Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms

Abstract

Chimeric antigen receptor (CAR) T cells are a cancer immunotherapy of extremes. Unprecedentedly effective, but complex and costly to manufacture, they are not yet a therapeutic option for all who would benefit. This disparity has motivated worldwide efforts to simplify treatment. Among the proposed solutions, the generation of CAR T cells directly in the patient, i.e., in vivo, is arguably simultaneously the most technically challenging and clinically useful approach to convert CAR therapy from a cell-based autologous medicinal product into a universally applicable off-the-shelf treatment. Here, we review the current state of the art of in vivo CAR therapy, focusing especially on the vector technologies used. These cover lentiviral vectors and adenovirus-associated vectors as well as synthetic polymer nanocarriers and lipid nanoparticles. Proof of concept, i.e., the generation of CAR cells directly in mouse models, has been demonstrated for all vector platforms. Receptor targeting of vector particles is crucial, as it can prevent CAR gene delivery into off-target cells, thus reducing toxicities. We discuss the properties of the vector platforms, such as their immunogenicity, potency, and modes of CAR delivery (permanent versus transient). Finally, we outline the work required to advance in vivo CAR therapy from proof of concept to a robust, scalable technology for clinical testing.

Keywords: AAV; LNP; T lymphocyte; lentiviral vector; nanoparticle; off the shelf.

Graphical abstract

Figure 1

Ex vivo versus in vivo CAR therapy The two strategies for converting T cells into CAR T cells are compared on the cellular level (A) and regarding their implications for clinical use (B). (A) Ex vivo generation of CAR T cells usually entails isolation of T cells from patient blood (1), followed by activation, transduction, and ex vivo expansion. After conditioning treatment (2), CAR T cells are infused into the patient (3). In the in vivo approach, vector particles (depicted as red dots) are infused directly into the patient, where they encounter T cells and selectively deliver genetic material encoding the CAR (red). (B) Due to their autologous nature, ex vivo-generated CAR cell products currently have to be prepared individually for each patient (left). The vector preparations currently under evaluation for in vivo CAR therapy constitute universally applicable off-the-shelf medicinal products (right).

Figure 2

Vector platforms used for in vivo CAR delivery The vector particles’ main features (genetic information shown in red) and their cell entry modes, including nuclear entry and potential genomic integration, are depicted from top to bottom, respectively. (A) LVs are enveloped particles containing one or more viral glycoproteins (blue) and two copies of a ssRNA genome packaged in a nucleocapsid. Depending on the glycoprotein, cell entry occurs directly at the cell membrane or is dependent on endocytosis. The transferred gene is reverse transcribed, shuttled into the nucleus, and integrated into the host genome. (B) AAVs consist of a ssDNA genome packaged into an icosahedral protein capsid. Cellular uptake by endocytosis is followed by release of the transferred gene into the nucleus, where it resides episomally, separate from host chromatin. (C) In synthetic vectors, CAR encoding nucleic acids are complexed with positively charged, biodegradable polymers (NCs) or lipids (LNPs). After endosomal escape, mRNA payloads (1) are available for translation. Packaged DNA (2) may reach the nucleus, where it can integrate into host chromatin when transposase is co-delivered.

Figure 3

Concepts for receptor targeting (A) LVs are pseudotyped with paramyxoviral (left) or alphavirus (right) glycoproteins. The receptor-binding proteins measles virus H, and Sindbis virus E1 are shown in complex with the membrane fusion proteins F and E2, respectively. Ablation of natural receptor binding by point mutations is indicated by red crosses. Target receptor binding is mediated through DARPins C-terminally fused to H protein by flexible linkers (left) or a tandem Fab serving as adapter between E2 protein and the target receptor (right). (B) An AAV particle displaying DARPins (red) at its 3-fold symmetry axis is shown. Zooming in to a single viral protein (VP), the DARPin is shown inserted into the GH2-GH3 loop. Residues in the GH12-GH13 loop mutated to ablate binding to the attachment receptor heparan-sulfate proteoglycan are labeled by the triangle. The ribbon structure was generated using ColabFold. Linker length was adapted manually to improve clarity. (C) Receptor targeting of NCs (left) and LNPs (right). Antibodies and antibody fragments used for targeting are shown in blue, the CAR encoding nucleic acid in red. Electrostatic coupling to the positively charged PBAE in NCs is enabled by conjugating antibodies to polyglutamic acid. In LNPs, antibodies functionalized with sulfhydryl groups are conjugated to the particles via thioether bonds.

Figure 4

Mechanisms detracting from the infused vector dose and potential solutions (A) Several mechanisms can detract from the administered vector dose, reducing the effective on-target dose (shown as darker pink fraction). Among them are the antibody-mediated incapacitation of particles, their uptake by phagocytes and off-target transduction. (B) Antibody-mediated immune responses against vector particles can be reduced by cleaving serum IgG before vector administration using IgG-degrading enzyme from Streptococcus pyogenes (IdeS). (C) LVs can be shielded against phagocytosis by incorporating CD47 into the LV envelope during production (left). CD47 engages signal-regulatory protein alpha (SIRPα) when LV particles (red dots) encounter myeloid cells. Through interaction of CD47 and SIRPα, phagocytosis is inhibited.