Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement

Abstract

Treatment of patients with adoptive T-cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single-cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Previous studies have evaluated artificial antigen presenting cells for their antigen-specific and costimulatory properties. We investigated engineered cells for costimulatory enhancement (ECCE) consisting of K562 cells that express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (P=0.001) from single-cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8CD62L and CD8CD27 T cells then comparable interleukin-2-expanded TIL and maintained antitumor reactivity. Moreover, ECCE improved TIL expansion from nonmelanoma-cell suspensions similar to that seen with melanoma tumors. These data demonstrate that the addition of ECCE to TIL production will enable the treatment of patients that are ineligible using current methods.

Figure 1. ECCE significantly improve TIL production from melanoma single-cell suspensions

3–5×10⁶ viable cells from cryopreserved (A) or freshly prepared (B) tumor single-cell suspensions were cultured in CM supplemented with IL-2 in the presence or absence of ECCE. Total viable TIL were evaluated throughout the culture. (C) The time required to achieve confluent lymphocyte growth (see material and methods) was determined.

Figure 2. 4-1BBL but not CD80 expression is necessary for augmented TIL production

3–5×10⁶ viable cells from cryopreserved tumor single-cell suspensions were cultured in CM supplemented with IL-2 only, ECCE, K562 cells or ECCE transduced to express CD80 (ECCE.CD80). Total viable TIL were evaluated throughout the culture.

Figure 3. ECCE enhance CD8⁺ T cell and natural killer cell expansion

(A) Age-matched TIL expanded with or without ECCE were evaluated for the frequency of CD3 and CD56 to determine natural killer (NK) cell frequency (left) or CD4⁺ and CD8⁺ T cells (right) by flow cytometry. (B) Pooled results from independent experiments showing frequency of CD3⁻CD56⁺ NK cells (n=27, left) or CD8⁺ of CD3⁺ T cells (n=31, right).

Figure 4. ECCE increase the absolute number of CD27⁺ and CD62L⁺ CD8⁺ T cells

(A) Fold expansion of TIL expanded with or without ECCE in media containing IL-2 for eleven days (n=11). Frequency (left, closed symbols) and total number (right, open symbols) of CD3⁺CD8⁺CD27⁺ (B) and CD3⁺CD8⁺CD62L⁺ (C) cells after 48 hours in media lacking IL-2.

Figure 5. ECCE-generated TIL vigorously expand with rapid expansion protocol (REP)

(A) Standard and ECCE-generated TIL were rapidly expanded with irradiated peripheral blood mononuclear feeder cells in media containing IL-2 and anti-CD3 (See materials and methods). Pooled results from independent experiments (n=17). Age-matched standard and ECCE-generated TIL were evaluated for frequency of (B) CD57⁺ or (C) CD279 (PD-1) after gating on CD3⁺CD8⁺.

Figure 6. ECCE and IL-2 expand TIL from non-melanoma tumor single-cell suspension

(A) Fold expansion was examined after a 14 day culture of 11 non-melanoma tumor digests with and without ECCE. (B–E) Flow cytometric analysis of expanded TIL: (B) Percentage of cells contained in forward-scatter versus side-scatter lymphocyte gate, (C) CD3⁻CD56⁺ NK cells, (D) CD3⁺CD8⁺ cells, and (E) CD3⁺CD4⁺cells.

Figure 6. ECCE and IL-2 expand TIL from non-melanoma tumor single-cell suspension

(A) Fold expansion was examined after a 14 day culture of 11 non-melanoma tumor digests with and without ECCE. (B–E) Flow cytometric analysis of expanded TIL: (B) Percentage of cells contained in forward-scatter versus side-scatter lymphocyte gate, (C) CD3⁻CD56⁺ NK cells, (D) CD3⁺CD8⁺ cells, and (E) CD3⁺CD4⁺cells.