Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Preclinical Development and Characterization of Novel Adeno-Associated Viral Vectors for the Treatment of Lipoprotein Lipase Deficiency.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing, and vector-related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-Good Laboratory Practice toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon-optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared with an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

Development of an Anti-Vascular Cell Adhesion Protein-1 Aptamer for Molecular Imaging and Inflammation Detection in Transgenic Mouse Model of Alzheimer's Disease.

Cerebrovascular inflammation is often involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). Non-invasive and sensitive molecular imaging of cerebrovascular inflammation biomarkers therefore represents a potential AD diagnostic and therapeutic monitoring method. Here, we describe the development of a novel aptamer-based near infrared fluorescence imaging probe targeting Vascular Cell Adhesion Molecule-1 (VCAM-1), an adhesion molecule overexpressed by the activated cerebrovasculature during inflammation. A SELEX-type screening of a random ssDNA library against human VCAM-1 identified a high-affinity ssDNA aptamer with a dissociation constant of 49 nM. We demonstrated that the Cy5.5-labeled aptamer binds to activated endothelial cells, with no affinity to non-activated cells. A scrambled aptamer labeled with Cy5.5 did not image activated and non-activated endothelial cells, confirming the sequence specificity of the targeting. In vivo, the aptameric imaging agent targeting VCAM-1 successfully identified inflammation associated with amyloid-ß plaques deposition in the vessels of the cerebellum of transgenic AD mice. It exhibited excellent retention by remaining bound to vessels 4 hours post-injection, indicating its effectiveness in in vivo imaging and its potential in early detection of cerebrovascular inflammation.

Low-temperature approach to highly emissive copper indium sulfide colloidal nanocrystals and their bioimaging applications.

We report our newly developed low-temperature synthesis of colloidal photoluminescent (PL) CuInS2 nanocrystals (NCs) and their in vitro and in vivo imaging applications. With diphenylphosphine sulphide (SDPP) as a S precursor made from elemental S and diphenylphosphine, this is a noninjection based approach in 1-dodecanethiol (DDT) with excellent synthetic reproducibility and large-scale capability. For a typical synthesis with copper iodide (CuI) as a Cu source and indium acetate (In(OAc)3) as an In source, the growth temperature was as low as 160 °C and the feed molar ratios were 1Cu-to-1In-to-4S. Amazingly, the resulting CuInS2 NCs in toluene exhibit quantum yield (QY) of ~23% with photoemission peaking at ~760 nm and full width at half maximum (FWHM) of ~140 nm. With a mean size of ~3.4 nm (measured from the vertices to the bases of the pyramids), they are pyramidal in shape with a crystal structure of tetragonal chalcopyrite. In situ (31)P NMR (monitored from 30 °C to 100 °C) and in situ absorption at 80 °C suggested that the Cu precursor should be less reactive toward SDPP than the In precursor. For our in vitro and in vivo imaging applications, CuInS2/ZnS core-shell QDs were synthesized; afterwards, dihydrolipoic acid (DHLA) or 11-mercaptoundecanoic acid (MUA) were used for ligand exchange and then bio-conjugation was performed. Two single-domain antibodies (sdAbs) were used. One was 2A3 for in vitro imaging of BxPC3 pancreatic cancer cells. The other was EG2 for in vivo imaging of a Glioblastoma U87MG brain tumour model. The bioimaging data illustrate that the CuInS2 NCs from our SDPP-based low-temperature noninjection approach are good quality.

Blood-brain barrier transport of amyloid beta peptides in efflux pump knock-out animals evaluated by in vivo optical imaging.

BACKGROUND: Aß transport (flux) across the blood-brain barrier (BBB) is thought to contribute to the pathogenesis of Alzheimer's disease as well as to elimination of toxic amyloid from the brain by immunotherapy. Several BBB transporters have been implicated in Aß exchange between brain parenchyma and the circulation, including efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2. Here we describe an application of in vivo optical imaging methods to study Aß transport across the BBB in wild-type or animals deficient in specific efflux transporters. METHODS/DESIGN: Synthetic human Aß1-40 or scrambled Aß40-1 peptides were labeled with the near-infrared fluorescent tracer, Cy5.5. The free tracer or Cy5.5-labeled peptides were injected intravenously into Abcb1-KO or Abcg2-KO mice or their corresponding wild-type controls. The animals were imaged prospectively at different time points over a period of 8 hours using eXplore Optix small animal imager. At the end of the observation, animals were sacrificed by perfusion, their brains were imaged ex-vivo and sectioned for immunofluorescence analyses. DISCUSSION: After appropriate circulation time, the fluorescence concentration in the head ROI measured in vivo was close to background values in both wild-type and Abcb1-KO or Abcg2-KO mice injected with either free dye or scrambled Aß40-1-Cy5.5. In animals injected with Aß1-40-Cy5.5, the deficiency in either Abcb1 or Abcg2 resulted in significant increases in fluorescence concentration in the head ROIs 2 hours after injection compared to wild-type animals. Fluorescence decay (elimination rate) over 2-8 hours after injection was similar between wild-type (t1/2 = 1.97 h) and Abcg2-KO (t1/2 = 2.34 h) and was slightly faster (t1/2 = 1.38 h) in Abcb1-KO mice. In vivo time-domain imaging method allows prospective, dynamic analyses of brain uptake/elimination of fluorescently-labeled compounds, including Aß. Deficiency of either of the two major efflux pumps, Abcb1 and Abcg2, implicated in Aß trafficking across the BBB, resulted in increased accumulation of peripherally-injected Aß1-40 in the brain.

Transport characteristics of a novel peptide platform for CNS therapeutics.

New and effective therapeutics that cross the blood-brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep-2 peptide, a sequence derived from ligands that bind to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep-2 conjugate and immunohistochemical studies of injected Angiopep-2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co-localization with the neuronal nuclei-selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [(¹²5I]-Angiopep-2 into brain endothelial cells occurred by a saturable mechanism involving LRP-1. The primary sequence and charge of Angiopep-2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.