Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody–DM1 Drug Conjugates

Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderate- to high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (ZHER2:2891)2–ABD–MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate–spacer–, (ZHER2:2891)2–ABD–E3–MC-DM1, or a hexaglutamate–spacer–, (ZHER2:2891)2–ABD–E6–MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (ZHER2:2891)2–ABD–MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

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Gold Nanoparticle-Based Fluorescent Theranostics for Real-Time Image-Guided Assessment of DNA Damage and Repair

International Journal of Molecular Sciences ◽

10.3390/ijms20030471 ◽

2019 ◽

Vol 20 (3) ◽

pp. 471 ◽

Cited By ~ 2

Author(s):

Shriya S. Srinivasan ◽

Rajesh Seenivasan ◽

Allison Condie ◽

Stanton L. Gerson ◽

Yanming Wang ◽

...

Keyword(s):

Molecular Imaging ◽

Real Time ◽

Targeted Delivery ◽

Specific Binding ◽

Imaging Techniques ◽

Cancer Cell Line ◽

Molecular Probe ◽

Biodistribution Studies

Chemotherapeutic dosing, is largely based on the tolerance levels of toxicity today. Molecular imaging strategies can be leveraged to quantify DNA cytotoxicity and thereby serve as a theranostic tool to improve the efficacy of treatments. Methoxyamine-modified cyanine-7 (Cy7MX) is a molecular probe which binds to apurinic/apyrimidinic (AP)-sites, inhibiting DNA-repair mechanisms implicated by cytotoxic chemotherapies. Herein, we loaded (Cy7MX) onto polyethylene glycol-coated gold nanoparticles (AuNP) to selectively and stably deliver the molecular probe intravenously to tumors. We optimized the properties of Cy7MX-loaded AuNPs using optical spectroscopy and tested the delivery mechanism and binding affinity using the DLD1 colon cancer cell line in vitro. A 10:1 ratio of Cy7MX-AuNPs demonstrated a strong AP site-specific binding and the cumulative release profile demonstrated 97% release within 12 min from a polar to a nonpolar environment. We further demonstrated targeted delivery using imaging and biodistribution studies in vivo in an xenografted mouse model. This work lays a foundation for the development of real-time molecular imaging techniques that are poised to yield quantitative measures of the efficacy and temporal profile of cytotoxic chemotherapies.

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Site-Specific Antibody–Drug Conjugates in Triple Variable Domain Fab Format

Biomolecules ◽

10.3390/biom10050764 ◽

2020 ◽

Vol 10 (5) ◽

pp. 764 ◽

Cited By ~ 1

Author(s):

Dobeen Hwang ◽

Christoph Rader

Keyword(s):

Tumor Tissue ◽

In Vitro Cytotoxicity ◽

Variable Domain ◽

Site Specific ◽

Antibody Drug Conjugates ◽

Drug Conjugates ◽

Solid Malignancies ◽

Antibody Drug

The interest in replacing the conventional immunoglobulin G (IgG) format of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) with alternative antibody and antibody-like scaffolds reflects a need to expand their therapeutic utility and potency while retaining their exquisite specificity, affinity, and low intrinsic toxicity. For example, in the therapy of solid malignancies, the limited tumor tissue penetration and distribution of ADCs in IgG format mitigates a uniform distribution of the cytotoxic payload. Here, we report triple variable domain Fab (TVD–Fab) as a new format that affords the site-specific and stable generation of monovalent ADCs without the Fc domain and a drug-to-antibody ratio (DAR) of 2. TVD–Fabs harbor three variable fragment (Fv) domains: one for tumor targeting and two for the fast, efficient, precise, and stable conjugation of two cargos via uniquely reactive lysine residues. The biochemical and in vitro cytotoxicity properties of a HER2-targeting TVD–Fab before and after conjugation to a tubulin inhibitor were validated. In vivo, the TVD–Fab antibody carrier revealed a circulatory half-life of 13.3 ± 2.5 h and deeper tumor tissue distribution compared to our previously reported dual variable domain (DVD)–IgG1 format. Taken together, the TVD–Fab format merits further investigations as an antibody carrier of site-specific ADCs targeting solid malignancies.

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A Therapeutic TCR Mimic Monoclonal Antibody for Intracellular PRAME Protein in Leukemias

Blood ◽

10.1182/blood.v126.23.2527.2527 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2527-2527 ◽

Cited By ~ 2

Author(s):

Aaron Chang ◽

Tao Dao ◽

Andrew Scott ◽

Leonid Dubrovsky ◽

Cheng Liu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Retinoic Acid ◽

Conflicts Of Interest ◽

Specific Binding ◽

Retinoic Acid Receptor ◽

Pharmacokinetic Studies ◽

Breast Cancers ◽

Biodistribution Studies

Abstract Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics. Disclosures No relevant conflicts of interest to declare.

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High non-specific binding of the β1-selective radioligand 2-125I-ICI-H

Nuklearmedizin ◽

10.1055/s-0038-1625187 ◽

2003 ◽

Vol 42 (04) ◽

pp. 173-180 ◽

Cited By ~ 6

Author(s):

M. P. Law ◽

K. Kopka ◽

St. Wagner ◽

S. Luthra ◽

V. W. Pike ◽

...

Keyword(s):

Specific Activity ◽

Single Photon ◽

Specific Binding ◽

Photon Emission ◽

Radiochemical Yield ◽

Emission Computed Tomography ◽

Biodistribution Studies ◽

Positron Emission

Summary: Aim: As results of cardiac biopsies suggest, myocardial β1-adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac β2-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess β-adrenoceptors non-invasively. Among the novel racemic analogues of the established β1-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to β1-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective β1-adrenergic radioligand in cardiac imaging. Methods: Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac β-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-125I-ICI-H from the silylated precursor 2-SiMe3-ICI-H was established. The specific activity was 80 GBq/µmol, the radiochemical yield ranged from 70 to 80%. Results: The unlabelled compound 2-I-ICI-H showed high β1-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac β-adrenoceptors. The radiolabelled counterpart 2-125I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac β1-adrenoceptors in vivo. Conclusion: Because of its high non-specific binding 2-125I-ICI-H is no suitable radiotracer for imaging in vivo.

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The Synergism of Platinum-Gold Bimetallic Nanoconjugates Enhances 5-Fluorouracil Delivery In Vitro

Pharmaceutics ◽

10.3390/pharmaceutics11090439 ◽

2019 ◽

Vol 11 (9) ◽

pp. 439 ◽

Cited By ~ 5

Author(s):

Vareessh Maney ◽

Moganavelli Singh

Keyword(s):

Cancer Cells ◽

Colloidal Stability ◽

Bimetallic Nanoparticles ◽

Therapeutic Potential ◽

Release Kinetics ◽

High Specificity ◽

In Vitro Cytotoxicity ◽

Oral Drug

Nanoparticle application has significantly impacted the field of medicine. The need to develop novel drugs with higher therapeutic potential has stimulated the development of innovative delivery strategies to mitigate the potent side effects associated with known chemotherapeutic drugs. This paper describes the synthesis of platinum-gold bimetallic nanoparticles (PtAuBNps), their functionalisation with chitosan, and entrapment of the anticancer drug 5-fluorouracil (5-FU). All PtAuBNps and their drug nanocomposites were physico-chemically characterised, displaying desirable properties with regards to shape, size (<120 nm) and colloidal stability. 5-FU binding and loading capacities in PtAuBNps were found to be 90.17% and 22.56%, respectively. In vitro cytotoxicity profiles determined using the MTT and SRB assays reflected up to 65% cell death in the MCF-7, HepG2 and Caco-2 cell lines. These nanocomposites exhibited excellent physiochemical attributes, high specificity towards cancer cells, with a pH-sensitive drug release in a simulated acidic tumour microenvironment through zero-order release kinetics. In addition, they possessed the potential to traverse the mucosal lining facilitating oral drug administration. Overall, 5-FU encapsulation improved the bioavailability of the drug in cancer cells, with the promise of enhancing its therapeutic effect, biocompatibility and safety. These positive results highlight PtAuBNps as promising in vitro delivery systems and merits future in vivo research.

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Coptis chinensisand Myrobalan (Terminalia chebula) Can Synergistically Inhibit Inflammatory Response In Vitro and In Vivo

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/510157 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Enhui Cui ◽

Xiaoyan Zhi ◽

Ying Chen ◽

Yuanyuan Gao ◽

Yunpeng Fan ◽

...

Keyword(s):

Nitric Oxide ◽

Therapeutic Potential ◽

In Vitro Cytotoxicity ◽

High Dose ◽

Dependent Manner ◽

Paw Edema ◽

Coptis Chinensis ◽

Anti Inflammatory

Objectives. To investigate the anti-inflammatory effect ofCoptis chinensisplus myrobalan (CM) in vitro and in vivo.Methods. The inflammation in mouse peritoneal macrophages was induced by lipopolysaccharide (LPS). Animal models were established by using ear swelling and paw edema of mouse induced by xylene and formaldehyde, respectively. In vitro, cytotoxicity, the phagocytosis of macrophages, the levels of nitric oxide (NO), induced nitric oxide synthase (iNOS), tumor necrosis factor-α(TNF-α), and interleukin-6 (IL-6) in cell supernatant were detected. In vivo, swelling rate and edema inhibitory rate of ear and paw were observed using CM-treated mice.Results. At 150–18.75 μg·mL−1, CM had no cytotoxicity and could significantly promote the growth and the phagocytosis of macrophages and inhibit the overproduction of NO, iNOS, TNF-α, and IL-6 in macrophages induced by LPS. In vivo, pretreatment with CM, the ear swelling, and paw edema of mice could be significantly inhibited in a dose-dependent manner, and the antiedema effect of CM at high dose was better than dexamethasone.Conclusion. Our results demonstrated thatCoptis chinensisand myrobalan possessed synergistically anti-inflammatory activities in vitro and in vivo, which indicated that CM had therapeutic potential for the prevention and treatment of inflammation-mediated diseases.

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A Proof-of-Concept Study on the Therapeutic Potential of Au Nanoparticles Radiolabeled with the Alpha-Emitter Actinium-225

Pharmaceutics ◽

10.3390/pharmaceutics12020188 ◽

2020 ◽

Vol 12 (2) ◽

pp. 188 ◽

Cited By ~ 4

Author(s):

Evangelia-Alexandra Salvanou ◽

Dimitris Stellas ◽

Charalampos Tsoukalas ◽

Barbara Mavroidi ◽

Maria Paravatou-Petsotas ◽

...

Keyword(s):

Gold Nanoparticle ◽

Radiation Treatment ◽

Therapeutic Potential ◽

In Vitro Cytotoxicity ◽

Alpha Particles ◽

Tumor Bearing ◽

Alpha Emitter ◽

Local Radiation

Actinium-225 (225Ac) is receiving increased attention for its application in targeted radionuclide therapy, due to the short range of its emitted alpha particles in conjunction with their high linear energy transfer, which lead to the eradication of tumor cells while sparing neighboring healthy tissue. The objective of our study was the evaluation of a gold nanoparticle radiolabeled with 225Ac as an injectable radiopharmaceutical form of brachytherapy for local radiation treatment of cancer. Au@TADOTAGA was radiolabeled with 225Ac at pH 5.6 (30 min at 70 °C), and in vitro stability was evaluated. In vitro cytotoxicity was assessed in U-87 MG cancer cells, and in vivo biodistribution was performed by intravenous and intratumoral administration of [225Ac]225Ac-Au@TADOTAGA in U-87 MG tumor-bearing mice. A preliminary study to assess therapeutic efficacy of the intratumorally-injected radio-nanomedicine was performed over a period of 22 days, while the necrotic effect on tumors was evaluated by a histopathology study. We have shown that [225Ac]225Ac-Au@TADOTAGA resulted in the retardation of tumor growth after its intratumoral injection in U87MG tumor-bearing mice, even though very low activities were injected per mouse. This gold nanoparticle radiopharmaceutical could be applied as an unconventional brachytherapy in injectable form for local radiation treatment of cancer.

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The effect of galactose oxidase treatment on the hepatic binding and biological activity of desialylated human chorionic gonadotrophin

Acta Endocrinologica ◽

10.1530/acta.0.0930234 ◽

1980 ◽

Vol 93 (2) ◽

pp. 234-242 ◽

Cited By ~ 1

Author(s):

Egenius C. Brand ◽

Jacobus Odink

Keyword(s):

Biological Activity ◽

Specific Binding ◽

Hepatic Uptake ◽

Galactose Oxidase ◽

High Dose ◽

Uptake Studies ◽

Radioligand Receptor Assay ◽

Galactosyl Residues

Abstract. 125I labelled desialylated hCG (asialo-hCG) was treated with galactose oxidase, in order to find out whether oxidation of the terminal galactosyl residues would diminish the hepatic uptake of asialo-hCG. Specific binding to the hepatic asialo-glycoprotein receptor was monitored in vitro by a rat liver radioligand receptor assay (RRA). Hormonal activities were compared by ovarian RRA and by in vitro bioassay. Uptake studies were done in superovulated immature rats. Galactose oxidase treatment had hardly any influence on the in vitro ovarian binding and biological activity of [125I]asialo-hCG. Binding in the liver RRA was virtually abolished. In vivo hepatic uptake, however, was considerably above the level of [125I]hCG, as was the uptake in the kidneys. The hepatic uptake was inhibited by the administration of a high dose of asialo-fetuin. It is concluded that oxidation of the terminal galactosyl residues reduces the binding of asialo-hCG to the hepatic asialo-glycoprotein receptor, without affecting its hormonal properties. The ovarian uptake in vivo, however, is still limited by the high hepatic and renal clearance.

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Preparation and Evaluation of Quercetin-Loaded MPEG-PLA Nanoparticles

Current Nanoscience ◽

10.2174/1573413714666180501094106 ◽

2018 ◽

Vol 14 (5) ◽

pp. 366-376 ◽

Cited By ~ 1

Author(s):

Hiraku Onishi ◽

Masashi Nakamura ◽

Masanaho Sasatsu

Keyword(s):

Organic Solvent ◽

Therapeutic Potential ◽

In Vitro Release ◽

Aqueous Solubility ◽

The Body ◽

In Vivo Studies ◽

Water Mixture ◽

Biodistribution Studies

Background: Quercetin (QE) is one of the flavonoids with various biological functions such as anti-oxidation, anti-inflammatory and antitumor. However, the low aqueous solubility and short half-life in the body reduce its in vivo efficacy. Therefore, the appropriate delivery techniques to solve those problems have drawn much attention. In the present study, methoxypolyethylene glycol- poly-DL-lactic acid (MPEG-PLA) nanoparticles loaded with quercetin (QE), called NP, were prepared, and their antitumor characteristics were investigated in vitro and in vivo. Method: NPs were produced by evaporating organic solvent from the organic solvent-water mixture in four formulations. The particle characteristics and in vitro release were examined for the obtained preparations (NP1 – NP4). The antitumor features were investigated in vivo with different administration schedules using mice inoculated subcutaneously with murine Sarcoma 180. In addition, the efficacy of co-administration of NP with a strong antitumor chemotherapeutic agent, irinotecan hydrochloride (CPT-11), was examined. Biodistribution studies were performed using the same animal models. Result: The NP with the higher drug content (0.58 % (w/w)) and gradual release profile, Preparation NP4, were chosen and used as NP in the in vivo studies. NP suppressed tumor growth better than QE solution in various dosing schedules (total dose = 2 mg/kg). In the combination therapy with CPT-11, NP exhibited antitumor efficacy in a nearly additive manner. No decrease in body weight observed with any administration. NP markedly enhanced the systemic distribution and tumor localization. Conclusion: These results indicated that the present NP should promote the efficacy of QE, and might have useful therapeutic potential in the treatment of solid tumors.

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H-CRRETAWAC-OH, a Lead Structure for the Development of Radiotracer Targeting Integrinα5β1?

BioMed Research International ◽

10.1155/2014/243185 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 6

Author(s):

Roland Haubner ◽

Simone Maschauer ◽

Jürgen Einsiedel ◽

Iris E. Eder ◽

Christine Rangger ◽

...

Keyword(s):

Specific Binding ◽

Radiochemical Yield ◽

Human Melanoma ◽

Cell Uptake ◽

Preclinical Research ◽

M Cell ◽

Biodistribution Studies ◽

Uptake Studies

Imaging of angiogenic processes is of great interest in preclinical research as well as in clinical settings. The most commonly addressed target structure for imaging angiogenesis is the integrinαvβ3. Here we describe the synthesis and evaluation of [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, a radiolabelled peptide designed to selectively target the integrinα5β1. Conjugation of 4-nitrophenyl-(RS)-2-[18F]fluoropropionate provided [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH in high radiochemical purity (>95%) and a radiochemical yield of approx. 55%. In vitro evaluation showedα5β1binding affinity in the nanomolar range, whereas affinity toαvβ3andαIIbβ3was >50 μM. Cell uptake studies using human melanoma M21 (αvβ3-positive andα5β1-negative), human melanoma M21-L (αvβ3-negative andα5β1-negative), and human prostate carcinoma DU145 (αvβ3-negative andα5β1-positive) confirmed receptor-specific binding. The radiotracer was stable in human serum and showed low protein binding. Biodistribution studies showed tumour uptake ranging from 2.5 to 3.5% ID/g between 30 and 120 min post-injection. However, blocking studies and studies using mice bearingα5β1-negative M21 tumours did not confirm receptor-specific uptake of [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, although this radiopeptide revealed high affinity and substantial selectivity toα5β1in vitro. Further experiments are needed to study the in vivo metabolism of this peptide and to develop improved radiopeptide candidates suitable for PET imaging ofα5β1expression in vivo.

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