Adoptive immunotherapy with haploidentical natural killer cells and Anti-GD2 monoclonal antibody m3F8 for resistant neuroblastoma: Results of a phase I study

Figures & data

Figure 1. Schema of protocol therapy. Abbreviations: NK = Natural Killer; VINC = Vincristine; CY = Cyclophosphamide; TO = Topotecan.

Table 1. Clinical features and results of genotyping on patients and donors.

		N (%)
MYCN status (n = 34)	Amplified	9 (26)
	Non-amplified	25 (74)
Prior ASCT	Yes	9 (26)
	No	25 (74)
Prior m3F8 therapy	Yes	13 (37)
	No	22 (63)
Disease status prior to study entry	Primary refractory	13 (37)
	Secondary refractory	13 (37)
	Progressive disease	9 (26)
“Missing KIR Ligand”	Yes	21 (60)
	No	14 (40)
“Missing Self”	Yes	12 (34.3)
	No	23 (65.7)
Donor FCGR3A polymorphisms	F/F	12 (34.3)
	F/V	21 (60)
	V/V	1 (2.9)
	Unknown	1 (2.9)
Host FCGR3A polymorphisms	F/F	21 (60)
	F/V	9 (25.7)
	V/V	4 (11.4)
	Unknown	1 (2.9)

Abbreviations: ASCT: autologous stem cell transplant; KIR: Killer immunoglobulin-like receptor.

¹ “Missing KIR ligand” denotes those patients who lack any HLA ligand for their donor's inhibitory KIR, regardless of HLA ligands in the donor.

² “Missing Self” denotes those patients who lack HLA ligands present in the donor.

Table 2. Planned and actual dosage of haploidentical NK cells administered.

Planned dose level (No. NK cells)	Planned No. of patients	Actual No. of patients treated	Planned No. of infusions	Actual No. of infusions	Mean±SD NK cells/infusion (million/kg)
0 (<1×10^6/kg)	0	3 (9%)	0	3	0.4 ± 0.27
I (1–4.99×10^6/kg)	6	11 (31%)	6	11	3.54 ± 1.45
2 (5–9.99×10^6/kg)	3	6 (17%)	6	11	8.53 ± 1.52
3 (10–30×10^6/kg)	6	14 (40%)	13	16	16.80 ± 5.38
4 (30–50×10^6/kg)	6	1 (3%)	9	1	32.6
Total	21	35	34	42

Abbreviations: NK: natural killer; No.: number; SD: standard deviation.

* raw value as mean could not be calculated.

Table 3. Numbers of patients experiencing toxicities.

	Toxicity Grade: numbers of patients
	1	2	3	4
Anemia		3 (9%)	27 (77%)	5 (14%)
Lymphopenia				35 (100%)
Neutropenia			6 (17%)	29 (83%)
Thrombocytopenia				35 (100%)
Hypotension	4 (11%)	4 (11%)
Non neutropenic fever	16 (46%)	3 (9%)	1 (3%)
Urticaria	3 (9%)	22 (63%)
Anorexia	8 (23%)	3 (9%)
Diarrhea	7 (20%)
Vomiting	7(20%)	20 (57%)	5 (14%)
Febrile neutropenia			32 (91%)
Elevated alanine aminotransferase	22 (63%)	5 (14%)	2 (6%)
Elevated aspartate aminotransferase	25 (71%)	1 (3%)	1 (3%)
Hypomagnesemia	9 (26%)
Hypokalemia	12 (46%)		1 (3%)
Hyponatremia	19 (54%)
Pain		35 (100%)
Cough	11 (31%)	1 (3%)
Hypertension	1 (3%)	7(20%)	1 (3%)
Flushing	1 (3%)	8 (23%)
Left ventricular dysfunction		1 (3%)
Hyperbilirubinemia	3 (9%)	2(6%)
Hyperkalemia	2(6%)
Fatigue	11(31%)
Bronchospasm	3 (9%)	3 (9%)
Hypoxia/Pneumonitis				1 (3%)

* Related to chemotherapy;

** Related to m3F8.

Table 4. Responses.

Response	CR	PR	NR	PD
Best response by INRC (n = 35)	5 (14%)	5 (14%)	17 (49%)	8 (23%)
Best site-specific responses
Bone marrow disease (involved by histology pre-treatment in 16 patients)	9 (56%)	N/A	7 (44%)	N/A
MIBG scan (n = 35)	5 (14%)	5 (14%)	11 (31%)	5 (14%)
Soft tissue disease (detected pre-treatment in 14 patients)	0	0	9 (64%)	5 (36%)

* Objective responses not amounting to PR were noted in 1/11 and 4/9 patients deemed to have NR on MIBG-avid skeletal and soft tissue disease respectively

Abbreviations: CR: complete remission; INRC: International Neuroblastoma Response Criteria; MIBG: metaiodobenzylguanidine; N/A: not applicable; NR: no response; PD: progressive disease; PR: partial response.

Table 5. Relationships between response and dose and other factors.

		Modified INRC			Bone Marrow Histology			MIBG scan (Reduction in MIBG score)
Factors tested		CR/PR n (%)	NR/PD n (%)	p-value	Complete response n(%)	No response n(%)	p-value	Median (Range) reduction in MIBG score	p-value
NK cell Dose Level	0–1	3 (21.4)	11 (78.6)	0.70	4 (28.6)	10 (71.4)	>0.95	−1 (−5 to +2)	0.71
	1–2	7 (33.3)	14 (66.7)		5 (26.3)	14 (73.7)		−1 (−18 to +5)
MYCN	Negative	8 (80)	19 (79.2)	>0.95	8 (88.9)	18 (78.3)	0.65	−2 (−18 to +5)	0.45
	Positive	2 (20)	5 (20.8)		1 (11.1)	5 (21.7)		−1 (−1 to 0)
Missing KIR Ligand	No	4 (40)	10 (40)	>0.95	3 (33.3)	9 (37.5)	>0.95	−1 (−18 to +5)	0.23
	Yes	6 (60)	15 (60)		6 (66.7)	15 (62.5)		0 (−2 to +2)
Missing Self	No	7 (70)	16 (64)	>0.95	5 (55.6)	16 (66.7)	0.69	−1 (−18 to +5)	0.56
	Yes	3 (30)	9 (36)		4 (44.4)	8 (33.3)		−1 (−10 to +2)
Donor Polymorphism	F/F	5 (50)	7 (29.2)	0.27	4 (44.4)	8 (34.8)	0.70	−1 (−18 to +5)	0.85
	F/V or V/V	5 (50)	17 (70.8)		5 (55.6)	15 (65.2)		−2 (+2 to −10)
Host Polymorphism	F/F	8 (80)	13 (54.2)	0.25	4 (44.4)	16 (69.6)	0.24	−2 (0 to −10)	0.14
	F/V or V/V	2 (20)	11 (45.8)		5 (55.6)	7 (30.4)		0 (−18 to +5)

Abbreviations: CR: complete remission; INRC: International Neuroblastoma Response Criteria; MIBG: metaiodobenzylguanidine; N/A: not applicable; NR: no response; PD: progressive disease; PR: partial response.

Figure 2. Complete response to protocol therapy in a 6-year old male with chemorefractory high-risk stage 4 neuroblastoma after 2 cycles of therapy at NK-cell dose level 3. Pre-therapy ¹²³I-MIBG scan showed uptake in multiple skeletal areas including pelvis, bilateral femora and spine. Post-therapy ¹²³I-MIBG scan showed complete resolution of skeletal uptake. Complete response was also noted on bone marrow histology.

Table 6. Correlation of NK cytotoxicity and donor-recipient HLA/KIR phenotyping.

				NKG2A	Global expression on total NK (%)			NK cytotoxic response to 3F8-mediated ADCC (%)
Pt#	Donor inhibitory KIR	Donor KIR ligands	Recipient KIR ligands	% NK cells	2DL1	2DL2/3	3DL1	2DL1	2DL2/3	3DL1
1	2DL2, 3DL1	C1, Bw4	C1, C2, Bw4	51	0	33.6	3.4	NP	22.5	35.3
2	2DL1, 2DL3, 3DL1	C1, C2, Bw4	C1	76.2	20.3	29.3	43.5	40.7	47.3	36.6
3	2DL1, 2DL3, 3DL1	C1, Bw4	C1, Bw4	63.7	10	18.9	14.5	18	44	42
4	2DL1, 2DL3, 3DL1	C1, C2, Bw4	C1, Bw4	58.7	17.5	18.5	29.5	44	43	48
5	2DL1, 2DL2, 2DL3, 3DL1	C1, Bw4	C1	45.6	15.5	33.5	18.5	26	35	37
6	2DL1, 2DL2, 3DL1	C1, C2, Bw4	C1, C2	63.5	3.51	41.6	34.6	31.6	30.1	43
7	2DL1, 2DL3, 3DL1	C1, C2	C1	54.5	29.4	28.7	15.5	18.8	21.3	24.4
8	2DL1, 2DL2, 2DL3, 3DL1	C1, C2, Bw4	C1	59.8	19.4	27.4	16.8	49.5	44	57.3
9	2DL1, 2DL3, 3DL1	C1	C1, C2, Bw4	55.4	15.3	27.4	15.2	38	33.5	33.1
10	2DL1, 2DL2, 2DL3, 3DL1	C1, C2, Bw4	C1	46	10	29.5	22.1	28.5	15.6	22.8
12	2DL1, 2DL2, 2DL3, 3DL1	C1, C2	C1, C2, Bw4	48.6	18.8	22	6.09	28.7	32.6	37.2
12	2DL1, 2DL2, 2DL3, 3DL1	C1, Bw4	C1, C2, Bw4	48.9	6.74	23.9	7.8	73.9	76.9	80

Abbreviations: ADCC: Antibody-dependent cell mediated cytotoxicity; NP: non performed; Pt: Patient.

Donor inhibitory KIR shown in bold face indicate self-HLA-specific inhibitory KIR predictive of licensed population; Global expression % depicted in bold face indicates missing self-HLA KIR+ NK cells; Italicized expression % indicates missing ligand KIR+ NK cells.

Figure 3. Donor and patient NK function. NK-cells were isolated from donors, the patient pre-treatment, and the patient post-treatment at indicated time points. NK response capacity was assessed by CD107a mobilization to (A) the NK-sensitive K562 cell line, or to (B) the neuroblastoma cell line LAN-1 in the presence of the m3F8 monoclonal antibody. 13 donors and 19 patients were assessed. Each bar represents the mean +/− SEM.

Figure 4. NKG2A expression among donors and patients. A high percentage of NKG2A expression was noted in the patient population pre- and post-infusion (A) compared to a healthy adult population (B).

Figure 5. HLA-E expression on NB selectively inhibits NKG2A-expressing NK cells. (A) HLA-E is readily upregulated on the cell surface of the NB cell line BE(2)N upon IFN-γ treatment. (B) m3F8-induced CD107a response among NKG2A+KIR- NK cells (left panel) or NKG2A-KIR- NK cells (right panel) to the IFN-γ treated or untreated BE(2)N target cell in the presence or absence of the anti-NKG2A blocking antibody. Similar results were noted with SKNLH cell line (data not shown)