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.

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.

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.

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.

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.

Background

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.

Methods

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).

Results

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.

Conclusions

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.

Impact

The ability to detect pancreatic adenocarcinoma at an early stage could provide for early therapeutic intervention with potentially improved patient outcomes.

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.

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.

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.

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.

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.

Methods

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.

Results

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.

Conclusions

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.

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.

Summation

Antibodies are highly versatile proteins with the ability to be used to target diverse compounds, such as radionuclides for imaging and therapy, or drugs and toxins for therapy, but also can be used unconjugated to elicit therapeutically beneficial responses, usually with minimal toxicity. This update describes a new procedure for forming multivalent and/or multispecific proteins, known as the dock-and-lock (DNL) technique. Developed as a procedure for preparing bispecific antibodies capable of binding divalently to a tumor antigen and monovalently to a radiolabeled hapten-peptide for pretargeted imaging and therapy, this methodology has the flexibility to create a number of other biologic agents of therapeutic interest. A variety of constructs, based on anti-CD20 and CD22 antibodies, have been made, with results showing that multispecific antibodies have very different properties from the respective parental monospecific antibodies. The technique is not restricted to antibody combination, but other biologics, such as interferon-α2b, have been prepared. These types of constructs not only allow small biologics to be sustained in the blood longer, but also to be selectively targeted. Thus, DNL technology is a highly flexible platform that can be used to prepare many different types of agents that could further improve cancer detection and therapy.

CD74, a transmembrane glycoprotein that associates with MHC II, is an important chaperone that regulates antigen presentation for immune response. In addition, CD74 is the receptor for macrophage migration-inhibitory factor which, when bound to CD74, initiates survival pathways and cell proliferation. Formalin fixed, paraffin embedded clinical specimens were evaluated by immunohistochemical procedures for expression of CD74. Overall, expression of CD74 within gastrointestinal carcinomas showed a statistically greater expression than in the normal tissue counterparts (P<0.001 or better). CD74 expression was observed in 95% of pancreatic carcinomas with the majority of cases presenting a mostly intense, diffuse labeling pattern. The results suggested a trend towards greater expression within the higher grade carcinomas (P=0.06). Colorectal and gastric carcinomas gave similar results with 60% and 86%, respectively, positive for CD74 with an intense, diffuse staining pattern. We hypothesized that precursor lesions would express levels of CD74 as high, or higher, than their respective carcinomas, since activation of survival pathways would be of particular importance at the early stages of neoplastic development. For PanIN lesions there was greater expression of CD74 within higher grade, PanIN-3 lesions, whereas the colonic adenomas showed no such trend, but overall, a higher frequency and intensity of CD74 labeling than was observed within the colon carcinomas. These findings are supportive of a role for CD74 in the development and maintenance of gastrointestinal neo-plasia, and provide a rationale for development of therapeutic agents that are able to block CD74 function, specifically within the tumor cell.

Introduction

Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27negative B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.

Methods

Epratuzumab binding specificity and the surface expression of adhesion molecules (CD62L, β7 integrin and β1 integrin) after culture with epratuzumab was studied on B-cell subsets of SLE patients by flow cytometry. In addition, in vitro transwell migration assays were performed to analyze the effects of epratuzumab on migration towards different chemokines such as CXCL12, CXCL13 or to CXCR3 ligands, and to assess the functional consequences of altered adhesion molecule expression.

Results

Epratuzumab binding was considerably higher on B-cells relative to other cell types assessed. No binding of epratuzumab was observed on T-cells, while weak non-specific binding of epratuzumab on monocytes was noted. On B-cells, binding of epratuzumab was particularly enhanced on CD27negative B-cells compared to CD27positive B-cells, primarily related to a higher expression of CD22 on CD27negative B-cells. Moreover, epratuzumab binding led to a decrease in the cell surface expression of CD62L and β7 integrin, while the expression of β1 integrin was enhanced. The effects on the pattern of adhesion molecule expression observed with epratuzumab were principally confined to a fraction of the CD27negative B-cell subpopulation and were associated with enhanced spontaneous migration of B-cells. Furthermore, epratuzumab also enhanced the migration of CD27negative B-cells towards the chemokine CXCL12.

Conclusions

The current data suggest that epratuzumab has effects on the expression of the adhesion molecules CD62L, β7 integrin and β1 integrin as well as on migration towards CXCL12, primarily of CD27negative B-cells. Therefore, induced changes in migration appear to be part of the mechanism of action of epratuzumab and are consistent with the observation that CD27negative B-cells were found to be preferentially reduced in the peripheral blood under treatment.

Purpose

To improve the efficacy and reduce the gastrointestinal toxicity of the cancer prodrug, CPT-11, we have developed immunoconjugates of its active form, SN-38, and an anti-CEACAM5 antibody for targeted chemotherapy.

Experimental Design

SN-38 conjugates of the anti-CEACAM5 mAb, labetuzumab (hMN-14), varying in the nature of the cross-linker attachment at the drug’s 20-hydroxyl position, were evaluated in vitro, in metastatic and/or subcutaneous (sc) human colonic and pancreatic cancer xenografts in nude mice using appropriate controls, and in a CEACAM5-negative tumor model.

Results

A pilot study in a sc LS174T model of human colonic carcinoma established the relative effectiveness of different conjugates. In the lung metastatic model of GW-39 human colonic carcinoma in nude mice, therapy with two specific labetuzumab-SN-38 conjugates, using 0.25 mg SN-38 equivalent/kg, q4d×8, significantly extended median survival time (MST) versus controls (P <0.002). In an expanded evaluation in the sc LS174T xenograft model, specific SN-38 conjugates produced significant tumor growth control and increases in MST versus other controls, including CPT-11 at a 33-fold greater cumulative dose (P <0.01). An improvement was also observed in the therapy of a sc human pancreatic tumor xenograft. In a CEACAM5-negative systemic lymphoma xenograft, one labetuzumab-SN-38 conjugate examined was ineffective, while the conjugate specific for the tumor model produced 100% survival.

Conclusions

The promising labetuzumab-SN-38 conjugates developed showed selective therapeutic efficacy in human tumor models at nontoxic doses that were a fraction of the CPT-11 doses used.

Purpose

The humanized anti-CD74 monoclonal antibody, milatuzumab, is in clinical evaluation for the therapy of multiple myeloma (MM). The ability of milatuzumab to increase the efficacy of bortezomib, doxorubicin, and dexamethasone was examined in three human CD74+ MM cell lines, CAG, KMS11, KMS12-PE, and one CD74− MM cell line, OPM-2.

Experimental Design

Activity of milatuzumab as a monotherapy and combined with the drugs was evaluated by studying in vitro cytotoxicity, signaling and apoptotic pathways, and in vivo therapeutic activity in SCID mouse models of MM.

Results

Given as a monotherapy, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects in CD74+ cells in vitro. The combination of crosslinked milatuzumab with bortezomib, doxorubicin, or dexamethasone caused more growth inhibition than either crosslinked milatuzumab or drug alone, producing significant reductions in the IC50 of the drugs when combined. Efficacy of combined treatments was accompanied by increased levels of apoptosis measured by increases of activated caspase-3 and hypodiploid DNA. Both milatuzumab and bortezomib affect the NF-κB pathway in CAG MM cells, although in opposite direction, with milatuzumab inhibiting and bortezomib stimulating phosphorylated NF-κB. In CAG and KMS11 SCID-mouse xenograft models of disseminated MM, milatuzumab more than doubled median survival time, compared to up to a 33% increase in median survival with bortezomib, but no significant benefit with doxorubicin. Moreover, combining milatuzumab and bortezomib increased survival significantly compared to either treatment alone.

Conclusions

The therapeutic efficacies of bortezomib, doxorubicin, and dexamethasone are enhanced in MM cell lines when given in combination with milatuzumab, suggesting testing these combinations clinically.

Abstract

There are many reports that cross-linking antibodies (Abs) bound to the surface of B-lymphoma cells can induce apoptosis and/or cell death, especially with anti-CD20 Abs. This study was intended to extend our understanding of these effects. To determine if CD20 is a unique target in this respect, or whether Abs to other antigens would have similar effects, six Abs were tested, with and without cross-linking with a secondary Ab, on three target cell lines. We utilized assays that distinguish between apoptotic, dead, and viable cells. Two assays were used: Annexin V plus propidium iodide, and JC-1 plus SYTOX® green (Molecular Probes, Eugene, OR). Most of the Abs tested induced a low level of apoptosis and cell death in Ramos cells, but not in the other two cell lines (Raji and RL). In general, the level of toxicity was correlated with the level of antigen expression, with Abs to high-density antigens having the strongest effects. However, since the majority of Ramos cells continued to multiply, it is questionable whether toxicity at this level can provide a significant clinical benefit. Unexpectedly, there was also a population of cells that stained weakly with Annexin V. These cells were distinct from classical apoptotic cells, and appeared to belong to the viable cell population. In these cells, Annexin V stained the region of the Ab cap, in contrast to the ringed staining of classical apoptotic cells. In conclusion: 1) Low-level induction of apoptosis was not unique for anti-CD20 Abs, but occurred similarly with other Abs, and 2) results of Annexin V staining experiments may need to be reevaluated. Further studies are required to explain why Annexin V binding sites are exposed in the region of an Ab cap.

CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation. Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38 derivatives were evaluated in vitro for stability in buffer and human serum, and for antigen-binding and cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16 and 17 were found promising for further development.