Chronic lymphocytic leukemia (CLL) is a malignancy of mature lymphocytes that is manifest by the progressive accumulation of transformed cells, mostly due to their decreased apoptosis. CD84 belongs to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors and has an as yet unknown function in normal B cells and CLL lymphocytes. We show that CD84 is over-expressed in CLL cells. Activation of cell surface CD84 initiates a signaling cascade, which enhances cell survival. Both immunoneutralization or blockade of CD84 induce cell death in vitro and in vivo. Thus, overexpression of CD84 from an early stage may be critical for the survival of CLL. These findings suggest novel therapeutic strategies based on the blockade of a CD84 dependent survival pathway.
Monoclonal antibody PAM4 has high specificity for pancreatic ductal adenocarcinoma (PDAC), as well as its precursor lesions, but is not reactive with normal and benign pancreatic tissues. Our purpose was to evaluate a PAM4-based serum-immunoassay alone, and in combination with the CA19-9 assay, for the detection of PDAC with particular attention to early-stage disease.
Sera from patients with confirmed PDAC (N=298), other cancers (N=99), benign disease of the pancreas (N=120), and healthy adults (N=79) were evaluated by specific enzyme-immunoassay for concentration of PAM4 and CA19-9 antigen levels by blinded analyses. All tests for statistical significance were two-sided.
Overall sensitivity for PAM4 detection of PDAC was 76%, with 64% of stage-1 patients also identified. The detection rate was considerably higher (85%) for advanced disease. The assay showed high specificity compared to benign pancreatic disease (85%), with a positive likelihood ratio (+LR) of 4.93. CA19-9 provided an overall sensitivity of 77%, and was positive in 58% of patients with stage-1 disease; however, specificity was significantly lower for CA19-9 (68%) with a +LR=2.85 (P=0.026 compared to PAM4). Importantly, a combined PAM4/CA19-9 assay demonstrated an improved sensitivity (84%) for overall detection of PDAC without significant loss of specificity (82%), as compared to either arm alone.
The PAM4-immunoassay identified approximately two-thirds of stage-1 PDAC patients with high discriminatory power with respect to benign, non-neoplastic pancreatic disease. These results provide a rationale for testing patient groups considered at high-risk for PDAC with a combined PAM4/CA19-9 biomarker assay for detection of early-stage PDAC.
pancreatic cancer; pancreatitis; diagnosis; early detection; PAM4; CA19-9
Corticosteroids are widely used for the treatment of B-cell malignancies, including non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74+ B-cell malignancies and explored its effect against the disease.
The targeting efficiency of milatuzumab-targeted liposomes to CD74+ cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexa-methasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo.
Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone-targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo.
These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies.
Objective. To evaluate health-related quality of life (HRQOL) and corticosteroid use in patients with moderate to severely active SLE enrolled in two international, multicentre, randomized controlled trials of epratuzumab (ALLEVIATE-1 and -2) and a long-term extension study (SL0006).
Methods. Ninety ALLEVIATE patients (43% BILAG A, mean BILAG score 13.2) were randomized to receive 360 mg/m2 (n = 42) or 720 mg/m2 (n = 11) epratuzumab or placebo (n = 37), plus standard of care, in 12-week cycles. Corticosteroid use, patient and physician global assessments of disease activity (PtGA and PGA) and 36-item Medical Outcomes Survey Short Form (SF-36) results were recorded at baseline and every 4 weeks. Both trials were prematurely discontinued due to a drug supply interruption; patients followed for ≥6 months were analysed. Twenty-nine patients continued in SL0006, with interim analysis at a median exposure of 120 (range 13–184) weeks.
Results. At week 12, proportions of patients with a PGA ≥20% above baseline or with a PtGA improvement greater than or equal to the minimum clinically important difference were higher in the epratuzumab arms than the placebo arm. PGA and PtGA improvements were sustained but did not reach statistical significance. At week 24, mean cumulative corticosteroid doses with epratuzumab 360 and 720 mg/m2 were 1051 and 1973 mg less than placebo (P = 0.034 and 0.081, respectively). At week 48, SF-36 scores approached or exceeded US age- and gender-matched norms in five domains with the 360 mg/m2 treatment. Improvements were maintained in SL0006 over ∼2 years.
Conclusion. Epratuzumab treatment produced clinically meaningful and sustained improvements in PGA, PtGA and HRQOL and reductions in corticosteroid doses.
epratuzumab; CD22; ALLEVIATE; lupus; SLE; HRQOL; SF-36; corticosteroids; clinical trial; monoclonal antibody
Radiolabeling compounds with positron-emitting radionuclides often involves a time-consuming, customized process. Herein, we report a simple lyophilized kit formulation for labeling peptides with 18F, based on the aluminum-fluoride procedure. The prototype kit contains IMP485, a NODA (1,4,7-triazacyclononane-1,4-diacetate)-MPAA (methyl phenylacetic acid)-di-HSG (histamine-succinyl-glycine) hapten-peptide, [NODA-MPAA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2], used for pretargeting, but we also examined a similar kit formulation for a somatostatin-binding peptide [IMP466, NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl] bearing a NOTA ligand to determine if the benefits of using a kit can be extended to other AlF-binding peptides. The NODA-MPAA ligand forms a single stable complex with (AlF)2+ in high yields. In order to establish suitable conditions for a facile kit, the formulation was optimized for pH, peptide to Al3+ ratio, bulking agent, radioprotectant, and the buffer. For optimal labeling, the kit was reconstituted with an aqueous solution of 18F− and ethanol (1:1), heated at 100–110 °C for 15 min, and then simply and rapidly purified using one of two equally effective solid-phase extraction (SPE) methods. Al18F-IMP485 was isolated as a single isomer complex, in high yield (45–97%) and high specific activity (up to 223 GBq/μmol), within 20 min. The labeled product was stable in human serum at 37 °C for 4 h and in vivo, urine samples showed the intact product was eliminated. Tumor targeting of the Al18F-IMP485 in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody showed very low uptake (0.06% ± 0.02 ID/g) in bone, further illustrating its stability. At 1 h, pretargeted animals had high Al18F-IMP485 tumor uptake (28.1% ± 4.5 ID/g), with ratios of 9 ± 4, 123 ± 38, 110 ± 43 and 120 ± 108 for kidney, liver, blood and bone, respectively. Tumor uptake remained high at 3 h post-injection, with increased tumor/nontumor ratios. The NOTA-somatostatin-binding peptide also was fluorinated with good yield and high specific activity in the same kit formulation. However, yields were somewhat lower than those achieved with IMP485 containing the NODA-MPAA ligand, likely reflecting this ligand's superior binding properties over the simple NOTA. These studies indicate that 18F-labeled peptides can be reproducibly prepared as stable Al-F complexes with good radiochemical yield and high specific activity using a simple, one-step, lyophilized kit followed by a rapid purification by SPE that provides the 18F-peptide, ready for patient injection within 30 min.
bispecific antibody; fluorine-18; pretargeting; peptides; antibodies; molecular imaging; PET; somatostatin
Cell fusion in vitro has been used to study cancer, gene mapping and regulation, and the production of antibodies via hybridomas. However, in-vivo heterosynkaryon formation by cell-cell fusion has received less attention. This investigation describes the spontaneous fusion of a human glioblastoma with normal hamster cells after xenogeneic transplantation, resulting in malignant cells that express both human and hamster genes and gene products, and retention of glioblastoma traits with an enhanced ability to metastasize. Three of 7 human genes found showed translation of their proteins during serial propagation in vivo or in vitro for years; namely, CD74, CXCR4, and PLAGL2, each implicated with malignancy or glioblastoma. This supports the thesis that genetic hybridization of cancer and normal cells can transmit malignancy and also, as first described herein, regulatory genes involved in the tumor’s organotypic morphology. Evidence also is increasing that even cell-free human cancer DNA can induce malignancy and transfer genetic information to normal cells. Hence, we posit that the transfer of genetic information between tumor and stromal cells, whether by cell-cell fusion or other mechanisms, is implicated in the progression of malignancy, and may further define the crosstalk between cancer cells and their stromal neighbors.
cell fusion; cancer stroma; metastasis; gene transfer; glioblastoma
The type III interferons (IFNs), comprising IFN-λ1, IFN-λ2, and IFN-λ3, behave similarly to IFN-α in eliciting antiviral, antitumor, and immune-modulating activities. Due to their more restricted cellular targets, IFN-λs are attractive as potential alternatives to existing therapeutic regimens based on IFN-αs. We have applied the DOCK-AND-LOCK™ method to improve the anti-proliferative potency of IFN-λ1 up to 1,000-fold in targeted cancer cell lines by tethering stabilized Fab dimers, derived from hRS7 (humanized anti-Trop-2), hMN-15 (humanized anti-CEACAM6), hL243 (humanized anti-HLA-DR), and c225 (chimeric anti-EGFR), to IFN-λ1 site-specifically, resulting in novel immunocytokines designated (E1)-λ1, (15)-λ1, (C2)-λ1, and (c225)-λ1, respectively. Targeted delivery of IFN-λ1 via (15)-λ1 or (c225)-λ1 to respective antigen-expressing cells also significantly increased antiviral activity when compared with non-targeting (C2)-λ1, as demonstrated in human lung adenocarcinoma cell line A549 by (15)-λ1 against encephalomyocarditis virus (EC50 = 22.2 pM versus 223 pM), and in human hepatocarcinoma cell line Huh-7 by (c225)-λ1 against hepatitis C virus (EC50 = 0.56 pM versus 91.2 pM). These promising results, which are attributed to better localization and stronger binding of IFN-λ1 to antibody-targeted cells, together with the favorable pharmacokinetic profile of (E1)-λ1 in mice (T1/2 = 8.6 h), support further investigation of selective prototypes as potential antiviral and antitumor therapeutic agents.
Targeted agents are increasingly used for treating cancer and other diseases, but patients may need to be carefully selected to maximize the potential for therapeutic benefit. One way to select patients is to bind an imaging radionuclide to a targeting agent of interest, so that its uptake in specific sites of disease can be visualized by positron-emission tomography (PET) or single-photon emission computed tomography.
18F is the most commonly used radionuclide for PET imaging. Its half-life of approximately 2 h is suited for same-day imaging of many compounds that clear quickly from the body to allow visualization of uptake in the intended target. A significant impediment to its use, however, is the challenging coupling of 18F to a carbon atom of the targeting agent. Because fluorine binds to aluminum, we developed a procedure where the Al18F complex could be captured by a chelate, thereby greatly simplifying the way that imaging agents can be fluorinated for PET imaging. This article reviews our experience with this technology.
Radiofluorination; Fluorine-18; Peptides; PET; Molecular imaging; Review
Advanced or metastatic renal cell carcinoma (RCC) has a poor prognosis, because it is relatively resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-α2b alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only a modest improvement in clinical outcome. One observation made with mTOR inhibitors is that carcinomas can overcome these inhibitory effects by activating the insulin-like growth factor-I (IGF-I) signaling pathway. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. In this work, we evaluate the anti-tumor activity of two novel IGF-1R-targeting agents against renal cell carcinoma given alone or in combination with an mTOR inhibitor.
hR1 was linked by the DOCK-AND-LOCK™ (DNL™) method to four Fabs of hR1, generating Hex-hR1, or to four molecules of interferon-α2b, generating 1R-2b. Eight human RCC cell lines were screened for IGF-1R expression and sensitivity to treatment with hR1 in vitro. Synergy with an mTOR inhibitor, temsirolimus, was tested in a cell line (ACHN) with low sensitivity to hR1.
Hex-hR1 induced the down-regulation of IGF-IR at 10-fold lower concentrations compared to the parental hR1. Sensitivity to growth inhibition mediated by hR1 and Hex-hR1 treatments correlated with IGF-1R expression (higher expression was more sensitive). The potency of 1R-2b to inhibit the in vitro growth of RCC was also demonstrated in two human cell lines, ACHN and 786-O, with EC50–values of 63 and 48 pM, respectively. When combined with temsirolimus, a synergistic growth-inhibition with hR1, Hex-hR1, and 1R-2b was observed in ACHN cells at concentrations as low as 10 nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b.
Both Hex-hR1 and 1R-2b proved to be more potent than parental hR1 in inhibiting growth of RCC in vitro. Synergy was achieved when each of the three hR1-based agents was combined with temsirolimus, suggesting a new approach for treating RCC.
Dock-and-Lock; Renal cell carcinoma; Insulin-like growth factor-I receptor; Hex-hR1; 1R-2b; mTOR inhibitors
We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5–6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.
A new ([18F]AlF)2+-binding ligand that contains 1,4,7-triazacyclononane-1,4-diacetate (NODA) attached to a methyl phenylacetic acid group (MPA) was conjugated to N-(2-aminoethyl)maleimide (EM) to form NODA-MPAEM. The NODA-MPAEM was labeled with ([18F]AlF)2+ at 105 °C in 49–82% yield and conjugated at room temperature to an antibody Fab’ fragment in 69–80% yield (total time ~ 50 min) and with retention of immunoreactivity. These data indicate that the rapid and simple [18F]AlF-labeling method can be easily adapted for preparing heat-sensitive compounds with 18F quickly and in high yields.
aluminum fluoride; antibody; CEA; 18fluorine; radiolabeling; PET
The coordination chemistry of a new pentadentate bifunctional chelator (BFC), NODA-MPAA 1, containing the 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif with a methyl phenyl acetic acid (MPAA) backbone, and its ability to form stable Al18F-chelates, was investigated. The organofluoroaluminates were easily accessible from the reaction of 1 and AlF3. X-ray diffraction studies revealed aluminum at the center of a slightly distorted octahedron, with fluorine occupying one of the axial positions. The tert-butyl protected prochelator 7, which can be synthesized in one step, is useful for coupling to biomolecules on solid phase or in solution. High yield (55–89%) aqueous 18F-labeling was achieved in 10–15 minutes with a tumor-targeting peptide 4 covalently linked to 1. Defluorination was not observed for at least 4 h in human serum at 37 °C. These results demonstrate the facile application of Al18F chelation using BFC 1 as a versatile labeling method for radiofluorinating other heat-stable peptides for positron emission imaging.
The carcinoembryonic antigen (CEA) was visualized in vitro in tissue from patients with colorectal cancer with trivalent bispecific antibody TF2 and two hapten molecules, [67/68Ga]Ga-IMP461 and [67/68Ga]Ga-IMP485 by means of pretargeting. Colorectal cancer tissue samples obtained from surgery at Uppsala University Hospital, were frozen fresh and cryosectioned. The two hapten molecules comprising 1,4,7-triazacyclononanetriacetic acid chelate moiety (NOTA) were labeled with 67Ga or 68Ga. The autoradiography was conducted by incubating the tissue samples with the bispecific antibody TF2, followed by washing and incubation with one of the radiolabeled hapten molecules. After washing, drying and exposure to phosphor imager plates, the autoradiograms were analyzed and compared to standard histochemistry (hematoxylin-eosin). Pronounced binding was found in the tissue from colorectal cancer using the bispecific antibody TF2 and either of the haptens [67/68Ga]Ga-IMP461 and [67/68Ga]Ga-IMP485. Distinct binding was also detected in the epithelium of most samples of neighboring tissue, taken at a minimum of 10 cm from the site of the tumor. It is concluded that pretargeting CEA with the bispecific antibody TF2 followed by the addition of 67/68Ga-labeled hapten is extremely sensitive for visualizing this marker for colorectal cancer. This methodology is therefore a very specific complement to other histochemical techniques in the diagnosis of biopsies or in samples taken from surgery. Use of the pretargeting technique in vivo may also be an advance in diagnosing patients with colorectal cancer, either using 67Ga and SPECT or 68Ga and PET.
Autoradiography; carcinoembryonic antigen; CEA; colorectal cancer; Ga-67; Ga-68; pretargeting
Targeting of radionuclides with antibodies, or radioimmunotherapy, has been an active field of research spanning nearly 50 years, evolving with advancing technologies in molecular biology and chemistry, and with many important preclinical and clinical studies illustrating the benefits, but also the challenges, which all forms of targeted therapies face. There are currently two radiolabeled antibodies approved for the treatment of non-Hodgkin lymphoma, but radioimmunotherapy of solid tumors remains a challenge. Novel antibody constructs, focusing on treatment of localized and minimal disease, and pretargeting are all promising new approaches that are currently under investigation.
bispecific antibody; cancer; monoclonal antibody; pretargeting; radioimmunotherapy; radionuclide
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.
bispecific antibody; cancer detection; pretargeting; radioimmunodetection; radioimmunotherapy.
Pancreatic adenocarcinoma is an almost universally lethal disease, in large part, due to our inability to detect early-stage disease. Monoclonal antibody PAM4 is reactive with a unique biomarker expressed by greater than 85% of pancreatic adenocarcinomas. In this report, we examined the ability of a PAM4-based immunoassay to detect early-stage disease.
The PAM4-based immunoassay was used to quantitate antigen in the serum of healthy volunteers (N=19), patients with known pancreatic adenocarcinoma (N=68), and patients with a primary diagnosis of chronic pancreatitis (N=29).
Sensitivity for detection of pancreatic adenocarcinoma was 82%, with a false-positive rate of 5% for healthy controls. Patients with advanced disease had significantly higher antigen levels than those with early-stage disease (P<0.01), with a diagnostic sensitivity of 91%, 86%, and 62% for stage 3/4 advanced disease, stage-2, and stage-1, respectively. We also evaluated chronic pancreatitis sera, finding 38% positive for antigen; however, this was discordant with immunohistochemical findings that suggest the PAM4-antigen is not produced by inflamed pancreatic tissue. Furthermore, several of the serum-positive pancreatitis patients, for whom tissue specimens were available for pathological interpretation, had evidence of neoplastic precursor lesions.
These results suggest the use of the PAM4-serum assay to detect early-stage pancreatic adenocarcinoma, and that positive levels of PAM4-antigen are not derived from inflamed pancreatic tissues, but rather may provide evidence of subclinical pancreatic neoplasia.
The ability to detect pancreatic adenocarcinoma at an early stage could provide for early therapeutic intervention with potentially improved patient outcomes.
pancreatic adenocarcinoma; early detection; immunoassay; biomarker; mucin
We recently reported a facile method based on the chelation of [18F]aluminum fluoride (Al18F) by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Here, we present a further optimization of the 18F labeling of NOTA-octreotide (IMP466). Octreotide was conjugated with the NOTA chelate and was labeled with 18F in a two-step, one-pot method. The labeling procedure was optimized with regard to the labeling buffer, ionic strength, peptide concentration, and temperature. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of 18F-IMP466 was studied in AR42J tumor-bearing mice. In addition, microPET/CT images were acquired. IMP466 was labeled with Al18F in a single step with 97% yield in the presence of 80% (v/v) acetonitrile or ethanol. The labeled product was purified by HPLC to remove unlabeled peptide and unbound Al18F. The radiolabeling, including purification, was performed for 45 min. Specific activities of 48,000 GBq/mmol could be obtained. 18F-IMP466 showed a high tumor uptake and excellent tumor-to-blood ratios at 2 h post-injection. In addition, the low bone uptake indicated that the Al18F–NOTA complex was stable in vivo. PET/CT scans revealed excellent tumor delineation and specific accumulation in the tumor. Uptake in receptor-negative organs was low. NOTA-octreotide could be labeled with 18F in quantitative yields using a rapid two-step, one-pot, method. The compound was stable in vivo and showed rapid accretion in SSTR2-receptor-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds such as RGD peptides, GRPR-binding peptides, and Affibody molecules with 18F.
Octreotide; Radiofluorination; NOTA; Peptide; PET; Aluminum fluoride
We reported previously the feasibility to radiolabel peptides with fluorine-18 (18F) using a rapid, one-pot, method that first mixes 18F− with Al3+, and then binds the (Al18F)2+ complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al18F NOTA complexes were stable in vitro in human serum and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100°C and the peptides could be labeled in as little as five minutes. The IMP467 peptide could be labeled up to 115 GBq/μmol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.
bispecific antibody; fluorine-18; pretargeting; molecular imaging; PET
The hallmark of chronic lymphocytic leukemia (CLL) is the relentless accumulation of mature lymphocytes, mostly due to their decreased apoptosis. CD74 was recently shown to serve as a survival receptor on CLL cells. In this study, we show that stimulation of CD74 with its natural ligand, migration inhibitory factor, initiates a signaling cascade that results in upregulation of TAp63, which directly regulates CLL survival. In addition, TAp63 expression elevates the expression of the integrin VLA-4, particularly during the advanced stage of the disease. Blocking of CD74, TAp63, or VLA-4 inhibits the in vivo homing of CLL cells to the bone marrow (BM). Thus, CD74 and its target genes TAp63 and VLA-4 facilitate migration of CLL cells back to the BM, where they interact with the supportive BM environment that rescues them from apoptosis. These results could form the basis of novel therapeutic strategies aimed at blocking homing of CLL cells in their return to the BM and attenuating their survival.
The selective delivery of therapeutic radionuclides is a promising approach for treating cancer. Antibody-targeted radionuclides are of particular interest, with two products approved for the treatment of certain forms of non-Hodgkin lymphoma. However, for many other cancers, radioimmunotherapy (RAIT) has been ineffective, being limited by prolonged exposure to the highly radiosensitive bone marrow. An alternative approach, known as pretargeting, separates radionuclide from the antibody, allowing the radiation to be delivered on a small molecule that can quickly and efficiently migrate into the tumor, and then rapidly clear from the body with minimal retention in tissues. Several pretargeting methods have been developed that differ in the way they selectively capture the radionuclide. This review focuses on the development of a novel form of bispecific monoclonal antibody (bsMAb) pretargeting that uses a unique radiolabeled hapten-peptide system that can be modified to bind a number of therapeutic and imaging radionuclides. Together with a specialized recombinant humanized bsMAb prepared with by a technique known as the Dock-and-Lock (DNL) method, this pretargeting procedure has been examined in a number of different animal models, showing a high level of sensitivity and specificity for localizing tumors, and improved efficacy with less hematologic toxicity associated with directly radiolabeled IgG. The bsMAb is a tri-Fab structure, having 2 binding arms for the tumor antigen and one capable of binding a hapten-peptide. Preclinical studies were preformed to support the clinical use of a bsMAb (TF2) and a hapten-peptide bearing a single DOTA moiety (IMP-288). A Phase 0 trial found an 131I-TF2 that targets carcinoembryonic antigen (CEA) was stable in vivo, quickly clears from the blood, and localizes known tumors. The first-in-patient pretargeting experience with the 111In-IMP-288 also observed rapid clearance and low tissue (kidney) retention, as well as localization of tumors, providing initial promising evidence for developing these materials for radioimmunotherapy.
bispecific antibody; carcinoembryonic antigen; pretargeting; radioimmunotherapy
18F-Fluorodeoxyglucose (18F-FDG) is the most common molecular imaging agent in oncology, with a high sensitivity and specificity for detecting a number of cancers. Antibodies could enhance specificity; therefore, procedures were developed for radiolabeling a small (∼1.5 kD) hapten-peptide with 68Ga or 18F to compare their specificity to 18F-FDG for detecting tumors using a pretargeting procedure. Mice were implanted with carcinoembryonic (CEA; CEACAM5)-expressing LS174T human colonic tumors, a CEA-negative tumor, or an inflammation was induced in thigh muscle. A bispecific monoclonal (bsMAb) anti-CEA × anti-hapten antibody was given to mice, and 16 h later, 5 MBq of 68Ga- or 18F-labeled hapten-peptides were administered intravenously. Within 1 h, tissues showed high and specific targeting of the 68Ga-IMP-288, with 10.7 ± 3.6% ID/g uptake in the tumor and very low uptake in normal tissues (e.g., tumor/blood 69.9 ± 32.3), in a CEA-negative tumor (0.35 ± 0.35% ID/g), and inflamed muscle (0.72 ± 0.20% ID/g). 18F-FDG localized efficiently in the tumor (7.42 ± 0.20% ID/g), but also in the inflamed muscle (4.07 ± 1.13% ID/g) and in a number of normal tissues; thus, pretargeted 68Ga-IMP-288 provided better specificity and sensitivity. PET/CT images reinforced the improved specificity of the pretargeting method. 18F-labeled IMP-449 distributed similarly in the tumor and normal tissues as the 68Ga-labeled IMP-288, indicating that either radiolabeled hapten-peptide could be used. Thus, pretargeted immunoPET performs exceptionally well with short-lived radionuclides, and is a highly sensitive procedure that is more specific than 18F-FDG-PET.
PET; pretargeting; bispecific antibody; gallium-68; fluorine-18; colorectal neoplasms; mice
Several methods have been developed to label peptides with fluorine-18. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of [18F]aluminum fluoride (“Al18F”) by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). The method is characterized by labeling NOTA-octreotide (IMP466) with 18F.
Octreotide was conjugated with the NOTA chelate and was labeled with 18F in a two-step, one-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of 18F-IMP466 was studied in AR42J tumor-bearing mice and compared to that of 68Ga-labeled IMP466. In addition, microPET/CT images were acquired.
IMP466 was labeled with “Al18F” in a single step with 50% yield. The labeled product was purified by HPLC to remove unbound “Al18F” and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol and the peptide was stable in serum for 4 h at 37° C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, HEPES and MES buffer and was optimal in sodium acetate buffer. The apparent IC50 of the 19F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h p.i. showed a high tumor uptake of 18F-IMP466 (28.3 ± 5.2 %ID/g, tumor-to-blood ratio: 300 ± 90), which could be blocked by an excess of unlabeled peptide (8.6 ± 0.7%ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of 68Ga-IMP466 was similar to that of 18F-IMP466. 18F-IMP466 was stable in vivo, since bone uptake was only 0.4 ± 0.2 %ID/g, whereas free “Al18F” accumulated rapidly in the bone (36.9 ± 5.0 %ID/g at 2 h p.i.). MicroPET/CT scans showed excellent tumor delineation and high preferential accumulation in the tumor.
NOTA-octreotide could be labeled rapidly and efficiently with 18F using a two-step, one-pot, method. The compound was stable in vivo and showed rapid accretion in SSTR2-receptor expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds with 18F.
octreotide; radiofluorination; NOTA; peptide; PET; aluminum fluoride
Radioimmunotherapy of non-Hodgkin lymphoma consists of a 90Y- or 131I-labeled murine anti-CD20 IgG, but both agents also include a substantial dose of unlabeled anti-CD20 IgG given immediately before the radioconjugate to reduce its uptake in the spleen (primary normal B-cell antigen sink); this extends its plasma half-life, and improves tumor visualization. Thus, these treatments combine an effective anti-CD20 radioconjugate with an unconjugated anti-CD20 antibody that is also therapeutically active, but the large anti-CD20 IgG pre-dose (~900 mg) may diminish the tumor localization of the radioimmunoconjugate (e.g., 10–35 mg). We have examined alternative approaches that enhance radionuclide targeting and improve anti-tumor responses. One uses a 90Y-labeled anti-CD22 IgG (epratuzumab) combined with an antibody therapy regimen of a humanized anti-CD20 IgG (veltuzumab). Pretargeted radionuclide therapy using a trivalent, humanized, recombinant bispecific anti-CD20 antibody with a 90Y-hapten-peptide is another highly effective method that is also less toxic than directly radiolabeled IgG. Finally, all approaches benefit from the addition of a consolidation-dosing regimen of the anti-CD20 IgG antibody. This article reviews these various options and discusses how some fundamental changes could potentially enhance the response and duration from radionuclide-targeted therapy.
CD20; CD22; non-Hodgkin lymphoma; pretargeting; radioimmunotherapy