Exploration of a F(ab') Fragment as the Targeting Agent of α-Radiation Therapy: A Comparison of the Therapeutic Benefit of Intraperitoneal and Intravenous Administered Radioimmunotherapy.

Refinement of treatment regimens enlisting targeted α-radiation therapy (TAT) is an ongoing effort. Among the variables to consider are the target molecule, radionuclide, dosage, and administration route. The panitumumab F(ab') fragment targeting epidermal growth factor receptor tolerated modification with the TCMC chelate as well as radiolabeling with Pb or Pb.

Comparative studies on the therapeutic benefit of targeted α-particle radiation therapy for the treatment of disseminated intraperitoneal disease.

Identification of the appropriate combination of radionuclide, target and targeting vehicle is critical for successful radioimmunotherapy. For the treatment of disseminated peritoneal diseases such as pancreatic or ovarian cancer, α-emitting radionuclides have been proposed for targeted radiation therapy. This laboratory has taken a systematic approach investigating targeted α-radiation therapy, allowing comparisons to now be made between At, Th, Bi and Pb.

Targeted α-Particle Radiation Therapy of HER1-Positive Disseminated Intraperitoneal Disease: An Investigation of the Human Anti-EGFR Monoclonal Antibody, Panitumumab.

Identifying molecular targets and an appropriate targeting vehicle, i.e., monoclonal antibodies (mAb) and their various forms, for radioimmunotherapy (RIT) remains an active area of research.

Cell Killing Mechanisms and Impact on Gene Expression by Gemcitabine and 212Pb-Trastuzumab Treatment in a Disseminated i.p. Tumor Model.

In pre-clinical studies, combination therapy with gemcitabine and targeted radioimmunotherapy (RIT) using 212Pb-trastuzumab showed tremendous therapeutic potential in the LS-174T tumor xenograft model of disseminated intraperitoneal disease. To better understand the underlying molecular basis for the observed cell killing efficacy, gene expression profiling was performed after a 24 h exposure to 212Pb-trastuzumab upon gemcitabine (Gem) pre-treatment in this model. DNA damage response genes in tumors were quantified using a real time quantitative PCR array (qRT-PCR array) covering 84 genes.

Mechanisms of Cell Killing Response from Low Linear Energy Transfer (LET) Radiation Originating from (177)Lu Radioimmunotherapy Targeting Disseminated Intraperitoneal Tumor Xenografts.

Radiolabeled antibodies (mAbs) provide efficient tools for cancer therapy. The combination of low energy β(-)-emissions (500 keVmax; 130 keVave) along with a γ-emission for imaging makes (177)Lu (T1/2 = 6.7 day) a suitable radionuclide for radioimmunotherapy (RIT) of tumor burdens possibly too large to treat with α-particle radiation.

Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial.

The first-in-human phase 1 clinical radioimmunotherapy (RIT) trial with 212Pb-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane-trastuzumab (212Pb-TCMC-trastuzumab) was completed in October 2014 as a joint effort at the University of Alabama (UAB) and the University of California San Diego Moores Cancer Center. The preliminary reports indicate that after five dose-levels of intraperitoneally administered 212Pb-TCMC-trastuzumab, patients with carcinomatosis experienced minimal agent-related toxicity. This report presents the data accumulated to date on the stability of the clinical grade, produced according to current good manufacturing practices (cGMP), TCMC-trastuzumab conducted in support of that clinical trial.

Toxicological Studies of 212Pb Intravenously or Intraperitoneally Injected into Mice for a Phase 1 Trial.

Faced with the novelty of a 212Pb-labeled monoclonal antibody (mAb) for clinical translation, concerns were expressed by the Food and Drug Administration (FDA) regarding 212Pb prematurely released from the mAb-chelate conjugate. The objective of this study was to simulate the worst case scenario of such a failure. Groups of Balb/c mice (n = 9-20) were administered 212Pb by intraperitoneal (0.

Synthesis and characterization of gadolinium-Peptidomimetic complex as an αvβ3 integrin targeted MR contrast agent.

There is growing interest in small and rigid peptidomimetic αvβ3 integrin antagonists that are readily synthesized and characterized and amenable to physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC) was successfully conjugated to DOTA, complexed with Gd(III) and radiolabeled with (153)Gd. Radioassay results demonstrated specificity of the labeled conjugate by blocking ∼95% binding with the addition of a 50-fold molar excess of cold IAC to the reaction solution.

Evaluation of cetuximab as a candidate for targeted α-particle radiation therapy of HER1-positive disseminated intraperitoneal disease.

Although the epidermal growth factor receptor (EGFR), also known as HER1, has been studied for over a decade, it continues to be a molecule of great interest and focus of investigators for development of targeted therapies. The marketed monoclonal antibody cetuximab binds to HER1, and thus might serve as the basis for creation of imaging or therapies that target this receptor. The potential of cetuximab as a vehicle for the delivery of α-particle radiation was investigated in an intraperitoneal tumor mouse model.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment.

In vitro and in vivo analysis of indocyanine green-labeled panitumumab for optical imaging-a cautionary tale.

Indocyanine green (IC-Green), the only FDA approved near-infrared (NIR) fluorophore for clinical use, is attractive to researchers for the development of targeted optical imaging agents by modification of its structure and conjugation to monoclonal antibodies (mAbs) or their fragments. IC-Green derivative, ICG-sulfo-OSu (ICG-sOSu), is frequently used for antibody conjugation. However, ICG-sOSu is amphiphilic and readily facilitates aggregation of mAbs that is not easily separable from the desired immunoconjugates.

Gene expression profiling upon (212) Pb-TCMC-trastuzumab treatment in the LS-174T i.p. xenograft model.

Recent studies have demonstrated that therapy with (212) Pb-TCMC-trastuzumab resulted in (1) induction of apoptosis, (2) G2/M arrest, and (3) blockage of double-strand DNA damage repair in LS-174T i.p. (intraperitoneal) xenografts.

Evaluation of platinum chemotherapy in combination with HER2-targeted α-particle radiation.

The studies described herein assess the potential of combining platinum-based chemotherapy with high-linear energy transfer (LET) α-particle-targeted radiation therapy using trastuzumab as the delivery vehicle. An initial study explored the combination of cisplatin with (213)Bi-trastuzumab in the LS-174T i.p.

Methodology for labeling proteins and peptides with lead-212 (212Pb).

Introduction: Alpha particles possess an exquisite degree of cytotoxicity when employed for targeted α-particle therapy (TAT) or radioimmunotherapy (RIT). (212)Pb, which acts as an in vivo generator of the α-emitting nuclide (212)Bi has shown great promise in pre-clinical studies when used to label the HER2 binding antibody, trastuzumab. Currently, the first RIT clinical trial employing (212)Pb radiolabeled trastuzumab is in progress.

Preclinical evaluation of NETA-based bifunctional ligand for radioimmunotherapy applications using 212Bi and 213Bi: radiolabeling, serum stability, and biodistribution and tumor uptake studies.

Introduction: Despite the great potential of targeted α-radioimmunotherapy (RIT) as demonstrated by pre-clinical and clinical trials, limited progress has been made on the improvement of chelation chemistry for (212)Bi and (213)Bi. A new bifunctional ligand 3p-C-NETA was evaluated for targeted α RIT using (212)Bi and (213)Bi.

Methods: Radiolabeling of 3p-C-NETA with (205/6)Bi, a surrogate of (212)Bi and (213)Bi, was evaluated at pH5.

Orthotopic pleural mesothelioma in mice: SPECT/CT and MR imaging with HER1- and HER2-targeted radiolabeled antibodies.

Purpose: To evaluate the potential of anti-human epidermal growth factor receptor (HER)1- and anti-HER2-targeted radiolabeled antibodies and magnetic resonance (MR) imaging for imaging of orthotopic malignant pleural mesothelioma (MPM) in mouse models.

Materials And Methods: Animal studies with 165 mice were performed in accordance with National Institutes of Health guidelines for the humane use of animals, and all procedures were approved by the institutional Animal Care and Use Committee. Flow cytometry studies were performed to evaluate HER1 and HER2 expression in NCI-H226 and MSTO-211H mesothelioma cells.

Sensitization of tumor to ²¹²Pb radioimmunotherapy by gemcitabine involves initial abrogation of G2 arrest and blocked DNA damage repair by interference with Rad51.

Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using (212)Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers.

Methods And Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by (212)Pb-TCMC-trastuzumab and compared with controls.

Results: Treatment with (212)Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours).

(111)In- and (203)Pb-Labeled Cyclic RGD Peptide Conjugate as an α(v)β(3) Integrin-Binding Radiotracer.

Methodology for site-specific modification and chelate conjugation of a cyclic RGD (cRGD) peptide for the preparation of a radiotracer molecular imaging agent suitable for detecting α(v)β(3) integrin is described. The method involves functionalizing the peptide with an aldehyde moiety and conjugation to a 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) derivative that possesses an aldehyde reactive aminooxy group. The binding assay of the (111)In-labeled peptide conjugate with α(v)β(3) integrin showed 60% bound when four equivalents of the integrin was added, a reasonable binding affinity for a mono-valent modified RGD peptide.

Synthesis and characterization of αvβ₃-targeting peptidomimetic chelate conjugates for PET and SPECT imaging.

There is growing interest in small peptidomimetic α(v)β(3) integrin antagonists that are readily synthesized and characterized and can be easily handled using physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC) was successfully conjugated to 1-(1-carboxy-3-carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-1,4,7-triazacyclononane (NODA-GA(tBu)(3)) and 1-(1-carboxy-3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)(4)) and radiolabeled with (111)In, (67)Ga and (203)Pb. Results of a radioimmunoassay demonstrated binding to purified α(v)β(3) integrin when 1-4equiv of integrin were added to the reaction.

(212)Pb-radioimmunotherapy induces G(2) cell-cycle arrest and delays DNA damage repair in tumor xenografts in a model for disseminated intraperitoneal disease.

In preclinical studies, targeted radioimmunotherapy using (212)Pb-TCMC-trastuzumab as an in vivo generator of the high-energy α-particle emitting radionuclide (212)Bi is proving an efficacious modality for the treatment of disseminated peritoneal cancers. To elucidate mechanisms associated with this therapy, mice bearing human colon cancer LS-174T intraperitoneal xenografts were treated with (212)Pb-TCMC-trastuzumab and compared with the nonspecific control (212)Pb-TCMC-HuIgG, unlabeled trastuzumab, and HuIgG, as well as untreated controls. (212)Pb-TCMC-trastuzumab treatment induced significantly more apoptosis and DNA double-strand breaks (DSB) at 24 hours.

Pre-Clinical Assessment of Lu-Labeled Trastuzumab Targeting HER2 for Treatment and Management of Cancer Patients with Disseminated Intraperitoneal Disease.

Studies from this laboratory have demonstrated the potential of targeting HER2 for therapeutic and imaging applications with medically relevant radionuclides. To expand the repertoire of trastuzumab as a radioimmunoconjugate (RIC) vector, use of (177)Lu was investigated. The combination of a 6.

PET and MRI of metastatic peritoneal and pulmonary colorectal cancer in mice with human epidermal growth factor receptor 1-targeted 89Zr-labeled panitumumab.

Unlabelled: Human epidermal growth factor receptor 1 (HER1) plays an important role in the pathogenesis of colorectal cancer. Panitumumab is an anti-HER1 monoclonal antibody approved for use in colorectal cancer. However, few data exist regarding HER1 status in the corresponding distant metastases, and little corresponding information is available regarding the localization of panitumumab at primary and metastatic lesions.

Preparation of cystamine core dendrimer and antibody-dendrimer conjugates for MRI angiography.

Herein we report the preparation along with the in vivo and in vitro MRI characterization of two generation four and five cystamine core dendrimers loaded with thirty and fifty-eight derivatized Gd-DOTA (G4SS30, G5SS58) respectively. Likewise the development and characterization of two half-dendrimers conjugated to the F(ab')(2) fragment of the monoclonal antibody (mAb) panitumumab functionalized with a maleimide conjugation functional group site (Ab-(G4S15)(4), Ab-(G5S29)(4)) are also described. The in vitro molar relaxivity of the Ab-(G4S15)(4) conjugate, measured at pH 7.

In Vitro and In Vivo Pre-Clinical Analysis of a F(ab')(2) Fragment of Panitumumab for Molecular Imaging and Therapy of HER1 Positive Cancers.

Background: The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')(2) fragment of panitumumab and to investigate its potential for imaging and radioimmunotherapy.

Methods: The panitumumab F(ab')(2) was generated by enzymatic pepsin digestion. After the integrity and immunoreactivity of the F(ab')(2) was evaluated, the fragment was radiolabeled.

Efficient bifunctional decadentate ligand 3p-C-DEPA for targeted α-radioimmunotherapy applications.

A new bifunctional ligand 3p-C-DEPA was synthesized and evaluated for use in targeted α-radioimmunotherapy. 3p-C-DEPA was efficiently prepared via regiospecific ring opening of an aziridinium ion and conjugated with trastuzumab. The 3p-C-DEPA-trastuzumab conjugate was extremely rapid in binding (205/6)Bi, and the corresponding (205/6)Bi-3p-C-DEPA-trastuzumab complex was stable in human serum.