Antigen-binding Capacity Research Articles

Comparison of Oriented and Random Antibody Immobilization Techniques on the Efficiency of Immunosensor

An immunosensor for bovine leukemia virus antigen (gp51) determination was developed using surface plasmon resonance (SPR) technique. During this work the SPR-chip coated with gold was used for immunosensor design. Different strategies of antibodies immobilization - oriented immobilization of specific antibodies fragments (frag-anti-gp51) on gold surface obtained after chemical reduction and random immobilization of intact antibodies (anti-gp51) on gold surface, were investigated. The immobilization based on the application of frag-anti-gp51 was found to be the most suitable for the design of SPR-immunosensor for gp51 detection. SPR sensor created using frag-anti-gp51 was tested in a range from 0.01 to 0.5 mg/mL of gp51 antigen concentration. Developed immunosensor exhibited sufficient antigen binding capacity, simplicity and low cost in respect to the currently evaluated techniques.

Biomimetic Engineering of Modular Bispecific Antibodies for Biomolecule Immobilization

Modular bispecific antibodies (BsAb's) that interact directly with a gold surface were engineered for immobilization on biosensing devices. The BsAb's consist of the variable fragments of antigold and antilysozyme antibodies connected via one of three linkers derived from naturally occurring proteins. The BsAb's were bound tightly to both the gold surface and to lysozyme, thus functioning as interface molecules between lysozyme and the gold surface without a substantial loss of antigen-binding activity. The antigen-binding capacity (the ratio of the amount of immobilized lysozyme to the amount of immobilized BsAb) on the gold surface reached 82%. An analysis of the correlation between binding capacity and linker characteristics indicated that the presence of a long, rigid linker sequence derived from a cellulase resulted in a higher antigen-binding capacity than did the presence of a long but relatively flexible glycine-rich linker. This result suggests a strategy for designing linkers suitable for BsAb-based biomolecular immobilization.

DNA-encoding to improve performance and allow parallel evaluation of the binding characteristics of multiple antibodies in a surface-bound immunoassay format.

High affinity capture agents against protein targets are essential components for immunoassays, regardless of specific analysis format. Here, we describe the use of DNA-encoded antibodies for rapidly screening the kinetic and equilibrium binding properties of twelve commercial antibodies in a parallel analysis format using a multiplexed array of microring optical resonators. We show that DNA-encoding offers advantages in terms of antigen binding capacity, compared to covalently tethered antibodies; we also demonstrate that this linkage modality facilitates the rapid self-assembly of multiplexed arrays on account of complementarity between the DNA sequences on the antibodies and sensor array, respectively. Furthermore, DNA-encoded antibodies also allow for sensor array regeneration and reprogramming, as chaotropic agents can be used to disrupt the DNA-DNA duplexes that link the capture agents to the sensor without harming the underlying DNA on the surface, which can subsequently be reloaded with antibodies either targeting the same or different antigens.

Role of antigen in later stages of B cell development (153.20)

Abstract B cell development occurs in the gut associated lymphoid tissue (GALT) of many mammals and birds. In chickens, the early stages of B cell development, including colonization of bursal follicles, B cell proliferation within bursal follicles and repertoire diversification by gene conversion, are supported by surface Ig receptor related constructs that lack antigen binding capacity. Thus early B cell development requires Ig receptor expression but not sIg ligation. In contrast later stages of bursal development, including cortico-medullary redistribution of B cells and their maintenance after hatch, is not supported by those constructs. The later stages of bursal B cell development occur in the presence of gut derived antigens suggesting the possibility that later stage may require antigen mediated sIg receptor ligation as opposed to simply receptor expression. To address this directly, we have introduced Ig receptor related constructs with defined antigen specificity into developing chick embryos. Expression of such constructs is sufficient to support early stages of B cell development, and in the absence of cognate antigen, is not sufficient to support the later stages of B cell development. Preliminary evidence suggests, however, that introduction of cognate antigen results in extended maintenance of receptor expressing B cells after hatch suggesting that ligation of the BCR may act as a survival signal in the later stages of B cell development in the bursa.

Nanocapsules incorporating IgG Fc-binding domain derived from Staphylococcus aureus protein A for displaying IgGs on immunosensor chips

To enhance the sensitivities and antigen-binding capacities of immunosensors, oriented immobilization of antibodies on the surface of the sensor chip is critical, but to date, this has not been adequately achieved. We describe a way of adsorbing immunoglobulin (Ig) proteins onto 32-nm bio-nanocapsules (BNCs) through IgG Fc-binding domains derived from Staphylococcus aureus protein A (ZZ-BNC). This arrangement permits ∼60 molecules of mouse total IgG bind to ZZ-BNC and all the IgG Fv regions to be displayed outwardly for the effective binding of antigens. ZZ-BNCs adsorbed onto the gold surface of the sensor chip of the quartz crystal microbalance (QCM) could markedly enhance the sensitivity and antigen-binding capacity of the chip. On the sensor chip of surface plasmon resonance (SPR), antibodies on the ZZ-BNCs showed higher affinities to each antigen than those on protein A. The BNC-coated sensor chip is very stable, and should prove useful for various immunosensor applications due to oriented immobilization of antibodies.

Preclinical and Clinical Binding Properties, Internalization Kinetics, and Clinicopathological Activity of Brentuximab Vedotin (SGN-35): A Novel Antibody Drug Conjugate for Anaplastic Large Cell Lymphoma and Classical Hodgkin Lymphoma

AbstractAbstract 1789 Background:Brentuximab vedotin (SGN-35) is a novel anti-CD30 antibody conjugated to the cytotoxic drug monomethyl auristatin E (MMAE) designed to selectively target and kill CD30 expressing neoplasms. This agent has demonstrated antitumor activity in classical Hodgkin lymphoma and CD30-positive T cell lymphoma, yet the binding properties, internalization kinetics, and clinicopathological findings have not been described in tumor specimens derived from treated patients. Therefore, we investigated the activity of SGN-35 on a patient with cutaneous manifestations of systemic ALK-negative anaplastic large cell lymphoma (ALCL), and correlated these results with studies of the activity of SGN-35 on cultured CD30-positive cells lines. Methods and Results:First, we confirmed (using flow cytometry) that SGN-35 and the anti-CD30 antibody clone BerH83 do not compete for binding to CD30. Next, in Karpas 299 (ALCL cell line) and KM-H2 (cell line derived from Hodgkin lymphoma) cells, we quantitatively monitored CD30 expression and cell-surface SGN-35 over time (measured as antigen binding capacity [ABC]/cell), demonstrating maximum antigen expression at 24–48 hours of incubation with 15 μg/ml SGN-35 followed by decrease at 120 hours, consistent with SGN-35-induced internalization (Table). Similar studies were performed on sequential tumor biopsies from a 68 year old male with a 5-year history of ALK-negative ALCL relapsing after 4 prior systemic regimens and radiation, now presenting with multiple pink ulcerated tumorous lesions on his lower extremities as well as bone and nodal involvement. H&E and anti-CD30 immunohistochemical stains of both pretreatment and 24 hrs post-treatment (after the first dose of SGN-35 (1.8mg/kg)-ClinicalTrials.gov identifier NCT01026415) skin punch biopsies showed a dense dermal infiltrate of large CD30-positive neoplastic cells. Biopsy at 48 hrs post-treatment demonstrated numerous apoptotic cells. Antigen density experiments on the patient biopsy specimens and clinical findings correlated with these morphologic results. CD30 antigen density was highest on the patient's cells pretreatment biopsy (1.01 × 105 ABC units/cell) and then decreased after 24 hrs (7.83 × 104 ABC units/cell) and 48 hrs (5.08 × 104 ABC units/cell). Bound SGN-35 was greatest at 24 hrs (2.26 × 103 ABC units/cell) and decreased at 48 hrs (1.40 × 103 ABC units/cell) (Table). The corresponding measured blood concentrations of SGN-35 at 24 hr, 48 hr, and 21 days were 10 μg/mL, 7 μg/mL, and 1 μg/mL, respectively. No CD30 positive cells were present in the day 21 biopsy, precluding evaluation of CD30 antigen density and bound SGN-35. Clinically, the size and prominence of the skin lesions were reduced at day 21 post treatment and biopsy at this time point showed no morphologic or immunohistochemical evidence of the neoplastic population (pathologic complete remission). After the second dose of SGN-35 the patient achieved an 81% reduction in all radiographically measurable lesions (partial response) and achieved a radiographic and cutaneous complete remission after the 5th infusion of this agent. Conclusion:These data are the first to suggest in both cell lines and patient-derived tissues that the internalization kinetics of SGN-35 is rapid with resultant reduction in CD30 expression within the first 48 hours and concurrent apoptosis induction within the targeted cells. While the measured SGN-35 occupancy of CD30 binding sites in the patient is lower than with the cell lines, these data highlight the potential antitumor activity of this agent. The results from the single patient need to be confirmed in a cohort of patients. Nevertheless, such results imply that even with subsaturating occupancy of CD30, thousands of molecules of SGN-35 (each with approximately 4 molecules of MMAE) are likely to be internalized by and potentially kill each targeted cell and may be sufficient to yield pathologic remissions and clinical activity. Disclosures:Fromm:Seattle Genetics: Research Funding. McEarchern:Seattle Genetics: Employment. Kennedy:Seattle Genetics: Employment. Shustov:Seattle Genetics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Gopal:Seattle Genetics: Research Funding.

Silencing the expression of platelet endothelial cell adhesion molecule‐1 prevents allogeneic T‐cell cytotoxicity

After solid organ transplantation, the endothelium is the first allorecognition checkpoint of the recipient's immune system. A major player at this interface is the platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-like glycoprotein, involved in white blood cell migration, cellular adhesion, and signal transduction. The potential of preventing allorecognition at this interface was explored by knocking down PECAM-1 expression using RNA interference. Lentiviral-based vectors encoding short-hairpin RNA sequences specific for PECAM-1 transcripts were used for the transduction of monocytic and endothelial cell lines. Expression of PECAM-1 decreased by up to 80% at mRNA and protein levels on monocytic and endothelial cell lines. Antigen-binding capacity assays likewise showed up to 80% reduction of PECAM-1 expression on cell surfaces. In allogeneic T-cell stimulation assays, T cells stimulated with PECAM-1-silenced monocytes had granzyme B mRNA levels up to 85% lower than those in T cells stimulated with monocytes expressing nonspecific shRNA. Also, T-cell cytotoxicity showed to decrease significantly against PECAM-1-silenced monocytes versus those nonsilenced for PECAM-1. Moreover, the former T cells did not secrete interferon-γ and exhibited reduced proliferation. These findings suggest that permanent silencing of PECAM-1 expression is feasible and contributes to efficiently inhibiting the cytotoxic T-cell response to allogeneic target cells. Thus, targeting both the immunologic synapse at the regulatory interface and the lymphocyte migration might provide a new strategy to overcome allogeneic transplant rejection.

Rapid detection of Aspergillus flavus in rice using biofunctionalized carbon nanotube field effect transistors

In the present study, we have used carbon nanotube field effect transistors (FET) that have been functionalized with protein G and IgG to detect Aspergillus flavus in contaminated milled rice. The adsorbed protein G on the carbon nanotubes walls enables the IgG anti-Aspergillus antibodies to be well oriented and therefore to display full antigen binding capacity for fungal antigens. A solution of Tween 20 and gelatine was used as an effective blocking agent to prevent the non-specific binding of the antibodies and other moulds and also to protect the transducer against the interferences present in the rice samples. Our FET devices were able to detect at least 10 μg/g of A. flavus in only 30 min. To evaluate the selectivity of our biosensors, Fusarium oxysporum and Penicillium chrysogenum were tested as potential competing moulds for A. flavus. We have proved that our devices are highly selective tools for detecting mycotoxigenic moulds at low concentrations in real samples.

Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor

A comparative study of four different antibody immobilization techniques that are suitable for modification of surface plasmon resonance (SPR) chip (SPR-chip) is reported. Antibodies against human growth hormone (anti-HGH) were used as the model system. The evaluated SPR-chip modification techniques were (i) random immobilization of intact anti-HGH (intact-anti-HGH) via self-assembled monolayer (SAM) based on 11-mercaptoundecanoic acid (MUA); (ii) random immobilization of intact-anti-HGH within carboxymethyl dextran (CMD) hydrogel by direct covalent amine coupling technique; (iii) oriented coupling of intact-anti-HGH via Fc-fragment to protein-G layer assembled on SAM consisting of MUA (MUA/pG); (iv) oriented immobilization of fragmented anti-HGH antibodies (frag-anti-HGH) via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the intact-anti-HGH was chemically "divided" into two frag-anti-HGH fragments by chemical reduction with 2-mercaptoethylamine (2-MEA). Optimal concentration of 2-MEA for preparation of anti-HGH was 15 mM. The surface concentration of immobilized antibodies and the antigen binding capacity for all four differently modified SPR-chips was evaluated and compared. The maximum surface concentration of immobilized intact-anti-HGH was obtained by immobilizing the antibody within CMD-hydrogel. The maximal antigen binding capacity was obtained by SPR-chip based on intact-anti-HGH immobilized via MUA/pG. The immobilization based on application of frag-anti-HGH was found to be the most suitable for design of SPR-immunosensor for HGH detection, due to its sufficient antigen binding capacity, simplicity, and low cost in respect to the currently evaluated techniques.

IgG Glycan Hydrolysis Attenuates ANCA-Mediated Glomerulonephritis

Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its pathogenicity by abolishing Fc receptor-mediated activation of leukocytes and complement. Here, we investigated whether IgG hydrolysis by the bacterial enzyme endoglycosidase S (EndoS) attenuates ANCA-mediated NCGN. In vitro, treatment of ANCA IgG with EndoS significantly attenuated ANCA-mediated neutrophil activation without affecting antigen-binding capacity. In a mouse model of anti-MPO IgG/LPS-induced NCGN, we induced disease with either unmodified or EndoS-treated (deglycosylated) anti-MPO IgG. In separate experiments, we administered EndoS systemically after disease induction with unmodified anti-MPO IgG. Pretreatment of anti-MPO IgG with EndoS reduced hematuria, leukocyturia, and albuminuria and attenuated both neutrophil influx and formation of glomerular crescents. After inducing disease with unmodified anti-MPO IgG, systemic treatment with EndoS reduced albuminuria and glomerular crescent formation when initiated after 3 but not 24 hours. In conclusion, IgG glycan hydrolysis by EndoS attenuates ANCA-induced neutrophil activation in vitro and prevents induction of anti-MPO IgG/LPS-mediated NCGN in vivo. Systemic treatment with EndoS early after disease induction attenuates the development of disease. Thus, modulation of IgG glycosylation is a promising strategy to interfere with ANCA-mediated inflammatory processes.

MNM-1A-2 鏡面・非鏡面X線反射率測定によるタンパク吸着膜構造の統計学的評価(セッション 1A マイクロ・ナノスケールバイオ計測と医用応用)

The purpose of this study is to investigate conformations of antibodies immobilized on three kinds of solid surfaces modified with APTES, AEAPTES or GA and their antigen-binding affinities. In the recent developments in biosensors, although the geometric structure of antibodies determines their sensitivity, few studies have focused on their conformations and its effect on their antigen-binding capacity due to a lack of applicable analysis techniques. In this work, specular and off-specular X-ray reflectivity measurement were employed, which enables us to analyze vertical and lateral multilayer structure. The results showed that the antigen-binding affinity on GA was highest among antibodies immobilized on three modified surfaces, although their conformations were almost the same as 'flat-on' analyzed by specular X-ray reflectivity. In contrast, lateral layer structure analysis by off-specular reflection revealed that protein layer on GA before antigen-binding had a relatively-long lateral correlation length and a low fractal dimension which would cause the reduction of steric hindrance for antigens' access to binding sites of antibodies.

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface p K a, antibody p I, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/ N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface p K a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab 2 antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

The effect of alkylhydroxybenzenes on the antigen-binding capacity of antibodies

The effect of the chemical analogues of microbial extracellular autoregulators belonging to alkylhydroxybenzenes (AHB), hexylresorcinol (HR), and methylresorcinol (MR), on the interactions between specific antibodies and the corresponding antigens was studied. Nonlinear dependency of the inhibition of binding of AHB-modified antibodies on the AHB chemical structures and concentrations was revealed by enzyme immunoassay. Hexylresorcinol was shown to decrease the antibody affinity and avidity indices and simultaneously increase the indices of nonspecific binding of AHB-modified antibodies to antigens, thereby promoting the formation of “false” antigen-antibody complexes. The nonspecificity of the influence of AHB on the antigenbinding capacity of antibodies is an important characteristic of these effects, which allows us to consider AHB as unique “superhaptenes”.

In this issue: Biotechnology Journal 9/2009

AbstractUsing plant hormones in biotechnologyCsukasi et al., Biotechnol. J. 2009, 4, 1293–130Plant hormones have determinant roles in the final value of agricultural products since plant domestication. Therefore, advances in deciphering their structure, synthesis, perception and signaling pathways have been a main focus for research. Application of basic information on plant hormones in food quality and production has established some landmarks in plant biotechnology, e.g. from the dramatic increase in rice yield to long life tomatoes. The review presented by Victoriano Valpuesta and colleagues from Málaga, Spain, compiles all these success events in plant biotechnology when hormones are involved. In addition, future targets for genetic improvement are suggested after summarizing the most recent discoveries in the field.BRET assays in all colorsKamal et al., Biotechnol. J. 2009, 4, 1337–1344G protein‐coupled receptors (GPCRs) play a central role in the signal transduction of an enormous array of biological stimuli. GPCR activation initiates their feedback desensitization mediated by GPCR kinases and β‐arrestin (β‐ARR) proteins. Researchers from France present and evaluate highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. Energy donors from Renilla luciferase (Rluc) were combined with the acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well‐established assays measuring ligand‐induced intramolecular rearrangements and recruitment to GPCRs. The results show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing β‐ARR assays and offer new options for the development of future BRET assays.Novel scFvs for biosensorsFalco et al., Biotechnol. J. 2009, 4, 1328–1336Single chain antibodies (scFvs) are synthetically constructed proteins that retain the antigen binding capacity of full‐length antibodies but are much smaller. Peter Berget and coworkers from Pittsburgh, USA have previously reported and characterized scFvs that are binding and activating several organic dyes, like thiazole orange or malachite green, causing them to fluoresce. Here they demonstrate that significant alteration of the fluorogenic dye activation can occur when different variable heavy (VH) or variable light (VL) domains are combined in the scFv format. Hybrid scFvs can be generated that have fluorogen‐activating domains that are completely inhibited by interactions with other domains. These new hybrid scFvs are members of a new class of two‐component fluoromolecules, which can be used to visualize cell‐surface proteins in mammalian cells. Moreover, they are excellent platforms for the development of genetically encoded, fluorescence‐generating biosensors.

Evaluation of Immobilized Metal-Ion Affinity Chromatography (IMAC) as a Technique for IgG1 Monoclonal Antibodies Purification: The Effect of Chelating Ligand and Support

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

Effect of flash-heat treatment on immunoglobulins in breast milk.

Heat-treated expressed breast milk is recommended by the World Health Organization as an option to reduce vertical HIV transmission in resource-poor regions. Flash-heat (FH) is a low technology pasteurization method developed for home use, but its effect on quantity and quality of breast milk immunoglobulins is unknown. To evaluate FH's effect on breast milk immunoglobulin levels and antigen-binding capacity. Fifty HIV+ mothers in South Africa provided breast milk. Part of each sample served as an unheated control; the remainder was flash-heated. Total and antigen-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) were measured by enzyme-linked immunosorbent assay. Paired t test was performed on log-transformed data. FH significantly decreased total IgA and IgG concentrations [geometric mean (geometric SD) 318.0 (1.9) vs. 398.2 (1.9) microg/mL and 89.1 (2.7) vs. 133.3 (2.5) microg/mL, P < 0.001 each]. Similar decreases in anti-HIV-1 gp120 IgG, anti-pneumococcal polysaccharide, and anti-poliovirus IgA occurred (P < 0.001 each). Although the latter was most affected, FH retained 66% of the antigen-binding ability. In contrast, binding capacity of IgA and IgG to influenza increased after FH (P = 0.029 and 0.025, respectively). Most breast milk immunoglobulin activity survives FH, suggesting flash-heated breast milk is immunologically superior to breast milk substitutes. Clinical significance of this decreased immunoglobulin activity needs evaluation in prospective trials.

Nanobodies - the new concept in antibody engineering

Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. The Nanobody technology was originally developed following the discovery that camelidae (camels and llamas) possess fully functional antibodies that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a perfectly stable polypeptide harboring the full antigen-binding capacity of the original heavy-chain antibody. These newly discovered VHH domains with their unique structural and functional properties form the basis of a new generation of therapeutic antibodies which were named Nanobodies. The aim of this paper is to show the properties of Nanobodies, their production and expression, applications and their clinical status.

Nanobodies - the new concept in antibody engineering

Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. The Nanobody technology was originally developed following the discovery that camelidae (camels and llamas) possess fully functional antibodies that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a perfectly stable polypeptide harboring the full antigen-binding capacity of the original heavy-chain antibody. These newly discovered VHH domains with their unique structural and functional properties form the basis of a new generation of therapeutic antibodies which were named Nanobodies. The aim of this paper is to show the properties of Nanobodies, their production and expression, applications and their clinical status.

131I anti-CD45 radioimmunotherapy effectively targets and treats T-cell non-Hodgkin lymphoma

AbstractRadioimmunotherapy (RIT) options for T-cell non-Hodgkin lymphomas (T-NHLs) are limited. We evaluated anti-CD45-RIT in human (h) and murine (m) T-NHL. CD45 was highly expressed on hT-NHL patient samples (median, 2.3 × 105 antigen-binding capacity units/cell) and hT-NHL cell lines (3.4 × 105 CD45 antigen-binding capacity units/cell). Biodistribution studies in hTNHL xenografts showed that 131I-labeled BC8 (anti-hCD45) delivered 154% (P = .01) and 237% (P = .002) more radioiodine to tumor sites over control antibodies at 24 hours and 48 hours, respectively. Importantly, tumor sites targeted with 131I-BC8 exhibited 2.5-fold (P = .05), 3.0-fold (P = .007), and 3.6-fold (P = .07) higher 131I retention over the nontarget organs of lungs, liver, and kidneys, respectively (24 hours). Because the clinical use of anti-hCD45 would target both T-NHL and other hematolymphoid tissues, we evaluated the ability of anti-mCD45 to target mT-NHL. mT-NHL grafts targeted with anti-mCD45 correspondingly retained 5.3 (P < .001), 5.4 (P < .001), and 8.7 (P < .001) times the radioactivity in tumor sites compared with nontarget organs of lung, liver, and kidney. 131I-labeled BC8 therapy yielded improved complete remission rates (75% vs 0%, P < .001) and progression-free survivals (median, 23 days vs 4.5 days, P < .001) compared with controls. These data indicate that the high CD45 expression of T-NHL allows reliable tumor targeting and disease control supporting anti-CD45 RIT for T-NHL patients.

Development and Characterization of Clinical-Grade 89Zr-Trastuzumab for HER2/neu ImmunoPET Imaging

The anti-human epidermal growth factor receptor 2 (HER2/neu) antibody trastuzumab is administered to patients with HER2/neu-overexpressing breast cancer. Whole-body noninvasive HER2/neu scintigraphy could help to assess and quantify the HER2/neu expression of all lesions, including nonaccessible metastases. The aims of this study were to develop clinical-grade radiolabeled trastuzumab for clinical HER2/neu immunoPET scintigraphy, to improve diagnostic imaging, to guide antibody-based therapy, and to support early antibody development. The PET radiopharmaceutical (89)Zr-trastuzumab was compared with the SPECT tracer (111)In-trastuzumab, which we have tested in the clinic already. Trastuzumab was labeled with (89)Zr and (for comparison) with (111)In. The minimal dose of trastuzumab required for optimal small-animal PET imaging and biodistribution was determined with human HER2/neu-positive or -negative tumor xenograft-bearing mice. Trastuzumab was efficiently radiolabeled with (89)Zr at a high radiochemical purity and specific activity. The antigen-binding capacity was preserved, and the radiopharmaceutical proved to be stable for up to 7 d in solvent and human serum. Of the tested protein doses, the minimal dose of trastuzumab (100 microg) proved to be optimal for imaging. The comparative biodistribution study showed a higher level of (89)Zr-trastuzumab in HER2/neu-positive tumors than in HER2/neu-negative tumors, especially at day 6 (33.4 +/- 7.6 [mean +/- SEM] vs. 7.1 +/- 0.7 percentage injected dose per gram of tissue). There were good correlations between the small-animal PET images and the biodistribution data and between (89)Zr-trastuzumab and (111)In-trastuzumab uptake in tumors (R(2) = 0.972). Clinical-grade (89)Zr-trastuzumab showed high and HER2/neu-specific tumor uptake at a good resolution.