Ewing sarcoma partial regression without GvHD by chondromodulin-I/HLA-A*02:01-specific allorestricted T cell receptor transgenic T cells

Figures & data

Figure 1. CHM1 and EZH2 relative gene expression in ES primary tumors and cell lines vs. controls. (A) Expression profile of CHM1 and EZH2 in ES primary samples, patient's-derived HLA-A*02:01⁺ cell lines ES-P#1, ES-P#2, ES-P#3, osteosarcoma primary samples, and normal body atlas (NBA) analyzed by microarray (GSE45544 and GSE73166). (B) Real-time RT-PCR confirms upregulation of CHM1 in patient-derived HLA-A*02:01⁺ cell lines ES-P#2, ES-P#3 in comparison to controls. EZH2 is upregulated in ES-P#1, ES-P#2, ES-P#3 in comparison to controls. A673, HLA-A*02:01⁺ ES cell line; SKNMC, SHSY5Y, and SIMA, neuroblastoma cell lines; SaOS, U2OS, osteosarcoma cell lines were used as controls. Analyses were performed as duplicates. Results were normalized to GAPDH and quantified by the ddCt-method.

Figure 2. Donor-derived HLA-A*02:01/CHM1³¹⁹ TCR transgenic T cells specifically recognize HLA-A*02⁺/CHM1⁺ cell lines in vitro. (A) CHM1³¹⁹-TCR-transgenic T cells specifically recognize CHM1³¹⁹-loaded T2 cells and (B) HLA-A*02:01⁺/CHM1⁺ Ewing sarcoma cell lines A673, TC-71, and the patient-derived cell line SB-KMS-GH in contrast to HLA-A*02:01⁻/CHM1⁺ SK-NMC, K562, and SB-KMS-KS1 control cell lines in IFNγ ELISpot assays. The E/T ratio for ELISpot assay was 1:4. Error bars represent standard deviation of triplicate experiments. Asterisks indicate significance levels.

Figure 3. Transferred HLA-A*02:01/CHM1³¹⁹ T cell receptor transgenic T cells home to bone marrow of patient #2 and persist. (A) Bone marrow (BM) aspirates from the left posterior iliacal spine (LPIS) stain positive for the CHM1³¹⁹ multimer at a stronger rate than BM of the right posterior iliacal spine (RPIS) at day 6 after first adoptive transfer. Peripheral blood (PB) cells do not stain CHM1³¹⁹ multimer positive. (B) At day 14 after first transfer BM aspirates and peripheral blood cells convert CHM1 multimer negative, demonstrating loss of adoptively transferred T cells after first adoptive transfer (x-axis; CHM1³¹⁶multimer⁺/irrelevant multimer⁺ CD8⁺ staining ratio). (C) and (D) At day 14 after second adoptive transfer BM aspirates show a stronger CHM1³¹⁹ multimer flourescence in RPIS but CHM³¹⁹ loss in LPIS. PB is CHM1³¹⁹ multimer positive and HLA-A*02:01/CHM1³¹⁹ T cell receptor transgenic T cells proliferate until last PB draw on day 27 after second (day 56 after first) adoptive transfer. An irrelevant EZH2⁶⁶⁶/HLA-A*02:01 restricted multimer serves as control (CD8⁺ gate).

Figure 4. PET-CT FDG-uptake reduction in multiple bone sites after second adoptive transfer. After second adoptive transfer, prior PET-CT positive sites (left panel) at the (A) left iliac spine, (B) right iliac spine and right acetabular roof, as well as (C) left femoral neck, left acetabular piller, and calvarium showed significant regression with respect to metabolic volume and SUV^max (right panel).

Table 1. Disease course of single PET-CT positive sites after first and second adoptive transfer of HLA-A*02:01/CHM1³¹⁹ TCR transgenic T cells compared with reference PET-CT before therapy (Patient #2).

	After first adoptive transfer			After second adoptive transfer
	Progressive sites	New metastases	Regressive sites	Progressive sites	New metastases	Regressive sites
Lower extremity	1	1	0	1	7	1
Pelvis	3	1	0	1	2	4
Upper extremity	0	0	0	0	1	0
Lungs	approx. 50	approx. 30	0	>50	>50	0
Skull	0	0	0	0	0	1