An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors

PurposeProstate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies.MethodsYeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors.ResultsThe A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo.ConclusionOf the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study

Functional characterisation of HLA-F

The work presented in this thesis is the functional characterisation of HLA-F, a human non-classical MHC class I molecule. HLA-F is thought to have evolved a specific immune function, similar to HLA-E and HLA-G, two other human non-classical class I molecules. HLA-F was first expressed and characterised in mammalian cell lines and bacteria. A prokaryotic system of expression was found to be more useful. Recombinant HLA-F heavy chain and beta-2 microglobulin proteins formed a stable complex when refolded in vitro in the absence of synthetic peptide. Furthermore, high-pressure liquid chromatography did not detect any bound peptides following acid elution of the refolded complex. This complex was used as an immunogen to produce a highly specific, high affinity monoclonal antibody (FGl) that was used to study the cell biology and tissue distribution of HLA-F. HLA-F was shown to have a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines, and from HUT-78, a T cell line. In B cell lines HLA-F was shown to bind TAP, but cell surface expression was only detected when these cells were grown at 26² C. HLA-F tetramers were generated to identify potential receptors for HLA-F. HLA- F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 (LIRl) and ILT4 (LIR2). Finally surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. At present, the function of HLA-F remains unclear however, we now have substantial insight into the biochemistry and intracellular interactions of HLA-F and the cell types, which express and may interact with it

A TCXO-less 100Hz-minimum-bandwidth transceiver for ultra-narrow-band sub-GHz IoT cellular networks

International audienceUltra-narrow-band (UNB) signaling is an enabling technology for low-power wide-area (LPWA) networks for the “Internet-of-Things”. Indeed, UNB signaling, based on spectrally efficient modulations such as DBPSK, simultaneously optimizes network capacity while maximizing the communication link budget. However, UNB signaling poses many technical challenges. In the receiver, carrier frequency offsets (CFO) can shift the desired signal from the expected channel. In the transmitter, the difficulty resides in generating the modulated signal with the required spectral purity. This work presents an 850-to-920 MHz RF transceiver dedicated to UNB communication systems employing the DBPSK/GFSK modulations. The receiver is resistant to CFO offsets and drifts of ±75Hz (i.e. 150% of the 100Hz channel) and 35Hz/s, respectively, with only 1dB sensitivity loss, thus allowing the circuit to function without a TCXO. In DBPSK 100b/s transmission mode, an error vector magnitude (EVM) better than 5% is measured for output powers up to 10dBm

Positron Emission Tomographic Imaging of Iodine 124 Anti–Prostate Stem Cell Antigen–Engineered Antibody Fragments in LAPC-9 Tumor–Bearing Severe Combined Immunodeficiency Mice

The humanized antibody (hu1G8) has been shown to localize to prostate stem cell antigen (PSCA) and image PSCA-positive xenografts. We previously constructed hu1G8 anti-PSCA antibody fragments and tested them for tumor targeting and the ability to image prostate cancer at early and late time points postinjection by positron emission tomography (PET). We now then compare the PET imaging and the radioactivity accumulation properties in prostate cancer tumors and nontarget tissues to determine the superior 124 I-labeled hu1G8 antibody format. 124 I-labeled diabody, minibody, scFv-Fc, scFv-Fc double mutant (DM), and parental IgG were administered into severe combined immunodeficiency (SCID) mice bearing LAPC-9 xenografts and followed by whole-body PET imaging of mice at preselected time points. Regions of interest were manually drawn around tumor and nontarget tissues and evaluated for radioactivity accumulation. The 124 I-hu1G8 IgG has its best time point for tumor high-contrast imaging at 168 hours postinjection. The 124 I-hu1G8 minibody at 44 hours postinjection results in superior tumor high-contrast imaging compared to the other antibody formats. The 124 I-hu1G8 minibody at 44 hours postinjection also has comparable percent tumor radioactivity compared to 124 I-hu1G8 IgG at 168 hours postinjection. The 124 I-hu1G8 minibody is the best engineered hu1G8 antibody format for imaging prostate cancer

HLA-Fatal attraction.

International audienceOpen conformers of the non-classical and monomorphic major histocompatibility complex (MHC) class I molecule HLA-F are ligands for the activating receptor KIR3DS1 and trigger the activation of natural killer (NK) cells