Supplementary MaterialsDocument S1. however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T?cell source and paralleled the CD4+-to-CD8+ T?cell ratio in the adoptive transfer product. CD4+ CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4+ CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity. expansion and cytokine production. These data spotlight how intrinsic properties of the CAR-T cell product can contribute to off-tumor toxicity. Results Second-Generation DARPin-Targeted Anti-HER2 CAR-T Cells Were Toxic effects, as the DARPin-BBz- and DARPin-z-T cells?displayed a similar functional avidity (Determine?S2), even though DARPin-BBz-T cells produced greater toxicity analysis of these T?cell products (Figures S7ACS7C) had not predicted the observed MAC014? LEUK001? MAC026 hierarchy of toxicity (Figures S7D and S7E). The only characteristic of the donor-variant DARPin-28z-T cell products that correlated with toxicity was the frequency of CD4+ T?cells in the adoptive transfer product (Physique?4B), where MAC014? LEUK001? MAC026. Open in a BRD-6929 separate window Physique?4 Differential Toxicity of DARPin-28z-T Cells Manufactured from Unique PBMC Donors Correlated with the Frequency of CD4+ T Cells in the Adoptive Transfer Product (A) OVCAR-3 tumor-bearing NRG mice were treated with 6.0? 106 or 1.7C2.0? 106 DARPin-28z-T cells produced from MAC026, LEUK001, or MAC014 PBMCs. Mice were monitored over time for changes in weight. Data were pooled from n?= x impartial experiments. For 6.0? 106 cells, MAC014, 2; LEUK001, 3; and MAC026, 4. For 2.0? 106 cells, MAC014, 1; LEUK001, 2; and MAC026, 1. Each line indicates data from one animal; curves end, indicating when mice succumbed to toxicity. (B) Composition of CD4+ or CD8+ cells in DARPin-28z-T cell products (days 13C14 post-activation) manufactured using starting PBMCs from donors as indicated and decided using flow cytometry (upstream gating strategy: lymphocytes singlets NGFR+). Error bars represent SD. Data from n?= x impartial experiments; MAC014, 5 (2 unique PBMC preparations); LEUK001, 6 (1 PBMC preparation); and MAC026, 12 (5 unique PBMC preparations). CD4+ T Cells in the DARPin-28z-T Cell Product Were the Crucial Drivers of Toxicity Given the correlation between the frequency of CD4+ T?cells in the DARPin-28z adoptive transfer product and the severity of toxicity culture period in a donor-specific manner. Unlike other donors, DARPin-28z-T cells generated from MAC026 PBMCs exhibited an increase in their CD4+:CD8+ ratio over time (Physique?S9A). Growth data for DARPin-28z-T cell cultures generated from purified CD4+ or CD8+ T?cells revealed that, while both MAC026 and MAC014 showed a similar proliferative capacity in their CD4+ T?cells, CD8+ T?cells from MAC026 had a diminished proliferative capacity (Physique?S9B). BRD-6929 Additional DARPin-28z-T Cell-Intrinsic Variables BRD-6929 Contributed to Donor-Specific Differences in Toxicity We postulated that, if the CD4+:CD8+ T?cell ratio of the adoptive transfer product was the sole driver of donor-specific variation in our toxicity model, normalizing the dose of Tnf CD4+ DARPin-28z-T cells should eliminate this variation. Purified CD4+ DARPin-28z-T cells were generated from a panel of five different PBMC donors and delivered to tumor-bearing NRG mice at equal doses. While doses of 6.0? 106 CD4+ DARPin-28z-T cells resulted in very similar toxicities, regardless of donor (Physique?S10), donor-specific differences in the toxicity of CD4+ T?cells were clearly resolved at the 2 2.0? 106 CAR-T cell dose level (Figures 6AC6C). MAC002-derived CD4+ DARPin-28z-T cells induced the most rapid toxicity and were uniformly lethal within 8?days of treatment. MAC026-, MAC014-, and MAC003-generated DARPin-28z-T cells all induced comparable onsets in toxicity (mice experienced weight loss by 10?days post-ACT1; the average percent change in weights were ?16.3%? 5.8%, ?16.2%? 9.3%, and ?16.0%? 3.6%,.