Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies

Abstract

Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies.

Keywords: Antibody-dependent cell-mediated cytotoxicity (ADCC); cytokine induced killer (CIK) cells; immunotherapy; monoclonal antibodies.

Figure 1.

Phenotypic characterization of CIK cells. CIK cells were generated in vitro and analyzed for their phenotype by flow cytometry throughout the culture period. In the figure, histograms refer to three distinct time points, namely day 0 (black), day 14 (gray) and day 28 (white). (A) Percentage of CD3⁺CD56⁺ CIK cells and CD3⁻CD56⁺ NK cells in the bulk cultures at the reported time points. (B) Expression of different cell surface markers on CD3⁺CD56⁺ cells at the reported time points. Results show the mean expression ± SD of 5 to 15 independent experiments performed on PBMCs and related cultures from distinct donors.

Figure 2.

CD16 expression on CIK cells. CIK cells were analyzed for CD3, CD56 and CD16 co-expression by flow cytometry. (A) Bulk cultures were first gated for CD3⁺ and CD56⁺ co-expression to identify the CIK subset (left panel), followed by analysis of CD16 expression (right panels). CIK cells were arbitrarily stratified as having a high (>25%), intermediate (<25% and >5%) or low (<5%) expression of CD16. (B) CD16 expression in CIK cells from different donors (n = 60) between the second and third week in vitro. Bar indicates mean value ± SD. (C) Comparison of the CD3⁺CD56⁺CD16⁺ component in distinct donors (n = 18, one symbol each) at different time points of culture (p > 0.5).

Figure 3.

CIK cells engage CD16 and exert ADCC. (A) Target cells were analyzed for Her2 (black line) and EGFR (dotted line) expression by flow cytometry analysis. Fluorochrome-conjugated secondary antibody alone (gray line) served as negative control. Results are representative of five experiments. (B) CIK cells were challenged against SKOV-3, IGROV-1, MDA-MB-231 and MDA-MB-468 tumor cell lines. Lytic activity was measured using bulk CIK cells (day 21 of culture) in the absence (open circles) or presence of trastuzumab (gray circles) or cetuximab (half-filled circles). The symbols refer to the specific lysis of individual CIK cell cultures from different donors at an E/T ratio of 100:1, and bars indicate mean values ± SD. Data were analyzed by Student's t-test (***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; not statistically significant (p > 0.05) if not indicated).

Figure 4.

CD16 engagement is critically required for CIK cell-mediated ADCC. CIK cells were challenged against SKOV-3 (A), IGROV-1 (B), MDA-MB-231 (C) and MDA-MB-468 (D) tumor cell lines. Lytic activity was measured using bulk CIK cells (day 21 of culture) in the absence (open circles) or presence of trastuzumab (gray circles) or cetuximab (half-filled circles), adding an anti-CD16 blocking antibody (open squares) or an isotype antibody (gray squares) to the corresponding specific mAb (trastuzumab in A, and cetuximab in B, C and D). An isotype antibody served as negative control (half-filled square). The symbols refer to the specific lysis of individual CIK cell cultures from different donors at an E/T ratio of 100:1, and bars indicate mean values ± SD. Data were analyzed by Student's t-test (§§§, p ≤ 0.001; §§, p ≤ 0.01; §, p ≤ 0.05; not statistically significant (p > 0.05) if not indicated).

Figure 5.

Correlation between CD16 expression and ADCC activity. CIK cells samples were arbitrarily stratified as low (<5%, circles), intermediate (>5 % and <25%, squares) and high (>25 %, triangles), according to their CD16 expressing levels. Their lytic activity against SKOV-3 (A), MDA-MB-231 (B) and MDA-MB-468 (C) tumor cell lines was measured in a standard cytotoxicity assay, in the presence of the corresponding specific mAb (trastuzumab in A, and cetuximab in B and C). The results are reported as the percentage of specific lysis at an E/T ratio of 100:1. Each symbol represents a different donor, and bars indicate mean values ± SD. Data were analyzed by Student's t-test. No statistical significant differences were found between different groups.

Figure 6.

ADCC is accountable to CD3⁺CD56⁺CD16⁺ CIK cells. (A) Flow cytometry analysis was performed in triple fluorescence to determine the absence of NKp46 expression on CD3⁺CD56⁺ cells. (B) NKp46⁺ NK cells were removed by magnetic beads depletion. The panels show one representative experiment out of 5 performed, where flow cytometry analysis was carried out before (left) and after (right) NK depletion. (C) CIK cells were challenged against SKOV-3, IGROV-1, MDA-MB-231 and MDA-MB-468 tumor cell lines. Lytic activity was measured using bulk CIK cells (circles) or NK-depleted CIK cells (triangles), in the absence (open symbols) or presence (gray symbols) of the corresponding mAb (trastuzumab for SKOV-3, and cetuximab for IGROV-1, MDA-MB-231 and MDA-MB-468). The symbols refer to the specific lysis of individual CIK cell cultures from different donors at an E/T ratio of 100:1, and bars indicate mean values ± SD. Data were analyzed by Student's t-test (##, p ≤ 0.01; #, p ≤ 0.05).

Figure 7.

CIK cell-mediated ADCC is independent of TCRγ/δ⁺ subpopulation. (A) Flow cytometry analysis was performed in quadruple fluorescence to determine the expression of CD16 in TCRα/β (n = 8) and TCRγ/δ (n = 14) subpopulations of CD3⁺CD56⁺ CIK cells from different donors. (B) TCRγ/δ⁺ cells were removed by magnetic beads depletion. The panels show one representative experiment out of 5 performed, in which flow cytometry analysis was carried out before (left) and after (right) depletion. (C) CIK cells were tested for cytotoxicity against SKOV-3 and MDA-MB-468 tumor cell lines. Lytic activity was measured using bulk CIK cells (circles) or TCRγ/δ-depleted CIK cells (triangles), in the absence (open symbols) or presence (black symbols) of the corresponding mAb (trastuzumab for SKOV-3, and cetuximab for MDA-MB-468). The symbols refer to the specific lysis of individual CIK cell cultures from different donors at an E/T ratio of 50:1, and bars indicate mean values ± SD. Data were analyzed by Student's t-test (+++, p ≤ 0.001; ++, p ≤ 0.01).

Figure 8.

Combining mAb and CIK cells improves the in vivo antitumor activity. (A) On day 0, mice received intraperitoneally 1 × 10⁶ SKOV-3 or IGROV-1 cells. Depending on the experimental group, treatments consisted of a combination of 1 mg trastuzumab or 1.5 mg cetuximab on day 3, 6, 9 and 1 × 10⁷ CIK cells on day 4, 7, 10 (n = 9 and n = 10, respectively), or mAbs only (n = 7) or CIK cells only (n = 9 and n = 8, respectively) at the same doses and on the same days of the combined treatment. The untreated group (n = 6) received three injections of PBS. (B) Kaplan–Meier survival curves of NSG mice bearing intraperitoneal SKOV-3 or IGROV-1 tumors. Combination therapy (black lines) significantly increased survival compared to any other treatment (mAbs only, gray-dashed lines; CIK only, black-dashed lines; untreated, gray lines), both in SKOV3 (P = 0.04) and IGROV-1-injected mice (p = 0.03).