(T1130-01-06) Development and Optimization of Next-Generation Lipid Nanoparticles for In Situ CAR-T Production

Purpose: Current chimeric antigen receptor (CAR) T cell therapy employees the ex-vivo engineering of T cells, which require expensive and complicated CAR-T cell and viral vector manufacturing facilities. In-situ engineering of T cells via synthetic lipid nanoparticles (LNPs) may obviate the needs for the complex manufacturing processes and facilities. However, targeting non-hepatic cells and tissues remain the major challenge for LNP-based therapeutics. Liver targeting of LNPs is a result of the shedding of poly(ethylene glycol) (PEG)-lipids and adsorption of apolipoprotein E (ApoE) on LNPs, leading to low-density lipoprotein receptor (LDLR)-mediated uptake by hepatocytes. PEG-lipid shedding occurs at a rate of 26%/h.² Despite the recent promise of antibody (Ab)-directed targeting of LNPs toward T cells³, anchoring the large Abs onto the LNP surface by the weak anchoring energy of lipid tails will result in partitioning the Ab-conjugate lipids off of the circulating LNPs even more rapidly than PEG-lipids. To reduce the liver targeting and improve the T cell engineering in vivo, a next-generation LNP (ngLNP) needs to be developed.

Methods: Here, we replace the lipid-PEG with poly(ε-caprolactone)-block-poly(ethylene glycol) (PLC-b-PEG) and optimize the formulation for T cell transfection. By anchoring PCL-b-PEG that has larger a hydrophobic block compared to lipid tails in PEG-lipids, “shedding off” event will be minimized even after the attachment of hydrophilic Abs. Using design of experiments (DOE), we selected 26 unique LNP formulations out of large LNP design space (3⁵ =243 possible formulations). LNPs are synthesized using a scalable nanoprecipitation process called Flash NanoPrecipitation (FNP). Physicochemical properties of LNPs and T cell transfection are evaluated. The optimal freeze-thaw condition is also screened.

Results: We identified the design space where the LNPs are smaller than 160 nm, achieve high encapsulation of pDNA ( >85%), and exhibit high capability to transfect T cells compared to standard LNPs (sLNPs). We also found the sucrose as the best cryoprotectant for freeze-thaw of ngLNPs as well as sLNPs.

Conclusion: The optimized ngLNP formulation did not compromise the in vitro T cell transfection. Sucrose improved the freeze-thaw stability of ngLNPs. Future study will involve the conjugation of anti-CD3 Abs onto ngLNPs; the biodistribution and the efficiency of in vivo CAR-T production will be evaluated in comparison to standard LNPs conjugated with Abs.

References: 1. Pan, Z.; Li, S.; Liu, Q.; Wang, Z.; Zhou, Z.; Di, R.; An, X.; Miao, B.; Wang, X.; Hu, W.; Guo, X.; Lv, S.; Li, F.; Ding, G.; Chu, M.; Li, Y., Rapid evolution of a retro-transposable hotspot of ovine genome underlies the alteration of BMP2 expression and development of fat tails. BMC Genomics 2019, 20 (1), 261.
2. Chen, S.; Tam, Y. Y. C.; Lin, P. J. C.; Sung, M. M. H.; Tam, Y. K.; Cullis, P. R., Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. J Control Release 2016, 235, 236-244.
3. Rurik, J. G.; Tombacz, I.; Yadegari, A.; Mendez Fernandez, P. O.; Shewale, S. V.; Li, L.; Kimura, T.; Soliman, O. Y.; Papp, T. E.; Tam, Y. K.; Mui, B. L.; Albelda, S. M.; Pure, E.; June, C. H.; Aghajanian, H.; Weissman, D.; Parhiz, H.; Epstein, J. A., CAR T cells produced in vivo to treat cardiac injury. Science 2022, 375 (6576), 91-96.


(A) Three levels of molar ratios of individual LNP component and three levels of PCL block size of PCL-b-PEG allow 243 (=35) possible LNP formulations.
(B) 26 unique formulations were selected for synthesis and screening of LNPs using a design of experiment (DOE) approach; definitive screening design (DSD) and augmentation were used. Each column represents the distribution of mole % of each lipid from selected formulations. Height of the bar indicates the frequency of corresponding lipids at specific mole%.


Identification of design space where the LNPs are smaller than 160 nm (A), achieve high encapsulation of pDNA (>85%) (B), and exhibit high capability to transfect T cells (C) compared to standard LNPs (sLNPs) (D).


The list of screened cryoprotectants (A) and change of LNP size pre- and post- freeze thaw (B).

Acknowledgements: I would like to thank Genentech for supporting this project.