CAR T cells obtained in situ (in vivo)

The path to cheaper and more widely available technology?

Evgeny Glukhanyuk, "Biomolecule"

"Scientists have cured cancer," as if deliberately escaped from a popular meme phrase pops up every now and then in the media. Eight out of ten such notes in recent days have been devoted to a really very powerful and interesting invention of scientists – lymphocytes with chimeric receptors – chimeric antigen receptor T cells, or CAR T cells for short.

What is a CAR T-cell? A genetic construct is delivered to T-lymphocytes using a viral vector or plasmid DNA by electroporation, forcing the lymphocyte to express a chimeric receptor. The extracellular part of such a receptor is an antibody scFv fragment and is able to recognize a specific target, and the intracellular part is a piece of the intracellular part of the T-cell receptor that activates the lymphocyte. The CAR T cell device is schematically shown in Figure 1 on the example of a CAR-T designed against the CD19 target receptor. CD19 is expressed on the surface of all B cells, including malignant cells in B-cell leukemias and lymphomas. (Biomolecule previously wrote about CAR T-cells in the article "T-cells are puppets, or how to reprogram T–lymphocytes to cure cancer" [1]).

Figure 1. The device of anti-CD19 CAR T-cells used for the therapy of B-cell oncohematological diseases. Drawing of the author of the article.

The traditional scheme for obtaining CAR T-cells is an ex vivo scheme: T–cells are extracted from the peripheral blood of patients themselves (in this case, they talk about autologous cells) or other donors (allogeneic cells), modified using a vector bearing a structure with a chimeric receptor, purified, tested and injected into the patient (Fig. 2). The production cycle requires very large logistical and human resources, quality and safety control at each stage and regulatory approval, which makes such therapy "boutique".

Figure 2. Ex vivo and in vivo cell therapy methods. Gene therapy FAQs, picture adapted.

Another, probably more important problem is the complexity of standardization of therapy, which does not allow considering CAR–T as the agent with which full-fledged multicenter studies can be performed: different centers can use different genetic constructs of chimeric receptors, different ways of delivering these genetic constructs to the nucleus, guided by different research designs. It turns out like in an equation in which there are a lot of variables – even if the therapy works great, you will never fully know why.

Some researchers go for a trick – you can create a supercenter in which CAR-T is centralized: you send them T-cells of patients, and they make CAR-T in two weeks, check and send it back to you [2]. Receive, sign – standard dose, standard conditioning-lymphodepletion before administration, standard criteria for evaluating efficacy and complications. Of course, the scheme is good in the case of strong inter-center communications, the absence of problems with delivery and technical equipment of the centers; it is quite feasible, for example, within the United States. But it's still not ideal. The "pharmacological" ideal can be easily measured, stored for a long time and simply transported without unnecessary headache, carry out the usual pharmacokinetic tests with it and theoretically extend it to centers devoid of a developed laboratory and technical base. It would be easier if you could make a CAR-T, dry it, pack it and sell it in powder – they say, just add water in such and such proportions. Fantasies are all this, but not really.

The authors of a recent article in Nature Nanotechnology [3] proposed an alternative to the classical scheme for obtaining CAR-T ex vivo – they came up with nanoparticles (Fig. 3) that are capable of producing CAR-T in vivo: when injected into the bloodstream, these nanoparticles attract the patient's T cells and, forcing them to express a chimeric receptor, turn them into CAR-T cells.T.

Figure 3. Obtaining a nanoparticle capable of producing anti-CD19 CAR-T cells in vivo. a is the nanoparticle diagram. The insert shows a micrograph of a nanoparticle obtained by transmission electron microscopy (scale – 100 nm). Diagrams of two plasmids encapsulated in nanoparticles are shown. One encodes the mouse 194-1BBz CAR, the other encodes the hyperactive iPB7 transposase. b is a scheme for manufacturing nanoparticles [3].

To do this, we had to solve several very interesting problems, which the researchers brilliantly coped with.

Task one: the nanoparticle must find the T-cell and penetrate it by endocytosis. To do this, the nanoparticle was coated with polyethylene glycol and functionalized with an anti-CD3 antibody fragment. CD3 is known to be a marker of all T cells.

Task two: a genetic construct encoding a chimeric receptor from a nanoparticle should get into the nucleus of a T-cell. To do this, negatively charged plasmid DNA with a genetic construct was mixed with a positively charged polymer PBAE 447, into which peptides containing a sequence of microtubule-associated sequences (MTAS) and a nuclear localization signal (NLS) were sewn, which provided rapid transport of the genetic construct along the "rails"-microtubules to the core and its unhindered penetration inside.

Task three: the genetic construct should be expressed more or less steadily. To do this, it needs to be embedded in the genome of the T-cell. In order for integration to take place, the structure was flanked with piggyBac traspozone sequences and an additional plasmid encoding the hyperactive iPB7 traspozase was introduced into the nanoparticle.

The nanoparticles were successfully tested on a mouse model of B-cell acute lymphoblastic leukemia and showed antileukemic activity and safety not inferior to CAR-T obtained by the traditional method – ex vivo.

It is clear that the presented technology is still far from clinical application, but the authors express great hope that it will become the basis for a relatively inexpensive and widely available application of CAR-T.

Literature

1. Biomolecule: Puppet T cells, or how to reprogram T lymphocytes to cure cancer;

2. Premal Lulla, Carlos A. Ramos. (2017). Expanding Accessibility to CD19-CAR T Cells: Commercializing a “Boutique” Therapy. Molecular Therapy. 25, 8-9;

3. Smith T.T., Stephan S.B., Moffett H.F., McKnight L.E., Ji W., Reiman D. et al. (2017). In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers. Nat. Nanotechnol.

Portal "Eternal youth" http://vechnayamolodost.ru  12.05.2017