Conditional ablation of astroglial CCL2 suppresses CNS accumulation of M1 macrophages and preserves axons in mice with MOG peptide EAE.

Current multiple sclerosis (MS) therapies only partially prevent chronically worsening neurological deficits, which are largely attributable to progressive loss of CNS axons. Prior studies of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG peptide), a model of MS, documented continued axon loss for months after acute CNS inflammatory infiltrates had subsided, and massive astroglial induction of CCL2 (MCP-1), a chemokine for CCR2(+) monocytes. We now report that conditional deletion of astroglial CCL2 significantly decreases CNS accumulation of classically activated (M1) monocyte-derived macrophages and microglial expression of M1 markers during the initial CNS inflammatory phase of MOG peptide EAE, reduces the acute and long-term severity of clinical deficits and slows the progression of spinal cord axon loss. In addition, lack of astroglial-derived CCL2 results in increased accumulation of Th17 cells within the CNS in these mice, but also in greater confinement of CD4(+) lymphocytes to CNS perivascular spaces. These findings suggest that therapies designed to inhibit astroglial CCL2-driven trafficking of monocyte-derived macrophages to the CNS during acute MS exacerbations have the potential to significantly reduce CNS axon loss and slow progression of neurological deficits.

Pyramidal neurons are generated from oligodendroglial progenitor cells in adult piriform cortex.

Previous studies have shown that oligodendroglial progenitor cells (OPCs) can give rise to neurons in vitro and in perinatal cerebral cortex in vivo. We now report that OPCs in adult murine piriform cortex express low levels of doublecortin, a marker for migratory and immature neurons. Additionally, these OPCs express Sox2, a neural stem cell marker, and Pax6, a transcription factor characteristic of progenitors for cortical glutamatergic neurons. Genetic fate-mapping by means of an inducible Cre-LoxP recombination system proved that these OPCs differentiate into pyramidal glutamatergic neurons in piriform cortex. Several lines of evidence indicated that these newly formed neurons became functionally integrated into the cortical neuronal network. Our data suggest that NG2(+)/PDGFRα(+) proteolipid protein promoter-expressing progenitors generate pyramidal glutamatergic neurons within normal adult piriform cortex.

Pharmacokinetic characterization in xenografted mice of a series of first-generation mimics for HLA-DR antibody, Lym-1, as carrier molecules to image and treat lymphoma.

UNLABELLED: Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (<2 kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab. METHODS: A lysine-polyethylene glycol (PEG) backbone was used to synthetically link 2 of the following ligands: deoxycholate, 5-leuenkephalin, triiodothyronine, thyronine, dabsyl-L-valine, and N-benzoyl-L-arginyl-4-amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelate attached to the linker. These SHALs have been assessed for their selectivity in binding to HLA-DR10-expressing cells and for their pharmacokinetics and tissue biodistribution in mice. Biotinylated versions of these SHALs discriminated cell lines positive for HLA-DR10 expression with near-nanomolar affinity. The DOTA versions of 4 SHALs were labeled with (111)In for pharmacokinetic studies in mice with HLA-DR10-expressing malignant Raji xenografts. RESULTS: The bidentate, biotinylated, and DOTA-SHALs were synthesized in high-purity, multimilligram amounts. Mean radiochemical and product yields and purities were 90%, 75%, and 90% at mean specific activities of 3.9 MBq/microg (105 microCi/microg) for the (111)In-labeled SHALs. As expected, rapid blood clearance and tumor targeting were observed. The pharmacokinetics of the SHALs was influenced by the component ligands. Biliary clearance, kidney localization, and serum receptor binding contributed to less favorable tumor targeting. CONCLUSION: A series of SHALs was readily synthesized in multimilligram amounts and showed the expected selective binding in vitro. Better selection of the SHAL components should provide second-generation SHALs with improved properties to fulfill the substantial potential of these novel molecular carriers for targeting.

Thermal dosimetry predictive of efficacy of 111In-ChL6 nanoparticle AMF--induced thermoablative therapy for human breast cancer in mice.

UNLABELLED: Antibody (mAb)-linked iron oxide nanoparticles (bioprobes) provide the opportunity to develop tumor specific thermal therapy (Rx) for metastatic cancer when inductively heated by an externally applied alternating magnetic field (AMF). To evaluate the potential of this Rx, in vivo tumor targeting, efficacy, and predictive radionuclide-based heat dosimetry were studied using (111)In-ChL6 bioprobes (ChL6 is chimeric L6) in a human breast cancer xenograft model. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 (DOTA is dodecanetetraacetic acid) was conjugated to polyethylene glycol-iron oxide-impregnated dextran 20-nm particles and purified as (111)In-bioprobes. (111)In doses of 740-1,110 kBq (20-30 muCi) (2.2 mg of bioprobes) were injected intravenously into mice bearing HBT3477 human breast cancer xenografts. Pharmacokinetic (PK) data were obtained at 1, 2, 3, and 5 d. AMF was delivered 72 h after bioprobe injection at amplitudes of 1,410 (113 kA/m), 1,300 (104 kA/m), and 700 (56 kA/m) oersteds (Oe) at 30%, 60%, and 90% "on" time (duty), respectively, and at 1,050 Oe (84 kA/m) at 50% and 70% duty over the 20-min treatment. Treated and control mice were monitored for 90 d. Tumor total heat dose (THD) from activated tumor bioprobes was calculated for each Rx group using (111)In-bioprobe tumor concentration and premeasured particle heat response to AMF amplitudes. Tumor growth delay was analyzed by Wilcoxon rank sum comparison of time to double, triple, and quintuple tumor volume in each group, and all groups were compared with the controls. RESULTS: Mean tumor concentration of (111)In-bioprobes at 48 h was 14 +/- 2 percentage injected dose per gram; this concentration 24 h before AMF treatment was used to calculate THD. No particle-related toxicity was observed. Toxicity was observed at the highest AMF amplitude-duty combination of 1,300 Oe and 60% over 20 min; 6 of 10 mice died acutely. Tumor growth delay occurred in all of the other groups, correlated with heat dose and, except for the lowest heat dose group, was statistically significant when compared with the untreated group. Electron microscopy showed (111)In-bioprobes on tumor cells and cell death by necrosis at 24 and 48 h after AMF. CONCLUSION: mAb-guided bioprobes (iron oxide nanoparticles) effectively targeted human breast cancer xenografts in mice. THD, calculated using empirically observed (111)In-bioprobe tumor concentration and in vitro nanoparticle heat induction by AMF, correlated with tumor growth delay.

Development of tumor targeting bioprobes ((111)In-chimeric L6 monoclonal antibody nanoparticles) for alternating magnetic field cancer therapy.

OBJECTIVES: (111)In-chimeric L6 (ChL6) monoclonal antibody (mAb)-linked iron oxide nanoparticle (bioprobes) pharmacokinetics, tumor uptake, and the therapeutic effect of inductively heating these bioprobes by externally applied alternating magnetic field (AMF) were studied in athymic mice bearing human breast cancer HBT 3477 xenografts. Tumor cell radioimmunotargeting of the bioprobes and therapeutic and toxic responses were determined. METHODS: Using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, (111)In-7,10-tetra-azacyclododecane-N, N',N'',N'''-tetraacetic acid-ChL6 was conjugated to the carboxylated polyethylene glycol on dextran-coated iron oxide 20 nm particles, one to two mAbs per nanoparticle. After magnetic purification and sterile filtration, pharmacokinetics, histopathology, and AMF/bioprobe therapy were done using (111)In-ChL6 bioprobe doses (20 ng/2.2 mg ChL6/ bioprobe), i.v. with 50 microg ChL6 in athymic mice bearing HBT 3477; a 153 kHz AMF was given 72 hours postinjection for therapy with amplitudes of 1,300, 1,000, or 700 Oe. Weights, blood counts, and tumor size were monitored and compared with control mice receiving nothing, or AMF or bioprobes alone. RESULTS: (111)In-ChL6 bioprobe binding in vitro to HBT 3477 cells was 50% to 70% of that of (111)In-ChL6. At 48 hours, tumor, lung, kidney, and marrow uptakes of the (111)In-ChL6 bioprobes were not different from that observed in prior studies of (111)In-ChL6. Significant therapeutic responses from AMF/bioprobe therapy were shown with up to eight times longer mean time to quintuple tumor volume with therapy compared with no treatment (P = 0.0013). Toxicity was only seen in the 1,300 Oe AMF cohort, with 4 of 12 immediate deaths and skin erythema. Electron micrographs showed bioprobes on the surfaces of the HBT 3477 cells of excised tumors and tumor necrosis 24 hours after AMF/bioprobe therapy. CONCLUSION: This study shows that mAb-conjugated nanoparticles (bioprobes), when given i.v., escape into the extravascular space and bind to cancer cell membrane antigen, so that bioprobes can be used in concert with externally applied AMF to deliver thermoablative cancer therapy.

Does paclitaxel (Taxol) given after (111)In-labeled monoclonal antibodies increase tumor-cumulated activity in epithelial cancers?

PURPOSE: Paclitaxel synergized radiolabeled monoclonal antibodies, enhancing therapeutic effect in studies in mice with human xenografts. Paclitaxel was also observed to increase tumor uptake in imaging studies of (111)In-DOTA-Gly3Phe-m170 in patients with breast and prostate cancers. Further evaluations of tissue-cumulated activities, therapeutic indices, and pharmacokinetics were done using data for patients with breast and prostate cancer and for mice with human breast cancer xenografts. EXPERIMENTAL DESIGN: In radioimmunotherapy trials, 12 patients with breast or prostate cancer were given two imaging doses (5 mCi each) of (111)In-DOTA-Gly3Phe-m170 1 week apart. Five of these patients were given a single dose of paclitaxel i.v. (75 mg/m2) 2 days after the second dose of (111)In. In a subsequent study, athymic mice with human breast cancer xenografts were given (111)In-DOTA-Gly3Phe-ChL6 alone, or in combination with daily paclitaxel i.p. (300 microg) one or more times. Pharmacokinetics were studied for at least 6 days in patients and 5 days in mice. Cumulated activities were determined for tumors and normal tissues. RESULTS: Tumor-cumulated activity for every patient in the paclitaxel-treated group increased for the second dose of (111)In-DOTA-Gly3Phe-m170. The median ratio of cumulated activities in tumors for imaging dose 2 to those for dose 1 was 1.0 (0.8-1.3) in patients that were not given paclitaxel and 1.3 (1.2-1.4) in patients given paclitaxel. Normal tissue-cumulated activities were not different for the two doses. Mice given paclitaxel 1 day after (111)In-DOTA-Gly3Phe-ChL6 also showed an increase in tumor-cumulated activity, 22.9 (+/- 1.3) versus 19.4 (+/- 3.3) microCi h/g/microCi (P = 0.05). Cumulated activities of normal tissues were similar for all groups of mice. CONCLUSIONS: Paclitaxel given 1 to 2 days after (111)In-DOTA-Gly3Phe-monoclonal antibody increased the tumor-cumulated activity in patients and in mice with epithelial cancers and did not alter cumulated activities in normal tissues.

Implantation of different malignant human cell lines in an athymic mouse does not alter success and growth rates of either xenograft.

PURPOSE: Human xenografts in athymic mice are frequently used as preclinical models of cancer to investigate the targeting of drugs. In order to distinguish specific from nonspecific targeting of the xenograft, the mice can be implanted with different malignant cell lines. We studied in xenograft success and growth rates after implantation of human lymphoma and breast cancer cells to begin an assessment of the validity of this approach for distinguishing specific from nonspecific targeting. Investigations were undertaken to determine the effect of two different cell-line xenografts, and prior radiation needed for one of the xenografts, on implantation success and growth rates. EXPERIMENTAL DESIGN: Female athymic mice were given 4 Gy of external beam radiation 4 days prior to subcutaneous (s.c.) abdominal implantation of 6 x 10(6) Raji human lymphoma cells. One week later, 3 x 10(6) hamster blood transfusion (HBT) 3477 human breast cancer cells were implanted s.c. in a contralateral abdominal site. Xenografts were evaluated frequently thereafter. Xenograft success and growth rates were compared to those observed in "historical" control groups, wherein only a single xenograft of each type was implanted. RESULTS: Raji xenografts developed from 73.7% of the implantations, and 100% of the HBT 3477 xenografts were successful in the experimental group. The "historical" Raji xenograft success rate was 74.1% (+/-9.3%), and the "historical" HBT 3477 xenografts success rate was 99.0% (+/-1.1%). HBT 3477 xenografts did not affect the growth rate of the Raji xenografts, and the mean doubling time for the experimental Raji xenografts was 6.3 days (+/-4.5 days), compared to the "historical" control group mean of 5.1 days (+/-3.9 days; p = 0.2). Similarly, the growth rates for the HBT 3477 xenografts were not affected by the Raji xenografts and the pre-radiation needed for this model. Mean doubling time for HBT 3477 xenografts in the presence of Raji xenografts was 9.2 days (+/-17.6 days), compared to a doubling time of 1.4 days (+/-15.2 days; p = 0.55 and 0.94 studies 1 and 2, respectively). Mean HBT 3477 xenograft doubling time for the "historical" control group was 4.4 days (+/-6.0 days). CONCLUSIONS: Implantation of Raji xenografts and HBT 3477 breast cancer xenografts in the same mouse did not affect xenograft success or growth rates, even when whole-body radiation of 4 Gy was used to promote Raji xenografts. These observations are not intended to imply an absence of differences in other biological parameters in this sytem or to encourage extrapolation of the conclusions indiscriminately to other preclinical models. Contrarily, our aim was to encourage other investigators to further validate these frequently used approaches.

Preclinical evaluation of cathepsin-degradable peptide linkers for radioimmunoconjugates.

PURPOSE: Immunoglobulins are catabolized in the hepatocytes, primarily by cathepsins. The liver becomes the likely dose-limiting tissue for radiometals, like (90)Y, in radioimmunoconjugates (RICs) used for radioimmunotherapy in combination with bone marrow support. To assess whether in vitro cathepsin-degradable peptide linkers between the chelated radiometal and the antibody decreased hepatic radiation dose, cumulated activity was used as a surrogate for radiation dose. EXPERIMENTAL DESIGN: Four different cathepsin-degradable peptides used to link 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-chelated (111)In to two different monoclonal antibodies were studied in athymic mouse models of human breast cancer or lymphoma. Measured concentrations of activity during 5 days were used to reflect pharmacokinetic behavior for normal tissues and tumor. With the use of linear regression to fit a monoexponential decay function, cumulated activities in the liver and xenografts were calculated. RESULTS: The pharmacokinetic behavior of the cathepsin-degradable peptide-linked RICs was similar to that for the 2-iminothiolane (2IT) nondegradable linked RICs except for the liver. The liver cumulated activities of peptide-linked RICs were significantly decreased from those of the corresponding 2IT-linked RICs, varying between reductions of 59 and 68%. Cumulated activities of peptide-linked RICs in the xenografts were as great as those of 2IT RICs, so that the therapeutic indices (tumor: liver cumulated activity ratios) were substantially better for cathepsin-degradable peptide-linked RICs. CONCLUSIONS: Cathepsin-degradable peptides used to link chelated radiometals to antibodies reduce liver radiation dose and improve the therapeutic index for radioimmunotherapy given in combination with bone marrow support.

Anti-CD22 ligand-blocking antibody HB22.7 has independent lymphomacidal properties and augments the efficacy of 90Y-DOTA-peptide-Lym-1 in lymphoma xenografts.

CD22 is a membrane glycophosphoprotein found on nearly all healthy B-lymphocytes and most B-cell lymphomas. Recent in vitro studies have identified several anti-CD22 monoclonal antibodies (mAbs) that block the interaction of CD22 with its ligand. One of these mAbs, HB22.7, has been shown to effectively induce apoptosis in several B-cell lymphoma cell lines. Lymphoma xenograft studies with Raji-xenograft mice were used to assess the toxicity and efficacy of HB22.7 alone and with combined modality immunotherapy (CMIT) with yttrium (90)Y-DOTA-peptide-Lym-1 radioimmunotherapy (RIT). The effect of the sequence of these agents on the combined treatment was assessed by administering HB22.7 24 hours before, simultaneously with, or 24 hours after RIT. Within the groups treated with RIT alone or with RIT and HB22.7 (CMIT), the reduction in tumor volume was the greatest when HB22.7 was administered simultaneously with and 24 hours after RIT, and in the RIT treatment groups, this translated into the greatest overall response and survival, respectively. Overall survival rates at the end of the 84-day CMIT trial were 67% and 50% in the groups treated with HB22.7 simultaneously and 24 hours after RIT, respectively. This compared favorably with the untreated and the RIT alone groups, which had survival rates of 38% and 43% at the end of the trial. Surprisingly, when compared with untreated controls and all other treatment groups, the greatest cure and overall survival rates were observed in the group treated with HB22.7 alone, with 47% cured and 76% surviving at the end of the 84-day trial. RIT clearance was not affected by treatment with HB22.7. When compared with RIT alone, there was no significant additional hematologic (white blood cell, red blood cell, or platelet count) toxicity when HB22.7 was added to RIT. Nonhematologic toxicity (assessed as change in body weight) was also unchanged when HB22.7 was added to RIT. Thus the anti-CD22 ligand-blocking antibody HB22.7 has independent lymphomacidal properties and augments the efficacy of (90)Y-DOTA-peptide-Lym-1 in lymphoma xenografts without significant toxicity.

Cilengitide targeting of alpha(v)beta(3) integrin receptor synergizes with radioimmunotherapy to increase efficacy and apoptosis in breast cancer xenografts.

Although metastatic breast cancer is responsive to radioimmunotherapy (RIT), a systemic targeted radiation modality, complete and permanent remissions are not typical with single-modality treatment. Antiangiogenic agents, which target normal, proliferating endothelial cells, have the potential to provide relatively nontoxic continuous inhibition of tumor growth by blocking new blood vessel growth and may synergize with RIT to increase efficacy. This study was designed to determine whether, and how, the cyclic Arg-Gly-Asp peptide Cilengitide (EMD 121974), which targets the alpha(v)beta(3) integrin receptor expressed on neovasculature, could increase systemic RIT efficacy of therapy in a human breast cancer tumor model having mutant p53 and expressing bcl-2. HBT 3477 breast cancer tumor response in nude mice was compared between groups of untreated mice (n = 24), Cilengitide-treated mice (n = 18), RIT (200-260 mu Ci (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-peptide ChL6; n = 46), and combined modality RIT (CMRIT) using RIT and six doses of Cilengitide (250 microg/dose; n = 41). Tumor size, survival, body weight, and blood counts were monitored for efficacy and toxicity of therapy. To clarify the mechanism of synergistic effect, tumors were evaluated at selected time points through 6 days for apoptosis, proliferation, and microvessel density. Cilengitide alone did not alter tumor growth when compared with untreated mice, but CMRIT with Cilengitide increased efficacy of treatment, with the cure rate for mice that received 260 mu Ci RIT increasing from 15 to 53% (P = 0.011). Lower-dose RIT (200 mu Ci) combined with Cilengitide resulted in less increase in cures (36 compared with 25% for RIT alone; P = 0.514). Combined analysis for high- and low-dose groups demonstrated increased efficacy of CMRIT (P = 0.020). Analysis of tumors from CMRIT mice indicated significantly increased apoptosis of tumor and endothelial cells 5 days after RIT compared with tumors from mice given RIT alone. Proliferation was decreased in CMRIT tumors compared with RIT tumors at 6 days (ANOVA, P < 0.05). Microvessel density in tumors from RIT and CMRIT mice was not different. No increased toxicity attributable to Cilengitide was observed based upon pooled blood sample and no statistical increase in mortality. In conclusion, CMRIT, combining Cilengitide and RIT, significantly increased the efficacy of therapy and increased apoptosis compared with single-modality therapy with either agent, in an aggressive, well-studied breast cancer model. The enhanced therapeutic synergy is of particular note, having been achieved without additional toxicity.

Combined modality radioimmunotherapy. Promise and peril.

BACKGROUND: Single-agent radioimmunotherapy (RIT), although potentially useful for slowing solid tumor growth, has not been effective in curing aggressive tumors, such as breast cancer. These cancers typically have p53 mutations and are less susceptible to apoptosis, the apparent mechanism of cell death from low dose-rate radiation. Thus, synergistic or combined modality radioimmunotherapy (CMRIT) agents are needed to increase radiosensitivity for therapeutic enhancement without additive toxicity. METHODS: To assess synergy in CMRIT in a breast cancer xenograft model, we evaluated RGD peptide EMD 121974, an inhibitor of alpha(v)beta(3) integrin; paclitaxel, an antimicrotubule; IMC-C225, a monoclonal antibody to epidermal growth factor receptor (EGFR); and bcl-2 antisense oligonucleotide G3139. Groups of mice received (90)Y-DOTA-ChL6 in combination with each agent. Tumor size, survival, and blood counts were monitored for efficacy and toxicity. Immunopathologic evaluation of apoptosis was performed at selected time points after RIT and RIT + RGD CMRIT. RESULTS: CMRIT with RGD peptide increased apoptosis and resulted in 57% cures, compared with 0% cures with RIT alone. CMRIT with paclitaxel after RIT increased cures to 88%, compared with 25% cures with RIT before paclitaxel administration. CMRIT with IMC-C225 resulted in up to 20% cures if given before RIT. A time-dependent increase in toxicity was observed with IMC-C225 after RIT. CMRIT with bcl-2 antisense G3139 resulted in no cures and an increased rate of regrowth compared with RIT alone. CONCLUSIONS: Some combined modality therapies resulted in higher numbers of cures, while others decreased cures and responses and increased toxicity compared with RIT alone. These results support the potential for CMRIT but illustrate the complexity of predicting the efficacy and toxicity and the importance of the relationship between dose and sequence of administration.

Combined modality radioimmunotherapy for human prostate cancer xenografts with taxanes and 90yttrium-DOTA-peptide-ChL6.

BACKGROUND: Therapy for prostate cancer in the PC3 tumor-nude mouse model with 90yttrium-(90Y)-DOTA-peptide-ChL6 (5.55 MBq;150 microCi) has resulted in durable responses. To make radioimmunotherapy (RIT) more effective, the radiation-enhancing drugs Taxol (paclitaxel) and Taxotere (docetaxel) were tested for synergy with 90Y-DOTA-peptide-ChL6. METHODS: Nude mice bearing human prostate cancer PC3 xenografts were treated with 90Y-DOTA-peptide-ChL6 (2.78 MBq; 75 microCi) and after 24 hr, paclitaxel (300 or 600 microg), or docetaxel (300 microg). Tumor size, survival, blood counts, and pharmacokinetics were monitored to assess efficacy and toxicity. RESULTS: Docetaxel plus RIT had a 67% cure rate, whereas no mice were cured among the RIT alone, chemotherapy alone, or untreated controls. Paclitaxel (600 microg) plus RIT produced a 100% response rate with 20% cures. Average tumor volume was reduced to a greater degree in the combined modality radioimmunotherapy (CMRIT) groups compared to controls and the anti-tumor response was durable. Myelotoxicity in the combined modality groups (RIT plus paclitaxel or RIT plus docetaxel) were similar to groups receiving the same dose of RIT alone. CONCLUSION: In the PC3-tumor nude mouse model, addition of paclitaxel or docetaxel to 90Y-DOTA-peptide-ChL6, in doses clinically achievable in humans, provided therapeutic synergy without increased or excessive toxicity.