Noninvasive Imaging of Immune Checkpoint Ligand PD-L1 in Tumors and Metastases for Guiding Immunotherapy

Immunotherapy holds great promise in cancer treatment. The challenges in advancing immunotherapies lie in patient stratification and monitoring therapy. Noninvasive detection of immune checkpoint ligand PD-L1 can serve as an important biomarker for guidance and monitoring of immunotherapy. Here in, we provide an overview of our efforts to develop clinically translatable PD-L1-specific imaging agents for quantitative and real-time assessment of PD-L1 expression in tumor microenvironment

PET imaging of distinct brain uptake of a nanobody and similarly-sized PAMAM dendrimers after intra-arterial administration

Introduction: We have recently shown that intracerebral delivery of an anti-VEGF monoclonal antibody bevacizumab using an intra-arterial (IA) infusion is more effective than intravenous administration. While antibodies are quickly emerging as therapeutics, their disadvantages such as large size, production logistics and immunogenicity motivate search for alternatives. Thus we have studied brain uptake of nanobodies and polyamidoamine (PAMAM) dendrimers. Methods: Nanobodies were conjugated with deferoxamine (DFO) to generate NB(DFO)(2). Generation-4 PAMAM dendrimers were conjugated with DFO, and subsequently primary amines were capped with butane-1,2-diol functionalities to generate G4(DFO)(3)(Bdiol)(110). Resulting conjugates were radiolabeled with zirconium-89. Brain uptake of (ZrNB)-Zr-89(DFO)(2) and (89)ZrG4(DFO)(3)(Bdiol)(110) upon carotid artery vs tail vein infusions with intact BBB or osmotic blood-brain barrier opening (OBBBO) with mannitol in mice was monitored by dynamic positron emission tomography (PET) over 30min to assess brain uptake and clearance, followed by whole-body PET-CT (computed tomography) imaging at 1h and 24h post-infusion (pi). Imaging results were subsequently validated by ex-vivo biodistribution. Results: Intravenous administration of (ZrNB)-Zr-89(DFO)(2) and (89)ZrG4(DFO)(3)(Bdiol)(110) resulted in their negligible brain accumulation regardless of BBB status and timing of OBBBO. Intra-arterial (IA) administration of (ZrNB)-Zr-89(DFO)(2) dramatically increased its brain uptake, which was further potentiated with prior OBBBO. Half of the initial brain uptake was retained after 24h. In contrast, IA infusion of (89)ZrG4(DFO)(3)(Bdiol)(110) resulted in poor initial accumulation in the brain, with complete clearance within 1h of administration. Ex-vivo biodistribution results reflected those on PET-CT. Conclusions: IA delivery of nanobodies might be an attractive therapeutic platform for CNS disorders where prolonged intracranial retention is necessary

Enhancing the Efficacy of Ara‑C through Conjugation with PAMAM Dendrimer and Linear PEG: A Comparative Study

1β-d-Arabinofuranosylcytosine (Cytarabine, Ara-C) is a key drug in the treatment of acute myeloid leukemia. Ara-C has a number of limitations such as a rapid deactivation by cytidine deaminase leading to the formation of a biologically inactive metabolite, Ara-U (1β-d-arabinofuranosyluracil), a low lipophilicity, and fast clearance from the body. To address these problems, we developed a conjugate in which hydroxyl-terminated PAMAM dendrimer, G4-OH [“D”] and PEG were used as carriers for the drug (Ara-C). The conjugates were synthesized using an efficient multistep protection/deprotection method resulting in the formation of a covalent bond between the primary hydroxyl group of Ara-C and dendrimer/PEG. The structure, physicochemical properties, and drug release kinetics were characterized extensively. ¹H NMR and MALDI-TOF mass spectrometry suggested covalent attachment of 10 Ara-C molecules to the dendrimer. The release profile of Ara-C in human plasma and in PBS buffer (pH 7.4) showed that the conjugates released the drug over 14 days in PBS, with the release sped up in plasma. In PBS, while most of the drug is released from PEG-Ara-C, the dendrimer continues to release the drug in a sustained fashion. The results also suggested that the formation of the inactive form of Ara-C (Ara-U) was delayed upon conjugation of Ara-C to the polymers. The inhibition of cancer growth by the dendrimer-Ara-C and PEG-Ara-C conjugates was evaluated in A549 human adenocarcinoma epithelial cells. Both dendrimer- and PEG-Ara-C conjugates were 4-fold more effective in inhibition of A549 cells compared to free Ara-C after 72 h of treatment

Structural Characterization and in Vivo Evaluation of β‑Hairpin Peptidomimetics as Specific CXCR4 Imaging Agents

The CXCR4 chemokine receptor is integral to several biological functions and plays a pivotal role in the pathophysiology of many diseases. As such, CXCR4 is an enticing target for the development of imaging and therapeutic agents. Here we report the evaluation of the POL3026 peptidomimetic template for the development of imaging agents that target CXCR4. Structural and conformational analyses of POL3026 and two of its conjugates, DOTA (POL-D) and PEG₁₂-DOTA (POL-PD), by circular dichroism, two-dimensional NMR spectroscopy and molecular dynamics calculations are reported. In silico observations were experimentally verified with in vitro affinity assays and rationalized using crystal structure-based molecular modeling studies. [¹¹¹In]-labeled DOTA conjugates were assessed in vivo for target specificity in CXCR4 expressing subcutaneous U87 tumors (U87-stb-CXCR4) through single photon emission computed tomography (SPECT/CT) imaging and biodistribution studies. In silico and in vitro studies show that POL3026 and its conjugates demonstrate similar interactions with different micelles that mimic cellular membrane and that the ε-NH₂ of lysine⁷ is critical to maintain high affinity to CXCR4. Modification of this group with DOTA or PEG₁₂-DOTA led to the decrease of IC₅₀ value from 0.087 nM for POL3026 to 0.47 nM and 1.42 nM for POL-D and POL-PD, respectively. In spite of the decreased affinity toward CXCR4, [¹¹¹In]­POL-D and [¹¹¹In]­POL-PD demonstrated high and significant uptake in U87-stb-CXCR4 tumors compared to the control U87 tumors at 90 min and 24 h post injection. Uptake in U87-stb-CXCR4 tumors could be blocked by unlabeled POL3026, indicating specificity of the agents in vivo. These results suggest POL3026 as a promising template to develop new imaging agents that target CXCR4