• Chimeric antigen receptors (CARs) displayed on extracellular vesicles and/or exosomes (EV-CARs) could offer a cell-free, scalable alternative to CAR-T therapies, with fewer side effects such as cytokine release syndrome (CRS).
• These targeted EVs can more easily penetrate solid tumors and avoid immune suppression, making them promising for hard-to-treat solid tumors or other wide-ranging disease indications.
• Mass production and stockpiling of EV-CARs from allogeneic cell sources could make treatments more affordable and accessible.
• Though still in early development, EV-CAR technology shows potential to revolutionize cancer treatment and beyond in a few years.

Let’s toggle the dial ahead on our imaginary time machine just a smidge, to the year 2030. Early in the next decade, we just might be hearing a lot about all kinds of new CARs. We’re not talking here about flying cars, although that would be fun. This blog is instead about “super” EV-CARs that will penetrate and destroy solid tumors without cytokine release syndromes, and can be dosed multiple times from an abundant and uniform allogeneic production source, but without serious allergic reaction or immune cell exhaustion. If you’re feeling curious, please take a moment to read on.

It’s been over a decade since the groups of U. Penn’s Dr. Carl June [1] and MSKCC’s Michel Sadelain [2] published their watershed reports on humans cured of their cancers with CAR-T, and six years since the FDA approvals of Kymriah and Yescarta. Today, CAR-T (chimeric antigen receptors with T cells) remains a suite of impressive technology that has treated thousands to date, and continues to innovate rapidly across diverse preclinical pipelines. Despite reaping dramatic benefits for many patients suffering from lymphomas, leukemias, and myelomas, there remain issues that continue to clip the wings of CAR-T. Yet now may be the time to start looking up. With apologies to the old Back to the Future franchise, a cheeky reply to this conundrum might well be: “Roads Cells?! Where we’re going, we don’t need roads cells!”

… OK now. Let’s cut to the chase. There may soon come a time when we might not need actual cells in the final, vialed CAR drug product. Yes, you heard that right…

From the very earliest days of extracellular vesicle (EV; aka “exosome”) domestication for therapy (e.g., “dexosomes”), [3, 4, 5] these lipid-bound particles that naturally bubble out from their parental cells have been advanced as potent immunomodulators, [6] either to vaccinate against tumors [7] or to cool down an immune system gone haywire, as per GvHD. [8] As this blog will briefly introduce, it was only a matter of time before intrepid investigators would begin to engineer extracellular vesicles (EVs)/exosomes with artificial cargoes and targeting moieties. [9, 10, 11, 12] Now, we’re learning what happens when we obtain them from cultures of CAR-T cells. [13, 14, 15] The latest in vivo results are compelling so far, and warrant expeditious clinical translation into human trials.

The “Trouble” with CAR-T

Technology enhances a prior idea that works, an upward whirlwind of innovation cycling and evolutionary idea breeding. Naturally, there are several areas where CAR-T technology could improve, standing on the shoulders of the original “giants.”

First, T after infusion, T cells may be suppressed by a tumor microenvironment and/or driven into an exhausted state [16] by factors such as TGF-beta, PD-L1, CTLA-4 ligands, IL-10, and even various micro-RNA species encapsulated in EVs. The result is a variable patient response rate that is highly dependent on a tumor’s “hot” or “cold” status. Such suppression could be found in many of today’s outcomes with CAR-T therapies directed against “liquid tumors,” [17] but it’s also an problem for encapsulated solid tumors and sites of metastasis. [18] Synthetic biology approaches to the CAR design and its integrated multigene system  [19] (e.g., expression of mIL-15 or IL-12, shRNA or CRISPR directed against PD-1) might cleverly address some of these issues. [20] However, many solid tumors are walled off physically and surrounded by layers of thick stromal tissue, as well as steeped in zones of poor oxygenation or Warburg effects and low pH. These physically harsh conditions also limit access by cytotoxic T cells and NK cells. [21] For this reason, approvals for approvals for CAR-T therapies against solid tumors lag behind, while therapies using cloned TCRs are only recently reaching fruition (e.g., anti-MAGE-A4, Tecelra™).

Another problem with CAR-T is the grueling cytokine release syndrome (CRS) [22] that’s often accompanied by neurotoxicity, and occasionally, mortality. [23] Physicians are getting better at managing CRS with infusions of agents such as dexamethasone, tocilizumab, and anakinra, [24] but there’s still a long road of optimization required. In addition, lymphodepletion is the usual practice prior to the CAR-T by agents such as cyclophosphamide and fludarabine, which flattens a patient’s immune system to carve out anatomical staging areas for expansion of combat-ready, CAR-expressing T cells. This pretreatment eliminates Tregs, CTLs, helpers, B cells, and NK cells alike. A secondary purpose of lymphodepletion is to minimize rejection the infused CAR-T cells that express non-native immunogens (e.g., murine sequences) on the CAR. Yet lymphodepletion’s “collateral damage” is not without risk, possibly leaving the patient susceptible to fatal infections and long-term chronic illnesses. [25]

A final issue with CAR-T is that treatments can cost more than $500K per patient. Sure enough, families want to spare no expense for their loved ones, but let’s be painfully honest: this price tag remains far out of reach for many. Loaded into this cost is the monumental effort of decades to develop CAR-T itself, the highly complex and weeks-long manufacturing process from a patient’s own cells, the personal customization of each therapy that demands stringent quality controls and regulatory oversight, and supply chain hiccups, etc. You can correctly surmise that these costs will begin to decline over time through innovations in CAR-T manufacturing, wherein the operations flow is streamlined and industrialized.  But how?

Imagine the Advantages of EV-CARs

It’s long known that cytotoxic T lymphocytes (CTLs) are “serial killers,” leaving the kiss of death on target cells via internal vesicle dependent fusion and release of secretory granule contents, which include FasL, perforin and granzyme B. [26] Yet, there’s something else clearly observed. Distinct from the secretory granules which fuse with the T cell’s own membrane, intact EVs are also launched from CTLs. These may also embody similar cytotoxic activities as granules and yet surface express fully functional CARs. [13, 14, 27, 28, 29]

Fu, et al (2019)’s pioneering work with Professor Shi Hu’s SMMU iGEM Team [13] showed that weekly (100 mcg) IV doses of such EV-CARs from T cells could exhibit potent and selective anti-tumor effects on mouse zenograft models without signs of suppression by PD-L1. Further, those EV-CARs did not induce a cytokine release syndrome in the animals. The concept continues to advance among a growing roster of investigators. [30] For example, in 2020, Xu, et al [31] showed that such CAR-targeted EVs could be produced by HEK293T cells and carry CRISPR/Cas9 leveraged against the MYC oncogene and related tumors, in vivo.  Table 1 (below) is a preliminary survey of the available evidence, although this nascent line of study has not (yet!) advanced into any human clinical trials.

	CAR-T Cells	EV-CARs
Cytokine Release Syndrome (CRS)	Common, must be monitored and managed in hospital, sometimes involving transfer to ICU, occasionally fatal [32]	Apparently much reduced risk on account of non-human, in vivo studies. [13, 33]
Requires pre-infusion lymphodepletion	Yes, for now. (Although multiple viable strategies [20] aim to minimize this step and its impact on patients)	Most likely reduced or eliminated due to molecular engineering of EV-CAR potency, lack of anti-EV immune responses, and ability for repeat dosing. [13]
In vivo Persistence	Up to months and decades. Potentially durable elimination of cancer cells through long-lived clones, [34] yet remote risk of CAR+ T cell lymphoma [35]	Highly transient. Eliminated in minutes to hours by the reticuloendothelial system (RES), [36] although artificial surface modifications can improve half-life in circulation, [37] or promote targeting/retention toward desired tissues or lesions. [38, 39]
Requires autologous cell source	For now, yes (…until TCR INDEL-edited or CMV- or EBV-specific allogeneic T cells are fully proven after ongoing human trials) [40]	No. In theory, could be sourced from immortalized & engineered primary cell lines. Regarding present-day, non-EV-CAR human trials in progress, there are no significant differences in serious adverse events between engineered or non-engineered EVs, or autologous or allogeneic EVs. [41]
Requires T Cells	Yes. (Although “CAR-NK” approaches in now in clinical development are exploring NK cells) [42]	No. In theory, any cell type could be engineered to express a chimeric antigen receptor system that displays on its EVs, and along with them, therapeutic internal cargos. [43, 44, 45]
Penetration of immunologic in vivo barriers	Challenging and variable between patients, requiring engineering of more functionality into CAR-T gene systems [16, 20]	Yes. EVs are not inhibited by signal transduction milieu of “cold” tumors and not weakened by presence of PD-L1; [13] being non-living nano-vesicles, EVs’ MOAs are not constrained in vivo by any cell-dependent expansion/viability. [27]
Penetration of physical in vivo barriers	Difficult for CAR-T+ cells (diameter = 10 microns) to penetrate many stroma-dense solid tumors [46]	Less challenging for EV-CARs to penetrate solid tumors (diameter is 1/100 of a T cell) [14]
Logistics for Mass Production	Timing is critical such that autologous CAR-T+ cells are expanded, cryopreserved, thawed, and recovered after approx. 2-8 weeks manufacturing. Must be administered to a patient in a tight “window” who remains strong enough to respond favorably, yet still under grave threat of the cancer.	Timing is also critical, but less constrained because large quantities of standard, ready-to-use, allogeneic-manufactured EV-CARs can be bio-produced, frozen, and stockpiled indefinitely for immediate shipping straight to point of care.
Role as Adjunctive Therapy for Super-Additive Effects on Potency	Not likely. CAR-T is instrumental as the dominant therapeutic agent as it is currently conceived.	Very plausible.  For example, EV-CARs can be engineered to act as Bispecific T Cell Engagers (e.g., as multivalent membrane-positioned BiTEs or “SMART-Exos” [47]) with CAR-Ts, enhancing CAR-T and/or CTL potency; or they could carry cargoes to soften tumor targets with RNAi, [10] gene editors, [11, 12] ligand-triggered iCaspases, [48] small molecules, etc.

Table 1 (Above). Comparing and contrasting features of CAR-T therapy vs. CARs administered cell-free, via extracellular vesicles (“EV-CARs”).

Bloviation through all of Table 1 would be tiresome to readers (above), so let’s instead imagine what it would be like if EV-CARs were fast on their way to BLAs…and the cures of tomorrow. This could be a new (but not unorthodox!) drug delivery modality that combines the best features of CAR-Ts, BiTEs, LNPs, antibody drug conjugates (ADCs), synthetic biology, RNA medicines, and scalable bioindustry manufacturing. [14, 49]

Specifically, a thriving EV-CAR industry would empower therapeutics developers with ability to selectively aim the immune system at solid tumor cancer, infectious disease, fibrotic injury, autoimmunity, or diseases of aging with minimal side effects. EV-CARs would not have to replace CAR-T, but could instead synergize with existing CAR-T approaches, [50] just as easily, perhaps facilitating smaller doses with less side effects. The targeting moieties displayed on the EV-CAR surface would be tethered to a small lipid bag that’s made of natural human cellular material, and is highly inert to the human immune system (unlike PEGlyated LNPs). Inside this ~100nm sized nano-capsule, there could numerous designer drug activities and features to increase the therapeutic’s potency and/or de-risk it with additional control, specificity and safety, such as genome editors, RNAi, mRNA, small molecules, AAV gene therapy vectors, or ligand switchable designer proteins. [48, 51, 52] The matrix of designs for this system could yield innumerable combinations of novel drug candidates, deterministically controlled by the “software of life” in their production host cells, and panned out with rapid high throughput screening (HTS) on the benchtops of smaller, academic-size labs. From these discovery units, a tech-transferred bioprocess to mass manufacture the semi-synthetic loaded EV doses (e.g., ~1e12 per large human) at low cost could be employed simply by way of massive scale transfection of the host cells, or the EVs, themselves.

An analogy to EVs and CAR-T cells might apply? A gallon of milk is about four bucks. A daily cow costs about $2000. That’s a 500-fold difference. “Why buy the cow when you can have the milk?” as others have said. [53] In other words, why bother dosing with T cells (or as many T cells) when you can still use the CARs?

A Bumpy Road for EV-CARs?

Any caveats here? Surprisingly, EV manufacture is not among them, because many if not most of the basic scalability and quality problems with “exosomes” have been solved [54, 55, 56] while cell engineers have been diligently cutting their teeth on mesenchymal stem/stromal (MSC) based cellular technology platforms. [57, 58, 59] Bioreactor cell expansion and EV collection [60] into 50L+ scales [61] are routinely demonstrated, and moreover, downstream process (DSP) innovations [62] (such as AgentV™) [63] to increase yield-to-COGS ratio are leveling up rapidly.

Is mRNA production for programmable “EV-CARgo” and/or surface CAR expression a bottleneck? Here are some back-of-envelope calculations. The COVID-19 pandemic amped up production capacity of mRNA vaccine doses such that economy of scale driven manufacturing costs were less than $5 per jab. [64] Billions of people worldwide have been injected with this product class since then. One dose of COMIRNATY contains 30 mcg of mRNA, which, in tissue culture, is good for high-efficiency transfection of between 3-30 million cells. One global pandemic of mRNA vaccines thus mobilized an approximate collective mass of product to transfect more than 1e16 bioreactor cells, which could conservatively yield ~1e19 harvested, post-DSP EVs. This would be sufficient for more than 10 million large (1e12 EVs) EV-CAR doses, where the mRNA material component would amount to approximately $1000-2000 per dose. This is not a tiny sum, but it’s hardly close to the sticker price of a CAR-T therapy. It would thus seem within reach of 2030s-era global economies to manufacture enough mRNA for a thriving emerging market of EV-CAR products for pipelines and approved drugs.

Is cGMP cell and gene therapy CDMO capacity limiting? A guesstimate here is more challenging to approach. However, anecdotes and press would unfortunately suggest “yes.” One emerging alternative to CAR-T’s time and expense is to use non-viral genetic material (e.g., electroporated Sleeping Beauty transposons) encoded in plasmid, [65] and to forego 2+ weeks of T cell expansion by engineering the CAR construct with built-in “fuel” for in vivo expansion and resilience to “cold” tumor microenvironment, e.g., mIL-15. This technology could theoretically enable a mere single night’s stay at a licensed hospital clinic. The CAR-T could be formulated and administered directly at the point of care in <24 hours, bypassing the CDMO. A decentralized model for EV-CARs might be similarly applicable and very cost effective, if the supply chain and production methods were adequately industrialized. Nevertheless, such would not be completely necessary, since allogeneic EV-CARs could be produced by cell lines in bioreactors, as per mAbs via CHO cells. If the product could be similarly stockpiled and cryopreserved, a just-in-time surplus would always be available for patients in need.

…One final caveat. At this point, despite more than 100 combined interventional EV/exosome trials posted to global clinical study databases, [66] there is not yet an approved EV drug on the market. It’s highly likely we’ll see the first EV-based drug approvals in 2-4 years, but there awaits a long runway for EV-CARs to launch, for which there are yet no known clinical trials. So, what are we waiting for? The future begins now.

♬ SuperCAR… SuperCAR ♬

Preclinical data suggest that cell-free EV “super”-CARs do not need primary, autologous T cells as their preliminary source. Today’s autologous CAR-T preparations [67] require a patient leukapheresis, cell isolation, T-cell activation, viral transduction, ex vivo expansion, formulation, and lot release; “vein to vein time” for the treatment is 1.5 to 3 weeks under optimal circumstances but can realistically extend past one month. Patients must wait while their personal leukopak grows to a lot size of almost 1 billion CAR+ cells, no trivial feat for either a severely ill person or the cell technicians!

What are plausible alternatives to T cells? NK cells, as well as the clinically tested NK-92 cell line, express EVs with anti-tumor cytotoxicity [68] despite their challenges in development as an off-the-shelf, allogeneic CAR therapy platform. [69] On the other hand, standardized, allogeneic MSCs for future EV-CAR production already demonstrate ready processes to expand in tens of liters of bioreactor culture—yielding 10-100-fold more cells than CAR-T preps—and trillions of purified EVs. 10¹⁴ or more EVs tallies up to hundreds of large human-size doses.

Approximately ten MSC-based (or “MSC-like”) cell therapies are approved outside of the USA, with others on the cusp of approval by the FDA, and have been safely administered in more than 1000 clinical trials. [70, 71] Since MSCs are known to be engineerable into “CAR-MSCs,” [45] it follows that this prolific cellular template [72] for EV production could be suitable for “EV-CARs.” In addition to MSCs, established cell lines with EV-adapted manufacturing protocols might also produce massive EV numbers for certain products, as these platforms were already proven to yield billions of COVID-19 vaccine doses consisting of 100-nm-size recombinant adenovirus particles.

Therapeutic CARs that are displayed on EVs need “software” to encode these effectors into the cellular “operating system,” i.e., the transcriptome. In accord, the genetic “software” demands the most efficient means of “upload.” CAR-T’s methods du jour are lentivirus or gammaretrovirus vectors. For these, the FDA recommends less than five integrated transgene copies per cell to minimize risk of insertional oncogenic mutation. [73]  It can thus be tricky to manage the best tradeoff between high T-cell transduction efficiency with low multiplicity of infection (MOI). Fortunately, specialized, complete media in GMP formats such as RoosterGEM™ can now enhance viral vector (e.g., AAV, adenovirus, lentivirus) gene transfer efficiency [74, 75] for a variety of primary cell types (including MSCs and T cells) that otherwise find it difficult to “drink” foreign nucleic acids. With henceforth lower MOIs, there’s easier compliance with regulatory guidelines. As a bonus, there’s less cost from depleting the CAR-expressing GMP virus stock.

Whereas CAR-T product profiles usually demand long-lived in vivo hunter-killer activity against tumors, EV-CARs wouldn’t involve doses of live cells. Therefore, preparations of EV-CARs may not need permanent genome modification of the production cells via an integrated lentivector. With a now supercharged global supply network that’s fully revved to fulfill needs of mRNA therapeutics developers (and possibly incentivize further breakthroughs on the way [76]), it might be more expedient to adopt an EV-CAR production system based on transient expression of the CAR systems. In contrast to lentivirus, when opting to use mRNA, cell expansion occurs first, and gene transfer is just prior to final EV collection and DSP operations. [77] Here, it’s imperative that a system for bulk transfection of large cell populations is accessible. For this very purpose, the RoosterGEM product system was just upgraded to include a 50x formulation in its online catalog. 50x concentration of this transfection/transduction enhancer enables the user to employ their own cell-optimized media systems, and then simply drop in as a process additive next to final dose filling.

In this blog, you’ve read about an amazing new technology in its infancy—a “super” one in fact! And hopefully, you’ve learned that major roadblocks to its rapid development are mostly cleared away. Is it time for EV-CAR “road tunes” to motivate your team to get going first, before the traffic is close behind?

I now close this blog with ChatGPT’s concoction of its own semi-cringey yet unironic and earnest lyrics, to be sung to the theme song of that old TV show, “Supercar” for your chuckling amusement:

♬ SuperCAR… SuperCAR,

Faster than a T-cell, in the blood it flows,

Bound to an exosome, where the magic grows.

No cytokine storm, no heavy payload,

SuperCAR on an exo, immune’s new road! ♬

 

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