In Vivo CAR-T Enters Clinical Validation: How It Works and What Comes Next

The development of in vivo chimeric antigen receptor therapies has accelerated rapidly. If 2025 was the year in vivo CAR-T became a strategic acquisition priority, 2026 is emerging as its first year of meaningful clinical validation.

During 2025, AstraZeneca acquired EsoBiotec, AbbVie acquired Capstan Therapeutics, Kite moved to acquire Interius BioTherapeutics, and Bristol Myers Squibb announced its acquisition of Orbital Therapeutics ^[1-4]. These transactions gave major pharmaceutical companies access to targeted lentiviral vectors, lipid nanoparticles, messenger RNA and circular RNA technologies designed to generate CAR-expressing immune cells directly inside the patient.

Figure 1. Major pharma acquisitions and early clinical data are accelerating the development and strategic value of in vivo CAR technologies.

The scientific case strengthened in 2026. A Phase 1 study of EsoBiotec's BCMA-directed ESO-T01 provided peer-reviewed evidence that functional CAR-T cells could be generated in patients with relapsed or refractory multiple myeloma without leukapheresis, ex vivo cell manufacturing or lymphodepleting chemotherapy. Four of five treated patients responded, including three stringent complete responses, although clinically significant adverse events and cytokine release syndrome underscored the need for careful safety assessment ^[5].

Kelonia's KLN-1010 program subsequently reported early responses in a larger multiple myeloma cohort, further supporting the biological feasibility of directly programming endogenous T cells ^[6]. Lilly's proposed acquisition of Kelonia—announced at up to $7 billion, including $3.25 billion upfront—illustrates how quickly credible human data can change the strategic value assigned to an in vivo cell-engineering platform ^[7]. As of July 23, 2026, the transaction remained subject to closing conditions.

In Vivo CAR-T at a Glance: The Patient as the Manufacturing Facility

Conventional ex vivo CAR-T therapy requires weeks of complex leukapheresis, centralized cell expansion, and highly variable production timelines. In stark contrast, in vivo CAR-T leverages targeted delivery systems—such as lentiviral vectors, lipid nanoparticles (LNPs), or circular RNA—to deliver genetic instructions directly into the patient. This paradigm-shifting approach transforms the patient's own body into the cell-manufacturing facility, reprogramming endogenous immune cells to express chimeric antigen receptors (CARs) capable of recognizing and eliminating target cells (e.g., CD19 or BCMA) in a fraction of the traditional timeframe.

Figure 2. Ex vivo CAR-T engineers patient T cells outside the body, whereas in vivo CAR-T delivers genetic instructions directly to immune cells within the patient ^[8].

The Preclinical Bottleneck: Why Standard Models Fall Short

As in vivo CAR therapies advance toward the clinic, drug developers face critical, pipeline-threatening questions: How selectively are the intended immune-cell subsets transduced? What is the true risk of off-target genetic modification? Can cytokine release syndrome (CRS) and neurotoxicity be accurately predicted?

Crucially, these questions cannot be answered by standard immunodeficient tumor models. In vivo CAR development hinges on complex interactions among delivery vehicles, human immune-cell subsets, and the surrounding tissue microenvironment. Because of profound species-specific differences in receptor expression and vector tropism, conventional murine immune systems are fundamentally inadequate for evaluating delivery technologies designed to target human immune cells.

The Solution: Bridging the Translational Gap with Cyagen’s HSC-Humanized Models

Meaningful preclinical evaluation of in vivo CAR therapies requires models that reproduce a functional, multi-lineage human immune system and allow therapeutic activity, cellular pharmacology, tissue distribution, and potential safety risks to be examined within the same biological context.

Cyagen’s enhanced HSC-humanized platforms, including the huHSC-NKG-ProF mouse, are generated through the engraftment of human CD34-positive hematopoietic stem cells. These models support the development of human T cells, B cells, NK cells, monocytes, macrophages, dendritic cells, and other myeloid populations, providing a broader human immune environment for studying immune-cell transduction, CAR expression, target-cell depletion, CAR-T-cell expansion and persistence, tissue distribution, immune phenotype, and vector tropism.

The translational utility of the huHSC-NKG-ProF platform was demonstrated in an exploratory in vivo CAR study in which human immune-cell engraftment was assessed before treatment and monitored throughout a 42-day observation period. Peripheral blood was collected weekly following vector administration, while the spleen and bone marrow were analyzed at the experimental endpoint. Human immune-cell engraftment remained detectable throughout the study, enabling treatment-related pharmacodynamic changes to be evaluated against an established human hematopoietic background.

Following in vivo CAR administration, treated mice showed sustained depletion of human CD19-positive B cells in peripheral blood. Both the proportion of hCD19-positive cells within the human leukocyte compartment and the absolute number of circulating hCD19-positive cells were reduced relative to the control group from Day 7 through Day 42. This depletion was accompanied by an increased relative proportion of human CD3-positive T cells, consistent with selective reduction of the targeted B-cell population rather than a general loss of human immune-cell engraftment.

Figure 3. Evaluation of the in vivo CAR-mediated B-cell depletion efficacy in peripheral blood.

In parallel with B-cell depletion, CAR-positive cells emerged within the human CD3-positive T-cell compartment and expanded dynamically in peripheral blood. Both the frequency and absolute number of circulating CAR-T cells increased after treatment, reaching their highest observed levels at approximately Day 14 before declining at subsequent time points. This temporal profile demonstrates that the model can capture the generation and expansion kinetics of CAR-T cells produced directly in vivo.

Figure 4. Dynamic expansion of in vivo-generated CAR-T cells in peripheral blood.

At the Day 42 endpoint, CAR-T cells remained detectable in both the spleen and bone marrow, demonstrating distribution and persistence beyond the peripheral circulation. The proportion of CAR-positive cells among human CD3-positive T cells was higher in the bone marrow than in the spleen. B-cell depletion was also observed in both tissues, although residual B cells with low CD19 expression remained detectable in the bone marrow of most animals. These findings illustrate the model’s ability to reveal tissue-dependent differences in CAR-T-cell distribution and target-cell depletion that may not be apparent from peripheral blood analysis alone.

Figure 5. Evaluation of B-cell depletion and in vivo-generated CAR-T-cell distribution in the spleen and bone marrow.

Beyond measuring CAR-T-cell abundance, the huHSC-NKG-ProF platform enabled characterization of their differentiation state. Analysis of CCR7 and CD45RA expression showed that in vivo-generated CAR-T cells were skewed toward effector-memory and terminal-effector phenotypes compared with control T cells. This pattern was observed in peripheral blood at the Day 14 expansion peak and remained evident in the spleen and bone marrow at the experimental endpoint, supporting the use of the model to examine the phenotypic evolution of CAR-T cells across circulation and tissues.

Figure 6. Effector and memory phenotypes of in vivo-generated CAR-T cells.

CAR-T cells also exhibited a higher proportion of PD-1-positive cells than control T cells in peripheral blood, spleen, and bone marrow. Because PD-1 expression may be associated with both recent activation and progressive functional exhaustion, this finding demonstrates the platform’s capacity to support the assessment of activation- and exhaustion-associated CAR-T-cell phenotypes during in vivo treatment.

Figure 7. PD-1 expression in in vivo-generated CAR-T cells.

Analysis of the CD4-positive and CD8-positive composition of the CAR-T-cell population provided an additional measure of cellular phenotype. In the bone marrow, CAR-T cells from the evaluated treatment groups contained a higher proportion of CD8-positive cells than control T cells, indicating that the tissue-resident CAR-T-cell compartment may differ from the broader human T-cell population in its CD4/CD8 distribution.

Figure 8. CD4-positive and CD8-positive composition of in vivo-generated CAR-T cells.

Importantly, CAR expression was not restricted to the intended T-cell population. At the endpoint, CAR-positive cells were detected among human monocytes and other hCD45-positive, CD3-negative immune-cell populations in the spleen and bone marrow. These other populations excluded phenotypically defined T cells, B cells, NK cells, and monocytes. The detection of CAR expression in non-T-cell compartments suggests potential off-target transduction following systemic vector administration and demonstrates why vector tropism should be evaluated alongside pharmacodynamic efficacy in preclinical in vivo CAR studies.

Figure 9. Flow-cytometric assessment of potential off-target CAR expression.

Together, these findings show how the huHSC-NKG-ProF platform can connect vector delivery with functional, phenotypic, biodistribution, and safety-relevant outcomes. Within a single model, researchers can determine which human immune-cell populations receive the CAR payload, characterize how CAR-T cells expand, differentiate, persist, and distribute across tissues, quantify target-cell depletion in multiple anatomical compartments, and identify unintended transduction of non-T-cell populations. This integrated analysis provides a more informative bridge between vector performance and the complex human immune responses that may shape the efficacy and safety of in vivo CAR therapies.

The acquisitions that defined 2025 and the emerging clinical results of 2026 signal that in vivo CAR is moving from platform investment toward clinical validation. As the field advances, translationally relevant humanized immune system models will be increasingly important for evaluating delivery selectivity, efficacy, and safety-related responses before broader clinical development.

👉 Explore Cyagen's HSC-humanized NKG mouse models for translational in vivo CAR research.

👉 Discover our preclinical study capabilities for evaluating immune-cell targeting, CAR expression, biodistribution, persistence, and pharmacodynamic activity.

Reference

[1] AbbVie. AbbVie to acquire Capstan Therapeutics, further strengthening commitment to transforming patient care in immunology [Internet]. North Chicago (IL): AbbVie; 2025 Jun 30 [cited 2026 Jul 23]. Available from: https://news.abbvie.com/2025-06-30-AbbVie-to-Acquire-Capstan-Therapeutics,-Further-Strengthening-Commitment-to-Transforming-Patient-Care-in-Immunology

[2] Gilead Sciences, Inc. Kite to acquire Interius BioTherapeutics to advance in vivo platform [Internet]. Foster City (CA): Gilead Sciences, Inc.; 2025 Aug 21 [cited 2026 Jul 23]. Available from: https://investors.gilead.com/news/news-details/2025/Kite-to-Acquire-Interius-BioTherapeutics-to-Advance-In-Vivo-Platform/default.aspx

[3] AstraZeneca. AstraZeneca to acquire EsoBiotec [Internet]. Cambridge (UK): AstraZeneca; 2025 Mar 17 [cited 2026 Jul 23]. Available from: https://www.astrazeneca.com/media-centre/press-releases/2025/astrazeneca-to-acquire-esobiotec.html

[4] Bristol Myers Squibb. Bristol Myers Squibb strengthens and diversifies cell therapy portfolio with acquisition of Orbital Therapeutics [Internet]. Princeton (NJ): Bristol Myers Squibb; 2025 Oct 10 [cited 2026 Jul 23]. Available from: https://news.bms.com/news/details/2025/Bristol-Myers-Squibb-Strengthens-and-Diversifies-Cell-Therapy-Portfolio-with-Acquisition-of-Orbital-Therapeutics/default.aspx

[5] An N, Wang D, Zhang P, Zhang J, Parone P, Hu J, Bao Y, Xu L, Ruan H, Wan Y, Wen X, Gao Y, Li C. In vivo generation of anti-BCMA CAR-T cells in relapsed or refractory multiple myeloma: a phase 1 study. Nat Med. 2026 Apr;32(4):1257-1266. doi: 10.1038/s41591-026-04244-6. Epub 2026 Mar 25. PMID: 41882404.

[6] Kelonia Therapeutics, Inc. Kelonia Therapeutics presents updated first-in-human data from Phase 1 inMMyCAR study of KLN-1010 in vivo BCMA CAR-T therapy at the 2026 American Society of Clinical Oncology (ASCO) Annual Meeting [Internet]. Boston (MA): Kelonia Therapeutics, Inc.; 2026 May 31 [cited 2026 Jul 23]. Available from: https://www.businesswire.com/news/home/20260531412768/en/Kelonia-Therapeutics-Presents-Updated-First-in-Human-Data-from-Phase-1-inMMyCAR-Study-of-KLN-1010-in-vivo-BCMA-CAR-T-Therapy-at-the-2026-American-Society-of-Clinical-Oncology-ASCO-Annual-Meeting

[7] Eli Lilly and Company. Lilly to acquire Kelonia Therapeutics to advance in vivo CAR-T cell therapies [Internet]. Indianapolis (IN): Eli Lilly and Company; 2026 Apr 20 [cited 2026 Jul 23]. Available from: https://investor.lilly.com/news-releases/news-release-details/lilly-acquire-kelonia-therapeutics-advance-vivo-car-t-cell

[8] Xu J, Chen Z, Su L, Ren A, Mei H. In vivo CAR cell therapy: from bench to bedside. J Hematol Oncol. 2025 Nov 20;18(1):105. doi: 10.1186/s13045-025-01759-2. PMID: 41261423; PMCID: PMC12632109.