiated LAPS' chip was biofunctionalized respectively by the antigens and antibodies of four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9) and Ferritin. Then unlabeled antibodies and antigens of these four biomarkers were detected by the proposed detection systems. Furthermore physical and measuring principles in this system were described, and qualitative understanding for experimental data were given. The measured response ranges were compared with their clinical cutoff values, and sensitivities were calculated by OriginLab. The results indicate that this bioinitiated LAPS based label-free detection system may offer a new choice for the realization of unlabeled multi tumor markers' clinical assay.

Pretargeted Molecular Imaging and Radioimmunotherapy

Pretargeting is a multi-step process that first has an unlabeled bispecific antibody (bsMAb) localize within a tumor by virtue of its anti-tumor binding site(s) before administering a small, fast-clearing radiolabeled compound that then attaches to the other portion of the bsMAb. The compound's rapid clearance significantly reduces radiation exposure outside of the tumor and its small size permits speedy delivery to the tumor, creating excellent tumor/nontumor ratios in less than 1 hour. Haptens that bind to an anti-hapten antibody, biotin that binds to streptavidin, or an oligonucleotide binding to a complementary oligonucleotide sequence have all been radiolabeled for use by pretargeting. This review will focus on a highly flexible anti-hapten bsMAb platform that has been used to target a variety of radionuclides to image (SPECT and PET) as well as treat tumors.

Noninvasive Detection of Breast Cancer Lymph Node Metastasis Using Carbonic Anhydrases IX and XII Targeted Imaging Probes

To develop targeted molecular imaging probes for the noninvasive detection of breast cancer lymph node metastasis. Six cell surface or secreted markers were identified by expression profiling and from the literature as being highly expressed in breast cancer lymph node metastases. Two of these markers were cell surface carbonic anhydrase isozymes (CAIX and/or CAXII) and were validated for protein expression by immunohistochemistry of patient tissue samples on a breast cancer tissue microarray containing 47 normal breast tissue samples, 42 ductal carcinoma in situ, 43 invasive ductal carcinomas without metastasis, 46 invasive ductal carcinomas with metastasis, and 49 lymph node macrometastases of breast carcinoma. Targeted probes were developed by conjugation of CAIX- and CAXII-specific monoclonal antibodies to a near-infrared fluorescent dye. Together, these two markers were expressed in 100% of the lymph node metastases surveyed. Selectivity of the imaging probes were confirmed by intravenous injection into nude mice-bearing mammary fat pad tumors of marker-expressing cells and nonexpressing cells or by preinjection of unlabeled antibody. Imaging of lymph node metastases showed that peritumorally injected probes detected nodes harboring metastatic tumor cells. As few as 1,000 cells were detected, as determined by implanting, under ultrasound guidance, a range in number of CAIX- and CAXII-expressing cells into the axillary lymph nodes. These imaging probes have potential for noninvasive staging of breast cancer in the clinic and elimination of unneeded surgery, which is costly and associated with morbidities.

Role of Thiol Isomerase ERp5 in Thrombus Formation

Abstract 370Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures:No relevant conflicts of interest to declare.

Epratuzumab with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy in patients with previously untreated diffuse large B-cell lymphoma

AbstractApproximately 60% of patients with diffuse large B-cell non-Hodgkin lymphoma (DLBCL) are curable with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemoimmunotherapy. Epratuzumab (E) is an unlabeled anti-CD22 monoclonal antibody with efficacy in relapsed DLBCL. This phase 2 trial tested the safety and efficacy of combining E with R-CHOP (ER-CHOP) in untreated DLBCL. A secondary aim was to assess the efficacy of interim positron emission tomography (PET) to predict outcome in DLBCL. Standard R-CHOP with the addition of E 360 mg/m2 intravenously was administered for 6 cycles. A total of 107 patients were enrolled in the study. Toxicity was similar to standard R-CHOP. Overall response rate in the 81 eligible patients was 96% (74% CR/CRu) by computed tomography scan and 88% by PET. By intention to treat analysis, at a median follow-up of 43 months, the event-free survival (EFS) and overall survival (OS) at 3 years in all 107 patients were 70% and 80%, respectively. Interim PET was not associated with EFS or OS. Comparison with a cohort of 215 patients who were treated with R-CHOP showed an improved EFS in the ER-CHOP patients. ER-CHOP is well tolerated and results appear promising as a combination therapy. This study was registered at www.clinicaltrials.gov as #NCT00301821.

Label-free Detection of Surface Markers on Stem Cells by Oblique-Incidence Reflectivity Difference Microscopy

Conventional fluorescence microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in often uncharacterized ways. Here we present a method using an oblique-incidence reflectivity difference (OI-RD) microscope for label-free, real-time detection of cell surface markers, and apply it to analysis of stage-specific embryonic antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces, and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies bind only to the surface of the stem cells and not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. Thus, the OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells; this information could be useful for determination of stem cell stages.