Localized therapy using anti-PD-L1 anchored and NIR-responsive hollow gold nanoshell (HGNS) loaded with doxorubicin (DOX) for the treatment of locally advanced melanoma.

Drug resistance and inefficient localization of chemotherapeutic agent limit the current treatment strategy in locally advanced melanoma (MEL), accounting to the 10-year survival rate from 24% to 68%. In this study we constructed anti-PD-L1 conjugated and doxorubicin loaded hollow gold nanoshell (T-HGNS-DOX) for targeted and localized chemo-photothermal therapy of MEL by the conjugation of LA-PEG-anti-PD-L1 antibody and short PEG chain on the surface of HGNS-DOX. Near infrared (NIR) as well as pH dependent drug release profile was observed. Significant uptake of DOX following NIR due to high PD-L1 receptors resulted in pronounced anticancer effect of T-HGNS-DOX. Following intratumoral administration, maximum nanoparticles retention with the significant reduction in tumor growth was observed as a result of elevated apoptosis marker (cleaved caspase-3, cleaved PARP) as well as downregulation of proliferative (Ki-67) and angiogenesis marker (CD31). Cumulatively, our system avoids the systemic toxicities of the nanosystem thereby providing maximum chemotherapeutic retention in tumor.

Trastuzumab Conjugated Superparamagnetic Iron Oxide Nanoparticles Labeled with 225Ac as a Perspective Tool for Combined α-Radioimmunotherapy and Magnetic Hyperthermia of HER2-Positive Breast Cancer.

It has been proven and confirmed in numerous repeated tests, that the use of a combination of several therapeutic methods gives much better treatment results than in the case of separate therapies. Particularly promising is the combination of ionizing radiation and magnetic hyperthermia in one drug. To achieve this objective, magnetite nanoparticles have been modified in their core with α emitter 225Ac, in an amount affecting only slightly their magnetic properties. By 3-phosphonopropionic acid (CEPA) linker nanoparticles were conjugated covalently with trastuzumab (Herceptin®), a monoclonal antibody that recognizes ovarian and breast cancer cells overexpressing the HER2 receptors. The synthesized bioconjugates were characterized by transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) measurement, thermogravimetric analysis (TGA) and application of 131I-labeled trastuzumab for quantification of the bound biomolecule. The obtained results show that one 225Ac@Fe3O4-CEPA-trastuzumab bioconjugate contains an average of 8-11 molecules of trastuzumab. The labeled nanoparticles almost quantitatively retain 225Ac (>98%) in phosphate-buffered saline (PBS) and physiological salt, and more than 90% of 221Fr and 213Bi over 10 days. In human serum after 10 days, the fraction of 225Ac released from 225Ac@Fe3O4 was still less than 2%, but the retention of 221Fr and 213Bi decreased to 70%. The synthesized 225Ac@Fe3O4-CEPA-trastuzumab bioconjugates have shown a high cytotoxic effect toward SKOV-3 ovarian cancer cells expressing HER2 receptor in-vitro. The in-vivo studies indicate that this bioconjugate exhibits properties suitable for the treatment of cancer cells by intratumoral or post-resection injection. The intravenous injection of the 225Ac@Fe3O4-CEPA-trastuzumab radiobioconjugate is excluded due to its high accumulation in the liver, lungs and spleen. Additionally, the high value of a specific absorption rate (SAR) allows its use in a new very perspective combination of α radionuclide therapy with magnetic hyperthermia.

The Impact of the Metal Interface on the Stability and Quality of a Therapeutic Fusion Protein.

Subvisible particle formation, which occurs after the sterile filtration step of the fill/finish process, is a challenge that may occur during the development of biotherapeutics with complex molecular structures. Here, we show that a stainless steel pump head from a rotary piston pump produces more protein aggregates, past the limit of the acceptable quality range for subvisible particle counts, in comparison to a ceramic pump head. The quartz crystal microbalance was used to quantify the primary layer, proteins irreversibly adsorbed at the solid-liquid interface, and the secondary diffuse gel-like layer interacting on top of the primary layer. The results showed that the mass of protein irreversibly adsorbed onto stainless steel sensors is greater than on an aluminum oxide surface (ceramic pump mimic). This suggests that the amount of adsorbed protein plays a role in surface-induced protein aggregation at the solid-liquid interface.

Tumor-Targeted Drug and CpG Delivery System for Phototherapy and Docetaxel-Enhanced Immunotherapy with Polarization toward M1-Type Macrophages on Triple Negative Breast Cancers.

Cancer immunotherapy has achieved promising clinical responses in recent years owing to the potential of controlling metastatic disease. However, there is a limited research to prove the superior therapeutic efficacy of immunotherapy on breast cancer compared with melanoma and non-small-cell lung cancer because of its limited expression of PD-L1, low infiltration of cytotoxic T lymphocytes (CTLs), and high level of myeloid-derived suppressor cells (MDSCs). Herein, a multifunctional nanoplatform (FA-CuS/DTX@PEI-PpIX-CpG nanocomposites, denoted as FA-CD@PP-CpG) for synergistic phototherapy (photodynamic therapy (PDT), photothermal therapy (PTT) included) and docetaxel (DTX)-enhanced immunotherapy is successfully developed. The nanocomposites exhibit excellent PDT efficacy and photothermal conversion capability under 650 and 808 nm irradiation, respectively. More significantly, FA-CD@PP-CpG with no obvious side effects can remarkably inhibit the tumor growth in vivo based on a 4T1-tumor-bearing mice modal. A low dosage of loaded DTX in FA-CD@PP-CpG can promote infiltration of CTLs to improve efficacy of anti-PD-L1 antibody (aPD-L1), suppress MDSCs, and effectively polarize MDSCs toward M1 phenotype to reduce tumor burden, further to enhance the antitumor efficacy. Taken together, FA-CD@PP-CpG nanocomposites offer an efficient synergistic therapeutic modality in docetaxel-enhanced immunotherapy for clinical application of breast cancer.

Broadening and Enhancing Functions of Antibodies by Self-Assembling Multimerization at Cell Surface.

Monoclonal antibody therapy has offered treatment benefits. Nonetheless, a lack of efficacy still exists, partially because monovalent binding of antibodies to specific receptors fails to translate into an active response. Here, we report a pretargeting-postassembly approach that exploits the selective Watson-Crick base pairing properties of oligonucleotides and multivalently tethers receptor-prebound antibodies to albumin at the cell surface. We demonstrate that this two-step self-assembling strategy allows sequential actions of receptor binding and clustering that broadens and strengthens the functions of antibodies. We show that anti-CD20 obinutuzumab (OBN) modified with one morpholino oligonucleotide (OBN-MORF1) maintains the feature of naked OBN antibody upon CD20 binding, and results in actin redistribution, homotypic adhesion, and lysosome-mediated cell death. Consecutive treatment with albumin grafted with multiple copies of a complementary morpholino oligonucleotide (HSA-(MORF2)x) hybridizes with surface-attached OBN-MORF1, manipulates CD20 clustering, and engages additional signals to induce calcium influx and caspase-related apoptosis. With the two types of different mechanisms collaborating in one system, the simple design exerted a notable survival extension of mice bearing disseminated B-cell lymphomas.

Cross-Interaction Chromatography-Based QSAR Model for Early-Stage Screening to Facilitate Enhanced Developability of Monoclonal Antibody Therapeutics.

Monoclonal antibodies (mAbs) constitute a rapidly growing biopharmaceutical sector. However, their growth is impeded by developability issues such as polyspecificity and lack of solubility, which leads to attrition as well as manufacturing failures. In this study a multitool hybrid quantitative structure-activity relationship (QSAR) model development framework is described. This framework uses four novel datasets derived from the primary sequences of IgG1-κ-humanized mAbs with varying degrees of resolutions. Unsupervised pattern recognition is first performed on the descriptor sets to visualize any intrinsic property-based clustering, followed by regression of descriptors against cross-interaction chromatography (CIC) retention times. Model optimization is performed via unsupervised variable reduction followed by supervised variable selection. Finally, the models and datasets are benchmarked based on the regression model performance metrics such as R2 , Q2 , and RMSE. The results show that datasets containing localized descriptors rather than averaged value over the entire protein have better predictive performance of CIC retention behavior with R2 > 0.8 and RMSE < 0.3. Furthermore, the results indicate the physicochemical, electronic, and topological properties of hypervariable regions of antibodies that contribute most to the CIC retention times. The results of these studies could contribute to early-stage screening and better design of mAbs.

In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.

Non invasive imaging assessment of the biodistribution of GSK2849330, an ADCC and CDC optimized anti HER3 mAb, and its role in tumor macrophage recruitment in human tumor-bearing mice.

The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced 'AccretaMab' monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled 89Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with 89Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of 89Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both 89Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.

Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Elotuzumab: a novel immune-stimulating therapy to treat multiple myeloma.

INTRODUCTION: Multiple myeloma (MM), constantly remains debilitating disease, consequently leading to death. Clinical trials involving drugs with different mechanisms of action, carry the expectancy for improvement of treatment outcomes. The results of the currently published studies on the monoclonal antibodies, in particular elotuzumab confirm previous expectations of improving treatment outcomes of such therapy in MM patients. AREAS COVERED: This humanized monoclonal antibody targeting surface glycoprotein CS1, expressed commonly on plasma cells and certain cells of the immune system, stimulates the immune system to fight against MM cells. Elotuzumab in the combination with len/dex has been approved by the FDA for treatment of relapsed/refractory MM patients who have received one to three prior therapies. Expert commentary: This review summarizes the chemistry, mechanism of action and preclinical and clinical studies, pharmacodynamics, pharmacokinetics, safety and toxicity of elotuzumab in terms of MM treatment and its potential application in the future.

Mechanistic investigation of the preclinical pharmacokinetics and interspecies scaling of PF-05231023, a fibroblast growth factor 21-antibody protein conjugate.

PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analog, was generated by covalently conjugating two engineered [des-His1, Ala129Cys]FGF21 molecules to a nontargeting human IgG1 κ scaffold. The pharmacokinetics (PK) of PF-05231023 after i.v. and s.c. administration was evaluated in rats and monkeys using two enzyme-linked immunosorbent assays with high specificity for biologically relevant intact N termini (NT) and C termini (CT) of FGF21. Intact CT of FGF21 displayed approximately 5-fold faster systemic plasma clearance (CL), an approximately 2-fold lower steady-state volume of distribution, and at least 5-fold lower bioavailability compared with NT. In vitro serum stability studies in monkeys and humans suggested that the principal CL mechanism for PF-05231023 was degradation by serum proteases. Direct scaling of in vitro serum degradation rates for intact CT of FGF21 underestimated in vivo CL 5-fold, 1.4-fold, and 2-fold in rats, monkeys, and humans, respectively. The reduced steady-state volume of distribution and the bioavailability for intact CT relative to NT in rats and monkeys were compatible with proteolytic degradation occurring outside the plasma compartment via an unidentified mechanism. Human CL and PK profiles for intact NT and CT of FGF21 were well predicted using monkey single-species allometric and Dedrick scaling. Physiologically based pharmacokinetic models incorporating serum stability data and an extravascular extraction term based on differential bioavailability of intact NT and CT of FGF21 in monkeys improved accuracy of human PK predictions relative to Dedrick scaling. Mechanistic physiologically based pharmacokinetic models of this nature may be highly valuable for predicting human PK of fusion proteins, synthetically conjugated proteins, and other complex biologics.

Feasibility and cardiac safety of trastuzumab emtansine after anthracycline-based chemotherapy as (neo)adjuvant therapy for human epidermal growth factor receptor 2-positive early-stage breast cancer.

PURPOSE: Trastuzumab emtansine (T-DM1), an antibody-drug conjugate comprising the cytotoxic agent DM1, a stable linker, and trastuzumab, has demonstrated substantial activity in human epidermal growth factor receptor 2 (HER2) -positive metastatic breast cancer, raising interest in evaluating the feasibility and cardiac safety of T-DM1 in early-stage breast cancer (EBC). PATIENTS AND METHODS: Patients (N = 153) with HER2-positive EBC and prechemotherapy left ventricular ejection fraction (LVEF) ≥ 55% received (neo)adjuvant doxorubicin plus cyclophosphamide or fluorouracil plus epirubicin plus cyclophosphamide followed by T-DM1 for four cycles. Patients could then receive three to four cycles of optional docetaxel with or without trastuzumab. T-DM1 was then resumed with optional radiotherapy (sequential or concurrent) for 1 year (planned) of HER2-directed therapy. The coprimary end points were rate of prespecified cardiac events and safety. RESULTS: Median follow-up was 24.6 months. No prespecified cardiac events or symptomatic congestive heart failures were reported. Four patients (2.7%) had asymptomatic LVEF declines (≥ 10 percentage points from baseline to LVEF < 50%), leading to T-DM1 discontinuation in one patient. Of 148 patients who received ≥ one cycle of T-DM1, 82.4% completed the planned 1-year duration of HER2-directed therapy. During T-DM1 treatment, 38.5% and 2.7% of patients experienced grade 3 and 4 adverse events, respectively. Approximately 95% of patients receiving T-DM1 plus radiotherapy completed ≥ 95% of the planned radiation dose with delay ≤ 5 days. CONCLUSION: Use of T-DM1 for approximately 1 year after anthracycline-based chemotherapy was feasible and generally well tolerated by patients with HER2-positive EBC, providing support for phase III trials of T-DM1 in this setting.

Self-assembled micellar nanocomplexes comprising green tea catechin derivatives and protein drugs for cancer therapy.

When designing drug carriers, the drug-to-carrier ratio is an important consideration, because the use of high quantities of carriers can result in toxicity as a consequence of poor metabolism and elimination of the carriers. However, these issues would be of less concern if both the drug and carrier had therapeutic effects. (-)-Epigallocatechin-3-O-gallate (EGCG), a major ingredient of green tea, has been shown, for example, to possess anticancer effects, anti-HIV effects, neuroprotective effects and DNA-protective effects. Here, we show that sequential self-assembly of the EGCG derivative with anticancer proteins leads to the formation of stable micellar nanocomplexes, which have greater anticancer effects in vitro and in vivo than the free protein. The micellar nanocomplex is obtained by complexation of oligomerized EGCG with the anticancer protein Herceptin to form the core, followed by complexation of poly(ethylene glycol)-EGCG to form the shell. When injected into mice, the Herceptin-loaded micellar nanocomplex demonstrates better tumour selectivity and growth reduction, as well as longer blood half-life, than free Herceptin.

Gold nanoparticles and radiofrequency in experimental models for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal and chemo-refractory cancers, clearly, alternative treatment strategies are needed. We utilized 10nm gold nanoparticles as a scaffold to synthesize nanoconjugates bearing a targeting antibody (cetuximab, C225) and gemcitabine. Loading efficiency of gemcitabine on the gold nanoconjugates was 30%. Targeted gold nanoconjugates in combination with RF were selectively cytotoxic to EGFR expressing Hep3B and SNU449 cells when compared to isotype particles with/without RF (P<0.05). In animal experiments, targeted gold nanoconjugates halted the growth of subcutaneous Hep3B xenografts in combination with RF exposure (P<0.05). These xenografts also demonstrated increased apoptosis, necrosis and decreased proliferation compared to controls. Normal tissues were unharmed. We have demonstrated that non-invasive RF-induced hyperthermia when combined with targeted delivery of gemcitabine is more effective and safe at dosages ~275-fold lower than the current clinically-delivered systemic dose of gemcitabine. FROM THE CLINICAL EDITOR: In a model of hepatocellular carcinoma, the authors demonstrate that non-invasive RF-induced hyperthermia applied with cetuximab targeted delivery of Au NP-gemcitabine conjugate is more effective and safe at dosages ~ 275-fold lower than the current clinically-used systemic dose of gemcitabine.

Ustekinumab : targeting the IL-17 pathway to improve outcomes in psoriatic arthritis.

INTRODUCTION: Psoriatic arthritis (PsA) is a clinically diverse inflammatory arthritis that can affect peripheral joints and the axial skeleton. About 25% of psoriasis patients develop PsA and many suffer from reduced function and quality of life. Anti-TNF agents have emerged as a pivotal treatment for many patients but the lack of alternative biologics for those who become unresponsive and or tolerate these medications remain a major unmet need. Recently, ustekinumab (UST) an agent that targets the 12 - 23/Th17 pathway was approved by the FDA for the treatment of active PsA. AREAS COVERED: Herein, we provide a comprehensive overview of the pharmacology and clinical efficacy and safety of UST in the treatment of PsA. In addition, the position of UST in the treatment of PsA is discussed. EXPERT OPINION: The lack of alternative therapies for patients who cannot tolerate or fail anti-TNF agents remains a major challenge for clinicians who treat PsA. UST, an agent that has proven efficacy in psoriasis, has recently been shown to also be effective for a number of the manifestations associated with PsA, including peripheral arthritis, dactylitis and enthesitis. This agent also inhibits radiographic progression. FDA approval of UST provides a much needed addition to the treatment options for the heterogeneous clinical features of PsA.

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: Prelude to Phase 1 clinical studies.

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with (89)Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake.   Studies with (89)Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44(+) or CD44(-)) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44(+), resp), PL45 (CD44(+), non-resp) and HepG2 (CD44(-), non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose. At 1, 2, 3, and 6 days after injection, (89)Zr-RG7356 uptake in MDA-MB-231 (CD44(+), resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44(-), non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of (89)Zr-RG7356 was similar in MDA-MB-231 (CD44(+), resp) and PL45 (CD44(+), non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. (89)Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356. (89)Zr-RG7356 selectively targets CD44(+) responsive and non-responsive tumors in mice and CD44(+) tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44(+) tumors may not be sufficient in itself to drive an anti-tumor response.

Photoimmunotherapy: comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor.

Photoimmunotherapy (PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. Herein we compare two commonly available anti-EGFR monoclonal antibodies, cetuximab and panitumumab, for their effectiveness as PIT agents in EGFR positive tumor models. A photosensitizer, IR-700, conjugated to either cetuximab (cet-IR700) or panitumumab (pan-IR700), was evaluated using EGFR-expressing A431 and MDAMB468-luc cells in 2D- and 3D-culture. PIT was conducted with irradiation of NIR light after exposure of the sample or animal to each conjugate. In vivo PIT was performed with fractionated exposure of NIR light after injection of each agent into A431 xenografts or a MDAMB468-luc orthotopic tumor bearing model. Cet-IR700 and pan-IR700 bound with equal affinity to the cells in 2D-culture and penetrated equally into the 3D-spheroid, resulting in identical PIT cytotoxic effects in vitro. In contrast, in vivo anti-tumor effects of PIT with cet-IR700 were inferior to that of pan-IR700. Assessment of the biodistribution showed lower accumulation into the tumors and more rapid hepatic catabolism of cet-IR700 compared to pan-IR700. Although cet-IR700 and pan-IR700 showed identical in vitro characteristics, pan-IR700 showed better therapeutic tumor responses than cet-IR700 in in vivo mice models due to the prolonged retention of the conjugate in the circulation, suggesting that retention in the circulation is advantageous for tumor responses to PIT. These results suggest that the choice of monoclonal antibody in photosensitizer conjugates may influence the effectiveness of PIT.

Preclinical evaluation of radiolabelled nimotuzumab, a promising monoclonal antibody targeting the epidermal growth factor receptor.

BACKGROUND: Radiolabelled monoclonal antibodies with affinity towards tumour-associated antigens may enhance the efficacy of cancer treatment with targeted radiotherapy. The humanized antibody nimotuzumab represents a promising vector to deliver radioactivity to tumours overexpressing epidermal growth factor receptor type 1 (ErbB1). We analysed the effect of radiolabelling nimotuzumab on its uptake in cancer cells and its biodistribution profile in preclinical experiments. METHODS: Nimotuzumab was labelled with (131) I by oxidative iodination and with (177) Lu using nimotuzumab conjugates with two different chelators (DTPA and DOTA) and two different spacers (p-SCN-Bn and NHS). For the receptor studies, two cell lines (HaCaT and A431) were used. Biodistribution studies were performed in male Wistar rats. RESULTS: The choice of radiolabel and the manner of its attachment to nimotuzumab had little effect on the internalization of the antibody into ErbB1-expressing cell lines. However, the type of radiolabel, the way in which it was attached to nimotuzumab and the radiolabelling procedure, significantly affected the blood clearance, liver uptake and liver persistence of radiolabelled nimotuzumab. (131) I-nimotuzumab had the longest elimination half-life and the lowest radioactivity uptake in the liver. (177) Lu-labelled nimotuzumab exhibited a shorter elimination half-life, high radioactivity and long-term retention in the liver.

Synthesis and characterization of lipid immuno-nanocapsules for directed drug delivery: selective antitumor activity against HER2 positive breast-cancer cells.

Lipid nanocapsules (LNC) are usually developed as nanocarriers for lipophilic drug delivery. The surface characteristics of these colloidal particles are determinant for a controlled and directed delivery to target tissues with specific markers. We report the development of immuno-nanocapsules, in which some antibody molecules with different immuno-specificity are conjugated to the nanocapsule surface, offering the standardization of a simple method to obtain vectorized nanosystems with specific recognition properties. Nanocapsules were prepared by a solvent-displacement technique, producing an oily core coated by a functional shell of different biocompatible molecules and surface carboxylic groups. Three different antibodies (one a specific HER2 oncoprotein antibody) were conjugated with these nanoparticles by the carbodiimide method, which allows the covalent immobilization of protein molecules through carboxylic surface groups. The immuno-nanocapsules were completely characterized physico-chemically via electrokinetic and colloidal stability experiments, confirming the correct immobilization of these antibody molecules on the colloidal nanoparticles. Also, additional immunological analyses verified that these IgG-LNC complexes showed the expected specific immuno-response. Finally, different healthy and tumoral breast-cell lines were cultured in vitro with Nile-Red-loaded and docetaxel-loaded HER2 immuno-nanocapsules. The results indicate that our immuno-nanocapsules can increase their uptake in HER2 overexpressing tumoral cell lines.

Development and preclinical characterization of a humanized antibody targeting CXCL12.

PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.