Hematologic Tumor Models Research Articles

Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

A phase I, open-label, dose-escalation study of the novel oral proteasome inhibitor (PI) ONX 0912 in patients with advanced refractory or recurrent solid tumors.

3075 Background: ONX 0912 is a potent, irreversible, orally bioavailable peptide epoxyketone PI. It is a structural analog of carfilzomib (CFZ), an intravenously administered PI in ph III trials. O...

A first-in-human phase I study of JNJ-26481585, a novel oral histone deacetylase inhibitor (HDACi), in patients with advanced cancer with evidence of target modulation and antitumor activity.

3024 Background: JNJ-26481585 is a potent, hydroxamate, pan-HDACi with broad activity in solid and hematologic tumor models. Methods: JNJ-26481585 was administered orally, once daily (QD) in 3-weekly cycles to advanced cancer patients (pts) using a 2-stage accelerated titration design. As QD dosing was poorly tolerated, three intermittent schedules were explored: 1) 4 days on, 3 days off; 2) every Mon, Wed, Fri (MWF); and 3) every Mon, Thurs (M-Th). Primary objectives were safety, maximum tolerated dose (MTD), and pharmacokinetics (PK). Secondary objectives were pharmacodynamic (PD) effects in normal and tumor tissues (acetylated Histone H3, Ki67), PK food effect and anti-tumor activity. Results: 73 patients (37 M, median age 56 yr) received JNJ-26481585. Number of pts, dose-limiting toxicities (DLTs), and MTD for each cohort and schedule are shown in the Table. Common grade 1-2 toxicities were fatigue, anorexia, and nausea. PK was dose-proportional with high volume of distribution and AUC (range 11-20 h.ng/mL) consistent with the exposure required for activity in xenograft models. There was no food effect. PD data showed increased acetylated Histone H3 in hair follicles, skin biopsies and tumor, confirming proof of mechanism. A partial response lasting 5 months (mo) was seen in one patient with melanoma. Stable disease was seen in 8 pts (duration 4 – 10.5 mo). Conclusions: The adverse event profile of JNJ-26481585 is comparable to other HDACi. Intermittent administration is better tolerated than QD. The recommended dose for phase II trials is 12 mg on the MWF schedule, based on tolerability, anti-tumor activity, PK predictions, and PD activity. Schedule. 2 mg 4 mg 6 mg 8 mg 10 mg 12 mg 15 mg 16 mg 19 mg QD N=2 N=2 N=6 1 DLT (bilirubin↑) N=8 1 DLT (NSVT*) 0 MTD - N=2 2 DLTs (NSVT*, fatigue) - - - 4 days on, 3 days off - - N=3 N=3 N=6 0..DLTs MTD N=7 2 DLTs (NSVT*, infection) - - - MWF - - N=3 N=3 - N=7 1 DLT (hypertension + troponin↑) MTD - N=6 0..DLTs*** - M-Th - - - N=3 - N=3 N=6 0..DLTs MTD - N=3 2 DLTs (T-wave inversion, SVT**) *Nonsustained ventricular tachycardia. **Supraventricular tachycardia. ***Palpitations + ventricular extrasystoles in 2 pts in cycle 2.

Potent and sustained inhibition of HIF-1α and downstream genes by a polyethyleneglycol-SN38 conjugate, EZN-2208, results in anti-angiogenic effects

Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFβ1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208’s superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-011-9209-1) contains supplementary material, which is available to authorized users.

Marizomib, a Proteasome Inhibitor for All Seasons: Preclinical Profile and a Framework for Clinical Trials

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique β-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

Phase I study of ACE-041, a novel inhibitor of vascular maturation, in patients with advanced solid tumors or relapsed/refractory multiple myeloma.

TPS179 Background: Activin receptor-like kinase 1 (ALK1) is a type I receptor belonging to the TGFβ superfamily of proteins that is transiently expressed on arterial endothelial cells during development and in response to injury or disease (Seki et al. Circ. Res. 2003). ALK1 signaling plays a pivotal role in the maturation phase of angiogenesis and is therefore critical to vasculature development. ACE-041 is a soluble receptor fusion protein consisting of the ligand-binding extracellular domain of human ALK1 linked to a human IgG1-Fc region. ACE-041 binds with high affinity to BMP9 and BMP10 but does not bind to either TGFβ1, 2 or 3; VEGF or bFGF. By inhibiting their interaction with ALK1, ACE-041 blocks ALK1-mediated signaling, prohibiting vascular endothelial cell maturation and further vascular development. Unlike other angiogenesis inhibitors that act on the initial proliferative phase, ACE-041 blocks a critical and convergent step in the maturation phase of angiogenesis. In vitro and in vivo studies demonstrated that ACE-041 inhibited vascular maturation regardless of which growth factor triggered the proliferative phase (e.g., VEGF, bFGF). ACE-041 has also been shown to inhibit tumor vascular development, resulting in potent antitumor activity in a variety of solid and hematological tumor models (Mitchell et al., MCT 2010, in press). Methods: A phase I study of ACE-041 is ongoing in the United States to evaluate the safety, tolerability, pharmacokinetics, and anti- tumor activity of ACE-041 in patients with advanced solid tumors or relapsed/refractory multiple myeloma. Cohorts of 3 to 6 patients will be enrolled at escalating dose levels and will receive ACE-041 subcutaneously once every 3 weeks for a total of 4 doses or until disease progression. Dose escalation is determined following review of safety data through 21 days from at least 3 patients at each dose level. The safety and efficacy data from this phase I first-in-human study of ACE-041 will be evaluated to plan additional monotherapy and combination studies in patients with solid tumors and hematologic malignancies. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Acceleron Pharma Acceleron Pharma Acceleron Pharma Acceleron Pharma

The Bcl-2 inhibitor ABT-263 enhances the response of multiple chemotherapeutic regimens in hematologic tumors in vivo

This study was designed to test the ability of the Bcl-2 family inhibitor ABT-263 to potentiate commonly used chemotherapeutic agents and regimens in hematologic tumor models. Models of B-cell lymphoma and multiple myeloma were tested in vitro and in vivo with ABT-263 in combination with standard chemotherapeutic regimens, including VAP, CHOP and R-CHOP, as well as single cytotoxic agents including etoposide, rituximab, bortezomib and cyclophosphamide. Alterations in Bcl-2 family member expression patterns were analyzed to define mechanisms of potentiation. ABT-263 was additive with etoposide, vincristine and VAP in vitro in the diffuse large B-cell lymphoma line (DLBCL) DoHH-2, while rituximab potentiated its activity in SuDHL-4. Bortezomib strongly synergized with ABT-263 in the mantle cell lymphoma line Granta 519. Treatment of DoHH-2 with etoposide was associated with an increase in Puma expression, while bortezomib upregulated Noxa expression in Granta 519. Combination of ABT-263 with cytotoxic agents demonstrated superior tumor growth inhibition and delay in multiple models versus cytotoxic therapy alone, along with significant improvements in tumor response rates. Inhibition of the Bcl-2 family of proteins by ABT-263 enhances the cytotoxicity of multiple chemotherapeutics in hematologic tumors and represents a promising addition to the therapeutic arsenal for treatment of these diseases.

Clinical trial of NPI-0052 in advanced malignancies including lymphoma and leukemia (advanced malignancies arm)

3582 Background: The novel structure (non-peptide based) of NPI-0052 (NPI) appears to lead to unique proteasome inhibition (PI), toxicology and signal transduction profiles. Preclinical research suggests improvements in therapeutic ratio and activity in hematologic and solid tumor models, leading to clinical trials in patients with myeloma, lymphomas, leukemias, and solid tumors. Methods: Patients with solid tumor, lymphoma or leukemia diagnoses without standard treatment options were treated with IV NPI on Days 1, 8 and 15 of 28-day cycles in a 3+3 design dose escalation to a Recommended Phase 2 Dose (RP2D). Enrollment then began in 10 patient lymphoma and CLL RP2D cohorts. PI (D1 and D15) and PK (D1 and D15) were assayed. Results: 30 patients were treated at doses ranging from 0.1 mg/m2 to 0.9 mg/m2. 0.7 mg/m2 was selected as the RP2D secondary to DLT of transient “hallucinations” (visual imprints when eyes closed) and dizziness/unsteady gait at 0.9 mg/m2. At the RP2D fatigue, parosmia/dysgeusia, transient peri-infusion site pain and lymphopenia were commonly ascribed to NPI. At the RP2D PK data showed: half-life = 31 ± 28 min; AUCtotal = 270 ± 219 ng/mL*min; Cmax = 33.4 ± 34.2 ng/mL; clearance = 7.17 ± 0.40 L/min; volume of distribution (Vz) = 223.3 ± 229.7 L. PI was assayed in blood, indicating a dose:response relationship with mean inhibition of chymotrypsin-like activity up to of 88% Day 1 and 100% Day 15, and inhibition of caspase-like and trypsin-like activity of up to 51% and 72%. PI remained between doses in whole blood (RBC), but recovered between doses in PBMC. Stable disease was induced in 31% of patients, including one each with mantle cell, Hodgkin's lymphoma, follicular lymphoma, sarcoma, prostate carcinoma, and two with melanoma. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range to an MTD, while producing a toxicity profile that is tolerable and dissimilar to bortezomib in spite of reaching higher PI levels. These data indicate potential for a greater range of uses than other proteasome inhibitors and lead to additional studies being initiated in hematologic malignancies and solid tumors alone and in combination. [Table: see text]

A novel liposomal formulation of flavopiridol

Flavopiridol has shown promising activities in hematologic and solid tumor models, as well as in clinical trials in chronic lymphocytic leukemia patients. Flavopiridol has relatively low solubility and high plasma protein-binding. To address these issues and to provide an alternative strategy to achieve clinical efficacy, we encapsulated flavopiridol into a liposomal carrier and characterized its physicochemical and pharmacokinetic properties. The liposomes, comprising hydrogenated soy phosphatidylcholine (HSPC), cholesterol and poly (ethylene glycol) 2000-distearoyl phosphatidylethanolamine (PEG-DSPE), were prepared by polycarbonate membrane extrusion and then loaded with flavopiridol by a pH-gradient driven remote loading procedure. The liposomes had a mean diameter of 120.7 nm and a flavopiridol entrapment efficiency of 70.4%. Pharmacokinetic study in mice after i.v. bolus injection showed that the liposomal flavopiridol had an increased elimination phase half-life ( T 1/2β, 339.7 min vs. 57.0 min), decreased clearance (CL, 0.012 L/min vs. 0.036 L/min), and increased area under the plasma concentration–time curve (AUC, 10.8 min μmol/L vs. 3.4 min μmol/L) compared to the free drug. This indicates a significant and potentially beneficial change in flavopiridol pharmacokinetics for the liposomal formulation. Further preclinical studies are warranted to define the toxicity and therapeutic efficacy of this novel formulation.

Phase I study of the novel proteasome inhibitor NPI-0052 in patients with lymphoma and solid tumors

3574 Background: NPI-0052 is a novel proteasome inhibitor that produces prolonged inhibition of all three catalytic activities of the 20S proteasome. Preclinical data suggest NPI-0052 may demonstrate an improved therapeutic ratio, with activity as a single agent or in combination in hematologic (myeloma, lymphoma, leukemia) and solid tumor models. Clinical trials are being conducted in patients with myeloma, lymphomas, leukemias and solid tumors. Methods: In this first-in-human study cohorts of 3 or more patients with solid tumor or lymphoma were treated with NPI-0052 administered weekly, for 3 weeks in 4-week cycles using a 3+3 design dose escalation. The dose of NPI-0052 was escalated in 50–100% increments dependent on observed adverse events (AE). In addition to regular safety monitoring, proteasome inhibition (PI) (baseline, D1–2, D8, D15–17, D22 and D29) and PK (D1 and D15) were assayed in blood. A Recommended Phase 2 Dose (RP2D) cohort of up to 20 patients will be enrolled at the RP2D. Results: 23 patients have been treated over a 20-fold dose range (0.0125 mg/m2 to 0.25 mg/m2) for up to 11 cycles without reaching an MTD. The AE profile has been unremarkable at the highest dose level assessed. Sixteen patients (73%) had no > Grade 1 drug-related AE. Preliminary PK data indicate an T1/2 of approximately 3–4 minutes, with clearance at 8–21 mL/min and Vz of 44–99L. PI has been assayed in blood, indicating a dose:response relationship with inhibition of up to 64% observed. No responses have been confirmed, however, 6 patients have had stable disease for at least 2 cycles, including one with melanoma (4 months), one with colorectal carcinoma (3 months), one with hepatocellular carcinoma (6 months), two with adenoid cystic carcinoma (4 months each), and one with cervical carcinoma (11 months). This patient (cervical carcinoma) underwent four intra-patient dose escalations, with increased PI observed at the higher dose assessed. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects at doses below the MTD. Combination studies are being initiated in solid tumor diagnoses with other cytotoxic / targeted agents. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Nereus Nereus Nereus

Pharmacokinetic and Pharmacodynamic Properties of SB1317, a Potent and Orally Active Multi-Kinase Inhibitor.

Key physicochemical properties such as solubility, lipophilicity (logD7.4, logP) and pKa (the negative log of the acid dissociation constant) can be used to predict protein binding, tissue distribution, and gastrointestinal (GI) absorption. More recent application of computational methods allows even better prediction of compound oral bioavailability from in vitro and/or in silico properties. During the lead optimization process in our multi-kinase inhibitor program a good correlation between physicochemical properties like logD and solubility (determined experimentally at pH 7) versus AUC(0-inf) (Area Under the Curve) was observed for a series of kinase inhibitors. A calculated logD*solubility value between 200 to 400 predicted a reasonably high oral AUC(0-inf) in mouse PharmacoKinetic (PK) studies for this series of compounds, with correlation coefficient of >0.9. The correlation of logD*solubility vs. AUC(0-inf) enabled us to prioritize compounds for PK studies. SB1317, a novel pyrimidine derivative, is an orally active multi-kinase inhibitor that evolved as a lead drug candidate from in vitro and in vivo pharmacokinetic studies. SB1317 is a highly permeable compound, does not undergo active P-glycoprotein transport, and has a solubility of 75 μg/ml. It is metabolically stable in dog and human liver microsomes and unstable in rodent liver microsomes. No P450 inhibition was observed up to 10 μM towards CYP3A4, CYP1A2, CYP2C9 and CYP2C19. In vivo pharmacokinetics in nude mice and Beagle dogs resulted in %F of 12 and 37% respectively. Tumor pharmacokinetics of SB1317 shows increased exposure in tumor than in plasma with a AUC(0-t) tumor/plasma ratio of 3. Excellent oral dose proportionality (10, 20 and 40mg/kg) was observed in both plasma and tumor. SB1317 has exhibited uniformly high plasma protein binding (>99%) across the preclinical species and human. It is the free or unbound portion of the compound in the plasma or at the tissue level that would yield effective anti-tumor activity. (AUC0-inf)unbound/GI50 and Cmax-unbound/GI50 were used as PK/PD surrogates for the measure of SB1317 efficacy. A factor of SB1317 unbound AUC (0-inf)/GI50 above 0.5 results in significant pharmacodynamic (PD) effects in hematological tumor models (MV4-11 and HL-60 engraft model). Linear pharmacokinetic extrapolation from preclinical species to human was determined by allometric scaling (predicted human % F = 36). PK/PD relationships established for SB1317 in preclinical species could form the basis of a PK/PD-driven clinical development program.

Translational In Vivo and In Vitro Studies in Patients (pts) with Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML) and Myeloproliferative Disease (MPD) Treated with MK-0457 (MK), a Novel Aurora Kinase, Flt3, JAK2, and Bcr-Abl Inhibitor.

Introduction: MK-0457 (VX-680) is a small molecule kinase inhibitor of the Aurora kinases A/B, wild type and mutant (T315I) Bcr-Abl, Flt3, and Jak2 (V617F) with preclinical activity in solid and hematological tumor models. We report correlative laboratory studies from pts with AML, CML and MPD enrolled in a Phase I/II study with MK-0457, on a 5-day continuous intravenous infusion (CIV), dose escalation schedule given at 2 to 3 week intervals.Methods: Specimens from 6 MPD (all Jak2 (V617F) positive), 6 CML pts in accelerated/blastic phase (3/6 positive for T315I) and 3 AML pts with at least two study time points were assessed for apoptosis (AnnexinV (AV)/PI by FACS) and cell cycle (PI) changes in vivo. A CD34+ progenitor sub-population was studied. PBMC isolated prior to therapy were cultured in vitro with MK-0457, either alone or in co-culture with human, BM-derived mesenchymal stem cells (MSC). Concentration and time dependent apoptosis and cell cycle changes were determined.Results: At day 5, there was no significant induction of apoptosis in 5/6 MPD pts compared to baseline (BL) (mean for total cell population 37 vs 27%; gated myeloid subpopulation 37 vs 38%). All pts had a hematological response and, except one, a slight decrease in Jak2 (V617F) transcript levels. In vivo, CML pts showed a heterogeneous response. 3/5 pts had an increase (7, 98, 103 %) in AV/PI positive cells and 2/5 pts a decrease (−28, −74%) at day 5, one of which in lymphoid BC, exhibited delayed massive apoptosis at day 11 (470% increase from BL). The other pt without evidence of apoptosis induction achieved a hematological response with disappearance of the T315I clone, relapsing with high Bcr-Abl transcript levels thereafter. A pt with partial cytogenetic and molecular response had high BL apoptosis (50%) that remained high (58%) at 90 days when taken off study due to side effects (muscle cramps/pain). In vitro, a dose dependent rise in specific apoptosis was seen in MPD cells between 48–144 hrs, reaching statistical significance at 50–400 nM MK (p=0.017−0.008, two tailed t-test, unequal variance) and in CML cells at 24, 48, 72 hrs (mean increase at 400nM MK of 17, 14, 28%, respectively in CML, p≤0.05 for mean of all time points). In AML, 2/3 pts had increased numbers of AV/PI positive cells on day 5, declining to BL levels on day 11 in one pt. In vitro, AML specimens (from non-study pts) showed less pronounced apoptosis than MPD and CML cells, with similar effects in CD34 cells. Co-incubation with MSC protected cells of AML and MPD pts at all drug concentrations, reaching significance at ≥100nM in MPD pts (p=0.03). In cell line experiments, HL-60 and OCI-AML cells showed striking G2/M block, polyploidization and apoptosis.Conclusion: In preliminary experiments, MK-0457 induces apoptosis in AML and CML pts in vivo, shows a time and dose dependent increase of apoptosis in vitro in MPD, CML and AML pts cells and leads to accumulation in G2/M phase. Co-culture with human MSC protect cells from MPD and AML pts in vitro. The clinical trial is ongoing and molecular profiling studies are underway.

Improving the Delivery of Radionuclides for Imaging and Therapy of Cancer Using Pretargeting Methods

The article reviews the background and current status of pretargeting for cancer imaging and therapy with radionuclides. Pretargeting procedures were introduced approximately 20 years ago as an alternative to directly radiolabeled antibodies. Because they were multistep processes, they were met with resistance but have since progressed to simple and improved procedures that could become the next generation of imaging and therapy with radionuclides. The separation of the radiolabeled compound from the antibody-targeting agent affords pretargeting procedures considerable flexibility in the radiolabeling process, providing opportunities for molecular imaging using gamma- or positron-emitting radionuclides and a variety of beta- and alpha-emitting radionuclides of therapeutic applications. Pretargeting methods improve tumor/nontumor ratios, exceeding that achieved with directly radiolabeled Fab' fragments, particularly within just a few hours of the radionuclide injection. In addition, tumor uptake exceeds that of a Fab' fragment by as much as 10-fold, giving pretargeting a greatly enhanced sensitivity for imaging. Advances in molecular biology have led to the development of novel binding proteins that have further improved radionuclide delivery in these systems. Studies in a variety of hematologic and solid tumor models have shown advantages of pretargeting compared with directly radiolabeled IgG for therapy, and there are several clinical studies under way that are also showing promising results. Thus, the next generation of targeting agents will likely employ pretargeting approaches to optimize radionuclide delivery for a wide range of applications.