CD44v6 as innovative sarcoma target for CAR-redirected CIK cells

Figures & data

Figure 1. Phenotype of antiCD44v6 CAR⁺.CIK cells. Mature CIK cells efficiently expressed anti-CD44v6CAR (A). The CD3⁺CD56⁺ subset of CIK cells was equally distributed between CAR⁺ (B) and CAR⁻ (C) cells. CAR⁺.CIK were mostly effector memory (EM / EM RA), followed in order by naive and central memory phenotype (D). The phenotype was comparable with that of unmodified NTD.CIK (unpaired t test, P > 0.05). Abbreviations: Chimeric Antigen Receptor (CAR); Not Transduced (NTD); Effector Memory (EM: CD45RA−/CD62 L-); Effector Memory RA (EM RA: CD45RA+/CD62 L-); Central Memory (CM: CD45RA−/CD62 L+); NAÏVE: CD45RA+/CD62 L+.

Table 1. CD44v6 expression in STS.

Histotype	CD44v6+ / Total
Liposarcoma	3/6
Fibrosarcoma	1/1
Leiomiosarcoma	1/2
UPS	4/6
GIST	2/12
Total number	11/27

Table 2. Phenotype of STS cell lines.

STS cell line	Histotype	MIC A/B (%)	ULBP1 (%)	ULBP2/5/6 (%)	ULBP3 (%)	HLA (%)	CD44v6 (RFI)
HT1080	fibrosarcoma	0	0	80	100	80	9
DMR	leiomyosarcoma	0	0	80	100	100	2,8
402.91	Liposarcoma (myxoid)	95	0	98	100	94	2,1
NARAH	UPS	20	0	100	90	78	1,2
S5	UPS	3	0	100	75	100	8,5
S1	UPS	13	0	100	90	100	2
S3	UPS	90	0	100	100	100	5,4
S23	Liposarcoma (retroperitoneal dedifferentiated)	0	0	98	5	100	1,7
S26	GIST	0	0	91	6	100	2,7
S25	GIST	10	1	75	9	100	2,4
S24	Liposarcoma (myxoid)	10	5	76	18	87,5	2,5
MEAN % (SEM%)		21,9 (±10,7)	0,5 (±0,4)	90,7 (±3,2)	63,0 (±13)	94,5 (±2,6)	3,6 (±0,8)

Histotype, CIK cell ligands, HLA class I expression and CD44v6 RFI of each CD44v6⁺ STS are reported in the table.

Figure 2. Sarcoma killing in vitro by antiCD44v6 CAR⁺.CIK Patient-derived antiCD44v6 CAR⁺.CIK (n = 12) efficiently killed STS (n = 11, autologous targets in 4/11). The specific killing by CAR⁺.CIK cells was significantly higher, especially at low E/T ratios, than that obtained with unmodified CIK generated from the same patients (A). Killing values (mean ± SEM) from 43 independent essays are reported, results were analyzed by two way ANOVA and Bonferroni post test analysis, statistical significance is reported as * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. The killing activity remained intense regardless the expression level (RFI high, intermediate, low) of CD44v6 on targets (B). Representative flow-cytometry histograms for each RFI level are reported. The addition of a blocking antibody against CD44v6 reduced the killing ability of CAR⁺.CIK cells to levels comparable to NTD.CIK. Blocking NKG2D receptor significantly reduced the tumor killing activity of unmodified NTD.CIK but did not impair CAR⁺.CIK (n = 4) (C). CAR⁺.CIK were moderately capable of killing monocytes at (E/T 5:1), the effect sensibly decreased and ceased at lower E:T ratios (n = 3) (D). Abbreviations: CAR Chimeric Antigen Receptor; NTD, Not Transduced; STS, Soft Tissue Sarcoma.

Figure 3. Cytokine production by CAR⁺.CIK. CAR⁺.CIK produced higher amounts of IL4, IFN-γ and IL6 compared with NTD.CIK. A trend toward lower production of IL5 and TGFβ was observed. Results are reported as optic density ratio (O.D. ratio) between samples and internal control (according to manufacturer instructions). Unpaired t test was adopted to compare CAR⁺.CIK with NTD.CIK. Significance is reported as * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Figure 4. In vivo anti-sarcoma activity of antiCD44v6 CAR⁺.CIK. CAR⁺.CIK were intravenously infused, every 3–4 days (total 8 infusions), in NOD/SCID mice bearing subcutaneous patient-derived UPS xenograft. Two different doses (4 × 10⁵ and 4 × 10⁶ per infusion) of CAR⁺.CIK were explored. We observed a significant delay of tumor growth in mice (n = 6) treated with CAR⁺.CIK cells (4 × 10⁵), compared with untreated controls (n = 6, p< 0.0001) or treated with equivalent doses of paired NTD.CIK cells (n = 6, p< 0.0001). A similar antitumor activity was observed with 1 log-higher dose of CAR⁺.CIK (4 × 10⁶). Arrows indicate CIK cell infusions. Results were analyzed by Two way Anova and correction for multiple comparisons test using Bonferroni method: Statistical significance is expressed as * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001 (A). In selected cases (1 representative mouse per group, n = 5) results were confirmed by a 3D imaging analysis based of fluorescent glucose uptake (B). Tumor homing of CAR⁺.CIK was confirmed by IHC in explanted tumors by staining with anti-human CD3 antibody (C). Abbreviations: CAR Chimeric Antigen Receptor; NTD, Not Transduced; NOD SCID mice, Non obese diabetic/severe combined immunodeficiency mice.