In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome

Abstract

Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B-cell malignancies. Notwithstanding, CAR T-cell manufacturing requires complex procedures impeding the broad supply chain. Here, we provide evidence that human CD19-CAR T cells can be generated directly in vivo using the lentiviral vector CD8-LV specifically targeting human CD8⁺ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8⁺ T cells and efficacious elimination of CD19⁺ B cells. Further, upon injection of CD8-LV into mice transplanted with human CD34⁺ cells, induction of CAR T cells and CD19⁺ B-cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue-invading CAR T cells and complete elimination of the B-lymphocyte-rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8⁺ CAR T cells active against CD19⁺ cells, yet with similar adverse effects currently notorious in the clinical practice.

Keywords: T‐cell targeting; cytokine release syndrome; gene delivery; humanized mouse.

Figure 1. CAR T‐cell generation in human PBMC‐transplanted mice

1. A

   Ex vivo generation of CAR T cells. Activated human PBMC were left untransduced or incubated with CD8‐LV^CD19CAR at an MOI of 2. Five days later, expression of CD19‐CAR and CD8 was determined on CD3⁺ cells. Numbers indicate the percentage of cells in the respective gate.

2. B

   Experimental outline for in vivo CAR generation. 1 × 10⁷ human PBMC were engrafted into naïve NSG mice or NSG mice that had been intraperitoneally (i.p.) injected with 5 × 10⁵ Raji cells (Raji⁺) 6 days before. One day later, 2 × 10⁶ t.u. of CD8‐LV^CD19CAR (filled circles) or CD8‐LV^RFP (gray triangles) were i.p. injected, respectively. As further control, another group of mice received PBS (open circles). Seven days later, mice were sacrificed and organs and cells were removed for further analysis.

3. C

   Detection of CAR T cells by vector copy numbers (VCN). Genomic DNA was isolated from peritoneal cavity, spleen, and blood cells. VCN were determined in technical duplicates by qPCR for two individual mice of each group. The presence of B cells in the transplanted PBMC is indicated below.

4. D–F

   Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD8⁺ in CD3⁺ cells (D), of CAR⁺ or RFP⁺ cells in the CD8⁺ and CD8⁻ fractions, respectively (E), and of human CD19⁺ cells (F) within the fraction of human CD45⁺ cells. Representative density plots are shown for the peritoneal cells. The gating strategy is represented in Appendix Fig S1A.

5. G

   Mice were transplanted with B‐cell‐depleted human PBMC and then received CD8‐LV^CD19CAR (filled circle) or PBS (open circle). As control, CD8‐LV^CD19CAR or PBS was injected into mice transplanted with non‐depleted PBMC.

Data information: Data represent mean ± SD for all groups (CD8‐LV^CD19CAR: n = 6 (+Raji) and n = 4 (−Raji) in (D), n = 4 (−B‐cells) in (G); CD8‐LV^RFP: n = 4; PBS: n = 4 in (G), all others n = 3). Statistical significance was determined using Mann–Whitney test; ns, not significant.

Figure EV1. Ex vivo CAR T‐cell generation

1. A

   Quantitative data of Fig 1A showing the percentages of CAR⁺ cells of three different donors. Mean values ± SD are shown with n = 3. Statistical evaluation of the data was performed using two‐tailed unpaired t‐test.

2. B

   CAR T cells eliminate CD19⁺ B cells. PBMC were activated for 3 days and incubated with CD8‐LV^CD19CAR. Expression of CD3 and CD19 was then analyzed by flow cytometry. The percentage of CD19⁺ B cells for n = 3 with mean ± SD is shown. Statistical significance was determined by two‐tailed unpaired t‐test.

3. C

   Selective killing of CD19⁺ Raji tumor cells by ex vivo‐generated CAR T cells (CAR) or untransduced T cells (UT) with CD19⁺ Raji cells, or as control CD19⁻ Hut78 cells. Mean values ± SD are shown with n = 3.

Figure EV2. Detection of CAR T cells in PBMC‐transplanted mice

Summary of CAR T‐cell detection in PBMC‐transplanted NSG by flow cytometry for the same two individual mice of each group shown in Fig 11C. Percentage of CAR⁺ cells of CD45⁺ cells is shown.

Figure EV3. Clonality and exhaustion of in vivo‐generated CAR T cells

1. A

   Clonality analysis by amplifying vector sequences integrated in genomic DNA. LM‐PCR detecting the integrated vector in genomic DNA purified from peritoneal and spleen cells harvested from PBMC‐transplanted mice injected with CD8‐LV^CD19CAR (CAR) in the presence or absence of B cells or CD8‐LV^RFP (RFP). Two mice of each group were analyzed. The internal control band is indicated as well as primer dimers in the water control (H₂O).

2. B

   Cells isolated from peritoneal cavity or spleen of human PBMC‐transplanted NSG mice (± i.p. transplanted Raji cells) treated with CD8‐LV^CD19CAR (CAR) or CD8‐LV^RFP (RFP) were analyzed for expression of exhaustion markers by flow cytometry. CD8⁺ cells from viable human CD3⁺ cells were gated for transgene‐positive (CAR⁺ or RFP⁺) and transgene‐negative (CAR⁻ or RFP⁻) cells. These two cell populations were then separately gated for expression of PD‐1, LAG‐3, and TIM‐3. For the four experimental groups, percentages of positive cells for each exhaustion marker are shown for transgene‐positive and transgene‐negative CD8⁺ cells. Mean values ± SD are shown. N = 3 in samples with closed circles, n = 4 in samples with open circles or closed triangles, while for samples with open triangles, n = 3 for peritoneum and n = 4 for spleen. Statistical evaluation of the data was performed using one‐way ANOVA test with Bonferroni correction.

Figure 2. CAR T‐cell generation in HSC‐transplanted mice

1. A

   Experimental outline. IL‐7 was injected intravenously into HSC‐humanized NSG mice before 2 × 10⁶ t.u. of CD8‐LV^CD19CAR (CAR), or PBS as control (PBS), were administered.

2. B

   CAR T‐cell levels in the CD8⁺ and CD8⁻ T cells harvested from blood, spleen, and bone marrow of PBS (PBS) or vector‐injected (CAR) mice determined by flow cytometry.

3. C

   Detection of CAR T cells by determining vector copy numbers (VCN) in genomic DNA isolated from CD8⁺‐enriched cells harvested from bone marrow.

4. D

   Detection of CAR T cells in bone marrow quantified by flow cytometry.

5. E

   CD19⁺ B‐cell levels in blood, spleen, and bone marrow determined by flow cytometry.

6. F

   Relative human CD19⁺ B‐cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration.

Data information: Distinct symbols for each individual animal are used throughout panels (B–F) (M16: lilac triangle; M19: blue triangle). Open symbols indicate animals from the vector‐injected group devoid of PCR‐detectable CAR T cells. Data represent mean ± SD for all groups. N = 6 in PBS group; n = 10 in CAR group. Statistical significance was determined using one‐way ANOVA test with Bonferroni correction.

Figure 3. Cytokines and histopathology

1. A

   Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig 2. Mean values ± SD of n = 2 technical replicates.

2. B

   Immunohistochemistry of paraffin‐embedded sections from the lungs, brain meninges, brain striatum, spleen, and liver of the vector‐injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads point at histiocytes, and the yellow line indicates B‐lymphocyte‐rich zones.

Figure EV4. Cytokines and organ infiltration

1. A

   Levels of further cytokines in plasma of individual mice obtained 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig 2. Mean values of n = 2 technical replicas.

2. B

   Hematoxylin/eosin staining of paraffin‐embedded sections from lung, liver, brain striatum, and bone marrow, of the PBS‐injected control mouse M32 or the CD8‐^LVCD19‐CAR‐injected M16 and M19 animals. Areas of hematopoietic cell depletion in bone marrow are indicated by the red‐dashed line and acellular debris by the yellow arrows. Infiltrating lymphocytes in the other tissues are labeled by black arrows.

Figure EV5. Analysis for signs of GvHD

1. A, B

   Hematoxylin/eosin staining of paraffin‐embedded sections from liver portal tracts (A) and ileum (B) of the PBS control mouse M32 and the CAR⁺ mouse M16. Arrows point to bile ducts (blue), arterial sinus (red), and portal vein (black).