Beyond autologous ex vivo CAR-expressing cell therapies: Toward allogeneic and nucleated cell-free delivery systems of CAR

Abstract

The success of ex vivo chimeric antigen receptor (CAR)-T cell therapies has transformed the treatment of hematologic malignancies but remains limited by individualized manufacturing complexity and cost. Emerging in situ and in vivo CAR engineering platforms aim to overcome these barriers by programming patient immune cells directly within the body. Targeted lipid nanoparticles, synthetic DNA nanocarriers, and viral and non-viral delivery systems have demonstrated the ability to generate functional CAR-T cells without ex vivo manipulation, enabling scalable, on-demand immunotherapy. In parallel, advances in allogeneic “off-the-shelf” cellular therapies provide donor-derived products that can be manufactured at scale, cryopreserved, and delivered rapidly. Additional cell types are also being explored as vehicles for in vivo CAR delivery. Hybrid approaches that leverage allogeneic cells as carriers for CAR constructs may combine the scalability of off-the-shelf products with the speed and flexibility of in vivo engineering. Despite this promise, challenges remain, including cell-specific targeting, control of transgene persistence, immunogenicity, and regulatory considerations. Together, these innovations signal a paradigm shift in the generation and deployment of CAR-based cellular immunotherapies.

Graphical abstract

CAR-T therapy is undergoing a fundamental redesign. This review highlights the shift from patient-specific ex vivo manufacturing toward in vivo programming and off-the-shelf delivery platforms, revealing how gene delivery, cellular carriers, and vesicle-based systems may transform CAR immunotherapy into a scalable and widely accessible cancer treatment.

Introduction

Over the past decade, chimeric antigen receptor (CAR)-T therapy has emerged as one of the most transformative innovations in cancer treatment, showcasing the immense therapeutic potential of the immune system when appropriately engineered. By redirecting autologous T cells to recognize and destroy malignant cells, CAR-T therapy has achieved remarkable success in hematological malignancies, particularly in refractory disease. However, despite its curative potential, the current model of patient-specific ex vivo CAR-T cell product manufacturing remains a major logistical and financial obstacle to widespread clinical implementation. Each product demands leukapheresis, viral transduction, and expansion under good manufacturing practices (GMP) conditions, typically spanning several weeks of manufacturing and incurring costs that approach half a million dollars per patient. This labor-intensive and highly individualized workflow has limited scalability and global accessibility of CAR-T cell therapy.

In contrast, in vivo and in situ CAR-T generation seeks to redefine this model. In vivo CAR-T therapy uses a delivery system to introduce the CAR gene directly into T cells inside the body, while in situ CAR-T therapy is a more specific form of in vivo therapy where the CAR gene is delivered directly to a particular tissue or tumor site to reprogram T cells there. Instead of engineering T cells outside the body, these approaches deliver genetic instructions directly into circulating lymphocytes, enabling the body itself to serve as the bioreactor. This approach effectively collapses the traditional work flow, enabling a streamlined, distributable therapy that could be administered like a biologic. The potential advantages are profound: shorter manufacturing timelines, controlled and consistent standardized dosing by delivery of a genetic payload rather than a live cell product with interpatient manufacturing variability, and, most importantly, expanded accessibility for patients with rapidly progressing disease. A central determinant of durable clinical responses to CAR-T therapy is the persistence of CAR-expressing immune cells over time. Accordingly, emerging in vivo and off-the-shelf platforms must be evaluated not only for scalability and accessibility but also for whether they induce transient versus long-term CAR expression and sustained immune cell persistence in patients.

Despite the profound advantages, significant challenges remain. Current in vivo gene delivery systems often exhibit undesirable biodistribution, with nanoparticle uptake mainly localizing to the liver rather than the lymphoid tissue. In addition, achieving precise lymphocyte targeting, ensuring controlled transgene expression and persistence, and establishing robust immunologic and regulatory safety frameworks remain active areas of investigation. Addressing these issues will be critical to translating preclinical advances into safe and clinically viable therapies.

Parallel to this development, allogeneic or “off-the-shelf” CAR-T products have emerged as another transformative innovation aimed to overcome the limitations of patient-specific manufacturing. While still ex vivo manufactured, allogeneic cell therapy products are manufactured ahead of time and in bulk from healthy donor material, cryopreserved, and distributed on demand from a centralized manufacturing site to local treatment centers. This approach eliminates the need for patient leukapheresis, accelerates delivery timelines, and circumvents challenges associated with manufacturing from heavily pretreated or immunocompromised patients. For many, this represents a pragmatic bridge from current practice to the long-term vision of in vivo reprogramming.

Despite recent successes, the use of off-the-shelf allogeneic CAR-T cells has generally been limited in success in the past due to challenges such as host rejection, graft-versus-host disease (GvHD), and limited persistence. While advances in genome editing, immune cloaking, and synthetic biology are mitigating these limitations, there is still progress to be made. Moreover, alternative effector cells, including CAR-engineered natural killer (NK) and γδ T cells and induced pluripotent stem cell (iPSC)-derived platforms,¹ expand the landscape of off-the-shelf cellular immunotherapies.

A compelling frontier is the convergence of in vivo CAR-T engineering with allogeneic platforms. Donor-derived cells, platelets, mesenchymal stromal cells (MSCs), red blood cells (RBCs), or immune cell-derived extracellular vesicles (EVs) can serve as vehicles for in vivo delivery of CAR constructs, combining the scalability and immediate availability of off-the-shelf products with the flexibility, targeting precision, and rapid deployment of in situ programming. These hybrid strategies could transform CAR-T therapy from a labor- and resource-intensive intervention into an accessible, distributable, and rapidly deployable therapeutic modality. However, to date, most of the research concerning these alternative cellular therapy systems has been restricted to autologous products. In the case of allogeneic systems, interventions such as MHC stripping or PEGylation to prevent alloimmunization in platelets; MHC knockout or CD47 engineering in the case of monocytes or T cells; and restriction to universal donor RBC membranes (O^– blood type) or engineered antigen masking in the case of erythrocyte-based carriers will require careful optimization.

This review will provide an overview of the latest advances in in vivo and in situ CAR-T cell generation across a spectrum of diseases, followed by a discussion of developments in allogeneic CAR-T cell products, and emerging strategies that integrate alternative cell sources that could be a future area to amalgamate and further enhance these paradigms. We highlight their complementary strengths, translational challenges, and the steps necessary to translate these innovations from preclinical experimentation to clinical reality, underscoring a transformative vision for the next generation of cellular immunotherapy.

In situ and in vivo CAR-T cells

In vivo CAR programming approaches are inherently heterogeneous with respect to durability, as viral vectors may support prolonged CAR expression in dividing lymphocytes, whereas non-viral platforms such as mRNA-loaded nanoparticles typically drive transient CAR expression. While transient expression may offer improved safety and tunability, it may also necessitate repeated dosing to maintain antitumor activity.

The foundational proof of concept for in vivo CAR-T generation predates nanoparticle systems and was first explored through viral vector platforms. Agarwal and colleagues demonstrated that a T cell-tropic lentiviral vector could selectively transduce human CD8⁺ and CD4⁺ lymphocytes in vivo, achieving targeted CAR expression and tumor elimination in vivo without prior cell isolation.^2,3 Zhou et al. and Jamali et al. further optimized lentiviral vectors for highly selective CAR gene transfer to CD4⁺ and CD8⁺ T cell subsets.^4,5 Subsequent work by Pfeiffer et al. and Michels et al. validated these findings, showing in vivo CD19-CAR-T cell generation with resultant B cell depletion and signs of cytokine release syndrome (CRS) in preclinical models.^6,7 Additionally, Nawaz and colleagues employed adeno-associated virus (AAV)-mediated delivery for in vivo CAR-T generation against human T cell leukemia, providing further evidence for the feasibility of viral-based approaches.⁸ Indeed, AAVs are among the most suitable tools for in vivo gene delivery as long-term expression can be achieved through episomal expression, without integration into the host genome, with good efficiency in both dividing and non-dividing cells, while they are considered one of the safest vectors for gene therapy.^9,10 To date, the direct in vivo engineering of NK cells with CAR-encoding nucleic acids has only been attempted by Andorko and colleagues, who designed a lentiviral vector encoding an anti-CD20 CAR pseudotyped with a novel binder to provide targeted transduction of CD7⁺ NK cells following intravenous delivery.^10,11 Collectively, these studies established that direct, in-body reprogramming of lymphocytes or NK cells is achievable, controllable, and therapeutically relevant.

Recent advances in non-viral delivery have enabled in vivo CAR-T cell generation using safer, transient, and scalable platforms. Rurik and colleagues demonstrated that lipid nanoparticles (LNPs), functionalized to target CD5 on T cells, could deliver mRNA encoding an antifibrotic CAR directly into circulating lymphocytes, ameliorating cardiac fibrosis in vivo.^12,13 Building upon this, Billingsley et al. engineered ionizable LNPs with extrahepatic tropism, capable of transfecting splenic T cells with CAR mRNA and inducing near-complete B cell depletion in murine models.^14,15,16 Rodriguez and co-workers extended these findings, showing that a similar targeted LNP system could reprogram T cells in humanized mice and non-human primates, achieving functional CAR expression without ex vivo manipulation.¹⁷ Most recently, Lemgart et al. reported the development and preclinical validation of a clinically scalable LNP formulation for in vivo CAR-T generation, demonstrating robust in vitro transfection efficiency and in vivo antitumor activity in murine models.¹⁸

A critical precursor to these studies came from Parayath and Stephan, who demonstrated in situ T cell programming using synthetic polymeric nanocarriers loaded with in-vitro-transcribed CAR mRNA, inducing transient antigen-specific cytotoxicity without off-target gene delivery.^19,20 These biodegradable nanocarriers, functionalized for T cell tropism, successfully induced transient CAR expression in circulating lymphocytes, resulting in antigen-specific cytotoxicity without off-target gene delivery. This work provided the first clear evidence that in vivo CAR-T generation was not only feasible but also controllable and reversible, a pivotal step toward the clinical translation of non-viral gene delivery for immunotherapy.

Zhao et al. further developed virus-mimetic fusogenic nanovesicles to produce CAR-T cells in vivo, highlighting additional delivery strategies that mimic viral entry while retaining non-viral safety advantages.²¹ Most recently, Hamilton et al. demonstrated that enveloped delivery vehicles (EDVs) can efficiently transduce human T cells in vivo, enabling the generation of functional CAR-T cells directly within the body. These EDVs are engineered with antibody-derived fragments for targeted delivery and can co-deliver Cas9 RNPs and CAR transgenes, allowing simultaneous genome editing and CAR expression. In humanized mouse models, EDV-generated CAR-T cells exhibited potent antitumor activity against CD19⁺ B cells while minimizing off-target delivery and systemic exposure. Compared with AAVs, EDVs offer more precise lymphocyte targeting, reduced liver tropism, and transient, controllable transgene expression, addressing key safety and biodistribution limitations of viral vectors. This work highlights EDVs as a versatile, precise, and potentially safer alternative to both viral and conventional lipid-based approaches for in vivo T cell engineering.²²

In parallel, other studies have also explored polymeric nanoparticles, cationic polymers, and combinations of biomaterials to optimize transfection efficiency and in vivo CAR-T generation.^{23,24,25,26,27}

While transient expression offers safety and reversibility, long-lasting immune control often demands more durable genetic modification. DNA-based nanocarriers and non-viral integrating systems have advanced this frontier. In a seminal contribution, Smith and colleagues achieved in situ programming of leukemia-specific T cells using synthetic DNA nanocarriers, leading to persistent CAR expression and potent antileukemic activity.²⁸ More recent advances employ hybrid delivery systems that co-encapsulate CAR DNA and transposase mRNA within T cell-targeted LNPs, generating stable, functional CAR-T populations in vivo.^29,30,31 Collectively, these approaches bridge the gap between transient mRNA-based therapies and traditional integrating viral vectors, offering programmable control over CAR expression durability tailored to disease context.

The biological sophistication of these technologies continues to evolve rapidly. For instance, preconditioning with interleukin-7 (IL-7) enhances mRNA translation and improves nanoparticle uptake by T cells, substantially boosting expression efficiency.³² Ligand-directed or antibody-decorated nanoparticles now allow selective delivery to CD3⁺, CD4⁺, or CD8⁺ subsets, while modified lipid chemistries increase extrahepatic biodistribution. Beyond oncology, Aera Therapeutics has adapted similar constructs to generate in vivo CAR-T cells targeting autoreactive B cells in autoimmune disease models.³³ In other translational efforts, Capstan Therapeutics has produced a similar novel product candidate (CPTX2309) for in vivo mRNA engineering of anti-CD19 CAR-T cells to target alloreactive B cells in autoimmune disease, utilizing novel CD8-targeted LNPs, demonstrating robust antigen-specific cytotoxicity in preclinical models, and highlighting the feasibility of systemic administration.³⁴ Together, these studies highlight the adaptability of in vivo CAR-T platforms across diverse immune-mediated conditions (Table 1).

Table 1.

Evolution of in vivo CAR-T cell generation platforms

Platform/delivery system	Mechanism/target cell	Key findings	Advantages	Limitations/challenges	Reference
T cell-tropic lentiviral vectors	lentivirus pseudotyped for T cell selectivity (e.g., CD4+/CD8+ subsets)	achieved selective in vivo transduction of CD4+/CD8+ T cells; functional CAR expression and tumor clearance in preclinical models	stable integration and long-term CAR expression	risk of insertional mutagenesis; CRS; scalability and biosafety concerns	Agarwal et al.2,3; Zhou et al.4; Jamali et al.5; Pfeiffer et al.6; Michels et al.7
AAV-based vectors	AAV tropism for lymphocytes	enabled in vivo CAR expression with episomal persistence; no genome integration	long-term expression, high safety profile	limited cargo size; pre-existing immunity; liver tropism	Nawaz et al.8; Naso et al.9; Pinto et al.10
mRNA lipid nanoparticles (LNPs)	CD5- or CD8-targeted LNPs deliver CAR mRNA to circulating T cells	transient in vivo programming of CAR-T cells inducing B cell depletion and antifibrotic or antitumor activity	transient, controllable expression; non-integrating; scalable	short-lived expression; need for repeated dosing; targeting efficiency	Rurik et al.12; Aghajanian et al.13; Billingsley et al.14,15,16; Rodriguez et al.17; Lemgart et al.18
Polymeric nanocarriers/synthetic systems	synthetic or virus-mimetic nanocarriers functionalized for T cell tropism	induced transient, antigen-specific CAR expression; demonstrated controlled in vivo cytotoxicity	fully synthetic, tunable degradation, and high safety	transient expression, lower efficiency than viral systems	Parayath et al.19; Moffett et al.20; Zhao et al.21
Enveloped delivery vehicles (EDVs)	enveloped nanoparticles displaying antibody fragments (e.g., CD3/CD8 binders)	achieved selective in vivo transduction of human T cells with functional CAR expression and Cas9 co-delivery	precise targeting; reduced liver tropism; lower off-target effects vs. AAV	manufacturing complexity; new regulatory pathway	Hamilton et al.22
Polymeric and cationic hybrid nanoparticles	DNA/mRNA complexation via ionizable polymers	enhanced in vivo transfection efficiency and CAR expression	modular, low-cost, and easily tunable	variability in biodistribution; cytotoxicity at high doses	Rai et al.23; Samal et al.24; Santo et al.25; Qin et al.26; Large et al.27
DNA nanocarrier/non-viral systems	DNA + transposase mRNA co-delivery via targeted LNPs	enabled durable CAR expression and sustained antitumor activity	programmable integration, hybrid stability	genotoxicity risk; complex design	Smith et al.28; Bimbo et al.29; Ashoti et al.30; Manuri et al.31
Immune-conditioning enhancers	IL-7 preconditioning	preconditioning with IL-7 enhances T cell translational capacity and nanoparticle uptake prior to in vivo CAR delivery	improves transfection efficiency, activation, and persistence	translational optimization of cytokine dosing, timing of kinetics, durability, and immune balance	Tilsed et al.32
B cell-based autoimmune disease	CD3/CD8 targeting, immune activation	demonstrated in vivo CAR-T generation for B cell depletion in autoimmunity models	broad disease applicability; improved targeting efficiency	limited publicly available clinical data on durability, manufacturability, and long-term safety in humans	Aera Therapeutics33,34

Allogeneic cellular therapy products

In addition to in vivo delivery systems, allogeneic CARs represent a promising strategy to overcome some of the logistical and biological hurdles of autologous CAR-T therapy. Specifically, these products offer benefits including rapid availability, for patients who cannot wait weeks for autologous manufacturing; consistent potency, derived from healthy donor-derived lymphocytes that generally possess superior proliferative capacity and cytotoxic function compared to the, often, senescent or functionally exhausted T cells of heavily pretreated and immunocompromised cancer patients; and scalable production that can support repeated dosing and more flexible infusion schedules for one or multiple patients from a single donor batch and that can be leveraged to support adaptive clinical trial designs and combination strategies. By leveraging healthy donor T cells, off-the-shelf products can be manufactured in bulk, cryopreserved, and rapidly delivered, providing treatment options for patients with aggressive or refractory disease, while also ensuring greater product consistency, quality control, cost efficiency, broader distribution, and reduced costs per dose. In addition, these treatments could theoretically improve the potency of autologous products due to enhanced graft-versus-leukemia (GvL) effects.

On the other hand, for the same reasons of alloreactivity, these products present challenges with increased risk of severe and even lethal GvHD. Another issue is host-versus-graft rejection (HvG) whereby the recipient’s immune system recognizes donor-derived CAR-T cells as foreign, leading to rapid clearance and limited persistence. These immune rejection phenomena remain a key limitation in achieving durable responses and tolerable safety profiles with allo-CARs.

To mitigate these challenges, genome editing approaches have been employed to eliminate TCR- and MHC-mediated responses. The approaches have employed TRAC disruption, for example, to eliminate TCR expression and/or β-2-macroglobulin (B2M) deletion to abrogate MHC-I expression to reduce TCR and MHC-I-dependent immune recognition. Other strategies grounded on selective host lymphodepletion by gene editing of donor cells to render them unsusceptible to destruction by targeted agents, such as anti-CD52 antibodies, have also been employed.

Safety control mechanisms have also been integrated into allogeneic platforms, including transient CAR expression and/or inducible suicide switches, such as inducible caspase-9 or suicide gene systems that enable for controlled ablation of the product in case of toxicity to further enhance clinical safety. Advanced editing tools such as TALENs, CRISPR-Cas9, and base editors are now increasingly being employed to produce universal CAR-T platforms that are functionally active but immunologically silent in terms of intolerable alloreactivity.

Beyond Tαβ cells, allogeneic CAR-NK cells and γδ T cell platforms are especially promising given that these cell types naturally lack conventional TCRs, reducing GvHD risk and simplifying production. Additional efforts to enhance persistence focus on immune cloaking and metabolic optimization through strategies such as overexpression of non-classical human leukocyte antigen (HLA) molecules (HLA-E or HLA-G) to inhibit NK-mediated rejection and secretion of immune-modulatory cytokines such as IL-7 or IL-15 to promote memory formation combined with engineering of orthogonal cytokine receptors to support selective in vivo expansion.

Allo-CAR-T and γδ CAR-T cells

Recent clinical studies have been successful and support the feasibility and safety of allogeneic, off-the-shelf CAR T cells for both solid and hematologic malignancies. Shahid et al. reported on an allogeneic CAR-T platform for patients with relapsed or refractory B cell malignancies, demonstrating that donor-derived CAR T cells could be safely infused with manageable toxicity, potent antitumor activity, and durable responses.³⁵ Specifically, this phase 1 trial evaluated an off-the-shelf allogeneic CAR T cell product derived from Epstein-Barr virus-specific T cells (EBV-VSTs) in patients with relapsed or refractory B cell malignancies. Donor EBV-VSTs were engineered to express a CD19-specific CAR and cryopreserved for rapid infusion. Notably, conventional HLA matching was not required due to the natural allorestricted properties of EBV-VSTs, which also reduced GvHD risk. The study demonstrated that the product was well tolerated, with robust engraftment, persistence, and objective antitumor responses, providing proof of concept for a scalable, off-the-shelf allogeneic approach in B cell malignancies.

Similarly, Roddie et al. developed an allogeneic matched donor CD19 CAR product for adult patients with B cell acute lymphoblastic leukemia (B-ALL) following failure of allogeneic stem cell transplantation.³⁶ Matching of donors was performed using standard HLA typing techniques. Donors (n = 17) underwent leukapheresis collection, and CAR-DLI (donor lymphocyte infusion) was manufactured and infused into 14 adult patients with B-ALL. The study evaluated the risks and benefits of pre-lymphodepleting chemotherapy and the efficacy of repeated CAR-DLI dosing per conventional DLI protocols. Patients were divided into two groups: one received CAR-T cell therapy alone, while the other received CAR-T cell therapy following lymphodepletion (fludarabine and cyclophosphamide). The results demonstrated that lymphodepletion prior to CAR-T cell infusion significantly improved CAR-DLI engraftment, expansion, and persistence of the CAR-T cells, leading to better event-free survival and overall survival rates. Importantly, this approach did not result in significant GvHD, highlighting the potential of allogeneic cells in delivering therapeutic payloads without inducing severe immune complications. Collectively, these studies underscore that allogeneic CAR-T cells represent increasingly clinically viable approaches for treating B cell malignancies.

The aforementioned studies have been predated by investigational products with mixed results. ALLO-501/ALLO-501A (Allogene Therapeutics) is a CD19-targeted CAR-T cell that incorporates TALEN-mediated TRAC knockout and CD52 knockout, enabling selective lymphodepletion with the anti-CD52 antibody (ALLO-647) of the host cells but preventing destruction of ALLO-501 by ALLO-647 to minimize GvHD and HvG.³⁷ The product is being evaluated in patients with relapsed/refractory large B cell lymphoma and follicular lymphoma in phase 1/2 trials and has produced early results showing favorable safety profiles, with manageable CRS and early antitumor responses, though persistence remains limited.³⁷ Notably, as of August 2025, the death of a trial patient due to disseminated adenovirus infection-related liver failure from profound lymphodepletion caused by the administration of ALLO-647 has resulted in the suspension of further trialing with the lymphodepleting agent ALLO-647.

UCART19 (Servier/Allogene) is one of the earliest allogeneic CAR-T constructs to have been investigated, also employing TALEN-based TCR and CD52 disruption.³⁸ UCART19 demonstrated proof-of-concept efficacy in pediatric and adult B-ALL; however, transient cell persistence highlighted the need for improved host conditioning and immune evasion strategies.³⁸

Allogeneic CAR-T products have also been studied in the context of multiple myeloma. ALLO-715 (Allogene Therapeutics) is an allogeneic, off-the-shelf CAR-T therapy targeting BCMA for relapsed/refractory multiple myeloma. It employs TALEN gene editing to disrupt the TRAC and CD52 loci, enabling universal donor use and selective lymphodepletion with the anti-CD52 antibody ALLO-647. In the phase 1 UNIVERSAL trial (NCT04093596), ALLO-715 demonstrated an overall response rate of 71%, with 25% of patients achieving complete or stringent complete responses. CRS occurred in 56% of patients (grade ≥3 in 2%), and no GvHD was reported. Ongoing studies are evaluating combination strategies with nirogacestat, a γ-secretase inhibitor that was added to enhance target density and clinical activity, and next-generation constructs to improve persistence and efficacy. ALLO-715 exemplifies the potential of gene-edited, universal CAR-T cells to deliver scalable, rapid, and clinically effective therapy in multiple myeloma.

Prior to ALLO-715, BCMA-UCART (NCT03752541) was investigated as another allogeneic, off-the-shelf CAR-T therapy targeting BCMA for relapsed/refractory multiple myeloma. The product, derived from healthy donor T cells, uses CRISPR-Cas9 gene editing to disrupt the TRAC locus and HLA class I and was administered following lymphodepleting chemotherapy (cyclophosphamide and fludarabine) in a phase 1/2 dose-escalation trial. Early data and trial design highlight the potential of BCMA-UCART; however, the trial has been temporarily suspended, and clinical outcome data remain limited. This construct exemplified early on the promise and ongoing challenges of universal, gene-edited CAR-T platforms in multiple myeloma.

Similarly, UCARTCS1 (NCT04142619), an allogeneic, off-the-shelf CAR-T cell therapy targeting CS1 (SLAMF7), developed by Cellectis for relapsed/refractory multiple myeloma, utilizes TALEN gene editing to disrupt the TCRαβ locus. A phase 1, open-label, dose-escalation trial (MELANI-01) evaluated the safety and efficacy of a single infusion of UCARTCS1A in heavily pretreated patients. Preliminary results indicated early antitumor activity, with detectable CAR-T cells and serum cytokine changes correlating with responses. Despite these findings, the trial was temporarily placed on clinical hold by the FDA in July 2020 following a fatal cardiac event. The hold was lifted in November 2020 after protocol adjustments were made to enhance patient safety. UCARTCS1 exemplifies the potential of gene-edited, off-the-shelf CAR-T therapies in treating multiple myeloma, though challenges remain in ensuring safety and long-term efficacy.

Precision BioSciences’ PBCAR0191 and PBCAR269A represent other important examples of genome-edited, off-the-shelf CAR-T cells advancing toward clinical translation. PBCAR0191 employs ARCUS nuclease-mediated gene editing to achieve precise and efficient disruption of the TRAC locus, thereby mitigating GvHD responses while enabling consistent CAR expression. This CD19-directed construct is manufactured from healthy donor T cells, expanding the feasibility of scalable, cryopreserved product generation for patients with relapsed or refractory B cell malignancies, including ALL and non-Hodgkin lymphoma (NHL).³⁹ Early-phase clinical evaluation has demonstrated manageable safety with low rates of severe CRS and neurotoxicity, accompanied by evidence of initial antitumor responses in heavily pretreated cohorts.^39,40 However, like other first-generation allogeneic CAR-T products, limited in vivo persistence has emerged as a major hurdle, prompting ongoing optimization of lymphodepletion and immune evasion strategies.

In parallel, PBCAR269A, another ARCUS-edited CAR-T program, extends this approach to BCMA-targeted multiple myeloma, further validating the modularity of the ARCUS platform for diverse antigenic targets. PBCAR269A was being explored clinically with or without combination therapy with nirogacestat (NCT04171843). The trial was terminated early due to lack of clinical activity. Nevertheless, together, these trials highlight both the feasibility and the current boundaries of allogeneic CAR-T therapy. While ARCUS editing confers high precision and favorable manufacturing consistency, achieving durable engraftment and long-term disease control remains a key translational challenge. In addition, recent innovations in immune evasion strategies are also reshaping the design of allogeneic CAR-T products.

Precision BioSciences’ PBCAR19B, a next-generation stealth allogeneic CD19 CAR-T cell therapy, incorporates gene edits that suppress immune recognition and enhance persistence.⁴¹ In early clinical evaluation, PBCAR19B demonstrated potent antitumor activity and durable B cell depletion in patients with relapsed/refractory B cell lymphoma, supporting the feasibility of sustained immune evasion without inducing GvHD. These data exemplify the shift toward rationally engineered, immune-cloaked allogeneic CAR-T platforms designed for both safety and long-term function in immune-competent hosts.

CRISPR Therapeutics’ CTX110, CTX120, and CTX130 represent complementary efforts to develop CRISPR-Cas9-based, TRAC- and/or MHC-edited allogeneic CAR-T cells targeting CD19, BCMA, and CD70, respectively. CTX110 has shown encouraging complete response rates in relapsed B cell lymphoma, with manageable toxicity and no GvHD, underscoring the feasibility of CRISPR-based universal CAR platforms.⁴² Preclinical studies have demonstrated that CTX120 exhibits desired in vivo persistence and antitumor effects in multiple myeloma mouse models.⁴³ Building upon these findings, a phase 1 clinical trial (NCT04244656) was conducted to evaluate the safety and efficacy of CTX120 in patients with relapsed/refractory multiple myeloma who have received at least two prior lines of therapy. The trial assessed various dose levels and included patient inclusion criteria similar to those for ALLO-715; however, the study was terminated early due to the death of a patient.

In solid tumors, CTX130 represents the first-in-human evaluation of an allogeneic CD70-directed CAR-T therapy for advanced clear cell renal cell carcinoma. In a phase 1 trial, CTX130 showed favorable safety with no dose-limiting toxicities, achieved disease control in 81% of patients, and induced a durable complete response in one patient lasting over 3 years. These results highlight the feasibility and potential of allogeneic CAR-T approaches in solid tumors.⁴⁴ CRISPR Therapeutics now is pivoting toward next-generation candidates (e.g., CTX112 and CTX131) with novel edits (e.g., Regnase-1 and TGFBR2) aimed at enhancing potency, reducing exhaustion, and improving persistence, building on findings from CTX110/CTX120/CTX130.

In the setting of acute myeloid leukemia (AML), off-the-shelf γδ CD33 CAR-T cells generated from healthy donor T cells by activation with zoledronic acid and IL-2 for 7–14 days have been explored preclinically.⁴⁵ These cells combine innate-like recognition with minimal risk of GvHD. The cells exhibited potent cytotoxicity and favorable safety attributes, supporting their translational potential as an allogeneic platform for myeloid malignancies.⁴⁵

Beyond cancer, allogeneic CAR-T platforms are now entering trials for autoimmune disease therapy. BMS-986515 is an allogeneic CD19-targeted CAR T cell derived from healthy donor T cells that is under phase 1 evaluation for severe, refractory autoimmune diseases (NCT07115745). The trial aims to assess the safety, tolerability, optimal dose, and preliminary efficacy of BMS-986515 and also involves co-administration of other drugs such as fludarabine, cyclophosphamide, and tocilizumab.

In summary, recent clinical studies have established that gene-edited, off-the-shelf CAR-T cells can be manufactured reproducibly, infused safely, and induce meaningful responses across hematologic malignancies. However, limited persistence, infection risk under profound lymphodepletion, and immune rejection remain central challenges. Ongoing refinements in genome editing, immune cloaking, and conditioning regimens are poised to define the next generation of allogeneic CAR platforms for oncology and beyond.

CAR-NK, iNKT, and γδ T cell platforms

Several investigational allogeneic CAR-NK cells, including invariant natural killer T (iNKT) and γδ T cell products, are in preclinical and clinical evaluation, targeting hematologic and solid malignancies. Notably, NK cells for such universal CAR products can be sourced from umbilical cord (UC) blood, peripheral blood (PB), or iPSCs, with each featuring different properties.

One of the pioneering research groups on NK-CARs is the Rezvani group at MD Anderson Cancer Center. In a pivotal phase 1/2 clinical trial, the Rezvani group evaluated UC-derived NK cells engineered to express an anti-CD19 CAR and IL-15 (CAR19/IL-15) in patients with CD19⁺ B cell malignancies. The study demonstrated an overall response rate of 48.6% at 30 days post-treatment, with 1-year progression-free survival and overall survival rates of 32% and 68%, respectively. Notably, the therapy exhibited an excellent safety profile, with no cases of severe CRS, neurotoxicity, or GvHD.⁴⁶ Additionally, this study identified predictors of response. Specifically, the most significant predictors for superior outcome were receiving CAR-NK cells from a cord blood unit (CBU) with nucleated RBCs ≤8 × 10⁷ and a collection-to-cryopreservation time of up to 24 h. These optimal CBUs yielded highly functional NK cells that were enriched in effector-related genes. In contrast, NK cells from suboptimal CBUs exhibited upregulation of inflammation, hypoxia, and cellular stress programs.⁴⁶

More recent findings highlight the transcription factor cyclic AMP response element modulator (CREM) as a critical regulator of CAR-NK cell function via IL-15 signaling.⁴⁷ Specifically, upregulation of CREM was found to limit the effectiveness of CAR-NK cells, and CREM targeting was found to enhance the persistence and antitumor activity of CAR-NK cells, providing a potential therapeutic strategy to improve the efficacy of these therapies.⁴⁷ Additionally, the group has developed the PreCiSE (Precise Cell Engineering for Solid Tumors) platform, a genome-wide CRISPR screening tool designed to identify genetic targets that can enhance the antitumor activity of CAR NK cells.⁴⁸ This platform has provided insights into key regulators and pathways tumors exploit to suppress NK cell function, enabling the engineering of CAR-NK cells that are more resilient to these tumor-induced suppressive mechanisms.⁴⁸

Building on these advances, a phase 1/2 trial pioneered from MD Anderson is investigating CD5-directed, IL-15-transduced UC blood-derived NK cells for relapsed/refractory T cell malignancies, mantle cell lymphoma, and chronic lymphocytic leukemia (NCT05110742), integrating lymphodepletion, CAR targeting, and an iC9 safety switch. Preclinical studies demonstrated potent cytotoxicity against CD5⁺ malignancies and enhanced NK cell survival.⁴⁹ This trial highlights the potential of off-the-shelf, gene-modified NK cells to overcome GvHD while providing scalable, rapid-access therapy for otherwise difficult-to-treat T cell malignancies.

Another research group at the forefront of CAR-NK cell therapy is the Karadimitris team at Imperial College London. Their research primarily revolves around the use of iNKT cells, a unique subset of immune cells that bridge innate and adaptive immunity. These cells offer several advantages over traditional NK and T cells, including potent antitumor activity, the ability to be used off-the-shelf without the risk of GvHD, and the potential for engineered enhancements. Both autologous and allogeneic iNKT cells engineered to express CARs targeting tumor-specific antigens have demonstrated robust antitumor activity that often exceeded that of conventional CAR-T cells in preclinical models, with several products being trialed clinically.⁵⁰ More recently, this group in collaboration with the University of Oxford has engineered an allogeneic off-the-shelf bispecific CAR-iNKT construct targeting CD19 and CD133 for high-risk KMT2A-rearranged ALL that eradicated leukemia cells, including those in the medullary and leptomeningeal compartments in relevant in vitro and in vivo models, supporting the translational potential of this product.^51,52 The bispecific design aids in overcoming tumor heterogeneity and preventing immune escape. Indeed, prior to these findings, promising preclinical results facilitated the licensing of the CAR-iNKT technology pioneered from the Karadimitris group to Arovella Therapeutics (formerly SUDA Pharmaceuticals), aiming to accelerate clinical translation and broaden patient access.⁵³ In addition to targeting B cell malignancies, the Karadimitris group has explored the application of CAR-iNKT cells for T cell lymphomas. Rowan and colleagues engineered CAR-iNKT cells to target clonal TCRVβ chains, providing a highly precise strategy to eliminate malignant T cells in vitro and in vivo while sparing healthy T cell populations.⁵⁴ Looking forward, the group is expanding applications to additional malignancies, including multiple myeloma.⁵⁵

Several additional allogeneic NK cell products are advancing through preclinical and early clinical testing. AgenT-797 (MiNK Therapeutics) is a non-genetically engineered, non-CAR, allogeneic, off-the-shelf iNKT cell therapy derived from healthy donors and expanded ex vivo. Early-phase clinical data demonstrate a favorable safety profile and signs of antitumor activity across solid tumors and hematologic malignancies (NCT04754100, NCT05108623, and NCT06251973), with no high-grade CRS or neurotoxicity.⁵⁶ In solid tumors refractory to PD-1 blockade, agenT-797 monotherapy and combination cohorts have shown disease stabilization and partial responses, with durable reductions in tumor burden observed in subsets of patients with advanced solid tumors.⁵⁷

Similarly, API-192 (Appia Bio) represents a notable example of a stem-progenitor-derived, off-the-shelf CAR-NK-T approach.⁵⁸ Developed from UC CD34⁺ hematopoietic progenitors differentiated into NK-T-like effector cells, API-192 carries dual CD19/CD20 CARs and is armored with soluble IL-15 to support in vivo persistence. Preclinical data show potent dual-antigen cytotoxicity and in vivo antitumor activity in B cell malignancy models, supporting Investigational New Drug (IND) development. These data support the rationale that progenitor-derived NK-T products may offer greater manufacturing consistency and fitness to mature donor lymphocytes. Key translational questions remain including optimal IL-15 dosing, dual-CAR signaling balance, and clinical persistence. Yet API-192 exemplifies a pragmatic route to scalable, multi-antigen allogeneic cellular therapy. Collectively, iNKT-based approaches such as agenT-797 and API-192 represent a scalable, low-toxicity alternative to conventional allogeneic CAR-T and CAR-NK therapies.

FT596 (Fate Therapeutics) is a CD19-targeted, iPSC-derived, off-the-shelf CAR-NK product engineered with a high-affinity, non-cleavable CD16 Fc receptor and an IL-15/IL-15 receptor fusion protein to enhance persistence.⁵⁹ The phase 1 trial assessed FT596 in patients with relapsed or refractory B cell lymphoma, demonstrating that the product was well tolerated, with manageable CRS and no observed neurotoxicity. The recommended phase 2 dose was identified as 1.8 × 10⁹ cells per cycle. Notably, the therapy induced deep and durable responses in patients with both indolent and aggressive lymphoma subtypes. These findings underscore the potential of iPSC-derived, gene-modified NK cells as a potent platform for cancer treatment, offering advantages such as scalable manufacturing and reduced risk of GvHD. NKX019 (Nkarta Therapeutics), a CD19-directed allogeneic NK cell product, is under phase 1 study (NCT05020678) in relapsed/refractory B cell malignancies to evaluate safety and antitumor activity. The therapy is engineered to co-express a humanized anti-CD19 CAR and membrane-bound IL-15 to enhance persistence and cytotoxic activity. Preclinical studies demonstrated robust in vitro and in vivo antitumor effects, with a rapid onset of activity and extended NK cell survival, supporting its potential as a scalable and safe alternative to autologous CAR-T therapy.⁶⁰

The Romee group, at Dana-Farber Cancer Institute (DFCI), in collaboration with the Massachusetts Institute of Technology (MIT) has pioneered an innovative strategy to further improve the persistence, safety, and antitumor activity of allogeneic CAR-NK cells via immunological clocking in a “one-step” construct.^61,62 In this study, the authors demonstrated that a single gene construct that combines a short hairpin RNA (shRNA) that selectively interferes with HLA class I but not HLA-E expression, a CAR, and PD-L1 or single-chain HLA-E (SCE) enables the one-step construction of allogeneic CAR-NK cells that evade host-mediated rejection both in vitro and in a xenograft mouse model. The expression of PD-L1, CD47, and HLA-E correlated with reduced clearance by host T and NK cells and enabled superior tumor control compared to unmodified CAR-NK cells. Importantly, this approach maintained safety by mitigating off-target immune activation. This study exemplifies a next-generation strategy CAR-NK cells that are both functionally potent and immunologically cloaked.

These findings complement ongoing clinical efforts with CAR-NK products such as FT596, NKX019, and AgenT-797, emphasizing the potential of combining gene editing with immune cloaking to overcome HvG and enhance therapeutic durability. Similarly, ADI-270 (Adicet Bio) represents an armored allogeneic γδ CAR T approach targeting CD70 that also expresses a dominant-negative TGF-β receptor II (dnTGFβRII), designed to enhance persistence and activity in relapsed/refractory renal cell carcinoma that has been studied in solid and hematologic malignancies.⁶³ AlloNK (Artiva Biotherapeutics) comprises an off-the-shelf, non-genetically modified NK cells aimed at augmenting antibody-dependent cellular cytotoxicity (ADCC) in combination with monoclonal antibodies, currently under evaluation for B cell malignancies (NCT04673617 and NCT05883449) and B cell-dependent rheumatologic diseases (NCT06991114 and NCT06265220) including rheumatoid arthritis, Sjögren’s Disease, systemic sclerosis, systemic lupus erythematosus, and lupus nephritis. This study highlights broader applications of NK-CAR cells for autoimmune indications.

In the setting of multiple myeloma, NK-CAR cells targeting CD138, CSI (SLAMF7), GPRC5D, and BCMA receptors are also being studied.⁶⁴ Maroto-Martin et al. performed a head-to-head comparison of NKG2D- versus BCMA-directed CAR-NK constructs, showing robust elimination of multiple myeloma targets while highlighting mechanistic differences in activation and persistence between these clinically relevant designs.⁶⁵ Building on this, Cao et al. developed an allogeneic CAR-NK platform targeting both BCMA and GPRC5D, demonstrating enhanced tumor control in preclinical models and supporting the feasibility of dual-antigen targeting to overcome antigen escape.⁶⁶ Together, these studies underscore the versatility and translational potential of CAR-NK therapies for multiple myeloma, providing a rationale for ongoing and future clinical evaluation.

Complementary efforts are also evaluating allogeneic dual-target CAR-T products (e.g., BCMA/GPRC5D). NCT05325801 investigates allogeneic CAR-T cells engineered to target both BCMA and GPRC5D in patients with relapsed or refractory multiple myeloma. Derived from healthy donor T cells, this off-the-shelf CAR-T product leverages gene editing to reduce T cell receptor-mediated alloreactivity, addressing the inherent risks of GvHD and host rejection. The trial’s primary objectives focus on safety and tolerability, with secondary endpoints assessing pharmacokinetics and preliminary antitumor activity. By simultaneously engaging two clinically relevant antigens, this dual-target approach aims to enhance tumor eradication, limit relapse due to single-antigen loss, and offer scalable, rapid-access cellular therapy to patients who might otherwise be ineligible for autologous CAR-T. Early preclinical data support robust cytotoxicity and persistence, underscoring the potential of allogeneic, dual-antigen CAR-T platforms to redefine adoptive immunotherapy for multiple myeloma.⁶⁷

Finally, next-generation constructs seek to improve persistence, immune evasion, and scalability. Gene-edited stem cell-derived CAR-T products are generated by engineering hematopoietic progenitor cells ex vivo to express CARs before differentiation into mature effector populations.⁶⁸ These stem cell-derived approaches offer theoretically limitless manufacturing capacity and the potential of long-term immune reconstitution following lymphodepletion.⁶⁸ Hybrid strategies integrating allogeneic products with in vivo CAR-T delivery, such as platelet- or NK cell-mediated nanocarrier systems, may accelerate CAR-T generation directly in the patient, combining the scalability of off-the-shelf products with the precision and efficiency of in situ gene transfer, bridging the gap between allogeneic and in vivo CAR-T paradigms (Table 2).

Table 2.

Summary of allogeneic CAR-T and CAR-NK products discussed

Category	Product/platform	Target antigen	Description	Cell source	Clinical stage/study	Key outcomes/notes
Allo-CAR-T (B cell malignancies)	EBV-VST CAR-T (Shahid et al.35)	CD19	derived from EBV-specific T cells; natural allorestriction	healthy donor T cells	phase 1	off-the-shelf infusion feasible without HLA matching; manageable toxicity, robust engraftment, and durable responses; reduced GvHD risk
	matched donor CAR-DLI (Roddie et al.36)	CD19	standard HLA-matched donor	healthy donor T cells	clinical study	lymphodepletion improved engraftment, expansion, persistence, and survival; no significant GvHD observed
	ALLO-501/ALLO-501A (Allogene)	CD19	TALEN: TRAC knockout, CD52 knockout; selective lymphodepletion	healthy donor T cells	phase 1/2	favorable safety; manageable CRS; persistence limited; trial paused due to ALLO-647 lymphodepletion-related death
	UCART19 (Servier/Allogene)	CD19	TALEN: TCR/CD52 knockout	healthy donor T cells	early clinical	proof-of-concept efficacy in pediatric/adult B-ALL; transient persistence; improved conditioning needed
	PBCAR0191 (Precision BioSciences)	CD19	ARCUS nuclease-mediated gene editing; TRAC knockout	healthy donor T cells	early-phase clinical	manageable CRS/neurotoxicity; limited persistence; initial anti-tumor responses in ALL/NHL
	PBCAR19B (Precision BioSciences)	CD19	ARCUS gene editing; TRAC knockout; miRNA β2Μ suppression	healthy donor T cells	phase 1 clinical trial	potent anti-tumor activity; durable B cell depletion; supports sustained immune evasion and safety without GvHD
Allo-CAR-T (multiple myeloma)	ALLO-715 (Allogene)	BCMA	TALEN: TRAC/CD52 knockout	healthy donor T cells	phase 1 UNIVERSAL trial	ORR 71%, 25% CR/sCR; CRS in 56% (grade ≥3: 2%); no GvHD; combination strategies ongoing
	BCMA-UCART (Bioray Laboratory)	BCMA	CRISPR-Cas9: TRAC and HLA-I knockout	healthy donor T cells	phase 1/2	early promising responses; trial temporarily suspended; limited persistence
	UCARTCS1 (Cellectis)	CS1 (SLAMF7)	TALEN: TCRαβ knockout	healthy donor T cells	phase 1 MELANI-01	early anti-tumor activity; temporary FDA hold due to fatal cardiac event; safety protocol improved
	PBCAR269A (Precision BioSciences)	BCMA	ARCUS nuclease-mediated gene editing; TRAC knockout	healthy donor T cells	early-phase clinical (terminated)	lack of clinical activity; illustrates challenges in persistence and efficacy
	CTX120 (CRISPR Therapeutics)	BCMA	CRISPR: TRAC/MHC edits	healthy donor T cells	phase 1 (terminated)	preclinical: good persistence and anti-tumor activity; trial terminated due to patient death
Allo-CAR-T (solid tumors)	CTX130 (CRISPR Therapeutics)	CD70	CRISPR: TRAC/MHC edits	healthy donor T cells	phase 1	favorable safety; 81% disease control; 1 durable CR > 3 years; solid tumor feasibility shown
Allo-CAR-T (acute myeloid leukemia)	γδ CAR-T (Dwivedi et al.45)	CD33	γδ CAR-T	healthy donor T cells	preclinical	potent cytotoxicity against AML; favorable safety profile, supporting translational potential
Allo-CAR-T (autoimmunity)	BMS-986515	CD19	not specified	healthy donor T cells	phase 1 (autoimmune diseases)	safety and preliminary efficacy under evaluation in severe, refractory autoimmunity
Allo-CAR-NK/iNKT/γδ T cell (solid tumors and hematologic malignancies)	CAR19/IL-15 (Rezvani, MD Anderson)	CD19	CAR + IL-15	UC NK cells	phase 1/2	ORR 48.6% at 30 days; 1-year PFS 32%, OS 68%; excellent safety, no severe CRS/neurotoxicity/GvHD
	CD5-directed IL-15 NK (Rezvani, MD Anderson)	CD5	CAR + IL-15 + iC9 safety switch	UC NK cells	phase 1/2	preclinical: potent cytotoxicity, enhanced NK survival; trial ongoing for T cell malignancies
	iNKT CAR (Karadimitris, Imperial College)	CD19/CD133,	CAR-engineered iNKT	allogeneic iNKT	preclinical/early clinical	robust antitumor activity; bispecific CAR addresses tumor heterogeneity; reduced GvHD risk
	iNKT CAR (Karadimitris, Imperial College)	TCRVβ	CAR-engineered iNKT	allogeneic iNKT	preclinical/early clinical	robust antitumor activity; reduced risk of GvHD
	AgenT-797 (MiNK Therapeutics)	non-CAR, invariant NKT	non-genetically modified iNKT cells	allogeneic iNKT	phase 1/2	early signals of disease control in solid tumors; favorable tolerability; combination cohorts with PD-1 inhibitors
	API-192 (Appia Bio)	CD19/CD20	CAR-iNKT cells; dual CD19/CD20 targeting; IL-15 armoring	allogeneic UC-derived CD34+ hematopoietic progenitors	preclinical/IND-enabling data	potent dual-antigen cytotoxicity and in vivo anti-tumor activity; progenitor-derived iNKT cells offer greater cell fitness and manufacturing consistency
	FT596 (Fate)	CD19	iPSC-derived NK, CAR + non-cleavable CD16 + IL-15/IL-15R	iPSC	phase 1	well tolerated; deep durable responses; scalable off-the-shelf therapy
	NKX019 (Nkarta)	CD19	CAR + membrane-bound IL-15	healthy donor NK cells	phase 1	robust preclinical anti-tumor activity; safe; extended NK survival
	one-step allo-CAR-NK (Romee and Chen, DFCI and MIT)	CD19 or mesothelin	CAR + HLA I knockdown + PD-L1 or HLA-E	healthy donor NK cells	preclinical	immunologically cloaked to reduce HvG rejection; enhanced persistence and tumor control; safe with minimal off-target effects
	ADI-270 (Adicet)	CD70	γδ CAR + dnTGFβRII	healthy donor γδ T cells	preclinical/early clinical	enhanced persistence; solid and hematologic malignancies
	AlloNK (Artiva)	not antigen-specific	non-genetically modified NK cells for ADCC	UC NK cells	preclinical/early clinical	evaluated in B cell malignancies and autoimmune diseases; off-the-shelf, combination with mAbs
	CAR-NK (MM dual-target BCMA + GPRC5D)	BCMA + GPRC5D	dual CAR, gene-edited	healthy donor T cells	phase 1 (ongoing)	early preclinical: robust cytotoxicity, enhanced persistence, dual-target reduces antigen escape
Next-generation/stem cell–derived	gene-edited stem cell CAR-T	various	CAR-expressing hematopoietic progenitors	stem cell-derived	preclinical	potential for limitless scalable manufacturing; long-term immune reconstitution; hybrid in vivo delivery integration

Cellular and vesicular products as gene delivery vehicles for in vivo CAR T cell therapy

Platelets, MSCs, RBCs, and immune cells, or their vesicles, can serve as in vivo CAR delivery vehicles. Most preclinical work to date discussed herein has used autologous or syngeneic sources; however, advances in immune cloaking, universal donor engineering, and membrane modification suggest that allogeneic versions could enable off-the-shelf applications. These strategies could bridge the gap between autologous CAR-T manufacturing and scalable in vivo gene delivery. Notably, carrier-based delivery systems, including engineered immune cells and EVs, are generally expected to induce transient CAR expression, positioning them as potentially safer but less durable alternatives that may require serial administration depending on tumor burden and immune cell turnover.

Platelet-based delivery vehicles for targeted immunotherapy

Emerging evidence suggests that platelets and platelet-derived microparticles can serve as natural delivery vehicles for targeted therapies, including in vivo CAR-T cell strategies. Allogeneic platelet membranes would require immune cloaking, through MHC stripping or PEGylation, to prevent alloimmunization. Platelets possess inherent homing capabilities to sites of vascular injury, inflammation, and tumors, which can be exploited to improve biodistribution and cell-specific targeting.^69,70 Their membranes can also be cloaked around synthetic nanoparticles or liposomes, providing immune evasion, prolonged circulation, and targeted tissue delivery.^71,72 The ability of platelets to travel directly to cancer sites and activate on site makes them a potentially attractive vehicle for targeting cancer therapeutics to the tumor environment.⁷³ Indeed, few groups have explored the utility of platelet-based systems for drug delivery. Hu and colleagues pioneered platelet membrane cloaking of nanoparticles, demonstrating enhanced targeting and reduced clearance by the reticuloendothelial system.⁶⁹ Wang et al. engineered platelet-membrane biomimetic nanoparticles for antitumor drug delivery, showing improved tumor accumulation and efficacy in preclinical models.⁷¹ Kailashiya et al. demonstrated that engineered platelet-derived microparticles can act as natural vectors for targeted drug delivery, highlighting their versatility as bioinspired carriers.⁷⁴

Beyond drug delivery, platelets can be leveraged for immunotherapy applications. Wang and colleaguesactivated platelets in situ with checkpoint inhibitors to enhance post-surgical antitumor immune responses, demonstrating the ability of platelet-based systems to mediate immunomodulatory effects.⁷⁵ Biomimetic liposomes hybridized with platelet membranes have been developed for targeted therapy of atherosclerosis, underscoring the broad potential of platelet-membrane hybrid platforms to deliver biologically active cargos in vivo.⁷²

In another example, Bahmani et al. developed engineered platelet-membrane-coated nanoparticles for localized delivery of a Toll-like receptor agonist for the treatment of cancer in a colorectal tumor model, which lead to enhanced local immune activation and complete tumor regression.⁷⁶ The potential of platelets genetically modified to express surface-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine that induces apoptosis specifically in tumor cells, was explored by Li and colleagues.⁷⁷ TRAIL-expressing modified platelets demonstrated in vitro killing activity and significantly reduced metastases in a mouse model of metastatic prostate cancer.

Collectively, these studies highlight the potential of platelet-inspired delivery systems as complementary or alternative vehicles for in vivo CAR strategies. By leveraging their natural targeting, circulation longevity, and biocompatibility, platelet-derived carriers may overcome limitations of viral vectors and conventional lipid nanoparticles, particularly regarding off-target tissue accumulation and immunogenicity. Integrating platelet-based approaches with allogeneic CAR platforms could provide a hybrid solution that combines the safety, scalability, and precision required for clinical translation.

RBCs/erythrocyte ghosts

RBCs and their erythrocyte-derived vesicles (“ghosts”) have gained attention as biocompatible carriers for therapeutic delivery. While RBCs are naturally immune compatible in autologous settings, allogeneic use would demand universal donor RBC membranes (O⁻ type) or engineered antigen masking. RBCs are abundant, long-circulating, and inherently non-immunogenic, providing a natural shield against immune recognition and rapid clearance.^78,79,80,81 Loading therapeutic cargo into RBCs or creating erythrocyte ghosts enables the delivery of proteins, enzymes, or nucleic acids while minimizing clearance by the reticuloendothelial system.^79,81,82,83 Preclinical studies have demonstrated the use of RBC-based carriers to deliver chemotherapeutics, immunomodulators, and genetic payloads to targeted tissues.^80,81,84,85 RBC membranes have been successfully employed as natural coatings for nanoparticles, enhancing immune evasion, circulation half-life, and tumor accumulation in preclinical models.^78,81,86 Moreover, RBCs and their membrane-derived nanocarriers can be engineered through surface functionalization with antibodies, ligands, or small molecules to achieve tissue-specific targeting and improved biodistribution.^86,87,88

Recent work has shown the potential of engineered RBCs for immunomodulation. Blagovic et al. demonstrated that antigen-loaded erythrocyte ghosts could stimulate antigen-presenting cells and induce robust CD8⁺ T cell activation, leading to tumor regression in mice.⁸⁰ Such findings underscore the versatility of RBC-based systems beyond passive drug delivery, extending to active immunotherapeutic applications. Hybrid platforms, such as liposome-erythrocyte composites, have further enhanced targeted drug delivery and blood circulation times compared with conventional formulations.^86,88

Despite these advantages, several challenges remain. Achieving efficient intracellular cargo loading without compromising RBC integrity, maintaining membrane functionality after engineering, and preventing splenic filtration or premature clearance are ongoing technical hurdles.^87,88,89,90 Nevertheless, due to their scalability, safety, and adaptability, RBCs and erythrocyte-derived carriers represent a promising, clinically translatable platform for systemic and targeted delivery in immunotherapy applications.

MSCs

MSCs have emerged as versatile delivery vehicles owing to their intrinsic tissue-homing properties, low immunogenicity, and ease of ex vivo engineering. MSCs naturally migrate to sites of inflammation, injury, and tumor microenvironments, guided by chemokine and adhesion molecule interactions such as the CXCR4/SDF-1 and platelet-derived growth factor (PDGF) signaling axes, making them ideal candidates for targeted delivery of therapeutic payloads, including cytokines, chemokines, and genetic constructs.^91,92

Engineered MSCs have been employed to deliver a wide range of biologics, from cytokines such as IL-2, CX3CL1, and IFN-β to oncolytic viruses and immune-modulating factors, achieving localized immune activation and antitumor effects while minimizing systemic toxicity.^92,93,94 In addition, MSCs function as biological “factories,” continuously secreting trophic and immunomodulatory molecules such as IDO and TGF-β, which can sustain local immune modulation and support tissue repair.^91,95 Their low immunogenicity and capacity to shield therapeutic cargos from immune clearance further enhance their translational utility.⁹⁶

Nonetheless, challenges remain, including variability in homing efficiency, risks of undesired differentiation, and potential pro-tumorigenic effects within certain microenvironments. Ongoing bioengineering strategies, such as surface modification, genetic programming, and the use of MSC-derived exosomes, are addressing these limitations and expanding the potential of MSCs as next-generation cellular delivery platforms for immunotherapy.^91,92

Immune cells (e.g., monocytes and T cells)

Autologous or allogeneic immune cells can serve as active delivery vehicles, leveraging their natural trafficking to lymphoid tissues and sites of inflammation. Allogeneic monocytes or T cells as carriers would necessitate MHC knockout or CD47 engineering. Monocytes and macrophages can infiltrate tumors and inflammatory sites, enabling localized delivery of therapeutic payloads such as nanoparticles, cytokines, or CAR constructs.^97,98,99 T cells themselves can be repurposed as vehicles for gene delivery or nanoparticle transport, combining homing specificity with cytotoxic function.^100,101 This “Trojan horse” strategy allows simultaneous immune-mediated tumor killing and local delivery of modulatory agents.^97,100 Challenges include maintaining cell viability and functionality after cargo loading, preventing off-target effects, and controlling immune activation to avoid CRS.^98,101 Despite these obstacles, immune cell-based delivery offers a highly specific, adaptable, and self-propagating platform for targeted immunotherapy.^{97,98,100,101}

Exosomes/EVs

Exosomes and EVs are naturally secreted nanoparticles that facilitate intercellular communication and molecular cargo transfer, including proteins, mRNA, and miRNA.^{102,103,104,105,106,107} Their small size, stability in circulation, and intrinsic ability to cross biological barriers make them promising cell-free delivery vehicles for immunotherapy.^102,103,104 EVs can be derived from immune cells, MSCs, endothelial cells, or platelets and engineered to carry CAR mRNA, immunomodulatory proteins, or therapeutic nanoparticles. Notably, CAR-T cell-derived exosomes have been shown to retain tumor-targeting specificity and cytotoxic potential, providing a cell-free alternative that delivers CAR-mediated antitumor signals while potentially reducing the risks of CRS and other toxicities associated with live CAR-T cells.¹⁰⁵ Moreover, CAR-T-derived exosomes can efficiently recognize tumor antigens such as mesothelin, reducing tumor growth in models of triple-negative breast cancer and demonstrating their utility as targeted, cell-free immunotherapeutics.¹⁰⁶ Preclinical studies have shown that EV-based CAR delivery can reprogram T cells or target tumor cells in vivo, reducing the risks associated with viral or lipid-based vectors.^{105,106,107,108,109} Advantages include low immunogenicity, biocompatibility, and potential for off-the-shelf availability.^{103,105,106,108,110} Limitations include heterogeneous cargo loading, production scalability, and rapid clearance, which are all active areas of investigation (Table 3).^{102,104,105,106,107,110}

Table 3.

Summary of alternative cellular and vesicular platforms for in vivo CAR-T delivery

Delivery vehicle	Key features and mechanism	Advantages	Limitations/challenges	Reference
Platelets/platelet-derived microparticles	platelets and platelet membranes naturally home to vascular injury, inflammation, and tumor sites; can be cloaked around nanoparticles or liposomes for immune evasion and targeted delivery	intrinsic homing to tumors/injury sites; biocompatible, low immunogenicity; enable targeted drug or gene delivery; extend circulation time when used as coatings	risk of alloimmunization (allogeneic use); require MHC stripping or PEGylatio; limited scalability for gene delivery applications; stress response activation in storage and manipulation induces nonreversible changes leading to procoagulability	Hu et al.69; Guo et al.70; Wang et al.71; Song et al.72; Kailashiya et al.74; Wang et al.75; Devine and Serrano111
Mesenchymal stromal cells (MSCs)	MSCs migrate to tumors/injury sites via chemokine-adhesion interactions (CXCR4/SDF-1); engineered to secrete cytokines, chemokines, or carry genetic constructs for local immunomodulation	natural tumor homing; low immunogenicity; continuous secretion of therapeutic factors; can deliver cytokines, oncolytic viruses, or CAR payloads	variability in homing efficiency; potential for pro-tumorigenic effects; risk of undesired differentiation	Lan et al.91; Yoon et al.92; Kolluri et al.93; McKenna et al.94; Merino et al.95; Xie et al.96
Red blood cells (RBCs) / erythrocyte ghosts	RBCs and their vesicles serve as immune-evasive, long-circulating carriers for proteins, enzymes, or nucleic acids; can be functionalized with ligands or antibodies for targeting	highly biocompatible and abundant; long circulation half-life; minimal immunogenicity (autologous); scalable and safe	cargo loading without damaging RBCs; potential clearance by spleen; allogeneic use requires antigen masking	Wang et al.78; Nguyen et al.79; Blagovic et al.80; Berikkhanova et al.81; Muzykantov82; Chen et al.83; Magnani et al.84; Koleva et al.85; Zhu et al.86; Sun et al.87; Zhu et al.88
Immune cells (monocytes, T cells)	immune cells naturally infiltrate inflamed or tumor tissues; can act as “Trojan horses” to deliver nanoparticles, cytokines, or genetic payloads while retaining immune activity	targeted migration to tumors; dual function: delivery + cytotoxicity; potential for self-amplifying effect; amenable to autologous or engineered allogeneic use	maintaining viability/function after cargo loading; risk of off-target immune activation or CRS; complex regulatory control for live-cell products	Choi et al.97; Dong et al.98; Liu et al.99; Huang et al.100; Cevaal et al.101
Exosomes / extracellular vesicles (EVs)	cell-derived vesicles carrying mRNA, miRNA, and proteins; can be engineered to deliver CAR mRNA or immunomodulatory payloads	cell-free, stable, and scalable; low immunogenicity; cross biological barriers; retain targeting of parent cells (e.g., CAR-T exosomes)	heterogeneous cargo loading; production and purification scalability; rapid clearance in vivo	Rani et al.102; Balaraman et al.103; Qiu et al.104; Sani et al.105; Yang et al.106; Wang et al.107; Gong et al.108; Tao et al.109; EL Andaloussi et al.110

Conclusion and future prospects

The convergence of in vivo CAR-T engineering with allogeneic and cellular delivery platforms represents a conceptual leap that could redefine the landscape of adoptive immunotherapy. If realized clinically, these systems could eliminate the need for costly and resource-prohibitive patient-bespoke cell manufacturing. The potential advantages are compelling: rapid generation of effector cells, simplified distribution, substantially reduced cost, and fewer infrastructure constraints (Figure 1). Practically, if a single infusion of targeted nanoparticles could yield functional CAR-T cells within days, bypassing current lengthy ex vivo manufacturing processes, this could be a new lifeline for patients for whom time is the most precious commodity and for whom disease can often progress more rapidly than these patients can obtain access to current ex vivo manufactured cell therapy products. Moreover, because these products can be formulated, stored, and distributed like standard biologics, they could democratize access to cell therapy globally, even in resource-limited settings.

Figure 1.

Summary of different approaches including autologous, allogeneic, and in vivo CAR T cell platforms as well as alternative cellular and vesicular systems

Figure produced by Biorender.

Allogeneic cellular therapy products have gained momentum as a pragmatic and scientifically robust response to the constraints of autologous manufacturing. By using healthy donor-derived cells, these platforms overcome the limitations of exhausted T cell phenotypes in heavily pretreated patients, shorten manufacturing time, and enable consistent product quality and scalable batch production. As shown in recent clinical trials, off-the-shelf CAR-T cells such as ALLO-501/501A, UCART19, and ALLO-715 have demonstrated encouraging safety and activity in B cell malignancies and multiple myeloma, while CAR-NK and γδ T cell approaches further mitigate GvHD risk and broaden applicability. Importantly, gene editing technologies, such as TALENs and CRISPR-Cas9, have enabled precise disruption of TCR and MHC loci to minimize GvHD and HvG immune responses, while cytokine support (e.g., IL-15 and IL-7) and bispecific targeting are enhancing persistence and durability. Despite successes, there are persistent challenges in this space, chiefly the limited in vivo persistence of current allogeneic CAR constructs and the safety implications.

In terms of alternative cellular and vesicular sources, these approaches exploit the natural homing and trafficking properties of donor-derived cells or extracellular carriers. Platelets, for example, possess inherent tropism for inflamed vasculature and tumor-associated endothelium. When engineered or coated with therapeutic nanoparticles, they can deliver CAR constructs directly to lymphoid tissues or the tumor microenvironment, enhancing efficacy while minimizing off-target exposure.^{69,70,71,72,74,75} MSCs offer another avenue, with intrinsic tissue-homing, low immunogenicity, and the ability to deliver cytokines, chemokines, or nucleic acids while sustaining local immune modulation.^{91,92,93,95,96} RBCs and erythrocyte-derived ghosts provide a highly biocompatible and long-circulating platform for protein, enzyme, or nucleic acid delivery, with surface functionalization enabling tissue-specific targeting and protection from immune clearance.^{78,79,80,82,83,84,85,86,87,88,89,90} Immune cells, including monocytes, macrophages, and T cells, can serve as “Trojan horses,” delivering nanoparticles or genetic payloads directly to target tissues, combining homing specificity with intrinsic cytotoxic function.^{97,98,99,100,101} Finally, EVs, naturally secreted nanoparticles from immune cells, MSCs, or platelets, can carry mRNA, proteins, or nanoparticles to target immune cells with low immunogenicity, offering an off-the-shelf, cell-free alternative for in vivo reprogramming.^{102,103,104,108,109,110}

The integration of these cellular and acellular delivery vehicles with allogeneic CAR-T platforms introduces a hybrid paradigm in which off-the-shelf donor-derived cells can function as programmable vectors for in vivo gene transfer. In this context, the allogeneic carrier serves as a transient, controlled conduit while the therapeutic payload is the effector being delivered, which may synergize with functions of the carrier self itself. Mechanistic advantages include targeted delivery to lymphoid tissues or tumors, reduced systemic exposure and toxicity, transient yet controllable CAR expression, and adjustable combinatorial engineering to enhance trafficking, retention, and payload release. This convergence enables complementation between the scalability of off-the-shelf products and the rapid, distributable, and precision-targeted nature of in vivo CAR-T programming, while the innate characteristics of the cell carrier may also synergize with the therapeutic being delivered or exert the therapeutic effect by itself, such as in the instance of allogeneic CAR-T cells.

Despite these advances, several translational challenges remain. Precision targeting must ensure that therapeutic payloads selectively transfect T cells while minimizing uptake by off-target populations such as hepatocytes or endothelial cells. Balancing transient expression for safety with sufficient persistence for durable efficacy remains a critical consideration. Indeed, evidence from current CAR-T cell approaches indicates that persistence of CAR-T cells over time is a critical parameter to achieve long-lasting tumor eradication. Newer modalities described may be hindered by limited expression or persistence, requiring repeated administration to sustain therapeutic benefit, which might also have cost implications, whereas durable in vivo reprogramming or long-lived CAR-T populations may enable more cost-effective, one-time interventions. Immune responses to the delivery vehicle or cargo, including cytokine activation or neutralizing antibodies, could limit repeated dosing. Optimizing nanoparticle or cargo loading without impairing carrier cell viability, controlling release kinetics, and embedding safety switches or suicide genes in carrier cells are all essential to mitigate potential risks. Scalable, GMP-compliant manufacturing and storage methods must be established for cellular carriers such as platelets, MSCs, or T cells, as well as for EV-based products. Each of these concerns demands rigorous preclinical characterization and standardized assay development before clinical deployment.¹¹²

Regulatory and ethical considerations will also be pivotal. Regulatory frameworks must also evolve in parallel. In vivo CAR-T manufacturing occupies an ambiguous regulatory space as it is neither fitting a traditional gene therapy nor a standard biologic. Establishing classification criteria and safety testing paradigms will require close collaboration between industry, academia, and regulators. Features such as inducible “off-switches,” suicide genes, or logic-gated CARs could be embedded to ensure controllability and mitigate unintended activation. Ethical considerations are equally critical. The potential irreversible nature of genomic integration necessitates long-term surveillance and robust informed consent frameworks, drawing on lessons from approved gene therapies for disorders such as sickle cell disease. Moreover, equitable access must be built into commercialization strategies from the beginning to avoid exacerbating disparities in advanced therapy access. The infrastructure developed for global mRNA vaccine distribution offers a ready-made foundation for scalable high-quality manufacturing and monitoring. Several years are expected to be required before these therapies are licensed for clinical use (Figure 2).

Figure 2.

Summary of the investigational stage of different approaches, including ex vivo and in vivo CAR T cell platforms, allogeneic cell therapies, as well as alternative cellular and vesicular systems, and their expected timeline in terms of years of research remaining to be licensed for clinical use

Figure produced by Biorender.

Looking ahead, several priorities are clear. The field must converge on standardized metrics for biodistribution, transfection efficiency, and persistence to ensure safety and efficacy.¹¹² Safety controls must be central in the development of in vivo CAR T cells and other in vivo cellular therapy products. Most importantly, early clinical trials must proceed with deliberate caution, beginning with low-risk indications, incorporating comprehensive immune monitoring, and maintaining patient safety as the paramount goal.

Notably, alternative approaches have also been employed for ultra-rapid CAR-T manufacturing without allogeneic donors, alternative cell sources, or in vivo gene delivery systems. The UF-Kure19 phase 1 study demonstrated the feasibility of an ultra-rapid (<24 h) near-instant anti-CD19 CAR-T manufacturing process that completely eliminates ex vivo expansion. Using minimally manipulated, freshly transduced autologous T cells enriched for naive and stem cell memory phenotypes, this approach achieved efficient in vivo expansion and clinical responses (80% overall response rate) with predominantly complete metabolic responses and a manageable safety profile (low-grade CRS and limited neurotoxicity) in relapsed/refractory non-Hodgkin lymphoma. Notably, CAR-T expansion kinetics were delayed but durable, with B cell aplasia persisting in responders. These findings highlight a new generation of on-demand, point-of-care “manufacture-to-infuse” CAR-T platforms that could dramatically reduce production time, cost, and logistical barriers while preserving functional potency.¹¹³

Nevertheless, as in situ CAR-T manufacturing moves from theoretical to tangible, it invites us to a future in which complex immunotherapies are accessible, affordable, and delivered on demand. The transition from ex vivo manufacturing to in vivo precision delivery, if successfully achieved, will be yet another transformative leap in the field of immunotherapy and, specifically, in cellular and genetic engineering-based therapeutic approaches.

Ultimately, the prospect of programming immune cells directly within the human body represents more than a technological milestone; it is a paradigm shift and conceptual redefinition of what cellular therapy can be. Moreover, by integrating allogeneic, platelet, MSC, RBC, immune cell, and EV-based delivery strategies, the field is moving toward a future in which potent, precise immunotherapies are rapidly generated, widely accessible, and deployable on demand. The transition from ex vivo, patient-specific manufacturing to in vivo, hybrid delivery platforms has the potential to democratize CAR-T therapy, accelerate timelines, and expand the therapeutic reach of adoptive immunotherapy across diverse disease contexts. Future comparative studies will be essential to define which delivery modalities achieve an optimal balance between persistence, safety, manufacturability, and cost, ultimately determining how next-generation CAR therapies are deployed in clinical practice.

Acknowledgments

The authors wish to acknowledge grant funding from the United States NCI (R01 CA237016, J.A.C.), NIDDK (R01 DK124115, J.A.C), NHLBI (5T32HL066987-23 and P01HL158688, K.S.R. and J.A.C., respectively), Blood Cancer United (TRP, J.A.C.), United States Department of Defense (J.A.C.), Blood Cancer United (Career Development Award, R.R.), and Parker Institute for Cancer Immunotherapy at Dana-Farber Cancer Institute (R.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

K.S.R., conceptualization, drafting, figures, and revisions; L.M.D., drafting and revisions; M.T., revisions; R.R., revisions and supervision; J.A.C., conceptualization, drafting, revisions, and supervision.

Declaration of interests

R.R. is a scientific co-founder of InnDura Therapeutics and serves on the scientific advisory board of Glycostem Therapeutics.