Background: We are developing cytotoxic immunoconjugates (CICs) to eliminate HIV-infected cells. We investigated the efficacy and kinetics of killing by different forms of CICs targeted by the same monoclonal antibody (mAb), an immunotoxin (IT), antibody-drug conjugate (ADC), and radioimmunoconjugate (RIC). Methods: We compared in vitro effects of CICs made by conjugating anti-gp41 mAb 7B2 to deglycosylated ricin A chain (7B2-dgA), the anthracycline derivative PNU-159682 (7B2-PNU), or the α-emitting isotope actinium-225 (7B2-²²⁵Ac). Kinetic analyses of cell growth were performed measuring electrical impedance every 15 min over a 7-day period using cells stably expressing the HIV envelope and Env-negative parent cells. Results: 7B2-dgA and 7B2-²²⁵Ac were more potent and acted more rapidly to kill cells than 7B2-PNU. Both the 7B2-PNU and 7B2-²²⁵Ac induced bystander-cell killing, whereas the IT did not and consequently allowed the outgrowth of Env-negative cells. Low dose or brief exposure to 7B2-PNU resulted in an increased rate of cell growth. Conclusions: An IT, ADC, and RIC showed substantial differences in the degree of specific toxicity, kinetics, and mechanisms of killing. The results of this side-by-side comparison have implications for the development of CICs to treat HIV, as well as other conditions.

We conducted an in vitro investigation of the selective cytotoxicity of alpha-emitting radioimmunoconjugates (α-RICs) directed against cells expressing HIV envelope (Env) proteins. It is well known that monoclonal antibody (mAb)-targeted α-emitting radionuclides can effectively kill antigen-expressing cells; however, the expected low-level expression of HIV antigens on latently infected cells poses an obstacle to all anti-HIV immune-based treatments, including α-RICs. This investigation tested the cytotoxicity of the HIV envelope antigen-binding mAbs, PGT126 (binding gp120) and 7B2 (binding gp41), conjugated with labeling chelators that bind the α-emitters astatine-211 (²¹¹At), actinium-225 (²²⁵Ac) or thorium-227 (²²⁷Th). High specific activity (SA) preparations of the α-RICs were made to increase the proportion of mAb conjugates carrying the α-emitting isotope. RIC cytolytic activity was evaluated against a cell line stably expressing the HIV envelope. ²¹¹At-labeled mAb conjugates did not demonstrate specific cell killing, while the longer lived radiometal α-RICs, ²²⁷Th and ²²⁵Ac, efficiently and specifically killed HIV envelope expressing cells. Potential explanations for these differential effects include the longer half-lives of ²²⁵Ac and ²²⁷Th compared to ²¹¹At and differences in the decay properties of radiometals compared to radiohalogens. These encouraging in vitro results suggest that in vivo evaluations of α-RIC in depleting the HIV harboring cells are warranted.

Background and Aims: Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs. Approach and Results: Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal’s distinctive serological status. Conclusions: This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type.