Enhanced NK cell-based targeting of HIV reservoirs through engagers and adcc engineering

The National Institute of Allergy and Infectious Diseases awarded The Wistar Institute of Anatomy and Biology $889,101 on August 21, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to develop enhanced natural killer cell-based strategies targeting HIV reservoirs.

The recipient will develop and test tri-specific NK cell engager molecules (TRIKEs) engineered to enhance NK-mediated antibody-dependent cellular cytotoxicity (ADCC) responses against HIV-infected cells. Specific work includes generating CD4 ecto-domain 1 (CD4ECD1) TRIKE and CD4/HSA TRIKE constructs enhanced with poly antigen cytokine complex (PACC) technology, and directly loading IgGFc killer engagers ex vivo onto CD64 gene-modified NK cells to extend half-life and anti-viral efficacy. The recipient will assess these constructs in IL-15 transgenic NSG humanized mouse models of HIV to test whether CD64-expressing NK cells administered during early antiretroviral therapy outperform soluble TRIKEs in reducing persistent HIV levels and viral rebound upon ART interruption.

Work is performed in Philadelphia, Pennsylvania. The period of performance extends from August 21, 2026 through July 31, 2031. The award is a Project Grant, an assistance type providing support for defined research objectives.

Abstract

Actively replicating HIV infected cells are the source of progeny virions and the basis of persistent reservoirs. We will develop and test innovative strategies to eliminate actively replicating cells by enhancing NK-mediated antibody dependent cellular cytotoxicity (adcc) responses against HIV. Recent evidence showing the ability of broadly neutralizing antibodies (bnabs) to control viremia highlights the potential to complement the adcc response with exogenous bnabs able to target diverse HIV isolates. BI-specific antibodies also enhance HIV-1 specific adcc by high affinity bridging between effector NK cells (through CD16 targeting) and HIV infected cells (through envelope targeting). TRI-specific NK cell engager molecules (trikes) offer even more advantages for mediating adcc against HIV-1 infected cells via the addition of an IL-15 cytokine moiety that stimulates NK cell activity while strengthening target cell engagement. Here, we will increase the half-life and anti-viral efficacy of HIV-specific trikes via poly antigen cytokine complex (pacc) enhancement and/or by directly loading similar iggfc killer engagers EX vivo onto CD64 gene-modified NK cells that sustain FC binding for days and are not subject to ADAM17-mediated negative regulation like CD16. Taken together, we hypothesize that CD4/HSA trike-pacc-IL-15 with an FC component loaded onto CD64 expressing NK cells administered during early art will be superior to soluble trikes in targeting HIV infected cells, reducing persistent HIV levels and viral rebound upon art interruption in humanized mice. Two specific aims will test this hypothesis: aim 1: generation of CD4 ecto-domain 1 (CD4ECD1) trike and CD4/HSA trike/pacc for half-life and anti- viral efficacy assessment in the IL-15 transgenic NSG humanized mouse model of HIV. Two versions of an HIV-specific CD4 ecto-domain 1 (CD4ECD1) targeting trike with IL-15 cytokine moiety as well as a single domain antibody (sdab/VHH) against CD16 will be created with and without a pacc complex containing the IL- 15 receptor alpha chain and human serum albumin (HSA) to enhance NK cell signaling and increase the molecule half-life, respectively. Activity will be tested in vitro and in vivo in an art-suppressed HIV-infected IL- 15 transgenic NSG humanized mouse model. Aim 2: demonstrate durable retention and anti-viral efficacy of a iggfc killer engagers pre-loaded EX vivo onto CD64 expressing NK cells in vitro and in vivo. NK cells will be genetically modified using mrna lnps to express the high-affinity FC receptor CD64 and then pre-loaded EX vivo with "iggfc killer engagers" containing a HIV-specific CD4ECD1 domain along with IL-15 and an FC component. Activity tested as in aim 1. The culmination of both aims will allow US to compare and identify the best strategy between the CD4 trike, CD4/HSA trike-pacc, or CD64 NK preloaded with iggfc killer engagers for anti-viral potency on HIV reservoir dynamics in the humanized mice. The best strategy identified will facilitate prioritizing a comprehensive single NK cell therapy strategy to advance into human phase i studies.