Jaspreet Singh Jaggi

Tumor Targeting with Antibody-Functionalized, Radiolabeled Carbon Nanotubes

Single-walled carbon nanotubes (CNT) are mechanically robust graphene cylinders with a high aspect ratio that are comprised of sp2-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. We hypothesized that novel prototype nanostructures consisting of biologics, radionuclides, fluorochromes, and CNT could be synthesized and designed to target tumor cells. Methods: Tumor-targeting CNT constructs were synthesized from sidewall-functionalized, water-soluble CNT platforms by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radiometal-ion chelates, and fluorescent probes. The constructs were characterized spectroscopically, chromatographically, and electrophoretically. The specific reactivity of these constructs was evaluated in vitro by flow cytometry and cell-based immunoreactivity assays and in vivo using biodistribution in a murine xenograft model of lymphoma. Results: A soluble, reactive CNT platform was used as the starting point to build multifunctional constructs with appended antibody, metal-ion chelate, and fluorescent chromophore moieties to effect specific targeting, to carry and deliver a radiometal-ion, and to report location, respectively. These nanoconstructs were found to be specifically reactive with the human cancer cells they were designed to target in vivo in a model of disseminated human lymphoma and in vitro by flow cytometry and cell-based immunoreactivity assays versus appropriate controls. Conclusion: The key achievement in these studies was the selective targeting of tumor in vitro and in vivo by the use of specific antibodies appended to a soluble, nanoscale CNT construct. The ability to specifically target tumor with prototype-radiolabeled or fluorescent-labeled, antibody-appended CNT constructs was encouraging and suggested further investigation of CNT as a novel delivery platform.

PET Imaging of Soluble Yttrium-86-Labeled Carbon Nanotubes in Mice

Background The potential medical applications of nanomaterials are shaping the landscape of the nanobiotechnology field and driving it forward. A key factor in determining the suitability of these nanomaterials must be how they interface with biological systems. Single walled carbon nanotubes (CNT) are being investigated as platforms for the delivery of biological, radiological, and chemical payloads to target tissues. CNT are mechanically robust graphene cylinders comprised of sp2-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. In order to evaluate the potential usefulness of this CNT scaffold, we undertook an imaging study to determine the tissue biodistribution and pharmacokinetics of prototypical DOTA-functionalized CNT labeled with yttrium-86 and indium-111 (86Y-CNT and 111In-CNT, respectively) in a mouse model. Methodology and Principal Findings The 86Y-CNT construct was synthesized from amine-functionalized, water-soluble CNT by covalently attaching multiple copies of DOTA chelates and then radiolabeling with the positron-emitting metal-ion, yttrium-86. A gamma-emitting 111In-CNT construct was similarly prepared and purified. The constructs were characterized spectroscopically, microscopically, and chromatographically. The whole-body distribution and clearance of yttrium-86 was characterized at 3 and 24 hours post-injection using positron emission tomography (PET). The yttrium-86 cleared the blood within 3 hours and distributed predominantly to the kidneys, liver, spleen and bone. Although the activity that accumulated in the kidney cleared with time, the whole-body clearance was slow. Differential uptake in these target tissues was observed following intraveneous or intraperitoneal injection. Conclusions The whole-body PET images indicated that the major sites of accumulation of activity resulting from the administration of 86Y-CNT were the kidney, liver, spleen, and to a much less extent the bone. Blood clearance was rapid and could be beneficial in the use of short-lived radionuclides in diagnostic applications.

124 I-huA33 Antibody PET of Colorectal Cancer

Humanized A33 (huA33) is a promising monoclonal antibody that recognizes A33 antigen, which is present in more than 95% of colorectal cancers and in normal bowel. In this study, we took advantage of quantitative PET to evaluate 124I huA33 targeting, biodistribution, and safety in patients with colorectal cancer. We also determined the biodistribution of 124I-huA33 when a large dose of human intravenous IgG (IVIG) was administered to manipulate the Fc receptor or when 124I-huA33 was given via hepatic arterial infusion (HAI). Methods: We studied 25 patients with primary or metastatic colorectal cancer; 19 patients had surgical exploration or resection. Patients received a median of 343 MBq (44.4–396 MBq) and 10 mg of 124I-huA33. Nineteen patients received the antibody intravenously and 6 patients via HAI, and 5 patients also received IVIG. Results: Ten of 12 primary tumors were visualized in 11 patients. The median concentration in primary colon tumors was 0.016% injected dose per gram, compared with 0.004% in normal colon. The PET-based median ratio of hepatic tumor uptake to normal-liver uptake was 3.9 (range, 1.8–22.2). Quantitation using PET, compared with well counting of serum and tissue, showed little difference. Prominent uptake in bowel hindered tumor identification in some patients. Pharmacokinetics showed that patients receiving IVIG had a significantly shorter serum half-time (41.6 ± 14.0 h) than those without (65.2 ± 9.8 h). There were no differences in clearance rates among the intravenous group, IVIG group, and HAI group, nor was there any difference in serum area under the curve, maximum serum concentration, or volume of distribution. Weak titers of human–antihuman antibodies were observed in 6 of 25 patients. No acute side effects or significant toxicities were associated with huA33. Conclusion: Good localization of 124I-huA33 in colorectal cancer with no significant toxicity has been observed. PET-derived 124I concentrations agreed well with those obtained by well counting of surgically resected tissue and blood, confirming the quantitative accuracy of 124I-huA33 PET. The HAI route had no advantage over the intravenous route. No clinically significant changes in blood clearance were induced by IVIG.

Efforts to Control the Errant Products of a Targeted In vivo Generator

Alpha-particle immunotherapy by targeted alpha-emitters or alpha-emitting isotope generators is a novel form of extraordinarily potent cancer therapy. A major impediment to the clinical use of targeted actinium-225 (225Ac) in vivo generators may be the radiotoxicity of the systemically released daughter radionuclides. The daughters, especially bismuth-213 (213Bi), tend to accumulate in the kidneys. We tested the efficacy of various pharmacologic agents and the effect of tumor burden in altering the pharmacokinetics of the 225Ac daughters to modify their renal uptake. Pharmacologic treatments in animals were started before i.v. administration of the HuM195-225Ac generator. 225Ac, francium-221 (221Fr), and 213Bi biodistributions were calculated in each animal at different time points after 225Ac generator injection. Oral metal chelation with 2,3-dimercapto-1-propanesulfonic acid (DMPS) or meso-2,3-dimercaptosuccinic acid (DMSA) caused a significant reduction (P < 0.0001) in the renal 213Bi uptake; however, DMPS was more effective than DMSA (P < 0.001). The results with DMPS were also confirmed in a monkey model. The renal 213Bi and 221Fr activities were significantly reduced by furosemide and chlorothiazide treatment (P < 0.0001). The effect on renal 213Bi activity was further enhanced by the combination of DMPS with either chlorothiazide or furosemide (P < 0.0001). Competitive antagonism by bismuth subnitrate moderately reduced the renal uptake of 213Bi. The presence of a higher target-tumor burden significantly prevented the renal 213Bi accumulation (P = 0.003), which was further reduced by DMPS treatment (P < 0.0001). Metal chelation, diuresis with furosemide or chlorothiazide, and competitive metal blockade may be used as adjuvant therapies to modify the renal accumulation of 225Ac daughters.

Selective alpha-particle mediated depletion of tumor vasculature with vascular normalization.

Background Abnormal regulation of angiogenesis in tumors results in the formation of vessels that are necessary for tumor growth, but compromised in structure and function. Abnormal tumor vasculature impairs oxygen and drug delivery and results in radiotherapy and chemotherapy resistance, respectively. Alpha particles are extraordinarily potent, short-ranged radiations with geometry uniquely suitable for selectively killing neovasculature. Methodology and Principal Findings Actinium-225 (225Ac)-E4G10, an alpha-emitting antibody construct reactive with the unengaged form of vascular endothelial cadherin, is capable of potent, selective killing of tumor neovascular endothelium and late endothelial progenitors in bone-marrow and blood. No specific normal-tissue uptake of E4G10 was seen by imaging or post-mortem biodistribution studies in mice. In a mouse-model of prostatic carcinoma, 225Ac-E4G10 treatment resulted in inhibition of tumor growth, lower serum prostate specific antigen level and markedly prolonged survival, which was further enhanced by subsequent administration of paclitaxel. Immunohistochemistry revealed lower vessel density and enhanced tumor cell apoptosis in 225Ac-E4G10 treated tumors. Additionally, the residual tumor vasculature appeared normalized as evident by enhanced pericyte coverage following 225Ac-E4G10 therapy. However, no toxicity was observed in vascularized normal organs following 225Ac-E4G10 therapy. Conclusions The data suggest that alpha-particle immunotherapy to neovasculature, alone or in combination with sequential chemotherapy, is an effective approach to cancer therapy.

Influence of the linker on the biodistribution and catabolism of actinium-225 self-immolative tumor-targeted isotope generators.

Current limitations to applications of monoclonal antibody (mAb) targeted isotope generators in radioimmunotherapy include the low mAb labeling yields and the nonspecific radiation of normal tissues by nontargeted radioimmunoconjugates (RIC). Radiotoxicity occurs in normal organs that metabolize radiolabeled proteins and peptides, primarily liver and kidneys, or in radiosensitive organs with prolonged exposure to the isotope from the blood, such as the bone marrow. Actinium-225 nanogenerators also have the problem of released agar-emitting daughters. We developed two new bifunctional chelating agents (BCA) in order to address these issues. Thiol-maleimide conjugation chemistry was employed to increase the efficiency of the mAb radiolabelings by up to 8-fold. In addition, one bifunctional chelating agent incorporated a cleavable linker to alter the catabolism of the alpha-particle-emitting mAb conjugate. This linker was designed to be sensitive to cathepsins to allow release and clearance of the chelated radiometal after internalization of the radioimmunoconjugate into the cell. We compared the properties of the cleavable conjugate (mAb-DOTA-G3FC) to noncleavable constructs (mAb-DOTA-NCS and mAb-DOTA-SH). The cleavable RIC was able to release 80% of its radioactive payload when incubated with purified cathepsin B. The catabolism of the constructs mAb-DOTA-G3FC and mAb-DOTA-NCS was investigated in vitro and in vivo. RIC integrity was retained at 85% over a period of 136 h in mouse serum in vivo. Both conjugates were degraded over time inside HL-60 cells after internalization and in mouse liver in vivo. While we found that the rates of degradation of the two RICs in those conditions were similar, the amounts of the radiolabeled product residues were different. The cleavable mAb-DOTA-G3FC conjugate yielded a larger proportion of fragments below 6kDa in size in mouse liver in vivo after 12 h than the DOTA-NCS conjugate. Biodistribution studies in mice showed that the mAb-DOTA-G3FC construct yielded a higher liver dose and prolonged liver retention of radioactivity compared to the mAb-DOTA-NCS conjugate. The accumulation in the liver seemed to be in part caused by the maleimide functionalization of the antibody, since the noncleavable mAb-DOTA-SH maleimide-functionalized control conjugate displayed the same biodistribution pattern. These results provide an insight into the catabolism of RICs, by demonstrating that the release of the radioisotope from a RIC is not a sufficient condition to allow the radioactive moiety to clear from the body. The excretion mechanisms of radiolabeled fragments seem to constitute a major limiting step in the chain of events leading to their clearance.

Natural Killer Cells License Dendritic Cell Cross-Presentation of B Lymphoma Cell–Associated Antigens

Purpose: Presentation of exogenous antigen by MHC class I molecules, or cross-presentation, is a property of dendritic cells, which is considered crucial for the priming of cytotoxic T-cell response to tumor antigens. However, the precise mechanisms of this process are not fully understood. Experimental Design and Results: We show here in a human in vitro system, using B lymphoma cells as a tumor model, that the cross-presentation of cell-associated antigens to T cells by dendritic cells requires “help” from natural killer cells. When autologous dendritic cells that had taken up apoptotic B lymphoma cells and induced to a fully mature state were used to stimulate nonadherent cells of peripheral blood mononuclear cells from healthy donors, they induced strong cytotoxicity against B lymphoma cells in a HLA-A0201-restricted manner. The cells failed to induce cytotoxicity, however, when purified T cells were used as effector cells. Depletion of CD56+ cells, but not CD14+ or CD19+ cells, abrogated the cytotoxicity of nonadherent cells, showing that the help was provided by natural killer cells. Further, when natural killer cells were present in the cultures, a strong and persistent production of interleukin-18, but not interleukin-12 and interleukin-15, was observed. Blocking interleukin-18 significantly reduced the cytotoxicity of nonadherent cells against B lymphoma cells. Conclusions: These results suggest that capture of tumor cells and a full maturation status of dendritic cells are not sufficient to cross-prime CD8 T cells. Effective cross-priming requires further activation of dendritic cells by natural killer cells and an abundant production of interleukin-18, which, along with other yet undefined mechanisms, contribute to the generation of CTL response against B-cell lymphoma.