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Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells

Nature Biotechnology volume 40, pages 1250–1258 (2022)Cite this article

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Abstract

Despite their clinical success, chimeric antigen receptor (CAR)-T cell therapies for B cell malignancies are limited by lengthy, costly and labor-intensive ex vivo manufacturing procedures that might lead to cell products with heterogeneous composition. Here we describe an implantable Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) that streamlines in vivo CAR-T cell manufacturing and reduces processing time to a single day. When seeded with human peripheral blood mononuclear cells and CD19-encoding retroviral particles, MASTER provides the appropriate interface for viral vector-mediated gene transfer and, after subcutaneous implantation, mediates the release of functional CAR-T cells in mice. We further demonstrate that in vivo-generated CAR-T cells enter the bloodstream and control distal tumor growth in a mouse xenograft model of lymphoma, showing greater persistence than conventional CAR-T cells. MASTER promises to transform CAR-T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy.

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Fig. 1
Fig. 2: MASTER promotes activation and retrovirus-mediated transduction of primary human T cells.
Fig. 3: MASTER-mediated gene transfer generates highly functional CAR-T cells.
Fig. 4: Subcutaneously implanted MASTER generates and releases fully functional CAR-T cells in a xenograft model of lymphoma.
Fig. 5: Subcutaneously implanted MASTER outperforms intravenously administered CAR-T cells in a rechallenge model of lymphoma.
Fig. 6: MASTER and conventional CAR-T cells exhibit equal anti-tumor efficacy against established tumor in vivo.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its extended data and supplementary information files. Source data are provided with this paper.

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Acknowledgements

This work was supported by the North Carolina Biotechnology Center Flash Grant 2019-FLG-3812; by the National Center for Advancing Translational Sciences and the National Institutes of Health through grant awards R37-CA260223, UL1-TR002489, R01-CA193140, R21-CA229938, T32-CA196589 and R25-NS094093; and by start-up funds provided by the University of North Carolina at Chapel Hill, North Carolina State University at Raleigh, the Lineberger Cancer Center and the Ross M. Lampe Endowed Chair. We thank the North Carolina State University College of Veterinary Medicine staff for proper care of animals used in experiments and valuable resources on training. We also thank the North Carolina State University flow cytometry core and J. Mohammed for training and guidance on flow cytometry analysis. We are grateful to T. Xianming for assistance with power analysis and to C. Clifford for evaluating histology samples. SEM and X-ray CT images were taken at the Analytical Instrumentation Facility at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation (award ECCS-1542015). The Analytical Instrumentation Facility is a member of the North Carolina Research Triangle Nanotechnology Network, a site in the National Nanotechnology Coordinated Infrastructure. The authors acknowledge the use of the Cellular and Molecular Imaging Facility at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation. Schematics were created with BioRender.

Author information

Authors and Affiliations

  1. Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC, USA

    Pritha Agarwalla, Kristen A. Froehlich, Frances S. Ligler & Yevgeny Brudno

  2. Comparative Medicine Institute, North Carolina State University, Raleigh, NC, USA

    Pritha Agarwalla, Frances S. Ligler & Yevgeny Brudno

  3. Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Edikan A. Ogunnaike

  4. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Edikan A. Ogunnaike, Gianpietro Dotti & Yevgeny Brudno

  5. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Sarah Ahn & Gianpietro Dotti

  6. Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA

    Kristen A. Froehlich

  7. Analytical Instrumentation Facility, North Carolina State University, Raleigh, NC, USA

    Anton Jansson

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Contributions

P.A. conceived of the study, designed and performed experiments, analyzed data and wrote the paper. Y.B. conceived of the study, analyzed data and wrote the paper. E.A.O., S.A., K.F. and A.J. prepared experimental materials and performed experiments. F.S.L. and G.D. contributed to the design of experiments and to writing and editing the paper. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding author

Correspondence to Yevgeny Brudno.

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Competing interests

P.A., G.D. and Y.B. are inventors on patents related to the use of biomaterials for generation of CAR-T cell therapeutics. Y.B. receives an industry-sponsored research grant related to CAR-T cell therapeutic technology (unrelated to this work). G.D. is a paid consultant for Bellicum Pharmaceuticals, Tessa Therapeutics and Catamaran. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Quantitative characterization of MASTER scaffold structure.

A) Scanned volume of MASTER (left) with a colored plane indicating the cross-section seen on the right. In the cross sections a brighter value indicates a higher density (scaffolds) and a darker value a lower density (air porosity). B) Relative frequency of pores of different dimensions. C) The aspect ratio of the pores showing most of the pores has an oblong shape. An aspect ratio of 1 corresponds to a sphere and close to 0 corresponds to a flat plane or stick. D) The surface area as a function of volume plotted. The total surface area inside of MASTER is roughly 810 mm2 E) Connectivity of sample showing most of the pores are connected to 0-3 other pores with a very few pores (around 6%) have more connections than 9.

Source data

Extended Data Fig. 2 Confocal images of GFP-expressing T cells within MASTER scaffold.

3D confocal micrograph showing distribution of GFP + T cells in AF647 labeled MASTER at 10X (A) and 40X (B) magnification. This experiment was repeated twice independently with similar results.

Source data

Extended Data Fig. 3 IL-2 loaded onto MASTER released in a sustained manner over five days in vitro and retained its bioactivity.

(A) Cumulative release of IL-2 from MASTER as quantified by ELISA assay. Data represent mean ± SD of three independent samples (B) Bioactivity of IL2 released at 24 hours as assessed by proliferation of CFSE stained T cells.

Source data

Extended Data Fig. 4 IL-2 promotes lymphocyte proliferation, but not transduction efficiency of MASTER.

(A) MASTER and MASTER without IL-2 was seeded with PBMCs and virus and number of cells were counted 5 days post transduction. *p < 0.05, two tailed unpaired t test, (B) CAR.19 expression in T cells 72 h post transduction. *p < 0.05, two-tailed unpaired t test. Data represent median of n = 3 biologically independent samples.

Source data

Extended Data Fig. 5 MASTER functions as an efficient T cell-release system.

A) Schematic of in vitro release study. B) Percent of cells released from scaffold. Data in represent mean ± SD of n = 3 independent samples.

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Extended Data Fig. 6 Biocompatibility of MASTER and its components.

Representative images of H&E-stained sections of five major organs and implantation site four weeks after subcutaneous implant of MASTER, MASTER + mouse PBMCs + GFP-encoding gamma retrovirus and untreated controls in C57Bl6/J immunocompetent mice. Data is representative of three biologically independent animals.

Extended Data Fig. 7 MASTER loaded with PBMCs and retrovirus does not transduce host cells.

A) In vitro transwell model mimicking the in vivo system. B) GFP expression in fibroblast cells seeded on the bottom of transwell plate. Data represent mean ± SEM of n = 3 biologically independent samples.

Source data

Extended Data Fig. 8 Characterization of host cells infiltrating MASTER.

A) Timeline of experiment B) Representative FACS plot showing efficient engraftment of human PBMCs (Hu-CD45 + CD3+) in blood. C-D) Different subsets of mouse and human cells that infiltrated MASTER. Cells were gated on live cells. Data in d-e represent mean ± SEM and median of three biologically independent samples. E) Phenotype of the engrafted human T cells that infiltrated into the scaffold. Cells were gated on human-CD45 + CD3 + cells. Data represent mean ± SEM of four biologically independent samples F) FACS plot showing no GFP expression in cells infiltrating MASTER, in blood and in the skin surrounding the scaffold.

Source data

Extended Data Fig. 9 Similar numbers of exhausted cells in blood of mice with conventional and MASTER-generated CAR-T cells.

Immunophenotypic composition of CAR-T cells in blood of mice treated with conventionally expanded CAR-T cells i.v. (A) or MASTER (B) at day 12 and 22 post tumor inoculation. Data in a represent mean ± SD of six experimental replicates. Data in b represent mean ± SEM of five experimental replicates.

Source data

Extended Data Fig. 10 Subcutaneously implanted MASTER exhibited better control of tumor growth and extended survival compared under stressed dose conditions.

A) Timeline of the study B) In vivo tumor bioluminescence imaging (BLI) of NSG mice (n = 6) treated with MASTER, conventional CAR-T cells, or control non-transduced (NT) cells. Mice were treated with 0.5 ×106 (left), 0.25 ×106 (middle), or 0.125 ×106 (right) CAR T cells. PBMCs seeded onto MASTER were normalized to transduction efficiency and both groups (MASTER and CAR T, i.v) were treated with equivalent number of CAR T cells. C-E) Survival of mice shown as Kaplan- Meier curves. **p < 0.01; ***p < 0.001 Log-rank (Mantel-Cox) test, Gehan-Breslow-Wilcoxon test.

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Agarwalla, P., Ogunnaike, E.A., Ahn, S. et al. Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells. Nat Biotechnol 40, 1250–1258 (2022). https://doi.org/10.1038/s41587-022-01245-x

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